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.

The gene encoding the conserved bacterial G protein CgtA (Obg) is essential for viability in every organism in which it has been studied. CgtA has been reported to be involved in several diverse bacterial functions, including ribosome assembly, DNA repair, sporulation, and morphological development. However, none of these functions have been identified as essential. Here we show that depletion of CgtA in Vibrio cholerae causes global changes in gene expression that are consistent with induction of a classical low nutrient stress response or ''stringent'' response. We show that depletion of CgtA leads to increased ppGpp levels that correlate with induction of the global stress response and cessation of growth. The enzyme RelA is responsible for synthesis of the alarmone ppGpp during the stringent response. We show that CgtA is no longer essential in a relA deletion mutant and thus conclude that the essentiality of CgtA is directly linked to its ability to affect ppGpp levels. The enzyme SpoT degrades ppGpp, and here we show that SpoT is essential in a RelA؉ CgtA؉ background but not in a relA deletion mutant. We also confirmed that CgtA interacts with SpoT in a two-hybrid assay. We suggest that the essential function of CgtA is as a repressor of the stringent response that acts by regulating SpoT activity to maintain low ppGpp levels when bacteria are growing in a nutrient-rich environment.alarmone ͉ cholera ͉ GTPase ͉ starvation ͉ transcription

YgbQ is a cell division protein in Escherichia coli and Vibrio cholerae. In E. coli the ygbQ gene was discovered as a result of a computer search of the E. coli genome designed to find potential interacting partners for cell division protein FtsL. In V. cholerae, ygbQ was identified as an essential gene by using a transposon that fuses genes to an arabinose promoter. The role of YgbQ in cell division is supported by the following. Cells depleted of YgbQ in both organisms form long filaments, but DNA segregation is not affected. YgbQ localizes to the constriction site in wild-type E. coli cells. Localization of E. coli YgbQ to the constriction site depends on cell division proteins FtsQ and FtsL but not FtsW and FtsI, placing YgbQ in the sequential dependency order of proteins localizing to the division site. Localization of green fluorescent protein-FtsL also depends on YgbQ, indicating that FtsL and YgbQ colocalize to the division site in E. coli. Our results show colocalization of proteins to the bacterial midcell in E. coli and raise the possibility that these proteins interact in a coiled-coil structure.C ell division in bacteria takes place at the midcell and occurs after the DNA has been duplicated and segregated into two daughter nucleoids. In Escherichia coli, this process requires a set of at least nine proteins that localize to the constriction site or septum. These proteins coordinate invagination of the cell membrane, inward growth of the peptidoglycan layer, and, finally, separation of daughter cells. The nine proteins, FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL, FtsW, FtsI, and FtsN, have been identified largely through genetic approaches. Because no systematic genetic approach for identifying such proteins has been performed or devised, there could be a number of thus-farundiscovered proteins that play a role in cell division. Information is available regarding function for only a few of these proteins (reviewed in refs. 1 and 2).Studies from a number of laboratories have led to a sequential dependency model for the assembly of this group of proteins at the cell septum (3). FtsZ arrives first at the cell septum, providing a scaffold for the recruitment of subsequent proteins. FtsA and ZipA localize to the septum independently of each other, but each depends on the presence of FtsZ for localization. The remaining proteins assemble at midcell in a strictly sequential dependency order as follows: FtsK-FtsQ-FtsL-FtsW-FtsIFtsN. The mechanisms for this order of recruitment are not understood.The work reported here arose from projects with different goals in the Beckwith and Mekalanos laboratories. The Beckwith laboratory has studied cell division in E. coli by focusing attention on three proteins, FtsQ, FtsL, and FtsI. These proteins have similar membrane topologies, i.e., a short amino-terminal domain facing the cytoplasm, a single transmembrane segment, and a carboxyl-terminal domain located in the periplasm. FtsL has a leucine zipper-like motif in its periplasmic domain, indicating that it may form coiled-co...

Identification of genes that encode essential products provides a promising approach to validation of new antibacterial drug targets. We have developed a mariner-based transposon, TnAraOut, that allows efficient identification and characterization of essential genes by transcriptionally fusing them to an outward-facing, arabinose-inducible promoter, PBAD, located at one end of the transposon. In the absence of arabinose, such TnAraOut fusion strains display pronounced growth defects. Of a total of 16 arabinose-dependent TnAraOut mutants characterized in Vibrio cholerae, four were found to carry insertions upstream of known essential genes (gyrB, proRS, ileRS, and aspRS) whereas the other strains carried insertions upstream of known and hypothetical genes not previously shown to encode essential gene products. One of the essential genes identified by this analysis appears to be unique to V. cholerae and thus may represent an example of a species-specific drug target.