Bacterial strains and culturing conditions
Escherichia coli strain JM109 [endA-1 recA-1 gyrA-96 thi hsdR-17(rk-, mk+) relA-1 supE-44 Δ(lac-proAB)(F' traD-36 proAB lacIqZΔM15)] , or TOP10 [F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ 80lacZΔM15 ΔlacX-74 recA-1 araD-139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA-1 nupG]  was cultured in LB medium at 37°C with shaking. Targeted gene disruption experiments were performed on G. sulfurreducens strain DL1 [60, 61] to produce strains DLCN29 (a kanamycin cassette insertion between GSU1888 and 1887 (rpoN)), DLCN32 (a kanamycin cassette insertion between GSU1887 and GSU1886) (Figure 1) and an rpoN diploid strain DLCN43. G. sulfurreducens strains were routinely cultured anaerobically in NB acetate-fumarate (NBAF) or freshwater acetate-Fe(III) citrate (FWAFC) medium at 30°C as previously described . NB and FW are the two basic mineral solutions, and differ mainly in buffering capaCity and trace element contents. Acetate (15 mM) and fumarate (40 mM) were the electron donor and electron acceptor, respectively, for the general propagation unless otherwise Stated. Both can be substituted with either lactate (20 mM) or hydrogen as an electron donor, or Fe(III) citrate (55 mM) as an electron acceptor when necessary (for a complete media composition please see references [61, 62]).
Genomic DNA was extracted with the Qiagen Genomic-tip 100/G. Plasmid DNA and PCR products were purified with the Qiagen mini plasmid purification kits and PCR purification kits, respectively (Qiagen). DNA cloning and other manipulations were carried out according to the methods outlined by Sambrook et al. . Restriction enzymes and other DNA-modifying enzymes were from New England Biolabs. Probes for Southern blot analyses were labeled with [α-32P]dCTP using the NEBlot kit (New England BioLabs). [α-32P]dCTP was from PerkinElmer Life and Analytical Sciences. Qiagen Taq DNA polymerase, unless otherwise Stated, was used for all PCR amplifications.
Single-step gene replacement
Sequences were deleted with single-step gene replacement as previously described . To disrupt the intergenic regions either upstream (between rpoN (GSU1887) and GSU1888) or downstream (between rpoN and GSU1886) of the rpoN gene, a linear DNA fragment was generated by recombinant PCR [64, 65] from three primary PCR products. For disruption of the intergenic region upstream of the rpoN gene, a 2.1 kb linear DNA fragment was composed of three PCR products: (1) the 3' end of GSU1888 (0.5 kb, amplified with primers rpoNU-1 and rpoNU-2); (2) 5' end of the rpoN gene (0.5 kb, amplified with primers rpoNU-5 and rpoNU-6); and (3) a kanamycin resistant cassette (KanR) (1.1 kb, amplified with primers rpoNU-3 and rpoNU-4). For disruption of the intergenic region downstream of the rpoN gene, three primary PCR reactions were performed to amplify a 2.1 kb linear DNA fragment: (1) the 3' end of rpoN [0.5 kb, position to position, amplified with primers rpoND-1 and rpoND-2); (2) 5' end of the GSU1886 gene (0.5 kb, amplified with primers rpoND-5 and rpoND-6); and (3) a KanR cassette (1.1 kb, amplified with primers rpoND-3 and rpoND-4). Recombinant PCR was performed with these three PCR products as templates with distal primer pairs, rpoNU-1/rpoNU-6 and rpoND-1/rpoND-6 for upstream or downstream intergenic region mutation respectively. PCR conditions were as previously described, except that the annealing temperature was 58°C . All primer sequences used in this work are listed in Additional file 7.
Electroporation, mutant isolation and genotype confirmation were performed as previously described [61, 64]. One of each of the mutants, designated DLCN29 and DLCN32, was chosen as the representative strain.
Construction of an rpoN diploid strain of G. sulfurreducens (DLCN43)
A 2.5 kb linear DNA fragment containing the chloramphenicol resistance cassette (CmR) followed by the coding region of the rpoN gene was constructed using cross-over PCR . The chloramphenicol resistance cassette was amplified with Cm-rpoNF1 (Cla I site) and Cm-rpoNR2 using pACYC184 as the template. The rpoN gene was amplified with C-rpoNF3 and C-rpoNR4. The two PCR products were joined together by cross-over PCR as described in [64, 65]. The resulted PCR product (Cm-rpoN) was Klenow filled-in and ligated to the Sma I-cut pLA01 (as described below), resulted in plasmid pLA03.
The plasmid pLA01 is a derivative of pCR2.1-TOPO that the 5'-end of the periplasmic c-type cytochrome gene (ppcA) which was amplified with primer pair: ppcAF1 and ppcAR2 was cloned into pCR2.1-TOPO using TOPO TA cloning kit (Invitrogen). Therefore, plasmid pLA03 contains the 5'-end ppcA followed by Cm-rpoN: the CmR resistance cassette and the rpoN gene, and the 3'-end of ppcA. The plasmid pLA03 was linearized and electroporated into G. sulfurreducens DL1 and CmR transformants were selected. The insertion of the Cm-rpoN construction within the ppcA gene was verified by PCR and the resultant strain was named DLCN43.
In order to interrupt any of the two copies of the rpoN gene in DLCN43, a linear PCR fragment containing the rpoN gene disrupted by the kanamycin resistance cassette was constructed with cross-over PCR. The 5' region of rpoN was amplified with primer pair: RpoNKmII-1 and RpoNKmII-2. The 3' region of rpoN was amplified with RpoNKmII-5 and RpoNKmII-6. The KanR cassette was amplified with RpoNKmII-3 and RpoNKmII-4. The recombinant PCR was carried out as described in the previous section and the resultant recombinant PCR product was electroporated into the strain DLCN43. A total of 15 KanR transformants were isolated. However, all 15 transformants had the KanR insertion within the Cm-rpoN locus.
Over-expression of rpoN in trans under the control of a lac or an IPTG-inducible taclac promoter
The complete rpoN coding sequence was amplified with primer sets RpoNfor-XbaI and RpoNrev-EcoRI for insertion to pJMG (lac promoter, gentamycin resistant) [66, 67] or RpoNfor-EcoRI and RpoNrev-HindIII for insertion to pCD341 (taclac promoter, kanamycin resistant)  using Phusion High-Fidelity DNA polymerase (New England Biolabs) under the following conditions: 98°C, 30 s followed by 30 cycles of 98°C,20 s; 58°C, 20 s; 72°C, 60 s; and a final extension at 72°C for 10 min. The PCR product of the rpoN coding sequence was digested with restriction enzyme sets of Xba I and EcoR I or EcoR I and Hind III and inserted into the Xba I and EcoR I sites of the vector pJMG or the EcoR I and Hind III sites of the vector pCD341 via ligation; the resulting plasmids were designated pJMG rpoN or pCD rpoN, respectively. The rpoN gene in pJMGrpoN or pCDrpoN was then sequenced to screen for PCR artifacts.
Following electroporation of strain DL1 with pJMGrpoN or pCDrpoN, a gentamycin-resistant transformant or a kanamycin-resistant transformant, was isolated and designated DL1/pJMGrpoN or DL1/pCDrpoN (RpoN+ for simplification), respectively. The presence of the plasmid in the DL1 strain was confirmed by plasmid purification and PCR.
The over-expression of rpoN for the strain containing pCDrpoN was achieved by adding 1 mM IPTG, a non-degradable analog of lactose to the medium. In the absence of lactose, transcription from the taclac promoter is inhibited by the lacZ repressor . Upon addition of lactose or IPTG, the lacZ repressor is inactivated, therefore inducing transcription of the rpoN operon.
Primer extension analyses
Total RNA was isolated from mid-exponential-phase cultures with RNeasy Midi kits (Qiagen) followed by treatment with RNase-free DNase (Ambion). Primer extension experiments were performed at 42°C using AMV reverse transcriptase (Roche) with primers GSU0364-06, GSU0420-04, GSU0777-04, GSU0938-06, GSU1836-04, GSU2005-02, GSU2302-04, GSU2490-02, GSU2751-02, GSU2806–08, GSU3046-02, and GSU3206-06, respectively for the corresponding promoter regions. The sequencing ladders presented in Figure 3 and Additional file 6 were also generated with these same primers using Thermo Sequenase Cycle sequencing kit (USB).
DNA microarray hybridization and statistical analysis
DNA microarray hybridization was carried out as previously described . Briefly, total RNA was extracted from three sets of identically treated batch cultures of the wild type harboring an empty vector (DL1/pCD341 or WTV for simplification) and the RpoN overexpressing strains (RpoN+). Ten micrograms of RNA from the wild type and the RpoN+ strain samples were chemically labeled with Cy3 or Cy5 fluorescent dyes respectively, using the MicroMax ASAP RNA Labeling Kit (Perkin Elmer), according to manufacturer's instructions. Labeled RNA was fragmented in a 20 μl volume at 70°C for 30 min using Ambion's Fragmentation Reagent and competitively hybridized to 12 K Arrays (Combimatrix) according to manufacturer's protocol. The arrays were scanned using a GenePix 4000B scanner (Molecular Devices), and analyzed using GenePix and Acuity 4.0 software. LIMMA mixed model analysis (R-package LIMMA ) was applied to the normalized Log2 expression ratios to identify differentially expressed genes. The P-value was then corrected for multiple comparisons according to Benjamini and Hochberg's procedure  to control the false discovery rate (FDR). Genes whose expression was significantly changed are listed in Additional files 1 & 2 according to their fold changes (≤ -1.5 for down-regulation and ≥ +1.5 for up-regulation) and the P-values (≤ 0.0005). A gene was considered differentially expressed if at least half of its probes had a P ≤ 0.0005 and a fold change ≤ -1.5 or ≥ +1.5.
Gene expression microarray data (raw data and statistically processed data files) for the G. sulfurreducens over-expressing RpoN strain are available from the NCBI GEO (Gene Expression Omnibus) database http://www.ncbi.nlm.nih.gov/geo/, with accession GSE8022.
Computational analysis of RpoN-regulated promoters and their target operons
RpoN-regulated promoters were predicted in the genome of G. sulfurreducens using the PromScan software . This software assigned scores representing the Kullback-Leibler distance for predicted RpoN sites in the G. sulfurreducens genome, based on 186 known RpoN promoter sites from 47 bacterial species . The predicted sequence elements were ranked according to their PromScan scores, and sequence elements with scores equal to or exceeding the default cutoff of 80 were selected for further consideration.
The operon organization of the G. sulfurreducens genome was predicted using a commercial version of the FGENESB software (V. Solovyev, A. Salamov, and P. Kosarev, unpublished; Softberry, Inc; 2003–2008). The reference June 1, 2004 version of operon annotation used in this study has been described previously . For all RpoN-regulated promoter elements predicted by PromScan, we compared their genome location and strand orientation relative to operons and singleton ORFs. Those sequence elements that were located upstream of and in the same direction with protein-coding genes were considered to be possible RpoN-regulated promoter elements. Only those elements that did not overlap with coding genes (according to gene boundaries predicted by the FGENESB software) were selected for further consideration.
To compare the predicted locations of RpoN-regulated promoters with experimental evidence, we identified predicted RpoN promoters located upstream of and in the same orientation with genes with significantly altered expression in the RpoN+ strain. This was achieved by comparing the list of suggested target genes located downstream of RpoN promoters (see Additional file 4) to the list of genes with significantly altered expression in the RpoN+ strain and identifying the genes present in both lists.
Consensus sequences of predicted RpoN promoters was computed using our software, CONSENS by J. Krushkal . Each nucleotide reported in the output consensus sequence represents the most frequent nucleotide. For ambiguous nucleotides co-occurring with equal highest frequencies, degenerate symbols were used according to the IUPAC-IUB ambiguity codes. Sequence logos of the predicted promoter sites were drawn using the WebLogo package v. 3 beta at http://weblogo.berkeley.edu/.
In silico analysis of G. sulfurreducens growth
In silico modeling was utilized to analyze the possible phenotypes of G. sulfurreducens mutants in which genes encoding enzymes for ammonia assimilation pathway were deleted. The constraint-based genome-scale metabolic model of G. sulfurreducens  was applied in simulating cell growth using flux balance analysis and linear optimization  in SimPheny (Genomatica, Inc., CA). Biomass synthesis was selected as the objective function to be maximized in growth simulations. The following external metabolites were allowed to freely enter and leave the network for simulations of anaerobic growth on minimal media: Ca2+, CO2, Fe2+, H+, H2O, K+, Mg2+, Na+, NH4+, PO43-, and SO42-. Acetate was supplied to the metabolic model as electron donor and Fe(III) or fumarate was supplied as electron acceptor for the simulations. All other external metabolites were only allowed to leave the system.
Preparation of antisera against RpoN
The rpoN coding region was amplified with primers pGEXrpoNEcoRIfor and pGEXrpoNXhoIrev, digested with EcoR I and Xho I, and inserted into the EcoR I-Xho I sites of pGEX-4T-1 (GE). Competent E. coli strain JM109 was transformed with the resulting plasmid, pGEXrpoN. The E. coli cell lysates containing the over-expressed GST-tagged RpoN was size-fractioned by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The band corresponding to RpoN protein was cut, crushed, and used to immunize New Zealand rabbits for antibody production against RpoN as described by Harlow and Lane .
Protein concentration was determined using the bicinchoninic acid method with bovine serum albumin as a standard . Western blot analyses were carried out by using antiserum against RpoN according to the protocol described by Ausubel et al . Immunoreactive bands were visualized using an alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (Pierce) and 1-step NBT/BCIP plus suppressor (Pierce) according to the manufacturer's instructions. Growth of fumarate cultures was monitored by measuring turbidity at 600 nm in a Genesys 2 spectrophotometer (Spectronic Instruments). Cell density of Fe(III)-grown cultures were determined using epifluorescence microscopy with acridine orange staining . Fe(II) concentrations were determined with the ferrozine assay as previously described . Agglutination assays were preformed as described by Reguera et al .