CdbA is a DNA-binding protein and c-di-GMP receptor important for nucleoid organization and segregation in Myxococcus xanthus.
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ABSTRACT:
SUBMITTER: Skotnicka D
PROVIDER: S-EPMC7156744 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The second messenger c-di-GMP has important functions in response to changing environmental and cellular cues in bacteria. Here, we report the identification of CdbA, a DNA binding protein of the ribbon-helix-helix family in Myxococcus xanthus, and show that it binds c-di-GMP in vitro. CdbA is essential for viability and its depletion caused defects in chromosome organization and segregation resulting in a block in cell division. CdbA binds multiple sites across the M. xanthus genome with moderate sequence specificity; however, its depletion only caused minor changes in transcription. C-di-GMP binding and DNA binding by CdbA are mutually exclusive and substitutions in the CdbA interfaces important for c-di-GMP binding not only abolished c-di-GMP binding but also DNA binding and rendered the mutant proteins non-functional in vivo. Our data are consistent with a model whereby CdbA functions as a nucleoid associated protein to organize the M. xanthus chromosome and the function of CdbA is modulated by c-di-GMP, thus, for the first time, establishing a link between c-di-GMP and chromosome organization and segregation.
2020-04-16 | PXD018028 | Pride
Project description:In bacteria, homologs of actin, tubulin, and intermediate filament proteins often act in concert with bacteria-specific scaffolding proteins to ensure the proper arrangement of cellular components. Among the bacteria-specific factors are the bactofilins, a widespread family of polymer-forming proteins whose biology is poorly investigated. Here, we study the three bactofilins BacNOP in the rod-shaped bacterium Myxococcus xanthus. We show that BacNOP co-assemble into elongated scaffolds that restrain the ParABS chromosome segregation machinery to the subpolar regions of the cell. The centromere (parS)-binding protein ParB associates with the pole-distal ends of these structures, whereas the DNA partitioning ATPase ParA binds along their entire length, using the newly identified protein PadC (MXAN_4634) as an adapter. The integrity of these complexes is critical for proper nucleoid morphology and chromosome segregation. BacNOP thus mediate a previously unknown mechanism of subcellular organization that recruits proteins to defined sites within the cytoplasm, far off the cell poles.
| S-EPMC5703909 | biostudies-other
Project description:The regulation of biofilm and motile states as alternate bacterial lifestyles has been studied extensively in flagellated bacteria, where the second messenger cyclic-di-GMP (cdG) plays a crucial role. However, much less is known about the mechanisms of such regulation in motile bacteria without flagella. The bacterial type IV pilus (T4P) serves as a motility apparatus that enables Myxococcus xanthus to move on solid surfaces. PilB, the T4P assembly ATPase, is, therefore, required for T4P-dependent motility in M. xanthus. Interestingly, T4P is also involved in the regulation of exopolysaccharide as the biofilm matrix material in this bacterium. A newly discovered cdG-binding domain, MshEN, is conserved in the N-terminus of PilB (PilBN) in M. xanthus and other bacteria. This suggests that cdG may bind to PilB to control the respective outputs that regulate biofilm development and T4P-powered motility. In this study, we aimed to validate M. xanthus PilB as a cdG effector protein. We performed a systematic mutational analysis of its cdG-binding domain to investigate its relationship with motility, piliation, and biofilm formation. Excluding those resulting in low levels of PilB protein, all other substitution mutations in PilBN resulted in pilB mutants with distinct and differential phenotypes in piliation and biofilm levels in M. xanthus. This suggests that the PilBN domain plays dual roles in modulating motility and biofilm levels, and these two functions of PilB can be dependent on and independent of each other in M. xanthus. IMPORTANCE The regulation of motility and biofilm by cyclic-di-GMP in flagellated bacteria has been extensively investigated. However, our knowledge regarding this regulation in motile bacteria without flagella remains limited. Here, we aimed to address this gap by investigating a non-flagellated bacterium with motility powered by bacterial type-IV pilus (T4P). Previous studies hinted at the possibility of Myxococcus xanthus PilB, the T4P assembly ATPase, serving as a cyclic-di-GMP effector involved in regulating both motility and biofilm. Our findings strongly support the hypothesis that PilB directly interacts with cyclic-di-GMP to act as a potential switch to promote biofilm formation or T4P-dependent motility. These results shed light on the bifurcation of PilB functions and its pivotal role in coordinating biofilm formation and T4P-mediated motility.
| S-EPMC10521364 | biostudies-literature
Project description: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.
| S-EPMC4877007 | biostudies-literature
Project description:Myxococcus xanthus has a nutrient-regulated biphasic life cycle forming predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. The second messenger 3'-5', 3'-5 cyclic di-GMP (c-di-GMP) is essential during both stages of the life cycle; however, different enzymes involved in c-di-GMP synthesis and degradation as well as several c-di-GMP receptors are important during distinct life cycle stages. To address this stage specificity, we determined transcript levels using transcriptome sequencing (RNA-seq) and transcription start sites using Cappable sequencing (Cappable-seq) during growth and development genome wide. All 70 genes encoding c-di-GMP-associated proteins were expressed, with 28 upregulated and 10 downregulated during development. Specifically, the three genes encoding enzymatically active proteins with a stage-specific function were expressed stage specifically. By combining operon mapping with published chromatin immunoprecipitation sequencing (ChIP-seq) data for MrpC (M. Robinson, B. Son, D. Kroos, L. Kroos, BMC Genomics 15:1123, 2014, http://dx.doi.org/10.1186/1471-2164-15-1123), the cAMP receptor protein (CRP)-like master regulator of development, we identified nine developmentally regulated genes as regulated by MrpC. In particular, MrpC directly represses the expression of dmxB, which encodes the diguanylate cyclase DmxB that is essential for development and responsible for the c-di-GMP increase during development. Moreover, MrpC directly activates the transcription of pmxA, which encodes a bifunctional phosphodiesterase that degrades c-di-GMP and 3',3'-cGAMP in vitro and is essential for development. Thereby, MrpC regulates and curbs the cellular pools of c-di-GMP and 3',3'-cGAMP during development. We conclude that temporal regulation of the synthesis of proteins involved in c-di-GMP metabolism contributes to c-di-GMP signaling specificity. MrpC is important for this regulation, thereby being a key regulator of developmental cyclic di-nucleotide metabolism in M. xanthus. IMPORTANCE The second messenger c-di-GMP is important during both stages of the nutrient-regulated biphasic life cycle of Myxococcus xanthus with the formation of predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. However, different enzymes involved in c-di-GMP synthesis and degradation are important during distinct life cycle stages. Here, we show that the three genes encoding enzymatically active proteins with a stage-specific function are expressed stage specifically. Moreover, we find that the master transcriptional regulator of development MrpC directly regulates the expression of dmxB, which encodes the diguanylate cyclase DmxB that is essential for development, and of pmxA, which encodes a bifunctional phosphodiesterase that degrades c-di-GMP and 3',3'-cGAMP in vitro and is essential for development. We conclude that temporal regulation of the synthesis of proteins involved in c-di-GMP metabolism contributes to c-di-GMP signaling specificity and that MrpC plays an important role in this regulation.
| S-EPMC8844925 | biostudies-literature
Project description:In Myxococcus xanthus the extracellular matrix is essential for type IV pili-dependent motility and starvation-induced fruiting body formation. Proteins of two-component systems including the orphan DNA binding response regulator DigR are essential in regulating the composition of the extracellular matrix. We identify the orphan hybrid histidine kinase SgmT as the partner kinase of DigR. In addition to kinase and receiver domains, SgmT consists of an N-terminal GAF domain and a C-terminal GGDEF domain. The GAF domain is the primary sensor domain. The GGDEF domain binds the second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP) and functions as a c-di-GMP receptor to spatially sequester SgmT. We identify the DigR binding site in the promoter of the fibA gene, which encodes an abundant extracellular matrix metalloprotease. Whole-genome expression profiling experiments in combination with the identified DigR binding site allowed the identification of the DigR regulon and suggests that SgmT/DigR regulates the expression of genes for secreted proteins and enzymes involved in secondary metabolite synthesis. We suggest that SgmT/DigR regulates extracellular matrix composition and that SgmT activity is regulated by two sensor domains with ligand binding to the GAF domain resulting in SgmT activation and c-di-GMP binding to the GGDEF domain resulting in spatial sequestration of SgmT.
| S-EPMC3509222 | biostudies-literature
Project description:UnlabelledIn the bacterium Myxococcus xanthus, starvation triggers the formation of multicellular fruiting bodies containing thousands of stress-resistant spores. Recent work showed that fruiting body development is regulated by a cascade of transcriptional activators called enhancer binding proteins (EBPs). The EBP Nla6 is a key component of this cascade; it regulates the promoters of other EBP genes, including a downstream-functioning EBP gene that is crucial for sporulation. In recent expression studies, hundreds of Nla6-dependent genes were identified, suggesting that the EBP gene targets of Nla6 may be part of a much larger regulon. The goal of this study was to identify and characterize genes that belong to the Nla6 regulon. Accordingly, a direct repeat [consensus, C(C/A)ACGNNGNC] binding site for Nla6 was identified using in vitro and in vivo mutational analyses, and the sequence was subsequently used to find 40 potential developmental promoter (88 gene) targets. We showed that Nla6 binds to the promoter region of four new targets (asgE, exo, MXAN2688, and MXAN3259) in vitro and that Nla6 is important for their normal expression in vivo. Phenotypic studies indicate that all of the experimentally confirmed targets of Nla6 are primarily involved in sporulation. These targets include genes involved in transcriptional regulation, cell-cell signal production, and spore differentiation and maturation. Although sporulation occurs late in development, all of the developmental loci analyzed here show an Nla6-dependent burst in expression soon after starvation is induced. This finding suggests that Nla6 starts preparing cells for sporulation very early in the developmental process.ImportanceBacterial development yields a remarkable array of complex multicellular forms. One such form, which is commonly found in nature, is a surface-associated aggregate of cells known as a biofilm. Mature biofilms are structurally complex and contain cells that are highly resistant to antibacterial agents. When starving, the model bacterium Myxococcus xanthus forms a biofilm containing a thin mat of cells and multicellular structures that house a highly resistant cell type called a myxospore. Here, we identify the promoter binding site of the transcriptional activator Nla6, identify genes in the Nla6 regulon, and show that several of the genes in the Nla6 regulon are important for production of stress-resistant spores in starvation-induced M. xanthus biofilms.
| S-EPMC4352671 | biostudies-literature
Project description:Our ChipSeq analysis show that while FrzCD does not bind DNA specific regions, ParB binds parS consistent with what as been previously shown. The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. FrzCD directly binds to the nucleoid and the FrzCD binding to the DNA leads to the formation of chemosensory complexes. This supra-molecular organization is required for cooperative interactions between clustered receptors, in turn important for the modulation of bacterial social behaviors.
2018-02-27 | GSE102724 | GEO
Project description:The Clp/Hsp100 proteins are chaperones that play a role in protein degradation and reactivation. In bacteria, they exhibit a high degree of pleiotropy, affecting both individual and multicellular phenotypes. In this article, we present the first characterization of a Clp/Hsp100 homolog in Myxococcus xanthus (MXAN_4832 gene locus). Deletion of MXAN_4832 causes defects in both swarming and aggregation related to cell motility and the production of fibrils, which are an important component of the extracellular matrix of a swarm. The deletion also affects the formation of myxospores during development, causing them to become sensitive to heat. The protein product of MXAN_4832 can act as a chaperone in vitro, providing biochemical evidence in support of our hypothesis that MXAN_4832 is a functional Clp/Hsp100 homolog. There are a total of 12 Clp/Hsp100 homologs in M. xanthus, including MXAN_4832, and, based on its mutational and biochemical characterization, they may well represent an important group.
| S-EPMC3302451 | biostudies-literature
Project description:During the developmental process of the Gram-negative soil bacterium Myxococcus xanthus, vegetatively growing rod cells differentiate to ultimately become metabolically quiescent and environmentally resistant myxospores encased within fruiting bodies. This program, initiated by nutrient deprivation, is propagated by both cell-autonomous and cell-nonautonomous signals. Our goal was to determine whether M. xanthus, like many other developmental systems, uses cell-cycle cues to regulate and control its developmental program. To address this question, the DNA replication cycle was used as a marker to monitor progression through the cell cycle in vegetative, stationary, and developing M. xanthus populations. Using flow cytometry, quantitative fluorescence microscopy, and FISH to establish the chromosome copy number of myxospores, it was determined that vegetatively growing cells contain one to two copies of the genome, but upon entry into stationary phase, the chromosome copy number drops to a single copy. Of particular interest, fruiting body-derived myxospores contain a specific two-chromosome DNA complement with both origin and terminus regions localized to the periphery of the myxospore. We speculate that this duplication of genetic information in the myxospore would help assure viability during germination by providing a second copy of each gene. The results of this study imply that not only is DNA replication tightly regulated during the developmental process of M. xanthus, but that there are also regulatory mechanisms to ensure that all myxospores acquire two copies of the chromosome.
| S-EPMC1228275 | biostudies-literature