- Volume 137, Issue 3, 1991

In Escherichia coli, the expression of the phenylalanine biosynthetic operon pheA is regulated by an attenuation mechanism. In the presence of excess phenylalanine, gene expression was decreased to 10% of the fully deattenuated level. To understand the factors that determine the basal level of pheA expression, we examined the role of ribosome release from the leader peptide stop codon UGA. The transcriptional readthrough from the pheA attenuator decreased by over 2-fold in the presence of the defective release factor 2. However, a release factor 1 (UAG and UAA specific) mutation did not influence the basal level of pheA expression. These results support the proposal that the release of translating ribosomes from the leader peptide stop codon in stem 2 of the pheA attenuator plays a crucial role in determining the basal level of expression of this gene.

Control of methionine biosynthesis in Escherichia coli K12 was reinvestigated by using methionine-analogue-resistant mutants. Norleucine (NL) and a-methylmethionine (MM) were found to inhibit methionine biosynthesis directly whereas ethionine (Et) competitively inhibited methionine utilization. Adenosylation of Et to generate S-adenosylethionine (AdoEt) by cell-free enzyme from E. coli K12 was demonstrated. Tolerance of increasing concentrations of NL by E. coli K12 mutants is expressed serially as phenotypes NLR, NLREtR, NLRMMR and finally NLREtRMMR. All spontaneous NLR mutants had a metK mutation, whereas NTG-induced mutants had mutations in both the metK and metJ genes. The kinetics of methionine adenosylation by the E. coli K12 cell-free enzyme were found to be similar to those reported for the yeast enzyme, showing the typical lag phase at low methionine concentration and disappearance of this phase when AdoMet was included in the incubation mixture. NL extended the lag phase, and lowered the rate of subsequent methionine adenosylation, but did not affect the shortening of the lag phase of adenosylation by AdoMet.

Between 1986 and 1988, multiresistant Klebsiella pneumoniae strains exhibiting high-level cefotaxime resistance were isolated from patient specimens particularly of the intensive care units of the Aachen Technical University Hospital. The resistance gene responsible was shown to be encoded on a conjugative 66 kb plasmid designated pZMPI. The MIC values for cefotaxime of the original isolates and the transconjugants were > 128 mg I-1 and 64 mg I-1. respectively, Isoelectric focusing of protein preparations from the transconjugants showed a β-lactamase with a pl of 7.6. A 3.6 kb BamHl fragment containing the β-lactamase gene was cloned into pLG339 resulting in the recombinant plasmid pZMP1-1. A restriction map of the cloned insert was established and Pst l subfragments of the insert were further subcloned into pBGS18. The nucleotide sequence of the complete 3.6 kb fragment was determined. Within 3663 bp an open reading frame of 858 kb was found to show 99% homology to the SHV-2 and -3 nucleotide sequences. The deduced amino acid sequence differed in one and two positions, respectively, from these established SHV enzymes. The 3′ noncoding sequence exhibited nearly perfect homology to that of SHV-2, but the 5′ upstream sequence showed homology of less than 50% to the corresponding SHV-2 sequence, indicating an altered promoter region of the variant SHV-enzyme. Kinetic analysis of the β-lactamase revealed a 50-100% elevated hydrolytic effectivity on cefotaxime in comparison to other SHV enzymes. Cefoxitin, ceftazidime, aztreonam and imipenem were not hydrolysed by the enzyme. The variant enzyme was inhibited by commonly available β-lactamase inhibitors. Clavulanic acid had the highest affinity for the enzyme and the greatest effectivity in blocking its action. Based on the genetic and kinetic data we propose to classify the enzyme as a new variant β-lactamase of the SHV-type and name it SHV-2a.

Membrane-bound chitin synthase from Fusarium graminearum A3/5 had a pH optimum of 6·5, an apparent temperature optimum of 30 °C and an apparent K m for UDP-N-acetylglucosamine (UDP-GlcNAc) of 2·5 mm. Digitonin-solubilized chitin synthase had a lower (about 25% of the original activity) and more variable activity than the membrane-bound chitin synthase from which it was prepared. The activity of solubilized chitin synthase was stabilized by incorporating Allosamidin (a chitinase inhibitor) into the reaction mixture, and an approximately 3·6-fold increase in activity was observed when the reaction mixture contained dimyristoylphosphatidylcholine. Concentrations of the organophosphorus fungicide edifenphos (Hinosan) up to 25 μm had no effect on the in vitro activity of membrane-bound chitin synthase, but between 25 and 145 μm, chitin synthase activity decreased with increase in fungicide concentration. Edifenphos caused non-competitive inhibition of chitin synthase activity with an apparent K i of 50 μm. Membrane-bound chitin synthase preparations from cultures of F. graminearum grown in 250 μm-edifenphos had a much lower in vitro activity than preparations from cultures grown in the absence of the fungicide. Pre-growth of F. graminearum in 250 μm (but not 80 μm) edifenphos also caused a reduction in incorporation of [3H]GlcNAc into chitin in vivo (washed biomass was incubated in [3H]GlcNAc-containing medium lacking fungicide).

Klebsiella pneumoniae catabolizes both 4-hydroxyphenylacetic acid and 3-hydroxyphenylacetic acid via meta-cleavage of 3,4-dihydroxyphenylacetic acid, ultimately yielding pyruvate and succinate. The organism can synthesize two hydroxylases catalysing 3,4-dihydroxyphenylacetic acid formation, which differ in substrte specificity, cofactor requirement, kinetics and regulation. Five enzymes sequentially involved in the catabolism of 3,4-dihydroxyphenylacetic acid are encoded on a 7 kbp fragment of the K. pneumoniae chromosome that has been isolated in a recombinant plasmid.

Glycerol is catabolized in Aspergillus nidulans by glycerol kinase and a mitochondrial FAD-dependent sn-glycerol-3-phosphate dehydrogenase. The levels of both enzymes are controlled by carbon catabolite repression and by specific introduction. Biochemical and genetical analyses show that dihydroxyacetone and d-glyceraldehyde are converted into glycerol and then catabolized by the same pathway, d-Glyceraldehyde can be reduced by NADP+-dependent glycerol dehydrogenase or by alcohol dehydrogenase I, while dihydroxyacetone is only reduced by the first enzyme. Three new glycerol non-utilizing mutants have been found. These three mutations define three hitherto unknown loci, glcE, glcF and glcG. The mutation in glcG leads to a greatly decreased sn-glycerol-3-phosphate dehydrogenase activity.

Glutathione-deficient mutants (gshA) of the yeast Saccharomyces cerevisiae, impaired in the first step of glutathione (GSH) biosynthesis were studied with respect to the regulation of enzymes involved in GSH catabolism and cysteine biosynthesis. Striking differences were observed in the content of the sulphur amino acids when gshA mutants were compared to wild-type strains growing on the same minimal medium. Furthermore, all mutants examined showed a derepression of γ-glutamyltranspeptidase (γ-GT), the enzyme initiating GSH degradation. However, γ-cystathionase and cysteine synthase were unaffected by the GSH deficiency as long as the nutrient sulphate source was not exhausted. The results suggest that the mutants are probably not impaired in the sulphate assimilation pathway, but that the γ-glutamyl cycle could play a leading role in the regulation of the sulphur fluxes. Studies of enzyme regulation showed that the derepression of γ-GT observed in the gshA strains was most probably due to an alteration of the thiol status. The effectors governing the biosynthesis of cysteine synthase and γ-cystathionase seemed different from those playing a role in γ-GT regulation and it was only under conditions of total sulphate deprivation that all these enzymes were derepressed. As a consequence the endogenous pool of GSH was used in the synthesis of cysteine. GSH might, therefore, fulfil the role of a storage compound.

Plasma membrane ATPase activity of Saccharomyces cerevisiae IGC 3507III grown in the presence of the lipophilic acid octanoic acid [4–50 mg I–1 (0·03–0·35 mM), pH 4·0] was 1·5-fold higher than that in cells grown in its absence. The K m for ATP, the pH profile and the sensitivity to orthovanadate of the basal and the activated forms of the membrane ATPase were identical. This activation was closely associated with a decrease in the biomass yield and an increase in the ethanol yield, and was rapidly reversed in vivo after removal of the acid. However, the activated level was preserved when membranes were extracted and subjected to manipulations which eliminated or decreased octanoic acid incorporation in the plasma membrane. The activity of the basal plasma membrane ATPase in the total membrane fraction was slightly increased by incubation at pH 6·5 with octanoic acid at 100 mg I–1 or less (2·4 mg acid form plus 97·6 mg octanoate ion I–1). However, destruction of the permeability barrier between the enzyme and its substrate could not explain the in vivo activation. A role for plasma membrane ATPase activation in the regulation of the intracellular pH (pHi) of cells grown with octanoic acid was not proven.