Deep sequencing-based analysis of the anaerobic stimulon in Neisseria gonorrhoeae

  • Vincent M Isabella1 and

    Affiliated with

    • Virginia L Clark1Email author

      Affiliated with

      BMC Genomics201112:51

      DOI: 10.1186/1471-2164-12-51

      Received: 19 November 2010

      Accepted: 20 January 2011

      Published: 20 January 2011

      Abstract

      Background

      Maintenance of an anaerobic denitrification system in the obligate human pathogen, Neisseria gonorrhoeae, suggests that an anaerobic lifestyle may be important during the course of infection. Furthermore, mounting evidence suggests that reduction of host-produced nitric oxide has several immunomodulary effects on the host. However, at this point there have been no studies analyzing the complete gonococcal transcriptome response to anaerobiosis. Here we performed deep sequencing to compare the gonococcal transcriptomes of aerobically and anaerobically grown cells. Using the information derived from this sequencing, we discuss the implications of the robust transcriptional response to anaerobic growth.

      Results

      We determined that 198 chromosomal genes were differentially expressed (~10% of the genome) in response to anaerobic conditions. We also observed a large induction of genes encoded within the cryptic plasmid, pJD1. Validation of RNA-seq data using translational- lacZ fusions or RT-PCR demonstrated the RNA-seq results to be very reproducible. Surprisingly, many genes of prophage origin were induced anaerobically, as well as several transcriptional regulators previously unknown to be involved in anaerobic growth. We also confirmed expression and regulation of a small RNA, likely a functional equivalent of fnrS in the Enterobacteriaceae family. We also determined that many genes found to be responsive to anaerobiosis have also been shown to be responsive to iron and/or oxidative stress.

      Conclusions

      Gonococci will be subject to many forms of environmental stress, including oxygen-limitation, during the course of infection. Here we determined that the anaerobic stimulon in gonococci was larger than previous studies would suggest. Many new targets for future research have been uncovered, and the results derived from this study may have helped to elucidate factors or mechanisms of virulence that may have otherwise been overlooked.

      Background

      N. gonorrhoeae was long considered to be an obligate aerobe until it was discovered that anaerobic growth was possible when nitrite or nitric oxide (NO) was used as a terminal electron acceptor [1, 2]. Anaerobic growth is accomplished through utilization of a truncated denitrification pathway, which is encoded within the gonococcal genome as a pair of divergently transcribed genes, aniA, encoding a nitrite reductase, and norB, encoding a nitric oxide reductase [3, 4]. Anaerobiosis is presumed to be a physiologically significant state during infection, as the gonococcus is often recovered from infected individuals in co-culture with obligate anaerobes such as Peptococcus and Bacteroides spp. [5]. Furthermore, gonococci have been shown to induce and repress the expression of several genes in response to anaerobiosis, and antibody to AniA, the major anaerobically induced outer membrane protein, can be found in sera from infected women, demonstrating that this protein is expressed in vivo [6, 7].

      The ability of gonococci to utilize this denitrification pathway to reduce NO may have immunomodulary effects during the course of infection. Some evidence suggests that the gonococcal reduction of host-produced NO may be responsible, at least in part, for the high incidence of asymptomatic disease [4, 8, 9]. In vitro, N. gonorrhoeae was shown to be capable of setting a NO steady-state level in the anti-inflammatory range [8]. Recently, in an in vitro cervical cell model of infection, it was shown that gonococcal activation of iNOS promoted bacterial survival. In this study, it was suggested that host-derived nitric oxide is not protective against gonococci, rather, nitric oxide may actually be required to sustain cervical bacterial disease [10].

      Previous research has shown that the genes involved in denitrification and/or adaptation to anaerobic growth in Neisseria spp. are subject to transcriptional regulation by the oxygen-sensitive regulator, FNR, the NO-sensitive repressor, NsrR, and the nitrite-insensitive two component system, NarQP [3, 4, 7, 11, 12]. Earlier data from a microarray-based approach suggested that the gonococcal FNR-regulon was composed of fourteen activated and six repressed transcripts, making the gonococcal FNR regulon much smaller than that of E. coli, where FNR was shown to regulate more than 100 operons [7, 13]. Discovery of NsrR and NarQP-regulated genes in Neisseria spp. has relied mainly on in silico analysis of intergenic chromosomal regions to identify similarity to previously defined regulator binding sites. As is the case with FNR, the neisserial NsrR and NarQP regulons, at least the currently defined members, are much smaller than their E. coli counterparts [3, 12, 14, 15].

      In this study we employ a powerful whole-genome approach, RNA-seq, to quantitatively sequence the complete gonococcal transcriptome. Using this method we were able to define global changes in gene expression in response to anaerobiosis. We show that the gonococcal anaerobic stimulon is not small, and that 198 chromosomal open reading frames (~10% of the genome) are differentially expressed. We present several novel findings that, taken together, support the view that anaerobic growth is an important facet of life for this organism, and should be considered when studying the host/pathogen interaction.

      Results and Discussion

      Sequencing the gonococcal transcriptome

      Whole genome mRNA sequencing is an attractive method of monitoring global changes in gene expression while overcoming many of the pitfalls of traditional DNA microarrays [16]. For the purposes of this study, anaerobic conditions were defined as anoxia, the presence of nitrite (an electron acceptor required for anaerobic growth) and the concomitant presence of nitric oxide (the product of nitrite reduction). In order to define the gonococcal anaerobic stimulon, RNA-seq was performed on two biological replicates of aerobically or anaerobically plate-grown gonococci using the ABI SOLiD™ system (see methods). Unique sequence reads from the RNA-seq data were mapped to the annotated FA1090 genome, and gene expression was quantified as reads per kilobase of coding sequence per million reads (RPKM) (See additional file 1 : Supplementary Table S1). When expression data for each replicate were plotted against each other, RPKM values were observed to be adequately reproducible (Figure 1A), though expectedly, the extent of reproducibility was slightly less in genes with low expression [17]. For this reason, we required mapped genes to have an RPKM ≥ 10 in order to be considered as a candidate for differential expression. However, genes that had an RPMK < 10 under one growth condition, but were highly induced in the other condition were accepted as candidates. Genes were considered to be differentially expressed if there was a three-fold or greater difference in RPKM between the two growth conditions.
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-51/MediaObjects/12864_2010_3190_Fig1_HTML.jpg
      Figure 1

      Reproducibility of expression level between replicates. (A) The RPKM of all genes with non-zero expression, (B) anaerobically induced, and (C) anaerobically repressed genes were plotted, with the RPKM of replicate 1 plotted on the x-axis, and the RPKM of replicate 2 plotted on the y-axis. Each spot represents a single gene. Blue circles represent genes expressed aerobically, and red squares represent genes expressed anaerobically. For (B) and (C) a line of m = 1 is included in the chart area.

      Analysis of RNA-seq data revealed that 198 chromosomal genes met the criteria for being differentially expressed, with 117 genes being anaerobically induced and 81 genes being anaerobically repressed (Table 1). Figure 1B and 1C illustrate a reasonable measure of reproducibility in expression level between replicates in the 198 differentially expressed genes.
      Table 1

      Chromosomal genes differentially expressed in response to anaerobic conditions a

      Gene

      ORF ID

      Rep 1 b

      Rep 2

      Definition c

      Regulon d

      Small molecule biosynthesis

      hda

      NGO0187

      4.1

      4.1

      Histone deacetylase-like protein/acetoin utilization protein

       

      pdxH

      NGO0658

      4.3

      5.5

      Pyridoxamine 5-phosphate oxidase

      MtrR

      mobA

      NGO0754

      3.5

      4.6

      Molybdopterin-guanine dinucleotide biosynthesis protein

      NsrR

      leu1

      NGO0848

      6.6

      4.5

      2-isopropylmalate synthase

       

      folA

      NGO1694

      3.8

      2.8

      Dihydrofolate reductase

      RpoH

      Transport and binding proteins

      fHBP

      NGO0033

      3.5

      4.2

      Factor H binding protein

       

      hecA

      NGO0706

      3.2

      4.9

      Filamentous hemagglutinin

       

      bfrA

      NGO0794

      14.1

      9.9

      Bacterioferritin A

       

      bfrB

      NGO0795

      9.7

      10.1

      Bacterioferritin B

      MtrR

      pil

      NGO1177

      3.5

      3.5

      Neisseria-specific type IV pilin-related protein

       
       

      NGO1440

      4.4

      2.8

      ABC-type amino acid transporter, periplasmic protein

       

      lecA

      NGO1981

      5.0

      3.9

      Adhesin protein

      Lrp

      Energy metabolism/Oxidation-Reduction

      cybP

      NGO0805

      3.2

      2.8

      Nickel-dependent hydrogenase, b-type cytochrome subunit

       

      npd

      NGO1024

      9.6

      7.9

      2-Nitropropane dioxygenase-like

      MtrR

      aniA

      NGO1276

      28.4

      28.0

      Nitrite reductase

      NsrR, FNR

      eftD

      NGO1396

      5.2

      5.1

      Electron transfer flavoprotein-ubiquinone oxidoreductase

       

      Macromolecular biosynthesis

      rl34

      NGO2182

      3.0

      3. 1

      50S ribosomal protein L34

       

      Regulation

           

      farR

      NGO0058

      4.0

      3.7

      MarR family transcriptional regulator

      Lrp, MtrR

      nmlR

      NGO0602

      7.2

      7.6

      Transcriptional regulator nmlR

      NmlR, FNR

      xre

      NGO0797

      3.2

      3.4

      XRE family transcriptional regulator

       

      marR

      NGO1244

      5.4

      15.2

      MarR-family transcriptional regulator

      RpoH, MtrR

      nosR a

      NGO1401/1402

      9.4

      9.5

      Regulator of nitrous oxide reductase pseudogenes

      FNR

      lexA

      NGO1427

      4.2

      5.3

      LexA-family repressor

      LexA

      arsR

      NGO1562

      6.1

      2.2

      ArsR-family transcriptional regulator

       

      Cell Structure

      ompA

      NGO1559

      3.7

      3.4

      Outer membrane protein

       

      lpcA

      NGO1986

      4.2

      6.1

      Phosphoheptose isomerase

       

      Adaptation and stress response

      grx3

      NGO0114

      3.3

      6.6

      Glutaredoxin 3

      Fur

      recN

      NGO0318

      4.7

      5.5

      Repair protein RecN

      Fur, LexA

      htpX

      NGO0399

      11.8

      8.6

      Heat shock protein, Peptidase family M48

      Lrp

      dnrN

      NGO0653

      8.9

      12.3

      Iron-sulfur cluster repair protein

      NsrR, FNR

       

      NGO0757

      2.6

      4.1

      Putative periplasmic Cpx-family sensor kinase involved in P pilus formation

       

      hslR

      NGO0822

      2.7

      5.0

      Heat shock protein HSP-15

       
       

      NGO1022

      3.8

      3.4

      DEDDh family exonuclease

       

      clpB

      NGO1046

      4.9

      9.4

      Endopeptidase Clp ATP-binding chain B (HSP- F84.1)

      RpoH, MtrR

      recX

      NGO1053

      3.6

      2.8

      RecX-family regulator of RecA function

       

      tehB

      NGO1161

      2.6

      4.0

      Tellurite resistance protein

       

      hsp33

      NGO1189

      6.1

      5.2

      Heat shock protein HSP-33 chaperonin

      MtrR

       

      NGO1245

      4.0

      8.5

      ABC-type multidrug transport system, ATPase/permease

       

      hit

      NGO1273

      2.3

      5.5

      Protein Kinase C Interacting protein belonging to the ubiquitous HIT family of hydrolases that act on alpha-phosphates of ribonucleotides

       

      nudH

      NGO1334

      3.3

      3.3

      Nudix hydrolase, dinucleoside polyphosphate hydrolase

       

      grpE

      NGO1422

      3.6

      2.7

      Heat shock protein (HSP-70 cofactor; nucleotide exchange factor)

      RpoH, MtrR

      erpA

      NGO1426

      2.8

      2.6

      Putative iron-sulfur cluster insertion protein

      RpoH, Lrp

      dnaK

      NGO1429

      2.7

      3.4

      Heat shock protein, chaperone

      RpoH

       

      NGO1566

      3.4

      3.1

      Nudix hydrolase, catalyzing the hydrolysis of nucleoside diphosphates

       

      ntrA

      NGO1702

      3.5

      20.1

      Nitroreductase-like family containing uncharacterized proteins similar to nitroreductase

       

      ccp

      NGO1769

      5.6

      12.1

      Cytochrome c peroxidase

       

      yhhF

      NGO1860

      3.7

      4.1

      Probable DNA methylase

       

      dnaJ

      NGO1901

      2.8

      5.1

      Heat shock protein HSP-40

      RpoH

      Hypothetical or Unassigned

       

      NGO0010

      5.1

      7.2

      Neisseria-specific protein, uncharacterized

       
       

      NGO0011

      3.2

      3.0

      Conserved hypothetical protein

       
       

      NGO0119

      5.4

      5.1

      Neisseria-specific protein, uncharacterized; possible ribonuclease

       
       

      NGO0165

      3.2

      5.9

      Neisseria-specific protein, uncharacterized

       
       

      NGO0569

      3.8

      2.1

      Conserved hypothetical protein (possible transcriptional regulator)

       
       

      NGO0570

      3.5

      3.4

      Possible DNA-binding CreA protein

      RpoH

       

      NGO0618

      3.3

      2.7

      Neisseria-specific protein, uncharacterized

       
       

      DUF331

      4.1

      7.8

      Uncharacterized protein family DUF331; the function of this family is unknown

       
       

      NGO0802

      3.7

      4.0

      Hypothetical protein (possible Neisseria-specific protein)

       
       

      NGO0854

      3.6

      8.4

      Hypothetical protein

       
       

      NGO0895

      6.4

      4.6

      Neisseria-specific protein, uncharacterized

       
       

      NGO0896

      4.1

      5.0

      Hypothetical protein

       
       

      NGO0995

      3.1

      3.3

      Neisseria-specific protein, uncharacterized

       
       

      NGO1033

      4.3

      6.8

      Probable transglycosylase

       
       

      NGO1037

      4.4

      5.2

      Hypothetical protein

       
       

      NGO1191

      3.5

      2.7

      Neisseria-specific protein, uncharacterized

       
       

      NGO1277

      6.6

      7.0

      Conserved hypothetical protein (NirV) FGE- sulfatase super family.

       
       

      NGO1428

      14.3

      8.4

      Neisseria-specific protein, uncharacterized

      LexA

       

      NGO1517

      4.8

      4.8

      Neisseria-specific protein, uncharacterized

      NsrR

       

      NGO1793

      6.4

      4.3

      Conserved hypothetical protein (probable integral membrane protein)

       
       

      NGO1987

      3.1

      4.4

      Uncharacterized protein family UPF0102; the function of this family is unknown

       
       

      NGO2023

      2.4

      7.3

      Conserved hypothetical protein

       

      Small RNA fnrS c

       

      24.9

      17.8

      Probable small regulatory RNA

      FNR

      Gene

      ORF ID

      Rep 1

      Rep 2

      Definition

      Regulon

      Ngo Φ 1 phage

       

      NGO0463

      9.2

      5.9

      Putative phage associated protein

       
       

      NGO0464

      12.3

      9.0

      Putative phage associated protein

       
       

      NGO0465

      15.4

      7.3

      Putative phage associated protein

       
       

      NGO0467

      23.6

      29.8

      Putative phage associated protein

       
       

      NGO0472

      94.6

      19.7

      Putative phage associated protein

      FNR

      dnaB

      NGO0485

      6.1

      4.2

      Replicative DNA helicase

       
       

      NGO0488

      4.2

      10.0

      Putative phage associated protein

       
       

      NGO0489

      10.2

      16.9

      Phage Holliday junction resolvase (RusA-like) protein

       
       

      NGO0491

      14.2

      5.1

      Putative phage associated protein

       
       

      NGO0492

      8.7

      6.6

      Putative phage associated protein

       
       

      NGO0494

      10.0

      10.9

      Putative phage associated protein

       
       

      NGO0495

      9.6

      7.6

      Putative phage associated protein

       
       

      NGO0496

      16.1

      12.9

      Putative phage associated protein

       
       

      NGO0497

      24.1

      17.0

      Putative phage associated protein

       
       

      NGO0498

      18.0

      9.5

      Putative phage associated protein

       
       

      NGO0499

      17.4

      12.9

      Putative phage associated protein

       
       

      NGO0500

      17.6

      6.6

      Putative phage associated protein

       
       

      NGO0501

      27.4

      9.1

      Putative phage associated protein

       
       

      NGO0502

      47.2

      6.8

      Putative phage associated protein

       
       

      NGO0503

      6.0

      4.8

      Putative phage associated protein

       
       

      NGO0504

      14.4

      5.2

      Putative phage associated protein

       
       

      NGO0506

      15.9

      11.3

      Putative phage associated protein

       
       

      NGO0507

      22.3

      12.1

      Putative phage associated protein

       
       

      NGO0508

      15.6

      11.1

      Putative phage associated protein

       
       

      NGO0509

      3.5

      3.6

      Putative phage associated protein

       
       

      NGO0510

      7.1

      5.6

      Putative phage associated protein

       
       

      NGO0512

      9.4

      13.2

      Putative phage associated protein

       
       

      NGO0513

      4.9

      5.2

      Putative phage associated protein

       
       

      NGO0514

      9.8

      6.2

      Putative phage associated protein

       
       

      NGO0522

      9.4

      5.6

      Putative phage associated protein

       

      Ngo Φ2 phage

       

      NGO1100

      4.1

      5.0

      Putative phage associated protein

       
       

      NGO1120

      75.5

      16.0

      Putative phage associated protein

       
       

      NGO1131

      19.4

      16.8

      Putative phage associated protein

       
       

      NGO1132

      11.3

      16.2

      Putative phage associated protein

       

      Ngo Φ3 phage

       

      NGO1614

      8.2

      11.0

      Putative phage associated protein

       
       

      NGO1615

      10.3

      7.2

      Conserved hypothetical protein (type I restriction enzyme related)

      FNR

       

      NGO1624

      34.0

      16.0

      Putative phage associated protein

       
       

      NGO1627

      6.6

      4.4

      Conserved hypothetical protein (probable phage origin)

       
       

      NGO1628

      7.6

      4.9

      Putative phage associated protein

       
       

      NGO1633

      26.9

      137.3

      Putative phage associated protein

       
       

      NGO1634

      27.4

      18.8

      Putative phage associated protein

       
       

      NGO1635

      58.1

      5.4

      Putative phage associated protein

       
       

      NGO1636

      26.4

      10.0

      Putative phage associated protein

       
       

      NGO1637

      16.6

      5.5

      Putative phage associated protein

       
       

      NGO1640

      22.4

      10.6

      Putative phage associated protein

       

      Ngo Φ5 phage

       

      NGO0731

      5.1

      7.3

      Putative phage associated protein

       
       

      NGO0732

      3.3

      2.2

      Putative phage associated protein

       

      Gene

      ID

      Rep 1

      Rep 2

      Definition

      Regulon

      Small molecule biosynthesis

      cysK

      NGO0340

      -2.8

      -6.7

      Cysteine synthase

      FNR

      hesB

      NGO0632

      -3.8

      -4.0

      Iron-sulfur cluster biosynthesis

       

      iscU

      NGO0633

      -4.8

      -5.3

      Iron-sulfur cluster assembly scaffold protein

       

      iscS

      NGO0636

      -4.8

      -5.6

      Cysteine desulferase

       

      hisD

      NGO1240

      -3.3

      -3.2

      Histidinol dehydrogenase

       

      pgsA

      NGO1247

      -3.8

      -7.1

      CDP-alcohol phosphatidyltransferase

       

      asd

      NGO1997

      -3.3

      -5.0

      Aspartate-semialdehyde dehydrogenase

       

      Transport and binding proteins

      gluP

      NGO0142

      -3.1

      -4.3

      Glucose/galactose transporter

       

      nhaC

      NGO0143

      -2.8

      -3.3

      Na+/H+ antiporter (NhaC)

       

      amtB

      NGO0198

      -3.7

      -5.3

      Ammonium Transporter Family

       

      fbp

      NGO0217

      -4.8

      -5.6

      ABC-type iron transporter, periplasmic binding protein

      Fur, Lrp

      oxiA

      NGO0372

      -6.7

      -20.0

      Bacterial extracellular solute-binding protein

      FarR

       

      NGO0373

      -3.7

      -11.1

      ABC-type arginine transport system, permease component

      FarR

      glnQ

      NGO0374

      -2.9

      -11.1

      ABC-type amino acid transporter, ATP-binding protein

      FarR, FNR

      citT

      NGO0377

      -5.6

      -9.1

      Di- and tri-carboxylate transporters (inorganic ion transport)

       

      cysA

      NGO0445

      -5.6

      -9.1

      ABC-type sulfate transporter, ATP-binding protein

      Lrp

      sbp

      NGO0877

      -4.8

      -25.0

      ABC-related sulfate-binding protein

       
       

      NGO1290

      -3.8

      -3.3

      Putative sodium/alanine symport protein

       

      hemO

      NGO1318

      -7.1

      -12.5

      Heme oxygenase/iron starvation protein

      Fur

      lldP

      NGO1361

      -9.1

      -8.3

      L-lactate permease (partial only)

       

      exbD

      NGO1377

      -3.4

      -3.6

      Membrane bound biopolymer transport protein ExbD/TolR

       

      exbB

      NGO1378

      -3.1

      -5.0

      Transport protein

       

      lctP

      NGO1449

      -4.0

      -14.3

      L-lactate permease

       

      potF

      NGO1494

      -7.7

      -20.0

      ABC-type spermidine/putrescine transporter, solute binding protein

      MtrR

      putP

      NGO1552

      -5.6

      -11.1

      Sodium/proline symport protein

       

      ompU

      NGO1688

      -2.6

      -3.7

      Putative iron uptake protein

      FNR

      yaaJ

      NGO1787

      -3.3

      -4.3

      Sodium/alanine symport protein

       
       

      NGO1807

      -2.7

      -3.8

      Amino-acid symport protein

       

      gltS

      NGO1890

      -3.1

      -5.0

      Sodium/glutamate symport protein

      MtrR

       

      NGO1954

      -3.6

      -6.3

      Di- and tri-peptide permease, PRT2

       

      sstT

      NGO1957

      -4.5

      -11.1

      Sodium/serine symport protein

       

      glnM

      NGO2011

      -6.7

      -12.5

      ABC-type amino acid transport system, permease component

      MtrR, FarR

      glnP

      NGO2012

      -10.0

      -10.0

      ABC-type amino acid transport system, permease component

      FarR

      glnQ

      NGO2013

      -6.3

      -11.1

      ABC-type amino acid transporter, ATP-binding protein

      MtrR, FarR

      apaA

      NGO2014

      -6.3

      -16.7

      Bacterial extracellular solute-binding proteins, family 3

      MtrR, FarR

       

      NGO2016

      -3.6

      -3.3

      Predicted permease

       
       

      NGO2096

      -6.3

      -12.5

      SNF family sodium-dependent transporter

       

      Energy metabolism/Oxidation-Reduction

       

      NGO0108

      -4.0

      -5.9

      NADPH-dependent FMN reductase

      Fur, Lrp

      lldD

      NGO0639

      -7.1

      -11.1

      L-lactate dehydrogenase

       

      eda

      NGO0713

      -3.0

      -5.6

      KHG-KDPG bifunctional aldolase

       

      rpeC

      NGO0758

      -4.8

      -3.8

      Ribulose-phosphate 3-epimerase

       

      fdx

      NGO0825

      -5.3

      -5.3

      Ferredoxin

       

      cisY

      NGO0918

      -3.6

      -3.8

      Type II citrate synthase

      Lrp

      sdhB

      NGO0920

      -2.9

      -4.2

      Succinate dehydrogenase, iron-sulfur protein

      Lrp

      sdhA

      NGO0921

      -3.7

      -3.4

      Succinate dehydrogenase, flavoprotein subunit

      Lrp

      sdhD

      NGO0922

      -2.9

      -3.4

      Succinate dehydrogenase, membrane anchor protein

       

      sdhC

      NGO0923

      -1.8

      -4.2

      Succinate dehydrogenase, cytochrome b556 chain

       

      fumC

      NG01029

      -2.9

      -6.3

      Fumarate hydratase

      Fur, MtrR

      gpmA

      NGO1258

      -4.0

      -5.6

      Phosphoglyceromutase

      Lrp

      nqrA

      NGO1413

      -4.0

      -7.7

      Na(+)-translocating NADH-quinone reductase subunit A

       

      nqrB

      NGO1414

      -5.0

      -8.3

      Na(+)-translocating NADH-quinone reductase subunit B

       

      nqrC

      NGO1415

      -4.2

      -8.3

      Na(+)-translocating NADH-quinone reductase subunit C

       

      nqrD

      NGO1416

      -3.8

      -16.7

      Na(+)-translocating NADH-quinone reductase subunit D

       

      nqrE

      NGO1417

      -4.3

      -12.5

      Na(+)-translocating NADH-quinone reductase subunit E

       

      nqrF

      NGO1418

      -3.7

      -9.1

      Na(+)-translocating NADH-quinone reductase subunit F

      MtrR

      dadA

      NGO1808

      -3.6

      -5.3

      D-amino acid dehydrogenase small subunit

       

      mqo

      NGO1980

      -3.7

      -5.0

      Malate:quinone oxidoreductase

       

      Macromolecular biosynthesis

      greB

      NGO0262

      -3.7

      -5.3

      Transcription elongation factor

       

      parC

      NGO1259

      -3.8

      -2.9

      DNA topoisomerase IV subunit A

       
       

      NGO1261

      -6.3

      -7.1

      S-adenosylmethionine-dependent methyltransferase

       

      rplP

      NGO1831.1

      -3.2

      -6.3

      50S ribosomal protein L16

      Lrp

      rpsC

      NGO1832

      -3.2

      -7.7

      30S ribosomal protein S3

      Lrp

      rplV

      NGO1833

      -2.9

      -10.0

      50S ribosomal protein L22

       

      rpsS

      NGO1834

      -2.8

      -12.5

      30S ribosomal protein S19

       

      rplD

      NGO1837

      -2.2

      -7.1

      50S ribosomal protein L4

       

      rspJ

      NGO1841

      -2.7

      -7.1

      30S ribosomal protein S10

      Lrp

      rpoC

      NGO1850

      -3.3

      -5.0

      DNA-directed RNA polymerase subunit beta'

       

      rpoB

      NGO1851

      -2.1

      -4.5

      DNA-directed RNA polymerase subunit beta

       

      Cell Structure

      nspA

      NGO0233

      -3.1

      -3.7

      Outer membrane protein (Probable Opa protein)

       

      GNA2132

      NGO1958

      -5.3

      -8.3

      Predicted lipoprotein GNA2132

       

      lgtG

      NGO2072

      -7.7

      -6.3

      Probable lipooligosaccharide glycosyl transferase G

       

      Adaptation and stress response

      cspA

      NGO0410

      -3.8

      -3.3

      Cold shock protein

       

      trxB

      NGO0580

      -2.6

      -4.8

      Thioredoxin reductase

      NmlR

      trx1

      NGO0652

      -5.3

      -6.3

      Thioredoxin I

      Fur

      cstA

      NGO1064

      -3.8

      -12.5

      Putative carbon starvation protein

       

      mtrF

      NGO1368

      -7.7

      -25.0

      Antibiotic resistance efflux pump component

      MtrR, FarR

      sspB

      NGO2131

      -4.5

      -3.6

      Protease specificity-enhancing factor

       

      Hypothetical or unassigned

       

      NGO0554

      -4.5

      -5.0

      Hypothetical protein

      Fur

       

      NGO0635

      -4.8

      -3.6

      Hypothetical protein

       
       

      NGO1065

      -4.8

      -7.7

      Hypothetical protein

       
       

      NGO2097

      -3.7

      -3.1

      Conserved hypothetical protein

       

      Small RNA

      nrrF e

       

      -4.8

      -7.7

      Fur-repressed small regulatory RNA

      Fur

      a Gonococci grown in an anaerobe chamber on plates containing 5 mM nitrite.

      b The fold change for each replicate was calculated by comparing RPKM values of anaerobically grown gonococci to RPKM values of aerobically grown gonococci. A positive value represents an induction of gene expression anaerobically, and a negative value represents a repression of gene expression anaerobically

      c Protein definitions are derived from the annotated FA1090 genome (NCBI), and/or the NCBI conserved domain database [33].

      d (Fur) Genes that have been shown to be directly regulated by Fur in Sebastian et al., (2002) [95] and/or Jackson et al., (2010) [75]. (RpoH) Genes reported to be upregulated during RpoH overexpression in Gunesekere et al., (2006) [23]. (LexA) Genes reported to be in the LexA regulon by Schook et al., (2010) [50]. (Lrp) Genes found to be differentially expressed in a N. meningitidis Δ lrp mutant (NMB0573, closest gonococcal homolog is NGO1407, NCBI) by microarray analysis in Ren et al., (2007) [46]. (MtrR) Genes found to be differentially expressed in a gonococcal Δ mtrR mutant by microarray analysis in FolsterΔ et al., (2009) [56]. (FarR) Genes reported to be in the FarR regulon by Friedrich et al., (2007) [54]. (NmlR) Genes reported to be in the NmlR regulon by Kidd et al., (2005) [47]. (FNR) Genes found to be differentially expressed microaerobically in a Δ fnr mutant with and without nitrite by microarray analysis in Whitehead et al., (2007) [7]. (NsrR) Genes reported to be in the NsrR regulon by Isabella et al., (2007) [3] and Isabella, (2010) [19].

      e Fur-repressed small regulatory RNA described by Ducey et al., (2009) [96].

      As expected, anaerobic expression levels cluster at higher RPKM values in the set of anaerobically induced genes (Figure 1B), while the opposite is true in anaerobically repressed genes (Figure 1C).

      Secondary verification of differentially expressed genes

      Many of the genes found to have been differentially expressed by RNA-seq have previously been determined to be members of the Neisserial FNR or NsrR regulons, validating the use of this technique to monitor transcriptome changes (Table 1) [3, 7, 18, 19]. To further corroborate the RNA-seq data, several genes shown to be differentially expressed were selected for secondary confirmation. Analysis of translational promoter- lacZ fusions or RT-PCR was used to accomplish this task (Figure 2 and 3). Analysis of translational promoter- lacZ fusions demonstrated that the genes encoding heat shock protein, ClpB (NGO1046), DNA repair enzyme, RecN (NGO0318), nitropropane dioxygenase, Npd (NGO1024), filamentous hemagglutinin, HecA (NGO0706), glutaredoxin, Grx3 (NGO0114), bacterioferritin, BfrA (NGO0794) and repressor protein LexA (NGO1427) were significantly upregulated anaerobically (Figure 2A-G). RT-PCR confirmed that 2-isopropylmalate synthase, leu1 (NGO0848), and adhesin, lecA (NGO1981), were also increased in expression anaerobically (Figure 3).
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-51/MediaObjects/12864_2010_3190_Fig2_HTML.jpg
      Figure 2

      Secondary verification of RNA-seq results by translational promoter-lacZfusion. On the right-hand side of each panel, translational lacZ fusions confirm that expression of (A) clpB, (B) recN, (C) npd, (D) hecA, (E) grx3, (F) brfA, and (G) lexA is anaerobically induced, and that expression of (H) fbpA and (I) oxiA is anaerobically repressed. For each panel, a prediction of -10 and -35 elements is given above the gene schematic. The predicted transcriptional start site, in parenthesis under the +1, is reported as the chromosomal location according to the annotated FA1090 genome (NCBI). Genes colored in grey are encoded on the positive strand, while genes colored in black are encoded on the negative strand. Above each gene schematic, raw RNA-seq data from.wig files are plotted. The base count is representative of the number of times each base was mapped by a 50 bp RNA sequence read from Replicate 1 (normalized to take into account slight differences in total mapped reads between the two samples). Blue bars represent aerobic base reads and β-galactosidase activity, while red bars represent anaerobic base reads and β-galactosidase activity. Genes are not drawn to the same scale. Results for β-galactosidase activity are presented as the mean + SD of 16 determinations. (*) indicates a p-value less than 0.001.

      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-51/MediaObjects/12864_2010_3190_Fig3_HTML.jpg
      Figure 3

      Secondary verification of RNA-seq results by RT-PCR. RT-PCR verifies that lecA and leu1 are anaerobically induced, and that NGO0108, lldD, trx1, hemO, and nqrA are anaerobically repressed. RT-PCR of the murA transcript (NGO1981), which was shown to have an equal expression level aerobically and anaerobically (See additional file 1 : Supplementary Table S1), was used as a loading control. (-RT) signifies that reverse transcriptase was not included in the reaction. For the anaerobically induced genes lexA/erpA and NG1428/dnaK, primers were used that amplified across the 3'end of the lexA coding region into the 5' end of the erpA coding region, and from the 3' end of the NG1428 coding region into 5'end of the dnaK coding region, respectively. Amplification across these intergenic regions suggests that these genes are cotranscribed.

      Translational promoter- lacZ fusions to the genes encoding the iron-binding protein, FbpA (NGO0217), and the solute binding protein, OxiA (NGO0372) confirmed that these genes are anaerobically repressed (Figure 2 H, I). RT-PCR confirmed that NGO0108, L-lactate dehydrogenase, lldD (NGO0639), thioredoxin, trx1 (NGO0652), heme oxygenase, hemO (NGO1318), and NADH dehydrogenase component, nqrA (NGO1413) were also anaerobically repressed (Figure 3).

      Analysis of raw RNA-seq data allows for the prediction of transcription start sites, -10, and -35 elements, as has been described in RNA-seq projects in both Helicobacter pylori and Listeria monocytogenes [20, 21]. Compared to E. coli, the use of alternative sigma factors in the gonococcus is very limited. Gonococci have no sigma-54 homolog, and only utilize sigma factors in the sigma-70 family. This class of sigma factors recognize -10 and -35 elements within bacterial promoters [22]. The predictions of transcriptional elements for the individual genes selected for secondary analysis are shown in Figure 2. These predictions were generally a high match to the E. coli consensus for transcriptional elements (-35, 5'-TTGACA, -10, 5'-TATAAT) or the α-proteobacteria consensus for RpoH-dependent transcriptional elements (-35, 5'-CTTG, -10, 5'-CC/TTATNTNNG) [23]. The ability to predict the location of these transcriptional elements in a large scale manner will prove very useful in the search for potential regulatory sites, and will aid in future work to define gonococcal transcriptional networks.

      Differentially expressed genes involved in macromolecular biosynthesis

      The genes found to be differentially expressed in response to anaerobic growth were broadly characterized according to their putative function (Table 1, Figure 4). Several genes encoding proteins involved in macromolecular synthesis displayed decreased expression under anaerobic conditions. The synthesis of ribosomal proteins is strongly related to growth rate, and ribosomal protein synthesis has been documented to decrease during times of energy deficiency [2426]. For facultative anaerobes, growth in an oxygen-limited environment is more energetically-deprived than in aerobic conditions. The reduced synthesis of ribosomal proteins is indicative of adaptation to a slower growth rate following recovery from a nutritional shift-down. The reduced expression of RNA polymerase β an β' subunits is also indicative of this (Table 1), and has been observed in E. coli as well [27].
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-51/MediaObjects/12864_2010_3190_Fig4_HTML.jpg
      Figure 4

      Functional categories of anaerobically repressed and anaerobically induced genes. The genes from Table 1 were broadly categorized according to their biological function. Each bar represents the actual number of genes. Blue bars indicate anaerobically repressed genes, and red bars represent anaerobically induced genes.

      Genes involved in transport and binding

      Many genes encoding proteins involved in transport and binding were found to be anaerobically repressed (Table 1, Figure 4). Most of these proteins are involved in the transport of amino acids, sugars, or other growth metabolites into the cell. This result comes as no surprise, as a slower anaerobic growth rate would result in decreased demand and slower utilization of such substrates. Proteins of this type were also shown to be downregulated anaerobically in E. coli, P. aeruginosa, and S. enterica [2830]. Three repressed genes, fbpA, hemO, and ompU, are involved in the acquisition and/or transport of iron into the cell. In S. enterica, expression of the gene encoding iron acquisition protein, SitA, was also found to be repressed anaerobically [30].

      While genes involved in iron acquisition and transport are repressed anaerobically, the genes encoding the iron-storage bacterioferritin proteins, BfrA and BfrB are induced (Table 1, Figure 2F). Three other proteins implicated in binding that were shown to be anaerobically induced, LecA (Figure 3), HecA (Figure 2D), and fHBP, may be involved directly in the host-pathogen interaction. The role of LecA in gonococci is unclear, but a LecA homolog in another member of the Neisseriaceae family, Eikenella corrodens, has been shown to be a part of an adhesin complex important in adherence to a variety of host tissues [31]. The gonococcal HecA protein contains a hemagglutination domain that, in other bacteria, has been shown to promote bacterial aggregation or host attachment [32, 33]. The role of Factor H binding protein (fHBP) as it relates to gonococcal pathogenesis has not been examined, however, in N. meningitidis, fHBP expression on the cell surface was shown to be a means of immune evasion that rendered meningococci resistant to complement-mediated killing [34].

      Genes involved in energy production/oxidation-reduction

      Unsurprisingly, numerous genes encoding proteins involved in energy metabolism were found to be anaerobically repressed (Table 1). Gonococci use a combination of the Entner-Douderoff and pentose phosphate pathways in the utilization of glucose [35]. Key enzymes involved in these pathways were found to be anaerobically repressed, as well as several enzymatic components of the Krebs cycle. Anaerobic repression of genes involved in central intermediary metabolism has also been observed in other bacteria, including E. coli, P. aeruginosa, and B. subtilus [28, 29, 36].

      Interestingly, gonococci contain two major NADH dehydrogenases capable of coupling the oxidation of NADH to the reduction of ubiquinone. One of these dehydrogenases is the prototypical H+-translocating 14-subunit complex encoded by the nuo operon (NGO1737-NGO1751). The second is a seven-subunit Na+-translocating complex encoded by the nqr operon (NGO1413-NGO1418, NCBI). Though genes in the nuo operon did not meet the criteria used in this study to define differential expression, there was an approximate two-fold level of anaerobic repression across its entire length (See additional file 1 : Supplementary Figure S1). The nqr operon, however, demonstrated a significant decrease in anaerobic expression (Table 1, Figure 3).

      Na+-translocating NADH dehydrogenases were originally discovered and characterized in marine bacteria, though it has now been revealed that several pathogenic species encode such complexes [37]. Transport of sodium ions into the periplasmic space through the Nqr complex generates an electrochemical membrane potential that plays a role in solute import, ATP synthesis, and, for organisms that are motile, flagellar rotation [37, 38]. The flow of electrons through the active portions of the electron transport chain would be expected to decrease anaerobically when: [1] oxygen is unavailable, rendering the terminal cytochrome oxidase (complex IV) inactive, [2] enzymatic components of the Krebs cycle are repressed, resulting in decreased production of reducing equivalents, and [3] growth is dependent on high potential alternative electron acceptors such as NO2 - and NO. Furthermore, neither the nitrite or nitric oxide reductases are thought to be capable of pumping protons across the membrane [39]. Downregulation of nqr would decrease the electrochemical potential across the membrane (ΔΨ), thus decreasing the proton motive force [40]. Because expression of the nuo operon is not affected to the same degree anaerobically, this would now result in a greater proportion of NADH oxidation being coupled to ATP production through H+ translocation. Also, many proteins involved in the transport of metabolites into the cell are repressed anaerobically. Downregulation of the Nqr complex may add another level of regulation to cellular transport. It stands to reason that the activity of solute transporters that rely on the sodium motive force would decrease when fewer Na+ ions are pumped across the membrane [38].

      The function and regulation of the most highly induced gene involved in anaerobic growth, the nitrite reductase aniA, has been thoroughly characterized, and has been shown to be required for anaerobic growth on nitrite [4]. Interestingly, gonococci contain a gene encoding a 2-nitropropane dioxygenase-like protein that was found to be induced anaerobically (NGO1024, Table 1, Figure 2C). This would seem unusual, as gonococci would not be expected to encounter the toxic nitroalkanes utilized by Npd proteins. Furthermore, molecular oxygen is consumed in the Npd reaction mechanism [41]. Further work should determine if this protein plays a novel role in gonococci, possibly microaerobically when oxygen would be present.

      Gonococci also showed increased anaerobic expression of a gene encoding electron transfer flavoprotein-ubiquinone oxidoreductase, EftD (NGO1396), a protein containing 57% identity (74% similarity) to human EftD. The presence of eftD in bacteria is rare, and its role has only been studied in plants and mammals. In Arabidopsis thaliana, EftD was shown to be upregulated in times of carbon starvation, when genes involved in glycolysis and the Krebs cycle were reduced in expression, as is observed when gonococci are grown anaerobically [42]. In A. thaliana, it was reported that EftD can act as an electron acceptor from various dehydrogenases involved in amino acid catabolism and fatty acid degradation. The reduction of ubiquinone by EtfD was proposed to be a mechanism of utilizing alternative respiratory substrates to feed into the electron transport chain [42]. EftD upregulation in N. gonorrhoeae could indicate a shift to the use of alternative growth substrates under anaerobic conditions.

      Genes involved in small molecule biosynthesis

      Anaerobically, several genes involved in the synthesis of amino acids were found to be repressed (Table 1), which has also been observed in other bacteria [28, 29]. Genes involved in the synthesis of iron-sulfur clusters were also repressed. This result is not entirely unexpected, as many iron-sulfur cluster containing proteins involved in respiration were shown to be downregulated anaerobically. The demand for iron-sulfur clusters may therefore be reduced under anaerobic conditions. Also, because it is common for the Fe-S clusters in Fe-S cluster-containing proteins to be oxygen-sensitive, a lower rate of oxygen-induced Fe-S cluster turnover would be expected under anaerobic conditions [43].

      Unlike other amino acid biosynthetic genes, the gene encoding Leu1, the first committed step in leucine biosynthesis, was found to be induced anaerobically (Table 1, Figure 3). This could be indicative of a decrease in intracellular leucine concentration, as leu1 (leuA) is repressed by leucine in E. coli and other organisms [44]. In E. coli, the anaerobic stimulon is intimately associated with global regulation through the leucine responsive regulatory protein, Lrp. [28, 45]. In N. meningitidis, the reported Lrp regulon demonstrated striking overlap to many of the genes in the gonococcal anaerobic stimulon (Table 1), particularly in regards to proteins involved in glucose utilization, the Krebs cycle, and ribosomal assembly [46]. Future work will examine whether gonococcal Lrp plays a role in control of the anaerobic stimulon.

      Genes involved in transcriptional regulation

      The expression of several regulatory proteins was found to be induced under anaerobic conditions, though no regulatory proteins appeared to be repressed (Table 1, Figure 4). The gene encoding ArsR (NGO1562), a regulator involved in norB repression in response to iron, was shown to be moderately upregulated [3]. It is unclear if ArsR regulates any other target genes. The FNR-regulated NmlR protein has been shown to act as both an activator and repressor of gene expression, and genes in the NmlR regulon are purported to be involved in resistance to nitrosative stress [47, 48]. Though there was an anaerobic increase in nmlR expression, none of the genes in the NmlR regulon, except for nmlR itself, were found to be upregulated. One member of the regulon, the thioredoxin reductase trxB (NGO580), was shown to be significantly downregulated (Table 1). Surprisingly, in a recent study, gonococcal trxB mutants were found to be susceptible to killing by NO, though this property was correlated with low expression of aniA and norB [49]. It stands to reason, therefore, that genes in the NmlR regulon may be important in adaptation to anaerobic conditions or in certain environments where reactive oxygen or nitrogen species are present, but they are possibly not as important once the anaerobic respiratory chain becomes highly induced. In this circumstance, NorB is likely able to reduce NO levels below a threshold needed to induce the activation function of NmlR (NO itself may not be a direct activating signal for NmlR) [47]. Under anaerobic conditions, and in the absence of oxygen or reactive oxygen species (ROS), NO may not need to be detoxified, as toxic NO-derived reactive nitrogen compounds will not be produced. In this instance NO would preferentially be used as an energy source.

      The gene encoding LexA was found to be upregulated anaerobically (Figure 2G). This transcriptional regulator was proposed to be important in regulating genes involved in defense against ROS [50]. LexA controls a small regulon in gonococci that includes itself, DNA repair protein, recN, and a gene of unknown function, NGO1428. LexA has been demonstrated to sense hydrogen peroxide through thiol modification of a single cysteine residue [50], though the anaerobic induction of the LexA regulon observed here suggests that LexA may also sense NO. Interestingly, analysis of the raw RNA-seq data and RT-PCR showed that under conditions where the LexA regulon was induced, significant transcriptional read-through occurred beyond lexA into the adjacent erpA gene (NGO1426), as well as past the divergently transcribed NGO1428 gene into the adjacent dnaK gene (NGO1429, Figure 3). Both erpA and dnaK are members of the RpoH regulon [23], suggesting that there may be crosstalk between the RpoH and LexA regulons.

      Though transcriptional read-through may account for part of the anaerobic induction of erpA and dnaK, our data suggest that RpoH activity itself was likely modulated anaerobically. Although there was no significant increase in the quantity of RpoH transcripts at the 24 hr time point at which the cells were harvested, control of rpoH at the transcriptional level plays a relatively minor role in maintaining RpoH levels in the cell [51]. A majority of RpoH regulation is posttranscriptional. Translation of RpoH is inhibited by secondary structure in the rpoH mRNA, and activity of RpoH can be further modulated by the chaperone DnaK [23]. Including erpA and dnaK, eight out of the twelve genes previously demonstrated to encompass the gonococcal RpoH regulon were found to be significantly upregulated anaerobically, including genes encoding a transcriptional regulator of unknown function, MarR (NGO1244), dihydrofolate reductase, FolA (NGO1694), putative DNA-binding protein, CreA (NGO0570), and chaperone proteins ClpB (NGO1046), GrpE (NGO1422), and DnaJ (NGO1901, Table 1, Figure 2A) [23]. The anaerobic induction of genes in the RpoH regulon may be of significance in vivo, as these genes have been shown to contribute to epithelial cell invasion [52].

      The gene encoding FarR (NGO0058) was found to be anaerobically induced (Table 1). FarR was originally described for its role in repression of the farAB operon, which is involved in fatty acid resistance [53]. Analysis of the RNA-seq data demonstrated that farAB expression was not altered anaerobically, suggesting that this operon may already be fully repressed under standard laboratory conditions. A recent study established that FarR, possibly indirectly, was also responsible for the repression of two ABC transporter cassettes (NGO372-NGO0374 and NGO2011-NGO2014) as well as the gene encoding the multiple transferable resistance protein, MtrF (NGO1368) [54]. All of these genes were found to be anaerobically repressed by RNA-seq analysis (Table 1, Figure 2I), suggesting that FarR is yet another player in the anaerobic lifestyle of N. gonorrhoeae. Interestingly, expression of FarR itself was determined to be controlled by repression through MtrR, a regulator of the MtrCDE efflux pump involved in resistance to antimicrobial agents [55]. MtrR was recently shown to act in a global fashion, regulating many genes, including RpoH [56]. Many of the genes in the reported MtrR regulon also appear to be part of the anaerobic stimulon in gonococci, suggesting a potential role for this regulator in anaerobic growth as well [56] (Table 1).

      Genes involved in adaptation and stress response

      In addition to the RpoH regulon, many genes involved in response and adaptation to stress were found to be anaerobically induced. Several genes encoding proteins involved in DNA repair were found to be upregulated anaerobically (Table 1). RecN is involved in recombinational repair of DNA damage, and has been shown to protect gonococci from both oxidative and nonoxidative mechanisms of killing [57, 58]. RecX (NGO1053) has been demonstrated to be involved in enhancement of RecA-mediated processes, including repair of DNA damage [59]. A gene of unknown function, NGO1022, was also upregulated anaerobically, and encodes a DEDDh-family protein with a high level of homology to DnaQ (NGO0973), the epsilon subunit of DNA polymerase III [33]. As the epsilon subunit of DNA polymerase III contains 3' → 5' exonuclease activity and is involved in DNA mismatch repair, this DnaQ-like protein may also function in the repair of DNA damage [60].

      Several genes typically involved in resistance to oxidative stress were found to be induced anaerobically, including those encoding glutaredoxin, Grx3, cytochrome C peroxidase, CCP (NGO1769), tellurite resistance protein, TehB (NGO1161), and nitroreductase-like protein, NtrA (NGO1702, Table 1, Figure 2E). CCP was shown to protect gonococci from hydrogen peroxide mediated damage, but not to be involved in protection from reactive nitrogen species [61, 62]. Activation of CCP by FNR suggests that gonococci may typically be exposed to ROS under anaerobic or microaerobic conditions in vivo. Proteins similar to TehB were originally described for their role in resistance to tellurite, however, gonococci do not inhabit an environment where tellurite would be encountered. In Haemophilus influenzae, it was recently shown that a deletion of tehB resulted in increased sensitivity to oxidizing agents including hydrogen peroxide, suggesting a dual role for this protein [63]. The protein encoded by NGO1702 was shown to contain a nitroreductase-like domain [33], and to contain 54% identity (66% similarity) to a novel nitroreductase in Staphylococcus aureus, NtrA. In S. aureus, NtrA was shown to contribute to nitrosative stress resistance through its S-nitrosoglutathione (GSNO) reductase activity [64]. Two other proteins, DnrN and ErpA, were also demonstrated to be upregulated anaerobically. DnrN has been implicated in the repair of iron sulfur clusters damaged by oxidative or nitrosative stress [65], and ErpA may be important in maintaining the iron-sulfur cluster status of specific proteins in cells growing under stressful conditions [66].

      Interestingly, genes encoding two Nudix-family hydrolases, NudH (NGO1334) and NGO1566, as well as a histidine triad family protein, Hit (NGO1273), were all found to be upregulated anaerobically (Table 1). Analysis of the conserved domains of these open reading frames reveals the greatest level of homology to proteins involved in metabolism/hydrolysis of diadenosine tetraphosphate (Ap4A) [33]. Ap4A is an important and ubiquitous signaling molecule in nature, and has been implicated in the maintenance and regulation of vital cellular functions [67, 68]. As a bacterial second messenger, Ap4A has been shown to be involved in modulating chaperone and heat shock protein activity, coupling DNA replication to cell division, and altering the ability of cells to use alternate carbohydrate sources, though these may be just a few of its functions [68, 69]. If allowed to accumulate unchecked, high Ap4A concentration can interfere with a number of ATP-dependent reactions [70]. In E. coli, Salmonella typhimurium, and Bartonella bacilliformis, deletion of the nudH gene resulted in gross deficiencies in the ability of these organisms to invade mammalian cells [68, 69]. The role of Ap4A as a second messenger in gonococci has yet to be evaluated thus far, though it appears that the metabolism of this molecule may be modulated anaerobically, making it an interesting target for future study.

      Hypothetical proteins regulated anaerobically

      Many genes encoding hypothetical proteins were found to be induced anaerobically (Table 1, Figure 4). One gene, listed as DUF331 in Table 1, was not found in the FA1090 genome annotation, rather it was discovered in proximity to the iron sulfur cluster assembly operon (isc) using raw RNA-seq data. Using this data, it was discovered that the isc operon spanned the iscR gene (NGO0637) through hesB (NGO0632), making it longer than the current annotation would suggest. NGO0634 probably does not encode a protein, and no reads were mapped on the positive strand at this location (See additional file 1 : Supplementary Table S1). A high level of transcription was found at an unannotated putative open reading frame with an ATG start codon on the positive strand at chromosomal coordinate 621,869 (NCBI). There was a high match to consensus -35, -10, and RBS sequences upstream of this open reading frame, and transcription of this gene partially overlapped the 3' end of the isc operon. This anaerobically induced gene encodes a protein with a conserved domain of unknown function (DUF331). This is just an example of how RNA-seq data can be used to locate and correct annotation errors in the gonococcal genome. The large quantity of hypothetical proteins that are differentially expressed in response to anaerobiosis may make appealing targets for future study. Many of these annotated proteins are small and Neisseria-specific. Future work should determine if these are akin to small proteins in E. coli that have been shown to accumulate in response to stress [71]. Alternatively, these small open reading frames could be mis-annotations, and may be involved in transcription of regulatory RNA. Current work involves integrating raw RNA-seq data in a genome-wide search for regulatory RNA.

      N. gonorrhoeae encodes an anaerobically induced sRNA

      In E. coli and several other Enterobacterial species, recent studies have described the anaerobic induction and function of a small regulatory RNA termed fnrS [72, 73]. In E. coli, the regulation of this sRNA was determined to be relatively complex. Maximal fnrS expression occurred anaerobically, however, the available carbon source and terminal electron acceptor present also impacted levels to a lesser extent. Anaerobic induction of fnrS was shown to be FNR-dependent and mediated by FNR binding to a class-II activation site centered at -41.5 with respect to the transcription start site [72]. No fnrS homolog has been observed in bacteria outside of the Enterobacteriaceae family, however, previous microarray analysis of the gonococcal FNR regulon identified a small FNR-activated transcript of unknown function. Coincidently, this transcript was also determined to be the most highly induced by FNR [7].

      This small FNR-induced transcript is located within the coding region and on the opposite strand of NGO0796, flanked on the 5' end by the bacterioferritin genes, brfA and bfrB, and on the 3' end by an Xre-family repressor (NGO0797) end by an (Figure 5A). Though not included the FA1090 genome annotation, analysis of the raw RNA-seq data confirms that this transcript is highly induced anaerobically (Table 1, Figure 5A). The region upstream of this transcript also contains a perfect match to the E. coli FNR consensus sequence (5'-TTGATnnnnATCAA) located at -41.5 with respect to the transcription start site. Secondary structure prediction of this 108 nt transcript was performed by the Mfold program http://​mfold.​rna.​albany.​edu/​, and the most stable predicted structure is shown in Figure 5B. This predicted structure is typical of a small regulatory RNA, with a 5' stem loop followed by an unstructured region and a 3' Rho-independent terminator (Terminator prediction performed at: http://​transterm.​cbcb.​umd.​edu).
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-51/MediaObjects/12864_2010_3190_Fig5_HTML.jpg
      Figure 5

      N. gonorrhoeaeencodes an anaerobically induced small RNA. (A) A schematic representation of the location of the fnrS coding region is given, with genes colored in grey encoded on the positive strand, and genes colored in black encoded on the negative strand (according to the NCBI annotation). Above the fnrS gene schematic, raw RNA-seq data from.wig files are plotted. The base count is representative of the number of times each base in the transcript was mapped by a 50 bp RNA sequence read from Replicate 1 (normalized to take into account slight differences in total mapped reads between the two samples). Blue bars represent aerobic base reads and red bars represent anaerobic base reads. A prediction of the -10 element and class-II FNR binding site is given to the left of the fnrS gene. The chromosomal location of the predicted transcriptional start site, in parenthesis under the +1, is reported. (B) The lowest energy structure of the fnrS transcript as predicted by the Mfold program http://​mfold.​rna.​albany.​edu/​ is displayed. (C) The β-galactosidase activity of an fnrS::lacZ transcriptional fusion is presented as the mean + SD of 16 determinations (p < 0.001).

      To confirm that this small RNA is expressed and induced anaerobically, a transcriptional fusion to lacZ was constructed. In this construct, the promoter region from the induced transcript (from -120 to -1) was cloned in front of the leader region of lacZ, which provided spacing and an RBS for efficient translation. Aerobic β-galactosidase activity from this transcriptional fusion was low, but was induced 260-fold anaerobically (Figure 5C), validating the RNA-seq data and the work of Whitehead, et al. (2007) [19]. To show that transcription was halted at the predicted Rho-independent terminator, a transcriptional fusion was generated in which the entire small RNA, including the promoter region (from -120 to +111), was fused to the leader region of lacZ. No β-galactosidase activity was observed from this fusion gene aerobically or anaerobically, confirming the terminator prediction and the raw RNA-seq data (data not shown, Figure 5). The first three nucleotides of this small transcript are AUG. To ensure that this transcript is indeed a small RNA and not a leaderless protein-coding message, a translational fusion was constructed. A leaderless lacZ gene was fused in-frame past nucleotide +9 in the transcript. No β-galactosidase activity was observed in this translational fusion under aerobic or anaerobic conditions (data not shown). For these reasons, we feel that this small anaerobically induced transcript should be termed fnrS. Much work needs to be completed to define the role, if any, of this gonococcal FnrS transcript in gene regulation, however, homologs of genes in the E. coli FnrS regulon that were found to be anaerobically repressed in gonococci, including gpmA (phosphoglycerol mutase) and mqo (malate:quinone oxidoreductase), will make interesting candidates to study for potential interactions [72]. Future work will employ a similar RNA-seq approach to define the prospective FnrS regulon in gonococci.

      Overlap of the anaerobic, iron, and hydrogen peroxide stimulons

      In vivo, N. gonorrhoeae will be faced with a multitude of environmental stresses, including oxygen and iron limitation, as well as exposure to reactive oxygen species. Previous studies have employed genome-wide approaches to define the gonococcal iron and hydrogen peroxide responsive stimulons [74, 75]. Genes differentially expressed in response to these stimuli were compared to the anaerobic stimulon to search for similarities in gene expression between these environmental signals (Figure 6, Table 2).
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-51/MediaObjects/12864_2010_3190_Fig6_HTML.jpg
      Figure 6

      Overlap of the anaerobic, iron, and hydrogen peroxide responsive stimulons inN. gonorrhoeae. Genes found to be differentially expressed in response to anaerobiosis, iron, and hydrogen peroxide were compared in order to discover anaerobically regulated genes that were responsive to additional environmental signals. A Venn diagram was constructed to display the extent of overlap between these stimulons. Data for the iron responsive stimulon was derived from Jackson, et al., (2010) [75]. Data for the hydrogen peroxide responsive stimulon was derived from Stohl, et al., (2005) [74].

      Table 2

      Comparison of the anaerobic stimulon with the iron and hydrogen peroxide responsive stimulon

      Gene

      ORF designation

         

      Definition

      Common elements of the anaerobic and iron responsive stimulon

        

      -O2/+O2a

      -Fe/+Feb

        

      farR

      NGO0058

      3.9

      -2.0

       

      MarR family transcriptional regulator

      gluP

      NGO0142

      -3.7

      2.9

       

      Glucose/galactose transporter

      nhaC

      NGO0143

      -3.1

      -2.3

       

      Na+/H+ antiporter (NhaC)

      amtB

      NGO0198

      -4.3

      -14.2

       

      Ammonium transporter AmtB

      nspA

      NGO0233

      -3.4

      2.6

       

      Outer membrane protein

      greB

      NGO0262

      -4.5

      -1.8

       

      Transcription elongation factor

       

      NGO0373

      -7.4

      -2.5

       

      Amino acid ABC transporter, permease protein

       

      NGO0377

      -7.4

      -150.4

       

      Probable transmembrane transport protein

      cspA

      NGO0410

      -3.6

      3.2

       

      Cold shock protein A

       

      NGO0492

      7.7

      1.8

       

      Putative phage associated protein

       

      NGO0506

      13.6

      -3.2

       

      Putative phage associated protein

       

      NGO0635

      -4.2

      4.2

       

      Hypothetical protein

      lldD

      NGO0639

      -9.1

      3.9

       

      L-lactate dehydrogenase

      pdxH

      NGO0658

      4.9

      1.8

       

      Pyridoxamine 5-phosphate oxidase

      eda

      NGO0713

      -4.3

      6.9

       

      KHG-KDPG bifunctional aldolase

       

      NGO0732

      2.8

      2.7

       

      Neisseria-specific protein

      mobA

      NGO0754

      4.1

      3.2

       

      Molybdopterin-guanine dinucleotide biosynthesis protein

       

      NGO0757

      3.4

      2.2

       

      Putative periplasmic Cpx-family sensor kinase

      bfrA

      NGO0794

      12.0

      -2.8

       

      Bacterioferritin A

      bfrB

      NGO0795

      9.9

      -1.9

       

      Bacterioferritin B

      hslR

      NGO0822

      3.9

      2.4

       

      Heat shock protein (Hsp15)

       

      NGO0895

      5.5

      1.8

       

      Neisseria-specific protein, uncharacterized

      sdhA

      NGO0921

      -3.6

      -2.3

       

      Succinate dehydrogenase flavoprotein subunit

      sdhC

      NGO0923

      -3.0

      -2.3

       

      Succinate dehydrogenase, cytochrome b556 chain

       

      NGO0995

      3.2

      1.9

       

      Neisseria-specific protein, uncharacterized

      hsp33

      NGO1189

      5.7

      -2.9

       

      Heat shock protein Hsp33 chaperonin

       

      NGO1261

      -6.7

      3.4

       

      S-adenosylmethionine-dependent methyltransferase

      lldP

      NGO1361

      -8.7

      -3.2

       

      L-lactate permease

      nqrB

      NGO1414

      -6.7

      -2.7

       

      Sodium-translocating NADH-ubiquinone reductase subunit B

      nqrC

      NGO1415

      -6.3

      -2.1

       

      Sodium-translocating NADH-ubiquinone reductase subunit C

      nqrE

      NGO1417

      -8.4

      -5.5

       

      Sodium-translocating NADH-ubiquinone reductase subunit E

      nqrF

      NGO1418

      -6.4

      -3.1

       

      Sodium-translocating NADH-ubiquinone reductase subunit F

      putP

      NGO1552

      -8.3

      -5.3

       

      Sodium/proline symporter PutP

      ompA

      NGO1559

      3.6

      2.5

       

      Probable outer membrane protein

      arsR

      NGO1562

      4.2

      -2.3

       

      ArsR family transcriptional regulator

       

      NGO1628

      6.3

      2.1

       

      Conserved hypothetical protein

       

      NGO1633

      82.1

      2.1

       

      Putative phage associated protein

       

      NGO1688

      -3.1

      3.7

       

      Conserved hypothetical protein

      folA

      NGO1694

      3.3

      -2.0

       

      Dihydrofolate reductase (FolA)

       

      NGO1807

      -3.3

      -3.0

       

      Amino-acid transporter

      dadA

      NGO1808

      -4.4

      -2.9

       

      D-amino acid dehydrogenase small subunit (DadA)

      yhhF

      NGO1860

      3.9

      2.2

       

      Probable DNA methylase Symport protein (possible sodium/dicarboxylate

       

      NGO1957

      -7.8

      -2.0

       

      symporter)

       

      NGO2023

      4.9

      1.9

       

      Conserved hypothetical protein

      sspB

      NGO2131

      -4.1

      3.3

       

      Stringent starvation protein B

       

      nrrF

      -6.2

      170†

       

      Fur regulated small regulatory RNA

      Common elements of the anaerobic and hydrogen peroxide responsive stimulon

        

      -O2/+O2

      +HP/-HPc

        

      recN

      NGO0318

      5.1

      3.0

       

      DNA repair protein RecN (recombination protein N)

      dnaB

      NGO0485

      5.2

      6.5

       

      Replicative DNA helicase

      nmlR

      NGO0602

      7.4

      -4.8

       

      Transcriptional regulator MerR-family

      nifU

      NGO0633

      -5.0

      4.6

       

      Fe-S scaffold protein

      iscS

      NGO0636

      -5.2

      4.1

       

      Cysteine desulferase

      leu1

      NGO0848

      5.6

      3.3

       

      2-isopropylmalate synthase

      aniA

      NGO1276

      28.2

      -3.6

       

      Copper-containing dissimilatory nitrite reductase

      grpE

      NGO1422

      3.2

      5.8

       

      Heat shock protein (HSP-70 cofactor)

      lexA

      NGO1427

      4.8

      6.3

       

      Transcriptional regulator, repressor

       

      NGO1428

      11.4

      3.9

       

      Neisseria-specific protein, uncharacterized

      dnaK

      NGO1429

      3.1

      10.8

       

      Heat shock protein (HSP-70 chaperone)

      ccp

      NGO1769

      8.9

      2.7

       

      Probable cytochrome c peroxidase

      dnaJ

      NGO1901

      4.0

      3.0

       

      Heat shock protein HSP-40/chaperone DnaJ

      Common elements of the anaerobic, iron, and hydrogen peroxide responsive stimulon

        

      -O2/+O2

      -Fe/+Fe

      +HP/-HP

       
       

      NGO0108

      -4.9

      4.7

      6.8

      Conserved hypothetical protein (possible oxidoreductase)

      glr3

      NGO0114

      5.0

      4.7

      4.8

      Glutaredoxin 3

      fbpA

      NGO0217

      -5.2

      8.7

      5.5

      Periplasmic iron-binding protein

       

      NGO0554

      -4.8

      24.5

      70.6

      Hypothetical protein

      trx1

      NGO0652

      -5.8

      3.2

      13.5

      Thioredoxin I

      fumC

      NGO1029

      -4.6

      6.4

      8.4

      Fumarate hydratase (fumarase C)

      clpB

      NGO1046

      7.2

      4.3

      23.8

      Endopeptidase ClpB (heat shock protein)

      pgm

      NGO1258

      -4.8

      2.0

      2.8

      Phosphoglycerate mutase

      hemO

      NGO1318

      -9.8

      8.4

      9.8

      Heme oxygenase/iron starvation protein

      exbD

      NGO1377

      -3.5

      3.9

      4.4

      Transport protein (ExbD)

      exbB

      NGO1378

      -4.1

      6.5

      4.7

      Transport protein (ExbB) Sodium-translocating NADH-ubiquinone reductase

      nqrA

      NGO1413

      -5.8

      -2.3

      -2.6

      subunit A

           

      Sodium-translocating NADH-ubiquinone reductase

      nqrD

      NGO1416

      -10.3

      -4.0

      -2.8

      subunit D

      lecA

      NGO1981

      4.5

      2.5

      2.5

      Adhesin protein

      a average of RPKM ratios from replicates in Table 1.

      b Data from Jackson et al., (2010) [75], taken from time point where iron depletion had the biggest effect on gene expression. A positive value represents an induction of gene expression under iron depleted conditions, and a negative value represents a repression of gene expression under iron depleted conditions.

      c Hydrogen peroxide (HP); Data from Stohl et al., (2005) [74]. A positive value represents induction of gene expression in the presence of hydrogen peroxide, and a negative value represents a repression in gene expression in the presence of hydrogen peroxide.

      † Data from Ducey, et al., (2009) [96].

      It should be noted that there are caveats in directly comparing these data sets due to the differences in experimental design that exist between each study. The use of microarrays in the iron and hydrogen peroxide studies may be less sensitive than the use of transcriptome sequencing, and therefore it is possible that some additional iron- or hydrogen peroxide-regulated genes may have been missed [16]. Differences in the cutoff level used to define differential expression, the growth medium utilized, and the growth phase of the cells used for RNA isolation between these studies may also impact the results. Though the iron and hydrogen peroxide studies utilized RNA from broth-grown cells in mid-log phase, the growth phase of anaerobically grown gonococcal cells is difficult to determine due to the requirement for growth on solid media, and consequently, this may also affect the comparison of these results with other studies [74, 75]. With these factors in mind, a significant overlap does exist between the members of these stimulons, and it should be noted that of the common members, the response is not always predictable. That is, genes regulated in response to these stimuli do not follow a common expression pattern between gene sets, underscoring the complexity of gonococcal gene regulation (Table 2). For example, the nqr operon and bfrA are both members of the iron stimulon and are both repressed under iron-depleted conditions, however, nqr and bfrA are repressed and induced, respectively, during anaerobic growth. From this comparison we hope to discover Neisserial genes that may play an important role in survival under a broad range of environmental stress. Analysis of this gene set may also help future work to define transcriptional networks at the regulator level.

      Gonococcal prophage genes are highly induced anaerobically

      Somewhat unexpectedly, the RNA-seq data showed that a large subset of the anaerobically induced genes were bacteriophage in origin (47 genes, Table 1). The dsDNA filamentous prophage, NgoΦ1, is believed to contain all of the coding regions necessary to produce functionally active phage particles. In a previous study, phage DNA from NgoΦ1, as well as phage particles that were likely NgoΦ1 -derived, were observed in gonococci, though plaques were not observed [76]. In this study, RNA-seq data showed dramatic anaerobic induction of the NgoΦ1 coding region.

      Though no plaques have been observed in anaerobically grown gonococci, this is not the first report of anaerobic prophage induction. In anaerobically grown Pseudomonas aeruginosa, prophage genes were shown to be among the most highly induced, and it was suggested that, more specifically, nitric oxide was likely involved in this apparent induction [29]. Evidence has also been provided to show that these anaerobically induced phage are involved in biofilm development and differentiation in P. aeruginosa [77]. Current work proposes that gonococcal biofilms are important in pathogenesis. It was recently shown that anaerobic metabolism occurs in the substratum of gonococcal biofilms, and that NO plays an important role in biofilm maintenance [78]. It is interesting to speculate that in the gonococcus, NgoΦ1 may play a role similar to that of anaerobically induced phage in P. aeruginosa.

      NgoΦ3, NgoΦ4, and NgoΦ5 encode incomplete phage genomes and are probably derived from NgoΦ1 or NgoΦ2 [76]. NgoΦ3, NgoΦ4, and NgoΦ5 were all shown to contain coding regions that were upregulated anaerobically (Table 1). The functions of these genes are unknown, but the presence of prophage sequences in bacterial genomes could have a profound effect on host cell fitness or pathogenicity. A diverse group of bacterial virulence factors, including toxins, have been found to be encoded by genes of phage origin. Some prophage can also contain regulatory proteins that affect expression of genes not encoded by phage [Reviewed in, [79]]. It is certainly possible that none of the anaerobically upregulated gonococcal prophage genes are involved in making active phage particles; alternatively, these genes may have acquired a novel function. Regardless, further research will be required to determine the role of these genes as they relate to anaerobiosis.

      Expression from the cryptic plasmid is induced anaerobically

      The gonococcal cryptic plasmid, pJD1, is 4,207 bp long and is found in approximately 96% of gonococcal strains. Despite its predominance, the function and replication mechanism of this plasmid remains largely unknown [80, 81]. This plasmid consists of two divergent transcripts, each consisting of five open reading frames (Table 3, Figure 7). Surprisingly, pJD1 transcripts were greatly increased in prevalence anaerobically (Table 3, Figure 7). It appears as though the increase in transcript quantity was not due to an increase in anaerobic copy number, as there was no significant difference in the quantity of plasmid recovered from equal numbers of aerobically or anaerobically grown cells (data not shown). This result implies that the two divergent cryptic plasmid promoters are subject to transcriptional regulation.
      Table 3

      Genes of the cryptic plasmid found to be differentially expressed in response to anaerobiosis.

      Gene

      Replicate 1 b

      Replicate 2

      Definition a

      Transcript 1

         ORF1

      12.8

      9.7

      Putative plasmid replicase, RepA

         ORF2

      4.5

      3.1

      Hypothetical protein

         ORF3

      4.5

      3.4

      Hypothetical protein

         ORF4

      4.5

      2.6

      Putative plasmid antitoxin, VapX

         ORF5

      5.1

      5.5

      Putative plasmid toxin, VapD

      Transcript 2

         ORF6

      18.2

      6.0

      Hypothetical protein

         ORF7

      13.3

      34.4

      Hypothetical protein

          cppC

      19.9

      41.6

      Putative plasmid mobilization protein, MobA

          cppB

      6.2

      7.9

      Putative plasmid mobilization protein, MobB, partial only

          cppA

      7.6

      8.2

      Cryptic plasmid protein A

      a Protein definitions are derived from Korch et al., (1985) [80] or the NCBI conserved domain database [33].

      b The fold change for each replicate was calculated by comparing total number of reads for each gene in anaerobically grown gonococci to number of reads for each gene in aerobically grown gonococci. Values were normalized to total number of chromosomal reads. A positive value represents an induction of gene expression anaerobically, and a negative value represents a repression of gene expression anaerobically.

      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-51/MediaObjects/12864_2010_3190_Fig7_HTML.jpg
      Figure 7

      Expression from the gonococcal cryptic plasmid is induced anaerobically. Schematic representations of (A) transcript-1 and (B) transcript-2 of the cryptic plasmid are displayed. Genes colored in grey are encoded on the positive strand, while genes colored in black are encoded on the negative strand. The predicted transcriptional start site, in parenthesis under the +1 for each transcript, is reported as the location on the plasmid according to the annotation of Korch et al., (1985) [80]. Above each gene schematic, raw RNA-seq data from.wig files are plotted. The base count is representative of the number of times each base was mapped by a 50 bp RNA sequence read from Replicate 1 (normalized to take into account slight differences in total mapped reads between the two samples). Blue bars represent aerobic base reads and red bars represent anaerobic base reads. A prediction of -10 and -35 elements is given to the left of each schematic. RT-PCR confirms that transcript-1 is induced anaerobically.

      In other plasmids, the essential RepA protein has been shown to be involved in regulating gene expression as well as the initiation of plasmid replication (RepA encoded by ORF1 in pJD1, Table 3) [82, 83]. In several organisms, DnaK has been shown to facilitate the dissociation of RepA dimers into monomers, resulting in derepression [84, 85]. Therefore it is possible that the anaerobic increase in DnaK concentration could result in a decrease in RepA-mediated autorepression (Table 1). Unfortunately, little is known about the replication cycle of pJD1 or the mechanism of RepA binding in gonococci, so this potential regulatory mechanism remains only speculative. An alternate possibility is that an as-of-yet unidentified chromosomally expressed transcription factor or factors acts at the divergent plasmid promoters to regulate their expression.

      The effect of increased cryptic plasmid expression on gonococcal physiology is unknown. Previous studies involving pJD1 have provided evidence that ORF1, ORF2, ORF4, ORF5, cppC, cppB, and cppA are capable of forming protein products, and several of these protein products have no homology to any other proteins in the NCBI conserved domain database (Table 3) [33, 80, 81]. It is uncertain if ORF3, ORF6, or ORF7 are translated. The high frequency and sequence conservation of this plasmid among gonococcal isolates suggests that the plasmid may be important in pathogenesis. The fact that transcription from this plasmid is induced anaerobically is perhaps suggestive that the gene products of this plasmid are important in an anaerobic niche.

      In transcript-1, ORF4 and ORF5 encode a putative toxin/antitoxin system analogous to the VapDX systems of Haemophilus influenzae and several species of Actinobacillus [86, 87]. It is assumed that the VapDX system is involved in stable maintenance of pJD1 [88]. Interestingly, in H. influenza e, the VapDX locus is located on the chromosome, and VapD has been characterized as a virulence factor. It has been proposed that the ability of VapD to cause an arrest of bacterial translation was responsible for an observed enhancement of bacterial survival within human cells compared to survival in a Δ vapD mutant [86]. VapD was postulated to facilitate persistent or chronic mucosal infections under stressful conditions. The role of VapD as it pertains to gonococcal virulence, if any, is unknown, though it may make an attractive target for future study.

      Conclusions

      The goal of this study was to define the gonococcal transcriptome in response to anaerobiosis. Unlike previous studies that have looked only at the contributions of individual transcriptional regulators, we used a powerful and sensitive methodology, RNA-seq [16], to look at the environmental condition of anaerobiosis as a whole. By using this method, we were able to show that the anaerobic stimulon in N. gonorrhoeae was larger than the FNR and NsrR regulons indentified in previous studies [7, 19]. The induction of prophage and cryptic plasmid genes was previously unknown to occur during anaerobiosis. Many new targets for future study have been uncovered, and we hope that other investigators will take an interest in working on these potential projects. Because anaerobic growth is assumed to be a physiologically significant state in vivo [4], the results of this study may help to elucidate factors or mechanisms of virulence that have previously been overlooked.

      Methods

      Growth of gonococcal strains

      All gonococcal strains were derived from strain F62 (See additional file 2 : Supplementary Table S2) and were grown on Difco™GC medium base (Becton, Dickinson and Co., Sparks, MD) plates with 1% Kellogg's supplement (GCK) [89]. A 5 mM concentration of nitrite was added to plates to be used for anaerobic cultures. For RNA isolation and β-galactosidase assays, overnight plate-grown cell cultures were suspended in GCP broth (proteose peptone #3 (15 g), soluble starch (1 g), KH2PO4 (4 g), K2HPO4 (1 g), NaCl (5 g)/L dH2O) to an OD600 of 1.0 and serially diluted tenfold to 10-4. A 100 μL volume of the 10-4 dilution was plated onto GCK plates with or without nitrite. Aerobic plate cultures were grown for 14-16 h in a 37°C incubator supplying 5% CO2. Anaerobic cultures were incubated in a Coy anaerobic chamber (Coy Laboratory Products, Grass Lake, MI) at 37°C for 20-24 h in an atmosphere of 85% N2, 5% H2, and 5% CO2. Cells were harvested from aerobically and anaerobically incubated plates when colonies were approximately the same size.

      RNA isolation and RT-PCR

      Plate-harvested cells were incubated shaking in tubes with 0.1 mm diameter silica beads in RNA pro ™solution, and RNA was extracted according to manufacturer's instructions (MP Biomedicals, CA). RNA was treated with DNase I (Invitrogen, instructions CA), and integrity was confirmed with a Bioanalyzer (Agilent). RT-PCR was performed with SuperScript® III One-step RT-PCR reagents (Invitrogen, CA). A 125 ng quantity of total RNA was used in each RT reaction, and primers were added at a concentration of 10 μM. Reactions were run as described: 20 min incubation at 45°C to complete the RT reaction, followed by 2 min incubation at 94°C, 23 to 26 subsequent cycles of 15 s at 94°C, 30 s at 55°C, and 30 s at 68°C, and a final incubation at 68°C for 5 min. Samples were electrophoresed on 1% agarose gels for visualization. All RT-PCR experiments were repeated a minimum of two times. Primers available upon request.

      RNA preparation and SOLiD™ RNA-seq

      For RNA to be used in RNA-seq, two rounds of mRNA enrichment were performed on total bacterial RNA using MICROBExpress Oligo MagBeads (Ambion, TX). The University of Rochester core facility performed the SOLiD sequencing. The SOLiD™ 3 Plus system was used to read short (50 nt) RNA sequences, and all RNA processing procedures were performed using a SOLiD™ Total RNA-seq Kit as recommended by Applied Biosystems (SOLiD™ Total RNA-seq Kit Protocol, Applied Biosystems, CA). Briefly, RNA was fragmented with RNase III, directionally hybridized and ligated with flanking adapters containing sequences for priming PCR amplification and sequencing reactions. After reverse transcription of the ligated RNA, the cDNA molecules were size selected by PAGE, amplified by PCR (limiting the number of cycles to minimize PCR bias), quantified by real-time PCR, and diluted to a concentration optimal for monoclonal amplification by emulsion PCR, during which copies of template molecules were attached to beads. Beads were deposited on slides upon which the sequential reactions and washing steps were done automatically by the SOLiD™ 3 instrument. DNA attached to beads was sequenced with 50 rounds of oligonucleotide annealing and ligation (five different primers, 10 sequential ligations per primer). During each round, an oligonucleotide labeled with a fluorescent dye was annealed and ligated to the 3' end of the primer or the oligo ligated in the previous round. The next two unoccupied nucleotides of the strand attached to the bead determined which oligo ("color") was ligated. Use of staggered primers permitted determination of the unique base sequences. BioScope (version 1.2, Applied Biosystems, CA) was used to map the color sequences of each bead to the annotated FA1090 reference genome obtained from the National Center for Biotechnology Information (NCBI). Sequences were counted if they matched a 25-color end of a reference sequence with no more than 2 mismatches (permitting some mismatches is necessary because of sequencing errors and actual variations from the reference sequence). The length of the match was then determined by extending the alignment through the full 50-color sequence, allowing for more mismatches with increasing length. If a sequence read aligned to more than one genomic locus, the best alignment was selected. If a sequence read aligned equally well to more than one genomic locus, it was discarded. Stringency was set so that any base in the gonococcal genome had to be counted at least 10 times in order to be considered mapped. After mapping, colors were converted to bases. Sequencing data can be accessed by Gene Expression Omnibus (GEO; http://​www.​ncbi.​nlm.​nih.​gov/​projects/​geo/​) with the accession number GSE26444.

      Analysis of raw data

      The Integrative Genomics Viewer (IGV) was utilized http://​www.​broadinstitute.​com/​igv to visualize raw sequencing data. The.wig files generated from RNA sequencing were loaded and compared against the gonococcal genome, allowing visualization of sequencing reads mapped outside of the annotated genome. This allowed for scanning of intergenic regions for detection of small RNAs and transcription start sites of genes.

      PCR

      Genomic DNA from gonococcal strain F62 was isolated for use as a PCR template. Promoter sequences for lac Z fusions were amplified with iProof™ High Fidelity Polymerase (Bio-Rad, Hercules, CA). Clones were screened by PCR for presence and orientation of the insert using Amplitaq® (Applied Biosystems, Foster City, CA). Primers available upon request

      Construction of lac Z fusions

      Translational and transcriptional lac Z fusions were constructed in pLES94 [90]. Genomic DNA from gonococcal strain F62 was used as template. PCR fragments and pLES94 were cut with BamHI. Digested insert and plasmid were ligated and cloned into E. coli DH10B. Transformants were selected on LB medium plates containing chloramphenicol at 25 μg ml-1 and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal, Invitrogen) at 40 μg ml-1. Plasmids were checked for the presence and orientation of the insert by PCR, and those plasmids that contained an insert in the correct orientation were used to transform F62. Colony PCR was performed on chloramphenicol resistant colonies to confirm the presence of the reporter construct. The PCR product was also sequenced to ensure the appropriate fusion was made.

      Gonococcal transformation

      Gonococci were transformed naturally. A light suspension of piliated cells was prepared in 1 ml of GCK broth containing 0.042% NaHCO3 and 10 mM MgCl2 [89]. Purified plasmid DNA was added and 100 μl of the suspension was plated on two GCK plates and incubated 6-9 hours at 37°C. Cells were then harvested from the plates and streaked on GCK plates containing chloramphenicol for selection of clones. Clones typically took 2 days to become visible on antibiotic plates.

      ß-Galactosidase assays

      Gene reporter activity was determined by ß-galactosidase assays from cultures grown aerobically or anaerobically with nitrite [91]. For gonococcal cultures, sterile swabs were used to harvest cells from overnight plate cultures and cells were resuspended in Z-buffer [91]. Cells were lysed with chloroform and 0.1% SDS and assayed as described [91]. Activity is reported in Miller units and the reported results are the average of at least three assays performed in duplicate from each day the cultures were grown.

      Oligonucleotide and DNA sequencing

      All synthesized oligonucleotides were obtained from Invitrogen, and confirmatory DNA sequencing was performed at ACGT Inc. (Wheeling, IL).

      Molecular biology techniques

      Cloning and PCR techniques were performed in accordance to standard protocols [9294]. Plasmid preparations were obtained with a QIAprep miniprep kit, and DNA fragments were purified with QIAquick PCR Purification or QIAquick Gel Extraction kits (QIAGEN, CA).

      Declarations

      Acknowledgements

      This study was supported by Public Health Service grant R21 AI 080912 from the National Institute of Allergy and Infectious Disease. In addition, we thank Michael Macguire for his technical assistance.

      Authors’ Affiliations

      (1)
      Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester

      References

      1. Knapp JS, Clark VL: Anaerobic growth of Neisseria gonorrhoeae coupled to nitrite reduction. Infect Immun 1984, 46 (1) : 176–181.PubMed
      2. Barth KR, Isabella VM, Clark VL: Biochemical and genomic analysis of the denitrification pathway within the genus Neisseria . Microbiology 2009, 155 (Pt 12) : 4093–4103.PubMedView Article
      3. Isabella V, Wright LF, Barth K, Spence JM, Grogan S, Genco CA, Clark VL: cis- and trans-acting elements involved in regulation of norB ( norZ ), the gene encoding nitric oxide reductase in Neisseria gonorrhoeae . Microbiology 2008, 154 (Pt 1) : 226–239.PubMedView Article
      4. Clark VL, Barth K, Overton T: Regulation and function of the Neisserial denitrification pathway: life with limited oxygen. In Neisseria: Molecular Mechanisms of Pathogenesis. Edited by: Wetzler CGaL. Norwich, UK: Horizon Scientific Press; 2009.
      5. Newkirk GR: Pelvic inflammatory disease: a contemporary approach. Am Fam Physician 1996, 53 (4) : 1127–1135.PubMed
      6. Clark VL, Knapp JS, Thompson S, Klimpel KW: Presence of antibodies to the major anaerobically induced gonococcal outer membrane protein in sera from patients with gonococcal infections. Microb Pathog 1988, 5 (5) : 381–390.PubMedView Article
      7. Whitehead RN, Overton TW, Snyder LA, McGowan SJ, Smith H, Cole JA, Saunders NJ: The small FNR regulon of Neisseria gonorrhoeae : comparison with the larger Escherichia coli FNR regulon and interaction with the NarQ-NarP regulon. BMC Genomics 2007, 8: 35.PubMedView Article
      8. Cardinale JA, Clark VL: Determinants of nitric oxide steady-state levels during anaerobic respiration by Neisseria gonorrhoeae . Mol Microbiol 2005, 58 (1) : 177–188.PubMedView Article
      9. Barth K, Clark VL: Differences in nitric oxide steady states between arginine, hypoxanthine, uracil auxotrophs (AHU) and non-AHU strains of Neisseria gonorrhoeae during anaerobic respiration in the presence of nitrite. Can J Microbiol 2008, 54 (8) : 639–646.PubMedView Article
      10. Edwards JL: Neisseria gonorrhoeae survival during primary human cervical epithelial cell infection requires nitric oxide and is augmented by progesterone. Infect Immun 2010, 78 (3) : 1202–1213.PubMedView Article
      11. Rock JD, Thomson MJ, Read RC, Moir JW: Regulation of denitrification genes in Neisseria meningitidis by nitric oxide and the repressor NsrR. J Bacteriol 2007, 189 (3) : 1138–1144.PubMedView Article
      12. Overton TW, Whitehead R, Li Y, Snyder LA, Saunders NJ, Smith H, Cole JA: Coordinated regulation of the Neisseria gonorrhoeae -truncated denitrification pathway by the nitric oxide-sensitive repressor, NsrR, and nitrite-insensitive NarQ-NarP. J Biol Chem 2006, 281 (44) : 33115–33126.PubMedView Article
      13. Constantinidou C, Hobman JL, Griffiths L, Patel MD, Penn CW, Cole JA, Overton TW: A reassessment of the FNR regulon and transcriptomic analysis of the effects of nitrate, nitrite, NarXL, and NarQP as Escherichia coli K12 adapts from aerobic to anaerobic growth. J Biol Chem 2006, 281 (8) : 4802–4815.PubMedView Article
      14. Tucker NP, Le Brun NE, Dixon R, Hutchings MI: There's NO stopping NsrR, a global regulator of the bacterial NO stress response. Trends Microbiol 2010, 18 (4) : 149–156.PubMedView Article
      15. Nakano MM, Geng H, Nakano S, Kobayashi K: The nitric oxide-responsive regulator NsrR controls ResDE-dependent gene expression. J Bacteriol 2006, 188 (16) : 5878–5887.PubMedView Article
      16. Marioni JC, Mason CE, Mane SM, Stephens M, Gilad Y: RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res 2008, 18 (9) : 1509–1517.PubMedView Article
      17. Bullard JH, Purdom E, Hansen KD, Dudoit S: Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments. BMC Bioinformatics 2010, 11: 94.PubMedView Article
      18. Oriente F, Scarlato V, Delany I: Expression of factor H binding protein of meningococcus responds to oxygen limitation through a dedicated FNR-regulated promoter. J Bacteriol 2010, 192 (3) : 691–701.PubMedView Article
      19. Isabella V: Genetic and Biochemical Analysis of NsrR, a Nitric Oxide-Responsive Repressor in Neisseria gonorrhoeae . PhD thesis. Rochester: University of Rochester; 2010:215.
      20. Oliver HF, Orsi RH, Ponnala L, Keich U, Wang W, Sun Q, Cartinhour SW, Filiatrault MJ, Wiedmann M, Boor KJ: Deep RNA sequencing of L. monocytogenes reveals overlapping and extensive stationary phase and sigma B-dependent transcriptomes, including multiple highly transcribed noncoding RNAs. BMC Genomics 2009, 10: 641.PubMedView Article
      21. Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S, Sittka A, Chabas S, Reiche K, Hackermuller J, Reinhardt R, Stadler PF, Vogel J: The primary transcriptome of the major human pathogen Helicobacter pylori . Nature 2010, 464 (7286) : 250–255.PubMedView Article
      22. Gruber TM, Gross CA: Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 2003, 57: 441–466.PubMedView Article
      23. Gunesekere IC, Kahler CM, Powell DR, Snyder LA, Saunders NJ, Rood JI, Davies JK: Comparison of the RpoH-dependent regulon and general stress response in Neisseria gonorrhoeae . J Bacteriol 2006, 188 (13) : 4769–4776.PubMedView Article
      24. Harvey RJ: Regulation of ribosomal protein synthesis in Escherichia coli . J Bacteriol 1970, 101 (2) : 574–583.PubMed
      25. Champney WS: Kinetics of ribosome synthesis during a nutritional shift-up in Escherischia coli K-12. Mol Gen Genet 1977, 152 (3) : 259–266.PubMedView Article
      26. Nomura M, Gourse R, Baughman G: Regulation of the synthesis of ribosomes and ribosomal components. Annu Rev Biochem 1984, 53: 75–117.PubMedView Article
      27. Iwakura Y, Ishihama A: Biosynthesis of RNA polymerase in Escherichia coli . II. control of RNA polymerase synthesis during nutritional shift up and down. Mol Gen Genet 1975, 142 (1) : 67–84.PubMed
      28. Salmon K, Hung SP, Mekjian K, Baldi P, Hatfield GW, Gunsalus RP: Global gene expression profiling in Escherichia coli K12. The effects of oxygen availability and FNR. J Biol Chem 2003, 278 (32) : 29837–29855.PubMedView Article
      29. Platt MD, Schurr MJ, Sauer K, Vazquez G, Kukavica-Ibrulj I, Potvin E, Levesque RC, Fedynak A, Brinkman FS, Schurr J, Hwang S, Lau GW, Limbach PA, Rowe JJ, Lieberman MA, Barraud N, Webb J, Kjelleberg S, Hunt DF, Hassett DJ: Proteomic, microarray, and signature-tagged mutagenesis analyses of anaerobic Pseudomonas aeruginosa at pH 6.5, likely representing chronic, late-stage cystic fibrosis airway conditions. J Bacteriol 2008, 190 (8) : 2739–2758.PubMedView Article
      30. Encheva V, Shah HN, Gharbia SE: Proteomic analysis of the adaptive response of Salmonella enterica serovar Typhimurium to growth under anaerobic conditions. Microbiology 2009, 155 (Pt 7) : 2429–2441.PubMedView Article
      31. Azakami H, Yumoto H, Nakae H, Matsuo T, Ebisu S: Molecular analysis of the gene encoding a protein component of the Eikenella corrodens adhesin complex that is close to the carbohydrate recognition domain. Gene 1996, 180 (1–2) : 207–212.PubMedView Article
      32. Rojas CM, Ham JH, Deng WL, Doyle JJ, Collmer A: HecA, a member of a class of adhesins produced by diverse pathogenic bacteria, contributes to the attachment, aggregation, epidermal cell killing, and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii seedlings. Proc Natl Acad Sci USA 2002, 99 (20) : 13142–13147.PubMedView Article
      33. Marchler-Bauer A, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, He S, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Liebert CA, Liu C, Lu F, Lu S, Marchler GH, Mullokandov M, Song JS, Tasneem A, Thanki N, Yamashita RA, Zhang D, Zhang N, Bryant SH: CDD: specific functional annotation with the Conserved Domain Database. Nucleic Acids Res 2009, (37 Database) : D205–210.
      34. Schneider MC, Exley RM, Chan H, Feavers I, Kang YH, Sim RB, Tang CM: Functional significance of factor H binding to Neisseria meningitidis . J Immunol 2006, 176 (12) : 7566–7575.PubMed
      35. Morse SA, Stein S, Hines J: Glucose metabolism in Neisseria gonorrhoeae . J Bacteriol 1974, 120 (2) : 702–714.PubMed
      36. Nakano MM, Zuber P, Sonenshein AL: Anaerobic regulation of Bacillus subtilis Krebs cycle genes. J Bacteriol 1998, 180 (13) : 3304–3311.PubMed
      37. Nunez C, Bogachev AV, Guzman G, Tello I, Guzman J, Espin G: The Na+-translocating NADH : ubiquinone oxidoreductase of Azotobacter vinelandii negatively regulates alginate synthesis. Microbiology 2009, 155 (Pt 1) : 249–256.PubMedView Article
      38. Bogachev AV, Verkhovsky MI: Na(+)-Translocating NADH:quinone oxidoreductase: progress achieved and prospects of investigations. Biochemistry (Mosc) 2005, 70 (2) : 143–149.View Article
      39. Zumft WG: Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 1997, 61 (4) : 533–616.PubMed
      40. Mulkidjanian AY, Dibrov P, Galperin MY: The past and present of sodium energetics: may the sodium-motive force be with you. Biochim Biophys Acta 2008, 1777 (7–8) : 985–992.PubMedView Article
      41. Gadda G, Francis K: Nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis. Arch Biochem Biophys 2010, 493 (1) : 53–61.PubMedView Article
      42. Ishizaki K, Larson TR, Schauer N, Fernie AR, Graham IA, Leaver CJ: The critical role of Arabidopsis electron-transfer flavoprotein:ubiquinone oxidoreductase during dark-induced starvation. Plant Cell 2005, 17 (9) : 2587–2600.PubMedView Article
      43. Py B, Barras F: Building Fe-S proteins: bacterial strategies. Nat Rev Microbiol 2010, 8 (6) : 436–446.PubMedView Article
      44. Cho BK, Barrett CL, Knight EM, Park YS, Palsson BO: Genome-scale reconstruction of the Lrp regulatory network in Escherichia coli . Proc Natl Acad Sci USA 2008, 105 (49) : 19462–19467.PubMedView Article
      45. Calvo JM, Matthews RG: The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli . Microbiol Rev 1994, 58 (3) : 466–490.PubMed
      46. Ren J, Sainsbury S, Combs SE, Capper RG, Jordan PW, Berrow NS, Stammers DK, Saunders NJ, Owens RJ: The structure and transcriptional analysis of a global regulator from Neisseria meningitidis . J Biol Chem 2007, 282 (19) : 14655–14664.PubMedView Article
      47. Kidd SP, Potter AJ, Apicella MA, Jennings MP, McEwan AG: NmlR of Neisseria gonorrhoeae : a novel redox responsive transcription factor from the MerR family. Mol Microbiol 2005, 57 (6) : 1676–1689.PubMedView Article
      48. Potter AJ, Kidd SP, Edwards JL, Falsetta ML, Apicella MA, Jennings MP, McEwan AG: Esterase D is essential for protection of Neisseria gonorrhoeae against nitrosative stress and for bacterial growth during interaction with cervical epithelial cells. J Infect Dis 2009, 200 (2) : 273–278.PubMedView Article
      49. Potter AJ, Kidd SP, Edwards JL, Falsetta ML, Apicella MA, Jennings MP, McEwan AG: Thioredoxin reductase is essential for protection of Neisseria gonorrhoeae against killing by nitric oxide and for bacterial growth during interaction with cervical epithelial cells. J Infect Dis 2009, 199 (2) : 227–235.PubMedView Article
      50. Schook P, Cahoon L, Stohl E, Criss A, Seifert HS: Neisseria gonorrhoeae responses and defenses against reactive oxygen species. [http://​neisseria.​org/​ipnc/​2010/​IPNC_​2010_​abstracts.​pdf] 17th International Pathogenic Neisseria Conference: 2010; Banff, CA 2010.
      51. Laskos L, Ryan CS, Fyfe JA, Davies JK: The RpoH-mediated stress response in Neisseria gonorrhoeae is regulated at the level of activity. J Bacteriol 2004, 186 (24) : 8443–8452.PubMedView Article
      52. Du Y, Arvidson CG: RpoH mediates the expression of some, but not all, genes induced in Neisseria gonorrhoeae adherent to epithelial cells. Infect Immun 2006, 74 (5) : 2767–2776.PubMedView Article
      53. Schielke S, Schmitt C, Spatz C, Frosch M, Schubert-Unkmeir A, Kurzai O: The transcriptional repressor FarR is not involved in meningococcal fatty acid resistance mediated by the FarAB efflux pump and dependent on lipopolysaccharide structure. Appl Environ Microbiol 2010, 76 (10) : 3160–3169.PubMedView Article
      54. Friedrich A, Arvidson CG, Shafer WM, Lee EH, So M: Two ABC transporter operons and the antimicrobial resistance gene mtrF are pilT responsive in Neisseria gonorrhoeae . J Bacteriol 2007, 189 (14) : 5399–5402.PubMedView Article
      55. Warner DM, Shafer WM, Jerse AE: Clinically relevant mutations that cause derepression of the Neisseria gonorrhoeae MtrC-MtrD-MtrE Efflux pump system confer different levels of antimicrobial resistance and in vivo fitness. Mol Microbiol 2008, 70 (2) : 462–478.PubMedView Article
      56. Folster JP, Johnson PJ, Jackson L, Dhulipali V, Dyer DW, Shafer WM: MtrR modulates rpoH expression and levels of antimicrobial resistance in Neisseria gonorrhoeae . J Bacteriol 2009, 191 (1) : 287–297.PubMedView Article
      57. Criss AK, Katz BZ, Seifert HS: Resistance of Neisseria gonorrhoeae to non-oxidative killing by adherent human polymorphonuclear leucocytes. Cell Microbiol 2009, 11 (7) : 1074–1087.PubMedView Article
      58. Reyes ED, Patidar PL, Uranga LA, Bortoletto AS, Lusetti SL: RecN is a cohesin-like protein that stimulates intermolecular DNA interactions in vitro . J Biol Chem 2010, 285 (22) : 16521–16529.PubMedView Article
      59. Gruenig MC, Stohl EA, Chitteni-Pattu S, Seifert HS, Cox MM: Less is more-- Neisseria gonorrhoeae RecX protein stimulates recombination by inhibiting RecA. J Biol Chem 2010, 285 (48) : 37188–37197.PubMedView Article
      60. Hamdan S, Carr PD, Brown SE, Ollis DL, Dixon NE: Structural basis for proofreading during replication of the Escherichia coli chromosome. Structure 2002, 10 (4) : 535–546.PubMedView Article
      61. Seib KL, Tseng HJ, McEwan AG, Apicella MA, Jennings MP: Defenses against oxidative stress in Neisseria gonorrhoeae and Neisseria meningitidis : distinctive systems for different lifestyles. J Infect Dis 2004, 190 (1) : 136–147.PubMedView Article
      62. Barth KR, Isabella VM, Wright LF, Clark VL: Resistance to peroxynitrite in Neisseria gonorrhoeae . Microbiology 2009, 155 (Pt 8) : 2532–2545.PubMedView Article
      63. Whitby PW, Seale TW, Morton DJ, VanWagoner TM, Stull TL: Characterization of the Haemophilus influenzae tehB gene and its role in virulence. Microbiology 2010, 156 (Pt 4) : 1188–1200.PubMedView Article
      64. Tavares AF, Nobre LS, Melo AM, Saraiva LM: A novel nitroreductase of Staphylococcus aureus with S-nitrosoglutathione reductase activity. J Bacteriol 2009, 191 (10) : 3403–3406.PubMedView Article
      65. Overton TW, Justino MC, Li Y, Baptista JM, Melo AM, Cole JA, Saraiva LM: Widespread distribution in pathogenic bacteria of di-iron proteins that repair oxidative and nitrosative damage to iron-sulfur centers. J Bacteriol 2008, 190 (6) : 2004–2013.PubMedView Article
      66. Loiseau L, Gerez C, Bekker M, Ollagnier-de Choudens S, Py B, Sanakis Y, Teixeira de Mattos J, Fontecave M, Barras F: ErpA, an iron sulfur (Fe S) protein of the A-type essential for respiratory metabolism in Escherichia coli . Proc Natl Acad Sci USA 2007, 104 (34) : 13626–13631.PubMedView Article
      67. Kisselev LL, Justesen J, Wolfson AD, Frolova LY: Diadenosine oligophosphates (Ap(n)A), a novel class of signalling molecules? FEBS Lett 1998, 427 (2) : 157–163.PubMedView Article
      68. Ismail TM, Hart CA, McLennan AG: Regulation of dinucleoside polyphosphate pools by the YgdP and ApaH hydrolases is essential for the ability of Salmonella enterica serovar typhimurium to invade cultured mammalian cells. J Biol Chem 2003, 278 (35) : 32602–32607.PubMedView Article
      69. McLennan AG: The Nudix hydrolase superfamily. Cell Mol Life Sci 2006, 63 (2) : 123–143.PubMedView Article
      70. Dunn CA, O'Handley SF, Frick DN, Bessman MJ: Studies on the ADP-ribose pyrophosphatase subfamily of the nudix hydrolases and tentative identification of trgB, a gene associated with tellurite resistance. J Biol Chem 1999, 274 (45) : 32318–32324.PubMedView Article
      71. Hemm MR, Paul BJ, Miranda-Rios J, Zhang A, Soltanzad N, Storz G: Small stress response proteins in Escherichia coli : proteins missed by classical proteomic studies. J Bacteriol 2010, 192 (1) : 46–58.PubMedView Article
      72. Durand S, Storz G: Reprogramming of anaerobic metabolism by the FnrS small RNA. Mol Microbiol 2010, 75 (5) : 1215–1231.PubMedView Article
      73. Boysen A, Moller-Jensen J, Kallipolitis B, Valentin-Hansen P, Overgaard M: Translational regulation of gene expression by an anaerobically induced small non-coding RNA in Escherichia coli . J Biol Chem 2010, 285 (14) : 10690–10702.PubMedView Article
      74. Stohl EA, Criss AK, Seifert HS: The transcriptome response of Neisseria gonorrhoeae to hydrogen peroxide reveals genes with previously uncharacterized roles in oxidative damage protection. Mol Microbiol 2005, 58 (2) : 520–532.PubMedView Article
      75. Jackson LA, Ducey TF, Day MW, Zaitshik JB, Orvis J, Dyer DW: Transcriptional and functional analysis of the Neisseria gonorrhoeae Fur regulon. J Bacteriol 2010, 192 (1) : 77–85.PubMedView Article
      76. Piekarowicz A, Klyz A, Majchrzak M, Adamczyk-Poplawska M, Maugel TK, Stein DC: Characterization of the dsDNA prophage sequences in the genome of Neisseria gonorrhoeae and visualization of productive bacteriophage. BMC Microbiol 2007, 7: 66.PubMedView Article
      77. Webb JS, Thompson LS, James S, Charlton T, Tolker-Nielsen T, Koch B, Givskov M, Kjelleberg S: Cell death in Pseudomonas aeruginosa biofilm development. J Bacteriol 2003, 185 (15) : 4585–4592.PubMedView Article
      78. Falsetta ML, McEwan AG, Jennings MP, Apicella MA: Anaerobic metabolism occurs in the substratum of gonococcal biofilms and may be sustained in part by nitric oxide. Infect Immun 2010, 78 (5) : 2320–2328.PubMedView Article
      79. Wagner PL, Waldor MK: Bacteriophage control of bacterial virulence. Infect Immun 2002, 70 (8) : 3985–3993.PubMedView Article
      80. Korch C, Hagblom P, Ohman H, Goransson M, Normark S: Cryptic plasmid of Neisseria gonorrhoeae : complete nucleotide sequence and genetic organization. J Bacteriol 1985, 163 (2) : 430–438.PubMed
      81. Sarandopoulos S, Davies JK: Genetic organization and evolution of the cryptic plasmid of Neisseria gonorrhoeae . Plasmid 1993, 29 (3) : 206–221.PubMedView Article
      82. Giraldo R, Diaz-Orejas R: Similarities between the DNA replication initiators of Gram-negative bacteria plasmids (RepA) and eukaryotes (Orc4p)/archaea (Cdc6p). Proc Natl Acad Sci USA 2001, 98 (9) : 4938–4943.PubMedView Article
      83. Giraldo R, Andreu JM, Diaz-Orejas R: Protein domains and conformational changes in the activation of RepA, a DNA replication initiator. EMBO J 1998, 17 (15) : 4511–4526.PubMedView Article
      84. Wickner S, Hoskins J, McKenney K: Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin. Proc Natl Acad Sci USA 1991, 88 (18) : 7903–7907.PubMedView Article
      85. Wickner S, Hoskins J, McKenney K: Function of DnaJ and DnaK as chaperones in origin-specific DNA binding by RepA. Nature 1991, 350 (6314) : 165–167.PubMedView Article
      86. Daines DA, Jarisch J, Smith AL: Identification and characterization of a nontypeable Haemophilus influenzae putative toxin-antitoxin locus. BMC Microbiol 2004, 4: 30.PubMedView Article
      87. Blanco M, Kadlec K, Gutierrez Martin CB, de la Fuente AJ, Schwarz S, Navas J: Nucleotide sequence and transfer properties of two novel types of Actinobacillus pleuropneumoniae plasmids carrying the tetracycline resistance gene tet(H) . J Antimicrob Chemother 2007, 60 (4) : 864–867.PubMedView Article
      88. Van Melderen L, Saavedra De Bast M: Bacterial toxin-antitoxin systems: more than selfish entities? PLoS Genet 2009, 5 (3) : e1000437.PubMedView Article
      89. Kellogg DS, Peacock WL, Deacon WE, Brown L, Pirkle DI: Neisseria Gonorrhoeae . I. Virulence Genetically Linked to Clonal Variation. J Bacteriol 1963, 85: 1274–1279.PubMed
      90. Silver LE, Clark VL: Construction of a translational lacZ fusion system to study gene regulation in Neisseria gonorrhoeae . Gene 1995, 166 (1) : 101–104.PubMedView Article
      91. Miller JH, (ed.): Experiments in Molecular Genetics. Cold Spring Harbor: Cold Spring Harbor Laboratory; 1972.
      92. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, (ed): Current Protocols in Microbiology. New York: John Wiley & Sons, Inc; 1987.
      93. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, (ed): Short Protocols in Microbiology. 2nd edition. New York: John Wiley & Sons, Inc; 1992.
      94. Sambrook J, Fritsch EF, Maniatis T, (ed): Molecular Cloning: a Laboratory Manual. 2nd edition. Cold Spring Harbor: Cold Spring Laboratory Press; 1989.
      95. Sebastian S, Agarwal S, Murphy JR, Genco CA: The gonococcal fur regulon: identification of additional genes involved in major catabolic, recombination, and secretory pathways. J Bacteriol 2002, 184 (14) : 3965–3974.PubMedView Article
      96. Ducey TF, Jackson L, Orvis J, Dyer DW: Transcript analysis of nrrf , a Fur repressed sRNA of Neisseria gonorrhoeae . Microb Pathog 2009, 46 (3) : 166–170.PubMedView Article

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