Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among Ϸ4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

To study the functions of the uncharacterized open reading frames identified in the Bacillus subtih genome, several vectors were constructed t o perform insertional mutagenesis in the chromosome. All the pMUTlN plasmids carry a lac2 reporter gene and an inducible Pspac promoter, which is tightly regulated and tan be induced about 1000-fold. The integration of a pMUTlN vector into the target gene has three consequences: (1) the target gene is inactivated; (2) lac2 becomes transcriptionally fused t o the gene, allowing its expression pattern t o be monitored; (3) the Pspac promoter controls the transcription of downstream genes in an IPTG-dependent fashion. This last feature is important because B. subti/is genes are often organized in operons. The potential polar effects generated by the integration of the vectors can be alleviated by addition of IPTG. Also, conditional mutants of essential genes can be obtained by integrating pMUTlN vectors upstream of the target gene. The vectors are currently being used for systematic inactivation of genes without known function within the B. subtilis European consortium. pMUTlN characteristics and the inactivation of eight genes in the resA-serA region of the chromosome are presented.

The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruitingbody-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.Formation of biofilms-surface-associated multicellular assemblages-is an important microbial survival strategy (4, 25). Relative to the rapid, continual, and extreme changes in environmental conditions that can characterize a planktonic existence, biofilms offer the constituent cells some shelter, enabling them to establish long-term relationships with each other and their immediate surroundings. Cells in different regions of a developing biofilm experience diverse environmental conditions, resulting in marked patterns of cellular differentiation (e.g., see references 5 and 16). Such spatiotemporal organization is particularly striking in biofilms formed by wild strains of the spore-forming bacterium Bacillus subtilis. Unlike strains that have been domesticated by decades of propagation in the laboratory (e.g., B. subtilis 168), which form thin and relatively undifferentiated biofilms, wild B. subtilis strains form elaborate multicellular communities that display conspicuous architectural features, such as fruiting-body-like aerial projections that extend from the surfaces of the biofilm. The tips of these fruiting bodies serve as preferential sites for spore formation (2).We are interested in discovering genes that are involved in biofilm and fruiting body formation. We have focused our efforts on a relatively undomesticated strain of B. subtilis, NCIB 3610 (hereafter referred to as "3610"), which forms robust and highly structured biofilms both in liquid and on solid medium (colonies). When inoculated into a standing culture of minimal medium, 3610 initially grows planktonically as motile, single cells. The ...

Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical and model-based data analyses of dynamic transcript, protein, and metabolite abundances and promoter activities. Adaptation to malate was rapid and primarily controlled posttranscriptionally compared with the slow, mainly transcriptionally controlled adaptation to glucose that entailed nearly half of the known transcription regulation network. Interactions across multiple levels of regulation were involved in adaptive changes that could also be achieved by controlling single genes. Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control programs.

A protein-interaction network centered on the replication machinery of Bacillus subtilis was generated by genome-wide two-hybrid screens and systematic specificity assays. The network consists of 91 specific interactions linking 69 proteins. Over one fourth of the interactions take place between homologues of proteins known to interact in other organisms, indicating the high biological significance of the other interactions we report. These interactions provide insights on the relations of DNA replication with recombination and repair, membrane-bound protein complexes, and signaling pathways. They also lead to the biological role of unknown proteins, as illustrated for the highly conserved YabA, which is shown here to act in initiation control. Thus, our interaction map provides a valuable tool for the discovery of aspects of bacterial DNA replication.T he replication of the bacterial chromosome is carried out by a large multiprotein machine, the replisome, in which the activities of individual polypeptides are highly coordinated to achieve efficient and faithful DNA replication. The components of bacterial replisomes have been characterized extensively, revealing the molecular mechanisms at work in a DNAreplication apparatus (1, 2). Localization studies indicated that the replication machinery is preferentially at midcell, suggesting a factory model of replication in which the DNA template moves through a rather stationary polymerase (3). In contrast, the origin regions of the chromosomes are moving toward the cell poles during cell-cycle progression (4, 5). However, other aspects of DNA replication still remain unclear. For instance, it is not known how the replication machinery coordinates its action with other cellular processes in a variety of environmental conditions or what the determinants that specify replisome or origin positions within the cell are. Mutants affected in these biological processes have not been reported yet, possibly because they display weak or inconsistent phenotypes caused by redundant functions.To gain insight into this unexplored area, we used genomewide yeast two-hybrid screens (6) to identify the proteins that physically associate with known replication proteins from the Gram-positive bacterium Bacillus subtilis. To circumvent one of the main limitations of the approach, the false-positive interactions, we verified experimentally the specificity of every potential interaction identified in the screens. The resulting protein network is composed of 91 specific interactions connecting 69 proteins. Over one fourth of the interactions were described previously in bacteria or eukaryotes, showing that our approach yields biologically significant interactions. The remaining interactions are previously uncharacterized, and in combination with data from the literature, many of their biological roles can be hypothesized. They link DNA replication with DNA recombination and repair, potential origin-and replisome-anchoring membrane complexes, signaling pathways, and numerous proteins of unkno...

Bacillus subtilis possesses two similar putative phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoding genes, gap (renamed gapA) and gapB. A gapA mutant was unable to grow on glycolytic carbon sources, although it developed as well as the wild-type strain on gluconeogenic carbon sources. A gapB mutant showed the opposite phenotype. Purified GapB showed a 50-fold higher GAPDHase activity with NADP ؉ than with NAD ؉ , with K m values of 0.86 and 5.7 mM, respectively. lacZ reporter gene fusions revealed that the gapB gene is transcribed during gluconeogenesis and repressed during glycolysis. Conversely, gapA transcription is 5-fold higher under glycolytic conditions than during gluconeogenesis. GAPDH activity assays in crude extracts of wild-type and mutant strains confirmed this differential expression pattern at the enzymatic level. Genetic analyses demonstrated that gapA transcription is repressed by the yvbQ (renamed cggR) gene product and indirectly stimulated by CcpA. Thus, the same enzymatic step is catalyzed in B. subtilis by two enzymes specialized, through the regulation of their synthesis and their enzymatic characteristics, either in catabolism (GapA) or in anabolism (GapB). Such a dual enzymatic system for this step of the central carbon metabolism is described for the first time in a nonphotosynthetic eubacterium, but genomic analyses suggest that it could be a widespread feature.Glycolysis is the main pathway for degradation of carbohydrates and is found in nearly all groups of organisms. The formation of the final product of glycolysis, pyruvate, from glucose is achieved by nine enzymatic steps, most of which function in the reverse direction during gluconeogenesis. The phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 1 occupies a pivotal role in the Embden-Meyerhoff pathway not only in glycolysis but also in gluconeogenesis because of the reversibility of the oxidation of glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (1,3dPG). In plants, two distinct types of phosphorylating GAPDH co-exist: (i) a strictly NAD-dependent cytoplasmic GAPDH involved in glycolysis and gluconeogenesis; and (ii) a chloroplastic GAPDH, which is involved in photosynthetic CO 2 assimilation and exhibits a dual coenzyme specificity with a preference for NADP (1, 2).Recently, two gap genes, named gap1 and gap2, have been characterized in the cyanobacterium Synechocystis sp. PPC 6803. The NAD-dependent enzyme Gap1 was reported to be essential for glycolytic glucose breakdown, whereas the enzyme Gap2, which exhibits dual coenzyme specificity, was shown to be operative in the photosynthetic Calvin cycle and in nonphotosynthetic gluconeogenesis (3). Thus, at least in some photoautotrophic bacterial species (in which the photosynthetic Calvin cycle and glycolysis/gluconeogenesis function in the same cellular compartment, in contrast to what happens in land plants and algae) two distinct GAPDHs, a strictly NAD-dependent one and the photosynthetic one, catalyze the two ...

The ability to use lactate as a sole source of carbon and energy is one of the key metabolic signatures of Shewanellae, a diverse group of dissimilatory metal-reducing bacteria commonly found in aquatic and sedimentary environments. Nonetheless, homology searches failed to recognize orthologs of previously described bacterial D-or L-lactate oxidizing enzymes (Escherichia coli genes dld and lldD) in any of the 13 analyzed genomes of Shewanella spp. By using comparative genomic techniques, we identified a conserved chromosomal gene cluster in Shewanella oneidensis MR-1 (locus tag: SO1522-SO1518) containing lactate permease and candidate genes for both D-and L-lactate dehydrogenase enzymes. The predicted D-LDH gene (dld-II, SO1521) is a distant homolog of FAD-dependent lactate dehydrogenase from yeast, whereas the predicted L-LDH is encoded by 3 genes with previously unknown functions (lldEGF, SO1520 -SO1518). Through a combination of genetic and biochemical techniques, we experimentally confirmed the predicted physiological role of these novel genes in S. oneidensis MR-1 and carried out successful functional validation studies in Escherichia coli and Bacillus subtilis. We conclusively showed that dld-II and lldEFG encode fully functional D-and L-LDH enzymes, which catalyze the oxidation of the respective lactate stereoisomers to pyruvate. Notably, the S. oneidensis MR-1 LldEFG enzyme is a previously uncharacterized example of a multisubunit lactate oxidase. Comparative analysis of >400 bacterial species revealed the presence of LldEFG and Dld-II in a broad range of diverse species accentuating the potential importance of these previously unknown proteins in microbial metabolism.central carbon metabolism ͉ genome context analysis ͉ lactate dehydrogenase M any aerobic and anaerobic bacteria are able to grow by using D-and/or L-lactate as a sole source of carbon and energy (1-4). Although lactate is a common product of carbohydrate fermentation (5, 6), it is rarely detected in environmental samples (7,8), suggesting that it is either a minor metabolic product or that its conversion rates are very high. In support of the latter possibility, Finke et al. (9) recently reported constant production and consumption of lactate in marine sediments, linking its high turnover rates with microbiological reduction of sulfate and metals.Among microorganisms actively coupling lactate oxidation to the reduction of multiple electron acceptors is a diverse and ubiquitous group of dissimilatory metal-reducing bacteria, which belong to the genus Shewanella (10). Shewanellae are commonly found in complex microbial communities within aquatic and sedimentary systems, many of which are subject to spatial and temporal variations in the type and concentration of organic and inorganic substrates that reflect redox gradients (10). The versatile flexibility of energygenerating pathways, which enables respiration of various electron acceptors including O 2 , Fe(III), Mn(IV), thiosulfate, elemental sulfur, and nitrate, contributes to the ability...