Genome annotation and access to information from large-scale experimental approaches at the genome level are essential to improve our understanding of living cells and organisms. This is even more the case for model organisms that are the basis to study pathogens and technologically important species. We have generated SubtiWiki, a database for the Gram-positive model bacterium Bacillus subtilis (http://subtiwiki.uni-goettingen.de/). In addition to the established companion modules of SubtiWiki, SubtiPathways and SubtInteract, we have now created SubtiExpress, a third module, to visualize genome scale transcription data that are of unprecedented quality and density. Today, SubtiWiki is one of the most complete collections of knowledge on a living organism in one single resource.

In most bacteria, fatty acid biosynthesis is an essential process that must be controlled by the availability of precursors and by the needs of cell division. So far, no mechanisms controlling synthesis of malonyl-coenzyme A (CoA), the committed step in fatty acid synthesis, have been identified in the Gram-positive model bacterium Bacillus subtilis. We have studied the localization and function of two highly expressed proteins of unknown function, YqhY and YloU. Both proteins are members of the conserved and widespread Asp23 family. While the deletion of yloU had no effect, loss of the yqhY gene induced the rapid acquisition of suppressor mutations. The vast majority of these mutations affect subunits of the acetyl-CoA carboxylase (ACCase) complex, the enzyme that catalyzes the formation of malonyl-CoA. Moreover, lack of yqhY is accompanied by the formation of lipophilic clusters in the polar regions of the cells indicating an increased activity of ACCase. Our results suggest that YqhY controls the activity of ACCase and that this control results in inhibition of ACCase activity. Hyperactivity of the enzyme complex in the absence of YqhY does then provoke mutations that cause reduced ACCase activity.

The threonine dehydratase IlvA is part of the isoleucine biosynthesis pathway in the Gram-positive model bacterium Bacillus subtilis. Consequently, deletion of ilvA causes isoleucine auxotrophy. It has been reported that ilvA pseudo-revertants having a derepressed hom-thrCB operon appear in the presence of threonine. Here we have characterized two classes of ilvA pseudo-revertants. In the first class the hom-thrCB operon was derepressed unmasking the threonine dehydratase activity of the threonine synthase ThrC. In the second class of mutants, threonine biosynthesis was more broadly affected. The first class of ilvA pseudo-revertants had a mutation in the Phom promoter (P*hom ), resulting in constitutive expression of the hom-thrCB operon. In the second class of ilvA pseudo-revertants, the thrR gene encoding a putative DNA-binding protein was inactivated, also resulting in constitutive expression of the hom-thrCB operon. Here we demonstrate that ThrR is indeed a DNA-binding transcription factor that regulates the hom-thrCB operon and the thrD aspartokinase gene. DNA binding assays uncovered the DNA-binding site of ThrR and revealed that the repressor competes with the RNA polymerase for DNA binding. This study also revealed that ThrR orthologs are ubiquitous in genomes from the Gram-positive phylum Firmicutes and in some Gram-negative bacteria.

of malonyl-CoA. This highly active compound could nonspecifically acylate many proteins of different pathways, leading to their inactivity and toxic effects for the cell. Although the precise role of YqhY remains elusive, the results of this work indicate a regulatory function in fatty acid synthesis. They provide possible ways how YqhY could be involved in this pathway and serve as a basis for future investigations.CoA to malonyl-CoA performed by the acetyl-CoA carboxylase (ACCase). This enzyme complex is built up by four subunits, AccA, AccB, AccC and AccD (further description below) (Cronan and Waldrop, 2002). Afterwards, the malonate group is transferred to the acyl carrier protein (ACP) by the malonyl transacylase FabD (Zhang and Rock, 2008). The produced malonyl-ACP is then formed to β-ketoacyl-ACP by FabH (Parsons and Rock, 2013). In Gram-negative bacteria FabH condenses acetyl-CoA with malonyl-ACP to produce straight-chain fatty acids. On the other hand, many Gram-positive bacteria like B. subtilis and S. aureus preferentially condense short-chain acyl-CoA with malonyl-ACP to create branched-chain fatty acids. B. subtilis possesses two FabH isozymes (FabHA and FabHB) that enable also the consumption of acetyl-CoA for the production of straightchain fatty acids (Choi et al., 2000). The differences in substrate specificity of FabH are determined by the hydrophobic binding pocket. Structural analyses revealed that in Escherichia coli FabH this pocket is only big enough to harbor acetyl-CoA or propionyl-CoA, whereas in S. aureus FabH is able to bind acyl-CoAs with up to five carbon atoms (Qiu et al., 2005). After the initiating step of FabH, β-ketoacyl-ACP is reduced by FabG in a NADPH dependent manner in the first reaction of the elongation cycle. The resulting product βhydroxyacyl-ACP is then dehydrated to trans-2-enoyl-ACP by FabA and FabZ in E. coli. Although both enzymes catalyze the same reaction, only FabA is able to perform the cistrans isomerase reaction needed to synthesize unsaturated fatty acids. Gram-positive ketoacyl-ACP synthase FabH condenses acyl-CoA with malonyl-ACP. The elongation cycle (blue) is a repeating process, in which FabG reduces β-ketoacyl-ACP in a first reaction. In the second step, the resulting β-hydroxyacyl-ACP is dehydrated to enoyl-ACP by FabZ. At the end, the enoyl-ACP reductases FabI or FabL complete the elongation. The resulting acyl-ACP is either condensed by FabF for a further round of elongation or it is used for phospholipid synthesis (green). PlsX and PlsY acylate glycerol-3-phosphate to 1acyl-glycerol-3-phosphate, to which another fatty acid is added to the 2-position by PlsC.There are two possibilities for the further utilization of the newly produced acyl-ACP.Either it is condensed by FabB or FabF for another elongation round or it has reached a sufficient chain length and is used for phospholipid formation. In the latter process, the peripheral membrane protein PlsX converts acyl-ACP to acyl-phosphate. This step is Gottesman, 2013). The AAA+ (ATPase associated wi...