In vivo functional analysis of a class A β-lactamase-related protein essential for clavulanic acid biosynthesis in Streptomyces clavuligerus

ABSTRACT Clavulanic acid is a potent inhibitor of β-lactamase enzymes and is of demonstrated value in the treatment of infections by β-lactam-resistant bacteria. Previously, it was thought that eight contiguous genes within the genome of the producing strainStreptomyces clavuligerus were sufficient for clavulanic acid biosynthesis, because they allowed production of the antibiotic in a heterologous host (K. A. Aidoo, A. S. Paradkar, D. C. Alexander, and S. E. Jensen, p. 219–236, In V. P. Gullo et al., ed., Development in industrial microbiology series, 1993). In contrast, we report the identification of three new genes, orf10 (cyp), orf11(fd), and orf12, that are required for clavulanic acid biosynthesis as indicated by gene replacement andtrans-complementation analysis in S. clavuligerus. These genes are contained within a 3.4-kb DNA fragment located directly downstream of orf9(cad) in the clavulanic acid cluster. While theorf10 (cyp) and orf11(fd) proteins show homologies to other knownCYP-150 cytochrome P-450 and [3Fe-4S] ferredoxin enzymes and may be responsible for an oxidative reaction late in the pathway, the protein encoded by orf12 shows no significant similarity to any known protein. The results of this study extend the biosynthetic gene cluster for clavulanic acid and attest to the importance of analyzing biosynthetic genes in the context of their natural host. Potential functional roles for these proteins are proposed.

Download Full-text

Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

Journal of Bacteriology ◽

10.1128/jb.186.19.6501-6507.2004 ◽

2004 ◽

Vol 186 (19) ◽

pp. 6501-6507 ◽

Cited By ~ 17

Author(s):

A. de la Fuente ◽

J. F. Martín ◽

A. Rodríguez-García ◽

P. Liras

Keyword(s):

Clavulanic Acid ◽

Streptomyces Clavuligerus ◽

Culture Conditions ◽

Transferase Activity ◽

Reaction Products ◽

Acid Biosynthesis ◽

Substrate Specificities ◽

Acetyltransferase Activity ◽

Standard Culture

ABSTRACT The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations.

Download Full-text

Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces clavuligerus.

Journal of Bacteriology ◽

10.1128/jb.177.5.1307-1314.1995 ◽

1995 ◽

Vol 177 (5) ◽

pp. 1307-1314 ◽

Cited By ~ 54

Author(s):

A S Paradkar ◽

S E Jensen

Keyword(s):

Functional Analysis ◽

Clavulanic Acid ◽

Streptomyces Clavuligerus ◽

Acid Biosynthesis ◽

Gene Encoding

Download Full-text

Five Additional Genes Are Involved in Clavulanic Acid Biosynthesis in Streptomyces clavuligerus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.1.192-202.2004 ◽

2004 ◽

Vol 48 (1) ◽

pp. 192-202 ◽

Cited By ~ 38

Author(s):

S. E. Jensen ◽

A. S. Paradkar ◽

R. H. Mosher ◽

C. Anders ◽

P. H. Beatty ◽

...

Keyword(s):

Cell Wall ◽

Clavulanic Acid ◽

Gene Cluster ◽

Acid Production ◽

Streptomyces Clavuligerus ◽

Open Reading Frames ◽

Acid Biosynthesis ◽

Penicillin Binding Proteins ◽

Total Absence ◽

Reading Frames

ABSTRACT An approximately 12.5-kbp region of DNA sequence from beyond the end of the previously described clavulanic acid gene cluster was analyzed and found to encode nine possible open reading frames (ORFs). Involvement of these ORFs in clavulanic acid biosynthesis was assessed by creating mutants with defects in each of the ORFs. orf12 and orf14 had been previously reported to be involved in clavulanic acid biosynthesis. Now five additional ORFs are shown to play a role, since their mutation results in a significant decrease or total absence of clavulanic acid production. Most of these newly described ORFs encode proteins with little similarity to others in the databases, and so their roles in clavulanic acid biosynthesis are unclear. Mutation of two of the ORFs, orf15 and orf16, results in the accumulation of a new metabolite, N-acetylglycylclavaminic acid, in place of clavulanic acid. orf18 and orf19 encode apparent penicillin binding proteins, and while mutations in these genes have minimal effects on clavulanic acid production, their normal roles as cell wall biosynthetic enzymes and as targets for β-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster.

Download Full-text

Deletion of the pyc Gene Blocks Clavulanic Acid Biosynthesis Except in Glycerol-Containing Medium: Evidence for Two Different Genes in Formation of the C3 Unit

Journal of Bacteriology ◽

10.1128/jb.181.22.6922-6928.1999 ◽

1999 ◽

Vol 181 (22) ◽

pp. 6922-6928 ◽

Cited By ~ 32

Author(s):

Rosario Pérez-Redondo ◽

Antonio Rodríguez-García ◽

Juan F. Martín ◽

Paloma Liras

Keyword(s):

Clavulanic Acid ◽

Gene Product ◽

Streptomyces Clavuligerus ◽

Defined Medium ◽

Thiamine Pyrophosphate ◽

Acid Biosynthesis ◽

Acid Protein ◽

In The Wild ◽

Lactamase Inhibitor ◽

Amino Acid Protein

ABSTRACT The β-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster ofStreptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of thepyc gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the pyc gene with theaph gene did not cause isoleucine-valine auxotrophy in the mutant. The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the β-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing d-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. Thepyc replacement mutant overproduces cephamycin.

Download Full-text

Faculty Opinions recommendation of Five additional genes are involved in clavulanic acid biosynthesis in Streptomyces clavuligerus.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1018831.215445 ◽

2004 ◽

Author(s):

Jean-Jacques Sanglier

Keyword(s):

Clavulanic Acid ◽

Streptomyces Clavuligerus ◽

Acid Biosynthesis

Download Full-text

An rplKDelta29-PALG-32 mutation leads to reduced expression of the regulatory genes ccaR and claR and very low transcription of the ceaS2 gene for clavulanic acid biosynthesis in Streptomyces clavuligerus

Molecular Microbiology ◽

10.1111/j.1365-2958.2006.05266.x ◽

2006 ◽

Vol 61 (3) ◽

pp. 758-770 ◽

Cited By ~ 11

Author(s):

Juan Pablo Gomez-Escribano ◽

Paloma Liras ◽

Agustin Pisabarro ◽

Juan F. Martin

Keyword(s):

Clavulanic Acid ◽

Streptomyces Clavuligerus ◽

Regulatory Genes ◽

Acid Biosynthesis

Download Full-text

In Vitro and In Vivo Activities of AM-112, a Novel Oxapenem

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.5.1652-1657.2003 ◽

2003 ◽

Vol 47 (5) ◽

pp. 1652-1657 ◽

Cited By ~ 16

Author(s):

Conor E. Jamieson ◽

Peter A. Lambert ◽

Iain N. Simpson

Keyword(s):

Clavulanic Acid ◽

Enterobacter Cloacae ◽

Penicillin Binding Proteins ◽

Class A ◽

Sepsis Model ◽

Class D ◽

Class C ◽

Lactamase Inhibitor

ABSTRACT AM-112 [(1′R,5R,6R)-3-(4-amino-1,1-dimethyl-butyl)-6-(1′-hydroxyethyl)oxapenem-3-carboxylate] is a novel oxapenem compound which possesses potent β-lactamase-inhibitory properties. Fifty-percent inhibitory concentrations (IC50s) of AM-112 for class A enzymes were between 0.16 and 2.24 μM for three enzymes, compared to IC50s of 0.008 to 0.12 μM for clavulanic acid. Against class C and class D enzymes, however, the activity of AM-112 was between 1,000- and 100,000-fold greater than that of clavulanic acid. AM-112 had affinity for the penicillin-binding proteins (PBPs) of Escherichia coli DC0, with PBP2 being inhibited by the lowest concentration of AM-112 tested, 0.1 μg/ml. Ceftazidime was combined with AM-112 at 1:1 and 2:1 ratios in MIC determination studies against a panel of β-lactamase-producing organisms. These studies demonstrated that AM-112 was effective at protecting ceftazidime against extended-spectrum β-lactamase-producing strains and derepressed class C enzyme producers, reducing ceftazidime MICs by 16- and 2,048-fold. Similar results were obtained when AM-112 was combined with ceftriaxone, cefoperazone, or cefepime in a 1:2 ratio. Protection of ceftazidime with AM-112 was maintained against Enterobacter cloacae P99 and Klebsiella pneumoniae SHV-5 in a murine intraperitoneal sepsis model. The 50% effective dose of ceftazidime against E. cloacae P99 and K. pneumoniae SHV-5 was reduced from >100 and 160 mg/kg of body weight to 2 and 33.6 mg/kg, respectively, when it was combined with AM-112 at a 1:1 ratio. AM-112 demonstrates potential as a new β-lactamase inhibitor.

Download Full-text