SummaryDespite over a century of research, tuberculosis remains a leading cause of infectious death worldwide. Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents. Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis , for optimal growth. These genes include those that can be assigned to essential pathways as well as many of unknown function. The genes important for the growth of M. tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae , indicating that nonessential functions have been selectively lost since this bacterium diverged from other mycobacteria. In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories.

The opportunistic pathogen Pseudomonas aeruginosa causes a variety of acute and chronic infections. We identified a gene whose inactivation results in attenuation of virulence due to premature activation of genes involved in biofilm formation and coordinate repression of genes required for initial colonization. This gene, retS, encodes a hybrid sensor kinase/response regulator with an unconventional arrangement of functional domains. Genome-wide transcriptional profiling indicates that the retS gene is required for expression of the Type III secretion system and other virulence factors and for repression of genes responsible for exopolysaccharide components of the P. aeruginosa biofilm matrix. These disparate phenotypes are suppressed by transposon insertions in genes encoding the GacS/GacA/rsmZ signal transduction pathway, a highly conserved system involved in the control of diverse adaptive functions. This study defines RetS as a pleiotropic regulator of multiple virulence phenotypes that orchestrates genes required for acute infection and genes associated with chronic persistence.

An increasing number of microbial genomes have been completely sequenced, and the identified genes are categorized based on their homology to genes of known function. However, the function of a large number of genes cannot be determined on this basis alone. Here, we describe a technique, transposon site hybridization (TraSH), which allows rapid functional characterization by identifying the complete set of genes required for growth under different conditions. TraSH combines high-density insertional mutagenesis with microarray mapping of pools of mutants. We have made large pools of independent transposon mutants in mycobacteria by using a marinerbased transposon and efficient phage transduction. By using TraSH, we have defined the set of genes required for growth of Mycobacterium bovis bacillus Calmette-Gué rin on minimal but not rich medium. Genes of both known and unknown functions were identified. Of the genes with known functions, nearly all were involved in amino acid biosynthesis. TraSH is a powerful method for categorizing gene function that should be applicable to a variety of microorganisms.

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.

Vitamin K epoxide reductase (VKOR) generates vitamin K hydroquinone to sustain γ-carboxylation of many blood coagulation factors. Here, we report the 3.6Å crystal structure of a bacterial homolog of VKOR from Synechococcus sp. The structure shows VKOR in complex with its naturally fused redox partner, a thioredoxin-like domain, and corresponds to an arrested state of electron transfer. The catalytic core of VKOR is a four transmembrane helix bundle that surrounds a quinone, connected through an additional transmembrane segment with the periplasmic thioredoxin-like domain. We propose a pathway for how VKOR uses electrons from newly synthesized proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulfide bridge formation. Our results have implications for the mechanism of the mammalian VKOR and explain how mutations can cause resistance to the VKOR inhibitor warfarin, the most commonly used oral anticoagulant.