Identification and molecular characterization of an endoglucanase gene, celS, from the extremely thermophilic archaeon Sulfolobus solfataricus.

A genomic region upstream of the alcohol dehydrogenase (Ssadh) gene was cloned and sequenced from a library of Sulfolobus solfataricus MT4 strain. The isolated 4,040-bp DNA fragment revealed an open reading frame (celS), lying in the opposite direction to Ssadh, which showed significant similarity to endo-beta-1,4-glucanases from Pyrococcus furiosus, Thermotoga maritima, and Thermotoga neapolitana. celS was shown to be a functional gene in vivo: a specific celS mRNA was detected by primer extension analysis showing a unique initiation transcription site coinciding with the ATG translation initiation codon. The specific gene product was detected as an extracellular cellulase after enzyme staining by carboxymethyl cellulose (CMC) SDS-PAGE, showing a molecular weight in agreement with that deduced from the open reading frame. Depending on growth conditions, different levels of cellulase activity and specific celS transcript were detected, revealing an inductive effect of CMC and suggesting a repressive role of glucose.

Amoxicillin/clavulanate-associated hepatic failure with progression to Stevens-Johnson syndrome.

OBJECTIVE: To describe a patient who developed hepatic failure, Stevens-Johnson syndrome (SJS), and died after receiving amoxicillin/clavulanate therapy. CASE SUMMARY: A 37-year-old white man without significant past medical history received a 10-day course of amoxicillin/clavulanate for treatment of pneumonia. Thirty-two days after starting amoxicillin/clavulanate, he developed jaundice, rash, pruritus, and increasing fatigue. On further evaluation, with the exclusion of toxicity from other drugs or diseases, the time course to development of cholestatic jaundice correlated with the use of amoxicillin/clavulanate. The patient consequently died with progressive hepatic failure, renal failure, and SJS. DISCUSSION: Hepatic injury has been reported with amoxicillin/clavulanate. Signs and symptoms of jaundice and pruritus may appear up to to six weeks after stopping therapy. Most cases of liver injury have been benign and reversible on discontinuation of the amoxicillin/clavulanate. Reported hepatic reactions have been mainly cholestatic, with some mixed cholestatic/hepatocellular liver function test abnormalities. CONCLUSIONS: Clinicians should be aware of amoxicillin/clavulanate as a drug capable of causing hepatitis with eventual systemic dysfunction. While recovery is usually complete following withdrawal of the drug, in patients with rash associated with hepatic dysfunction, renal insufficiency, or other unusual symptoms, earlier consideration of initiating systemic steroids or liver transplantation referral, in hopes of avoiding progressive systemic response, might be worthwhile.

Proline biosynthesis in Streptococcus thermophilus: characterization of the proBA operon and its products.

The presence of proline in the medium was not essential for growth of Streptococcus thermophilus, indicating that there is a proline biosynthetic pathway in this organism. Genetic and biochemical analysis identified and characterized this pathway. Two genes, designated proB and proA, were cloned, sequenced and characterized. Biochemical analysis of the proB- and proA-encoded enzymes showed that the proline biosynthetic pathway of S. thermophilus is similar to the one previously described in Escherichia coli. The deduced amino acid sequence of a 2-408 kb DNA region containing the genes revealed the similarity of the S. thermophilus gene products to ProB and ProA of E. coli and Serratia marcescens, and to the corresponding N- and C-terminal domains of the bifunctional plant enzyme delta 1-pyrroline-5-carboxylate synthetase of Vigna aconitifolia. Northern blot analysis showed that the two genes in S. thermophilus are organized in a single operon with proB proximal and proA distal to the promoter; primer extension analysis indicated that proBA transcription is not under repressive control by exogenously supplied proline.

Characterization of the rho genes of Neisseria gonorrhoeae and Salmonella typhimurium.

We have cloned and sequenced the genomic regions encompassing the rho genes of Neisseria gonorrhoeae and Salmonella typhimurium. Rho factor of S. typhimurium has only three amino acid differences with respect to the Escherichia coli homolog. Northern (RNA) blots and primer extension experiments were used to characterize the N. gonorrhoeae rho transcript and to identify the transcription initiation and termination elements of this cistron. The function of the Rho factor of N. gonorrhoeae was investigated by complementation assays of rho mutants of E. coli and S. typhimurium and by in vivo transcription assays in polar mutants of S. typhimurium.

Further characterization of the histidine gene cluster of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis of hisD.

We have further characterized the genomic region of Streptomyces coelicolor A3(2) that contains genes involved in the biosynthesis of histidine. A 2,357-base pair fragment contained in plasmid pSCH3328 that complemented hisD mutations has been sequenced. Computer analysis revealed an open reading frame that encodes a protein with significant homology to the Escherichia coli, Salmonella typhimurium and Mycobacterium smegmatis hisD product, Saccharomyces cerevisiae HIS4C, and Neurospora crassa his3 gene products. Two other contiguous open reading frames oriented divergently with respect to hisD did not show significant similarity with any of the his genes or to other sequences included in the gene bank. S1 nuclease mapping and primer extension experiments indicate that the transcription initiation site of the his-specific mRNA coincides with the GUG translation initiation codon of the hisD cistron.

Intrathecal clonidine and the response to hemorrhage.

Intraspinally administered alpha 2-adrenergic agonists are being examined for postoperative analgesia, yet their effects on the hemodynamic response to acute hemorrhage have not been examined. In this study chronically prepared conscious sheep received thoracic intrathecal saline or clonidine 300 micrograms followed in 15 min by rapid removal of 1,000 ml blood. In saline-treated ewes blood pressure was maintained and heart rate steadily increased during hemorrhage of up to 700 ml blood, with further blood removal resulting in rapid decreases in both variables. In contrast, heart rate never increased and blood pressure was maintained only up to 400 ml blood loss in animals receiving intrathecal clonidine. Compared to saline controls, clonidine did not alter blood pressure or heart rate at the end of hemorrhage or during blood pressure restitution during the next hour. Clonidine inhibited the increase in plasma epinephrine at the end of hemorrhage without altering plasma norepinephrine, vasopressin, renin, or atrial natriuretic factor. Intrathecal idazoxan, a specific alpha 2-adrenergic antagonist, reversed clonidine's effect on blood pressure during hemorrhage. Intravenous DG-5128, a poorly lipid-soluble alpha 2-adrenergic antagonist, also reversed clonidine's effect and additionally completely blocked any reduction in blood pressure and heart rate during hemorrhage. These data suggest that intrathecal clonidine interferes with maintenance of blood pressure during hemorrhage, likely because of a spinal sympatholytic effect, but does not affect the ultimate decrease in blood pressure after rapid removal of 1,000 ml blood. This difference in effect during the two phases of hemorrhage can be explained by the relative importance of the sympathetic nervous system in each.

A consensus motif common to all Rho-dependent prokaryotic transcription terminators.

We have characterized at the molecular level several polar mutations in four different cistrons of the his operon of S. typhimurium. An analysis of the his-specific transcripts produced in vivo in the mutant strains, together with in vitro transcription assays, led to the identification of several cryptic Rho-dependent transcription termination elements within the his operon that are activated by the uncoupling of transcription and translation. Common features of these elements were sought and found with a computer program. We have identified a consensus motif, consisting of a cytosine-rich and guanosine-poor region, that is located upstream of the heterogeneous 3' endpoints of the prematurely terminated in vivo transcripts and that is present in all the Rho-dependent transcription terminators described thus far.

Cloning and characterization of the histidine biosynthetic gene cluster of Streptomyces coelicolor A3(2).

Biochemical and genetic data indicate that in Streptomyces coelicolor A3(2) the majority of the genes involved in the biosynthesis of histidine are clustered in a small region of the chromosome [Carere et al., Mol. Gen. Genet. 123 (1973) 219-224; Russi et al., Mol. Gen. Genet. 123 (1973) 225-232]. To investigate the structural organization and the regulation of these genes, we have constructed genomic libraries from S. coelicolor A3(2) in pUC vectors. Recombinant clones were isolated by complementation of an Escherichia coli hisBd auxotroph. A recombinant plasmid containing a 3.4-kb fragment of genomic DNA was further characterized. When cloned in the plasmid vector, pIJ699, this fragment was able to complement S. coelicolor A3(2) hisB mutants. Overlapping clones spanning a 15-kb genomic region were isolated by screening other libraries with labeled DNA fragments obtained from the first clone. Derivative clones were able to complement mutations in four different cistrons of the his cluster of S. coelicolor A3(2). Nucleotide sequence analysis of a 4-kb region allowed the identification of five ORFs which showed significant homology with the his gene products of E. coli. The order of the genes in S. coelicolor A3(2) (5'--hisD-hisC-hisBd-hisH-hisA-3') is the same as in the his operon of E. coli.

Enhanced acetohydroxy acid synthase III activity in an ilvH mutant of Escherichia coli K-12.

The acetohydroxy acid synthase III isozyme, which catalyzes the first common step in the biosynthesis of isoleucine, leucine, and valine in Escherichia coli K-12, is composed of two subunits, the ilvI and ilvH gene products. A missense mutation in ilvH (ilvH612), which reduced the sensitivity of the enzyme to the end product inhibition by valine, also increased its specific activity and lowered the Km for alpha-acetolactate synthesis. The mutation increased the sensitivity of acetohydroxy acid synthase III to dialysis and heat treatment and reduced the requirement for thiamine pyrophosphate addition to the assay mixture for activity. A strain carrying the ilvH612 mutation grew better than a homologous ilvH+ strain in the presence of leucine. The data indicate that this is a consequence of a more active acetohydroxy acid synthase III isozyme rather than the result of an alteration of the leucine-mediated repression of the ilvIH operon.

Correlation of beta-lactamase stability and antibacterial activity of beta-lactams in beta-lactamase-producing bacteria and respective transconjugants.

A total of 343 Gram-negative bacteria were isolated and identified from urine specimens of patients with urinary tract infections. All the bacteria were investigated for their production of beta-lactamases by the nitrocefin test. beta-lactamase-producing strains were tested by the Datta method to detect any transfer of beta-lactamase production to a receiving E. coli K12 RN-strain. MICs of six beta-lactamase-stable compounds (ceftazidime, ceftriaxone, cefamandole, cefoxitin, cefotaxime, cefuroxime) were determined against all the beta-lactamase transferring bacteria and their respective transconjugants by a miniaturized dilution broth method. beta-lactamases produced by donors and transconjugants were purified and identified by determination of the isoelectric point by focusing; their hydrolytic activity was assessed by a spectrophotometric method using cytochrome c reduction. A total of 129 bacteria out of 343 produced beta-lactamases and 27 of these transferred the beta-lactamase production by conjugation. The beta-lactamases isolated from donors and transconjugants had the same pl and the same substrate profile. Ceftazidime was more stable to all the beta-lactamases isolated and more active against all the bacteria examined than the other compounds.

Comparative activity of ceftizoxime and four other cephalosporins against gram negative bacteria and their sensitivity to beta-lactamases.

The in vitro activity of ceftizoxime compared with other beta-lactamase stable compounds was assessed against recent urinary gram negative isolates. 343 bacterial strains were isolated from patients affected by UTI, identified by standard bacteriological methods and investigated for their production of beta-lactamases by Nitrocefin test. MICs and MBCs of ceftizoxime, ceftriaxone, cefamandole, cefoxitin and cefotaxime were determined by a miniaturized dilution broth method against all beta-lactamase producing bacteria (129 out of 343). Sensitivity of antibiotics to beta-lactamases isolated and semi-purified by ultrasonic disruption and high speed centrifugation was assessed by a spectrophotometric method. In vitro antibacterial activity of each antibiotics was correlated to their sensitivity to isolated beta-lactamases. Ceftizoxime showed lower MIC and MBC values and lower MBC/MIC ratio than the other compounds against all the bacteria including Pseudomonas. Ceftizoxime was not hydrolized by any isolated beta-lactamase.

Comparative activity of ceftazidime and four other cephalosporins against gram-negative bacteria and their sensitivity to beta-lactamases.

The in vitro activity of ceftazidime compared with other beta-lactamase stable compounds was assessed against recent Gram-negative isolates. Three hundred forty-three bacterial strains were isolated from patients affected with UTI, identified by standard bacteriological methods and investigated for their production of beta-lactamases by the Nitrocefin test. MICs and MBCs (minimum inhibitory concentrations and minimum bactericidal concentration) of ceftazidime, cefamandole, cefoxitin, cefotaxime and cefuroxime were determined by a miniaturized dilution broth method against all beta-lactamase producing bacteria (129 out of 343). Sensitivity of the antibiotics to beta-lactamases isolated and semipurified by ultrasonic disruption and high-speed centrifugation was assessed by a spectrophotometric method. The in vitro antibacterial activity of each antibiotic was correlated to its sensitivity to isolated beta-lactamases. Ceftazidime showed lower MIC and MBC values and lower MBC/MIC ratios than the other compounds against all the bacteria including Pseudomonas spp. In addition, ceftazidime was not hydrolyzed significantly by any isolated beta-lactamases.

Comparative in vitro activity of norfloxacin and four other chemotherapeutics against urinary gram-negative isolates.

Three hundred seventy-five Gram-negative bacterial strains were isolated from urine specimens of as many patients affected by symptomatic or asymptomatic urinary tract infections. Susceptibility of bacteria to five chemotherapeutics (norfloxacin, oxolinic acid, pipemidic acid, nalidixic acid and nitrofurantoin) was studied determining minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of each compound by a miniaturized dilution broth method. Norfloxacin, a quinoline carboxylic acid compound structurally related to nalidixic acid, showed a much higher antibacterial activity against all bacterial strains under examination including Pseudomonas sp. The best activity of norfloxacin was expressed either by lower MICs and lower MBCs with respect to those of the other compounds, or by a low MBC/MIC ratio, which represents an important advantage in clinical practice.