Functional investigation of the two ClpPs and three ClpXs in Myxococcus xanthus DK1622.

ClpXP is a protease complex that plays important roles in protein quality control and cell cycle regulation, but the functions of multiple ClpXs and multiple ClpPs in M. xanthus remain unknown. The genome of Myxococcus xanthus DK1622 contains two clpPs and three clpXs. The clpP1 and clpX1 genes are cotranscribed and are both essential, while the other copies are isolated in the genome and are deletable. The deletion of clpX2 caused the mutant to be deficient in fruiting body development, while the clpX3 gene is involved in resistance to thermal stress. Both ClpPs possess catalytic active sites, but only ClpP1 shows in vitro peptidase activity on the typical substrate Suc-LY-AMC. All of these clpP and clpX genes exhibit strong transcriptional upregulation in the stationary phase, and the transcription of the three clpX genes appears to be coordinated. Our results demonstrated that multiple ClpPs and multiple ClpXs are functionally divergent and may assist in the environmental adaptation and functional diversification of M. xanthus.IMPORTANCEClpXP is an important protease complex of bacteria and is involved in various physiological processes. Myxococcus xanthus DK1622 possesses two ClpPs and three ClpXs with unclear functions. We investigated the functions of these genes and demonstrated the essential roles of clpP1 and clpX1. Only ClpP1 has in vitro peptidase activity on Suc-LY-AMC, and the isolated clpX copies participate in distinct cellular processes. All of these genes exhibited significant transcriptional upregulation in the stationary phase. Divergent functions appear in multiple ClpPs and multiple ClpXs in M. xanthus DK1622.

Redundancy in microbiota-mediated suppression of the soybean cyst nematode.

BACKGROUND: Soybean cyst nematodes (SCN) as animal parasites of plants are not usually interested in killing the host but are rather focused on completing their life cycle to increase population, resulting in substantial yield losses. Remarkably, some agricultural soils after long-term crop monoculture show a significant decline in SCN densities and suppress disease in a sustainable and viable manner. However, relatively little is known about the microbes and mechanisms operating against SCN in such disease-suppressive soils. RESULTS: Greenhouse experiments showed that suppressive soils (S) collected from two provinces of China and transplantation soils (CS, created by mixing 10% S with 90% conducive soils) suppressed SCN. However, SCN suppressiveness was partially lost or completely abolished when S soils were treated with heat (80 °C) and formalin. Bacterial community analysis revealed that the specific suppression in S and CS was mainly associated with the bacterial phylum Bacteroidetes, specifically due to the enrichment of Chitinophaga spp. and Dyadobacter sp., in the cysts. SCN cysts colonized by Chitinophaga spp. showed dramatically reduced egg hatching, with unrecognizable internal body organization of juveniles inside the eggshell due to chitinase activity. Whereas, Dyadobacter sp. cells attached to the surface coat of J2s increased soybean resistance against SCN by triggering the expression of defence-associated genes. The disease-suppressive potential of these bacteria was validated by inoculating them into conducive soil. The Dyadobacter strain alone or in combination with Chitinophaga strains significantly decreased egg densities after one growing cycle of soybeans. In contrast, Chitinophaga strains alone required more than one growing cycle to significantly reduce SCN egg hatching and population density. CONCLUSION: This study revealed how soybean monoculture for decades induced microbiota homeostasis, leading to the formation of SCN-suppressive soil. The high relative abundance of antagonistic bacteria in the cyst suppressed the SCN population both directly and indirectly. Because uncontrolled proliferation will likely lead to quick demise due to host population collapse, obligate parasites like SCN may have evolved to modulate virulence/proliferation to balance these conflicting needs. Video Abstract.

DnaK duplication and specialization in bacteria correlates with increased proteome complexity.

The 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 ß 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 ß 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 ß substrate-binding domain (SBDß), 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.

A Dual-Functional Orphan Response Regulator Negatively Controls the Differential Transcription of Duplicate groELs and Plays a Global Regulatory Role in Myxococcus.

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.

Bioinformatic and Functional Characterization of Hsp70s in Myxococcus xanthus.

Hsp70 proteins are among the most ubiquitous chaperones and play important roles in maintaining proteostasis and resisting environmental stress. Multiple copies of Hsp70s are widely present in eukaryotic cells with redundant and divergent functions, but they have been less well investigated in prokaryotes. Myxococcus xanthus DK1622 is annotated as having many hsp70 genes. In this study, we performed a bioinformatic analysis of Hsp70 proteins and investigated the functions of six hsp70 genes in DK1622, including two genes that encode proteins with the conserved PRK00290 domain (MXAN_3192 and MXAN_6671) and four genes that encode proteins with the cl35085 or cd10170 domain. We found that only MXAN_3192 is essential for cell survival and heat shock induction. MXAN_3192, compared with the other hsp70 genes, has a high transcriptional level, far exceeding that of any other hsp70 gene, which, however, is not the reason for its essentiality. Deletion of MXAN_6671 (sglK) led to multiple deficiencies in development, social motility, and oxidative resistance, while deletion of each of the other four hsp70 genes decreased sporulation and oxidative resistance. MXAN_3192 or sglK, but not the other genes, restored the growth deficiency of the E. colidnaK mutant. Our results demonstrated that the PRK00290 proteins play a central role in the complex cellular functions of M. xanthus, while the other diverse Hsp70 superfamily homologues probably evolved as helpers with some unknown specific functions.IMPORTANCE Hsp70 proteins are highly conserved chaperones that occur in all kingdoms of life. Multiple copies of Hsp70s are often present in genome-sequenced prokaryotes, especially taxa with complex life cycles, such as myxobacteria. We investigated the functions of six hsp70 genes in Myxococcus xanthus DK1622 and demonstrated that the two Hsp70 proteins with the PRK00290 domain play a central role in complex cellular functions in M. xanthus, while other Hsp70 proteins probably evolved as helpers with some unknown specific functions.