Why and how bacteria communicate.
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Bacterial Physiological Phenomena↗
Biomedical subjects
R Losick
Publications and source records attributed to R Losick.
Localization of the Escherichia coli cell division protein Ftsl (PBP3) to the division site and cell pole.
FtsI, also known as penicillin-binding protein 3, is a transpeptidase required for the synthesis of peptidoglycan in the division septum of the bacterium, Escherichia coli. FtsI has been estimated to be present at about 100 molecules per cell, well below the detection limit of immunoelectron microscopy. Here, we confirm the low abundance of FtsI and use immunofluorescence microscopy, a highly sensitive technique, to show that FtsI is localized to the division site during the later stages of cell growth. FtsI was also sometimes observed at the cell pole; polar localization was not anticipated and its significance is not known. We conclude (i) that immunofluorescence microscopy can be used to localize proteins whose abundance is as low as approximately 100 molecules per cell; and (ii) that spatial and temporal regulation of FtsI activity in septum formation is achieved, at least in part, by timed localization of the protein to the division site.
Alkaline Phosphatase↗
Localization of the sporulation protein SpoIIE in Bacillus subtilis is dependent upon the cell division protein FtsZ.
SpollE is an integral membrane protein that governs the establishment of cell-specific gene transcription during the process of sporulation in Bacillus subtilis. Synthesis of SpollE commences shortly after the onset of sporulation, after which the protein localizes at sites of potential cell division near both ends of the sporangium. We now show that, within the limits of resolution of immunofluorescence microscopy, this bipolar pattern of localization observed in early-sporulating cells was superimposable with the bipolar pattern of localization of the cell division protein FtsZ. The localization of SpollE was dependent upon FtsZ because little or no localization was observed along the length of filaments that were generated by depleting sporulating cells for the cell division protein. In contrast, SpollE and FtsZ were found to co-localize at regularly spaced intervals in filaments generated by the use of a temperature-sensitive mutant of the cell division gene divlC. Finally, in cells engineered to synthesize SpollE during growth, SpollE localized at the mid-cell position, coincident with the position of FtsZ, which exhibits a medial pattern of localization in cells undergoing binary fission. These results suggest that the bipolar pattern of localization of SpollE is dictated by the sporulation-induced switch in the position of FtsZ or of other, FtsZ-associated, cell division proteins. Thus, it appears that B. subtilis has co-opted the cell division machinery as a means of localizing a cell fate determinant to the polar septum during sporulation.
Bacillus subtilis↗
Assembly of the cell division protein FtsZ into ladder-like structures in the aerial hyphae of Streptomyces coelicolor.
In the filamentous bacterium Streptomyces coelicolor, the cell division protein FtsZ is required for the conversion of multinucleoidal aerial hyphae into chains of uninucleoidal spores, although it is not essential for viability. Using immunofluorescence microscopy, we have shown that FtsZ assembles into long, regularly spaced, ladder-like arrays in developing aerial hyphae, with an average spacing of about 1.3 microm. Within individual hyphae, ladder formation was relatively synchronous and extended for distances over 100 microm. These ladders were present only transiently, decreasing in intensity as chromosomes separated into distinct nucleoids and disappearing upon the completion of septum formation. Evidence from the overall intensity of immunofluorescence staining suggested that ladder formation was regulated in part at the level of the accumulation and degradation of FtsZ within individual aerial hyphae. Finally, FtsZ ladder formation was under developmental control in that long arrays of FtsZ rings could not be detected in certain so-called white mutants (whiG, whiH and whiB), which are blocked in spore formation. The assembly of FtsZ into ladders represents the earliest known molecular manifestation of the process of spore formation, and its discovery provides insight into the role of whi genes in the conversion of aerial hyphae into chains of spores. We have also described a novel use of a cell wall-staining technique to visualize apical tip growth in vegetatively growing hyphae.
Antibodies, Bacterial↗
Disappearance of the sigma E transcription factor from the forespore and the SpoIIE phosphatase from the mother cell contributes to establishment of cell-specific gene expression during sporulation in Bacillus subtilis.
We used immunofluorescence microscopy to investigate mechanisms governing the establishment of cell-specific gene transcription during sporulation in the bacterium Bacillus subtilis. The transcription factors sigma E and sigma F are synthesized shortly after the start of sporulation but do not become active in directing gene transcription until after polar division, when the activity of sigma E is confined to the mother cell and the activity of sigma F is restricted to the forespore. We show that shortly after septation, sigma E and its proprotein precursor pro-sigma E appear to be absent from the forespore and that a null mutation in spoIIIE, a gene known to be required for the translocation of a chromosome into the forespore, allows sigma E and/or pro-sigma E to persist and sigma E to become active in the forespore. These findings suggest that the loss of sigma E/pro-sigma E from the forespore contributes to the compartmentalization of sigma E-directed gene transcription. We also investigated the distribution of SpoIIE, a regulatory phosphatase required for the activation of sigma F which exhibits a bipolar pattern of localization shortly after the start of sporulation. Normally, SpoIIE rapidly disappears from the sporangium, first from the mother-cell pole and then from the forespore pole. Here we show that a null mutation in spoIIIE causes the SpoIIE phosphatase to persist at both poles. The persistence of the SpoIIE phosphatase at the mother-cell pole could explain the lack of compartmentalization of sigma F activity observed in a spoIIIE null mutant. We conclude that the establishment of cell-specific gene transcription involves the loss of sigma E/pro-sigma E from the forespore and the loss of the SpoIIE phosphatase from the mother-cell pole and that both processes are dependent upon the SpoIIIE protein.
Bacillus subtilis↗
A Bacillus subtilis gene encoding a protein similar to nucleotide sugar transferases influences cell shape and viability.
Bacillus subtilis gene ypfP, which is located at 196 degrees on the genetic map, shows similarity to both the monogalactosyldiacylglycerol synthase gene of Cucumis sativus, which encodes a galactosyltransferase, and the murG genes of B. subtilis, Escherichia coli, Haemophilus influenzae, and Synechocystis sp. strain PCC6803, which encode N-acetylglucosaminyltransferases involved in peptidoglycan biosynthesis. Cells containing a null mutation of ypfP are shorter and rounder than wild-type cells during growth in Luria-Bertani medium and glucose minimal medium. In addition, the mutant cells preferentially undergo lysis when grown on solid Luria-Bertani medium.
Acyltransferases↗
Translation of the mRNA for the sporulation gene spoIIID of Bacillus subtilis is dependent upon translation of a small upstream open reading frame.
We report the existence of a small open reading frame (usd) that is located between the promoter and coding sequence for the sporulation gene spoIIID in Bacillus subtilis. The mRNA from the usd-spoIIID operon contains an inverted repeat sequence that is predicted to form a stem-loop structure that would sequester the ribosome binding site for spoIIID. A mutation eliminating the ribosome binding site for the upstream open reading frame caused an oligosporogenous phenotype and interfered with the translation, but not the transcription, of the downstream gene spoIIID. We propose that efficient synthesis of SpoIIID requires that the putative stem-loop structure be disrupted by translation through the upstream open reading frame.
Amino Acid Sequence↗
Establishment of cell type in a primitive organism by cell-specific elimination and proteolytic activation of a transcription factor.
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Bacillus subtilis↗
Three sites of contact between the Bacillus subtilis transcription factor sigmaF and its antisigma factor SpoIIAB.
The developmental regulatory protein sigmaF of Bacillus subtilis, a member of the sigma70-family of RNA polymerase sigma factors, is regulated negatively by the antisigma factor SpoIIAB, which binds to sigmaF to form an inactive complex. Complex formation between SpoIIAB, which contains an inferred adenosine nucleotide binding pocket, and sigmaF is stimulated strongly by the presence of ATP. Here we report that SpoIIAB contacts sigmaF at three widely spaced binding surfaces corresponding to conserved regions 2.1, 3.1, and 4.1 of sigma70-like sigma factors. This conclusion is based on binding studies between SpoIIAB and truncated portions of sigmaF, the isolation of mutants of sigmaF that were partially resistant to inhibition by SpoIIAB in vivo and were defective in binding to the antisigma factor in vitro, and the creation of alanine substitution mutants of regions 2.1, 3.1, or 4.1 of sigmaF that were impaired in complex formation. Because the interaction of SpoIIAB with all three binding surfaces was stimulated by ATP, we infer that ATP induces a conformational change in SpoIIAB that is needed for tight binding to sigmaF. Finally, we discuss the possibility that another antisigma factor, unrelated to SpoIIAB, may interact with its respective sigma factor in a similar topological pattern of widely spaced binding surfaces located in or near conserved regions 2.1, 3.1, and 4.1.
Adenosine Triphosphate↗
SpoIIAA governs the release of the cell-type specific transcription factor sigma F from its anti-sigma factor SpoIIAB.
The Bacillus subtilis transcription factor sigma F is a cell-type specific regulatory protein whose activity is governed by SpoIIAB and SpoIIAA and the nucleotides ATP and ADP. SpoIIAB is an anti-sigma factor that binds to sigma F in a manner that is stimulated by ATP, thereby trapping sigma F in an inactive complex. Alternatively, SpoIIAB binds to SpoIIAA in a manner that is stimulated by ADP to form a SpoIIAB.SpoIIAA complex in which SpoIIAB is sequestered from sigma F. SpoIIAB is also a protein kinase that uses ATP to phosphorylate, and thereby inactivate, SpoIIAA. Thus, ATP inhibits sigma F activity both by promoting formation of the SpoIIAB.sigma F complex and by phosphorylation of SpoIIAA. In extension of previous results, we use affinity chromatography to show that SpoIIAB is capable of forming long-lived complexes with sigma F and SpoIIAA and that the formation of these complexes is dependent on ATP and ADP, respectively. Using a DNA template lacking adenosine residues on the non-transcribed strand, we demonstrate that ATP is required for SpoIIAB-mediated inhibition of sigma F-directed RNA synthesis and that this inhibition is prevented by SpoIIAA in a manner that is stimulated by ADP. We show that ADP acts by protecting SpoIIAA from phosphorylation by SpoIIAB and that a mutant protein bearing an amino acid substitution at the site of phosphorylation in SpoIIAA is capable of preventing the inhibition of sigma F in a manner that does not depend on ADP. A principal finding from the investigation is that SpoIIAA restores activity to sigma F that had previously been inhibited by SpoIIAB. This is demonstrated both by the capacity of SpoIIAA to reverse SpoIIAB-mediated inhibition of sigma F-directed RNA synthesis and by its capacity to interact with and disrupt the SpoIIAB. sigma F complex. The results are consistent with a model in which sigma F is controlled by the cellular concentration of unphosphorylated SpoIIAA.
Adenosine Diphosphate↗
Role of adenosine nucleotides in the regulation of a stress-response transcription factor in Bacillus subtilis.
The RNA polymerase sigma factor sigma B is a stress-response regulatory protein in Bacillus subtilis. The activity of sigma B is controlled in part by RsbW, a protein that inhibits sigma B, and RsbV, a protein that counteracts this inhibition. We now demonstrate that purified RsbW is capable of forming alternative complexes with either sigma B or RsbV. Sigma B in the RsbW. sigma B complex was transcriptionally inactive. RsbV reversed this inhibition by sequestering RsbW in a RsbW-RsbV complex, thereby allowing sigma B to remain free and active. In contrast to interactions among the components of the homologous regulatory system for the sporulation transcription factor sigma F, the binding of RsbW to RsbV and sigma B did not require adenosine nucleotides. Experiments involving the exchange of proteins between the two regulatory systems demonstrated that RsbW and its homolog in the sigma F system, SpoIIAB, exhibit strong preference in binding to RsbV and sigma B, and SpoIIAA and sigma F, respectively, and that the difference in nucleotide-dependence of binding between these two systems is attributable to a difference between RsbW and SpoIIAB. In confirmation and extension of previous results, we show that RsbW is also a protein kinase that uses ATP to phosphorylate RsbV, thereby blocking the capacity of RsbV to bind to RsbW and activate transcription. A close correlation was observed between the concentration of ATP required for efficient RsbW-mediated phosphorylation of RsbV, inhibition of RsbW.RsbV comlex formation, and inhibition of sigma B-directed transcription. These results are consistent with the hypothesis that activation of sigma B under certain stress condition is due to a decrease in cellular ATP levels.
Adenosine Diphosphate↗
SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis.
Cell-specific activation of the transcription factor sigma F during sporulation in Bacillus subtilis is controlled by a regulatory pathway involving the proteins SpoIIE, SpoIIAA, and SpoIIAB. SpoIIAB is an antagonist of sigma F, and SpoIIAA, which is capable of overcoming SpoIIAB-mediated inhibition of sigma F, is an antagonist of SpoIIAB. SpoIIAA is, in turn, negatively regulated by SpoIIAB, which phosphorylates SpoIIAA on serine 58. SpoIIAA is also positively regulated by SpoIIE, which dephosphorylates SpoIIAA-P, the phosphorylated form of SpoIIAA. Here, isoelectric focusing and Western blot analysis were used to examine the phosphorylation state of SpoIIAA in vivo. SpoIIAA was found to be largely in the phosphorylated state during sporulation in wild-type cells but a significant portion of the protein that was unphosphorylated could also be detected. Consistent with the idea that SpoIIE governs dephosphorylation of SpoIIAA-P, SpoIIAA was entirely in the phosphorylated state in spoIIE mutant cells. Conversely, overexpression of spoIIE led to an increase in the ratio of unphosphorylated SpoIIAA to SpoIIAA-P and caused inappropriate activation of sigma F in the predivisional sporangium. We also show that a mutant form of SpoIIAA (SpoIIAA-S58T) in which serine 58 was replaced with threonine was present exclusively as SpoIIAA-P, a finding that confirms previous biochemical evidence that the mutant protein is an effective substrate for the SpoIIAB kinase but that SpoIIAA-S58T-P cannot be dephosphorylated by SpoIIE. We conclude that SpoIIE plays a crucial role in controlling the phosphorylation state of SpoIIAA during sporulation and thus in governing the cell-specific activation of sigma F.
Bacillus subtilis↗
Transcription factor Spo0A switches the localization of the cell division protein FtsZ from a medial to a bipolar pattern in Bacillus subtilis.
Entry into sporulation by the Gram-positive bacterium Bacillus subtilis is governed by two transcription factors, Spo0A and sigma H, and involves a switch in the site of division from a medial to a polar location. We report that at the onset of sporulation, assembly of the cell division protein FtsZ shifts from midcell to potential division sites near both poles. The switch to a bipolar pattern of FtsZ localization is dependent on Spo0A. Additionally, synthesis of an activated form of Spo0A during growth artificially activates the switch in FtsZ localization and results in the formation of polar septa. The sigma H factor, on the other hand, is dispensable for the switch in the position of the FtsZ assembly site, although it is required for formation of the polar septum. Our results suggest that during the transition from growth to sporulation, Spo0A induces the expression of genes that suppress FtsZ assembly at the midcell site and activate sites at both poles, whereas sigma H induces genes required for a subsequent step in cytokinesis.
Bacillus subtilis↗
Subcellular localization of proteins governing the proteolytic activation of a developmental transcription factor in Bacillus subtilis.
BACKGROUND: Spore formation in Bacillus subtilis takes place in a sporangium consisting of two compartments called the forespore and the mother cell. Late in development, when the forespore is wholly contained within the mother cell, gene transcription is coordinated between the compartments by an intercellular signal transduction pathway. This pathway operates at the level of proteolytic processing of the proprotein precursor (pro-sigma K to the mother-cell transcription factor sigma K. The conversion of pro-sigma K to sigma K is governed by the putative processing enzyme SpoIVFB and its negative regulator SpoIVFA, which are produced in the mother cell. RESULTS: We used fluorescence microscopy in conjunction with antibodies against SpoIVFA and SpoIVFB and a fusion of SpoIVFB to the Green Fluorescent Protein from Aquorea victoria to visualize these proteins in the sporangium. Both proteins were found to co-localize with the forespore region of the sporangium, a finding consistent with the idea that SpoIVFA and SpoIVFB, which are inferred to be integral membrane proteins, are located in the mother cell membrane that surrounds the forespore. CONCLUSIONS: We conclude that SpoIVFA and SpoIVFB are situated at the boundary between the forespore and the mother cell, at which location SpoIVFB could be activated by a signalling protein produced in the forespore.
Antibodies, Bacterial↗
An oligopeptide permease responsible for the import of an extracellular signal governing aerial mycelium formation in Streptomyces coelicolor.
Morphological differentiation in the filamentous bacterium Streptomyces coelicolor is believed to involve a mechanism of extracellular signalling that culminates with the formation of an aerial mycelium. We have identified a gene cluster designated bldK in which insertional and deletion mutations cause a block in aerial mycelium formation. Extracellular complementation experiments indicate that bldK defines a step in a cascade of extracellular signals; colonies of a bldK-mutant strain extracellularly complement bld261-mutant colonies, and are themselves extracellularly complemented by bldA- and bldH-mutant colonies. The bldK locus, which is located at 5 o'clock on the genetic map and within Asel fragment "N' on the physical map, consists of five adjacent open reading frames. These genes specify homologues of the subunits of the oligopeptide-permease family of ATP-binding cassette (ABC) membrane-spanning transporters. Because bldK mutations confer resistance to the toxic tripeptide bialaphos, it is inferred that BldK is an oligopeptide importer. We propose that the BldK transporter is responsible for the import of an extracellular signalling molecule produced under the control of the wild-type product of the bld261 gene. The BldK-imported signal, in turn, causes the production of a second extracellular signal molecule that depends on the products of bldA and bldH for its action.
ATP-Binding Cassette Transporters↗
Identification of additional genes under the control of the transcription factor sigma F of Bacillus subtilis.
We describe the identification of five transcriptional units under the control of the sporulation transcription factor sigma F in Bacillus subtilis. These are csfA, csfB, csfC, csfD, and csfF, located at approximately 230 degrees, 2 degrees, 316 degrees, 205 degrees, and approximately 290 degrees, respectively, on the genetic map. Null mutations in csfA, csfB, csfC, or csfD, either alone or together, do not cause a noticeable defect in sporulation or germination.
Bacillus subtilis↗
Cell division gene ftsQ is required for efficient sporulation but not growth and viability in Streptomyces coelicolor A3(2).
We show that the cell division gene ftsQ of Streptomyces coelicolor A3(2) is dispensable for growth and viability but is needed during development for the efficient conversion of aerial filaments into spores. Combined with our previous demonstration that ftsZ of S. coelicolor is not needed for viability, these findings suggest that cell division has been largely co-opted for development in this filamentous bacterium. This makes S. coelicolor an advantageous system for the study of cell division genes.
Amino Acid Sequence↗
Molecular genetics of sporulation in Bacillus subtilis.
The process of sporulation in the bacterium Bacillus subtilis proceeds through a well-defined series of morphological stages that involve the conversion of a growing cell into a two-cell-chamber sporangium within which a spore is produced. Over 125 genes are involved in this process, the transcription of which is temporally and spatially controlled by four DNA-binding proteins and five RNA polymerase sigma factors. Through a combination of genetic, biochemical, and cell biological approaches, regulatory networks have been elucidated that explicitly link the activation of these sigma factors to landmark events in the course of morphogenesis and to each other through pathways of intercellular communication. Signals targeting proteins to specific subcellular localizations and governing the assembly of macromolecular structures have been uncovered but their nature remains to be determined.
Bacillus subtilis↗