Updated genome assembly and annotation of Paenibacillus larvae, the agent of American foulbrood disease of honey bees

  • Queenie WT Chan1,

    Affiliated with

    • R Scott Cornman2,

      Affiliated with

      • Inanc Birol3,

        Affiliated with

        • Nancy Y Liao3,

          Affiliated with

          • Simon K Chan3,

            Affiliated with

            • T Roderick Docking3,

              Affiliated with

              • Shaun D Jackman3,

                Affiliated with

                • Greg A Taylor3,

                  Affiliated with

                  • Steven JM Jones3,

                    Affiliated with

                    • Dirk C de Graaf4, 5,

                      Affiliated with

                      • Jay D Evans2 and

                        Affiliated with

                        • Leonard J Foster1Email author

                          Affiliated with

                          BMC Genomics201112:450

                          DOI: 10.1186/1471-2164-12-450

                          Received: 21 February 2011

                          Accepted: 16 September 2011

                          Published: 16 September 2011

                          Abstract

                          Background

                          As scientists continue to pursue various 'omics-based research, there is a need for high quality data for the most fundamental 'omics of all: genomics. The bacterium Paenibacillus larvae is the causative agent of the honey bee disease American foulbrood. If untreated, it can lead to the demise of an entire hive; the highly social nature of bees also leads to easy disease spread, between both individuals and colonies. Biologists have studied this organism since the early 1900s, and a century later, the molecular mechanism of infection remains elusive. Transcriptomics and proteomics, because of their ability to analyze multiple genes and proteins in a high-throughput manner, may be very helpful to its study. However, the power of these methodologies is severely limited without a complete genome; we undertake to address that deficiency here.

                          Results

                          We used the Illumina GAIIx platform and conventional Sanger sequencing to generate a 182-fold sequence coverage of the P. larvae genome, and assembled the data using ABySS into a total of 388 contigs spanning 4.5 Mbp. Comparative genomics analysis against fully-sequenced soil bacteria P. JDR2 and P. vortex showed that regions of poor conservation may contain putative virulence factors. We used GLIMMER to predict 3568 gene models, and named them based on homology revealed by BLAST searches; proteases, hemolytic factors, toxins, and antibiotic resistance enzymes were identified in this way. Finally, mass spectrometry was used to provide experimental evidence that at least 35% of the genes are expressed at the protein level.

                          Conclusions

                          This update on the genome of P. larvae and annotation represents an immense advancement from what we had previously known about this species. We provide here a reliable resource that can be used to elucidate the mechanism of infection, and by extension, more effective methods to control and cure this widespread honey bee disease.

                          Background

                          Paenibacillus larvae is a spore-forming, Gram-positive bacterium, studied for the past century due to its ability to cause American foulbrood (AFB), a larval disease of honey bees [1]. The host is most vulnerable during an approximately 48-h window in the life cycle - the early larval stage - where arguably an undeveloped immune system and/or a lack of energy stores result in death. During this period, the oral LD50 ingestion is 8.49 spores [2]; death occurs due to systemic infection after the germinated bacterial spores proliferate in the midgut and then breach the midgut epithelium via a paracellular route [3]. The antibiotics oxytetracycline and tylosin are used both prophylactically and to treat symptoms; however, widespread drug resistance is evident [4] and their registered use is being withdrawn in many countries since residues can show up in honey. Even in susceptible isolates though, it is extremely difficult to completely eliminate from a hive, so without definitive knowledge of the molecular mechanism of pathogenesis, the design of specific treatments is significantly hindered. In 2006, a draft of the P. larvae genome was published at an estimated 5-6x coverage [5]; here we extend this coverage and further annotate the genome sequence with a combination of bioinformatics and proteomics.

                          Results and Discussion

                          Assembly of the P. larvae genome

                          Using the Illumina GAII platform and the ABySS assembler, together with the previously collected Sanger reads [5]; we achieved 182x coverage of the P. larvae genome and generated an initial assembly with a contig N50 of 49.6 kb. This statistic describes the contiguity of an assembly, and denotes that 50% of the reconstructed genome is contained in contigs equal to or larger than the given value. Contigs were joined into scaffolds using ABySS and Anchor (version 0.2.7, http://​www.​bcgsc.​ca/​platform/​bioinfo/​software/​anchor/​releases/​0.​2.​7) (Docking et al., in preparation) to achieve a scaffold N50 of 83.2 kb. The largest contig size was 261,601 base pairs. There were 184 contigs with a size less than 1 kb, 136 contigs with length between 1 kb and 10 kb, 57 contigs with length between 10 kb and 100 kb and 11 contigs larger than 100 kb. The total length of the assembled contigs was 4,505,771 base pairs. Assembly statistics are listed in Table 1.
                          Table 1

                          Summary of the P. larvae genome assembly.

                          Read pairs

                          8,212,402

                          Read length

                          50 bp

                          Fold coverage

                          182x

                          Contig N50

                          49.6 kb

                          Scaffold N50

                          83.2 kb

                          Number of contigs

                          388

                          Total length of assembled contigs

                          4,505,771 bp

                          Average GC content per contig

                          44.04%

                          Mean contig size

                          11.6 kb

                          Largest contig

                          261,601 bp

                          This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession ADZY00000000, with the latest version being ADZY02000000. Genome annotation and downstream analysis described below is based on the first version, ADZY01000000. Despite high sequence coverage, the assembly was relatively fragmented (Figure 1a). This fragmentation appears to be due primarily to long genomic repeats that could not be bridged by our sequencing strategy, as indicated by a preponderance of repetitive sequence occurring at contig ends (example shown in Figure 1b). Many of these repeats are similar to known bacterial insertion sequences and exceed 1 kb in length (data not shown). As a result, the majority of contigs were 1-10 kb in length.
                          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-450/MediaObjects/12864_2011_3602_Fig1_HTML.jpg
                          Figure 1

                          Self-alignment of 353P. larvaecontigs of assembly ADZY01000000.(a) MUMmer-generated graph of the P. larvae contigs aligned with themselves under default parameters. Red dots indicate sense matches and blue dots represent antisense matches. (b) Enlarged view of a sample region to demonstrate the common occurrence of alignments at the ends of contigs.

                          Given the fragmented nature of the assembly and the potential contribution of transposable elements to chromosome re-arrangements, we used tBLASTx and MUMmer to investigate the level of synteny between P. larvae and the congeneric soil bacterium P. JDR2. Although the completely sequenced P. JDR2 genome is 7.2 Mbp, substantially larger than our assembly (4.4 Mbp), the species share ~93% identity at 16S ribosomal loci. Figure 2a shows that, despite the difference in assembly size, gene-level synteny is generally conserved with P. JDR2 across the entirety of many P. larvae contigs. Some regions have excellent conservation (example in Figure 2b). This suggests that P. JDR2 can be a useful reference for comparative genomics and to order P. larvae contigs for genome finishing. Divergent regions between the two genomes are also of interest because they may harbor species-specific genes that are ecologically important in soil and beehive environments, respectively. For example, contig Seq24, which lacks synteny with P. JDR-2 (Figure 2c), contains two gene regions not present elsewhere in P. JDR-2 that are potential virulence factors (Table 2). Interesting, the same contig was also poorly conserved in another bacterium P. vortex (Figure 2d), thereby strengthening the claim that genes in this contig are unique to P. larvae. P. vortex is a pattern-forming soil bacterium with a 6.4 Mbp genome [6], so as expected, its overall level of synteny with P. larvae is quite high (Figure 2e), though slightly lower than observed when compared against P. JDR-2.
                          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-450/MediaObjects/12864_2011_3602_Fig2_HTML.jpg
                          Figure 2

                          Comparative genomics for aligningP. larvaecontigs.MUMmer-generated dot-plot showingP. larvaecontigs (y-axis) aligned with the fully-sequenced genomes of (a)P. JDR2and (b)P. vortex(x-axes). Proteins sequences were used for matching. Red dots indicate same-direction matches and blue dots represent antisense matches. Magnified views of regions from (a) exemplify P. larvae contigs that are (c) highly conserved and (d) poorly conserved with P. JDR2; the same contigs are also compared in P. vortex (e).

                          Table 2

                          Potential P. larvae virulence factors in genomic regions not found in P. JDR2.

                          ORF

                          Strand

                          Start

                          Stop

                          Best match E-value

                          Best match taxon

                          Best match accession

                          Description

                          EFX43964

                          minus

                          1200

                          2189

                          1.00E-024

                          Bacillus subtilis

                          NP_389716.2

                          plipastatin synthetase

                          EFX43965

                          minus

                          2207

                          7786

                          1.00E-110

                          Bacillus subtilis

                          NP_389598.3

                          polyketide synthase of type I

                          EFX43966

                          minus

                          7790

                          12370

                          1.00E-107

                          Streptococcus mutans

                          BAH88686.1

                          NcpA protein

                          EFX43967

                          minus

                          12556

                          15906

                          0

                          Bacillus subtilis

                          NP_389714.1

                          plipastatin synthetase

                          EFX43968

                          minus

                          15902

                          19897

                          0

                          Bacillus subtilis

                          NP_388230.2

                          surfactin synthetase

                          EFX43969

                          minus

                          19920

                          25211

                          0

                          Bacillus subtilis

                          NP_389714.1

                          plipastatin synthetase

                          EFX43970

                          minus

                          25232

                          34267

                          0

                          Bacillus subtilis

                          NP_388230.2

                          surfactin synthetase

                          EFX43971

                          minus

                          34309

                          42072

                          0

                          Bacillus subtilis

                          NP_389598.3

                          polyketide synthase of type I

                          EFX44032

                          minus

                          110449

                          111363

                          3.00E-090

                          Bacillus subtilis

                          NP_388265.1

                          putative iron-siderophore ABC transporter (binding lipoprotein)

                          EFX44033

                          minus

                          111493

                          112248

                          1.00E-097

                          Bacillus subtilis

                          NP_388264.1

                          putative iron-siderophore ABC transporter (ATP-binding protein)

                          EFX44034

                          minus

                          112245

                          113180

                          9.00E-094

                          Bacillus subtilis

                          NP_388263.1

                          putative iron-siderophore ABC transporter (permease)

                          EFX44035

                          minus

                          113188

                          114057

                          8.00E-095

                          Bacillus subtilis

                          NP_388262.1

                          putative iron-siderophore ABC transporter (permease)

                          Within the regions unique to P. larvae, one includes several open reading frames (ORFs) with strong homology to the synthetases of the antibiotic plipastatin, which inhibits phospholipase A2 [7] and surfactin [8], which possess hemolytic activity [9]. The region also contains putative type I polyketide synthetases whose products are secondary metabolites, some of which have antibiotic, immunosuppresant, or toxic effects [10]. The second region encodes homologs of a putative iron-siderophore ABC transporter, which enables iron uptake from the medium, an important process for many bacterial infections [11].

                          Annotation of putative P. larvae proteins

                          Using GLIMMER to predict genes from the P. larvae assembly and BLAST [12] searches against Bacillus or Streptococcus to provide annotation information (Additional File 1), we identified 3,568 gene models (final list in Additional File 2). Using shotgun proteomics methods to analyze P. larvae lysates, we identified 1262 proteins (1% false discovery rate), thus confirming the expression of 35% of the predicted proteins. Details regarding the identities of these proteins can be found in Additional File 3. As a major aim of this project is to find potential virulent factors and unique genes for this niche-specific bacterium, we describe below the genome content of P. larvae for several pathways considered to be important in this regard. The putative functions of the proteins were predicted by searching for matches against the Conserved Domain Database (CDD), and the complete results are tabulated in Additional File 4.

                          Flagellar system

                          P. larvae is a flagellated bacterium and 41 genes associated with this system are detected in this assembly (Table 3), based on comparisons to two other fully-sequenced organisms. This group includes motor/switch, flagellar rings, rod hook and filament proteins, along with proteins involved in regulation and as chaperones. When compared against the Bacillus subtilis (Figure 3a, based on [13]) and Escherichia coli (Figure 3b, based on [14]) flagella, P. larvae has orthologs for nearly all of the genes. Those that are missing, such as the L and P ring proteins FlgH and FlgI in the outer membrane of the Gram-negative E. coli, are not necessary for the Gram-positive P. larvae since it only has one membrane [15]. However, it appears to be missing some players needed for assembly of the flagellar hook, a structure that acts both as a joint and motor for each individual flagellum. The hook itself, made of monomers, requires the monomer scaffolding protein FlgD [16]; this was not found in the P. larvae genome. Also missing is FliK, which acts essentially as a checkpoint for the flagellum's correct length prior to export. This may mean that this species has evolved ways to proceed with hook assembly despite their omission, or the enzymes were simply not detected by our current methods; as with many negative results, however, our inability to detect such genes does not imply P. larvae is completely incapable of quality control functions. Besides the flagellar structure, another important aspect is the control of its movement. Directionality is largely dictated by the Che gene family and methyl-accepting chemotaxis proteins [17], which can also be found in the P. larvae genome. The base of the flagellum consists of assembly and regulatory components, which are made of Fli and Mot gene families in E. coli [18]. Again, orthologs for most of these can be seen in P. larvae.
                          Table 3

                          Putative P. larvae flagellar proteins

                           

                          Gene

                          Protein, functional information from CDD

                          P. larvae protein accession

                          Two component system chemotaxis proteins

                          cheA

                          CheA, sensor kinase [EC:2.7.13.3]

                          EFX46098; EFX46097

                           

                          cheW

                          CheW, purine-binding protein

                          EFX46097

                           

                          cheX

                          CheX

                          Not found

                           

                          cheC

                          CheC

                          EFX44110; EFX46096

                           

                          cheD

                          CheD [EC:3.5.1.44]

                          EFX46095

                           

                          cheR

                          CheR, methyltransferase [EC:2.1.1.80]

                          EFX46532

                           

                          cheB

                          CheB, response regulator [EC:3.1.1.61]

                          EFX46099

                           

                          cheY

                          CheY, response regulator

                          EFX46498; EFX46108

                           

                          cheZ

                          CheZ

                          EFX46498

                           

                          cheV

                          CheV, response regulator

                          EFX46097; EFX46099

                          Methyl-accepting chemotaxis proteins

                          mcp

                          methyl-accepting chemotaxis protein

                          EFX44725; EFX44051; EFX45552; EFX46097

                           

                          tsr

                          McpI, serine sensor receptor

                          EFX44051; EFX44725

                           

                          tar

                          McpII, aspartate sensor receptor

                          EFX44725; EFX46877

                           

                          trg

                          McpIII, ribose and galactose sensor receptor

                          EFX44725

                           

                          tap

                          McpIV, peptide sensor receptor

                          Not found

                           

                          aer

                          Aerotaxis receptor

                          EFX44725; EFX46877

                           

                          hemAT

                          haem-based aerotactic transducer

                          EFX44725; EFX46877

                          Type-III secretion proteins for flagellar assembly

                          fliH

                          FliH

                          EFX46029

                           

                          fliI

                          FliI, flagellum-specific ATP synthase [EC:3.6.3.14]

                          EFX46028

                           

                          fliZ, fliO

                          FliO/FliZ

                          EFX46107

                           

                          fliP

                          FliP, flagellar biosynthetic protein

                          EFX46106

                           

                          fliQ

                          FliQ, flagellar biosynthetic protein

                          EFX46105

                           

                          fliR

                          FliR, flagellar biosynthetic protein

                          EFX46104

                           

                          flhA

                          FlhA, flagellar biosynthesis protein

                          EFX46102

                           

                          flhB

                          FlhB, flagellar biosynthetic protein

                          EFX46103

                           

                          flhE

                          FlhE

                          Not found

                           

                          flhF

                          FlhF, flagellar biosynthetic protein

                          EFX46101

                          Motor/Switch

                          motA

                          MotA, chemotaxis protein

                          EFX45489

                           

                          motB

                          MotB, chemotaxis protein

                          EFX45490

                          C-ring

                          fliG

                          FliG, flagellar motor switch protein

                          EFX46030

                           

                          fliM

                          FliM, flagellar motor switch protein

                          EFX46110

                           

                          fliNY, fliN

                          FliN/FliY, flagellar motor switch protein

                          EFX46109

                          M, S, P and L rings

                          fliF

                          FliF, flagellar M-ring protein

                          EFX46031

                           

                          flgI

                          FlgI, flagellar P-ring protein precursor

                          not found

                           

                          flgA

                          FlgA, flagella basal body P-ring formation protein

                          not found

                           

                          flgH

                          FlgH, flagellar L-ring protein precursor

                          not found

                           

                          fliF

                          FliF, flagellar M-ring protein

                          EFX46031

                           

                          flgA

                          FlgA, flagella basal body P-ring formation protein

                          not found

                           

                          flgH

                          FlgH, flagellar L-ring protein precursor

                          not found

                          Rod, hook, and filament

                          flgB

                          FlgB, flagellar basal-body rod protein

                          EFX46034

                           

                          flgC

                          FlgC, flagellar basal-body rod protein

                          EFX46033

                           

                          flgD

                          FlgD, flagellar basal-body rod modification protein

                          not found

                           

                          flgF

                          FlgF, flagellar basal-body rod protein

                          EFX46113; EFX45368

                           

                          flgG

                          FlgG, flagellar basal-body rod protein

                          EFX46113; EFX45367

                           

                          flgJ

                          FlgJ, flagellar protein

                          not found

                           

                          flgE

                          FlgE, flagellar hook protein

                          EFX46113

                           

                          fliE

                          FliE, flagellar hook-basal body complex protein

                          EFX46032

                           

                          flgK

                          FlgK, flagellar hook-associated protein 1

                          EFX46010

                           

                          flgL

                          FlgL, flagellar hook-associated protein 3

                          EFX46011

                           

                          fliD

                          FliD, flagellar hook-associated protein 2

                          EFX46016

                           

                          fliK

                          FliK, flagellar hook-length control protein

                          not found

                           

                          fliL

                          FliL, flagellar protein

                          EFX46111

                           

                          fliC

                          Flagellin

                          EFX46015

                           

                          flaF

                          FlaF, flagellar protein

                          EFX46015

                           

                          flaG

                          FlaG, flagellar protein

                          not found

                           

                          flbA

                          FlbA, flagellar protein

                          EFX46102

                           

                          flbB

                          FlbB, flagellar protein

                          not found

                           

                          flbC

                          FlbC, flagellar protein

                          not found

                           

                          flbD

                          FlbD, flagellar protein

                          not found

                           

                          flbT

                          FlbT, flagellar protein

                          not found

                          Regulation

                          flhC

                          FlhC, flagellar transcriptional activator

                          not found

                           

                          flhD

                          FlhD, flagellar transcriptional activator

                          not found

                           

                          fliA

                          FliA, RNA polymerase sigma factor for flagellar operon

                          EFX44501;EFX46093; EFX43745; EFX44502

                           

                          flgM

                          FlgM, negative regulator of flagellin synthesis

                          not found

                          Chaperones

                          flgN

                          FlgN, flagella synthesis protein

                          not found

                           

                          fliJ

                          FliJ, flagellar protein

                          EFX46027

                           

                          fliS

                          FliS, flagellar protein

                          EFX46017

                           

                          fliT

                          FliT, flagellar protein

                          not found

                           

                          fliW

                          FliW, flagellar assembly factor

                          EFX46013

                           

                          fliY

                          FliY, cystine transport system substrate-binding protein

                          EFX46109

                           

                          fliZ

                          FliZ

                          EFX46107

                          Archaeal flagellar proteins

                          flaA-A, flaA

                          FlaA, archaeal flagellin

                          EFX46015

                           

                          flaB-A, flaB

                          FlaB, archaeal flagellin

                          EFX46015

                           

                          flaC-A, flaC

                          FlaC

                          EFX46015

                           

                          flaD-A, flaD

                          FlaD

                          EFX46015

                           

                          flaE-A, flaE

                          FlaE

                          EFX46113

                           

                          flaF-A, flaF

                          FlaF

                          EFX46015

                           

                          flaG-A, flaG

                          FlaG

                          not found

                           

                          flaH-A, flaH

                          FlaH

                          not found

                           

                          flaI-A, flaI

                          FlaI

                          not found

                           

                          flaJ-A, flaJ

                          FlaJ

                          not found

                           

                          flaK-A, flaK

                          FlaK, archaeal preflagellin peptidase

                          not found

                          http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-12-450/MediaObjects/12864_2011_3602_Fig3_HTML.jpg
                          Figure 3

                          Flagellar proteins. Distribution of flagellar proteins (excluding chemotaxis proteins) among flagellated Gram-positive (A) and Gram-negative (B) bacteria. The proteins encoded by the P. larvae genome are boxed in green (the ones that are lacking: in red).

                          Toxins

                          Our search for P. larvae genes that can encode toxins returned three matches to sixteen proteins (Table 4). A domain of the Clostridum perfringens epsilon toxin (pfam03318) was observed in EFX46729 and EFX45732, which forms pores on host cells [19] leading to cell death. P. larvae appear to possess classic binary toxins: the first factor is a membrane component that makes the host cell permeable to a second factor - the enzymatic component. Seven proteins matched the Clostridial binary toxin B domain (pfam03495), the membrane component. We also saw that four proteins matched the VIP2 domain (cd00233), which is the enzymatic component of a Bacillus toxin capable of ADP ribosylation on actin. In both the Clostridial and Bacillus toxins, the associated partners of these binary toxin-pairs were not observed.
                          Table 4

                          Putative P. larvae toxins.

                          Protein (E-value)

                          Domain, functional information from CDD

                          EFX46729 (8.00E-20),

                          EFX45732 (1.00E-18)

                          ETX_MTX2 [pfam03318]: Clostridium epsilon toxin ETX/Bacillus mosquitocidal toxin MTX2

                          EFX43878 (4.00E-32),

                          EFX45096 (3.00E-22),

                          EFX46664 (1.00E-17),

                          EFX46330 (1.00E-10)

                          VIP2 [cd00233]: A member of the vegetative insecticidal protein family. An actin-ADP-ribosylating toxin. A binary toxin.

                          EFX43879 (2.00E-102),

                          Binary_toxB [pfam03495]: Clostridial binary toxin B/anthrax toxin PA. A binary toxin.

                          EFX45058 (1.00E-83),

                           

                          EFX45095 (2.00E-48),

                           

                          EFX44748 (3.00E-37),

                           

                          EFX46332 (2.00E-29),

                           

                          EFX44749 (6.00E-19),

                           

                          EFX46331 (1.00E-16),

                           

                          EFX44747 (4.00E-6),

                           

                          EFX44951 (6.00E-5),

                           

                          EFX46847 (6.00E-5),

                           

                          *the listed match is not the one with the lowest E-value; it is displayed because it is the most informative

                          Hemolysins

                          In the mid to late stage of the P. larvae infection cycle, bacteria circulate in bee hemolymph which contains hemocytes. Hemolysins are a class of bacterial toxins with lytic activity against blood cells. Four hemolysin domains were matched by proteins from the P. larvae genome. (Table 5). Three proteins contained regions that were highly similar to the hemolytic domain TylC (COG1253) and concomitantly to other domains such as CBS_pair_CorC_HylC_assoc and DUF21 (data not shown), implying that these are very similar proteins. EFX46836 was matched to the hlyIII domain, an integral membrane protein from Bacillus cereus which forms channels on host cells [20]. EFX45686 appears to be related to hemolysin TlyA (TIGR00478) with an indirect pore-forming role. One protein (EFX45724) showed similarity to DUF37 with unpublished claims of hemolysin activity in Aeromonas hydrophila.
                          Table 5

                          Proteins with domains related to hemolysins or cytolytic activity.

                          Protein (E-value)

                          Domain, functional information from CDD

                          EFX44255 (8.00E-69),

                          EFX44544 (8.00E-72),

                          EFX46152 (3.00E-69)

                          TylC [COG1253]: Hemolysins and related proteins

                          EFX46836 (2.00E-23)

                          hlyIII [pfam03006]: channel-forming cytolysin

                          EFX45724 (1.00E-22)

                          DUF37 [cl00506]: found in various bacteria, a member from Aeromonas hydrophila has been found to have hemolytic activity (unpublished).

                          EFX45686 (2E-56*)

                          tly [TIGR00478]: hemolysin TlyA family protein; at least two members of this protein family have been characterized indirectly as pore-forming hemolysins.

                          *the listed match is not the one with the lowest E-value; it is displayed because it is the most informative

                          Proteases

                          The occurrence of proteases in larval scales (remains of P. larvae-infected bee larvae) has already been well documented [21]. A more recent study hints on a definite possibility that one or more such proteins can act as the virulence factor [22]. Since it has been shown that vegetative rods residing in the host midgut can traverse the epithelium by a paracellular route, P. larvae may generate proteolytic activity that can disrupt epithelial cell-to-cell junctions. We searched the proteins for domains with this function, and after excluding ones with roles that are not directly implicated in pathogenesis (e.g. signal peptidase, SOS response for DNA repair), we list the matches in Table 6. Based on the information provided by the CDD, several proteins may conceivably serve as virulence factors. The Clp protease complex, in particular, has been linked to virulence factor production in Gram-positive bacteria, as well as degradation of misfolded proteins [23, 24]. Proteins EFX45851, EFX44792 may be capable of cleaving the transmembrane regions of substrate proteins (matching domains cd06161, cd06158). Given that tight junctions between epithelial cells are mainly composed of occludin and claudin proteins, both of which have transmembrane domains [25], they may be specific substrates for P. larvae proteases. These points, however, are merely speculative, and the many proteases in Table 6 await experimental evidence to prove whether or not they actually contribute a real role in virulence.
                          Table 6

                          Putative P. larvae based on matches to conserved domains.

                          Protein (E-value)

                          Domain, functional information from CDD

                          EFX44344 (2E-37)

                          AmpS [COG2309]: Leucyl aminopeptidase (aminopeptidase T)

                          EFX45451 (6E-13)

                          APP-like [cd01092]: Prolidase and Aminopeptidase P

                          EFX47002 (0)

                          clpC [CHL00095]: Clp protease ATP binding subunit

                          EFX46058 (4E-5)

                          ClpP [COG0740]: Protease subunit of ATP-dependent Clp proteases

                          EFX44161 (1E-85),

                          EFX45289 (1E-82)

                          clpP [PRK00277]: Clp protease proteolytic subunit

                          EFX45288 (7E-170)

                          clpX [PRK05342]: Clp protease ATP-binding subunit

                          EFX45551 (7E-82),

                          COG0826 [COG0826]: Collagenase and related proteases

                          EFX45550 (4E-48),

                           

                          EFX45481 (3E-19),

                           

                          EFX44736 (1E-28)

                           

                          EFX44399 (9E-8*)

                          COG1310 [COG1310]: Predicted metal-dependent protease of the PAD1/JAB1 superfamily

                          EFX46420 (8E-149)

                          COG2317 [COG2317]: Zn-dependent carboxypeptidase

                          EFX44401 (9E-45)

                          COG2738 [COG2738]: Predicted Zn-dependent protease

                          EFX44533 (2E-9),

                          EFX45796 (2E-9)

                          COG2856 [COG2856]: Predicted Zn peptidase

                          EFX46883 (2E-31)

                          COG3740 [COG3740]: Phage head maturation protease

                          EFX45850 (6E-11)

                          COG4942 [COG4942]: Membrane-bound metallopeptidase

                          EFX46250 (1E-34),

                          EFX44926 (2E-64)

                          degP_htrA_DO [TIGR02037]: periplasmic serine protease, Do/DeqQ family

                          EFX47184 (5E-45)

                          DegQ [COG0265]: Trypsin-like serine proteases, typically periplasmic

                          EFX46573 (5E-91),

                          FtsH_fam [TIGR01241]: ATP-dependent metalloprotease FtsH

                          EFX44535 (5E-20*),

                           

                          EFX44172 (3E-17*)

                           

                          EFX47022 (0)

                          HflB [COG0465]: ATP-dependent Zn proteases

                          EFX45319 (1E-36*),

                          EFX44073 (5E-8*)

                          HflC [COG0330]: membrane protease subunits, stomatin/prohibitin homologs

                          EFX46036 (4E-157)

                          HslU [COG1220]: ATP-dependent protease HslVU (ClpYQ), ATPase subunit

                          EFX46037 (8E-65)

                          HslV [COG5405]: ATP-dependent protease HslVU (ClpYQ), peptidase subunit

                          EFX43995 (2E-97),

                          LasB [COG3227]: Zinc metalloprotease (elastase)

                          EFX45639 (1E-78),

                           

                          EFX45177 (9E-58)

                           

                          EFX45284 (0)

                          Lon [COG0466]: ATP-dependent Lon protease

                          EFX43687 (8E-131)

                          M3_fam_3 [TIGR02290]: oligoendopeptidase, pepF/M3 family

                          EFX44444 (2E-74)

                          M14_Endopeptidase_I [cd06229]: Peptidase M14-like domain of Gamma-D-glutamyl-L-diamino acid endopeptidase 1

                          EFX44181 (2E-45)

                          MrcA [COG5009]: Membrane carboxypeptidase/penicillin-binding protein

                          EFX45553 (4E-10*),

                          MrcB [COG0744]: Membrane carboxypeptidase (penicillin-binding protein)

                          EFX46407 (3E-97*),

                           

                          EFX44298 (3E-75*),

                           

                          EFX46175 (2E-122*),

                           

                          EFX44515 (1E-12*),

                           

                          EFX44516 (4E-11*)

                           

                          EFX45371 (7E-9), EFX45028 (2E-7), EFX45493 (4E-25)

                          NlpD [COG0739]: Membrane proteins related to metalloendopeptidases

                          EFX44427 (8E-144)

                          pepF [TIGR00181]: oligoendopeptidase F

                          EFX46173 (4E-9)

                          PepN [COG0308]: Aminopeptidase N

                          EFX45105 (1E-66)

                          PepP [COG0006]: Xaa-Pro aminopeptidase

                          EFX47190 (3E-18)

                          Peptidase_M23 [pfam01551]: Peptidase family M23

                          EFX46425 (8E-126)

                          Peptidase_M6 [pfam05547]: Immune inhibitor A peptidase M6, a metallopeptidase from Bacillus thuringiensis that cleaves host antibacterial proteins

                          EFX47085 (4E-29)

                          Peptidase_M9_N [pfam08453]: Peptidase family M9 N-terminal

                          EFX46343 (8E-6*)

                          Peptidase_S24 [pfam00717]: Peptidase S24-like

                          EFX43847 (9E-13)

                          Peptidase_S41_CPP [cd07560]: C-terminal processing peptidase; serine protease family S41

                          EFX43943 (3E-27*)

                          Peptidase_S66 [pfam02016]: LD-carboxypeptidase; this cleaves the bond between an L- and a D-amino acid

                          EFX45480 (6E-38), EFX45479 (2E-8)

                          Peptidase_U32 [pfam01136]: Peptidase family U32

                          EFX43960 (8E-78),

                          EFX45983 (2E-59)

                          Peptidases_S8_11 [cd04843]: Peptidase S8 family domain, uncharacterized subfamily 11

                          EFX46806 (8E-36)

                          Peptidases_S8_Lantibiotic_specific_protease [cd07482]: Peptidase S8 family domain in Lantiobiotic (lanthionine-containing antibiotics) specific proteases

                          EFX44534 (5E-26), EFX45795 (1E-20*), EFX44174 (8E-25)

                          Peptidases_S8_Subtilisin_like_2 [cd04847]: Peptidase S8 family domain in Subtilisin-like proteins

                          EFX45938 (8E-46), EFX45020 (3E-62), EFX46180 (2E-50), EFX45070 (2E-20)

                          Peptidases_S8_Subtilisin_subset [cd07477]: Peptidase S8 family domain in Subtilisin proteins

                          EFX44839 (5E-67),

                          EFX44567 (5E-47),

                          EFX44567 (4E-55)

                          Peptidases_S8_Thermitase_like [cd07484]: Peptidase S8 family domain in Thermitase-like proteins

                          EFX47149 (7E-172)

                          peptidase-T [TIGR01882]: peptidase T

                          EFX45664 (5E-87)

                          PepT-like [TIGR01883]: peptidase T-like protein

                          EFX46054 (6E-33),

                          EFX46055 (2E-7),

                          EFX46066 (2E-67)

                          PqqL [COG0612]: Predicted Zn-dependent peptidases

                          EFX45492 (3E-73)

                          prc [TIGR00225]: C-terminal peptidase (prc)

                          EFX44827 (3E-16*)

                          PRK00016 [PRK00016]: putative metalloprotease

                          EFX44779 (1E-110)

                          PRK02858 [PRK02858]: germination protease

                          EFX45078 (5E-118)

                          PRK07318 [PRK07318]: dipeptidase PepV

                          EFX44480 (4E-11*)

                          PRK08554 [PRK08554]: peptidase

                          EFX45711 (2E-73)

                          PRK09795 [PRK09795]: aminopeptidase

                          EFX45631 (4E-24*),

                          EFX44757 (1E-22*)

                          PRK13914 [PRK13914]: invasion associated secreted endopeptidase

                          EFX44490 (3E-22*)

                          PRK14791 [PRK14791]: lipoprotein signal peptidase

                          EFX46689 (1E-42)

                          S14_ClpP_1 [cd07016]: Caseinolytic protease (ClpP)

                          EFX44792 (2E-28)

                          S2P-M50_like_1 [cd06158]: Uncharacterized homologs of Site-2 protease (S2P), zinc metalloproteases (MEROPS family M50) which cleave transmembrane domains of substrate proteins, regulating intramembrane proteolysis (RIP) of diverse signal transduction mechanisms

                          EFX44503 (2E-42)

                          spore_II_GA [TIGR02854]: sigma-E processing peptidase SpoIIGA

                          EFX45851 (1E-12)

                          S2P-M50_SpoIVFB [cd06161]: SpoIVFB Site-2 protease (S2P), a zinc metalloprotease (MEROPS family M50B), regulates intramembrane proteolysis (RIP)

                          EFX47001 (2E-8*)

                          spore_lon_C[TIGR02903]: ATP-dependent protease, Lon family

                          EFX45285 (0)

                          spore_lonB [TIGR02902]: ATP-dependent protease LonB

                          EFX46081 (7E-45)

                          TIGR00054 [TIGR00054]: RIP metalloprotease RseP

                          EFX44693 (1E-61)

                          trio_M42_hydro [TIGR03106]: hydrolase, peptidase M42 family

                          *the listed match is not the one with the lowest E-value; it is displayed because it is the most informative

                          Antibiotic resistance

                          P. larvae infections have been treated with tetracycline for decades. It has been shown that resistance against this antibiotic can be conferred by the pMA67 plasmid [4]. More recently, the macrolide tylosin has now been registered as a new antibiotic in the U.S. for use when tetracycline is no longer effective [26]. Since antibiotic resistance is inevitable [27], new therapeutics will eventually be required. Realizing that P. larvae appears to encode a plethora of drug efflux proteins (Table 7) may help in the selection of useful drugs.
                          Table 7

                          Putative P. larvae antibiotic resistance proteins based on matches to conserved domains

                          Protein (E-value)

                          Domain, functional information from CDD

                          EFX43850 (3E-37),

                          EFX47127 (3E-26),

                          EFX43889 (2E-44),

                          ABC_BcrA_bacitracin_resist [cd03268]: The BcrA subfamily represents ABC transporters involved in peptide antibiotic resistance; bacitracin is an antibiotic produced by B. licheniformis and B. subtilis with potent antibiotic activity against gram-positive bacteria

                          EFX46602 (1E-57),

                           

                          EFX44842 (2E-50),

                           

                          EFX46272 (4E-41),

                           

                          EFX45233 (9E-52)

                           

                          EFX46614 (4E-25),

                          ABC_DR_subfamily_A [cd03230]: This family of ATP-binding proteins belongs to a multisubunit transporter involved in drug resistance (BcrA and DrrA), nodulation, lipid transport, and lantibiotic immunity

                          EFX44699 (4E-16),

                           

                          EFX45065 (1E-50)

                          ABC_DrrA [cd03265]: DrrA is the ATP-binding protein component of a bacterial exporter complex that confers resistance to the antibiotics daunorubicin and doxorubicin

                          EFX46465 (1E-55),

                          ABC_drug_resistance_like [cd03264]: ABC-type multidrug transport system, ATPase component

                          EFX45467 (3E-61),

                           

                          EFX45475 (2E-48*)

                           

                          EFX43892 (1E-42),

                          ABC_putative_ATPase [cd03269]: This subfamily is involved in drug resistance, nodulation, lipid transport, and bacteriocin and lantibiotic immunity

                          EFX46872 (1E-35),

                           

                          EFX47099 (4E-20*),

                           

                          EFX45045 (1E-71*)

                           

                          EFX46236 (3E-6*)

                          ABCC_MRP_Like [cd03228]: The MRP (Mutidrug Resistance Protein)-like transporters are involved in drug, peptide, and lipid export; they belong to the subfamily C of the ATP-binding cassette (ABC) superfamily of transport proteins.

                          EFX44443 (3E-26)

                          ACR_tran [pfam00873]: AcrB/AcrD/AcrF family. Members of this family are integral membrane proteins. Some are involved in drug resistance

                          EFX44702 (4E-33),

                          CcmA [COG1131]: ABC-type multidrug transport system, ATPase component

                          EFX46006 (5E-34),

                           

                          EFX44595 (4E-16),

                           

                          EFX43880 (8E-54),

                           

                          EFX47052 (7E-40),

                           

                          PL1_2983 (1E-33),

                           

                          EFX46146 (6E-40),

                           

                          EFX45253 (3E-22),

                           

                          EFX45810 (9E-18),

                           

                          EFX45945 (1E-18),

                           

                          EFX47082 (9E-21*),

                           

                          EFX44360 (3E-15*),

                           

                          EFX46805 (6E-30*),

                           

                          EFX45180 (5E-27*)

                           

                          EFX43881 (3E-4*)

                          COG0842 [COG0842]: ABC-type multidrug transport system, permease component

                          EFX44713 (1E-52)

                          COG2409 [COG2409]: Predicted drug exporters of the RND superfamily

                          EFX47096 (4E-18)

                          COG4152 [COG4152]: ABC-type uncharacterized transport system, ATPase component

                          EFX46793 (6E-34)

                          COG4586 [COG4586]: ABC-type uncharacterized transport system, ATPase component

                          EFX46612 (2E-58),

                          drrA [TIGR01188]: daunorubicin resistance ABC transporter ATP-binding subunit

                          EFX43786 (8E-63),

                           

                          EFX45226 (4E-54),

                           

                          EFX44634 (3E-36*)

                           

                          EFX43785 (4E-5)

                          drrB [TIGR01247]: daunorubicin resistance ABC transporter membrane protein; the protein associated with effux of the drug, daunorubicin

                          EFX44398 (8E-11),

                          EFX47139 (1E-59)

                          efflux_Bcr_CflA [TIGR00710]: drug resistance transporter, Bcr/CflA subfamily

                          EFX46169 (4E-40),

                          efflux_EmrB [TIGR00711]: drug resistance transporter, EmrB/QacA subfamily

                          EFX44387 (7E-37),

                           

                          EFX44446 (8E-09),

                           

                          EFX46307(5E-24),

                           

                          EFX46126 (1E-53),

                           

                          EFX44956 (3E-9*)

                           

                          EFX46708 (3E-14)

                          emrE [PRK09541]: multidrug efflux protein

                          EFX44177 (2E-4*)

                          Glyoxalase [pfam00903]: Glyoxalase/Bleomycin resistance protein/Dioxygenase superfamily

                          EFX45543 (4E-10),

                          HTH_MARR [smart00347]: helix_turn_helix multiple antibiotic resistance protein

                          EFX43861 (8E-11),

                           

                          EFX46148 (6E-10),

                           

                          EFX46170 (4E-12),

                           

                          EFX44712 (3E-12),

                           

                          EFX45783 (3E-6*),

                           

                          EFX46306 (1E-8*),

                           

                          EFX46227 (3E-4*),

                           

                          EFX44716 (5E-9*)

                           

                          EFX45439 (2E-48)

                          Lant_dehyd_C [pfam04738]: Lantibiotic dehydratase, C terminus

                          EFX45438 (3E-31)

                          LanC [cd04793]: LanC is the cyclase enzyme of the lanthionine synthetase. Lanthinoine is a lantibiotic, a unique class of peptide antibiotics

                          EFX46802 (4E-77*),

                          EFX46803 (4E-12*)

                          LcnDR2 [COG4403]: Lantibiotic modifying enzyme

                          EFX45784 (1E-84),

                          MdlB [COG1132]: ABC-type multidrug transport system; ATPase and permease components

                          EFX45333 (6E-107),

                           

                          EFX44415 (7E-47*),

                           

                          EFX44416 (5E-44*),

                           

                          EFX45434 (1E-26*)

                           

                          EFX45348 (3E-36)

                          NorM [COG0534]: Na+-driven multidrug efflux pump

                          EFX45532 (1E-114)

                          PRK01766 [PRK01766]: multidrug efflux protein

                          EFX46249 (6E-16)

                          PRK03545 [PRK03545]: sugar efflux transporter

                          EFX46262 (2E-15),

                          EFX44433 (8E-82)

                          PRK09874 [PRK09874]: drug efflux system protein MdtG

                          EFX45329 (5E-21)

                          PRK10054 [PRK10054]: putative MFS-type transporter YdeE

                          EFX47111 (5E-4*)

                          PRK10504 [PRK10504]: multidrug efflux system protein MdtE

                          EFX45115 (3E-58*)

                          PRK10522 [PRK10522]: multidrug transporter membrane component/ATP-binding component

                          EFX47080 (1E-129)

                          PRK10789 [PRK10789]: putative multidrug transporter membrane\ATP-binding components

                          EFX47079 (5E-98)

                          PRK10790 [PRK10790]: putative multidrug transporter membrane\ATP-binding components

                          EFX45606 (2E-13)

                          PRK11102 [PRK11102]: bicyclomycin/multidrug efflux system

                          EFX46841 (4E-19),

                          EFX46842 (3E-13)

                          PRK11431 [PRK11431]: quaternary ammonium compound-resistance protein SugE

                          EFX43805 (8E-7*)

                          PRK11646 [PRK11646]: multidrug resistance protein MdtH

                          EFX47100 (4E-6)

                          RND_mfp [TIGR01730]: RND family efflux transporter, MFP subunit

                          *the listed match is not the one with the lowest E-value; it is displayed because it is the most informative

                          It is known that the bee midgut contains a vast array of bacteria, some of which can inhibit P. larvae growth in vitro [2830], presumably by the production of lantibiotics. In our search we were able to find a number of different drug and lantibiotic efflux pumps and modifying enzymes (Table 7), and it is likely that these proteins are enlisted by P. larvae during interactions with co-occurring antagonistic bacteria.

                          Conclusions

                          In this article we report an update on the P. larvae genome to 182-fold coverage, and estimated the ordering of contigs based on comparison against the P. JDR2 genome. We predicted more than 3500 genes and provided these with functional annotation. Of great interest are enzymes that allow the bacterium to traverse the midgut epithelium after ingestion, a process which can contribute to its virulence. Proteases have been thought of as a key factor [22], and this is supported by the large repertoire of such enzymes which we have seen from the predicted gene products. Damage to the host is likely the result of mixed effects from the hemolysins and toxins that can be encoded by P. larvae. Its ability to survive in the host by evading the immune system, as well as protecting itself against other gut bacteria, is likely made possible due to the antibiotic proteins that may be expressed.

                          This improved version of the P. larvae genome and the more detailed annotations will be tremendously useful for improving our understanding of this species; for example, in designing primers to clone genes, or mutate them by site-directed mutagenesis. Especially for the field of proteomics, which often relies heavily on a database of protein sequences to make identifications, the current genome update will be a very powerful tool. Computer-based modeling techniques can be employed to predict the structure of virulence factors, which can guide drug design. Ultimately, the genome will pave the way to more effective prevention; or better yet, a cure for AFB. Given the bee population is under severe strain from the widely publicized Colony Collapse Disorder, attributable to multiple biotic and abiotic factors, it is important to continually improve our knowledge of the key threats that burden worldwide bee health.

                          Methods

                          Preparation of genomic DNA

                          P. larvae strain BRL-230010, stored at -80°C was cultured for 5 d in a shaking incubator at 35°C in 25 mL of media. This media contained 1% Yeast Extract (Difco), 1% soluble starch which was prepared in 10 mM KH2PO4 and adjusted to pH 6.6 with KOH (Recipe #4 from [31]). Genomic DNA was extracted using the DNeasy kit (Qiagen), following the manufacturer's protocol for Gram-positive bacteria.

                          Genome sequencing

                          8,212,402 2 × 50 bp paired-end reads with a mean fragment size of 182 bp were sequenced at the Genome Sciences Centre, Vancouver, Canada using the Illumina GAIIx platform as per manufacturer's instructions. This yielded approximately 182-fold coverage of the estimated genome size. In addition, 24,768 Sanger capillary-derived sequences were generated at the Human Genome Sequencing Centre, Baylor College of Medicine, using the ABI3730 sequencing platform. This provided an additional 3-fold coverage. Raw Sanger sequences were aligned against the UniVec database (NCBI http://​www.​ncbi.​nlm.​nih.​gov) using BLAST. Those Sanger sequences that had good BLAST hits (e-value < 1e-10) were filtered from the dataset. Then, vector sequences were trimmed from the raw Sanger reads using cross_match (Cross_match http://​www.​phrap.​org/​) against the UniVec database. We then aligned all the Illumina reads to the Sanger sequences using MAQ version 0.7.1 [32]. Due to observed Honey Bee DNA contamination arising within the Sanger sequences, only sequences where at least 30% of the length aligned with Illumina reads were retained.

                          In the first stage of the assembly process, the Illumina reads were assembled into contigs using ABySS version 1.2.7 [33]. Assemblies were attempted using several different values for the ABySS sequence overlap parameter k (which determines the k-mer length used to construct the assembly graph), with the best assembly found at k = 37. Other modified ABySS parameter values were s = 100, n = 10, and d = 50. All other ABySS parameters were set to their default values. Following the initial assembly, contigs were joined into scaffolds by aligning the long-insert Sanger sequence data to the assembly, and merged using the scaffolding module of ABySS (Jackman et al., in preparation). Where possible, scaffold gaps were filled, and contigs were extended, using the local reassembly tool Anchor (version 0.2.7, http://​www.​bcgsc.​ca/​platform/​bioinfo/​software/​anchor/​releases/​0.​2.​7) (Docking et al., in preparation).

                          Genome annotation

                          ORFs were predicted with GLIMMER [34] using the long-ORF training script provided with the software package. ORFs were annotated as predicted proteins if they have at least 100 amino acids or at least 50 with a BLAST hit at a threshold of E = 10-5 to Bacillus or Streptococcus (Additional File 1). Annotation terms were transferred from FASTA headers where appropriate, and in accordance with NCBI Prokaryotic Genome Submission Guidelines (full list of proteins in Additional File 2).

                          Genomic dot-plot comparisons were made with the program MUMmer [35]. To produce Figure 2, P. larvae contigs were ordered manually based on BLAST hit coordinates to P. JDR2 and P. vortex as well as the graphical output of MUMmer alignments.

                          The genome annotation as described above was accomplished using the first version of the assembly from this sequencing (ADZY01000000), which had a total N50 of 56,120 base pairs, a total length of 4,352,422, and a mean contig size of 12,329 bp. When we repeated this analysis with the latest assembly, all but 53 of the 3,568 gene models were identical. Of the 53, most were strong partial hits. All downstream analysis was done using ADZY01000000.

                          Shotgun proteomics of P. larvae lysates

                          P. larvae culture was prepared as above. Bacteria were collected by centrifugation for 5 minutes at 16100 relative centrifugal force and the pellet was washed twice in PBS. For an in-gel digestion, the bacteria were lysed in 100 μl of 4× LDS and heated for 15 minutes at 90°C, passed 5 times through a 26 G needle fitted with a syringe before heating for another 10 minutes at 90°C. Lysis was observed under the microscope. Protein concentration was estimated by Coomassie Plus staining, and 200 μg of protein was divided among all 10 wells of a 2-12% NuPAGE precast MES mini gel set up according to the manufacturer's instructions and the in-gel digestion proceeded essentially as described in [36]. For in-solution digestions, bacteria was lysed by either 100 μl of 6 M urea, 2 M thiourea, 20 mM Tris-HCl, pH 8.0 or 0.2 μl RapiGest in 100 μl NH4HCO3 pH 8 and boiled for 6 minutes. Bacteria from both samples were lysed by the needle and syringe method as above. In a third sample, bacterial surface proteins were stripped by adding 100 μL of 100 mM glycine pH 2 and proteins precipitated by ethanol before being denatured using urea/thiourea as above. Proteins were reduced, alkylated, digested with LysC (urea sample only) and trypsin and purified as described in [36]. Proteins from all three samples, as well as gel fragments, were fractionated by strong cation exchange STAGE tips [37]. Peptides were subjected to liquid chromatography-tandem mass spectrometry on an LTQ-Orbitrap and FT-ICR (Thermo) according to procedures described in [36]. The raw data were searched against the new P. larvae protein set derived as above using MaxQuant (v1.1.1.25) [38], considering up to two missed cleavages, Cys carbamidomethylation as a fixed modification and Met oxidation and N-terminal acetylation as variable modifications. The overall mass accuracy of the data set was 0.99 ppm and the false discovery rate, at the protein level, was set to 1%.

                          Bioinformatic annotation of predicted proteins

                          Predicted proteins were BLAST-searched against selected amino acid sequences using the stand-alone BLAST-software package [12]. For the defined targets, i.e. 'flagellar system', 'toxins', 'hemolysins' and 'proteases', query files of representative protein sequences in FASTA format were composed by key word searching the 'protein' database of the National Center of Biotechnology Information (NCBI) at http://​www.​ncbi.​nlm.​nih.​gov/​. The outcome of each BLASTp-search was verified by cross searching the found hits on the online BLASTp tool (NCBI) and by an in depth, manual comparison of their conserved domain architecture and sequence identity. To expand our search for relevant domains, we used BLASTp to search the CDD [39] which contain domain sequences from various sources (Pfam v 24, COG v1, SMART v5.1, Entrez protein clusters database, TIGRFAM v9.0, NCBI, Entrez multi-model superfamilies), with the maximum E-value set at 10-3. Where there are matches to more than one domain, we only highlight the one with the lowest E-value in the manuscript. Full results are shown in Additional File 4.

                          Declarations

                          Acknowledgements

                          The authors wish to thank the members of their respective groups for helpful guidance and discussions; in particular, Nikolay Stoynov for technical assistance with mass spectrometry. This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant to LJF. LJF is the Canada Research Chair in Quantitative Proteomics. The infrastructure used in this work was supported, in part, by the Canada Foundation for Innovation, the BC Knowledge Development Fund and the BC Proteomics Network.

                          Authors’ Affiliations

                          (1)
                          Department of Biochemistry ž Molecular Biology, Centre for High-throughput Biology, University of British Columbia
                          (2)
                          United States Department of Agriculture, Bee Research Laboratory, Agricultural Research Service
                          (3)
                          Michael Smith Genome Sciences Centre, British Columbia Cancer Agency
                          (4)
                          Ghent University, Laboratory of Zoophysiology
                          (5)
                          Institute of Marine Sciences Kiel

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                          © Chan et al; licensee BioMed Central Ltd. 2011

                          This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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