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Biomedical subjects

Richard Losick

Publications and source records attributed to Richard Losick.

47 records · Page 3Linked to original sources
PubMed2003

Additional targets of the Bacillus subtilis global regulator CodY identified by chromatin immunoprecipitation and genome-wide transcript analysis.

Additional targets of CodY, a GTP-activated repressor of early stationary-phase genes in Bacillus subtilis, were identified by combining chromatin immunoprecipitation, DNA microarray hybridization, and gel mobility shift assays. The direct targets of CodY newly identified by this approach included regulatory genes for sporulation, genes that are likely to encode transporters for amino acids and sugars, and the genes for biosynthesis of branched-chain amino acids.

Bacillus subtilis↗
PubMed2002

RacA, a bacterial protein that anchors chromosomes to the cell poles.

Eukaryotic chromosomes are anchored to a spindle apparatus during mitosis, but no such structure is known during chromosome segregation in bacteria. When sister chromosomes are segregated during sporulation in Bacillus subtilis, the replication origin regions migrate to opposite poles of the cell. If and how origin regions are fastened at the poles has not been determined. Here we describe a developmental protein, RacA, that acts as a bridge between the origin region and the cell poles. We propose that RacA assembles into an adhesive patch at a centromere-like element near the origin, causing chromosomes to stick at the poles.

Bacillus subtilis↗
PubMed2002

Generating and exploiting polarity in bacteria.

Bacteria are often highly polarized, exhibiting specialized structures at or near the ends of the cell. Among such structures are actin-organizing centers, which mediate the movement of certain pathogenic bacteria within the cytoplasm of an animal host cell; organized arrays of membrane receptors, which govern chemosensory behavior in swimming bacteria; and asymmetrically positioned septa, which generate specialized progeny in differentiating bacteria. This polarization is orchestrated by complex and dynamic changes in the subcellular localization of signal transduction and cytoskeleton proteins as well as of specific regions of the chromosome. Recent work has provided information on how dynamic subcellular localization occurs and how it is exploited by the bacterial cell. The main task of a bacterial cell is to survive and duplicate itself. The bacterium must replicate its genetic material and divide at the correct site in the cell and at the correct time in the cell cycle with high precision. Each kind of bacterium also executes its own strategy to find nutrients in its habitat and to cope with conditions of stress from its environment. This involves moving toward food, adapting to environmental extremes, and, in many cases, entering and exploiting a eukaryotic host. These activities often involve processes that take place at or near the poles of the cell. Here we explore some of the schemes bacteria use to orchestrate dynamic changes at their poles and how these polar events execute cellular functions. In spite of their small size, bacteria have a remarkably complex internal organization and external architecture. Bacterial cells are inherently asymmetric, some more obviously so than others. The most easily recognized asymmetries involve surface structures, e.g., flagella, pili, and stalks that are preferentially assembled at one pole by many bacteria. "New" poles generated at the cell division plane differ from old poles from the previous round of cell division. Even in Escherichia coli, which is generally thought to be symmetrical, old poles are more static than new poles with respect to cell wall assembly (1), and they differ in the deposition of phospholipid domains (2). There are many instances of differential polar functions; among these is the preferential use of old poles when attaching to host cells as in the interaction of Bradyrhizobium with plant root hairs (3) or the polar pili-mediated attachment of the Pseudomonas aeruginosa pathogen to tracheal epithelia (4). An unusual polar organelle that mediates directed motility on solid surfaces is found in the nonpathogenic bacterium Myxococcus xanthus. The gliding motility of this bacterium is propelled by a nozzle-like structure that squirts a polysaccharide-containing slime from the pole of the cell (5). Interestingly, M. xanthus, which has nozzles at both poles, can reverse direction by closing one nozzle and opening the other in response to end-to-end interactions between cells.

Bacillus subtilis↗
PubMed2002

Does RNA polymerase help drive chromosome segregation in bacteria?

In contrast to eukaryotic cells, bacteria segregate their chromosomes without a conspicuous mitotic apparatus. Replication of bacterial chromosomes initiates bidirectionally from a single site (oriC), and visualization of the region of the chromosome containing oriC in living cells reveals that origins rapidly move apart toward opposite poles of the cell during the cell cycle. The motor that drives this poleward movement is unknown. An attractive candidate is RNA polymerase, which is a powerful and abundant molecular motor. If, as has been suggested for other macromolecular complexes, the movement of RNA polymerase is restricted in the cell, then transcription would translocate the DNA template, thereby providing the motive force to separate replicating chromosomes. A coordinated effect of many transcribing RNA polymerases could result from the widely conserved global bias of gene orientation away from oriC. By using fluorescence microscopy of living Bacillus subtilis cells, we demonstrate that an inhibitor of RNA polymerase acts to inhibit separation of newly duplicated DNAs near the origin of replication. We propose that the force exerted by RNA polymerase contributes to chromosome movement in bacteria, and that this force, coupled with the biased orientation of transcription units, helps to drive chromosome segregation.

Aminoglycosides↗
PubMed2002

A widely conserved bacterial cell division protein that promotes assembly of the tubulin-like protein FtsZ.

Cell division in bacteria is mediated by the tubulin-like protein FtsZ, which assembles into a structure known as the Z ring at the future site of cytokinesis. We report the discovery of a Z-ring-associated protein in Bacillus subtilis called ZapA. ZapA was found to colocalize with the Z ring in vivo and was capable of binding to FtsZ and stimulating the formation of higher-order assemblies of the cytokinetic protein in vitro. The absence of ZapA alone did not impair cell viability, but the absence of ZapA in combination with the absence of a second, dispensable division protein EzrA caused a severe block in cytokinesis. The absence of ZapA also caused lethality in cells producing lower than normal levels of FtsZ or lacking the division-site-selection protein DivIVA. Conversely, overproduction of ZapA reversed the toxicity of excess levels of the division inhibitor MinD. In toto, the evidence indicates that ZapA is part of the cytokinetic machinery of the cell and acts by promoting Z-ring formation. Finally, ZapA is widely conserved among bacteria with apparent orthologs in many species, including Escherichia coli, in which the orthologous protein exhibited a strikingly similar pattern of subcellular localization to that of ZapA. Members of the ZapA family of proteins are likely to be a common feature of the cytokinetic machinery in bacteria.

Amino Acid Sequence↗
PubMed2002

Asymmetric cell division in B. subtilis involves a spiral-like intermediate of the cytokinetic protein FtsZ.

A fundamental feature of development in the spore-forming bacterium Bacillus subtilis is the switch from medial to asymmetric division. The switch is brought about by a change in the location of the cytokinetic Z ring, which is composed of the tubulin-like protein FtsZ, from the cell middle to the poles during sporulation. We report that the medial Z ring is replaced by a spiral-like filament of FtsZ that grows along the long axis of the cell. We propose that the filament mediates the switch by redeploying FtsZ to the poles. Spiral formation and the switch to polar Z rings are largely caused by a sporulation-specific increase in transcription of the gene for FtsZ and activation of the gene for the FtsZ-associated protein SpoIIE.

Bacillus subtilis↗
PubMed2002

A sporulation membrane protein tethers the pro-sigmaK processing enzyme to its inhibitor and dictates its subcellular localization.

The developmental transcription factor sigmaK is derived from the inactive precursor protein pro-sigmaK by regulated proteolysis during the process of sporulation in the bacterium Bacillus subtilis. The putative pro-sigmaK processing enzyme SpoIVFB is a member of a family of membrane-embedded metalloproteases and is held inactive by two other integral membrane proteins, SpoIVFA and BofA. Herein we show that the processing enzyme and its two regulators exist in a multimeric complex that localizes to the membrane surrounding the developing spore (the forespore). We further show that one of the regulators, SpoIVFA, plays a central role in both the formation of this complex and its subcellular localization. Evidence is presented in support of a model in which SpoIVFA acts as a platform for bringing BofA and SpoIVFB together, whereby BofA inhibits pro-sigmaK processing until a signal has been received from the forespore.

Bacillus subtilis↗
PubMed2002

An investigation into the compartmentalization of the sporulation transcription factor sigmaE in Bacillus subtilis.

Sporulation in Bacillus subtilis involves the formation of a polar septum, which divides the sporangium into a mother cell and a forespore. The sigmaE factor, which is encoded within the spoIIG operon, is a cell-specific regulatory protein that directs gene transcription in the mother cell. SigmaE is synthesized as an inactive proprotein pro-sigmaE, which is converted to the mature factor by the putative processing enzyme SpoIIGA. Processing of pro-sigmaE does not commence until after asymmetric division when sigmaE is largely confined to the mother cell. Processing depends on the signalling protein SpoIIR, which delays proteolysis until after polar septation, but the mechanism by which sigmaE is confined to the mother cell is not understood. Previous work favoured a model in which pro-sigmaE localizes to the mother cell face of the polar septum, such that sigmaE would be selectively released into mother cell cytoplasm. Based on the use of green fluorescent protein (GFP) fusions, we now report that pro-sigmaE is distributed approximately uniformly along all membrane surfaces and is not confined to the mother- cell face of the septum. Rather, our results are consistent with a model in which preferential and persistent transcription of the spoIIG operon in the mother cell and degradation of sigmaE in the forespore contribute to the selective accumulation of sigmaE in the mother cell. Persistent transcription of spoIIG after polar septation also contributes to the proper timing of pro-sigmaE processing.

Artificial Gene Fusion↗
PubMed2002

FtsA mutants of Bacillus subtilis impaired in sporulation.

Spore formation in Bacillus subtilis involves a switch in the site of cell division from the midcell to a polar position. Both medial division and polar division are mediated in part by the actin-like, cytokinetic protein FtsA. We report the isolation of an FtsA mutant (FtsA(D265G)) that is defective in sporulation but is apparently unimpaired in vegetative growth. Sporulating cells of the mutant reach the stage of asymmetric division but are partially blocked in the subsequent morphological process of engulfment. As judged by fluorescence microscopy and electron microscopy, the FtsA(D265G) mutant produces normal-looking medial septa but immature (abnormally thin) polar septa. The mutant was unimpaired in transcription under the control of Spo0A, the master regulator for entry into sporulation, but was defective in transcription under the control of sigmaF, a regulatory protein whose activation is known to depend on polar division. An amino acid substitution at a residue (Y264) adjacent to D265 also caused a defect in sporulation. D265 and Y264 are conserved among endospore-forming bacteria, raising the possibility that these residues are involved in a sporulation-specific protein interaction that facilitates maturation of the sporulation septum and the activation of sigmaF.

Amino Acid Sequence↗
PubMed2002

Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis.

Sigma-H is an alternative RNA polymerase sigma factor that directs the transcription of many genes that function at the transition from exponential growth to stationary phase in Bacillus subtilis. Twenty-three promoters, which drive transcription of 33 genes, are known to be recognized by sigma-H-containing RNA polymerase. To identify additional genes under the control of sigma-H on a genome-wide basis, we carried out transcriptional profiling experiments using a DNA microarray containing >99% of the annotated B. subtilis open reading frames. In addition, we used a bioinformatics-based approach aimed at the identification of promoters recognized by RNA polymerase containing sigma-H. This combination of approaches was successful in confirming most of the previously described sigma-H-controlled genes. In addition, we identified 26 putative promoters that drive expression of 54 genes not previously known to be under the direct control of sigma-H. Based on the known or inferred function of most of these genes, we conclude that, in addition to its previously known roles in sporulation and competence, sigma-H controls genes involved in many physiological processes associated with the transition to stationary phase, including cytochrome biogenesis, generation of potential nutrient sources, transport, and cell wall metabolism.

Bacillus subtilis↗