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ABSTRACT The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) system widely occurs in prokaryotic organisms to recognize and destruct genetic invaders. Systematic collation and characterization of endogenous CRISPR-Cas systems are conducive to our understanding and potential utilization of this natural genetic machinery. In this study, we screened 39 complete and 692 incomplete genomes of myxobacteria using a combined strategy to dispose of the abridged genome information and revealed at least 19 CRISPR-Cas subtypes, which were distributed with a taxonomic difference and often lost stochastically in intraspecies strains. The cas genes in each subtype were evolutionarily clustered but deeply separated, while most of the CRISPRs were divided into four types based on the motif characteristics of repeat sequences. The spacers recorded in myxobacterial CRISPRs were in high G+C content, matching lots of phages, tiny amounts of plasmids, and, surprisingly, massive organismic genomes. We experimentally demonstrated the immune and self-target immune activities of three endogenous systems in Myxococcus xanthus DK1622 against artificial genetic invaders and revealed the microhomology-mediated end-joining mechanism for the immunity-induced DNA repair but not homology-directed repair. The panoramic view and immune activities imply potential omnipotent immune functions and applications of the endogenous CRISPR-Cas machinery.IMPORTANCEServing as an adaptive immune system, clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) empower prokaryotes to fend off the intrusion of external genetic materials. Myxobacteria are a collective of swarming Gram-stain-negative predatory bacteria distinguished by intricate multicellular social behavior. An in-depth analysis of their intrinsic CRISPR-Cas systems is beneficial for our understanding of the survival strategies employed by host cells within their environmental niches. Moreover, the experimental findings presented in this study not only suggest the robust immune functions of CRISPR-Cas in myxobacteria but also their potential applications.

ABSTRACTThe chaperone 70 kDa heat shock protein (Hsp70) is important for cells from bacteria to humans to maintain proteostasis, and all eukaryotes and several prokaryotes encode Hsp70 paralogs. Although the mechanisms of Hsp70 function have been clearly illuminated, the function and evolution of Hsp70 paralogs is not well studied. DnaK is a highly conserved bacterial Hsp70 family. Here, we show that dnaK is present in 98.9% of bacterial genomes, and 6.4% of them possess two or more DnaK paralogs. We found that the duplication of dnaK is positively correlated with an increase in proteomic complexity (proteome size, number of domains). We identified the interactomes of the two DnaK paralogs of Myxococcus xanthus DK1622 (MxDnaKs), which revealed that they are mostly nonoverlapping, although both prefer α and β domain proteins. Consistent with the entire M. xanthus proteome, MxDnaK substrates have both significantly more multi-domain proteins and a higher isoelectric point than that of Escherichia coli, which encodes a single DnaK homolog. MxDnaK1 is transcriptionally upregulated in response to heat shock and prefers to bind cytosolic proteins, while MxDnaK2 is downregulated by heat shock and is more associated with membrane proteins. Using domain swapping, we show that the nucleotide-binding domain and the substrate-binding β domain are responsible for the significant differences in DnaK interactomes, and the nucleotide binding domain also determines the dimerization of MxDnaK2, but not MxDnaK1. Our work suggests that bacterial DnaK has been duplicated in order to deal with a more complex proteome, and that this allows evolution of distinct domains to deal with different subsets of target proteins.IMPORTANCEAll eukaryotic and ~40% of prokaryotic species encode multiple 70 kDa heat shock protein (Hsp70) homologs with similar but diversified functions. Here, we show that duplication of canonical Hsp70 (DnaK in prokaryotes) correlates with increasing proteomic complexity and evolution of particular regions of the protein. Using the Myxococcus xanthus DnaK duplicates as a case, we found that their substrate spectrums are mostly nonoverlapping, and are both consistent to that of Escherichia coli DnaK in structural and molecular characteristics, but show differential enrichment of membrane proteins. Domain/region swapping demonstrated that the nucleotide-binding domain and the β substrate-binding domain (SBDβ), but not the SBDα or disordered C-terminal tail region, are responsible for this functional divergence. This work provides the first direct evidence for regional evolution of DnaK paralogs.

ABSTRACT Different habitats harbor different microbial communities with elusive assembly mechanisms. This study comprehensively investigated the global assembly mechanisms of microbial communities and effects of community-internal influencing factors using the Earth Microbiome Project (EMP) data set. We found that deterministic and stochastic processes contribute approximately equally to global microbial community assembly, and, specifically, deterministic processes generally play a major role in free-living and plant-associated (but not plant corpus) environments, while stochastic processes are the major contributor in animal-associated environments. In contrast with the assembly of microorganisms, the assembly of functional genes, predicted from PICRUSt, is mainly attributed to deterministic processes in all microbial communities. The sink and source microbial communities are normally assembled using similar mechanisms, and the core microorganisms are specific to different environment types. On a global scale, deterministic processes are positively related to the community alpha diversity, microbial interaction degree and bacterial predatory-specific gene abundance. Our analysis provides a panoramic picture and regularities of global and environment-typical microbial community assemblies. IMPORTANCE With the development of sequencing technologies, the research topic of microbial ecology has evolved from the analysis of community composition to community assembly, including the relative contribution of deterministic and stochastic processes for the formation and maintenance of community diversity. Many studies have reported the microbial assembly mechanisms in various habitats, but the assembly regularities of global microbial communities remain unknown. In this study, we analyzed the EMP data set using a combined pipeline to explore the assembly mechanisms of global microbial communities, microbial sources to construct communities, core microbes in different environment types, and community-internal factors influencing assembly. The results provide a panoramic picture and rules of global and environment-typical microbial community assemblies, which enhances our understandings of the mechanisms globally controlling community diversity and species coexistence.

ABSTRACT Differential transcription of functionally divergent duplicate genes is critical for bacterial cells to properly and competitively function in the environment, but the transcriptional regulation mechanisms remain in mystery. Myxococcus xanthus DK1622 possesses two duplicate groELs with divergent functions. Here, we report that MXAN_4468, an orphan gene located upstream of groEL2, encodes a response regulator (RR) and is responsible for the differential expression regulation of duplicate groELs. This RR protein realizes its negative regulatory role via a novel dual-mode functioning manner: binding to the transcription repressor HrcA to enhance its transcriptional inhibition of duplicate groELs and binding to the 3′ end of the MXAN_4468 sequence to specifically decrease the transcription of the following groEL2. Phosphorylation at the conserved 61st aspartic acid is required to trigger the regulatory functions of MXAN_4468. Pull-down experiment and mutation demonstrated that two noncognate CheA proteins, respectively belonging to the Che8 and Che7 chemosensory pathways, are involved in the protein phosphorylation. A transcriptome analysis, as well as the pull-down experiment, suggested that MXAN_4468 plays a global negative regulatory role in M. xanthus. This study elucidates, for the first time, the regulatory mechanism of differential transcription of bacterial duplicate groELs and suggests a global regulatory role of a dual-functional orphan RR. IMPORTANCE Multiply copied groELs require precise regulation of transcriptions for their divergent cellular functions. Here, we reported that an orphan response regulator (RR) tunes the transcriptional discrepancy of the duplicate groELs in Myxococcus xanthus DK1622 in a dual-functional mode. This RR protein has a conserved phosphorylation site, and the phosphorylation is required for the regulatory functions. Transcriptomic analysis, as well as a pull-down experiment, suggests that the RR plays a global regulatory role in M. xanthus. This study highlights that the dual-functional orphan RR might be involved in conducting the transcriptional symphony to stabilize the complex biological functions in cells.

ABSTRACT Protein toxins secreted by prokaryotes have been found to affect the pathogenicity of pathogens or directly mediate antagonistic interactions between prokaryotes. PAAR proteins are important carriers of toxic effectors and are located at the forefront of either the type VI secretion system (T6SS) or the extracellular contractile injection system (eCIS). This study systematically investigated PAAR homologues and related toxic effectors. We found that PAAR homologues were divided into 8 types and 16 subtypes and distributed in 23.1% of bacterial genomes and 7.8% of archaeal genomes. PAAR proteins of all types fold into a highly similar conical structure, even from relatively diverse underlying sequences. PAAR homologues associated with different secretion systems display a mixed phylogenetic relationship, indicating that PAAR proteins from such a subtype can be assembled on either a T6SS or an eCIS. More than 1,300 PAAR-related toxic effector genes were identified; one PAAR subtype can be associated with toxins of over 40 families, and toxins from one family can be associated with more than 10 PAAR subtypes. A large-scale comparison of Earth Microbiome Project data and prokaryotic genomes revealed that prokaryotes encoding PAAR genes are widely present in diverse environments worldwide, and taxa encoding multiple PAAR gene copies exhibit a wider distribution in environments than other taxa. Overall, our studies highlighted that PAAR proteins are versatile clips loaded with antimicrobial toxin bullets for secretion weapons (T6SS and eCIS), greatly enriching the weapon arsenal of prokaryotes, which, often together with VgrG, help prokaryotes fight for survival advantages in crowded environments. IMPORTANCE Infectious diseases caused by microbial pathogens are severe threats to human health and economic development. To respond to these threats, it is necessary to understand how microorganisms survive in and adapt to complex environments. Microorganic toxins, which are widely distributed in nature, are the key weapons in life domain interactions. PAAR proteins are important carriers of prokaryotic toxic effectors. We reveal the versatility of PAAR proteins between secretory systems and the massive diversity of toxic effectors carried by PAAR proteins, which helps prokaryotes enrich their arsenal and expand their ability to attack their neighbors. A large number of PAAR homologues and related toxic effectors enhance the survival competitiveness of prokaryotic populations. In conclusion, our work provides an example for large-scale analysis of the global distribution and ecological functions of prokaryotic functional genes.

ABSTRACT Vegetation represents probably the most crucial step for the ecosystem functions of wetlands, but it is unclear how microbial populations and functions shift along with vegetation. In this study, we found that the richness and diversity of soil bacteria increased with vegetation levels and that the community composition was distinctly shifted from bare to vegetative places. The bare land displayed an extremely high abundance of Cyanobacteria as a monospecies genus, while a Gemmatimonadetes genus was predominant as multiple species in all the vegetative wetlands, suggesting their important ecosystem functions and potential mechanisms. Expression of the genes related to photosynthesis was enriched exclusively in bare land. Genes involved in biological organic carbon metabolism and the cycling of main elements (C, N, S, and P) were highly expressed in vegetative wetlands and were mostly included in the metagenome-assembled genome (MAG) of Gemmatimonadetes. Some compounds identified from soil metabolomic results also corresponded to pathways involving these key active genes. Cyanobacteria is thus responsible for the carbon sink in early infertile wetlands, and Gemmatimonadetes plays a crucial role in ecosystem functions in vegetative wetlands. Our results highlight that the soil microbial populations execute ecosystem functions for wetlands and that vegetation is the determinant for the population and functional shifts in the coastal estuarine wetland of the Yellow River Delta. IMPORTANCE Vegetation probably represents the most crucial step for the ecosystem functions of wetlands, but it is unclear how microbial populations and functions shift in pace with the colonization and succession of vegetation. In this study, we found that a Cyanobacteria monospecies genus and a Gemmatimonadetes multispecies genus are fastidiously predominant in the bare and vegetative wetlands of the Yellow River Delta, respectively. Consistently, photosynthesis genes were enriched exclusively in bare land, while genes involved in biological organic carbon metabolism and the cycling of main elements were highly expressed in vegetative wetlands, were mostly included in the MAG of Gemmatimonadetes, and were consistent with soil metabolomic results. Our results provide insight into the adaptive succession of predominant bacterial species and their ecosystem functions in response to the presence of vegetation.