It has long been hypothesized that subcellular positioning of chromosomal loci in bacteria may be influenced by gene function and expression state. Here we provide direct evidence that membrane protein expression affects the position of chromosomal loci in Escherichia coli. For two different membrane proteins, we observed a dramatic shift of their genetic loci toward the membrane upon induction. In related systems in which a cytoplasmic protein was produced, or translation was eliminated by mutating the start codon, a shift was not observed. Antibiotics that block transcription and translation similarly prevented locus repositioning toward the membrane. We also found that repositioning is relatively rapid and can be detected at positions that are a considerable distance on the chromosome from the gene encoding the membrane protein (>90 kb). Given that membrane protein-encoding genes are distributed throughout the chromosome, their expression may be an important mechanism for maintaining the bacterial chromosome in an expanded and dynamic state.bacterial nucleoid | chromosome-membrane association | transertion S tudies of several different bacteria have revealed that their chromosomes are organized structures, with genetic loci occupying relatively defined positions along the long axis of the cell (1-7). However, the potential impact of gene function and expression state on chromosome organization and on subcellular positioning of specific loci remains relatively unexplored. Such modulation of the spatial organization of the chromosome may affect numerous cellular processes, including the regulation of gene expression, gross transcriptional control, assembly of macromolecular complexes and domains, and the generation of cellular asymmetry (8)(9)(10)(11)(12)(13)(14).One long-standing hypothesis posits that genes actively expressing membrane proteins are localized to regions proximal to the membrane (8,11,15). To date, however, there is no direct evidence for membrane protein expression affecting the subcellular positions of chromosomal loci. We therefore sought to test directly whether expressing a membrane protein affects the localization of its encoding gene in Escherichia coli. We tested two loci and found that induction of membrane protein expression rapidly results in a dramatic repositioning toward the membrane. We also show that the positions of chromosomal loci as far away as 90 kb from the induced gene are affected. This shift in position is a significant perturbation on the scale of the cell and may therefore be a major determinant of chromosome conformation. ResultsWe first tested the effect of inducing the lac operon on the intracellular position of the chromosomal lac locus. The operon lacZYA encodes the membrane protein lactose permease (LacY), in addition to two cytoplasmic proteins, beta-galactosidase (LacZ) and galactoside acetyltransferase (LacA). To follow the intracellular position of lacZYA, we inserted an array of Tet repressor binding sites (tetO) in the gene cynX, which is adjacent to lacA ...

Most bacteria contain both eukaryotic-like Ser/Thr kinases (eSTKs) and eukaryotic-like Ser/Thr phosphatases (eSTPs). Their role in bacterial physiology is not currently well understood in large part because the conditions where the eSTKs are active are generally not known. However, all sequenced Gram-positive bacteria have a highly conserved eSTK with extracellular PASTA repeats that bind cell wall derived muropeptides. Here, we report that in the Gram-positive bacterium Bacillus subtilis, the PASTA-containing eSTK PrkC and its cognate eSTP PrpC converge with the essential WalRK two-component system to regulate WalR regulon genes involved in cell wall metabolism. By continuously monitoring gene expression throughout growth, we consistently find a large PrkC-dependent effect on expression of several different WalR regulon genes in early stationary phase, including both those that are activated by WalR (yocH) as well as those that are repressed (iseA, pdaC). We demonstrate that PrkC phosphorylates WalR in vitro and in vivo on a single Thr residue located in the receiver domain. Although the phosphorylated region of the receiver domain is highly conserved among several B. subtilis response regulators, PrkC displays specificity for WalR in vitro. Consistently, strains expressing a nonphosphorylatable WalR point mutant strongly reduce both PrkC dependent activation and repression of yocH, iseA, and pdaC. This suggests a model where the eSTK PrkC regulates the essential WalRK two-component signaling system by direct phosphorylation of WalR Thr101, resulting in the regulation of WalR regulon genes involved in cell wall metabolism in stationary phase. As both the eSTK PrkC and the essential WalRK two-component system are highly conserved in Gram-positive bacteria, these results may be applicable to further understanding the role of eSTKs in Gram-positive physiology and cell wall metabolism.

β-lactams represent one of the most important classes of antibiotics discovered to date. These agents block Lipid II processing and cell wall biosynthesis through inactivation of Penicillin-Binding Proteins (PBPs). PBPs enzymatically load cell wall building blocks from Lipid II carrier molecules onto the growing cell wall scaffold during growth and division. Lipid II, a bottleneck in cell wall biosynthesis, is the target of some of the most potent antibiotics in clinical use. Despite the immense therapeutic value of this biosynthetic pathway, the PBP-Lipid II association has not been established in live cells. To determine this key interaction, we designed an unnatural d-amino acid dipeptide that is metabolically incorporated into Lipid II molecules. By hijacking the peptidoglycan biosynthetic machinery, photoaffinity probes were installed in combination with click partners within Lipid II, thereby allowing, for the first time, demonstration of PBP interactions in vivo with Lipid II.

Isogenic populations of cells exhibit phenotypic variability that has specific physiological consequences. Individual bacteria within a population can differ in antibiotic tolerance, but whether this variability can be regulated or is generally an unavoidable consequence of stochastic fluctuations is unclear. Here we report that a gene encoding a bacterial (p)ppGpp synthetase in Bacillus subtilis, sasA, exhibits high levels of extrinsic noise in expression. We find that sasA is regulated by multisite phosphorylation of the transcription factor WalR, mediated by a Ser/Thr kinase-phosphatase pair PrkC/PrpC, and a Histidine kinase WalK of a two-component system. This regulatory intersection is crucial for controlling the appearance of outliers; rare cells with unusually high levels of sasA expression, having increased antibiotic tolerance. We create a predictive model demonstrating that the probability of a given cell surviving antibiotic treatment increases with sasA expression. Therefore, multisite phosphorylation can be used to strongly regulate variability in antibiotic tolerance.