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Two systems for conditional gene expression in Myxococcus xanthus inducible by isopropyl-?-D-thiogalactopyranoside or vanillate.

ABSTRACT: Conditional expression of a gene is a powerful tool to study its function and is typically achieved by placing the gene under the control of an inducible promoter. There is, however, a dearth of such inducible systems in Myxococcus xanthus, a well-studied prokaryotic model for multicellular development, cell differentiation, motility, and light response and a promising source of secondary metabolites. The few available systems have limitations, and exogenously based ones are unavailable. Here, we describe two new, versatile inducible systems for conditional expression of genes in M. xanthus. One employs isopropyl-?-d-thiogalactopyranoside (IPTG) as an inducer and is inspired by those successfully applied in some other bacteria. The other requires vanillate as an inducer and is based on the system developed originally for Caulobacter crescentus and recently adapted for mammalian cells. Both systems are robust, with essentially no expression in the absence of an inducer. Depending on the inducer and the amounts added, expression levels can be modulated such that either system can conditionally express genes, including ones that are essential and are required at high levels such as ftsZ. The two systems operate during vegetative growth as well as during M. xanthus development. Moreover, they can be used to simultaneously induce expression of distinct genes within the same cell. The conditional expression systems we describe substantially expand the genetic tool kit available for studying M. xanthus gene function and cellular biology.

SUBMITTER: Iniesta AA

PROVIDER: S-EPMC3486104 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Two systems for conditional gene expression in Myxococcus xanthus inducible by isopropyl-β-D-thiogalactopyranoside or vanillate.

Iniesta Antonio A AA García-Heras Francisco F Abellón-Ruiz Javier J Gallego-García Aránzazu A Elías-Arnanz Montserrat M

Journal of bacteriology 20120824 21

Conditional expression of a gene is a powerful tool to study its function and is typically achieved by placing the gene under the control of an inducible promoter. There is, however, a dearth of such inducible systems in Myxococcus xanthus, a well-studied prokaryotic model for multicellular development, cell differentiation, motility, and light response and a promising source of secondary metabolites. The few available systems have limitations, and exogenously based ones are unavailable. Here, w ...[more]

PMID: 22923595

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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: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.

Dataset Information

Two systems for conditional gene expression in Myxococcus xanthus inducible by isopropyl-?-D-thiogalactopyranoside or vanillate.

Publications

Two systems for conditional gene expression in Myxococcus xanthus inducible by isopropyl-β-D-thiogalactopyranoside or vanillate.

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