RESUMO
C-di-GMP is a bacterial second messenger that regulates diverse processes in response to environmental or cellular cues. The nucleoid-associated protein (NAP) CdbA in Myxococcus xanthus binds c-di-GMP and DNA in a mutually exclusive manner in vitro. CdbA is essential for viability, and CdbA depletion causes defects in chromosome organization, leading to a block in cell division and, ultimately, cell death. Most NAPs are not essential; therefore, to explore the paradoxical cdbA essentiality, we isolated suppressor mutations that restored cell viability without CdbA. Most mutations mapped to cdbS, which encodes a stand-alone c-di-GMP binding PilZ domain protein, and caused loss-of-function of cdbS. Cells lacking CdbA and CdbS or only CdbS were fully viable and had no defects in chromosome organization. CdbA depletion caused post-transcriptional upregulation of CdbS accumulation, and this CdbS over-accumulation was sufficient to disrupt chromosome organization and cause cell death. CdbA depletion also caused increased accumulation of CsdK1 and CsdK2, two unusual PilZ-DnaK chaperones. During CdbA depletion, CsdK1 and CsdK2, in turn, enabled the increased accumulation and toxicity of CdbS, likely by stabilizing CdbS. Moreover, we demonstrate that heat stress, possibly involving an increased cellular c-di-GMP concentration, induced the CdbA/CsdK1/CsdK2/CdbS system, causing a CsdK1- and CsdK2-dependent increase in CdbS accumulation. Thereby this system accelerates heat stress-induced chromosome mis-organization and cell death. Collectively, this work describes a unique system that contributes to regulated cell death in M. xanthus and suggests a link between c-di-GMP signaling and regulated cell death in bacteria.
Assuntos
Proteínas de Bactérias , Myxococcus xanthus , Proteínas de Bactérias/metabolismo , Myxococcus xanthus/genética , Proteínas de Transporte/genética , Chaperonas Moleculares/genética , Morte Celular , Cromossomos/metabolismo , GMP Cíclico/metabolismo , Ligação ProteicaRESUMO
In bacteria, the nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pilus-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pilus-dependent and gliding motility in a Frz-dependent manner as well as development. The acetyltransferase domain is required and sufficient for function during growth, while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility independently of the Frz system, likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA, and PixB act in independent pathways and have distinct functions in regulation of motility. IMPORTANCE c-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts in a manner that depends on the Frz chemosensory system to regulate motility via the acetyltransferase domain, while the intact protein and c-di-GMP binding are essential for PixB to support development. In contrast, PixA acts in a Frz-independent manner to regulate motility. Taking our results together with previous observations, we conclude that PilZ domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Domínios Proteicos , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Ligação ProteicaRESUMO
Members of the Rhizobiales (class of α-proteobacteria) display zonal peptidoglycan cell wall growth at one cell pole, contrasting with the dispersed mode of cell wall growth along the sidewalls of many other rod-shaped bacteria. Here we show that the seven-transmembrane receptor (7TMR) protein RgsP (SMc00074), together with the putative membrane-anchored peptidoglycan metallopeptidase RgsM (SMc02432), have key roles in unipolar peptidoglycan formation during growth and at mid-cell during cell division in Sinorhizobium meliloti. RgsP is composed of a periplasmic globular 7TMR-DISMED2 domain, a membrane-spanning region, and cytoplasmic PAS, GGDEF and EAL domains. The EAL domain confers phosphodiesterase activity towards the second messenger cyclic di-GMP, a key regulatory player in the transition between bacterial lifestyles. RgsP and RgsM localize to sites of zonal cell wall synthesis at the new cell pole and cell divison site, suggesting a role in cell wall biogenesis. The two proteins are essential for cell wall biogenesis and cell growth. Cells depleted of RgsP or RgsM had an altered muropeptide composition and RgsM binds to peptidoglycan. RgsP and RgsM orthologs are functional when interchanged between α-rhizobial species pointing to a conserved mechanism for cell wall biogenesis/remodeling within the Rhizobiales. Overall, our findings suggest that RgsP and RgsM contribute to the regulation of unipolar cell wall biogenesis in α-rhizobia.
Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/química , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana/metabolismo , Sistemas do Segundo Mensageiro/genética , Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte , GMP Cíclico/genética , GMP Cíclico/metabolismo , Proteínas de Membrana/genética , Microscopia Eletrônica de Transmissão , Peptidoglicano/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , beta-Lactamas/farmacologiaRESUMO
Cyclic dimeric GMP (c-di-GMP) has emerged as a key regulatory player in the transition between planktonic and sedentary biofilm-associated bacterial lifestyles. It controls a multitude of processes including production of extracellular polysaccharides (EPSs). The PilZ domain, consisting of an N-terminal "RxxxR" motif and a ß-barrel domain, represents a prototype c-di-GMP receptor. We identified a class of c-di-GMP-responsive proteins, represented by the AraC-like transcription factor CuxR in plant symbiotic α-proteobacteria. In Sinorhizobium meliloti, CuxR stimulates transcription of an EPS biosynthesis gene cluster at elevated c-di-GMP levels. CuxR consists of a Cupin domain, a helical hairpin, and bipartite helix-turn-helix motif. Although unrelated in sequence, the mode of c-di-GMP binding to CuxR is highly reminiscent to that of PilZ domains. c-di-GMP interacts with a conserved N-terminal RxxxR motif and the Cupin domain, thereby promoting CuxR dimerization and DNA binding. We unravel structure and mechanism of a previously unrecognized c-di-GMP-responsive transcription factor and provide insights into the molecular evolution of c-di-GMP binding to proteins.
Assuntos
Proteínas de Bactérias/metabolismo , GMP Cíclico/análogos & derivados , Polissacarídeos Bacterianos/biossíntese , Sinorhizobium meliloti/metabolismo , Transativadores/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Fator de Transcrição AraC/química , Fator de Transcrição AraC/genética , Fator de Transcrição AraC/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência Conservada , Cristalografia por Raios X , GMP Cíclico/metabolismo , Modelos Moleculares , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Sinorhizobium meliloti/genética , Transativadores/química , Transativadores/genéticaRESUMO
Generally, the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates the switch between motile and sessile lifestyles in bacteria. Here, we show that c-di-GMP is an essential regulator of multicellular development in the social bacterium Myxococcus xanthus. In response to starvation, M. xanthus initiates a developmental program that culminates in formation of spore-filled fruiting bodies. We show that c-di-GMP accumulates at elevated levels during development and that this increase is essential for completion of development whereas excess c-di-GMP does not interfere with development. MXAN3735 (renamed DmxB) is identified as a diguanylate cyclase that only functions during development and is responsible for this increased c-di-GMP accumulation. DmxB synthesis is induced in response to starvation, thereby restricting DmxB activity to development. DmxB is essential for development and functions downstream of the Dif chemosensory system to stimulate exopolysaccharide accumulation by inducing transcription of a subset of the genes encoding proteins involved in exopolysaccharide synthesis. The developmental defects in the dmxB mutant are non-cell autonomous and rescued by co-development with a strain proficient in exopolysaccharide synthesis, suggesting reduced exopolysaccharide accumulation as the causative defect in this mutant. The NtrC-like transcriptional regulator EpsI/Nla24, which is required for exopolysaccharide accumulation, is identified as a c-di-GMP receptor, and thus a putative target for DmxB generated c-di-GMP. Because DmxB can be-at least partially-functionally replaced by a heterologous diguanylate cyclase, these results altogether suggest a model in which a minimum threshold level of c-di-GMP is essential for the successful completion of multicellular development in M. xanthus.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Myxococcus xanthus/genética , Fósforo-Oxigênio Liases/genética , Esporos Bacterianos/genética , Proteínas de Bactérias/biossíntese , GMP Cíclico/análogos & derivados , GMP Cíclico/genética , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Myxococcus xanthus/crescimento & desenvolvimento , Esporos Bacterianos/crescimento & desenvolvimento , InaniçãoRESUMO
UNLABELLED: The nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes. Myxococcus xanthus undergoes major lifestyle changes in response to nutrient availability, with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP in M. xanthus and show that this bacterium synthesizes c-di-GMP during growth. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase correlated with defects in type IV pilus (T4P)-dependent motility, whereas gliding motility was unaffected. An increased level of c-di-GMP correlated with reduced transcription of the pilA gene (which encodes the major pilin of T4P), reduced the assembly of T4P, and altered cell agglutination, whereas a decreased c-di-GMP level correlated with altered cell agglutination. The systematic inactivation of the 24 genes in M. xanthus encoding proteins containing GGDEF, EAL, or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation, or binding, identified three genes encoding proteins important for T4P-dependent motility, whereas all mutants had normal gliding motility. Purified DmxA had diguanylate cyclase activity, whereas the hybrid histidine protein kinases TmoK and SgmT, each of which contains a GGDEF domain, did not have diguanylate cyclase activity. These results demonstrate that c-di-GMP is important for T4P-dependent motility in M. xanthus. IMPORTANCE: We provide the first direct evidence that M. xanthus synthesizes c-di-GMP and demonstrate that c-di-GMP is important for T4P-dependent motility, whereas we did not obtain evidence that c-di-GMP regulates gliding motility. The data presented uncovered a novel mechanism for regulation of T4P-dependent motility, in which increased levels of c-di-GMP inhibit transcription of the pilA gene (which encodes the major pilin of T4P), ultimately resulting in the reduced assembly of T4P. Moreover, we identified an enzymatically active diguanylate cyclase that is important for T4P-dependent motility.
Assuntos
GMP Cíclico/análogos & derivados , Fímbrias Bacterianas/fisiologia , Myxococcus xanthus/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , GMP Cíclico/genética , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Movimento , Estrutura Terciária de ProteínaRESUMO
UNLABELLED: Sinorhizobium meliloti undergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3',5'-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG in S. meliloti Rm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation of N-Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of the S. meliloti genes pleD, SMb20523, SMb20447, SMc01464, and SMc03178 encoding putative DGCs and of SMb21517 encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, an S. meliloti strain that did not produce detectable levels of cdG (cdG(0)) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. The SMc01790-SMc01796 locus, homologous to the Agrobacterium tumefaciens uppABCDEF cluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility. IMPORTANCE: We present the first systematic genome-wide investigation of the role of 3',5'-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a symbiotic alpha-rhizobial species. Phenotypes of an S. meliloti strain unable to produce cdG (cdG(0)) demonstrated that this second messenger is not essential for root nodule symbiosis but may contribute to acid tolerance. Our data further suggest that enhanced levels of cdG promote sessility of S. meliloti and uncovered a single-domain PilZ protein as regulator of motility.
Assuntos
GMP Cíclico/análogos & derivados , Regulação Bacteriana da Expressão Gênica/fisiologia , Sinorhizobium meliloti/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/genética , GMP Cíclico/metabolismo , Mutação , Polissacarídeos Bacterianos/biossíntese , Sinorhizobium meliloti/genéticaRESUMO
Here we study the influence of the putative fatty acid biosynthesis (FAB) regulator FabT (originally called RmaG [Llmg_1788]) on gene transcription in Lactococcus lactis MG1363. A strain with a knockout mutation of the putative regulator was constructed, and its transcriptome was compared to that of the wild-type strain. Almost all FAB genes were significantly upregulated in the knockout. Using electrophoretic mobility shift assays (EMSAs) and DNase I footprinting, the binding motif of the regulator and the binding locations in the genome were characterized. Fatty acid composition analysis revealed that a strain lacking FabT contained significantly more saturated acyl chains in its phospholipids. This observation demonstrates that the vital pathway of FAB in L. lactis is regulated by the repressor FabT.
Assuntos
Proteínas de Bactérias/metabolismo , Ácidos Graxos/biossíntese , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Proteínas de Bactérias/genética , Pegada de DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Desoxirribonuclease I/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Ácidos Graxos/química , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Lipídeos de Membrana/química , Mutação , Fosfolipídeos/química , Regiões Promotoras Genéticas , Proteínas Repressoras/genéticaRESUMO
Cyclic di-GMP (c-di-GMP) is a second messenger that modulates multiple responses to environmental and cellular signals in bacteria. Here we identify CdbA, a DNA-binding protein of the ribbon-helix-helix family that binds c-di-GMP in Myxococcus xanthus. CdbA is essential for viability, and its depletion causes defects in chromosome organization and segregation leading to a block in cell division. The protein binds to the M. xanthus genome at multiple sites, with moderate sequence specificity; however, its depletion causes only modest changes in transcription. The interactions of CdbA with c-di-GMP and DNA appear to be mutually exclusive and residue substitutions in CdbA regions important for c-di-GMP binding abolish binding to both c-di-GMP and DNA, rendering these protein variants non-functional in vivo. We propose that CdbA acts as a nucleoid-associated protein that contributes to chromosome organization and is modulated by c-di-GMP, thus revealing a link between c-di-GMP signaling and chromosome biology.
Assuntos
Proteínas de Bactérias/metabolismo , Núcleo Celular/metabolismo , Segregação de Cromossomos , GMP Cíclico/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Myxococcus xanthus/metabolismo , Proteínas de Bactérias/química , Sequência de Bases , Cromossomos Bacterianos/metabolismo , GMP Cíclico/metabolismo , DNA Bacteriano/metabolismo , Loci Gênicos , Modelos Moleculares , Multimerização Proteica , Estrutura Secundária de Proteína , Transcrição GênicaRESUMO
The nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates multiple processes in bacteria including cellular motility. The rod-shaped Myxococcus xanthus cells move in the direction of their long axis using two distinct motility systems: type IV pili (T4P)-dependent motility and gliding motility. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase causes defects in T4P-dependent motility without affecting gliding motility. As both an increased and a decreased level of c-di-GMP affect T4P-dependent motility, M. xanthus represents a good model system to assess enzyme activity of diguanylate cyclases and phosphodiesterases using T4P-dependent motility as a readout. Here, we describe the assay, which allows correlating diguanylate cyclase and phosphodiesterase activity with T4P-dependent motility in M. xanthus.