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1.
mBio ; : e0131124, 2024 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-39287436

RESUMEN

Bacterial shape and division rely on the dynamics of cell wall assembly, which involves regulated synthesis and cleavage of the peptidoglycan. In ovococci, these processes are coordinated within an annular mid-cell region with nanometric dimensions. More precisely, the cross-wall synthesized by the divisome is split to generate a lateral wall, whose expansion is insured by the insertion of the so-called peripheral peptidoglycan by the elongasome. Septum cleavage and peripheral peptidoglycan synthesis are, thus, crucial remodeling events for ovococcal cell division and elongation. The structural DivIVA protein has long been known as a major regulator of these processes, but its mode of action remains unknown. Here, we integrate click chemistry-based peptidoglycan labeling, direct stochastic optical reconstruction microscopy, and in silico modeling, as well as epifluorescence and stimulated emission depletion microscopy to investigate the role of DivIVA in Streptococcus pneumoniae cell morphogenesis. Our work reveals two distinct phases of peptidoglycan remodeling during the cell cycle that are differentially controlled by DivIVA. In particular, we show that DivIVA ensures homogeneous septum cleavage and peripheral peptidoglycan synthesis around the division site and their maintenance throughout the cell cycle. Our data additionally suggest that DivIVA impacts the contribution of the elongasome and class A penicillin-binding proteins to cell elongation. We also report the position of DivIVA on either side of the septum, consistent with its known affinity for negatively curved membranes. Finally, we take the opportunity provided by these new observations to propose hypotheses for the mechanism of action of this key morphogenetic protein.IMPORTANCEThis study sheds light on fundamental processes governing bacterial growth and division, using integrated click chemistry, advanced microscopy, and computational modeling approaches. It addresses cell wall synthesis mechanisms in the opportunistic human pathogen Streptococcus pneumoniae, responsible for a range of illnesses (otitis, pneumonia, meningitis, septicemia) and for one million deaths every year worldwide. This bacterium belongs to the morphological group of ovococci, which includes many streptococcal and enterococcal pathogens. In this study, we have dissected the function of DivIVA, which is a structural protein involved in cell division, morphogenesis, and chromosome partitioning in Gram-positive bacteria. This work unveils the role of DivIVA in the orchestration of cell division and elongation along the pneumococcal cell cycle. It not only enhances our understanding of how ovoid bacteria proliferate but also offers the opportunity to consider how DivIVA might serve as a scaffold and sensor for particular membrane regions, thereby participating in various cell cycle processes.

2.
Nat Commun ; 15(1): 5625, 2024 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-38987237

RESUMEN

Competence for natural transformation is a central driver of genetic diversity in bacteria. In the human pathogen Streptococcus pneumoniae, competence exhibits a populational character mediated by the stress-induced ComABCDE quorum-sensing (QS) system. Here, we explore how this cell-to-cell communication mechanism proceeds and the functional properties acquired by competent cells grown under lethal stress. We show that populational competence development depends on self-induced cells stochastically emerging in response to stresses, including antibiotics. Competence then propagates through the population from a low threshold density of self-induced cells, defining a biphasic Self-Induction and Propagation (SI&P) QS mechanism. We also reveal that a competent population displays either increased sensitivity or improved tolerance to lethal doses of antibiotics, dependent in the latter case on the competence-induced ComM division inhibitor. Remarkably, these surviving competent cells also display an altered transformation potential. Thus, the unveiled SI&P QS mechanism shapes pneumococcal competence as a health sensor of the clonal population, promoting a bet-hedging strategy that both responds to and drives cells towards heterogeneity.


Asunto(s)
Antibacterianos , Proteínas Bacterianas , Percepción de Quorum , Streptococcus pneumoniae , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/fisiología , Antibacterianos/farmacología , Percepción de Quorum/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Transformación Bacteriana
3.
iScience ; 26(9): 107563, 2023 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-37664601

RESUMEN

In a scenario where the discovery of new molecules to fight antibiotic resistance is a public health concern, ribosomally synthesized and post-translationally modified peptides constitute a promising alternative. In this context, the Gram-positive human gut symbiont Ruminococcus gnavus E1 produces five sactipeptides, Ruminococcins C1 to C5 (RumC1-C5), co-expressed with two radical SAM maturases. RumC1 has been shown to be effective against various multidrug resistant Gram-positives clinical isolates. Here, after adapting the biosynthesis protocol to obtain the four mature RumC2-5 we then evaluate their antibacterial activities. Establishing first that both maturases exhibit substrate tolerance, we then observed a variation in the antibacterial efficacy between the five isoforms. We established that all RumCs are safe for humans with interesting multifunctionalities. While no synergies where observed for the five RumCs, we found a synergistic action with conventional antibiotics targeting the cell wall. Finally, we identified crucial residues for antibacterial activity of RumC isoforms.

4.
Annu Rev Microbiol ; 76: 533-552, 2022 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-35671533

RESUMEN

RNA degradosomes are multienzyme complexes composed of ribonucleases, RNA helicases, and metabolic enzymes. RNase E-based degradosomes are widespread in Proteobacteria. The Escherichia coli RNA degradosome is sequestered from transcription in the nucleoid and translation in the cytoplasm by localization to the inner cytoplasmic membrane, where it forms short-lived clusters that are proposed to be sites of mRNA degradation. In Caulobacter crescentus, RNA degradosomes localize to ribonucleoprotein condensates in the interior of the cell [bacterial ribonucleoprotein-bodies (BR-bodies)], which have been proposed to drive the concerted degradation of mRNA to nucleotides. The turnover of mRNA in growing cells is important for maintaining pools of nucleotides for transcription and DNA replication.Membrane attachment of the E. coli RNA degradosome is necessary to avoid wasteful degradation of intermediates in ribosome assembly. Sequestering RNA degradosomes to C. crescentus BR-bodies, which exclude structured RNA, could have a similar role in protecting intermediates in ribosome assembly from degradation.


Asunto(s)
Caulobacter crescentus , Endorribonucleasas , Escherichia coli , Complejos Multienzimáticos , Nucleótidos , Polirribonucleótido Nucleotidiltransferasa , ARN Helicasas , Estabilidad del ARN , ARN Mensajero , Caulobacter crescentus/enzimología , Caulobacter crescentus/genética , Endorribonucleasas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Nucleótidos/metabolismo , Polirribonucleótido Nucleotidiltransferasa/genética , Polirribonucleótido Nucleotidiltransferasa/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo
5.
Elife ; 92020 09 23.
Artículo en Inglés | MEDLINE | ID: mdl-32965219

RESUMEN

The spread of antimicrobial resistance and vaccine escape in the human pathogen Streptococcus pneumoniae can be largely attributed to competence-induced transformation. Here, we studied this process at the single-cell level. We show that within isogenic populations, all cells become naturally competent and bind exogenous DNA. We find that transformation is highly efficient and that the chromosomal location of the integration site or whether the transformed gene is encoded on the leading or lagging strand has limited influence on recombination efficiency. Indeed, we have observed multiple recombination events in single recipients in real-time. However, because of saturation and because a single-stranded donor DNA replaces the original allele, transformation efficiency has an upper threshold of approximately 50% of the population. The fixed mechanism of transformation results in a fail-safe strategy for the population as half of the population generally keeps an intact copy of the original genome.


Asunto(s)
Recombinación Homóloga , Streptococcus pneumoniae/genética , Farmacorresistencia Bacteriana/genética , Análisis de la Célula Individual
6.
Genes (Basel) ; 11(6)2020 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-32575751

RESUMEN

Natural genetic transformation is a programmed mechanism of horizontal gene transfer in bacteria. It requires the development of competence, a specialized physiological state during which proteins involved in DNA uptake and chromosomal integration are produced. In Streptococcus pneumoniae, competence is transient. It is controlled by a secreted peptide pheromone, the competence-stimulating peptide (CSP) that triggers the sequential transcription of two sets of genes termed early and late competence genes, respectively. Here, we used a microfluidic system with fluorescence microscopy to monitor pneumococcal competence development and transformation, in live cells at the single cell level. We present the conditions to grow this microaerophilic bacterium under continuous flow, with a similar doubling time as in batch liquid culture. We show that perfusion of CSP in the microfluidic chamber results in the same reduction of the growth rate of individual cells as observed in competent pneumococcal cultures. We also describe newly designed fluorescent reporters to distinguish the expression of competence genes with temporally distinct expression profiles. Finally, we exploit the microfluidic technology to inject both CSP and transforming DNA in the microfluidic channels and perform near real time-tracking of transformation in live cells. We show that this approach is well suited to investigating the onset of pneumococcal competence together with the appearance and the fate of transformants in individual cells.


Asunto(s)
Proteínas Bacterianas/genética , Transferencia de Gen Horizontal/genética , Infecciones Neumocócicas/genética , Streptococcus pneumoniae/genética , Cromosomas/genética , Competencia de la Transformación por ADN/genética , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica/genética , Microfluídica/métodos , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/patogenicidad , Transformación Bacteriana/genética
7.
Methods Mol Biol ; 1968: 63-78, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30929206

RESUMEN

The ability of Streptococcus pneumoniae (the pneumococcus) to transform is particularly convenient for genome engineering. Several protocols relying on sequential positive and negative selection strategies have been described to create directed markerless modifications, including deletions, insertions, or point mutations. Transformation with DNA fragments carrying long flanking homology sequences is also used to generate mutations without selection but it requires high transformability. Here, we present an optimized version of this method. As an example, we construct a strain harboring a translational fusion ftsZ-mTurquoise at the ftsZ locus. We provide instructions to produce a linear DNA fragment containing the chimeric construction and give details of the conditions to obtain optimal pneumococcal transformation efficiencies.


Asunto(s)
Cromosomas Bacterianos/genética , ADN Bacteriano/genética , Streptococcus pneumoniae/genética , Mutagénesis Insercional , Mutación/genética , Recombinación Genética/genética
8.
PLoS Genet ; 14(11): e1007753, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30403663

RESUMEN

During the morphological process of sporulation in Bacillus subtilis two adjacent daughter cells (called the mother cell and forespore) follow different programs of gene expression that are linked to each other by signal transduction pathways. At a late stage in development, a signaling pathway emanating from the forespore triggers the proteolytic activation of the mother cell transcription factor σK. Cleavage of pro-σK to its mature and active form is catalyzed by the intramembrane cleaving metalloprotease SpoIVFB (B), a Site-2 Protease (S2P) family member. B is held inactive by two mother-cell membrane proteins SpoIVFA (A) and BofA. Activation of pro-σK processing requires a site-1 signaling protease SpoIVB (IVB) that is secreted from the forespore into the space between the two cells. IVB cleaves the extracellular domain of A but how this cleavage activates intramembrane proteolysis has remained unclear. Structural studies of the Methanocaldococcus jannaschii S2P homolog identified closed (substrate-occluded) and open (substrate-accessible) conformations of the protease, but the biological relevance of these conformations has not been established. Here, using co-immunoprecipitation and fluorescence microscopy, we show that stable association between the membrane-embedded protease and its substrate requires IVB signaling. We further show that the cytoplasmic cystathionine-ß-synthase (CBS) domain of the B protease is not critical for this interaction or for pro-σK processing, suggesting the IVB-dependent interaction site is in the membrane protease domain. Finally, we provide evidence that the B protease domain adopts both open and closed conformations in vivo. Collectively, our data support a substrate-gating model in which IVB-dependent cleavage of A on one side of the membrane triggers a conformational change in the membrane-embedded protease from a closed to an open state allowing pro-σK access to the caged interior of the protease.


Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Expresión Génica , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Conformación Proteica , Estabilidad Proteica , Transporte de Proteínas , Proteolisis , Esporas
9.
Nat Commun ; 8(1): 1621, 2017 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-29158515

RESUMEN

Competence for genetic transformation is a differentiation program during which exogenous DNA is imported into the cell and integrated into the chromosome. In Streptococcus pneumoniae, competence develops transiently and synchronously in all cells during exponential phase, and is accompanied by a pause in growth. Here, we reveal that this pause is linked to the cell cycle. At least two parallel pathways impair peptidoglycan synthesis in competent cells. Single-cell analyses demonstrate that ComM, a membrane protein induced during competence, inhibits both initiation of cell division and final constriction of the cytokinetic ring. Competence also interferes with the activity of the serine/threonine kinase StkP, the central regulator of pneumococcal cell division. We further present evidence that the ComM-mediated delay in division preserves genomic integrity during transformation. We propose that cell division arrest is programmed in competent pneumococcal cells to ensure that transformation is complete before resumption of cell division, to provide this pathogen with the maximum potential for genetic diversity and adaptation.


Asunto(s)
Genoma Bacteriano , Streptococcus pneumoniae/citología , Streptococcus pneumoniae/genética , Transformación Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , División Celular , Streptococcus pneumoniae/metabolismo
10.
Mol Microbiol ; 106(5): 832-846, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28960579

RESUMEN

The peptidoglycan is a rigid matrix required to resist turgor pressure and to maintain the cellular shape. It is formed by linear glycan chains composed of N-acetylmuramic acid-(ß-1,4)-N-acetylglucosamine (MurNAc-GlcNAc) disaccharides associated through cross-linked peptide stems. The peptidoglycan is continually remodelled by synthetic and hydrolytic enzymes and by chemical modifications, including O-acetylation of MurNAc residues that occurs in most Gram-positive and Gram-negative bacteria. This modification is a powerful strategy developed by pathogens to resist to lysozyme degradation and thus to escape from the host innate immune system but little is known about its physiological function. In this study, we have investigated to what extend peptidoglycan O-acetylation is involved in cell wall biosynthesis and cell division of Streptococcus pneumoniae. We show that O-acetylation driven by Adr protects the peptidoglycan of dividing cells from cleavage by the major autolysin LytA and occurs at the septal site. Our results support a function for Adr in the formation of robust and mature MurNAc O-acetylated peptidoglycan and infer its role in the division of the pneumococcus.


Asunto(s)
Pared Celular/metabolismo , Peptidoglicano/metabolismo , Streptococcus pneumoniae/metabolismo , Acetilación , Acetilglucosamina/metabolismo , División Celular , Bacterias Gramnegativas/metabolismo , Ácidos Murámicos/metabolismo , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo
11.
PLoS Genet ; 10(4): e1004275, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24722178

RESUMEN

Despite years of intensive research, much remains to be discovered to understand the regulatory networks coordinating bacterial cell growth and division. The mechanisms by which Streptococcus pneumoniae achieves its characteristic ellipsoid-cell shape remain largely unknown. In this study, we analyzed the interplay of the cell division paralogs DivIVA and GpsB with the ser/thr kinase StkP. We observed that the deletion of divIVA hindered cell elongation and resulted in cell shortening and rounding. By contrast, the absence of GpsB resulted in hampered cell division and triggered cell elongation. Remarkably, ΔgpsB elongated cells exhibited a helical FtsZ pattern instead of a Z-ring, accompanied by helical patterns for DivIVA and peptidoglycan synthesis. Strikingly, divIVA deletion suppressed the elongated phenotype of ΔgpsB cells. These data suggest that DivIVA promotes cell elongation and that GpsB counteracts it. Analysis of protein-protein interactions revealed that GpsB and DivIVA do not interact with FtsZ but with the cell division protein EzrA, which itself interacts with FtsZ. In addition, GpsB interacts directly with DivIVA. These results are consistent with DivIVA and GpsB acting as a molecular switch to orchestrate peripheral and septal PG synthesis and connecting them with the Z-ring via EzrA. The cellular co-localization of the transpeptidases PBP2x and PBP2b as well as the lipid-flippases FtsW and RodA in ΔgpsB cells further suggest the existence of a single large PG assembly complex. Finally, we show that GpsB is required for septal localization and kinase activity of StkP, and therefore for StkP-dependent phosphorylation of DivIVA. Altogether, we propose that the StkP/DivIVA/GpsB triad finely tunes the two modes of peptidoglycan (peripheral and septal) synthesis responsible for the pneumococcal ellipsoid cell shape.


Asunto(s)
División Celular/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Streptococcus pneumoniae/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , División Celular/genética , Pared Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Morfogénesis/fisiología , Peptidoglicano/metabolismo , Fosforilación/genética , Fosforilación/fisiología , Mapas de Interacción de Proteínas/fisiología , Streptococcus pneumoniae/genética
12.
Trends Microbiol ; 22(3): 113-9, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24508048

RESUMEN

Streptococcus pneumoniae (the pneumococcus) is an important human pathogen. Natural genetic transformation, which was discovered in this species, involves internalization of exogenous single-stranded DNA and its incorporation into the chromosome. It allows acquisition of pathogenicity islands and antibiotic resistance and promotes vaccine escape via capsule switching. This opinion article discusses how recent advances regarding several facets of pneumococcal transformation support the view that the process has evolved to maximize plasticity potential in this species, making the pneumococcus le transformiste of the bacterial kingdom and providing an advantage in the constant struggle between this pathogen and its host.


Asunto(s)
Transferencia de Gen Horizontal , Streptococcus pneumoniae/genética , Transformación Bacteriana , Adaptación Biológica
13.
PLoS Pathog ; 9(9): e1003596, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24039578

RESUMEN

Genetic transformation, in which cells internalize exogenous DNA and integrate it into their chromosome, is widespread in the bacterial kingdom. It involves a specialized membrane-associated machinery for binding double-stranded (ds) DNA and uptake of single-stranded (ss) fragments. In the human pathogen Streptococcus pneumoniae, this machinery is specifically assembled at competence. The EndA nuclease, a constitutively expressed virulence factor, is recruited during competence to play the key role of converting dsDNA into ssDNA for uptake. Here we use fluorescence microscopy to show that EndA is uniformly distributed in the membrane of noncompetent cells and relocalizes at midcell during competence. This recruitment requires the dsDNA receptor ComEA. We also show that under 'static' binding conditions, i.e., in cells impaired for uptake, EndA and ComEA colocalize at midcell, together with fluorescent end-labelled dsDNA (Cy3-dsDNA). We conclude that midcell clustering of EndA reflects its recruitment to the DNA uptake machinery rather than its sequestration away from this machinery to protect transforming DNA from extensive degradation. In contrast, a fraction of ComEA molecules were located at cell poles post-competence, suggesting the pole as the site of degradation of the dsDNA receptor. In uptake-proficient cells, we used Cy3-dsDNA molecules enabling expression of a GFP fusion upon chromosomal integration to identify transformed cells as GFP producers 60-70 min after initial contact between DNA and competent cells. Recording of images since initial cell-DNA contact allowed us to look back to the uptake period for these transformed cells. Cy3-DNA foci were thus detected at the cell surface 10-11 min post-initial contact, all exclusively found at midcell, strongly suggesting that active uptake of transforming DNA takes place at this position in pneumococci. We discuss how midcell uptake could influence homology search, and the likelihood that midcell uptake is characteristic of cocci and/or the growth phase-dependency of competence.


Asunto(s)
Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/metabolismo , ADN de Cadena Simple/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de la Membrana/metabolismo , Streptococcus pneumoniae/metabolismo , Transformación Bacteriana/fisiología , Factores de Virulencia/metabolismo , Proteínas Bacterianas/genética , Cromosomas Bacterianos/genética , ADN de Cadena Simple/genética , Endodesoxirribonucleasas/genética , Humanos , Proteínas de la Membrana/genética , Streptococcus pneumoniae/genética , Factores de Virulencia/genética
14.
PLoS Pathog ; 9(6): e1003473, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23825953

RESUMEN

Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.


Asunto(s)
ADN Bacteriano/metabolismo , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Streptococcus pneumoniae/metabolismo , Transformación Bacteriana/fisiología , ADN Bacteriano/genética , ADN Bacteriano/inmunología , Resistencia a Medicamentos/fisiología , Proteínas Fimbrias/genética , Proteínas Fimbrias/inmunología , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/inmunología , Humanos , Evasión Inmune/fisiología , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/patogenicidad
15.
Proc Natl Acad Sci U S A ; 109(37): E2466-75, 2012 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-22904190

RESUMEN

Transformation promotes genome plasticity in bacteria via RecA-driven homologous recombination. In the gram-positive human pathogen Streptococcus pneumoniae, the transformasome a multiprotein complex, internalizes, protects, and processes transforming DNA to generate chromosomal recombinants. Double-stranded DNA is internalized as single strands, onto which the transformation-dedicated DNA processing protein A (DprA) ensures the loading of RecA to form presynaptic filaments. We report that the structure of DprA consists of the association of a sterile alpha motif domain and a Rossmann fold and that DprA forms tail-to-tail dimers. The isolation of DprA self-interaction mutants revealed that dimerization is crucial for the formation of nucleocomplexes in vitro and for genetic transformation. Residues important for DprA-RecA interaction also were identified and mutated, establishing this interaction as equally important for transformation. Positioning of key interaction residues on the DprA structure revealed an overlap of DprA-DprA and DprA-RecA interaction surfaces. We propose a model in which RecA interaction promotes rearrangement or disruption of the DprA dimer, enabling the subsequent nucleation of RecA and its polymerization onto ssDNA.


Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Conformación Proteica , Rec A Recombinasas/metabolismo , Streptococcus pneumoniae/metabolismo , Transformación Bacteriana/fisiología , Proteínas Bacterianas/química , Western Blotting , Cristalización , ADN/metabolismo , Cartilla de ADN/genética , Dimerización , Proteínas de la Membrana/química , Mutagénesis Sitio-Dirigida , Transformación Bacteriana/genética , Técnicas del Sistema de Dos Híbridos
16.
Mol Microbiol ; 75(6): 1513-28, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20180906

RESUMEN

A secreted competence-stimulating peptide (CSP), encoded by comC, constitutes, together with the two-component system ComD-ComE, the master switch for competence induction in Streptococcus pneumoniae. Interaction between CSP and its membrane-bound histidine-kinase receptor, ComD, is believed to lead to autophosphorylation of ComD, which then transphosphorylates the ComE response regulator to activate transcription of a limited set of genes, including the comCDE operon. This generates a positive feedback loop, amplifying the signal and co-ordinating competence throughout the population. On the other hand, the promoter(s) and proteins important for basal comCDE expression have not been defined. We now report that CSP-induced and basal comCDE transcription both initiate from the same promoter, P(E); that basal expression necessitates the presence of both ComD and a phosphate-accepting form of ComE, but not CSP; and that overexpression of ComE(R120S) triggers ComD-dependent transformation in the absence of CSP. These observations suggest that self-activation of ComD is required for basal comCDE expression. We also establish that transcriptional readthrough occurs across the tRNA(Arg5) terminator and contributes significantly to comCDE expression. Finally, we demonstrate by various means, including single-cell competence analysis with GFP, that readthrough is crucial to avoid the stochastic production of CSP non-responsive cells lacking ComD or ComE.


Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Quinasas/metabolismo , Transducción de Señal , Streptococcus pneumoniae/fisiología , ADN Bacteriano/metabolismo , Orden Génico , Genes Bacterianos , Histidina Quinasa , Operón , Regiones Promotoras Genéticas , Streptococcus pneumoniae/genética , Transcripción Genética , Transformación Bacteriana
17.
J Biol Chem ; 283(8): 4975-82, 2008 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-18077456

RESUMEN

During the process of spore formation in Bacillus subtilis, many membrane proteins localize to the polar septum where they participate in morphogenesis and signal transduction. The forespore membrane protein SpoIIQ plays a central role in anchoring several mother-cell membrane proteins in the septal membrane. Here, we report that SpoIIQ is also responsible for anchoring a membrane protein on the forespore side of the sporulation septum. Co-immunoprecipitation experiments reveal that SpoIIQ resides in a complex with the polytopic membrane protein SpoIIE. During the early stages of sporulation, SpoIIE participates in the switch from medial to polar division and co-localizes with FtsZ at the polar septum. We show that after cytokinesis, SpoIIE is released from the septum and transiently localizes to all membranes in the forespore compartment. Upon the initiation of engulfment, it specifically re-localizes to the septal membrane on the forespore side. Importantly, the re-localization of SpoIIE to the engulfing septum requires SpoIIQ. These results indicate that SpoIIQ is required to anchor membrane proteins on both sides of the division septum. Moreover, our data suggest that forespore membrane proteins can localize to the septal membrane by diffusion-and-capture as has been described for membrane proteins in the mother cell. Finally, our results raise the intriguing possibility that SpoIIE has an uncharacterized function at a late stage of sporulation.


Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/metabolismo , División Celular/fisiología , Membrana Celular/metabolismo , Bacillus subtilis/citología , Transporte de Proteínas/fisiología , Esporas Bacterianas
18.
PLoS Genet ; 3(7): e117, 2007 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-17630835

RESUMEN

Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving two paralogous tyrosine recombinases, XerC and XerD, and a 28-bp recombination site (dif) located at the junction of the two replication arms. Xer recombination is tightly controlled by the septal protein FtsK. XerCD recombinases and FtsK are found on most sequenced eubacterial genomes, suggesting that the Xer recombination system as described in E. coli is highly conserved among prokaryotes. We show here that Streptococci and Lactococci carry an alternative Xer recombination machinery, organized in a single recombination module. This corresponds to an atypical 31-bp recombination site (dif(SL)) associated with a dedicated tyrosine recombinase (XerS). In contrast to the E. coli Xer system, only a single recombinase is required to recombine dif(SL), suggesting a different mechanism in the recombination process. Despite this important difference, XerS can only perform efficient recombination when dif(SL) sites are located on chromosome dimers. Moreover, the XerS/dif(SL) recombination requires the streptococcal protein FtsK(SL), probably without the need for direct protein-protein interaction, which we demonstrated to be located at the division septum of Lactococcus lactis. Acquisition of the XerS recombination module can be considered as a landmark of the separation of Streptococci/Lactococci from other firmicutes and support the view that Xer recombination is a conserved cellular function in bacteria, but that can be achieved by functional analogs.


Asunto(s)
Lactococcus/genética , Lactococcus/metabolismo , Recombinasas/genética , Recombinasas/metabolismo , Recombinación Genética , Streptococcus/genética , Streptococcus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , ADN Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genómica , Integrasas/genética , Integrasas/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Filogenia , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo
19.
J Bacteriol ; 189(16): 6021-7, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17557826

RESUMEN

The proteolytic activation of the mother cell transcription factor pro-sigma(K) is controlled by a signal transduction pathway during sporulation in the bacterium Bacillus subtilis. The pro-sigma(K) processing enzyme SpoIVFB, a membrane-embedded metalloprotease, is held inactive by two other integral membrane proteins, SpoIVFA and BofA, in the mother cell membrane that surrounds the forespore. Two signaling serine proteases, SpoIVB and CtpB, trigger pro-sigma(K) processing by cleaving the regulatory protein SpoIVFA. The SpoIVB signal is absolutely required to activate pro-sigma(K) processing and is derived from the forespore compartment. CtpB is necessary for the proper timing of sigma(K) activation and was thought to be a mother cell signal. Here, we show that the ctpB gene is expressed in both the mother cell and forespore compartments but that synthesis in the forespore under the control of sigma(G) is both necessary and sufficient for the proper timing of pro-sigma(K) processing. We further show that SpoIVB cleaves CtpB in vitro and in vivo but that this cleavage does not appear to be necessary for CtpB activation. Thus, both signaling proteins are made in the forespore and independently target the same regulatory protein.


Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/metabolismo , Factor sigma/metabolismo , Transducción de Señal/fisiología , Esporas Bacterianas/crecimiento & desarrollo , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/fisiología , Factor sigma/genética , Esporas Bacterianas/fisiología , Factores de Transcripción
20.
Mol Cell ; 23(1): 25-35, 2006 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-16818230

RESUMEN

The proteolytic activation of the membrane-associated transcription factor pro-sigma(K) is controlled by a signal transduction pathway during sporulation in the bacterium Bacillus subtilis. The pro-sigma(K) processing enzyme SpoIVFB, a membrane-embedded metalloprotease, is held inactive by two other integral-membrane proteins, SpoIVFA and BofA. We demonstrate that the signaling protease SpoIVB (IVB) triggers pro-sigma(K) processing by cleaving the extracellular domain of the SpoIVFA regulator at multiple sites. In vitro, these cleavages do not disrupt the interactions between SpoIVFA, SpoIVFB, and BofA, suggesting that IVB-dependent activation of the processing enzyme results from a conformational change in this complex. Our data further suggest that when IVB is unable to cleave SpoIVFA, it can still activate pro-sigma(K) processing through a second protease, CtpB. Finally, we demonstrate that CtpB, like IVB, triggers pro-sigma(K) processing by cleaving SpoIVFA. We propose that IVB regulates intramembrane proteolysis through two proteolytic pathways, both of which converge on the same regulator.


Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Péptido Hidrolasas/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Bacillus subtilis/citología , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Endopeptidasas/metabolismo , Proteínas de la Membrana/genética , Modelos Biológicos , Mutación , Proteínas Represoras/metabolismo , Factor sigma/genética , Factor sigma/fisiología , Esporas Bacterianas/metabolismo , Regulación hacia Arriba
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