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 β-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.

Origins of the β-lactam rings in natural products.

Naturally occurring β-lactam compounds fall into four basic structural groups, the penicillins/cephalosporins, the clavams, the carbapenems and the monocyclic β-lactams. Biosynthetic studies have clarified the steps involved in the formation of the β-lactam ring for the first three of these groups, but the corresponding process or processes for the monocyclic β-lactams remains obscure. Isopenicillin N synthase is responsible for formation of the β-lactam ring in all penicillin/cephalosporin compounds, and the reaction catalyzed is completely separate from that of β-lactam synthetase, the enzyme responsible for ring formation in all clavam compounds.

Latent viral immune inflammatory response model for chronic multisymptom illness.

A latent viral immune inflammatory response (LVIIR) model is presented which integrates factors that contribute to chronic multisymptom illness (CMI) in both the veteran and civilian populations. The LVIIR model for CMI results from an integration of clinical experience with a review of the literature in four distinct areas: (1) studies of idiopathic multisymptom illness in the veteran population including two decades of research on Gulf War I veterans with CMI, (2) new evidence supporting the existence of chronic inflammatory responses to latent viral antigens and the effect these responses may have on the nervous system, (3) recent discoveries concerning the role of vitamin D in maintaining normal innate and adaptive immunity including suppression of latent viruses and regulation of the immune inflammatory response, and (4) the detrimental effects of extreme chronic repetitive stress (ECRS) on the immune and nervous systems. The LVIIR model describes the pathophysiology of a pathway to CMI and presents a new direction for the clinical assessment of CMI that includes the use of neurological signs from a physical exam, objective laboratory data, and a new proposed latent viral antigen-antibody imaging technique for the peripheral and central nervous system.

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.

Biosynthesis of clavam metabolites.

Naturally occurring clavam metabolites include the valuable β-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.

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 β-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 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.

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.

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.

Clavulanic acid biosynthesis and genetic manipulation for its overproduction.

Clavulanic acid, a β-lactamase inhibitor, is used together with β-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 β-lactam compounds.

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.

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.

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.

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.

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.

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.

The Longitudinal Elder Initiative: helping students learn to care for older adults.

This paper describes an educational innovation called the Longitudinal Elder Initiative (LEI), which was developed by a BSN program to improve students' knowledge about the health needs and nursing care of older adults. In the LEI, new nursing students are paired with older adults in the community and develop a relationship with them over the duration of the nursing program. Students complete a variety of assignments designed to facilitate learning gerontological nursing concepts.

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.

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.

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.

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.

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.

Prediction and functional analysis of the replication origin of the linear plasmid pSCL2 in Streptomyces clavuligerus.

pSCL2 (120 kb), one of the linear plasmids found in Streptomyces clavuligerus NRRL3585, was isolated and partially sequenced. Computational analysis of the central region of pSCL2 revealed the presence of two open reading frames that appear to encode proteins highly homologous to RepL1 and RepL2, replication proteins from pSLA2-L, the large linear plasmid in Streptomyces rochei. The S.

The linkage between psychological capital and commitment to organizational mission: a study of nurses.

A variety of studies demonstrate a clear linkage between the positive psychological capacities of employees and desirable workplace outcomes. However, to date, little empirical research exists which has examined this relationship. Given current and projected challenges in healthcare industry, the need for nurses who are committed to the mission of their organization has never been more critical.