1 Center of Excellence for Vaccine Research, The University of Connecticut, Storrs, CT 06269-3089, USA
2 Department of Pathobiology and Veterinary Science, The University of Connecticut, Storrs, CT 06269-3089, USA
3 Plum Island Animal Disease Center, US Department of Agriculture, Greenport, NY 11944, USA
4 Department of Veterinary Science, University of Melbourne, Parkville, Victoria 3010, Australia
5 Department of Medicine, University of Washington, Seattle, WA 98195, USA
6 Regulome Corporation, Seattle, WA 98195, USA
7 The Institute for Systems Biology, Seattle, WA 98103-8904, USA
Correspondence
Steven J. Geary
geary{at}uconnvm.uconn.edu
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AE015450.
The online version of this paper (at http://mic.sgmjournals.org) contains a supplementary table showing additional information concerning the features and homologies of the M. gallisepticum strain Rlow genome.
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INTRODUCTION |
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Mycoplasmas have long been considered model systems for defining the minimal set of genes required for a living cell (Morowitz, 1984). For this reason, it was not surprising when Mycoplasma genitalium (580 kb) was selected as one of the first targets for complete genome sequencing (Fraser et al., 1995
). Since this initial report, the genomes of four additional mycoplasmas have been sequenced, Mycoplasma pneumoniae (816 kb; Dandekar et al., 2000
; Himmelreich et al., 1996
), Ureaplasma urealyticum (752 kb; Glass et al., 2000
), Mycoplasma pulmonis (964 kb; Chambaud et al., 2001
) and Mycoplasma penetrans (1358 kb; Sasaki et al., 2000
). Theoretical and experimental approaches have estimated the minimum number of essential mycoplasma genes to be between 265 and 350 (Hutchison et al., 1999
; Mushegian & Koonin, 1996
).
Mycoplasma gallisepticum is an avian pathogen involved in chronic respiratory disease in chickens resulting in considerable economic losses in poultry production. Infection with this bacterium is spread by aerosol exposure and egg transmission. Outbreaks spread rapidly through flocks, establish chronic infections, and are difficult to control with antimicrobial therapy. The chronic nature of mycoplasma infections demonstrates a failure of the host immune system to deal effectively with these organisms. Antigenic variation of surface proteins allows M. gallisepticum to evade the host's immune response through the generation of escape variants (Glew et al., 2000; Gorton & Geary, 1997
; Levisohn et al., 1995
). Intracellular invasion and survival within eukaryotic cells by M. gallisepticum may contribute to this organism's resistance to the host's immune response and antimicrobial therapy (Winner et al., 2000
).
M. gallisepticum colonizes the respiratory system of chickens, but has also been isolated from the reproductive organs, brain and eyes of several avian species. Cytadherence to the epithelial surfaces of these host tissues is a requirement for successful colonization. Research into the molecular mechanisms of M. gallisepticum cytadherence has identified a coordinate action between the primary cytadhesin, GapA, and at least one cytadherence-related molecule, CrmA (Papazisi et al., 2002).
Whole-genome sequencing establishes a solid foundation from which to begin a myriad of experimental projects directed at examining the pathogenic mechanisms of an organism. Here we present the complete sequence and initial analysis of the M. gallisepticum genome.
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METHODS |
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Sequencing and assembly.
Each quadrant of a stock 384-well plate was freshly inoculated into a deep-well 96-well plate containing 1 ml 2x YT medium (Difco) supplemented with 100 µg carbenicillin ml-1. These inoculated plates were shaken at 250 r.p.m. for 14 h at 37 °C followed by centrifugation at 3000 r.p.m. for 10 min. Plasmid preparations were processed automatically using an Eppendorf PerfectPrep-96 VAC Robotic Workstation. Sequencing reactions were carried out using 1 µl DNA (350 ng), 4 pmol forward and reverse primers (-21M13 and M13Reverse) and 3 µl ABI Prism Big Dye Terminators (Applied Biosystems/Perkin Elmer) in a total volume of 5 µl. Reactions were carried out in the ABI GeneAmp PCR System 9700 (Applied Biosystems/Perkin Elmer). The DNA from sequencing reactions was precipitated with 2-propanol, resuspended in 10 µl sterile distilled H2O, and loaded onto an ABI 3700 capillary electrophoresis sequencer. Sequence data were assembled and analysed using the Phred/Phrap/Consed assembly package (Ewing & Green, 1998; Ewing et al., 1998
; Gordon et al., 1998
) and CAP3 (Huang & Madan, 1999
). CAP3 uses forward and reverse sequence reads from the same plasmid sequences to place constraints on assembled sequence and is useful to break false joins and assure correct sequence assembly (Huang & Madan, 1999
). Additional reactions were performed to close gaps in the assembly. Custom primers were designed near gaps and sequence was obtained from plasmid clones that spanned the gaps. Where no clone was available to span the gap, PCR products were generated using custom primers on genomic DNA and the subsequent PCR products were sequenced and added to the assembly. Sequencing coverage, of both strands, was 11x (or 22 reads per kb), done to an error rate less than 1 per 10 kb.
The sequence data have been submitted to the NCBI database under accession number AE015450.
Identification of CDSs and annotation.
Coding DNA sequences (CDSs), of at least 195 nt in length, were initially identified using ORF Finder software (with genetic code 4) provided by the National Center for Biotechnology information (NCBI; http://www.ncbi.nlm.nih.gov/). GLIMMER (Delcher et al., 1999) was trained to recognize a potential CDS whose translated product had at least 33 amino acids and did not overlap more than 30 nt with neighbouring CDSs. BLAST (Zhang et al., 1998
) and FASTA (Pearson, 1999
) searches were performed on the NCBI non-redundant protein databases, conserved orthologous groups (COGs) database (Tatusov et al., 2001
), and proteins from the mycoplasma genomes sequenced to date. A CDS was assigned a designation based on homology when its translated product presented a BLAST score of at least 100 in the COG database and 150 in the non-redundant database. Scores of 300 or more were considered definitive. MSP-Crunch (Sonnhammer & Durbin, 1994
) was used to filter low-complexity sequences in order to eliminate spurious homologies. FAStRNA (http://bioweb.pasteur.fr/seqanal/interfaces/fastrna.html) (el-Mabrouk & Lisacek, 1996
) was used for finding tRNA coding sequences. The PROSITE (Falquet et al., 2002
), Pfam (Bateman et al., 2000
), BLOCKS (Henikoff et al., 1999
) and TIGERfam (http://www.tigr.org/) databases were searched to identify motifs and other shared features of proteins. Specific motif searches were performed against the M. gallisepticum genome using FUZZPRO from the Jemboss software package (http://www.hgmp.mrc.ac.uk/Software/EMBOSS/Jemboss/index.html). Protein topology and location were predicted using TMPred (Hofmann & Stoffel, 1993
), MEMSAT (Jones et al., 1994
), PSORT (http://psort.nibb.ac.jp/) and SAPS (Brendel et al., 1992
).
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RESULTS AND DISCUSSION |
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In silico analysis localized the predicted oriC region of M. gallisepticum based on several criteria. First, despite an apparent overall lack of conserved gene order in the mollicute oriC region (Cordova et al., 2002), the gene order in the oriC region does appear to be conserved within the phylogenetic cluster containing M. pneumoniae, M. genitalium and M. gallisepticum (Cordova et al., 2002
; Hilbert et al., 1996
; Zou & Dybvig, 2002
). These three mycoplasmas have the gyrA, gyrB, dnaJ, dnaN and soj genes upstream of the dnaA gene, and ABC transporter genes, rpl34 and rpnA, downstream of dnaA. As in M. pneumoniae, there is a unique hypothetical coding region immediately upstream of the dnaA gene in M. gallisepticum. Second, we identified putative DnaA boxes in the vicinity of the dnaA gene (Fig. 2
). The region around dnaA was searched for 9 nt long sequences resembling a consensus sequence (5'-TTWTMHAMA-3') based on DnaA box sequences from M. pulmonis, M. capricolum and M. pneumoniae (Chambaud et al., 2001
; Cordova et al., 2002
; Hilbert et al., 1996
; Zou & Dybvig, 2002
). Finally, the region between dnaN and soj contains higher than average AT base pair frequency (80 %) and contains AT-rich repeats. These criteria are considered to be characteristic of the origin of replication in prokaryotes (Baker & Wickner, 1992
).
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To establish consistency, and in accordance with standard nomenclature, we have annotated this family of genes as vlhA (Noormohammadi et al., 1998). This family contains 43 genes, constituting a total of 103 kb or 10·4 % of the genome. The 43 vlhA genes are distributed among five loci containing 8, 2, 9, 12 and 12 genes, respectively, and have been numbered according to their locus and position (e.g. vlhA1.01) (Fig. 3
). Of the 43 genes, 38 possess the signature vlhA gene features, which include a GAA repeat motif 5' of a GTG start codon and conserved regions flanking the start codon (Markham et al., 1994
). The sequence identity among these 38 genes ranges between 41 and 99 %. Five of the genes (1.05, 2.01, 2.02, 5.01 and 5.02) possess sequence homology to vlhA but lack the vlhA signature motifs. The genes within each of the five loci are in the same transcriptional orientation, with the exception of vlhA1.05. In addition to the lack of vlhA signature motifs, a putative transposase (MGA_0073) has been identified adjacent to vlhA1.05, suggesting that rearrangements by a transposon may account for the differences observed in this region. Five of the genes (vlhA1.01, 1.08, 3.01, 4.03 and 5.01) have been interrupted by mutations that introduce shifts in the reading frames.
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Adhesin-related proteins
A paralogous family of five adhesin-like proteins has been identified in the M. gallisepticum genome. Papazisi et al. (2002) have recently complemented a cytadherence-deficient, GapA- CrmA- isolate of M. gallisepticum strain R with a functional gapAcrmA operon (MGA_0934 and MGA_0939), and have demonstrated that expression of both genes is required for cytadherence and pathogenesis of M. gallisepticum. The gapA gene was initially characterized as the M. gallisepticum orthologue of the M. pneumoniae cytadhesin P1 (Goh et al., 1998
). Attachment inhibition assays using anti-GapA Fab fragments established the role of GapA in M. gallisepticum cytadherence (Goh et al., 1998
). A comparative analysis of virulent and avirulent isolates of M. gallisepticum strain R identified a frameshift mutation, resulting in a premature termination codon, in the gapA gene of an avirulent, GapA-deficient isolate (Papazisi et al., 2000
). With the initial characterization of gapA, a potential CDS was identified and partially sequenced immediately downstream of gapA (Goh et al., 1998
). During the characterization of a cytadherence-deficient isolate of M. gallisepticum strain R, Papazisi et al. (2000)
matched this partial CDS with amino acid data from a 116 kDa protein that was expressed in a cytadherence-positive isolate of strain R, but not expressed in the cytadherence-deficient isolate. This CDS (designated crmA) was sequenced and found to encode a protein that exhibits 41 % amino acid identity with the M. pneumoniae and M. genitalium ORF6 proteins (Papazisi et al., 2000
). These proteins have been shown to play an accessory role in M. pneumoniae cytadherence (Krause & Balish, 2001
; Krause et al., 1982
; Layh-Schmitt & Harkenthal, 1999
; Seto et al., 2001
).
Immediately downstream of the gapAcrmA operon there are two CDSs (MGA_0943 and MGA_0945; designated crmB and C) that, although not showing significant nucleotide homology, are predicted to encode proteins sharing homology to GapA and CrmA. Analysis shows that the majority of the similarity lies within the C-terminal region of these four proteins. A fifth paralogue (MGA_1117) shares similarity with the N-terminus of GapA and CrmB.
Membrane-associated proteins
Motif analysis of the M. gallisepticum genome has predicted a large repertoire of membrane-associated proteins. Of these proteins, 149 contain multiple transmembrane domains and 18 possess ten or more transmembrane domains. These 18 proteins include molecules involved in amino acid transport (PotE), phosphate transport (Pts) and protein translocation (SecY), as well as five conserved hypothetical and two unique hypothetical proteins. Based on the prokaryotic membrane lipoprotein lipid attachment site motif (PROSITE PS00013), 80 CDSs are predicted to encode lipoproteins (10·8 % of all CDSs). The second largest paralogous gene family in M. gallisepticum consists of 24 ATP-binding proteins belonging to the ABC transporter superfamily. This family accounts for nearly one-third of the 75 proteins predicted to be involved in transport of biomolecules.
Components of the membrane protein secretion and translocation pathways were found in M. gallisepticum. The Sec pathway of M. gallisepticum consists of SecA (MGA_670), SecE (MGA_0474), SecY (MGA_0740), YidC (MGA_0631), trigger factor (Tig, MGA_1297) and DnaK (MGA_0279). The signal recognition particle pathway contains FtsY (MGA_0919) and Ffh (MGA_1143). Although the Sec pathway is incomplete when compared to the secretion pathway in E. coli (Cao & Saier, 2003; Driessen et al., 2001
; van Wely et al., 2001
), these proteins are consistent with the minimal set of secretion and translocation proteins proposed by Mushegian & Koonin (1996)
.
Signal peptidase I (LepB; SPase I) was identified in M. gallisepticum (MGA_1091) and was found to possess two motifs similar (one mismatch) to PROSITE SPase I motifs (PS00501; PS00761). The only other known SPase I orthologue in mycoplasmas is in M. pulmonis (MYPU_6300). Phylogenetic analysis using FASTA shows that these two mycoplasma signal peptidases cluster separately from other prokaryotic LepB proteins (data not shown).
Prolipoprotein signal peptidase (LspA; SPase II) was also identified in M. gallisepticum (MGA_0997). Orthologues of this protein have been identified in all mycoplasmas sequenced to date. Although these proteins have been annotated as signal peptidase type 2 based on the homology that they share with their prokaryotic homologues, all of the mycoplasma LspAs appear to form a divergent phylogenetic cluster using FASTA analysis (data not shown). Additionally, the SPase II motif for all mycoplasmas is similar, but not identical, to the PROSITE SPase II motif (data not shown).
A potential cleavage motif (PXRX05SS, with 1 mismatch; for review see Krause, 1998) was found in GapA, 26 of the VlhA proteins, and three high-molecular-mass proteins (MGA_0306, MGA_0928 and MGA_0205), which have homology to M. pneumoniae proteins HMW1, HMW3 and P200, respectively.
Collectively, these findings indicate that post-translational modification of membrane proteins in M. gallisepticum, and perhaps other mycoplasma species, occurs in a divergent manner from that of other prokaryotes.
Transposases
Twelve putative transposases were identified within the M. gallisepticum genome. Ten of these have homology to transposases found in both Gram-positive and Gram-negative bacteria, while two (MGA_1109 and MGA_1329) have homology with the transposases found in M. mycoides subsp. mycoides Small Colony type and M. hyopneumoniae. In eight cases the presence of a transposase was found to be associated with the rearrangement or disruption of a neighbouring gene. An example of this is seen in the rearrangement found in the vlhA locus 1 (Fig. 3). Six of the putative transposases have a motif similar to PROSITE PS01007 transposase/mutator (one mismatch). However, it remains to be proven if any of the 12 M. gallispticum predicted transposases are part of any active mobile element.
PvpA
PvpA is a putative haemagglutinin that undergoes phase-variable expression and exhibits size variation among strains of M. gallisepticum (Boguslavsky et al., 2000; Liu et al., 2001
; Yogev et al., 1994
). Analysis of the pvpA gene from the genomic sequence revealed a duplication of 37 nt compared to the M. gallisepticum strain R sequence submitted as GenBank accession number AF224059 (Boguslavsky et al., 2000
). The region encompassing the pvpA gene has been amplified by PCR and sequenced to confirm this duplication. The 37 nt duplication results in a frameshift and predicted premature termination of PvpA expression.
Virulence factors
Putative virulence factors in M. gallisepticum were identified based on protein motif analysis. Nine of the 20 proteases were found to possess between one and five transmembrane domains; three of them appear to be zinc metalloproteases. In addition, three nucleases and a lipase-like (GDSL motif) protein were found to possess between one and four transmembrane domains. We found 133 membrane-associated proteins containing putative lectin-binding motifs (Elgavish & Shaanan, 1997; Loris et al., 1998
); 51 of them are lipoproteins and 34 belonged to the VlhA family. This suggests that there are a number of membrane-associated proteins that may bind sugar moieties for the purpose of nutrient uptake or cytadherence. Two proteins, MGA_0090 and MGA_0091, showed similarity to phospholipid-binding proteins. These findings indicate that M. gallisepticum, in addition to GapA, CrmA and the VlhAs, has a wide array of proteins predicted to be involved in binding biomolecules. A motif similar to the aerolysin motif (PROSITE PS00274, one mismatch) was found in four membrane proteins: GapA (MGA_0934), two hypotheticals (MGA_0226 and MGA_1162) and VlhA5.04 (MGA_1243). Although this remains speculative, it may help to explain the reported invasiveness of GapA+ variants (Much et al., 2002
; Winner et al., 2000
). Proteins MGA_0313 and MGA_0931 were found to contain a motif similar to the staphylococcal/streptococcal pyrogenic exotoxin signature motif (PROSITE PS00277). A number of heat-shock proteins were identified, including GroEL (MGA_0152), GroES (MGA_0153), Clp (MGA_0178), DnaK (MGA_0279), GrpE (MGA_1232), and seven DnaJ-class proteins (MGA_0617, MGA_0877, MGA_0885, MGA_1131, MGA_1135, MGA_1228, MGA_1324). The number of DnaJ-class proteins is higher than in other mycoplasmas (e.g. M. pneumoniae and M. genitalium each have three DnaJ-class proteins).
Regulatory proteins
The reduction of the mycoplasma genome has resulted in biosynthetic limitations affecting alterations in regulatory pathways. No component of any classical bacterial two-component regulatory system was found in M. gallisepticum. However, a search for motifs identified several potential regulatory proteins. It is known that regulatory proteins that bind DNA possess helixturnhelix (HTH) motifs. We found 21 proteins (excluding sigma 70, recombinases, helicases, and other enzymes involved in nucleic acid base modification) that contained HTH motifs similar to the PROSITE AraC (PS00041), LysR (PS00044), GntR (PS00043) and LuxR (PS00622) motifs. Another protein, MGA_1295, was found to share homology with the Fur-like class of regulatory proteins involved in iron/zinc uptake (Escolar et al., 1998). Two multi-transmembrane proteins, MGA_1037 and MGA_0337, were found to share homology with chemoreceptors. A motif similar to the PROSITE sigma 54 interaction domain (PS00675) was found in several proteins, including PhnL and HatB (MGA_0655 and MGA_1018, respectively, ATPases that are components of two different ABC transporter sytems), UvrC (MGA_1269; recombinase), OppF (MGA_0218 and MGA_0230; oligopeptide/dipeptide uptake transporter), Gmk (MGA_0462; guanylate kinase) and a HprK (MGA_0599; predicted serine kinase). This finding indicates that, in the absence of identified alternative sigma factors in mycoplasmas, proteins exhibiting similar function may exist.
Conclusions
M. gallisepticum strain R contains a 996 kb genome with 742 CDSs. Among the 742 CDSs, function has been assigned to 469 genes, while approximately one-third of the genes remain undefined in terms of function. Nearly 17 % of the genes appear to be unique to M. gallisepticum.
We identified all of the members of the pMGA family, and have renamed this family vlhA in accordance with standard nomenclature. The vlhA family, totalling 43 genes, constitutes the largest paralogous set of genes in M. gallisepticum. Our analysis has identified three putative adhesin-related molecules that share homology with the GapA and CrmA cytadherence molecules of M. gallisepticum. We have found that 10 % of the CDSs are putative lipoproteins, and nearly 20 % of the CDSs contain multiple transmembrane domains. With 24 CDSs, ABC transporter molecules make up the second-largest paralogous family in M. gallisepticum. A large percentage of the M. gallisepticum genome is devoted to membrane-associated molecules.
The completion of this sequence has spawned multiple projects directed at defining the biological basis of M. gallisepticum. We have developed microarray chips covering the entire genome of M. gallisepticum and are currently performing experiments to analyse gene expression patterns. Experiments are currently under way to examine the proteome profile of M. gallisepticum. This sequence is greatly aiding our transposon mutagenesis studies of mycoplasma pathogenicity.
The most salient feature arising from this sequencing project is the large number (36 %) of CDSs annotated as unique (126) or conserved (151) hypothetical, a total of 277 CDSs whose functions remain speculative at this time. This emphasizes the vast amount we do not know about the inner workings of this pathogen. Further analysis of these CDSs and the functions of the proteins that they encode will add significantly to our ever-expanding body of knowledge regarding virulence mechanisms in M. gallisepticum.
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ACKNOWLEDGEMENTS |
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Received 16 April 2003;
revised 9 June 2003;
accepted 16 June 2003.