Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells. We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media. We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions. Functional context analysis of these data allows individual functional assignments to be refined. Evolutionary context analysis demonstrates a significant tendency of essential E. coli genes to be preserved throughout the bacterial kingdom. Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.Sequencing and comparative analysis of multiple diverse genomes is revolutionizing contemporary biology by providing a framework for interpreting and predicting the physiologic properties of an organism. A variety of emerging postgenomic techniques such as genome-wide expression profiling and monitoring of macromolecular complex formation can reveal the detailed molecular compositions of cells. New computational approaches to exploring the inherent organization of cellular networks, the mode and dynamics of interactions among cellular constituents, are in early stages of development (14,22,23). These techniques allow us to begin unraveling a major paradigm of cellular biology: how biological properties arise from the large number of components making up an individual cell.

Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets. Our strategy is based on genetic footprinting in Escherichia coli followed by metabolic context analysis of essential gene orthologs in various species. Genes required for viability of E. coli in rich medium were identified on a whole-genome scale using the genetic footprinting technique. Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data. Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets. Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host. The most promising targets are validated by direct knockouts in model pathogens. The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide. Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of the E. coli genes nadD, coaD, and ribF), are discussed in detail.

Microorganisms respond to environmental changes by reprogramming their metabolism primarily through altered patterns of gene expression. DNA microarrays provide a tool for exploiting microorganisms as living sensors of their environment. The potential of DNA microarrays to reflect availability of nutrient components during fermentations on complex media was examined by monitoring global gene expression throughout batch cultivation of Escherichia coli MG1655 on Luria-Bertani (LB) medium. Gene expression profiles group into pathways that clearly demonstrate the metabolic changes occurring in the course of fermentation. Functional analysis of the gene expression related to metabolism of sugars, alcohols, and organic acids revealed that E. coli growing on LB medium switches from a sequential mode of substrate utilization to the simultaneous one in the course of the growth. Maltose and maltodextrins are the first of these substrates to support growth. Utilization of these nutrients associated with the highest growth rate of the culture was followed by simultaneous induction of enzymes involved in assimilation of a large group of other carbon sources including D-mannose, melibiose, D-galactose, L-fucose, L-rhamnose, D-mannitol, amino sugars, trehalose, L-arabinose, glycerol, and lactate. Availability of these nutrients to the cells was monitored by induction of corresponding transport and/or catabolic systems specific for each of the compounds.

Expression profiles of genes related to stress responses, substrate assimilation, acetate metabolism, and biosynthesis were obtained by monitoring growth of Escherichia coli MG1655 in Luria-Bertani (LB) medium with transcriptional microarrays. Superimposing gene expression profiles on a plot of specific growth rate demonstrates that the cells pass through four distinct physiological states during fermentation before entering stationary phase. Each of these states can be characterized by specific patterns of substrate utilization and cellular biosynthesis corresponding to the nutrient status of the medium. These data allow the growth phases of the classical microbial growth curve to be redefined in terms of the physiological states and environmental changes commonly occurring during bacterial growth in batch culture on LB medium.

A chemostat coculture of the sulfate-reducing bacterium Desulfovibrio oxyclinae together with a facultative aerobe heterotroph tentatively identified as Marinobacter sp. strain MB was grown under anaerobic conditions and then exposed to a stepwise-increasing oxygen influx (0 to 20% O 2 in the incoming gas phase). The coculture consumed oxygen efficiently, and no residual oxygen was detected with an oxygen supply of up to 5%. Sulfate reduction persisted at all levels of oxygen input, even at the maximal level, when residual oxygen in the growth vessel was 87 M. The portion of D. oxyclinae cells in the coculture decreased gradually from 92% under anaerobic conditions to 27% under aeration. Both absolute cell numbers and viable cell counts of the organism were the same as or even higher than those observed in the absence of oxygen input. The patterns of consumption of electron donors and acceptors suggest that aerobic incomplete oxidation of lactate to acetate is performed by D. oxyclinae under high oxygen input. Both organisms were isolated from the same oxic zone of a cyanobacterial mat where they have to adapt to daily shifts from oxic to anoxic conditions. This type of syntrophic association may occur in natural habitats, enabling sulfate-reducing bacteria to cope with periodic exposure to oxygen.The photic zone of cyanobacterial mats is characterized by extreme shifts in oxygen and sulfide concentrations from oxygen supersaturation during the light period to anoxic, sulfideenriched conditions in the dark (24). These changes are a challenge for the organisms living in such a habitat. Daily exposure to high oxygen concentrations may be deleterious to obligately anaerobic organisms, such as sulfate-reducing bacteria (SRB) (13,18,24). High numbers of SRB were found in the oxic zone of a cyanobacterial mat from the Solar Lake (Sinai) throughout the diurnal cycle (10,21,22,25,27), indicating that these organisms can deal with exposure to elevated oxygen concentrations of even up to 1.5 mM. Moreover, high rates of sulfate reduction were found in the oxic layers of these microbial mats (4, 5, 9), as well as in microbial mats from Baja California, Mexico (2).None of the SRB isolated so far can either grow aerobically or reduce sulfate under high concentrations of oxygen. Desulfovibrio vulgaris Hildenborough was found to be capable of slow linear aerobic growth under very low concentrations of oxygen. Oxygen concentrations of only 0.07% were toxic to this organism (14). The oxygen-tolerant SRB Desulfovibrio oxyclinae isolated in our laboratory (16) was demonstrated to persist in continuous culture gassed with 1% O 2 , although flocculation occurred after exposure to oxygen (25).It was previously demonstrated that SRB in the upper layer of cyanobacterial mats can form close associations with oxygen-scavenging bacteria (12, 26). Such consortia may be responsible, at least in part, for the observed in situ sulfatereducing activity under aerobic conditions.In this report, we present the effects of exposure to oxygen on the ...