Type II Secretion System

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.

Individuals with the genetic disease cystic fibrosis (CF) accumulate mucus or sputum in their lungs. This sputum is a potent growth substrate for a range of potential pathogens, and the opportunistic bacterium Pseudomonas aeruginosa is generally most difficult of these to eradicate. As a result, P. aeruginosa infections are frequently maintained in the CF lung throughout life, and are the leading cause of death for these individuals. While great effort has been expended to better understand and treat these devastating infections, only recently have researchers begun to rigorously examine the roles played by specific nutrients in CF sputum to cue P. aeruginosa pathogenicity. This chapter summarizes the current state of knowledge regarding how P. aeruginosa metabolism in CF sputum affects initiation and maintenance of these infections. It contains an overview of CF lung disease and the mechanisms of P. aeruginosa pathogenicity. Several model systems used to study these infections are described with emphasis on the challenge of replicating the chronic infections observed in humans with CF. Nutrients present in CF sputum are surveyed, and the impacts of these nutrients on the infection are discussed. The chapter concludes by addressing the future of this line of research including the use of next-generation technologies and the potential for metabolism-based therapeutics.

This chapter starts with a description of the general characteristics of the genome sequences of Legionella pneumophila and Legionella longbeachae. It then highlights the characteristic features and common traits of the two main human-pathogenic Legionella species. Emphasis is given to putative virulence and Legionella life cycle-related functions. In the second part, the focus is on the comparison of these genome sequences, in order to learn about the plasticity of the Legionella genomes and the possible mechanisms involved. In the third part, the possible evolution of the identified virulence factors are analyzed and discussed. Finally, future perspectives in Legionella genomics are presented. L. longbeachae NSW 150 encodes four T4SS, a rather exceptional number and L. longbeachae D-4968 encodes two T4SS. The authors found that IcmE/DotG of L. longbeachae (1,525 amino acids) is 477 amino acids larger than that of L. pneumophila (1,048 amino acids). Most of the T4SS are located on regions of genome plasticity; some even show plasticity on the gene/protein level, as shown above for DotG. The authors undertook a phylogenetic analysis for the L. longbeachae protein Llo2643, which contains PPR repeats, a protein family typically present in plants. In the last few years, genome analyses, as well as comparative and functional genomics, have demonstrated that genome plasticity plays a major role in differences in host cell exploitation and niche adaptation of Legionella.

In this chapter, the knowledge of the genome of E. coli O157:H7 may be used in the control of serious foodborne pathogen, E. coli strains are associated with gastrointestinal and extraintestinal illness. The E. coli O157:H7 genome provides us with information about the evolution and emergence of pathogen and the diversity that exists within populations of E. coli O157:H7. Most of E. coli prophage are defective and cannot form infectious phage. This chapter provides a brief review of subtyping methods used to characterize E. coli O157:H7 strains. Differences in biochemical utilization can be used to distinguish large categories of E. coli strain. Following the generation of cDNA from the bacteria’s mRNA using the enzyme reverse transcriptase, the level of gene expression is inferred from the intensity of the label signal from gene-specific microarray spots following hybridization. This procedure is the foundation of the new science of transcriptomics. Typically, quantitative PCR assays are also carried out on selected genes to measure mRNA levels to verify the significant changes in expression of genes observed in microarray-based transcriptomics studies. It is also evident that our understanding of E. coli O157:H7 gene regulatory systems will be enhanced through genomic sciences. E. coli O157:H7 and other bacterial pathogens have evolved through the acquisition of gene clusters borne on plasmids, bacteriophages, and genomic islands. Finally, with the arrival of the genomics revolution researchers are able to examine the "pan-genome" of the species and specific groups within species such as enterohemorrhagic E. coli(EHEC) and E. coli O157:H7.

This chapter integrates unpublished data with the published reports on the synergism between veillonellae and streptococci. It speculates on how the genome sequence of veillonellae will help one understand its biology and potential role in the pathogenicity of the dental biofilm community. It was discovered that hydrogen peroxide (H2O2) resistance in Streptococcus mutans was increased 100 to 1,000-fold over monospecies culture in cocultures with Veillonella sp. strain PK1910. This chapter focuses on some important findings from the Veillonella sp. strain PK1910 genome. In the 1964 description of Veillonellasp. by Rogosa, the species V. alcalescens was distinguished from V. parvula by the ability to decompose H2O2. In the 1982 revision of the species, the former subspecies in V. alcalescens were classified as three species: V. ratti, V. criceti, and V. dispar. Veillonella species are among the most prevalent early colonizers of oral biofilm. In-depth investigations on the mechanism of interactions of veillonellae with other oral microbial species will contribute significantly to one's overall understanding of the ecology of oral biofilms in the human host. Different from a metagenomics approach, metatranscriptomics focuses on community member functions at a particular time under a particular condition.

Tom Whittam's pioneering research into the structure of Escherichia coli populations not only advanced the field of microbial evolution and genetics, but it also provided a contextual framework for investigating variation in the epidemiology and virulence of bacterial populations. This chapter highlights the work of his laboratory involving the use of various population genetic strategies for characterizing E. coli pathotypes, particularly enterohemorrhagic E. coli (EHEC) O157:H7, while focusing on contributions to epidemiology and public health. To determine whether specific genotypes are associated with human enteric disease, multilocus enzyme electrophoresis (MLEE) was used to examine 1,300 E. coli strains representing 16 serotypes, including EHEC O157:H7 from patients with hemorrhagic colitis and hemolytic uremic syndrome (HUS). Similar to MLEE and multilocus sequence typing (MLST) data, systematic analysis of single nucleotide polymorphisms (SNPs) is amenable to both population genetic and phylogenetic analyses. SNPs data can also be used to examine epidemiological associations between bacterial genotypes and clinical disease. By using the SNP genotyping phylogeny as a framework, it is possible to investigate virulence gene diversity, allelic variation, and gene expression differences among genotypes to identify bacterial genomic determinants of colonization, pathogenesis, and transmissibility. The chapter describes differences in stress resistance properties among clades, nonrandom distribution of stx variants, variation in adherence to epithelial cells, and differential expression among shared genes.

Typically, the biotechnological basis for strain improvement to enhance recombinant protein production relies on the permanent implementation of desirable traits into the production strain to stimulate both cell growth and functional expression of the target gene during the cultivation. This chapter reviews the major technical issues associated with Escherichia coli strain engineering to enhance recombinant protein production and directs the reader to protocols appropriate for specific applications. Theoretically, strategies based on enhancing the limiting step can lead to an overall improvement in recombinant protein production. Stationary-phase genes encode proteins that may lead to a reduction in cellular and metabolic activity, which can negatively affect recombinant protein production, and as such, these genes are targets for strain improvement. The recently commercialized recombineering protocol from Gene Bridges, also based on λ Redmediated recombination, allows versatile chromosomal engineering, including gene disruption, deletion, insertion, point mutation, modification, and even promoter fine-tuning, and can serve as a versatile manipulation tool for strain improvement and even optimization. The general guidelines for strain improvement are (i) to ensure the genetic stability of the host/vector system, (ii) to maximize the synthesis fluxes for all the gene expression steps (i.e., transcription, translation, and posttranslational processing steps), (iii) to ensure the flux balance of these protein synthesis steps, (iv) to stabilize all the expression intermediates and final products, and (v) to minimize the physiological impact associated with high-level gene expression and high-cell density cultivation.

One of the first bacterial species for which N-acylhomoserine lactone (AHL) quorum sensing (QS) was described was Erwinia carotovora. Since then, QS has been well studied in the soft-rot erwinias, where, as described in this chapter, QS plays a key role in the regulation of secreted plant cell wall-degrading enzymes (PCWDEs) production and hence in virulence. In certain strains, a well-defined AHL QS system also controls production of β-lactam antibiotic, carbapenem. In addition, it must be emphasized that QS is only one of many regulatory inputs into virulence factor production in Erwinia. The majority of the key secreted virulence factors of E. carotovora and E. chrysanthemi, including multiple Pels, Peh, Cel, and Svx, are secreted by a type II secretion system known as the Out system. There have been two reports describing the existence of AHL QS in E. amylovora. First, production of a single AHL, most likely 3-oxo-C6-HSL, was described for several Italian strains of E. amylovora; for one strain, production of AHL was observed in planta. Second, AHL activity was detected in the culture supernatant of a Swiss strain of E. amylovora. Both reports describe the detection and partial sequencing of pairs of convergent luxIR homologues, named eamIR.

Proteinaceous, nonflagellar surface appendages constitute a variety of structures, including those known variably as fimbriae or pili. Constructed by distinct assembly pathways resulting in diverse morphologies, fimbriae have been described to mediate functions including adhesion, motility, and DNA transfer. As these structures can represent major diversifying elements among Escherichia and Salmonella isolates, multiple fimbrial classification schemes have been proposed and a number of mechanistic insights into fimbrial assembly and function have been made. Herein we describe the classifications and biochemistry of fimbriae assembled by the chaperone/usher, curli, and type IV pathways.

This chapter provides an overview of how genome sequence data are changing our understanding of the mechanisms and evolution of plant pathogenesis, and discusses the opportunities and challenges genome data present for future research. The majority of plant pathogenic bacteria contain large numbers of genomic islands, and it is clear that in many cases they play an essential role in disease. Genes encoding the P. syringae phytotoxins coronatine and syringomycin reside on PIs, as do their recently discovered counterparts in E. carotovora subsp. atroseptica SCR1043. Pathogenicity and virulence factors have received the greatest focus in the genomic analysis of plant pathogens. Protein secretion systems are essential for pathogenesis in most plant pathogenic bacteria and have been extensively studied, particularly in proteobacterial pathogens. The phytohormones indole-3-acetic acid (IAA or auxin) is produced by many phytopathogenic bacteria. IAA production has been shown to contribute to in planta and epiphytic growth, virulence, and the regulation of syringomycin in P. syringae. Genome analyses of plant pathogens have highlighted three aspects of physiological adaptation to life on plants: specialization, innovation, and flexibility. A largely uninvestigated element in understanding the content and function of plant pathogen genomes, particularly the genomes of facultative plant pathogens, rests on understanding what plant pathogenic bacteria do when not causing disease.