RESUMEN
In rod-shaped bacteria, type IV pili (Tfp) promote twitching motility by assembling and retracting at the cell pole. In Myxococcus xanthus, a bacterium that moves in highly coordinated cell groups, Tfp are activated by a polar activator protein, SgmX. However, while it is known that the Ras-like protein MglA is required for unipolar targeting, how SgmX accesses the cell pole to activate Tfp is unknown. Here, we demonstrate that a polar beacon protein, FrzS, recruits SgmX at the cell pole. We identified two main functional domains, including a Tfp-activating domain and a polar-binding domain. Within the latter, we show that the direct binding of MglA-GTP unveils a hidden motif that binds directly to the FrzS N-terminal response regulator (CheY). Structural analyses reveal that this binding occurs through a novel binding interface for response regulator domains. In conclusion, the findings unveil the protein interaction network leading to the spatial activation of Tfp at the cell pole. This tripartite system is at the root of complex collective behaviours in this predatory bacterium.
Asunto(s)
Proteínas Bacterianas , Myxococcus xanthus , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Myxococcus xanthus/metabolismo , Fimbrias Bacterianas/químicaRESUMEN
Type Four Pili (T4P) are extracellular appendages mediating several bacterial functions such as motility, biofilm formation and infection. The ability to adhere to substrates is essential for all these functions. In Myxococcus xanthus, during twitching motility, the binding of polar T4P to exopolysaccharides (EPS), induces pilus retraction and the forward cell movement. EPS are produced, secreted and weakly associated to the M. xanthus cell surface or deposited on the substrate. In this study, a genetic screen allowed us to identify two factors involved in EPS-independent T4P-dependent twitching motility: the PilY1.1 protein and the HsfBA phosphorelay. Transcriptomic analyses show that HsfBA differentially regulates the expression of PilY1 proteins and that the down-regulation of pilY1.1 together with the accumulation of its homologue pilY1.3, allows twitching motility in the absence of EPS. The genetic and bioinformatic dissection of the PilY1.1 domains shows that PilY1.1 might be a bi-functional protein with a role in priming T4P extension mediated by its conserved N-terminal domain and roles in EPS-dependent motility mediated by an N-terminal DUF4114 domain activated upon binding to Ca2+. We speculate that the differential transcriptional regulation of PilY1 homologs by HsfBA in response to unknown signals, might allow accessorizing T4P tips with different modules allowing twitching motility in the presence of alternative substrates and environmental conditions.
Asunto(s)
Proteínas Fimbrias , Myxococcus xanthus , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Movimiento Celular/genética , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Myxococcus xanthus/fisiologíaRESUMEN
Type IV pili (Tfp) are highly conserved macromolecular structures that fulfill diverse cellular functions, such as adhesion to host cells, the import of extracellular DNA, kin recognition, and cell motility (twitching). Outstandingly, twitching motility enables a poorly understood process by which highly coordinated groups of hundreds of cells move in cooperative manner, providing a basis for multicellular behaviors, such as biofilm formation. In the social bacteria Myxococcus xanthus, we know that twitching motility is under the dependence of the small GTPase MglA, but the underlying molecular mechanisms remain elusive. Here we show that MglA complexed to GTP recruits a newly characterized Tfp regulator, termed SgmX, to activate Tfp machines at the bacterial cell pole. This mechanism also ensures spatial regulation of Tfp, explaining how MglA switching provokes directional reversals. This discovery paves the way to elucidate how polar Tfp machines are regulated to coordinate multicellular movements, a conserved feature in twitching bacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Fimbrias Bacterianas/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Myxococcus xanthus/fisiología , Proteínas Bacterianas/genética , Polaridad Celular/fisiología , Myxococcus xanthus/citología , Myxococcus xanthus/genética , Polimorfismo de Nucleótido Simple , Secuenciación Completa del GenomaRESUMEN
The development of multicellularity is a key evolutionary transition allowing for differentiation of physiological functions across a cell population that confers survival benefits; among unicellular bacteria, this can lead to complex developmental behaviors and the formation of higher-order community structures. Herein, we demonstrate that in the social δ-proteobacterium Myxococcus xanthus, the secretion of a novel biosurfactant polysaccharide (BPS) is spatially modulated within communities, mediating swarm migration as well as the formation of multicellular swarm biofilms and fruiting bodies. BPS is a type IV pilus (T4P)-inhibited acidic polymer built of randomly acetylated ß-linked tetrasaccharide repeats. Both BPS and exopolysaccharide (EPS) are produced by dedicated Wzx/Wzy-dependent polysaccharide-assembly pathways distinct from that responsible for spore-coat assembly. While EPS is preferentially produced at the lower-density swarm periphery, BPS production is favored in the higher-density swarm interior; this is consistent with the former being known to stimulate T4P retraction needed for community expansion and a function for the latter in promoting initial cell dispersal. Together, these data reveal the central role of secreted polysaccharides in the intricate behaviors coordinating bacterial multicellularity.
Asunto(s)
Myxococcus xanthus/citología , Myxococcus xanthus/metabolismo , Polisacáridos Bacterianos/metabolismo , Acetilación , Vías Biosintéticas/genética , Espectroscopía de Resonancia Magnética con Carbono-13 , Membrana Celular/metabolismo , Familia de Multigenes , Myxococcus xanthus/genética , Polisacáridos Bacterianos/química , Espectroscopía de Protones por Resonancia Magnética , Tensoactivos/metabolismoRESUMEN
Chemosensory systems are highly organized signaling pathways that allow bacteria to adapt to environmental changes. The Frz chemosensory system from M. xanthus possesses two CheW-like proteins, FrzA (the core CheW) and FrzB. We found that FrzB does not interact with FrzE (the cognate CheA) as it lacks the amino acid region responsible for this interaction. FrzB, instead, acts upstream of FrzCD in the regulation of M. xanthus chemotaxis behaviors and activates the Frz pathway by allowing the formation and distribution of multiple chemosensory clusters on the nucleoid. These results, together, show that the lack of the CheA-interacting region in FrzB confers new functions to this small protein.
Asunto(s)
Quimiotaxis , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Myxococcus xanthus/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Movimiento Celular , Regulación Bacteriana de la Expresión Génica , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Myxococcus xanthus/genética , Operón , Fenotipo , Transducción de SeñalRESUMEN
Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.
Asunto(s)
Bacillus subtilis/enzimología , Membrana Celular/enzimología , Fosfatidato Fosfatasa/química , Fosfatidato Fosfatasa/metabolismo , Bacillus subtilis/genética , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/metabolismo , Genes Bacterianos , Prueba de Complementación Genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfatidato Fosfatasa/genética , Fosfatidilgliceroles/metabolismo , Solubilidad , Especificidad por SustratoRESUMEN
Signal transduction systems and ABC transporters often contribute jointly to adaptive bacterial responses to environmental changes. In Bacillus subtilis, three such pairs are involved in responses to antibiotics: BceRSAB, YvcPQRS and YxdJKLM. They are characterized by a histidine kinase belonging to the intramembrane sensing kinase family and by a translocator possessing an unusually large extracytoplasmic loop. It was established here using a phylogenomic approach that systems of this kind are specific but widespread in Firmicutes, where they originated. The present phylogenetic analyses brought to light a highly dynamic evolutionary history involving numerous horizontal gene transfers, duplications and lost events, leading to a great variety of Bce-like repertories in members of this bacterial phylum. Based on these phylogenetic analyses, it was proposed to subdivide the Bce-like modules into six well-defined subfamilies. Functional studies were performed on members of subfamily IV comprising BceRSAB from B. subtilis, the expression of which was found to require the signal transduction system as well as the ABC transporter itself. The present results suggest, for the members of this subfamily, the occurrence of interactions between one component of each partner, the kinase and the corresponding translocator. At functional and/or structural levels, bacitracin dependent expression of bceAB and bacitracin resistance processes require the presence of the BceB translocator loop. Some other members of subfamily IV were also found to participate in bacitracin resistance processes. Taken together our study suggests that this regulatory mechanism might constitute an important common antibiotic resistance mechanism in Firmicutes. [Supplemental material is available online at http://www.genome.org.].
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adaptación Fisiológica/genética , Bacillus subtilis/fisiología , Farmacorresistencia Microbiana , Transducción de Señal , Bacterias , Proteínas Bacterianas/fisiología , Evolución Molecular , Histidina Quinasa , Filogenia , Proteínas Quinasas , Multimerización de ProteínaRESUMEN
The Bacillus subtilis BceAB ABC transporter involved in a defense mechanism against bacitracin is composed of a membrane-spanning domain and a nucleotide-binding domain. Induction of the structural bceAB genes requires the BceR response regulator and the BceS histidine kinase of a signal transduction system. However, despite the presence of such a transduction system and of bacitracin, no transcription from an unaltered bceA promoter is observed in cells lacking the BceAB transporter. Expression in trans of the BceAB transporter in these bceAB cells restores the transcription from the bceA promoter. Cells possessing a mutated nucleotide-binding domain of the transporter are also no longer able to trigger transcription from the bceA promoter in the presence of bacitracin, although the mutated ABC transporter is still bound to the membrane. In these cells, expression of the bceA promoter can no longer be detected, indicating that the ABC transporter not only must be present in the cell membrane, but also must be expressed in a native form for the induction of the bceAB genes. Several hypotheses are discussed to explain the simultaneous need for bacitracin, a native signal transduction system, and an active BceAB ABC transporter to trigger transcription from the bceA promoter.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antibacterianos/farmacología , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bacitracina/farmacología , Farmacorresistencia Bacteriana/genética , Regulación Bacteriana de la Expresión Génica , Transportadoras de Casetes de Unión a ATP/genética , Bacillus subtilis/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Genes Bacterianos/genética , Mutación , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
The genome of Bacillus subtilis, like those of some other AT-rich Gram-positive bacteria, has the uncommon feature of containing several copies of arrangements in which the genes encoding two-component and cognate ABC transporter systems are adjacent. As the function of one of these systems, the product of the yxd locus, is still unknown, it was analysed further in order to get some clues on the physiological role of the gene products it encodes. The yxdJ gene was shown to encode a DNA-binding protein that directly controls transcription of the neighbouring operon encoding the ABC transporter YxdLM. Primer extension and DNase protection experiments allowed precise definition of the yxdLM transcription start and controlling region. Two putative direct repeats were identified that are proposed to be the YxdJ response regulator binding sites. Whole-cell transcriptome analyses revealed that the YxdJ regulon is extremely restricted. In addition to the yxdJKLMyxeA operon, only a few genes involved in modifications of the bacterial cell wall were shown to be regulated by YxdJ.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Transportadoras de Casetes de Unión a ATP/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Proteínas Portadoras/metabolismo , ADN Bacteriano/genética , Expresión Génica , Genoma Bacteriano , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Operón , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Eliminación de Secuencia , Transcripción GenéticaRESUMEN
The Bacillus subtilis yts, yxd and yvc gene clusters encode a putative ABC transporter and a functionally coupled two-component system. When tested for their sensitivity towards a series of antibiotics, null yts mutants were found to be sensitive to bacitracin. Real-time polymerase chain reaction (PCR) experiments demonstrated that the presence of bacitracin in the growth medium strongly stimulates the expression of the ytsCD genes encoding the ABC transporter and that this stimulation strictly depends on the YtsA response regulator. The ywoA gene encodes a protein known to confer some resistance to bacitracin on the bacterium. When it was mutated in a null yts background, the ywoA yts double mutant was found to be five times more sensitive than the yts one. We propose that (i) the YtsCD ABC transporter exports the bacitracin; (ii) YwoA, the protein that contains an acidPPc (PAP2 or PgpB) domain, is not part of an ABC transporter but competes with bacitracin for the dephosphorylation of the C55-isoprenyl pyrophosphate (IPP); (iii) the two resistance mechanisms are independent and complementary.