Surfactin, a lipopeptide produced by Bacillus subtilis, is one of the most powerful biosurfactants known. This molecule consists of a cyclic heptapeptide linked to a β-hydroxy fatty acid chain. The isomery and the length of the fatty acid (FA) chain are responsible for the surfactin's activities. In this study, the gene codY, which encode for the global transcriptional regulator and the gene lpdV, located in the bkd operon (lpdV, bkdAA, bkdAB and bkdB genes), which is responsible for the last step of the branched chain amino acid (BCAA) degradation in acyl-CoA were deleted. The influence of these deletions on the quantitative and qualitative surfactin production was analysed. The surfactin production was quantified by RP-HPLC and the surfactin isoforms were characterized using LC-MS-MS and GC-MS analysis. The results obtained in the mutants showed an enhancement of surfactin specific production by a factor of 5.8 for the codY mutant and 1.4 for lpdV mutant. Moreover qualitative analysis of the lpdV mutant reveals that it mainly produced surfactin C isoform (2 fold more than the wild type) with linear FA chain. Complete analysis of the extracellular metabolites using H quantitative NMR reveals a reduced production of acetoin in this mutant. This work demonstrates for the first time an original approach to overproduce specifically surfactin with C FA chain.

The peptidoglycan layer is responsible for maintaining bacterial cell shape and permitting cell division. Cell wall growth is facilitated by peptidoglycan synthases and hydrolases and is potentially modulated by components of the central carbon metabolism. In Bacillus subtilis, UgtP synthesises the glucolipid precursor for lipoteichoic acid and has been suggested to function as a metabolic sensor governing cell size. Here we show that ugtP mutant cells have increased levels of cell wall precursors and changes in their peptidoglycan that suggest elevated dl-endopeptidase activity. The additional deletion of lytE, encoding a dl-endopeptidase important for cell elongation, in the ugtP mutant background produced cells with severe shape defects. Interestingly, the ugtP lytE mutant recovered normal rod-shape by acquiring mutations that decreased the expression of the peptidoglycan synthase PBP1. Together our results suggest that cells lacking ugtP must re-adjust the balance between peptidoglycan synthesis and hydrolysis to maintain proper cell morphology.

Upon competence-inducing nutrient-limited conditions, only part of the Bacillus subtilis population becomes competent. Here, we separated the two subpopulations by fluorescence-assisted cell sorting (FACS). Using RNA-seq, we confirmed the previously described ComK regulon. We also found for the first time significantly downregulated genes in the competent subpopulation. The downregulated genes are not under direct control by ComK but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell division and replication are halted. By investigating the proteome during competence, we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post-transcriptional level. In the competent subpopulation, yhfW was newly identified as being highly upregulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK-binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ΔyhfW mutant. RNA-seq analysis of ΔyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC.

Pyruvate is a key intermediate of diverse metabolic pathways of central carbon metabolism. In addition to being the end product of glycolysis, pyruvate is an essential carbon distribution point to oxidative metabolism, amino acid and fatty acid syntheses, and overflow metabolite production. Hence, a tight regulation of pyruvate kinase (Pyk) activity is of great importance. This study aimed to analyze targeted metabolites from several pathways and possible changes in Bacillus subtilis lacking Pyk. Wild type and Δpyk cells were cultivated in chemically defined medium with glucose and pyruvate as carbon sources, and the extracted metabolites were analyzed by 1H-NMR, GC-MS, HPLC-MS, and LC-MS/MS. The results showed that the perturbation created in the pyruvate node drove an adaptation to new conditions by altering the nutritional compounds’ consumption. In Δpyk, pyruvate, which is subject to glucose-dependent carbon catabolite repression, did not comply with the hierarchy in carbon source utilization. Other metabolic alterations were observed such as the higher secretion of the overflow metabolites acetoin and 2,3-butanediol by Δpyk. Our results help to elucidate the regulatory transport of glucose and pyruvate in B. subtilis and possible metabolic reroute to alternative pathways in the absence of Pyk.

The conserved WhiA protein family is present in most Gram-positive bacteria and plays a role in cell division. WhiA contains a DNA-binding motive and has been identified as a transcription factor in actinomycetes. In Bacillus subtilis, the absence of WhiA influences cell division and chromosome segregation, however, it is still unclear how WhiA influences these processes, but the protein does not seem to function as transcription factor in this organism. To further investigate the function of WhiA in B. subtilis, we performed a yeast two-hybrid screen to find interaction partners, and a Hi-C experiment to reveal possible changes in chromosome conformation. The latter experiment indicated a reduction in short range chromosome interactions, but how this would affect either cell division or chromosome segregation is unclear. Based on adjacent genes, a role in carbon metabolism was put forward. To study this, we measured exometabolome fluxes during growth on different carbon sources. This revealed that in ΔwhiA cells the pool of branched-chain fatty acid precursors is lower. However, the effect on the membrane fatty acid composition was minimal. Transcriptome data could not link the metabolome effects to gene regulatory differences.