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3rd ASM and TIGR Conference on Microbial Genomes New Orleans, USA, 29 January – 1 February 2003
I am involved in a collaboration between SCRI and the Sanger Institute, which has just completed sequencing the genome of Erwinia carotovora subsp atroseptica (Eca, which causes blackleg and soft rot on potato). So my attendance at the Microbial Genomes conference, part funded through BSPP, was very timely. The conference provided an ideal opportunity for us to present initial findings from the genome sequence, to speak with our collaborators (especially those involved in sequencing Erwinia chrysanthemi (Ech)), and to hear of post-genomic studies in other pathogens such as Pseudomonas syringae and Xanthomonas spp.
One very relevant body of work was a talk and several posters from Alan Collmer s group (Cornell, USA) and their collaborators who work on P. syringae. The genome of P. syringae pv. tomato DC3000 is complete (but unpublished) whilst that of P. syringae pv. syringae has been draft sequenced. P. syringae is a stealth pathogen that can grow asymptomatically in compatible host plants, and proteins translocated by the type III secretion system (TTSS or hrp system) into the host have a key role in pathogenesis. In incompatible reactions, some of these proteins betray the pathogen and initiate the host s hyper-sensitive response (HR). Thus, a major thrust of the P. syringae genomic research is to identify the full complement of hrp-secreted proteins. This involves a combination of bioinformatic mining of the genome data (e.g. to identify genes with possible hrp box promoter sequences), and microrarray / proteomic analysis of hrp-regulated transcripts / proteins. An elegant calmodulin reporter gene system is also being used to verify the presence of putative translocated proteins within the host cells. Virulence functions of hrpsecreted proteins are also being demonstrated by their ability to suppress an HR when produced by a strain of the non-pathogenic P. fluorescens carrying a functional TTSS. Alan also explained how hrp-secreted proteins are now thought to fall in to two classes: helpers (or accessory proteins) and effectors (TTSEs). Harpins (heat stable, glycine rich, cysteine lacking acid proteins, often secreted in relatively large amounts and causing HR in non-hosts) are now regarded as helper proteins that assist in the translocation of the true effector proteins into the host via the hrp pilus. In addition, although different helper and effector proteins may not superficially resemble one another in terms of their similarity by a BLAST search for example, there are often certain shared features that become apparent on closer inspection. This work is bearing fruit with the identification of a large number of hrp-regulated genes, some of which have been verified as harpins or effectors.
Nicole Perna, who heads the Erwinia chrysanthemi genome sequencing project, gave a brief update on progress: only a few gaps remain in the genome sequence and it should be finished shortly. It is very similar in size to the Eca genome but sequence divergence is reasonably high, with around 85% nucleotide identity. Systematic comparisons will be revealing. Meanwhile Ching Hong Yang and staff at UC Riverside are using various screens for novel pathogenicity determinants. In one study, GFP was used as a reporter in an IVET promoter trapping screen of a library of 10 000 cloned fragments of Ech DNA. In 61 of these clones genes upregulated in planta were identified.
Mutants for some of these genes, including several hrp genes, were found to have reduced virulence in planta. In another study functional cloning , where a library of cloned sequences (with a GFP reporter), co- transformed into E. coli along with a plasmid bearing the hrp gene cluster, was used to identify novel hrp-regulated genes. These new genes were then added to a training set to allow bioinformatic predictions of further candidate hrp-regulated genes from the genome sequence.
The Brazilian consortium that sequenced the first phytopathogen genome (Xylella fastidiosa) 3 years ago has now sequenced 3 different xanthomonad genomes. The sequences of X. axopodis pv. citri (Xac) and X. campestris pv. campestris (Xcc) were published last year, whilst that of X. axopodis pv. aurantifolli (Xaa) was completed recently. Analysis of the Xac and Xcc genomes has revealed many shared virulence factors that are almost identical (e.g. xanthan gum synthesis, Types I, II, III and IV secretion systems and adhesins), some shared factors that differ significantly (e.g. the complement of plant cell wall degrading enzymes) and some that are unique to one species or other (such as some TTSEs).
These differences can be postulated to account for differences in pathogenesis e.g. the smaller number of plant cell degrading enzymes in Xac probably accounts for its limited lesions, whilst more putative TTSEs in Xcc may partially account for its ability to infect systemically, as might nitrate assimilation genes (since the xylem is a nitrogen- poor habitat). In Xaa high throughput in planta screens of both random and directed mutants is being used to investigate candidate pathogenicity genes. Furthermore, two additional strains of Xaa are almost completely sequenced. The three Xaa strains show variations in host specificity on different citrus fruit and it is hoped that comparative genomics will reveal the molecular bases for this.
Away from phytopathogens, but staying with a theme of how examining gene content in related species can account for variation in pathogenicity, Julian Parkhill of the Sanger Institute compared the genomes of three Bordetella species: pertusis, parapertusis and bronchoseptica. B. pertusis and parapertusis are host specific pathogens whereas B. bronchiseptica is a generalist. The specialists have lost many more small molecule uptake genes relative to the others, which fits with the idea of niche specialisation. Also B. pertusis has a large number of pseudogenes: this fits with observations of decay of genes into pseudogenes being associated (cause or effect?) with host-specialisation as seen in other pathogens e.g. Salmonella typhi. This finding also illustrates the value of a complete genome sequence as fragmentary genomic information, e.g. from draft shotgun sequencing, might miss pseudogenes. Another surprising finding was that in B. parapertusis, the pertusis toxin gene has decayed into a pseudogene whereas in B. bronchispetica the toxin gene is intact. The suggestion is that in B. pertusis expression of the toxin gene has become de-regulated, thus causing it to be far more pathogenic.
This creates a problem for the pathogen, i. e. killing of the host, but the symptoms of the whooping cough provide a transmission mechanism to a new host. Thus, the idea that simple acquisition and loss of key genes will account for all variations in pathogenic phenotype is rather simplistic. More subtle effects on regulation need to be considered also.
I thank BSPP for providing funds to enable me to attend this conference.