Bacterial Mobile Genetic Elements

Escherichia coli and other Gram-negative and -positive bacteria employ type IV secretion systems (T4SSs) to translocate DNA and protein substrates, generally by contact-dependent mechanisms, to other cells. The T4SSs functionally encompass two major subfamilies, the conjugation systems and the effector translocators. The conjugation systems are responsible for interbacterial transfer of antibiotic resistance genes, virulence determinants, and genes encoding other traits of potential benefit to the bacterial host. The effector translocators are used by many Gram-negative pathogens for delivery of potentially hundreds of virulence proteins termed effectors to eukaryotic cells during infection. In E. coli and other species of Enterobacteriaceae, T4SSs identified to date function exclusively in conjugative DNA transfer. In these species, the plasmid-encoded systems can be classified as the P, F, and I types. The P-type systems are the simplest in terms of subunit composition and architecture, and members of this subfamily share features in common with the paradigmatic Agrobacterium tumefaciens VirB/VirD4 T4SS. This review will summarize our current knowledge of the E. coli systems and the A. tumefaciens P-type system, with emphasis on the structural diversity of the T4SSs. Ancestral P-, F-, and I-type systems were adapted throughout evolution to yield the extant effector translocators, and information about well-characterized effector translocators also is included to further illustrate the adaptive and mosaic nature of these highly versatile machines.

This chapter reviews pulsed-field gel electrophoresis (PFGE) as an epidemiological tool, considering (i) factors that influence the electrophoretic process, (ii) methodological streamlining, (iii) the troubleshooting of common problems, (iv) quality assurance, (v) use of PFGE for continuous surveillance, and (vi) issues of data interpretation. To be suitable for reliable PFGE analysis, intact chromosomal DNA must be isolated in a protected environment free from mechanical, chemical, and enzymatic degradation to yield a clear and reproducible macrorestriction fragment pattern. As PFGE analysis is applied to larger study populations, the need for computer-assisted analysis (CAA) of banding patterns becomes increasingly evident. At the laboratory level the quality assurance/ quality control (QA/QC) system consists of strict adherence to each of the PFGE standard operating procedures (SOPs) as described in the laboratory QA/QC manual. It is important to emphasize that the successful establishment of dynamic databases is dependent on strict adherence to well-defined QA and QC criteria. An important component of the protocol standardization and QA/QC program for PulseNet is the annual update meeting. Molecular typing, along with a variety of other microbiological assays is clearly moving toward sequence-based analysis. However, this approach is still being validated for a variety of applications including strain typing. Thus far, none of the new sequence-based typing methods are as broadly applicable as PFGE. Therefore, while this problem will undoubtedly be solved in the future, at present PFGE will clearly continue to provide meaningful epidemiological data on molecular typing in a variety of important settings for years to come.

Horizontally transferred genes were frequently disseminated among bacterial populations as components of mobile genetic elements, such as plasmids, phages, and transposons. This chapter focuses on bacterial pathogens, and in particular, on the role of sequence-specific mechanisms for intercellular gene transfer in the evolution of such organisms. It concentrates on virulence factors encoded by genes on mobilizable or formerly mobilizable genetic elements, especially plasmids, bacteriophages, and pathogenicity islands (PAIs). Finally, analyses based on the different classes of mobile elements can highlight the processes by which virulence genes have been transported and illuminate the evolutionary relationships between types of mobile elements. In the chapter, the last-mentioned approach is used, and bacteriophages, plasmids, and PAIs are sequentially discussed. Finally, some plasmids, such as the large Yersinia virulence plasmid and the enterohemorrhagic Escherichia coli (EHEC) virulence plasmid, contain remnants of DNA transfer systems, suggesting that they were previously capable of self-mobilization. Although the virulence genes acquired as components of mobile elements may not have been subject to host regulatory processes immediately after acquisition, they clearly have not remained autonomous agents, independent of host functions. Mobile genetic elements have clearly distributed a diverse collection of virulence genes and thereby played essential roles in the evolution of bacterial pathogens. However, a subset of virulence factors does not seem to confer any advantages on the bacterial hosts, so the forces underlying their continued production are a mystery.