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Genome Update |
Center for Biological Sequence Analysis, BioCentrum-DTU, Building 208, The Technical University of Denmark, DK-2800 Kgs, Lyngby, Denmark
Correspondence
David W. Ussery
(dave{at}cbs.dtu.dk)
Genomes of the month
Eight new microbial genomes have been published since the last ‘Genome Update’ column was written. The collection of this month's genomes is shown in Table 1
. The published genomes represent four bacterial phyla: one from the Actinobacteria (Mycobacterium avium subsp. paratuberculosis), one from the Chlamydiae/Verrucomicrobia (Chlamydia trachomatis A/HAR-13), two from the Firmicutes (Staphylococcus saprophyticus subsp. saprophyticus ATCC 15305 and Streptococcus agalactiae A909) and four from the Proteobacteria (‘Candidatus Pelagibacter ubique’ HTCC1062, Pseudoalteromonas haloplanktis TAC125, Pseudomonas syringae pv. phaseolicola 1448A and Xanthomonas campestris pv. vesicatoria 85-10).
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sequenced and investigated the MAP strain K-10 which consists of a single circular chromosome of around 4·8 Mbp. The genome contains 
3000 genes with homologues to Mycobacterium tuberculosis and 161 unique genomic regions that encode 39 new map genes. Also noteworthy, a possible explanation for mycobactin dependence of MAP has been proposed, based on the genome sequence. (Mycobactin is a siderophore responsible for the binding or transport of iron into cells.) A truncation in a specific domain of a salicyl-AMP ligase, which is the first gene in the mycobactin biosynthesis gene cluster, has been identified.
Streptococcus agalactiae is a commensal bacterium colonizing the intestinal tract of a significant proportion of the human population. However, it is the major cause of invasive bacterial disease, including septicaemia, meningitis and pneumonia, in the neonatal period. Tettelin et al. (2005) sequenced six strains of Streptococcus agalactiae covering the five major disease-causing serotypes and compared these newly sequenced genomes with those of two already available strains. Analysis of these eight genomes shows that S. agalactiae contains a pan-genome consisting of a core genome shared by all investigated isolates, accounting for 80 % of any single genome and a dispensable genome composed of partially shared and strain-specific genes. Surprisingly, even after analysing eight genomes, unique genes were still detected and mathematical extrapolation predicts that more should be expected even after sequencing many more strains.
Chlamydia trachomatis is the causative agent of the disease chlamydia. Infection with Chlamydia trachomatis may result in urethritis, epididymitis, cervicitis, acute salpingitis or other syndromes when sexually transmitted. Isolates exist as 15 serovariants that are separated according to different pathobiotypes. Carlson et al. (2005) have sequenced the genome of an oculotropic trachoma isolate (A/HAR-13) and compared it to the already available genome of a genitotropic (D/UW-3) isolate. Analysis showed that the genomes share a remarkable sequence identity of 99·6 % and single-nucleotide polymorphism investigations between the two strains confirmed the minimal genetic variation. An important outcome was the identification of a diagnostic marker that allows differentiation between genitotropic and oculotropic strains and between invasive and non-invasive serovars.
Pseudoalteromonas haloplanktis strain TAC125 is a psychrophilic gammaproteobacterium found in Antarctica (Médigue et al., 2005). It is adapted to fast growth, suggesting that it lives in areas rich in nutrients (i.e. plenty of plankton debris). Since P. haloplanktis lacks the necessary activities resulting in reactive oxygen species, it could prove useful for expression of foreign proteins in the cold and might be a useful tool in biotechnology. Médigue et al. (2005) provide an in-depth analysis of P. haloplanktis strain TAC125, reporting that it contains around 3·85 Mbp in two circular chromosomes. There is evidence that the smaller chromosome, chrII, was initially a plasmid that had been recruited to become a chromosome encoding essential genes and that it does not display a standard GC skew. Manual annotation identified 3488 protein coding genes (CDS). A relatively high number of tRNA genes, 106, is consistent with the findings in other Gammaproteobacteria.
Pseudomonas syringae pv. phaseolicola 1448A is a plant pathogen causing halo blight disease in the common bean (Phaseolus vulgaris), resulting in severe problems in developing countries. A comparative analysis between this new genome and the four other published Pseudomonas genomes identified a set of 3567 (67 %) core Pseudomonas ORFs (Joardar et al., 2005). Comparison with P. syringae pv. tomato DC3000 shows that most (81 %) of the identified virulence factors of DC3000 are present in 1448A as well (Joardar et al., 2005).
Staphylococcus saprophyticus subsp. saprophyticus strain ATCC 15305 is known to cause uncomplicated urinary tract infections (UTIs) in women (Kuroda et al., 2005). The circular genome of S. saprophyticus is 2 156 575 bp long with an A+T content of 67 % and has six rRNA operons and 60 tRNAs for all amino acids. A comparison of S. saprophyticus to Staphylococcus aureus and Staphylococcus epidermidis, the two other Staphylococcus species recognized as major human pathogens, revealed the absence of virulence factors in S. saprophyticus, such as extracellular matrix-binding proteins which are found in the other two species. This offers a reason as to why S. saprophyticus does not cause a bacterial infection quite as severe as those that arise from the other virulent Staphylococcus species. Further studies also indicated the presence of a single unique ORF product in S. saprophyticus with the ability to mediate cell-wall-anchoring in adhesion to uroepithelial cells. This can be compared to S. aureus and S. epidermidis, where 20 and 11 such proteins are present, respectively. The complete genome sequence of S. saprophyticus confirms this bacterium as an important uropathogenic species in relation to UTIs.
‘Candidatus Pelagibacter ubique’ strain HTCC1062 (originally named SAR11) is a marine bacterium that is possibly the most numerous bacterium in sea water and accounts for about 25 % of all microbial plankton cells. This bacterium plays a key role in the carbon cycle by oxidizing the dissolved organic carbon in the oceans. Giovannoni et al. (2005) sequenced the ‘Candidatus P. ubique’ genome to discover an extremely compact and efficient genome, containing only 1·3 Mbp, which is considered to be the smallest among free-living organisms. Moreover, there are no pseudogenes, viral genes, transposons or junk DNA of any kind. Even the intergenic spacers are considered the shortest yet observed. Despite the small number of genes encoded (1354), it has complete biosynthetic pathways for all 20 amino acids and all but a few cofactors. The streamlined genome, minimizing the costs of cellular replication, is apparently a great advantage when replicating in hostile environments. ‘Candidatus P. ubique’ is a unique member of this phylum since it is free-living and the loss of functional capabilities is minimal.
Xanthomonas campestris is a Gram-negative plant-pathogenic bacterium that some use as a model organism in the study of Gram-negative bacteria protein secretion systems. It is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. It also produces an exopolysaccharide called xanthan which can be used in many areas such as spinning and paper making. X. campestris pv. vesicatoria strain 85-10 is the third X. campestris (and the fifth Xanthomonas) genome to be published. X. campestris pv. vesicatoria can cause bacterial spot disease in pepper and tomato plants, but strain 85-10, described by Thieme et al. (2005), is pathogenic only for pepper plants. The genome of X. campestris pv. vesicatoria strain 85-10 consists of one circular chromosome of 5 178 466 bp and four plasmids. Like other Xanthomonas strains, its chromosome has a low AT content (37 %), and this number varies between 43 and 40 % among the plasmids. The whole genome contains 4726 CDS, including two rRNA operons (in the order 16S–23S–5S) and 54 tRNA genes. However, 697 out of the 4726 CDS are conserved hypothetical CDS and 949 CDS have unknown function. A comparison to three other Xanthomonas genomes revealed 548 CDS (12·2 %) unique to X. campestris pv. vesicatoria. Controlled by two key regulatory proteins, HrpG (an OmpR family regulator) and HrpX (an AraC-type regulator controlled by HrpG), a type III protein secretion system (TTSS) is responsible for the pathogenicity of X. campestris pv. vesicatoria. Additionally, strain 85-10 has all other types of protein secretion systems known in Gram-negative bacteria. The largest plasmid encodes a putative type IV secretion system similar to the Icm/Dot system of human pathogens Legionella pneumophila and Coxiella burnetii. This is the first report of a putative Icm/Dot like type IV secretion system in a plant-pathogenic bacterium. Also, six novel type III effector proteins and several other virulence factors were predicted through comparisons with other completely sequenced plant pathogens.
Method of the month – bias of purines and alternating pyrimidine/purine stretches
The highlighted method chosen this time is used for comparison of microbial genomes by looking for stretches of purines (R) and alternating pyrimidine/purines (YR) in sequenced chromosomes or genomes. A length of 10 bp or longer was chosen, as this represents roughly at least one complete turn of the DNA helix. The expected fraction of a chromosome with a sequence of 10 bp or longer of either purines (RR) or alternating YR stretches is about 1·07 % (Ussery et al., 2002). We have examined 287 totally sequenced chromosomes from bacterial genomes in 13 phyla. Links to the individual chromosome sequences and references can be found on our ‘Genome Atlas' web page (http://www.cbs.dtu.dk/services/GenomeAtlas/).
Fig. 1(a) shows that the observed fraction of purine tracts in the various phyla differs significantly. Indeed, each phylum seems to have its own distinct distribution. On average, nearly all phyla have more purine stretches than would be expected. Only the Proteobacteria and Actinobacteria have significant numbers of chromosomes with lower than expected numbers. Analysis of the proteobacterial and actinobacterial genomes with the largest bias show that many of these are GC-rich. The thermophiles in Deinococcus/Thermus, with only three organisms sequenced (or four chromosomes), show the greatest breadth of deviation from the mean. They are also GC-rich. However, Spirochaetes, with six organisms sequenced (or eight chromosomes), have a much tighter distribution and are AT-rich. A link to a web-based table showing the fraction for all sequenced bacterial chromosomes is provided in the supplemental web page (http://www.cbs.dtu.dk/services/GenomeAtlas/suppl/GenUp020/) and can be sorted by the fraction of RR or YR stretches, as well as by the genus name, taxonomic group, length or percentage AT.
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the fraction of YR tracts is plotted for various phyla. Although the expected values are the same (about 1 % of the chromosome), here most organisms have fewer than expected, although the distributions are not quite as diverse as for the purine tracts. Again, as in Fig. 1(a), a significant fraction of proteobacterial and actinobacterial genomes show the opposite trend, with a large number of ‘outliers’. These tend to be the same organisms which have less purine stretches, and are also often GC-rich.
Why do we see such a trend? Although at this stage we cannot say for certain, there are several possible explanations, in terms of DNA structures and stability. In broad terms, the purine tracts tend to be more thermodynamically stable, whilst alternating pyrimidine/purine tracts require less energy to melt (with sequences like TATA melting quite readily, for example). Some purine tracts can stabilize an A-DNA type of structure, which is also the type of helix adopted by RNA–DNA hybrids; it is thought that ‘normal’ DNA in cells consists of a mixture of A-DNA and B-DNA conformations. Furthermore, short runs of phased A-tracts can form stable DNA curvature, which could play an important biological role in DNA interaction with proteins. It is interesting to note that most of the genomes with the highest bias towards purine stretches tend to be AT-rich, whilst the genomes with less purine stretches tend to be GC-rich. The opposite trend seems to hold for YR stretches, which can be seen in Fig. 2. Alternating CG runs can form left-handed Z-DNA under the right conditions, and certain Burkholderia genomes (which are the most biased toward YR tracts) tend to have more CG dinucleotides than would be expected based on the mononucleotide composition. ‘Aquifex aeolicus’ has been noted previously to be ‘unique’ in its overrepresentation or preference for polypurines in protein-coding regions (Raghavana et al., 2000). In general, most soil bacteria tend to have considerably less purine tracts and more YR tracts than would be expected. These findings hint at the possibility of being able to find some general structures or maybe even sequence signatures that would be indicative of the environment in which an organism lives.
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. In contrast, Proteobacteria tend to have G and T residues on the leading strand, resulting in less bias towards purine stretches, but more bias towards YR stretches, as can be seen in Fig. 1(b). The bias of A residues towards the leading strand is correlated with the presence of the polC gene; in the absence of polC there is a general tendency for the A residues to be on the lagging strand (Worning et al., 2005). Finally, we have calculated the bias in the intergenic regions to test whether it is coming from the coding sequences (90 % of the DNA in many bacterial genomes). One could argue that most of the bias might come from choice of codon usage, for example. If this were true, in general one should see means closer to the expected value of around 1 % for the intergenic regions, which is the opposite of what is seen for most of the genomes in Fig. 1 (with the exception of Spirochaetes). Currently, we cannot offer a simple explanation for why this trend is seen.
Supplemental web pages
Access to additional web pages containing supplemental material related to this article can be obtained via the following URL: http://www.cbs.dtu.dk/services/GenomeAtlas/suppl/GenUp020/
Acknowledgements
This work was supported by a grant from the Danish Center for Scientific Computing. We also thank the Sanger Centre (http://www.sanger.ac.uk/Projects/) and the Joint Genomes Initiative (http://genome.jgi-psf.org/finished_microbes/) for making their genome sequences and preliminary annotations available to the public.
REFERENCES
Carlson, J. H., Porcella, S. F., McClarty, G. & Caldwell, H. D. (2005). Comparative genomic analysis of Chlamydia trachomatis oculotropic and genitotropic strains. Infect Immun 73, 6407–6418.
Giovannoni, S. J., Tripp, H. J., Givan, S. & 11 other authors (2005). Genome streamlining in a cosmopolitan oceanic bacterium. Science 309, 1242–1245.
Joardar, V., Lindeberg, M., Jackson, R. W. & 29 other authors (2005). Whole-genome sequence analysis of Pseudomonas syringae pv. phaseolicola 1448A reveals divergence among pathovars in genes involved in virulence and transposition. J Bacteriol 187, 6488–6498.
Kuroda, M., Yamashita, A., Hirakawa, H. & 10 other authors (2005). Whole genome sequence of Staphylococcus saprophyticus reveals the pathogenesis of uncomplicated urinary tract infection. Proc Natl Acad Sci U S A 102, 13272–13277.
Li, L., Bannantine, J. P., Zhang, Q., Amonsin, A., May, B. J., Alt, D., Banerji, N., Kanjilal, S. & Kapur, V. (2005). The complete genome sequence of Mycobacterium avium subspecies paratuberculosis. Proc Natl Acad Sci U S A 102, 12344–12349.
Médigue, C., Krin, E., Pascal, G. & 21 other authors (2005). Coping with cold: the genome of the versatile marine Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. Genome Res 15, 1325–1335.
Raghavana, S., Hariharanb, R. & Brahmachari, S. K. (2000). Polypurine polypyrimidine sequences in complete bacterial genomes: preference for polypurines in protein-coding regions. Gene 242, 275–283.[Medline]
Tettelin, H., Masignani, V., Cieslewicz, M. J. & 43 other authors (2005). Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial ‘pan-genome’. Proc Natl Acad Sci U S A 102, 13950–13955.
Thieme, F., Koebnik, R., Bekel, T. & 26 other authors (2005). Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence. J Bacteriol 187, 7254–7266.
Ussery, D., Soumpasis, D. M., Brunak, S., Staerfeldt, H. H., Worning, P. & Krogh, A. (2002). Bias of purine stretches in sequenced chromosomes. Comput Chem 26, 531–541.[CrossRef][Medline]
Worning, P., Jensen, L. J., Hallin, P. F., Stærfeldt, H.-H. & Ussery, D. W. (2005). Origin of replication in circular prokaryotic chromosomes. Environ Microbiol (in press). doi:10.1111/j.1462-2920.2005.00917.x
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