RESUMO
Successful treatment of bacterial infections requires the timely administration of appropriate antimicrobial therapy. The failure to initiate the correct therapy in a timely fashion results in poor clinical outcomes, longer hospital stays, and higher medical costs. Current approaches to antibiotic susceptibility testing of cultured pathogens have key limitations ranging from long run times to dependence on prior knowledge of genetic mechanisms of resistance. We have developed a rapid antimicrobial susceptibility assay for Staphylococcus aureus based on bacterial cytological profiling (BCP), which uses quantitative fluorescence microscopy to measure antibiotic induced changes in cellular architecture. BCP discriminated between methicillin-susceptible (MSSA) and -resistant (MRSA) clinical isolates of S. aureus (n = 71) within 1-2 h with 100% accuracy. Similarly, BCP correctly distinguished daptomycin susceptible (DS) from daptomycin non-susceptible (DNS) S. aureus strains (n = 20) within 30 min. Among MRSA isolates, BCP further identified two classes of strains that differ in their susceptibility to specific combinations of beta-lactam antibiotics. BCP provides a rapid and flexible alternative to gene-based susceptibility testing methods for S. aureus, and should be readily adaptable to different antibiotics and bacterial species as new mechanisms of resistance or multidrug-resistant pathogens evolve and appear in mainstream clinical practice.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Farmacorresistência Bacteriana , Staphylococcus aureus Resistente à Meticilina/citologia , Antibacterianos/farmacologia , Daptomicina/farmacologia , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacosRESUMO
During the stage of engulfment in the Bacillus subtilis spore formation pathway, the larger mother cell engulfs the smaller forespore. We have tested the role of forespore-specific gene expression in engulfment using two separate approaches. First, using an assay that unambiguously detects sporangia that have completed engulfment, we found that a mutant lacking the only forespore-expressed engulfment protein identified thus far, SpoIIQ, is able to efficiently complete engulfment under certain sporulation conditions. However, we have found that the mutant is defective, under all conditions, in the expression of the late-forespore-specific transcription factor sigma(G); thus, SpoIIQ is essential for spore production. Second, to determine if engulfment could proceed in the absence of forespore-specific gene expression, we made use of a strain in which activation of the mother cell-specific sigma factor sigma(E) was uncoupled from forespore-specific gene expression. Remarkably, engulfment occurred in the complete absence of sigma(F)-directed gene expression under the same conditions permissive for engulfment in the absence of SpoIIQ. Our results demonstrate that forespore-specific gene expression is not essential for engulfment, suggesting that the machinery used to move the membranes around the forespore is within the mother cell.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Expressão Gênica , Genes Bacterianos , Fator sigma/genética , Fator sigma/metabolismo , Esporos Bacterianos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
A key step in the Bacillus subtilis spore formation pathway is the engulfment of the forespore by the mother cell, a phagocytosis-like process normally accompanied by the loss of peptidoglycan within the sporulation septum. We have reinvestigated the role of SpoIIB in engulfment by using the fluorescent membrane stain FM 4-64 and deconvolution microscopy. We have found that spoIIB mutant sporangia display a transient engulfment defect in which the forespore pushes through the septum and bulges into the mother cell, similar to the situation in spoIID, spoIIM, and spoIIP mutants. However, unlike the sporangia of those three mutants, spoIIB mutant sporangia are able to complete engulfment; indeed, by time-lapse microscopy, sporangia with prominent bulges were found to complete engulfment. Electron micrographs showed that in spoIIB mutant sporangia the dissolution of septal peptidoglycan is delayed and spatially unregulated and that the engulfing membranes migrate around the remaining septal peptidoglycan. These results demonstrate that mother cell membranes will move around septal peptidoglycan that has not been completely degraded and suggest that SpoIIB facilitates the rapid and spatially regulated dissolution of septal peptidoglycan. In keeping with this proposal, a SpoIIB-myc fusion protein localized to the sporulation septum during its biogenesis, discriminating between the site of active septal biogenesis and the unused potential division site within the same cell.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto , Regulação Bacteriana da Expressão Gênica , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , Western Blotting , Microscopia Eletrônica , Microscopia de Fluorescência , Microscopia de Vídeo/métodos , Peptidoglicano/metabolismo , Fenótipo , Esporos Bacterianos/fisiologia , Esporos Bacterianos/ultraestruturaRESUMO
Shortly after the synthesis of the two cells required for sporulation in Bacillus subtilis, the membranes of the larger mother cell begin to migrate around and engulf the smaller forespore cell. At the completion of this process the leading edges of the migrating membrane meet and fuse, releasing the forespore into the mother cell cytoplasm. We developed a fluorescent membrane stain-based assay for this membrane fusion event, and we isolated mutants defective in the final stages of engulfment or membrane fusion. All had defects in spoIIIE, which is required for translocation of the forespore chromosome across the polar septum. We isolated one spoIIIE mutant severely defective in chromosome translocation, but not in membrane fusion; this mutation disrupts the ATP/GTP-binding site of SpoIIIE, suggesting that ATP binding and hydrolysis are required for DNA translocation but not for the late engulfment function of SpoIIIE. We also correlated relocalization of SpoIIIE-green fluorescent protein from the sporulation septum to the forespore pole with the completion of membrane fusion and engulfment. We suggest that SpoIIIE is required for the final steps of engulfment and that it may regulate or catalyze membrane fusion events.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/fisiologia , Fusão de Membrana , Fator sigma , Fatores de Transcrição , Proteínas de Bactérias/química , DNA Bacteriano/metabolismo , Esporos Bacterianos/fisiologia , Translocação GenéticaRESUMO
Cell-specific activation of transcription factor sigmaF during sporulation in Bacillus subtilis requires the formation of the polar septum and the activity of a serine phosphatase (SpoIIE) located in the septum. The SpoIIE phosphatase indirectly activates sigmaF by dephosphorylating a protein (SpoIIAA-P) in the pathway that controls the activity of the transcription factor. By use of a SpoIIE-GFP fusion protein in time-course and time-lapse experiments and by direct visualization of septa in living cells, we show that SpoIIE is present in the predivisional sporangium, where it often localizes near both cell poles in structures known as E-rings. We also present evidence consistent with the view that SpoIIE is present in both progeny cells after polar division. These findings are incompatible with a model for the control of sigmaF activity in which the phosphatase is simply sequestered to one cell. Instead, we conclude that the function of SpoIIE is subject to regulation, and we present evidence that this occurs in two stages. The first stage, which involves the phosphatase function of SpoIIE, depends on the cell division protein FtsZ and could correspond to the FtsZ-dependent assembly of SpoIIE into E-rings. The second stage occurs after the dephosphorylation of SpoIIAA-P and is dependent on the later-acting, cell-division protein DivIC. Evidence based on the use of modified and mutant forms of the phosphatase protein indicates that SpoIIE blocks the capacity of unphosphorylated SpoIIAA to activate sigmaF until formation of the polar septum is completed.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto , Fator sigma/metabolismo , Fatores de Transcrição , Bacillus subtilis/citologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Divisão Celular , Polaridade Celular , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Fosforilação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Esporos Bacterianos/fisiologiaRESUMO
At the onset of sporulation in Bacillus subtilis, two potential division sites are assembled at each pole, one of which will be used to synthesize the asymmetrically positioned sporulation septum. Using the vital stain FM 4-64 to label the plasma membrane of living cells, we examined the fate of these potential division sites in wild-type cells and found that, immediately after the formation of the sporulation septum, a partial septum was frequently synthesized within the mother cell at the second potential division site. Using time-lapse deconvolution microscopy, we were able to watch these partial septa first appear and then disappear during sporulation. Septal dissolution was dependent on sigma E activity and was partially inhibited in mutants lacking the sigma E-controlled proteins SpoIID, SpoIIM and SpoIIP, which may play a role in mediating the degradation of septal peptidoglycan. Our results support a model in which sigma E inhibits division at the second potential division site by two distinct mechanisms: inhibition of septal biogenesis and the degradation of partial septa formed before sigma E activation.
Assuntos
Bacillus subtilis/fisiologia , Membrana Celular/metabolismo , Corantes Fluorescentes/metabolismo , Regulação Bacteriana da Expressão Gênica , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo Celular , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Fotomicrografia , Fator sigma/metabolismo , Esporos Bacterianos/ultraestrutura , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas do Citoesqueleto , Proteínas de Escherichia coli , Escherichia coli/citologia , Escherichia coli/genética , Proteínas Quinases/genética , Proteínas de Bactérias/análise , Divisão Celular , Deleção Cromossômica , Cromossomos Bacterianos , Escherichia coli/ultraestrutura , Proteínas de Ligação ao GTP/biossíntese , Genótipo , Microscopia Eletrônica , Óperon , Transdução de SinaisRESUMO
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.
Assuntos
Proteínas de Transporte , Proteínas do Citoesqueleto , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Hexosiltransferases/metabolismo , Complexos Multienzimáticos/metabolismo , Muramilpentapeptídeo Carboxipeptidase , Peptidoglicano Glicosiltransferase , Peptidil Transferases/metabolismo , Fosfatase Alcalina/metabolismo , Especificidade de Anticorpos , Proteínas de Bactérias/genética , Western Blotting , Divisão Celular/genética , Clonagem Molecular , Escherichia coli/citologia , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Genótipo , Hexosiltransferases/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Complexos Multienzimáticos/imunologia , Proteínas de Ligação às Penicilinas , Peptidil Transferases/imunologiaRESUMO
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.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Fosfoproteínas Fosfatases/genética , Fator sigma/genética , Fatores de Transcrição/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Compartimento Celular/genética , Mutagênese Insercional , Fosfoproteínas Fosfatases/metabolismo , Fator sigma/fisiologia , Esporos Bacterianos/enzimologia , Esporos Bacterianos/genética , Fatores de Transcrição/fisiologiaRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte , Divisão Celular , Proteínas do Citoesqueleto , Escherichia coli/citologia , Hexosiltransferases/metabolismo , Complexos Multienzimáticos/metabolismo , Muramilpentapeptídeo Carboxipeptidase , Peptidil Transferases/metabolismo , Cefalexina/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Hexosiltransferases/antagonistas & inibidores , Substâncias Macromoleculares , Complexos Multienzimáticos/antagonistas & inibidores , Proteínas de Ligação às Penicilinas , Peptidil Transferases/antagonistas & inibidores , Piperacilina/farmacologia , Ligação ProteicaRESUMO
We describe the application of immunofluorescence microscopy to visualization of the subcellular localization of proteins involved in coat morphogenesis and chromosome packaging during the process of sporulation in Bacillus subtilis. In confirmation and extension of previous findings, we show that SpolVA, which is responsible for guiding coat formation to the surface of the outer membrane that surrounds the developing spore, assembles into a shell that is located close to or on the surface of this enveloping membrane. CotE, which is responsible for the formation of the outer layer of the coat, assembles into a second shell of apparently larger diameter. Assembly of SpolVA could be detected as early as the morphological stage of polar septation and closely followed the enveloping membrane of the mother cell during the stage of engulfment, thereby providing a sensitive and diagnostic marker for this phagocytic-like process. Surprisingly, the chromosome of the developing spore and the small, acid-soluble proteins, known as alpha/beta-type SASPs, that are known to coat the spore chromosome, were found to co-localize to a doughnut-like ring of approximately 1 micrometer in diameter. The use of a double mutant lacking the alpha/beta-type SASP demonstrated that these high abundance, DNA-binding proteins are responsible for packaging the chromosome of the developing spore into this unusual structure. We conclude that sporulation in B. subtilis is a fertile system for addressing cell biological problems in a bacterium and that immunofluorescence microscopy provides a sensitive method for visualizing protein subcellular localization at high resolution.
Assuntos
Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Microscopia de Fluorescência , Fator sigma , Esporos Bacterianos , Fatores de Transcrição , Bacillus subtilis/química , Bacillus subtilis/fisiologia , Compartimento Celular , Estrutura Molecular , beta-Galactosidase/metabolismoRESUMO
Asymmetric division in Bacillus subtilis generates progeny cells with dissimilar fates. SpoIIE, a membrane protein required for the establishment of cell type, was shown to localize near sites of potential polar division. SpoIIE initially localizes in a bipolar pattern, coalescing at marks in the cell envelope at which asymmetric division can take place. Then, during division, SpoIIE becomes restricted to the polar septum and is lost from the distal pole. Thus, when division is complete, SpoIIE sits at the boundary between the progeny from which it dictates cell fate by the activation of a cell-specific transcription factor.
Assuntos
Bacillus subtilis/química , Bacillus subtilis/fisiologia , Proteínas de Bactérias/análise , Divisão Celular , Esporos Bacterianos/química , Fatores de Transcrição , Bacillus subtilis/citologia , Bacillus subtilis/genética , Proteínas de Bactérias/fisiologia , Membrana Celular/química , Expressão Gênica , Microscopia de Fluorescência , Mutação , Proteínas Recombinantes de Fusão/análise , Fator sigma/fisiologiaRESUMO
We have adapted immunofluorescence microscopy for use in Bacillus subtilis and have employed this procedure for visualizing cell-specific gene expression at early to intermediate stages of sporulation. Sporangia were doubly stained with propidium iodide to visualize the forespore and mother cell nucleoids and with fluorescein-conjugated antibodies to visualize the location of beta-galactosidase produced under the control of the sporulation RNA polymerase sigma factors sigma E and sigma F. In confirmation and extension of earlier reports, we found that expression of a lacZ fusion under the control of sigma E was confined to the mother cell compartment of sporangia at the septation (II) and engulfment (III) stages of morphogenesis. Conversely, sigma F-directed gene expression was confined to the forespore compartment of sporangia at postseptation stages of development. Little indication was found for sigma E- or sigma F-directed gene expression prior to septation or in both compartments of postseptation sporangia. Gene expression under the control of the forespore sigma factor sigma G also exhibited a high level of compartmentalization. A high proportion of sporangia exhibited fluorescence in our immunostaining protocol, which should be suitable for the subcellular localization of sporulation proteins for which specific antibodies are available.
Assuntos
Bacillus subtilis/fisiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Clonagem Molecular , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Microscopia de Contraste de Fase , Fator sigma/fisiologia , Esporos Bacterianos/metabolismo , beta-Galactosidase/biossínteseRESUMO
The secD operon of Escherichia coli is required for the efficient export of proteins. We have characterized this operon, and found that, in addition to secD and secF, it contains the upstream gene yajC, but not the genes queA or tgt, in contrast to previous reports. An analysis of yajC mutations constructed in vitro and recombined onto the chromosome indicates that yajC is neither essential nor a sec gene. The secD operon is not induced in response to either secretion defects or temperature changes. TnphoA fusions have been used to analyze the topology of SecD in the inner membrane; the protein contains six transmembrane stretches and a large periplasmic domain. TnphoA fusions to SecD and SecF have also been recombined onto the chromosome and used to determine the level of these proteins within the cell. Our results indicate that there are fewer than 30 SecD and SecF molecules per cell.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Proteínas de Membrana Transportadoras , Sequência de Aminoácidos , Sequência de Bases , Expressão Gênica , Ligação de Hidrogênio , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutagênese Insercional , Conformação de Ácido Nucleico , Sondas de Oligonucleotídeos/química , Óperon , RNA Bacteriano/genética , RNA Mensageiro/genética , TemperaturaRESUMO
We have found that temperature can have a striking effect upon protein export in Escherichia coli, suggesting that there is a cold-sensitive step in the protein export pathway. Cs mutations comprise the largest class of mutations affecting the membrane-localized Sec proteins SecD, SecE, SecF and SecY. Although some of these mutations could encode cold-labile proteins, this is unlikely to account for the Cs phenotype of most export mutants, as mutations which simply produce lower amounts of SecE protein have the same phenotype. Certain signal sequence mutations affecting maltose binding protein are also cold sensitive for export. These effects appear to arise by a specific interaction of cold with certain export defects. We believe that the Cs sec mutations are representative of a large class of conditional lethal mutations, whose conditional phenotype reflects an underlying thermal sensitivity of the process in which they are involved.