Search PubMed⌕ Search

Biomedical subjects

S E Jensen

Publications and source records attributed to S E Jensen.

At least 19 recordsLinked to original sources
PubMed2004

Five additional genes are involved in clavulanic acid biosynthesis in Streptomyces clavuligerus.

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 beta-lactam antibiotics, together with their clustered location, suggest that they are part of the clavulanic acid gene cluster.

Anti-Bacterial Agents↗
PubMed2001

Applications of gene replacement technology to Streptomyces clavuligerus strain development for clavulanic acid production.

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine epsilon-aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.

Cephamycins↗
PubMed2001

Prognostic value of left ventricular diastolic function and association with heart rate variability after a first acute myocardial infarction.

OBJECTIVE: To study the prognostic value of left ventricular (LV) diastolic function and its relation with autonomic balance expressed by heart rate variability (HRV) in patients after a first acute myocardial infarction. DESIGN: The study population consisted of 64 consecutive patients with first acute myocardial infarction and 31 control subjects. Long and short term HRV indices were evaluated by 24 hour Holter monitoring, and LV systolic and diastolic function were assessed by two dimensional and Doppler echocardiography before discharge. Patients were divided into two groups: those with restrictive LV filling characteristics (deceleration time </= 140 ms) and those with non-restrictive LV filling characteristics (deceleration time > 140 ms). RESULTS: Both long and short term HRV indices were significantly reduced in patients with restrictive LV filling compared with the non-restrictive group and control subjects. Mitral deceleration time and isovolumetric relaxation time correlated weakly but significantly with all indices of HRV whereas ejection fraction correlated weakly with the long term HRV indices. The mean follow up time was 14.9 (8.7) months. Multivariate analysis showed that mitral deceleration time (chi(2) = 6.4, p < 0.001) and ejection fraction </= 40% (chi(2) = 4.4, p < 0.05) were independent predictors of cardiac death and readmission to hospital with congestive heart failure. CONCLUSIONS: A restrictive LV filling pattern was found to be the strongest predictor of adverse outcome independent of HRV and ejection fraction during follow up after a first acute myocardial infarction. Patients with restrictive LV filling characteristics had more reduced HRV than those with non-restrictive diastolic filling.

Adult↗
PubMed2000

Structure-based design guides the improved efficacy of deacylation transition state analogue inhibitors of TEM-1 beta-Lactamase(,).

Transition state analogue boronic acid inhibitors mimicking the structures and interactions of good penicillin substrates for the TEM-1 beta-lactamase of Escherchia coli were designed using graphic analyses based on the enzyme's 1.7 A crystallographic structure. The synthesis of two of these transition state analogues, (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboronic acid (1) and (1R)-1-acetamido-2-(3-carboxy-2-hydroxyphenyl)ethylboronic acid (2), is reported. Kinetic measurements show that, as designed, compounds 1 and 2 are highly effective deacylation transition state analogue inhibitors of TEM-1 beta-lactamase, with inhibition constants of 5.9 and 13 nM, respectively. These values identify them as among the most potent competitive inhibitors yet reported for a beta-lactamase. The best inhibitor of the current series was (1R)-1-phenylacetamido-2-(3-carboxyphenyl)ethylboronic acid (1, K(I) = 5.9 nM), which resembles most closely the best known substrate of TEM-1, benzylpenicillin (penicillin G). The high-resolution crystallographic structures of these two inhibitors covalently bound to TEM-1 are also described. In addition to verifying the design features, these two structures show interesting and unanticipated changes in the active site area, including strong hydrogen bond formation, water displacement, and rearrangement of side chains. The structures provide new insights into the further design of this potent class of beta-lactamase inhibitors.

Acylation↗
PubMed2000

Relationship between serum amino-terminal propeptide of type III procollagen and changes of left ventricular function after acute myocardial infarction.

BACKGROUND: The amino-terminal propeptide of type III procollagen (PIIINP) is a marker of type III collagen synthesis, which has previously been shown to correlate with infarct size in nonthrombolyzed myocardial infarction (MI) and to provide prognostic information after MI. METHODS AND RESULTS: The relationship between PIIINP and changes of left ventricular (LV) function was studied in 47 consecutive patients with first acute MI and 16 control subjects. Serum PIIINP analysis was measured daily during hospitalization and on days 90, 180, and 360. LV function was assessed by echocardiography on days 1, 5, 90, and 360. Patients with MI were stratified according to their serum PIIINP value at day 4 (group A, </=5.0 microg/L; group B, >5.0 microg/L). On arrival, LV function and size were comparable between groups A (n=31) and B (n=16). LV ejection fraction, initially depressed (day 1: group A, 47+/-7% versus group B, 47+/-8%; P=NS), increased significantly in group A (day 360: 54+/-8%, P<0.001) but was unchanged in group B (day 360: 43+/-8%, P=NS). LV volumes increased significantly in group B (P<0. 05) but not in group A. Furthermore, patients in group B developed signs of restrictive LV diastolic filling. Multivariate regression analysis identified PIIINP >5.0 microg/L and deceleration </=140 ms as independent predictors of cardiac death or complicating heart failure during follow-up. CONCLUSIONS: PIIINP assessed in the subacute phase of MI relates to long-term changes of LV function and provides clinical prognostic information.

Acute Disease↗
PubMed2000

Serial changes and prognostic implications of a Doppler-derived index of combined left ventricular systolic and diastolic myocardial performance in acute myocardial infarction.

The purpose of this study was to investigate the serial changes and prognostic value of a nongeometric Doppler-derived index of myocardial function that combines systolic and diastolic time intervals of the left ventricle in acute myocardial infarction (AMI). The Doppler index was measured in 60 consecutive patients with AMI and in 30 patients admitted to hospital with suspected but disproved AMI who served as controls. The patients were studied at days 1, 5, 90, and 360 after arrival in the coronary care unit. The index was defined as the sum of isovolumetric contraction time, and isovolumetric relaxation time divided by ejection time was measured from mitral inflow and left ventricular outflow Doppler velocity profiles. The index was significantly higher in patients with AMI than in control subjects at days 1 and 360 (day 1, 0.58 +/- 0.09 vs 0.41 +/- 0.08, p <0.0001; day 360, 0.50 +/- 0.09 vs 0.39 +/- 0.07, p <0.01, respectively). The index decreased significantly in patients with AMI during follow-up (p <0.01). The index was significantly higher in patients who developed congestive heart failure or died compared with survivors who were free of congestive heart failure (day 1, 0.63 +/- 0.10 vs 0.53 +/- 0.10, p <0.01; day 360, 0.56 +/- 0.08 vs 0.48 +/- 0.10, p <0.01, respectively). During 20.2 +/- 8.5 months' follow-up, 10 patients died of cardiac causes and 13 developed congestive heart failure. Univariate analyses demonstrated that the Doppler index > or =0.60 (chi-square 8.3, p <0.0001), deceleration time < or =140 ms (chi-square 8.5, p <0.0001), ejection fraction < or =0.40% (chi-square 3.3, p <0.005), anterior wall AMI (chi-square 3.2, p <0.01), and age (chi-square 1.06/ year increase, p <0.01) were significant predictors of outcome. Multivariate stepwise analysis showed that the index < or =0.60 (chi-square 3.4, p <0.05), deceleration time < or =140 ms (chi-square 4.2, p <0.02), and age (chi-square 1.06/year increase, p <0.02) were independent predictors of outcome. The Doppler index reflects severity of left ventricular function and has incremental prognostic value in patients with AMI.

Age Factors↗
PubMed2000

The Bloch equations in high-gradient magnetic resonance force microscopy: theory and experiment.

We report theory and observations of paramagnetic resonance in a measured field gradient of 44,000 T per meter by the technique of magnetic resonance force microscopy (MRFM). Resonance was induced in a dilute solid solution of diphenylpicrylhydrazyl in polystyrene at 77 and 10 K by an amplitude-modulated microwave field. This modulated the force between resonant sample spins and a micrometer-scale SmCo magnetic tip on a force microscope cantilever. The force signals were typically of order 10 fN, and were detected above a thermal noise floor of 80 aN per root hertz at 10 K, equivalent to a magnetic moment noise of 200 micro(B) per root hertz of bandwidth. Resonance saturation was readily observed. Starting with the Bloch equations, we derived simple analytic expressions for the predicted cantilever signal amplitudes and T(1)-dependent phase lags, valid at low microwave power levels. For power levels below saturation, the data were in good agreement with the Bloch equation predictions, while above saturation the measured force increased more slowly with power than predicted. Several ESR mechanisms which might lead to non-Bloch dynamics in the MRFM environment are reviewed. Spin-relaxation mechanisms are also reviewed. A detailed description of the experimental apparatus is offered.

Electron Spin Resonance Spectroscopy↗
PubMed2000

Improvement of exercise capacity and left ventricular diastolic function with metoprolol XL after acute myocardial infarction.

BACKGROUND: Left ventricular (LV) diastolic function predicts and correlates with exercise capacity. Beta-blockers improve exercise capacity and LV diastolic function in patients with severe LV systolic dysfunction in dilated cardiomyopathy. However, information on the effect of metoprolol XL on exercise capacity in relation to LV diastolic function in patients with mild to moderate LV systolic dysfunction after acute myocardial infarction is limited. METHODS: In a randomized, double-blind, placebo-controlled study of 77 patients, a subgroup of 59 patients with mild to moderate LV systolic dysfunction after acute myocardial infarction were given metoprolol XL (n = 29) or placebo (n = 30). The effects of metoprolol XL on exercise capacity in relation to effects on LV diastolic filling were studied. Two-dimensional Doppler echocardiography and maximal symptom limited bicycle test were performed on days 5 through 7 and after 3 months. RESULTS: Maximal exercise capacity increased in the metoprolol XL group (124 +/- 30 W vs 135 +/- 29 W) compared with placebo (125 +/- 31 W vs 126 +/- 34 W) (P <.01). E/A ratio decreased, A peak velocity increased, reverse pulmonary flow decreased, and deceleration time was significantly prolonged in the metoprolol XL group. A significant correlation was found between the changes of deceleration time (metoprolol XL: rho = 0.69, P <.0001; placebo: rho = 0.31, P = not significant) and A peak velocity (metoprolol XL: rho = 0.71, P <.0001; placebo: rho = -0.15, not significant) in relation to changes of exercise capacity. CONCLUSION: Metoprolol XL increases exercise capacity after 3 months, and this change seems related to improvement of LV diastolic filling after acute myocardial infarction.

Adrenergic beta-Antagonists↗
PubMed2000

Value of the Doppler index of myocardial performance in the early phase of acute myocardial infarction.

Prospective assessment of a nongeometric Doppler-derived index of combined systolic and diastolic myocardial performance was performed in 64 patients with acute myocardial infarction (MI) within 1 hour after their arrival to the hospital and in 39 age-matched healthy subjects. The index is defined as the sum of isovolumetric contraction time and relaxation time divided by ejection time, and is obtained by Doppler measurement from the mitral inflow and left ventricular outflow velocity-time intervals. The index was significantly higher in patients with MI compared with healthy subjects (P <.0001). In patients with MI and in-hospital congestive heart failure (CHF), the index was significantly higher compared with patients without CHF. In a multivariate regression analysis, the index >0.45 was the strongest independent predictor of the development of CHF. This simply obtained nongeometric Doppler index, assessed in the early phase of MI, detected and graded left ventricular dysfunction and identified patients at risk for the development of CHF.

Adult↗
PubMed2000

Enzymes catalyzing the early steps of clavulanic acid biosynthesis are encoded by two sets of paralogous genes in Streptomyces clavuligerus.

Genes encoding the proteins required for clavulanic acid biosynthesis and for cephamycin biosynthesis are grouped into a "supercluster" in Streptomyces clavuligerus. Nine open reading frames (ORFs) associated with clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, including six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavulanic acid biosynthesis is unclear, and one ORF not previously reported. Evidence for the involvement of these ORFs in clavulanic acid production was obtained by generating mutants and showing that all were defective for clavulanic acid production when grown on starch asparagine medium. However, when five of the nine mutants, including mutants defective in known clavulanic acid biosynthetic enzymes, were grown in a soy-based medium, clavulanic acid-producing ability was restored. This ability to produce clavulanic acid when seemingly essential biosynthetic enzymes have been mutated suggests that paralogous genes encoding functionally equivalent proteins exist for each of the five genes but that these paralogues are expressed only in the soy-based medium. The five genes that have paralogues encode proteins involved in the early steps of the pathway common to the biosynthesis of both clavulanic acid and the other clavam metabolites produced by this organism. No evidence was seen for paralogues of the four remaining genes involved in late, clavulanic acid-specific steps in the pathway.

Biological Assay↗
PubMed2000

Early cephamycin biosynthetic genes are expressed from a polycistronic transcript in Streptomyces clavuligerus.

A polycistronic transcript that is initiated at the lat promoter has been implicated in the expression of the genes involved in early steps of cephamycin C biosynthesis in Streptomyces clavuligerus. pcbC is also expressed as a monocistronic transcript from its own promoter. However, an alternative interpretation involving expression via three separate yet interdependent transcripts has also been proposed. To distinguish between these possibilities, mutants lacking the lat promoter and containing a transcription terminator within the lat gene (Deltalat::tsr/term mutants) were created. This mutation eliminated the production of lysine-epsilon-aminotransferase (the lat gene product) but also affected the expression of downstream genes, indicating an operon arrangement. Production of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) (the pcbAB gene product) was eliminated in Deltalat::tsr/term mutants, while production of isopenicillin N synthase (IPNS) (the pcbC gene product) was greatly reduced. The provision of alpha-aminoadipate to the Deltalat::tsr/term mutants, either via exogenous feeding or via lat gene complementation, did not restore production of ACVS or IPNS. Analysis of RNA isolated from the Deltalat::tsr/term mutants confirmed that the polycistronic transcript was absent but also indicated that monocistronic pcbC transcript levels were greatly decreased. In contrast, Deltalat mutants created by in-frame internal deletion of lat maintained the polycistronic transcript and allowed production of wild-type levels of both ACVS and IPNS.

Cephamycins↗
PubMed2000

Mechanisms associated with methiocarb resistance in Frankliniella occidentalis (Thysanoptera: Thripidae).

Biochemical mechanisms associated with methiocarb resistance were examined in laboratory-selected and field populations of the western flower thrips, Frankliniella occidentalis (Pergande). Seven populations were examined and they differed in their susceptibility to methiocarb by 30 times. Including the synergists piperonyl butoxide, a cytochrome P-450 monooxygenase inhibitor, or S,S,S-tributylphosphorotrithioate, an esterase inhibitor, in the methiocarb bioassays partially suppressed resistance in the most resistant populations. In vitro assays of general esterase, glutathione S-transferase, and acetylcholinesterase activities showed increased activity in some of the resistant populations and increased activity of the enzymes after methiocarb selection on one of the populations. Assays of acetylcholinesterase sensitivity to inhibition by methiocarb, dichlorvos, and eserine suggested insensitive acetylcholinesterase in two of the resistant populations. These results indicate that methiocarb resistance in F. occidentalis was polyfactorial and involved detoxification and altered target site. None of the biochemical assays showed interpopulation enzymatic differences strongly correlated with the level of methiocarb resistance. The possibilities for developing rapid biochemical diagnostic assays to detect methiocarb resistance in F. occidentalis are discussed.

Animals↗
PubMed1999

Effects of long-term adrenergic beta-blockade on left ventricular diastolic filling in patients with acute myocardial infarction.

BACKGROUND: Left ventricular (LV) systolic and diastolic function are known to be affected in the wake of a myocardial infarction (MI). beta-Adrenergic blocking agents have demonstrated improvement of LV systolic and diastolic function in patients with dilated cardiomyopathy and theoretically would have same beneficial effects in MI. beta-Adrenergic blocking agents are widely used in MI; however only few reports on changes of LV systolic and diastolic function during long-term treatment after acute MI are available. METHODS: Two-dimensional and Doppler echocardiography were used to evaluate LV diastolic filling in 77 patients randomly assigned to placebo (n = 38) or metoprolol (n = 39). The patients were randomly assigned at day 5 to 7 (baseline) after acute MI and were treated for 12 months. LV diastolic filling was assessed by pulsed Doppler measurements of transmitral and pulmonary venous flow. RESULTS: Mitral E-wave deceleration time was prolonged in the metoprolol group (baseline vs 12 months: 167 +/- 51 ms to 218 +/- 36 ms; P =. 01) versus the placebo group (baseline vs 12 months: placebo 174 +/- 46 ms to 189 +/- 41 ms), which implies a less restrictive filling of the LV in the metoprolol group. This was supported by a decrease of E/A ratio (baseline vs 12 months: placebo, 1.06 +/- 0.40 to 0.96 +/- 0.29; metoprolol, 1.09 +/- 0.33 to 0.80 +/- 0.21; P =.05) and prolongation of the isovolumetric relaxation time in the metoprolol treated group (baseline vs 12 months: placebo, 83 +/- 19 ms to 95 +/- 20 ms; metoprolol, 82 +/- 23 ms to 117 +/- 22 ms; P =.01). The difference between mitral A wave and pulmonary venous flow reversal duration was significantly changed during follow-up (baseline vs 12 months: placebo, 22 +/- 11 ms to 24 +/- 11; metoprolol, 11 +/- 21 to 32 +/- 17 ms; P =.02). Patients with normal LV filling pattern at baseline in the metoprolol group preserved a normal LV filling pattern during the study, and patients with restrictive LV filling pattern in the metoprolol group had a nonrestrictive LV filling pattern develop. Maximal or near maximal changes of the diastolic Doppler measurements occurred by 3 months of follow-up, whereas a significant increase in LV ejection fraction was noted after 12 months treatment with metoprolol. CONCLUSIONS: Long-term treatment with the beta-blocking agent metoprolol seems to improve LV diastolic filling after acute MI. Less restrictive LV filling was noted during beta-blockade indicated by a significant prolongation of the mitral E deceleration time, which was predominantly noted in patients with restrictive LV filling. This observation might have prognostic implications because this LV filling pattern is known to be associated with poor outcome. The changes of LV diastolic filling occurred during the first 3 months, whereas systolic recovery was seen at up to 12 months of treatment.

Adrenergic beta-Antagonists↗
PubMed1999

Longitudinal changes and prognostic implications of left ventricular diastolic function in first acute myocardial infarction.

BACKGROUND: Left ventricular (LV) diastolic dysfunction contributes to signs and symptoms of clinical heart failure and may be related to prognosis in heart diseases. LV diastolic dysfunction is reported to be present in acute myocardial infarction (MI); however, little is known about the time course of changes in LV diastolic function and its relation to prognosis after acute MI. METHODS AND RESULTS: Two-dimensional and Doppler echocardiographic examinations were performed in 58 consecutive patients with first acute MI. The patients were studied serially within 1 hour and at days 5, 90, and 360 after arrival to the coronary care unit. LV diastolic function was assessed by Doppler measurements of transmitral and pulmonary venous flow. On the basis of mitral inflow, patients with MI were stratified at baseline to 3 LV diastolic filling patterns: normal, impaired relaxation, or pseudonormal/restrictive. Patients with MI were observed for development of congestive heart failure (Killip class >I) during hospitalization and for death during 1-year follow-up, and these complications were related to LV diastolic function. LV diastolic dysfunction was present in the very early phase of acute MI, with signs of impaired relaxation or restrictive LV filling dynamics in 38% and 24% of the patients, respectively, whereas 38% had normal LV filling characteristics. Impaired relaxation of the LV was most pronounced and found in 60% after 1-year follow-up. In-hospital congestive heart failure (Killip class >I) was found in 50% of the patients with initial impaired LV relaxation and in 71% of the patients with initially pseudonormal or restrictive LV filling dynamics, whereas patients with normal LV filling were free of heart failure. Patients with initial impaired relaxation and restrictive LV filling dynamics demonstrated a significant LV dilation during 1-year follow-up. Patients with initial pseudonormal/restrictive LV filling pattern were more frequently readmitted to the hospital for heart failure and had significant higher New York Heart Association class score compared with patients with normal or impaired relaxation during follow-up. Cardiac death was (n = 6) only observed in patients with pseudonormal or restrictive LV filling pattern. In a multivariate stepwise regression analysis, mitral E deceleration time </=140 ms and age were identified as independent variables related to development of in-hospital congestive heart failure and cardiac death during 12 months of follow-up. CONCLUSIONS: LV diastolic dysfunction is present in the very early phase of MI. LV remodeling and development of in-hospital congestive heart failure appear in patients with very early signs of LV diastolic dysfunction. Furthermore, mitral E deceleration time </=140 ms best identified patients at risk of development of in-hospital congestive heart failure and cardiac death after MI.

Adult↗
PubMed1999

Genes specific for the biosynthesis of clavam metabolites antipodal to clavulanic acid are clustered with the gene for clavaminate synthase 1 in Streptomyces clavuligerus.

Portions of the Streptomyces clavuligerus chromosome flanking cas1, which encodes the clavaminate synthase 1 isoenzyme (CAS1), have been cloned and sequenced. Mutants of S. clavuligerus disrupted in cvm1, the open reading frame located immediately upstream of cas1, were constructed by a gene replacement procedure. Similar techniques were used to generate S. clavuligerus mutants carrying a deletion that encompassed portions of the two open reading frames, cvm4 and cvm5, located directly downstream of cas1. Both classes of mutants still produced clavulanic acid and cephamycin C but lost the ability to synthesize the antipodal clavam metabolites clavam-2-carboxylate, 2-hydroxymethyl-clavam, and 2-alanylclavam. These results suggested that cas1 is clustered with genes essential and specific for clavam metabolite biosynthesis. When a cas1 mutant of S. clavuligerus was constructed by gene replacement, it produced lower levels of both clavulanic acid and most of the antipodal clavams except for 2-alanylclavam. However, a double mutant of S. clavuligerus disrupted in both cas1 and cas2 produced neither clavulanic acid nor any of the antipodal clavams, including 2-alanylclavam. This outcome was consistent with the contribution of both CAS1 and CAS2 to a common pool of clavaminic acid that is shunted toward clavulanic acid and clavam metabolite biosynthesis.

Amino Acid Sequence↗
PubMed1998

A pathway-specific transcriptional activator regulates late steps of clavulanic acid biosynthesis in Streptomyces clavuligerus.

A Streptomyces clavuligerus gene (designated claR) located downstream from the gene encoding clavaminate synthase in the clavulanic acid biosynthetic gene cluster is involved in regulation of the late steps in clavulanic acid biosynthesis. Nucleotide sequence analysis and database searching of ClaR identified a significant similarity to the helix-turn-helix motif (HTH) region of LysR transcriptional regulators. A gene replacement mutant disrupted in claR was unable to produce clavulanic acid, suggesting that claR is essential for clavulanic acid biosynthesis. Furthermore, the accumulation of clavaminic acid in the claR mutant suggested that ClaR regulates the late steps in the clavulanic acid pathway, i.e. those involved in the conversion of clavaminic acid to clavulanic acid. Transcriptional analysis using RNA isolated from the wild type and the claR mutant showed that the expression of the putative late genes, but not the early genes, was regulated by ClaR. High-resolution S1 nuclease analysis of claR suggested that it is expressed as a monocistronic transcript and also as a bicistronic transcript along with the late gene orf-9. The transcription start site of the monocistronic claR transcript was identified as a C residue 155 nucleotides upstream from the claR start codon.

Amino Acid Sequence↗
PubMed1998

Investigation of the Streptomyces clavuligerus cephamycin C gene cluster and its regulation by the CcaR protein.

As part of a search for transcriptional regulatory genes, sequence analysis of several previously unsequenced gaps in the cephamycin biosynthetic cluster has revealed the presence in Streptomyces clavuligerus of seven genes not previously described. These include genes encoding an apparent penicillin binding protein and a transport or efflux protein, as well as the CmcI and CmcJ proteins, which catalyze late reactions in the cephamycin biosynthetic pathway. In addition, we discovered a gene, designated pcd, which displays significant homology to genes encoding semialdehyde dehydrogenases and may represent the gene encoding the long-sought-after dehydrogenase involved in the conversion of lysine to alpha-aminoadipate. Finally, two genes, sclU and rhsA, with no obvious function in cephamycin biosynthesis may define the end of the cluster. The previously described CcaR protein displays homology to a number of Streptomyces pathway-specific transcriptional activators. The ccaR gene was shown to be essential for the biosynthesis of cephamycin, clavulanic acid, and non-clavulanic acid clavams. Complementation of a deletion mutant lacking ccaR and the adjacent orf11 and blp genes showed that only ccaR was essential for the biosynthesis of cephamycin, clavulanic acid, and clavams and that mutations in orf11 or blp had no discernible effects. The lack of cephamycin production in ccaR mutants was directly attributable to the absence of biosynthetic enzymes responsible for the early and middle steps of the cephamycin biosynthetic pathway. Complementation of the ccaR deletion mutant resulted in the return of these biosynthetic enzymes and the restoration of cephamycin production.

Actinomyces↗