Changing views on the nature of the bacterial cell: from biochemistry to cytology.
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Bacteria↗
Biomedical subjects
R Losick
Publications and source records attributed to R Losick.
A four-dimensional view of assembly of a morphogenetic protein during sporulation in Bacillus subtilis.
We report the use of a fusion to the green fluorescent protein to visualize the assembly of the morphogenetic protein SpoIVA around the developing forespore during the process of sporulation in the bacterium Bacillus subtilis. Using a deconvolution algorithm to process digitally-collected optical sections, we show that SpoIVA, which is synthesized in the mother cell chamber of the sporangium, assembled into a spherical shell around the outer surface of the forespore. Time-lapse fluorescence microscopy showed that this assembly process commenced at the time of polar division and seemed to continue after engulfment of the forespore was complete. SpoIVA remained present throughout the late stages of morphogenesis and was present as a component of the fully mature spore. Evidence indicates that assembly of SpoIVA depended on the extreme C-terminal region of the protein and an additional region that directly or indirectly facilitated interaction among SpoIVA molecules. The N- and C-terminal regions of SpoIVA, including the extreme C terminus, are highly similar to the corresponding regions of the homologous protein from the distantly related endospore-forming bacterium Clostridium acetobutylicum, attesting to their importance in the function of the protein. Finally, we show that proper localization of SpoIVA required the expression of one or more genes which, like spoIVA, are under the control of the mother cell transcription factor sigmaE. One such gene was spoVM, whose product was required for efficient targeting of SpoIVA to the outer surface of the forespore.
Amino Acid Sequence↗
Evidence for common sites of contact between the antisigma factor SpoIIAB and its partners SpoIIAA and the developmental transcription factor sigmaF in Bacillus subtilis.
The activity of the developmental transcription factor sigmaF in Bacillus subtilis is governed by a switch involving the dual function protein SpoIIAB. SpoIIAB is an antisigma factor that forms complexes with sigmaF and with an alternative partner protein SpoIIAA. SpoIIAB is also a protein kinase that can inactivate SpoIIAA by phosphorylating it on a serine residue. We sought to identify amino acids in SpoIIAB that are involved in the formation of the SpoIIAB-SpoIIAA complex by screening for mutants that were defective in the activation of sigmaF. This genetic screen, in combination with biochemical analysis and the construction of loss-of-side-chain (alanine substitution) mutants, led to the identification of amino acid side-chains in the N-terminal region of SpoIIAB that could contact SpoIIAA. Unexpectedly, the same amino acid side-chains (R20 and N50) that appear to touch SpoIIAA are required for binding to, and may represent sites of contact with, sigmaF. We propose that the N-terminal region of SpoIIAB forms a binding surface that is responsible for the formation of both the SpoIIAB-SpoIIAA and the SpoIIAB-sigmaF complexes, and that in some cases the same amino acid side-chains contact both partner proteins. N50 is also the defining residue of a region of amino acid sequence homology known as the N-box that is shared by SpoIIAB and related serine protein kinases, as well as by members of a mechanistically dissimilar family of protein kinases that undergo autophosphorylation at a histidine residue. We discuss the implications of this finding for the mechanism of histidine autophosphorylation.
Alanine↗
The kinase activity of the antisigma factor SpoIIAB is required for activation as well as inhibition of transcription factor sigmaF during sporulation in Bacillus subtilis.
The activity of the developmental transcription factor sigmaF in the spore-forming bacterium Bacillus subtilis is controlled by SpoIIAB, which sequesters sigmaF in an inactive complex. sigmaF is released from the SpoIIAB-sigmaF complex by the action of SpoIIAA, which triggers the dissociation of the complex. SpoIIAB is also a protein kinase that phosphorylates SpoIIAA on serine residue 58 (S58). This phosphorylation inactivates SpoIIAA and thus indirectly prevents the activation of sigmaF. Here, we report the identification of a patch of amino acid residues located in the vicinity of the adenosine nucleotide binding pocket of SpoIIAB that is required for the phosphorylation of SpoIIAA. A lysine substitution (E104K) at one of these residues (Glu104) markedly impaired the capacity of SpoIIAB to phosphorylate SpoIIAA in vitro as well as during sporulation. Kinetic analysis and evidence from the construction of alanine substitution mutants indicates that the side-chains of these amino acids could be contact sites for the SpoIIAA substrate during the phosphorylation reaction. Importantly, E104K and other kinase mutants blocked the activation of sigmaF during sporulation. This is paradoxical, because a mutant of SpoIIAA (S58A) that cannot be phosphorylated is known to cause higher than normal levels of sigmaF activity during sporulation. In resolution of this paradox, we present biochemical evidence indicating that SpoIIAA directly attacks the SpoIIAB-sigmaF complex and that SpoIIAA is phosphorylated as a result of this reaction. Consistent with this idea, mutations impairing kinase function of SpoIIAB were found to be epistatic to a mutation causing the S58A substitution in SpoIIAA; that is, cells producing mutant forms of both proteins were blocked in the activation of sigmaF. We conclude that phosphorylation of SpoIIAA plays a dual role in the sigmaF pathway, and that the kinase function of SpoIIAB is required for the activation as well as the inhibition of sigmaF during sporulation.
Alanine↗
DNA replication. Bringing the mountain to Mohammed.
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Bacillus subtilis↗
Chromosome arrangement within a bacterium.
BACKGROUND: The contour length of the circular chromosome of bacteria is greater than a millimeter but must be accommodated within a cell that is only a few micrometers in length. Bacteria do not have nucleosomes and little is known about the arrangement of the chromosome inside a prokaryotic cell. RESULTS: We have investigated the arrangement of chromosomal DNA within the bacterium Bacillus subtilis by using fluorescence microscopy to visualize two sites on the chromosome simultaneously in the same cell. Indirect immunofluorescence with antibodies against the chromosome partition protein Spo0J were used to visualize the replication origin region of the chromosome. Green fluorescent protein fused to the lactose operon repressor Lacl was used to decorate tandem copies of the lactose operon operator lacO. A cassette of tandem operators was separately inserted into the chromosome near the origin (359 degrees), near the replication terminus (181 degrees), or at two points in between (90 degrees and 270 degrees). The results show that the layout of the chromosome is dynamic but is principally arranged with the origin and terminus maximally apart and the quarter points of the chromosome in between. CONCLUSIONS: The use of cytological methods to visualize two chromosomal sites in the same cell has provided a glimpse of the arrangement of a bacterial chromosome. We conclude that, to a first approximation, the folding of the bacterial chromosome is consistent with, and may preserve, the linear order of genes on the DNA.
Bacillus subtilis↗
One for all and all for one.
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4-Butyrolactone↗
Negative regulation of the proteolytic activation of a developmental transcription factor in Bacillus subtilis.
The sporulation transcription factor sigmaK of Bacillus subtilis is controlled by a signal transduction pathway that operates at the level of the proteolytic processing of the inactive precursor protein pro-sigmaK. The conversion of pro-sigmaK to sigmaK requires the putative processing enzyme SpoIVFB and is governed by the regulatory proteins SpoIVFA and BofA. We engineered vegetative cells to carry out processing of pro-sigmaK by inducing the synthesis of the proprotein, a modified form of the putative processing enzyme, and its two regulators during growth. The results showed that (i) modified SpoIVFB was the only sporulation protein necessary to achieve processing of pro-sigmaK; (ii) SpoIVFA stimulated processing, apparently by protecting the processing enzyme from degradation; (iii) BofA inhibited processing in a manner that did not involve degradation of SpoIVFB; and (iv) the inhibition of SpoIVFB by BofA was dependent on SpoIVFA. We conclude that BofA and SpoIVFA act synergistically and are the only two sporulation proteins needed to inhibit the function of SpoIVFB. Our results are consistent with the idea that activation of pro-sigmaK occurs by a reversal of the BofA/SpoIVFA-mediated inhibition of the processing enzyme.
Bacillus subtilis↗
Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis.
We describe the use of time-lapse fluorescence microscopy to visualize the movement of the DNA replication origin and terminus regions on the Bacillus subtilis chromosome during the course of the cell cycle. The origin and terminus regions were tagged with a cassette of tandem lac operator repeats and visualized through the use of a fusion of the green fluorescent protein to the LacI repressor. We have discovered that origin regions abruptly move apart towards the cell poles during a brief interval of the cell cycle. This movement was also seen in the absence of cell wall growth and in the absence of the product of the parB homologue spo0J. The origin regions moved apart an average distance of 1.4 microm in an 11 min period of abrupt movement, representing an average velocity of 0.17 microm min(-1), and reaching a maximum velocity of greater than 0.27 microm min(-1). The terminus region also exhibited a striking pattern of movement but not as far or a rapid as the origin region. These results provide evidence for a mitotic-like motor that is responsible for segregation of the origin regions of the chromosomes.
Bacillus subtilis↗
Summary: three decades after sigma.
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Animals↗
Aberrant cell division and random FtsZ ring positioning in Escherichia coli cpxA* mutants.
In Escherichia coli, certain mutations in the cpxA gene (encoding a sensor kinase of a two-component signal transduction system) randomize the location of FtsZ ring assembly and dramatically affect cell division. However, deletion of the cpxRA operon, encoding the sensor kinase and its cognate regulator CpxR, has no effect on division site biogenesis. It appears that certain mutant sensor kinases (CpxA*) either exhibit hyperactivity on CpxR or extend their signalling activity to one or more noncognate response regulators involved in cell division.
Bacterial Proteins↗
Subcellular localization of Bacillus subtilis SMC, a protein involved in chromosome condensation and segregation.
We have investigated the subcellular localization of the SMC protein in the gram-positive bacterium Bacillus subtilis. Recent work has shown that SMC is required for chromosome condensation and faithful chromosome segregation during the B. subtilis cell cycle. Using antibodies against SMC and fluorescence microscopy, we have shown that SMC is associated with the chromosome but is also present in discrete foci near the poles of the cell. DNase treatment of permeabilized cells disrupted the association of SMC with the chromosome but not with the polar foci. The use of a truncated smc gene demonstrated that the C-terminal domain of the protein is required for chromosomal binding but not for the formation of polar foci. Regular arrays of SMC-containing foci were still present between nucleoids along the length of aseptate filaments generated by depleting cells of the cell division protein FtsZ, indicating that the formation of polar foci does not require the formation of septal structures. In slowly growing cells, which have only one or two chromosomes, SMC foci were principally observed early in the cell cycle, prior to or coincident with chromosome segregation. Cell cycle-dependent release of stored SMC from polar foci may mediate segregation by condensation of chromosomes.
Animals↗
Purification of an extracellular signaling molecule involved in production of aerial mycelium by Streptomyces coelicolor.
We have extensively purified a factor from conditioned medium that restores aerial mycelium formation to a mutant of Streptomyces coelicolor that is defective in morphological differentiation. Response to this factor is shown to depend on the presence of the BldK oligopeptide import system. We suggest that this substance acts at the first step in a putative cascade of developmental regulatory signals.
Biological Transport↗
Panspermia, spores and the Bacillus subtilis genome.
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Bacillus subtilis↗
Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechanisms.
We have investigated DNA segregation in E. coli by inserting multiple lac operator sequences into the chromosome near the origin of replication (oriC), in the hisC gene, a terminus marker, and into plasmids P1 and F. Expression of a GFP-LacI fusion protein allowed visualization of lac operator localization. oriC was shown to be specifically localized at or near the cell poles, and when duplicated, one copy moved to the site of new pole formation near the site of cell division. In contrast, P1 and F localized to the cell center and on duplication appeared to move rapidly to the quarter positions in the cell. Our analysis suggests that different active processes are involved in movement and localization of the chromosome and of the two plasmids during segregation.
Cell Cycle↗
Protein localization and cell fate in bacteria.
A major breakthrough in understanding the bacterial cell is the discovery that the cell is highly organized at the level of protein localization. Proteins are positioned at particular sites in bacteria, including the cell pole, the incipient division plane, and the septum. Differential protein localization can control DNA replication, chromosome segregation, and cytokinesis and is responsible for generating daughter cells with different fates upon cell division. Recent discoveries have revealed that progression through the cell cycle and communication between cellular compartments are mediated by two-component signal transduction systems and signaling pathways involving transcription factor activation by proteolytic processing. Asymmetric cell division in Caulobacter crescentus and sporulation in Bacillus subtilis are used as paradigms for the control of the cell cycle and cellular morphogenesis in bacterial cells.
Bacillus subtilis↗
Bipolar localization of the replication origin regions of chromosomes in vegetative and sporulating cells of B. subtilis.
To investigate chromosome segregation in B. subtilis, we introduced tandem copies of the lactose operon operator into the chromosome near the replication origin or terminus. We then visualized the position of the operator cassettes with green fluorescent protein fused to the Lac1 repressor. In sporulating bacteria, which undergo asymmetric cell division, origins localized near each pole of the cell whereas termini were restricted to the middle. In growing cells, which undergo binary fission, origins were observed at various positions but preferentially toward the poles early in the cell cycle. In contrast, termini showed little preference for the poles. These results indicate the existence of a mitotic-like apparatus that is responsible for moving the origin regions of newly formed chromosomes toward opposite ends of the cell.
Bacillus subtilis↗
Inactivation of FtsI inhibits constriction of the FtsZ cytokinetic ring and delays the assembly of FtsZ rings at potential division sites.
A universally conserved event in cell division is the formation of a cytokinetic ring at the future site of division. In the bacterium Escherichia coli, this ring is formed by the essential cell division protein FtsZ. We have used immunofluorescence microscopy to show that FtsZ assembles early in the division cycle, suggesting that constriction of the FtsZ ring is regulated and supporting the view that FtsZ serves as a bacterial cytoskeleton. Assembly of FtsZ rings was heterogeneously affected in an ftsI temperature-sensitive mutant grown at the nonpermissive temperature, some filaments displaying a striking defect in FtsZ assembly and others displaying little or no defect. By using low concentrations of the beta-lactams cephalexin and piperacillin to specifically inhibit FtsI (PBP3), an enzyme that synthesizes peptidoglycan at the division septum, we show that FtsZ ring constriction requires the transpeptidase activity of FtsI. Unconstricted FtsZ rings are stably trapped at the midpoint of the cell for several generations after inactivation of FtsI, whereas partially constricted FtsZ rings are less effectively trapped. In addition, FtsZ rings are able to assemble in newborn cells in the presence of cephalexin, suggesting that newborn cells contain a site at which FtsZ can assemble (the nascent division site) and that the transpeptidase activity of FtsI is not required for assembly of FtsZ at these sites. However, aside from this first round of FtsZ ring assembly, very few additional FtsZ rings assemble in the presence of cephalexin, even after several generations of growth. One interpretation of these results is that the transpeptidase activity of FtsI is required, directly or indirectly, for the assembly of nascent division sites and thereby for future assembly of FtsZ rings.
Bacterial Proteins↗