Homologs of the vancomycin resistance d-Ala-d-Ala dipeptidase VanX in Streptomyces toyocaensis, Escherichia coli and Synechocystis: attributes of catalytic efficiency, stereoselectivity and regulation with implications for function

349

409

Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes͞operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an ntrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, Ϸ2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and 70 -dependent promoters.

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Nitrogen regulatory protein C-controlled genes of Escherichia coli : Scavenging as a defense against nitrogen limitation

Zimmer

Soupène

Lee

et al. 2000

349

409

“…It has been shown that PBP3, which is involved in septum formation, is down-regulated by RpoS upon entry into stationary phase. RpoS is also implicated in remodeling of crosslinks in the cell wall of stationary phase E. coli cells (38). The frequency of direct crosslinks between two diaminopimelate residues on adjacent peptidoglycan strands increases 6-fold in stationary phase, a change paralleled by a decrease in D-Ala-D-Ala crosslinks (39).…”

Section: Discussionmentioning

confidence: 99%

A role for mechanosensitive channels in survival of stationary phase: Regulation of channel expression by RpoS

Stokes

Murray

Subramaniam

et al. 2003

111

101

The mechanosensitive (MS) channels MscS and MscL are essential for the survival of hypoosmotic shock by Escherichia coli cells. We demonstrate that MscS and MscL are induced by osmotic stress and by entry into stationary phase. Reduced levels of MS proteins and reduced expression of mscL-and mscS-LacZ fusions in an rpoS mutant strain suggested that the RNA polymerase holoenzyme containing S is responsible, at least in part, for regulating production of MS channel proteins. Consistent with the model that the effect of S is direct, the MscS and MscL promoters both use RNA polymerase containing S in vitro. Conversely, clpP or rssB mutations, which cause enhanced levels of S , show increased MS channel protein synthesis. RpoS null mutants are sensitive to hypoosmotic shock upon entry into stationary phase. These data suggest that MscS and MscL are components of the RpoS regulon and play an important role in ensuring structural integrity in stationary phase bacteria.

“…We have previously demonstrated the presence of the vanH, vanA, and vanX resis- tance genes in S. toyocaensis and other glycopeptide producing bacteria, precisely in the same orientation and with the same enzymatic activities as the orthologous genes that confer highlevel glycopeptide resistance in Enterococci (VRE) (35). The genes for vanHst, an ␣-ketoacid reductase required for D-lactate biosynthesis (36), vanAst, a D-Ala-D-lactate ligase (13,37) and vanXst, a D-Ala-D-Ala-specific dipeptidase (38), located at the 5Ј-end of the A47934 biosynthetic cluster, represent the first time antibiotic resistance genes have been unambiguously coupled to the biosynthesis genes in a glycopeptide-producing organism. These genes are both necessary and sufficient for host resistance to A47934 by predicted conversion D-Ala-D-Ala in the peptidoglycan layer to D-Ala-D-lactate.…”

Section: The Organization Of the E Domains Predicts A Stereochemistrymentioning

confidence: 99%

Assembling the glycopeptide antibiotic scaffold: The biosynthesis of from Streptomyces toyocaensis NRRL15009

Pootoolal

Thomas

Marshall

et al. 2002

Self Cite

172

163

The glycopeptide antibiotics vancomycin and teicoplanin are vital components of modern anti-infective chemotherapy exhibiting outstanding activity against Gram-positive pathogens including members of the genera Streptococcus, Staphylococcus, and Enterococcus. These antibiotics also provide fascinating examples of the chemical and associated biosynthetic complexity exploitable in the synthesis of natural products by actinomycetes group of bacteria. We report the sequencing and annotation of the biosynthetic gene cluster for the glycopeptide antibiotic A47934 from Streptomyces toyocaensis NRRL15009, the first complete sequence for a teicoplanin class glycopeptide. The cluster includes 34 ORFs encompassing 68 kb and includes all of the genes predicted to be required to synthesize A47934 and regulate its biosynthesis. The gene cluster also contains ORFs encoding enzymes responsible for glycopeptide resistance. This role was confirmed by insertional inactivation of the D-Ala-D-lactate ligase, vanAst, which resulted in the predicted A47934-sensitive phenotype and impaired antibiotic biosynthesis. These results provide increased understanding of the biosynthesis of these complex natural products.G lycopeptide antibiotics (GPAs) have been mainstays of antimicrobial chemotherapy since their discovery in the mid 1950s. These antibiotics act exclusively on Gram-positive bacteria by forming a tight and specific noncovalent complex with the D-Ala-D-Ala terminus of the peptidoglycan, inhibiting cell wall growth and crosslinking (1, 2). Clinical resistance to GPAs was first described in the enterococci in 1988 (3), and resistance has now manifested itself in the more virulent streptococci and staphylococci (reviewed in ref. 4). GPA resistance is now a significant worldwide phenomenon that has severely impacted the health care sector both in increased mortality and morbidity, and economically (5). The predominant mechanism of resistance is the synthesis of cell wall peptidoglycan terminating in D-Ala-D-lactate, which dramatically decreases the affinity of these antibiotics for their target (6).GPAs are exclusively obtained through fermentation, yet despite their importance, their biosynthesis is not well understood. They are comprised of a heptapeptide core consisting of both common and unusual amino acids (4). Crosslinking of the amino acids through aryl ether and carbon-carbon bonds provides rigidity to the peptide. There are two major structural classes of GPAs based on the identity of the core peptide, and the two clinically used GPAs, vancomycin and teicoplanin, exemplify both classes (Fig. 1). An additional class of structurally homologous secondary metabolites with anticomplement activity is exemplified by complestatin (Fig. 1). Structural diversity in these natural products is achieved through changes in the peptide backbone, and selective amino acid halogenation, glycosylation, lipidation, methylation, and sulfonylation. In principle, understanding of the genetic and mechanistic basis of these modifications could lead ...