δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS): discovery and perspectives.

The δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) tripeptide is the first dedicated intermediate in the biosynthetic pathway leading to the penicillin and cephalosporin classes of ß-lactam natural products in bacteria and fungi. It is synthesized nonribosomally by the ACV synthetase (ACVS) enzyme, which has been purified and partially characterized from many sources. Due to its large size and instability, many details regarding the reaction mechanism of ACVS are still not fully understood. In this review we discuss the chronology and associated methodology that led to the discovery of ACVS, some of the main findings regarding its activities, and some recent/current studies being conducted on the enzyme. In addition, we conclude with perspectives on what can be done to increase our understating of this very important protein in the future.

Origins of the ß-lactam rings in natural products.

Naturally occurring ß-lactam compounds fall into four basic structural groups, the penicillins/cephalosporins, the clavams, the carbapenems and the monocyclic ß-lactams. Biosynthetic studies have clarified the steps involved in the formation of the ß-lactam ring for the first three of these groups, but the corresponding process or processes for the monocyclic ß-lactams remains obscure. Isopenicillin N synthase is responsible for formation of the ß-lactam ring in all penicillin/cephalosporin compounds, and the reaction catalyzed is completely separate from that of ß-lactam synthetase, the enzyme responsible for ring formation in all clavam compounds. Conversely, carbapenam synthetase, the enzyme responsible for ß-lactam ring formation for all carbapenem compounds, shows clear relatedness to ß-lactam synthetase, despite differences in the substrates and the products for the two enzymes. The mechanism of ring formation has not yet been clarified for any of the monocyclic ß-lactams, but a third distinct mechanism of ß-lactam ring formation seems likely, and this group includes such a diverse collection of structures that even more new ring-forming reactions may be involved.

Carboxyethylarginine synthase genes show complex cross-regulation in Streptomyces clavuligerus.

Carboxyethylarginine synthase is the first dedicated enzyme of clavam biosynthesis in Streptomyces clavuligerus and is present in two isoforms encoded by two separate genes. When grown on a liquid soy medium, strains with ceaS1 deleted showed only a mild reduction of clavam biosynthesis, while disruption of ceaS2 abolished all clavam biosynthesis. Creation of an in-frame ceaS2 deletion mutant to avoid polarity did not restore clavam production, nor did creation of a site-directed mutant altered only in a single amino acid residue important for activity. Reverse transcriptase PCR analyses of these mutants indicated that the failure to produce clavam metabolites could be traced to reduced or abolished transcription of ceaS1 in the ceaS2 mutants, despite the location of ceaS1 on a replicon completely separate from that of ceaS2. Western analyses further showed that the CeaS1 protein (as well as the CeaS2 protein) was absent from the ceaS2 mutants. Complementation experiments were able to restore clavam production partially, but only by virtue of restoring CeaS2 production. CeaS1 was still absent from the complemented strains. While this dependence of CeaS1 production on the expression of ceaS2 from its native chromosomal location was seen in all of the ceaS2 mutants, the effect was limited to growth in liquid medium. When the same mutants were grown on solid soy medium, clavam production was restored and CeaS1 was produced, albeit at low levels compared to the wild type.

Biosynthesis of clavam metabolites.

Naturally occurring clavam metabolites include the valuable ß-lactamase inhibitor, clavulanic acid, as well as stereochemical variants with side-chain modifications, called the 5S clavams. Because of the clinical importance of clavulanic acid, most studies of clavam biosynthesis are based on the industrial producer species Streptomyces clavuligerus. Well-characterized early steps in clavam biosynthesis are outlined, and less well understood late steps in 5S clavam biosynthesis are proposed. The complex genetic organization of the clavam biosynthetic genes in S. clavuligerus is described and, where possible, comparisons with other producer species are presented.

5S clavam biosynthesis is controlled by an atypical two-component regulatory system in Streptomyces clavuligerus.

Streptomyces clavuligerus produces a collection of five clavam metabolites, including the clinically important ß-lactamase inhibitor clavulanic acid, as well as four structurally related metabolites called 5S clavams. The paralogue gene cluster of S. clavuligerus is one of three clusters of genes for the production of these clavam metabolites. A region downstream of the cluster was analyzed, and snk, res1, and res2, encoding elements of an atypical two-component regulatory system, were located. Mutation of any one of the three genes had no effect on clavulanic acid production, but snk and res2 mutants produced no 5S clavams, whereas res1 mutants overproduced 5S clavams. Reverse transcriptase PCR analyses showed that transcription of cvm7p (which encodes a transcriptional activator of 5S clavam biosynthesis) and 5S clavam biosynthetic genes was eliminated in snk and in res2 mutants but that snk and res2 transcription was unaffected in a cvm7p mutant. Both snk and res2 mutants could be complemented by introduction of cvm7p under the control of an independently regulated promoter. In vitro assays showed that Snk can autophosphorylate and transfer its phosphate group to both Res1 and Res2, and Snk-H365, Res1-D52, and Res2-D52 were identified as the phosphorylation sites for the system. Dephosphorylation assays indicated that Res1 stimulates dephosphorylation of Res2∼P. These results suggest a regulatory cascade in which Snk and Res2 form a two-component system controlling cvm7p transcription, with Res1 serving as a checkpoint to modulate phosphorylation levels. Cvm7P then activates transcription of 5S clavam biosynthetic genes.

A comparison of the clavam biosynthetic gene clusters in Streptomyces antibioticus Tü1718 and Streptomyces clavuligerus.

The production of clavam metabolites has been studied previously in Streptomyces clavuligerus , a species that produces clavulanic acid as well as 4 other clavam compounds, but the late steps of the pathway leading to the specific end products are unclear. The present study compared the clavam biosynthetic gene cluster in Streptomyces antibioticus , chosen because it produces only 2 clavam metabolites and no clavulanic acid, with that of S. clavuligerus. A cosmid library of S. antibioticus genomic DNA was screened with a clavaminate synthase-specific probe based on the corresponding genes from S. clavuligerus, and 1 of the hybridizing cosmids was sequenced in full. A clavam gene cluster was identified that shows similarities to that of S. clavuligerus but also contains a number of novel genes. Knock-out mutation of the clavaminate synthase gene abolished clavam production in S. antibioticus, confirming the identity of the gene cluster. Knock-out mutation of a novel gene encoding an apparent oxidoreductase also abolished clavam production. A potential clavam biosynthetic pathway consistent with the genes in the cluster and the metabolites produced by S. antibioticus, and correspondingly different from that of S. clavuligerus, is proposed.

Paenibacillus polymyxa PKB1 produces variants of polymyxin B-type antibiotics.

Polymyxins are cationic lipopeptide antibiotics active against many species of Gram-negative bacteria. We sequenced the gene cluster for polymyxin biosynthesis from Paenibacillus polymyxa PKB1. The 40.8 kb gene cluster comprises three nonribosomal peptide synthetase-encoding genes and two ABC transporter-like genes. Disruption of a peptide synthetase gene abolished all antibiotic production, whereas deletion of one or both transporter genes only reduced antibiotic production. Computational analysis of the peptide synthetase modules suggested that the enzyme system produces variant forms of polymyxin B (1 and 2), with D-2,4-diaminobutyrate instead of L-2,4-diaminobutyrate in amino acid position 3. Two antibacterial metabolites were resolved by HPLC and identified by high-resolution mass spectrometry and MS/MS sequencing as the expected variants 3 and 4 of polymyxin B(1) (1) and B(2) (2). Stereochemical analysis confirmed the presence of both D-2,4-diaminobutyrate and L-2,4-diaminobutyrate residues.

Draft genome sequence of Streptomyces clavuligerus NRRL 3585, a producer of diverse secondary metabolites.

Streptomyces clavuligerus is an important industrial strain that produces a number of antibiotics, including clavulanic acid and cephamycin C. A high-quality draft genome sequence of the S. clavuligerus NRRL 3585 strain was produced by employing a hybrid approach that involved Sanger sequencing, Roche/454 pyrosequencing, optical mapping, and partial finishing. Its genome, comprising four linear replicons, one chromosome, and four plasmids, carries numerous sets of genes involved in the biosynthesis of secondary metabolites, including a variety of antibiotics.

Clavulanic acid biosynthesis and genetic manipulation for its overproduction.

Clavulanic acid, a ß-lactamase inhibitor, is used together with ß-lactam antibiotics to create drug mixtures possessing potent antimicrobial activity. In view of the clinical and industrial importance of clavulanic acid, identification of the clavulanic acid biosynthetic pathway and the associated gene cluster(s) in the main producer species, Streptomyces clavuligerus, has been an intriguing research question. Clavulanic acid biosynthesis was revealed to involve an interesting mechanism common to all of the clavam metabolites produced by the organism, but different from that of other ß-lactam compounds. Gene clusters involved in clavulanic acid biosynthesis in S. clavuligerus occupy large regions of nucleotide sequence in three loci of its genome. In this review, clavulanic acid biosynthesis and the associated gene clusters are discussed, and clavulanic acid improvement through genetic manipulation is explained.

Crystal structures of an oligopeptide-binding protein from the biosynthetic pathway of the beta-lactamase inhibitor clavulanic acid.

Clavulanic acid (CA) is a clinically important beta-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 A resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis.

Use of the native flp gene to generate in-frame unmarked mutations in Streptomyces spp.

The generation of unmarked deletion mutants free from polar effects on downstream genes is typically a lengthy and arduous process in Streptomyces spp. The use of FLP recombinase can greatly facilitate this process when combined with established polymerase chain reaction (PCR)-targeting techniques. In vivo production of FLP within Streptomyces cells would streamline the process further, but expression of flp in Streptomyces spp. has proven difficult to achieve. Two Escherichia coli-Streptomyces shuttle plasmids that constitutively express native flp within Streptomyces cells were constructed and tested within Streptomyces clavuligerus and Streptomyces coelicolor to produce in-frame mutations in genes associated with antibiotic production. Only one of the flp-expressing plasmids was functional in S. clavuligerus, but both functioned in S. coelicolor and both were easily lost from cells. Although a separate study has recently shown successful expression of a synthetic flp gene in Streptomyces, this is the first report of expression of the native flp gene within Streptomyces spp. Through the use of these plasmids to generate unmarked deletion mutants, C7p was shown to be essential for production of 5S clavams in S. clavuligerus, and RedJ was demonstrated to be important for optimal undecylprodigiosin biosynthesis in S. coelicolor but traces of the antibiotic were still produced in a DeltaredJ mutant.

Insights into positive and negative requirements for protein-protein interactions by crystallographic analysis of the beta-lactamase inhibitory proteins BLIP, BLIP-I, and BLP.

Beta-lactamase inhibitory protein (BLIP) binds a variety of beta-lactamase enzymes with wide-ranging specificity. Its binding mechanism and interface interactions are a well-established model system for the characterization of protein-protein interactions. Published studies have examined the binding of BLIP to diverse target beta-lactamases (e.g., TEM-1, SME-1, and SHV-1). However, apart from point mutations of amino acid residues, variability on the inhibitor side of this enzyme-inhibitor interface has remained unexplored. Thus, we present crystal structures of two likely BLIP relatives: (1) BLIP-I (solved alone and in complex with TEM-1), which has beta-lactamase inhibitory activity very similar to that of BLIP; and (2) beta-lactamase-inhibitory-protein-like protein (BLP) (in two apo forms, including an ultra-high-resolution structure), which is unable to inhibit any tested beta-lactamase. Despite categorical differences in species of origin and function, BLIP-I and BLP share nearly identical backbone conformations, even at loop regions differing in BLIP. We describe interacting residues and provide a comparative structural analysis of the interactions formed at the interface of BLIP-I.TEM-1 versus those formed at the interface of BLIP.TEM-1. Along with initial attempts to functionally characterize BLP, we examine its amino acid residues that structurally correspond to BLIP/BLIP-I binding hotspots to explain its inability to bind and inhibit TEM-1. We conclude that the BLIP family fold is a robust and flexible scaffold that permits the formation of high-affinity protein-protein interactions while remaining highly selective. Comparison of the two naturally occurring, distinct binding interfaces built upon this scaffold (BLIP and BLIP-I) shows that there is substantial variation possible in the subnanomolar binding interaction with TEM-1. The corresponding (non-TEM-1-binding) BLP surface shows that numerous favorable backbone-backbone/backbone-side-chain interactions with a protein partner can be negated by the presence of a few, strongly unfavorable interactions, especially electrostatic repulsions.

A gene located downstream of the clavulanic acid gene cluster in Streptomyces clavuligerus ATCC 27064 encodes a putative response regulator that affects clavulanic acid production.

Three open reading frames denoted as orf21, orf22, and orf23 were identified from downstream of the currently recognized gene cluster for clavulanic acid biosynthesis in Streptomyces clavuligerus ATCC 27064. The new orfs were annotated after in silico analysis as genes encoding a putative sigma factor, a sensor kinase, and a response regulator. The roles of the individual genes were explored by disruption of the corresponding orfs, and the morphological and antibiotic production phenotypes of the resulting mutants were compared. In orf21 and orf22 mutants, no growth or morphological differences were noted, but modest reduction of cephamycin C (orf21), or both cephamycin C and clavulanic acid production (orf22) compared with wild-type, were observed. In orf23 mutant, cell growth and sporulation was retarded, and clavulanic acid and cephamycin C production were reduced to 40 and 47% of wild-type levels, respectively. Conversely, overexpression of orf23 caused precocious hyperproduction of spores on solid medium, and antibiotic production was increased above the levels seen in plasmid control cultures. Transcriptional analyses were also carried out on orf23 and showed that mutation had little effect on transcription of genes associated with the early stages of cephamycin C or clavulanic acid production but transcription of claR, which regulates the late stages of clavulanic acid production, was reduced in orf23 mutants. These observations suggest that the orf23 product may enable S. clavuligerus to respond to environmental changes by altering cell growth and differentiation. In addition, the effects of ORF23 on growth might indirectly regulate the biosynthesis of secondary metabolites such as clavulanic acid and cephamycin C.

Alanylclavam biosynthetic genes are clustered together with one group of clavulanic acid biosynthetic genes in Streptomyces clavuligerus.

Streptomyces clavuligerus produces at least five different clavam metabolites, including clavulanic acid and the methionine antimetabolite, alanylclavam. In vitro transposon mutagenesis was used to analyze a 13-kb region upstream of the known paralogue gene cluster. The paralogue cluster includes one group of clavulanic acid biosynthetic genes in S. clavuligerus. Twelve open reading frames (ORFs) were found in this area, and mutants were generated in each using either in vitro transposon or PCR-targeted mutagenesis. Mutants with defects in any of the genes orfA, orfB, orfC, or orfD were unable to produce alanylclavam but could produce all of the other clavams, including clavulanic acid. orfA encodes a predicted hydroxymethyltransferase, orfB encodes a YjgF/YER057c/UK114-family regulatory protein, orfC encodes an aminotransferase, and orfD encodes a dehydratase. All of these types of proteins are normally involved in amino acid metabolism. Mutants in orfC or orfD also accumulated a novel clavam metabolite instead of alanylclavam, and a complemented orfC mutant was able to produce trace amounts of alanylclavam while still producing the novel clavam. Mass spectrometric analyses, together with consideration of the enzymes involved in its production, led to tentative identification of the novel clavam as 8-OH-alanylclavam, an intermediate in the proposed alanylclavam biosynthetic pathway.

Functional effects of increased copy number of the gene encoding proclavaminate amidino hydrolase on clavulanic acid production in Streptomyces clavuligerus ATCC 27064.

The effect of increasing levels of proclavaminate amidino hydrolase (Pah) on the rate of clavulanic acid production in Streptomyces clavuligerus ATCC 27064 was evaluated by knock-in a gene (pah2) encoding Pah. A strain (SMF5703) harboring a multicopy plasmid containing the pah2 gene showed significantly retarded cell growth and reduced clavulanic acid production, possibly attributable to the deleterious effects of the multicopy plasmid. In contrast, a strain (SMF5704) carrying a single additional copy of pah2 introduced into chromosome via an integrative plasmid showed enhanced production of clavulanic acid and increased levels of pah2 transcripts. Analysis of transcripts of other genes involved in the clavulanic acid biosynthetic pathway revealed a pattern similar to that seen in the parent. From these results, it appears that clavulanic acid production can be enhanced by duplication of pah2 through integration of a second copy of the gene into chromosome. However, increasing the copy number of only one gene, such as pah2, does not affect the expression of other pathway genes, and so only modest improvements in clavulanic acid production can be expected. Flux controlled by Pah did increase when the copy number of pah2 was doubled, suggesting that under these growth conditions, Pah levels may be a limiting factor regulating the rate of clavulanic acid biosynthesis in S. clavuligerus.

Nonribosomal biosynthesis of fusaricidins by Paenibacillus polymyxa PKB1 involves direct activation of a D-amino acid.

Paenibacillus polymyxa PKB1 produces fusaricidins, a family of lipopeptide antibiotics that strongly inhibits the growth of many plant pathogenic fungi. The fusaricidin biosynthetic gene cluster was cloned and sequenced, and it spans 32.4 kb, including an open reading frame (fusA) encoding a six-module nonribosomal peptide synthetase. The second, fourth, and fifth modules of fusaricidin synthetase each contain an epimerization domain, consistent with the structure of fusaricidins. However, no epimerization domain is found in the sixth module, corresponding to D-Ala. This sixth adenylation domain was produced at a high level in Escherichia coli and is shown to activate D-Ala specifically, providing evidence for direct activation of a D-amino acid by a prokaryotic peptide synthetase. The fusaricidin gene cluster also includes genes involved in the biosynthesis of the lipid moiety, but no genes for resistance, regulation, or transport functions were encountered.

Involvement of beta-lactamase inhibitory protein, BLIP-II, in morphological differentiation of Streptomyces exfoliatus SMF19.

The beta-lactamase inhibitory protein, BLIP-II, found in the culture supernatant of Streptomyces exfoliatus SMF19, shows no discernible sequence identity with other beta-lactamase inhibitory proteins identified in Streptomyces spp. A null mutant of the gene encoding BLIP-II (bliB::hygr) showed a bald appearance on solid media. Although BLIP-II was initially isolated from the supernatant of submerged cultures, sites of BLIP-II accumulation were seen in the cell envelope. The mutation of bliB was also associated with changes in the formation of septa and condensation of the chromosomal DNA associated with sporulation. The bliB mutant exhibited infrequent septa, showing dispersed chromosomal DNA throughout the mycelium, whereas the condensed chromosomes of the wild-type were separated by regularly spaced septa giving the appearance of a string of beads. Therefore, on the basis of these results, it is suggested that BLIP-II is a regulator of morphological differentiation in S. exfoliatus SMF19.

pcd mutants of Streptomyces clavuligerus still produce cephamycin C.

Biosynthesis of cephamycin C in Streptomyces clavuligerus involves the initial conversion of lysine to alpha-aminoadipic acid. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase carry out this two-step reaction, and genes encoding each of these enzymes are found within the cephamycin C gene cluster. However, while mutation of the lat gene causes complete loss of cephamycin production, pcd mutants still produce cephamycin at 30% to 70% of wild-type levels. Cephamycin production by pcd mutants could be restored to wild-type levels either by supplementation of the growth medium with alpha-aminoadipic acid or by complementation of the mutation with an intact copy of the pcd gene. Neither heterologous PCR nor Southern analyses showed any evidence for the presence of a second pcd gene. Furthermore, cell extracts from pcd mutants lack detectable PCD activity. Cephamycin production in the absence of detectable PCD activity suggests that S. clavuligerus must have some alternate means of producing the aminoadipyl-cysteinyl-valine needed for cephamycin biosynthesis.

Use of PCR-targeted mutagenesis to disrupt production of fusaricidin-type antifungal antibiotics in Paenibacillus polymyxa.

Paenibacillus polymyxa (formerly Bacillus polymyxa) PKB1 has been identified as a potential agent for biocontrol of blackleg disease of canola, caused by the pathogenic fungus Leptosphaeria maculans. The factors presumed to contribute to disease suppression by strain PKB1 include the production of fusaricidin-type antifungal metabolites that appear around the onset of bacterial sporulation. The fusaricidins are a family of lipopeptide antibiotics consisting of a beta-hydroxy fatty acid linked to a cyclic hexapeptide. Using a reverse genetic approach based on conserved motifs of nonribosomal peptide synthetases, a DNA fragment that appears to encode the first two modules of the putative fusaricidin synthetase (fusA) was isolated from PKB1. To confirm the involvement of fusA in production of fusaricidins, a modified PCR targeting mutagenesis protocol was developed to create a fusA mutation in PKB1. A DNA fragment internal to fusA was replaced by a gene disruption cassette containing two antibiotic resistance genes for independent selection of apramycin resistance in Escherichia coli and chloramphenicol resistance in P. polymyxa. Inclusion of an oriT site in the disruption cassette allowed efficient transfer of the inactivated fusA allele to P. polymyxa by intergeneric conjugation. Targeted disruption of fusA led to the complete loss of antifungal activity against L. maculans, suggesting that fusA plays an essential role in the nonribosomal synthesis of fusaricidins.

5S clavam biosynthetic genes are located in both the clavam and paralog gene clusters in Streptomyces clavuligerus.

The Streptomyces clavuligerus clavam gene cluster was examined to identify genes specifically involved in 5S clavam biosynthesis. A reduction/loss of 5S clavam production was seen in cvm2 and cvm5 gene mutants, and a clavam metabolite not previously observed, 2-carboxymethylideneclavam, accumulated in the cvm5 mutant. Disruption of additional genes from the region of the clavam cluster did not have any effect on 5S clavam production. Examination of the paralog gene cluster region for 5S clavam biosynthetic genes led to the identification of cvm6P and cvm7P, which encode a putative aminotransferase and a transcriptional regulator, respectively. Mutants defective in cvm6P and cvm7P were completely blocked in 5S clavam but not clavulanic acid production. The loss of 5S clavam production in cvm7P mutants suggests that this gene encodes a transcriptional regulator specific for 5S clavam metabolite biosynthesis.