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Characterization of bcsA mutations that bypass two distinct signaling requirements for Myxococcus xanthus development.

ABSTRACT: The BsgA protease is required for starvation-induced development in Myxococcus xanthus. Bypass suppressors of a bsgA mutant were isolated to identify genes that may encode additional components of BsgA protease-dependent regulation of development. Strain M951 was isolated following Tn5 mutagenesis of a bsgA mutant and was capable of forming fruiting bodies and viable spores in the absence of the BsgA protease. The Tn5Omega951 insertion was localized to a gene, bcsA, that encodes a protein that has significant amino acid similarity to a group of recently described flavin-containing monooxygenases involved in styrene catabolism. Mutations in bcsA bypassed the developmental requirements for both extracellular B and C signaling but did not bypass the requirement for A signaling. Bypass of the B-signaling requirement by the bcsA mutation was accompanied by restored expression of a subset of developmentally induced lacZ fusions to the BsgA protease-deficient strain. bcsA mutant cells developed considerably faster than wild-type cells at low cell density and altered transcriptional levels of a developmentally induced, cell-density-regulated gene (Omega4427), suggesting that the bcsA gene product may normally act to inhibit development in a cell-density-regulated fashion. Bypass of the requirements for both B and C signaling by bcsA mutations suggests a possible link between these two genetically, biochemically, and temporally distinct signaling requirements.

SUBMITTER: Cusick JK

PROVIDER: S-EPMC135330 | biostudies-literature | 2002 Sep

REPOSITORIES: biostudies-literature

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Characterization of bcsA mutations that bypass two distinct signaling requirements for Myxococcus xanthus development.

Cusick John K JK Hager Elizabeth E Gill Ronald E RE

Journal of bacteriology 20020901 18

The BsgA protease is required for starvation-induced development in Myxococcus xanthus. Bypass suppressors of a bsgA mutant were isolated to identify genes that may encode additional components of BsgA protease-dependent regulation of development. Strain M951 was isolated following Tn5 mutagenesis of a bsgA mutant and was capable of forming fruiting bodies and viable spores in the absence of the BsgA protease. The Tn5Omega951 insertion was localized to a gene, bcsA, that encodes a protein that h ...[more]

PMID: 12193631

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Project description:UNLABELLED:Myxococcus xanthus produces several extracellular signals that guide fruiting body morphogenesis and spore differentiation. Mutants defective in producing a signal may be rescued by codevelopment with wild-type cells or cell fractions containing the signal. In this paper, we identify two molecules that rescue development of the E signal-deficient mutant LS1191 at physiological concentrations, iso15:0 branched-chain fatty acid (FA) and 1-iso15:0-alkyl-2,3-di-iso15:0-acyl glycerol (TG1), a development-specific monoalkyl-diacylglycerol. The physiological concentrations of the bioactive lipids were determined by mass spectrometry from developing wild-type cells using chemically synthesized standards. Synthetic TG1 restored fruiting body morphogenesis and sporulation and activated the expression of the developmentally regulated gene with locus tag MXAN_2146 at physiological concentrations, unlike its nearly identical tri-iso15:0 triacylglycerol (TAG) counterpart, which has an ester linkage instead of an ether linkage. iso15:0 FA restored development at physiological concentrations, unlike palmitic acid, a straight-chain fatty acid. The addition of either lipid stimulates cell shortening, with an 87% decline in membrane surface area, concomitantly with the production of lipid bodies at each cell pole and in the center of the cell. We suggest that cells produce triacylglycerol from membrane phospholipids. Bioactive lipids may be released by programmed cell death (PCD), which claims up to 80% of developing cells, since cells undergoing PCD produce lipid bodies before lysing. IMPORTANCE:Like mammalian adipose tissue, many of the M. xanthus lipid body lipids are triacylglycerols (TAGs), containing ester-linked fatty acids. In both systems, ester-linked fatty acids are retrieved from TAGs with lipases and consumed by the fatty acid degradation cycle. Both mammals and M. xanthus also produce lipids containing ether-linked fatty alcohols with alkyl or vinyl linkages, such as plasmalogens. Alkyl and vinyl linkages are not hydrolyzed by lipases, and no clear role has emerged for lipids bearing them. For example, plasmalogen deficiency in mice has detrimental consequences to spermatocyte development, myelination, axonal survival, eye development, and long-term survival, though the precise reasons remain elusive. Lipids containing alkyl- and vinyl-linked fatty alcohols are development-specific products in M. xanthus. Here, we show that one of them rescues the development of E signal-producing mutants at physiological concentrations.

Project description:BackgroundThe Myxococcales are well known for their predatory and developmental social processes, and for the molecular complexity of regulation of these processes. Many species within this order have unusually large genomes compared to other bacteria, and their genomes have many genes that are unique to one specific sequenced species or strain. Here, we describe RNAseq based transcriptome analysis of the FruA regulon of Myxococcus xanthus and a comparative RNAseq analysis of two Myxococcus species, M. xanthus and Myxococcus stipitatus, as they respond to starvation and begin forming fruiting bodies.ResultsWe show that both species have large numbers of genes that are developmentally regulated, with over half the genome showing statistically significant changes in expression during development in each species. We also included a non-fruiting mutant of M. xanthus that is missing the transcriptional regulator FruA to identify the direct and indirect FruA regulon and to identify transcriptional changes that are specific to fruiting and not just the starvation response. We then identified Interpro gene ontologies and COG annotations that are significantly up- or down-regulated during development in each species. Our analyses support previous data for M. xanthus showing developmental upregulation of signal transduction genes, and downregulation of genes related to cell-cycle, translation, metabolism, and in some cases, DNA replication. Gene expression in M. stipitatus follows similar trends. Although not all specific genes show similar regulation patterns in both species, many critical developmental genes in M. xanthus have conserved expression patterns in M. stipitatus, and some groups of otherwise unstudied orthologous genes share expression patterns.ConclusionsBy identifying the FruA regulon and identifying genes that are similarly and uniquely regulated in two different species, this work provides a more complete picture of transcription during Myxococcus development. We also provide an R script to allow other scientists to mine our data for genes whose expression patterns match a user-selected gene of interest.

Project description:Myxococcus xanthus arranges into two morphologically distinct biofilms depending on its nutritional status, i.e., coordinately spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. A secreted polysaccharide, referred to as exopolysaccharide (EPS), is a structural component of both biofilms and is also important for type IV pilus-dependent motility and fruiting body formation. Here, we characterize the biosynthetic machinery responsible for EPS biosynthesis using bioinformatics, genetics, heterologous expression, and biochemical experiments. We show that this machinery constitutes a Wzx/Wzy-dependent pathway dedicated to EPS biosynthesis. Our data support that EpsZ (MXAN_7415) is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for the initiation of the repeat unit synthesis. Heterologous expression experiments support that EpsZ has galactose-1-P transferase activity. Moreover, MXAN_7416, renamed WzxEPS, and MXAN_7442, renamed WzyEPS, are the Wzx flippase and Wzy polymerase responsible for translocation and polymerization of the EPS repeat unit, respectively. In this pathway, EpsV (MXAN_7421) also is the polysaccharide copolymerase and EpsY (MXAN_7417) the outer membrane polysaccharide export (OPX) protein. Mutants with single in-frame deletions in the five corresponding genes had defects in type IV pilus-dependent motility and a conditional defect in fruiting body formation. Furthermore, all five mutants were deficient in type IV pilus formation, and genetic analyses suggest that EPS and/or the EPS biosynthetic machinery stimulates type IV pilus extension. Additionally, we identify a polysaccharide biosynthesis gene cluster, which together with an orphan gene encoding an OPX protein make up a complete Wzx/Wzy-dependent pathway for synthesis of an unknown polysaccharide.IMPORTANCE The secreted polysaccharide referred to as exopolysaccharide (EPS) has important functions in the social life cycle of M. xanthus; however, little is known about how EPS is synthesized. Here, we characterized the EPS biosynthetic machinery and showed that it makes up a Wzx/Wzy-dependent pathway for polysaccharide biosynthesis. Mutants lacking a component of this pathway had reduced type IV pilus-dependent motility and a conditional defect in development. These analyses also suggest that EPS and/or the EPS biosynthetic machinery is important for type IV pilus formation.

Project description: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. coli dnaK 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.

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Characterization of bcsA mutations that bypass two distinct signaling requirements for Myxococcus xanthus development.

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Characterization of bcsA mutations that bypass two distinct signaling requirements for Myxococcus xanthus development.

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