Type III secretion systems are toxin delivery systems that are present in a large number of pathogens. A hallmark of all type III secretion systems studied to date is that expression of one or more of their components is induced upon cell contact. It has been proposed that this induction is controlled by a negative regulator that is itself secreted by means of the type III secretion machinery. Although candidate proteins for this negative regulator have been proposed in a number of systems, for the most part, a direct demonstration of their role in regulation is lacking. Here, we report the discovery of ExsE, a negative regulator of type III secretion gene expression in Pseudomonas aeruginosa . Deletion of exsE deregulates expression of the type III secretion genes. We provide evidence that ExsE is itself secreted by means of the type III secretion machinery and physically interacts with ExsC, a positive regulator of the type III secretion regulon. Taken together, these data demonstrate that ExsE is the secreted negative regulator that couples triggering of the type III secretion machinery to induction of the type III secretion genes.

Historically, the first six recorded cholera pandemics occurred between 1817 and 1923 and were caused by Vibrio cholerae O1 serogroup strains of the classical biotype. Although strains of the El Tor biotype caused sporadic infections and cholera epidemics as early as 1910, it was not until 1961 that this biotype emerged to cause the 7th pandemic, eventually resulting in the global elimination of classical biotype strains as a cause of disease. The completed genome sequence of 7th pandemic El Tor O1 strain N16961 has provided an important tool to begin addressing questions about the evolution of V. cholerae as a human pathogen and environmental organism. To facilitate such studies, we constructed a V. cholerae genomic microarray that displays over 93% of the predicted genes of strain N16961 as spotted features. Hybridization of labeled genomic DNA from different strains to this microarray allowed us to compare the gene content of N16961 to that of other V. cholerae isolates. Surprisingly, the results reveal a high degree of conservation among the strains tested. However, genes unique to all pandemic strains as well as genes specific to 7th pandemic El Tor and related O139 serogroup strains were identified. These latter genes may encode gain-of-function traits specifically associated with displacement of the preexisting classical strains in South Asia and may also promote the establishment of endemic disease in previously cholera-free locations.

The relationship among ( i ) the local incidence of cholera, ( ii ) the prevalence in the aquatic environment of Vibrio cholerae , and ( iii ) bacterial viruses that attack potentially virulent O1 and O139 serogroup strains of this organism (cholera phages) was studied in Dhaka, Bangladesh. Over nearly a 3-year period, we found that significantly more environmental water samples contained either a phage or a phage-susceptible V. cholerae strain than both ( P < 0.00001). The number of cholera patients varied seasonally during this period and frequently coincided with the presence of pathogenic V. cholerae strains in water samples that otherwise lacked detectable cholera phages. Interepidemic periods were characterized by water samples containing cholera phages but no viable bacteria. Our data support the conclusion that cholera phages can influence cholera seasonality and may also play a role in emergence of new V. cholerae pandemic serogroups or clones.

A high-density transposon mutagenesis strategy was applied to the Haemophilus influenzae genome to identify genes required for growth or viability. This analysis detected putative essential roles for the products of 259 ORFs of unknown function. Comparisons between complete genomes defined a subset of these proteins in H. influenzae having homologs in Mycobacterium tuberculosis that are absent in Saccharomyces cerevisiae , a distribution pattern that favors their use in development of antimicrobial therapeutics. Three genes within this set are essential for viability in other bacteria. Interfacing the set of essential gene products in H. influenzae with the distribution of homologs in other microorganisms can detect components of unrecognized cellular pathways essential in diverse bacteria. This genome-scale phenotypic analysis identifies potential roles for a large set of genes of unknown function.

Vibrio cholerae is the etiologic bacterial agent of cholera, a severe diarrheal disease endemic in much of the developing world. The V. cholerae genome contains 3,890 genes distributed between a large and a small chromosome. Although the large chromosome encodes the majority of recognizable gene products and virulence determinants, the small chromosome carries a disproportionate number of hypothetical genes. Thus, little is known about the role of the small chromosome in the biology of this organism or other Vibrio species. We have used the rabbit ileal loop model of V. cholerae infection to obtain in vivo -grown cells under near midexponential conditions in the small-intestinal environment. We compared the global transcriptional pattern of these in vivo -grown cells to those grown to midexponential phase in rich medium under aerobic conditions. Under both conditions, the genes showing the highest levels of expression reside primarily on the large chromosome. However, a shift occurs in vivo that results in many more small chromosomal genes being expressed during growth in the intestine. Our analysis further suggests that nutrient limitation (particularly iron) and anaerobiosis are major stresses experienced by V. cholerae during growth in the rabbit upper intestine. Finally, relative to in vitro growth, the intestinal environment significantly enhanced expression of several virulence genes, including those involved in phenotypes such as motility, chemotaxis, intestinal colonization, and toxin production.