PubMed · 10873814
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B Dixon. 2000-06-15. Coliform confusion.. https://doi.org/10.1016/s0960-9822(00)00547-9
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Pangenome graphs are increasingly used to represent population-scale bacterial diversity, yet construction methods span fundamentally different representation paradigms whose outputs and sensitivities to assembly quality remain poorly quantified. We systematically reviewed microbial pangenome graph tools and benchmarked seven representative methods spanning gene-cluster, compacted coloured de Bruijn graph, one hybrid approach and one multiple sequence alignment method. Using a repeat-rich Escherichia coli O157:H7 dataset with complete genomes and matched short-read data, we constructed graphs from identical inputs and observed orders-of-magnitude differences in graph size and fragmentation, indicating that global topology is driven by representation strategy. Varying completeness composition revealed that assembly fragmentation is a first-order determinant of graph structure: gene-cluster graphs contracted as draft assemblies replaced complete genomes, whereas compacted coloured de Bruijn graphs expanded, with distinct degree-prevalence fingerprints across tools. In contrast, the multiple sequence alignment method could not be evaluated across fragmented inputs because it did not run reliably on draft-assembly datasets. Computational cost mirrored these shifts and depended strongly on completeness composition, including a pronounced runtime penalty for one compacted coloured de Bruijn graph implementation on all-draft inputs. Finally, analysis of Shiga toxin loci showed that pangenome-level reconciliation by gene-cluster-based tools does not reliably correct assembly artefacts at challenging multi-copy genes and that performance varies by locus. Together, these findings show that pangenome graphs are representation-dependent models of bacterial diversity, and that, in this repeat-rich O157:H7 benchmark dataset, assembly completeness is a primary determinant of their topology, scalability, and locus-level accuracy.
BACKGROUND: Previous research has identified the potential for the existence of two separate lineages of Escherichia coli O157:H7. Clinical isolates tended to cluster primarily within one of these two lineages. To determine if there are virulence related genes differentially expressed between the two lineages we chose to utilize microarray technology to perform an initial screening. RESULTS: Using a 610 gene microarray, designed against the E. coli O157 EDL 933 transcriptome, targeting primarily virulence systems, we chose 3 representative Lineage I isolates (LI groups mostly clinical isolates) and 3 representative Lineage II isolates (LII groups mostly bovine isolates). Using standard dye swap experimental designs, statistically different expression (P < 0.05) of 73 genes between the two lineages was revealed. Result highlights indicate that under in vitro anaerobic growth conditions, there is up-regulation of stx2b, ureD, curli (csgAFEG), and stress related genes (hslJ, cspG, ibpB, ibpA) in Lineage I, which may contribute to enhanced virulence or transmission potential. Lineage II exhibits significant up-regulation of type III secretion apparatus, LPS, and flagella related transcripts. CONCLUSION: These results give insight into comparative regulation of virulence genes as well as providing directions for future research. Ultimately, evaluating the expression of key virulence factors among different E. coli O157 isolates has inherent value and the interpretation of such expression data will continue to evolve as our understanding of virulence, pathogenesis and transmission improves.
Diarrhoeagenic enterohaemorrhagic Escherichia coli and enteropathogenic E. coli attach to human intestinal epithelium and efface brush-border microvilli, forming an A/E (attaching and effacing) lesion. These human pathogens are phenotypically similar to the mouse pathogen Citrobacter rodentium. Genetically, they all have a homologous set of virulent genes involved in the A/E lesion, and these genes are organized on a LEE (locus of enterocyte effacement), a pathogenicity island. This island comprises 41 specific open reading frames, of which most are organized at five operons, LEE1, LEE2, LEE3, LEE4 and tir (LEE5). The expression of the LEE genes is regulated in a complicated manner, and current knowledge is that there are at least two positive regulators, Ler (LEE-encoded regulator) and GrlA (global regulator of LEE activator), and one negative regulator, called GrlR (global regulator of LEE repressor). In enterohaemorrhagic E. coli, GrlA is encoded by l0043, whereas GrlR is encoded by l0044. Here we report a fourth regulatory gene located in LEE3, namely l0036. Its expression is tightly controlled. When overexpressed, this factor, named Mpc (multiple point controller), interacts with Ler and suppresses the expression of the LEE proteins. When the translation is not initiated or terminated before maturation, the type III secretion of effectors is completely abolished. Therefore, together with the fact that several cis elements reside in the region that l0036 spans, l0036 appeared to have multiple functions in the regulation of LEE expression.