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Characterization of the regulatory region of a cell interaction-dependent gene in Myxococcus xanthus.

ABSTRACT: omega 4403 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C-signaling, are required for expression of Tn5 lac omega 4403. We have cloned DNA upstream of the omega 4403 insertion site, localized the promoter, and identified a potential open reading frame. From the deduced amino acid sequence, the gene disrupted by Tn5 lac omega 4403 appears to encode a serine protease that is dispensable for development. The gene begins to be expressed between 6 and 12 h after starvation initiates development, as determined by measuring mRNA or beta-galactosidase accumulation in cells containing Tn5 lac omega 4403. The putative transcriptional start site was mapped, and sequences centered near -10 and -35 bp relative to this site show some similarity to the corresponding regions of promoters transcribed by Escherichia coli sigma70 RNA polymerase. However, deletions showed that an essential promoter element lies between -80 and -72 bp, suggesting the possible involvement of an upstream activator protein. DNA downstream of -80 is sufficient for C-signal-dependent activation of this promoter. The promoter is not fully expressed when fusions are integrated at the Mx8 phage attachment site in the chromosome. Titration of a limiting factor by two copies of the regulatory region (one at the attachment site and one at the native site) can, in part, explain the reduced expression. We speculate that the remaining difference may be due to an effect of chromosomal position. These results provide a basis for studies aimed at identifying regulators of C-signal-dependent gene expression.

SUBMITTER: Fisseha M

PROVIDER: S-EPMC177977 | biostudies-other | 1996 May

REPOSITORIES: biostudies-other

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Characterization of the regulatory region of a cell interaction-dependent gene in Myxococcus xanthus.

Fisseha M M Gloudemans M M Gill R E RE Kroos L L

Journal of bacteriology 19960501 9

omega 4403 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C-signaling, are required for expression of Tn5 lac omega 4403. We have cloned DNA upstream of the omega 4403 insertion site, localized the promoter, and identified a potential open reading frame. From the deduced amino acid sequence, the gene disrupted by Tn5 lac omega 4403 appears to en ...[more]

PMID: 8626320

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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.

Dataset Information

Characterization of the regulatory region of a cell interaction-dependent gene in Myxococcus xanthus.

Publications

Characterization of the regulatory region of a cell interaction-dependent gene in Myxococcus xanthus.

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