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Molecular cloning, sequence analysis, and characterization of a penicillin-resistant DD-carboxypeptidase of Myxococcus xanthus.

ABSTRACT: We have cloned a gene, pdcA, from the genomic library of Myxococcus xanthus with an oligonucleotide probe representing conserved regions of penicillin-resistant DD-carboxypeptidases. The amino- and carboxy-terminal halves of the predicted pdcA gene product showed significant sequence similarity to N-acetylmuramoyl-L-alanine amidase and penicillin-resistant DD-carboxypeptidase, respectively. The pdcA gene was expressed in Escherichia coli, and the characteristics of the gene product were similar to those of DD-carboxypeptidase (VanY) of vancomycin-resistant enterococci. No apparent changes in cell growth, sporulation, or germination were observed in pdcA deletion mutants.

SUBMITTER: Kimura Y

PROVIDER: S-EPMC103608 | biostudies-literature | 1999 Aug

REPOSITORIES: biostudies-literature

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Molecular cloning, sequence analysis, and characterization of a penicillin-resistant DD-carboxypeptidase of Myxococcus xanthus.

Kimura Y Y Takashima Y Y Tokumasu Y Y Sato M M

Journal of bacteriology 19990801 15

We have cloned a gene, pdcA, from the genomic library of Myxococcus xanthus with an oligonucleotide probe representing conserved regions of penicillin-resistant DD-carboxypeptidases. The amino- and carboxy-terminal halves of the predicted pdcA gene product showed significant sequence similarity to N-acetylmuramoyl-L-alanine amidase and penicillin-resistant DD-carboxypeptidase, respectively. The pdcA gene was expressed in Escherichia coli, and the characteristics of the gene product were similar ...[more]

PMID: 10419975

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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.

Project description:Penicillin-binding protein 5 (PBP 5) of Escherichia coli hydrolyzes the terminal D-Ala-D-Ala peptide bond of the stem peptides of the cell wall peptidoglycan. The mechanism of PBP 5 catalysis of amide bond hydrolysis is initial acylation of an active site serine by the peptide substrate, followed by hydrolytic deacylation of this acyl-enzyme intermediate to complete the turnover. The microscopic events of both the acylation and deacylation half-reactions have not been studied. This absence is addressed here by the use of explicit-solvent molecular dynamics simulations and ONIOM quantum mechanics/molecular mechanics (QM/MM) calculations. The potential-energy surface for the acylation reaction, based on MP2/6-31+G(d) calculations, reveals that Lys47 acts as the general base for proton abstraction from Ser44 in the serine acylation step. A discrete potential-energy minimum for the tetrahedral species is not found. The absence of such a minimum implies a conformational change in the transition state, concomitant with serine addition to the amide carbonyl, so as to enable the nitrogen atom of the scissile bond to accept the proton that is necessary for progression to the acyl-enzyme intermediate. Molecular dynamics simulations indicate that transiently protonated Lys47 is the proton donor in tetrahedral intermediate collapse to the acyl-enzyme species. Two pathways for this proton transfer are observed. One is the direct migration of a proton from Lys47. The second pathway is proton transfer via an intermediary water molecule. Although the energy barriers for the two pathways are similar, more conformers sample the latter pathway. The same water molecule that mediates the Lys47 proton transfer to the nitrogen of the departing D-Ala is well positioned, with respect to the Lys47 amine, to act as the hydrolytic water in the deacylation step. Deacylation occurs with the formation of a tetrahedral intermediate over a 24 kcal x mol(-1) barrier. This barrier is approximately 2 kcal x mol(-1) greater than the barrier (22 kcal x mol(-1)) for the formation of the tetrahedral species in acylation. The potential-energy surface for the collapse of the deacylation tetrahedral species gives a 24 kcal x mol(-1) higher energy species for the product, signifying that the complex would readily reorganize and pave the way for the expulsion of the product of the reaction from the active site and the regeneration of the catalyst. These computational data dovetail with the knowledge on the reaction from experimental approaches.

Dataset Information

Molecular cloning, sequence analysis, and characterization of a penicillin-resistant DD-carboxypeptidase of Myxococcus xanthus.

Publications

Molecular cloning, sequence analysis, and characterization of a penicillin-resistant DD-carboxypeptidase of Myxococcus xanthus.

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