- Volume 135, Issue 5, 1989

The HXK2 gene product has an important role in controlling carbon catabolite repression in Saccharomyces cerevisiae. We have raised specific antibodies against the hexokinase PII protein and have demonstrated that it is a 58 kDa phosphoprotein with protein kinase activity. The predict amino acid sequence of the HXK2 gene product has significant homology to the catalytic domain of mammalian and yeast protein kinases. Protein kinase activity was located in a different domain of the protein from the hexose-phosphorylating activity. The hexokinase PII protein level remained unchanged in P2T22D mutant cells (hxk1 HXK2 glk1) growing in a complex medium with glucose. The protein kinase activity of hexokinase PII is regulated by the glucose concentration of the culture medium. Exit from the carbon catabolite repression phase and entry into the derepression phase may be controlled, in part, by modulation of the 58 kDa protein kinase activity by changes in cyclic AMP concentration.

In a clinical isolate of Serratia marcescens different states of low and high resistance to different β-lactam antibiotics considered to be β-lactamase-stable, viz. cefotaxime, ceftizoxime, ceftazidime, aztreonam, cefoxitin and imipenem, were found to be connected with the presence of constitutively overproduced, chromosomally encoded β-lactamase at concentrations in the bacterial periplasm of 0·4 and 0·9 mm, respectively. All the antibiotics were degraded by the β-lactamase. However, kinetic constants varied widely:K m from 92 to 0·012 µm, and k cat from 3·4 to 2 × 10−4 s−1. The relative contributions to resistance by the functioning of periplasmic β-lactamase, resynthesis of this enzyme, and limitation of antibiotic penetration by the bacterial outer membrane were analysed by computer simulations according to steady-state and non-steady-state models of interactions in the periplasm. Results for cefotaxime, ceftizoxime, ceftazidime, aztreonam and latamoxef revealed overproduced β-lactamase as the sole cause of the state of low resistance while antibiotic permeability was the same as in non-resistant S. marcescens strains. In contrast, high resistance was due to β-lactamase action and decreased permeability of antibiotics. For resistance to aztreonam, only, immobilization of the antibiotic as covalent acyl-enzyme by newly synthesized β-lactamase was essential. For cefoxitin, ampicillin and imipenem the analyses indicated that additional resistance factors may play a role, e.g. induction of β-lactamase.

The l-leucine dehydrogenase (LeuDH, EC 1.4.1.9) from the extremely thermophilic Bacillus caldolyticus (DSM 405) has been purified to homogeneity and crystallized. Its physical and biochemical properties, such as subunit structure, M r, amino acid composition, isoelectric point, heat stability, pH-optima, K m values for coenzymes and substrates, and pattern of inhibition have been determined. The native enzyme is an octamer (M r 320000) composed of identical subunits (M r 39000) which contain one intrachain disulphide bridge. Only aliphatic amino acids were accepted as substrates and a preference was exhibited for branched-chain residues. Inhibition occurred only upon incubation with thiol reagents such as HgCl2 and p-mercuribenzoate, and pyridoxal 5'-phosphate. The LeuDH was very thermostable, with 50% residual activity remaining after 30 min incubation at 80 C. Its properties, as well as amino acid composition and N-terminal sequence, were compared with those of the phylogenetically related LeuDH from the mesophile Bacillus cereus. Both enzymes displayed very similar properties: the quaternary structures were identical, amino acid composition alike, the N-terminal sequence showed 62% homology for the first 19 residues, and K m values for the different substrates differed by a factor of two or less, indicating that the active centres had a similar structure. Thus, the only major difference observed was the much higher stability of the thermophilic enzyme to denaturation by heat and organic solvents.

Pseudomonas strain CF600 is able to utilize phenol and 3,4-dimethylphenol as sole carbon and energy source. We demonstrate that growth on these substrates is by virtue of plasmid-encoded phenol hydroxylase and a meta-cleavage pathway. Screening of a genomic bank, with DNA from the previously cloned catechol 2,3-dioxygenase gene of the TOL plasmid pWW0, was used in the identification of a clone which could complement a phenol-hydroxylase-deficient transposon insertion mutant. Deletion mapping and polypeptide production analysis identified a 1·2 kb region of DNA encoding a 39·5 kDa polypeptide which mediated this complementation. Enzyme activities and growth properties of Pseudomonas strains harbouring this fragment on a broad-host-range expression vector indicate that phenol hydroxylase is a multicomponent enzyme containing the 39·5 kDa polypeptide as one component.

6-Hydroxy-d-nicotine oxidase (6-HDNO) of Arthrobacter oxydans, an enzyme inducible by dl-nicotine, contains FAD covalently bound via an 8a-N(3)His linkage. Expression of the gene encoding 6-HDNO and flavinylation of the protein were studied in Bacillus subtilis. In this heterologous system the following findings were made. 1. An enzymically active covalently flavinylated 6-HDNO of normal size can be expressed in B. subtilis. 2. The natural promoter of the 6-HDNO gene appeared inefficient in B. subtilis. The B. subtilis sdh promoter, when inserted upstream of the A. oxydans promoter, increased 6-HDNO expression >50-fold. 3. Expression of the 6-HDNO gene from plasmids in B. subtilis was, independently of the promoter construct used, stimulated more than fivefold by dl-nicotine in the growth medium. It is concluded that flavinylation of 6-HDNO is possibly autocatalytic and mediated by factors generally found in bacterial cells.

Selection by sulphonamides was investigated in Neisseria gonorrhoeae because a sulphonamide-resistant (Sulr), methionine-requiring (Met−) phenotype that was common in the era of sulphonamide therapy became rare in the penicillin era. Cultures of wild-type (SulrMet+) gonococci on a conventional medium containing sulphadiazine (210 g ml-1) yielded numerous, nonidentical mutations of two met genes. The requirement of MetI- mutants was satisfied only by methionine, whereas MetI-1 mutants utilized either homocysteine or methionine. My theory that increased resistance to sulphonamides is a pleiotropic effect of methionine auxotrophy was confirmed by the return of sulphonamide susceptibility in all Met+ spontaneous mutants. Furthermore, the SulrMet- traits were introduced or eliminated together by DNA-mediated transformation. Sulphonamides are known to inhibit dihydropteroate synthase; consequently, they interrupt the entire sequence of reactions in the folate pathway including the methyl group transfer from N 5-methyltetrahydrofolate to homocysteine to form methionine. The increased sulphonamide resistance of these Met- mutants is discussed in terms of conservation of the pool of essential tetrahydrofolate derivatives. The ease with which spontaneous forward and reverse met mutations can be obtained is unique among gonococcal genes.

The nucleotide sequence of the pilin gene from Bacteroides nodosus strain 351, currently classified as serogroup H, subgroup 2 (H2) has been determined. The gene encodes a single polypeptide (prepilin) of 160 amino acids and M r 17 150. However, pilin isolated from B. nodosus 351 migrates as two distinct bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, due to an internal peptide bond cleavage. Amino acid sequence studies of pilin from B. nodosus 351 have established that the cleavage occurs between 72Ala and 73Ser of the mature protein sequence. Comparisons of gene and amino acid sequences of pilin from B. nodosus 351 with the corresponding sequences from strains of serogroups D and H1 indicate that these sequences share a close relationship. However, the level of sequence identity between B. nodosus 351 pilin and pilin from strain 265 of serogroup H1 is lower than anticipated for strains within a serogroup and suggests that B. nodosus 265 and B. nodosus 351 should not be classified within the same serogroup.

Examination of 12 Chlamydia psittaci strains recovered from nine different host species (three avian and six mammalian) revealed the presence of a 7·5 kb plasmid in all isolates except two ovine abortion strains, the human strain IOL207 and the Cal 10 strain. Restriction mapping analysis distinguished four different plasmids that were associated with avian, feline, equine and guinea-pig C. psittaci isolates, respectively. The restriction maps of these four C. psittaci plasmid types all differed from that of the plasmid recovered from C. trachomatis L2/434. Despite this plasmid diversity, which is likely to be of taxonomic importance, all four plasmids identified within the species C. psittaci were found to share some sequence homology, which was mapped to two separate regions in the plasmid molecules. One region, which showed a high degree of homology between C. psittaci plasmids and also detectable homology with the C. trachomatis plasmid, may represent a common replication control region for plasmids of this genus.

Most strains of Candida albicans are capable of switching spontaneously and at high frequency between a number of phenotypes distinguishable by colony morphology. The switching frequency of Candida albicans strain WO-1 between two predominant phenotypes, ‘white’ and ‘opaque’, and a minor phenotype, ‘fuzzy’, increased dramatically with low doses of ultraviolet irradiation that killed less than 20% of the population. The ultraviolet irradiation effect continued to be expressed over many generations as evidenced by stimulated sectoring. Ultraviolet irradiation stimulated switching in both the white-to-opaque and opaque-to-white direction, suggesting that a common mechanism functions in both directions.

Effects of three mutant genes, CAT1-2d, cat2-1 and hex2-3, on catabolite repression of mitochondrial cytochromes and the first two enzymes of haem biosynthesis were compared. The CAT1-2d mutation gave no resistance to glucose, whereas cat2-1 endowed both cytochromes and 5-aminolaevulinate dehydratase with resistance, but did not alter the effect of glucose on 5-aminolaevulinate synthase. The hex2-3 mutation caused repression resistance of cytochromes and of the two haem biosynthetic enzymes. hex2-3 strains also accumulated intracellular 5-aminolaevulinate. Co-inheritance of the latter traits, sensitivity to maltose inhibition and ability to grow on raffinose in the presence of 2-deoxyglucose, demonstrated that the pleiotropic phenotype is a function of the single gene hex2-3. Revertants which grew on maltose regained sensitivity to deoxyglucose and exhibited normal sensitivity of cytochromes and haem biosynthesis enzymes to repression. Addition of the hex1-18 mutation, which renders cytochromes resistant to repression, to a cat2-1 strain did not produce the same effect on 5-aminolaevulinate synthase as hex2-3. It is concluded that repression patterns of haem and cytochrome biosynthesis are substantially affected by hex2-3 and cat2-1 but not by CAT1-2d .

The dha regulon of Klebsiella pneumoniae specifying fermentative dissimilation of glycerol was mobilized by the broad-host-range plasmid RP4:mini Mu and introduced conjugatively into Escherichia coli. The recipient E. coli was enabled to grow anaerobically on glycerol without added hydrogen acceptors, although its cell yield was less than that of K. pneumoniae. The reduced cell yield was probably due to the lack of the coenzyme-B12-dependent glycerol dehydratase of the dha system. This enzyme initiates the first step in an auxiliary pathway for disposal of the extra reducing equivalents from glycerol. The lack of this enzyme would also account for the absence of 1,3-propanediol (a hallmark fermentation product of glycerol) in the spent culture medium. In a control experiment, a large quantity of this compound was detected in a similar culture medium following the growth of K. pneumoniae. The other three known enzymes of the dha system, glycerol dehydrogenase, dihydroxyacetone kinase and 1,3-propanediol oxidoreductase, however, were synthesized at levels comparable to those found in K. pneumoniae. Regulation of the dha system in E. coli appeared to follow the same pattern as in K. pneumoniae: the three acquired enzymes were induced by glycerol, catabolite repressed by glucose, and glycerol dehydrogenase was post-translationally inactivated during the shift from anaerobic to aerobic growth. The means by which the E. coli recipient can achieve redox balance without formation of 1,3-propanediol during anaerobic growth on glycerol remains to be discovered.

An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4·5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 107 to 4 x 108 transformants per µg DNA. The transformation frequency (transformants per regenerant) was 0·5 to 1·0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of Bacillus subtilis were transformed with pLW05 [2·5 x 105 transformants (µg DNA)-1] and pTHT15 [1·8 x 105 transformants (µg DNA)-1], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cmr was not stably maintained in cultures growing at temperatures between 50 and 65 °C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70 °C. In contrast, thermophilic plasmid pTHT15 Tcr was stable in cultures growing at temperatures up to 60 °C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50,60, and 65 °C was 69,18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 °C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 °C.

A protocol designed to isolate mutants with thermosensitive (Ts) synthesis of the bacteriophage 𝜙29 receptor, which includes the major wall teichoic acid in Bacillus subtilis 168, yielded a significant enrichment for Ts growth mutants among colonies surviving 𝜙29 treatment. Nine mutants, Ts for both 𝜙29 susceptibility and cell growth, harboured mutations which were located in the tag locus by PBS1 transduction and recombination index with the tag-1 marker. Physical mapping revealed that they were distributed on a segment of more than 4 kb. Chemical analysis of cell walls showed a marked reduction in phosphate relative to diaminopimelic acid content of all mutants at the non-permissive temperature. Differences between mutants were correlated with the distribution of tag mutations on the genetic map. We conclude (i) that the newly identified markers affect several genes involved in poly(glycerol phosphate) synthesis, and (ii) that on phosphate-rich media, cell growth cannot occur without the synthesis of the latter polymer.

The expression of the spo0A-lacZ fusion gene was partially repressed in the presence of an excess of glucose. Expression was restored either by the mutation sigA47 (crsA47) or by addition of decoyinine, an inhibitor of GMP synthetase, to the medium. By constructing a lacZ fusion with a smaller fragment of the spo0A gene, we observed a β-galactosidase profile in which expression was completely repressed by an excess of glucose. This expression was restored by the addition of decoyinine. These results indicate that the expression of the spo0A gene is regulated by at least two different mechanisms, one sensitive to glucose, the other not. Furthermore, the glucose-sensitive regulation was shown to reside at the transcriptional level. It is likely that the reduced expression of the spo0A gene in the presence of glucose at an early stage of sporulation causes the repression of sporulation.

The transformation of uninucleate amoebae of Physarum polycephalum into multinucleate plasmodia is under the control of a multiallelic mating-type locus, matA. Plasmodium formation usually occurs only when amoebal fusion brings together two different matA alleles within a single cell; such plasmodium formation is termed crossing. Mutations (gadA) in the matA locus permit haploid amoebae to self, that is to form plasmodia in clonal cultures without amoebal fusion. By constructing diploid amoebae heterozygous for gadA alleles, it was shown that the mutant alleles gadA5 and gadA111 were each dominant to gadA +. Non-selfing revertants of gadA mutants may be generated as a result of further mutations (npf) within the matA locus. Sixty-one revertants of this type were studied, derived from three matA3 gadA mutants. Some revertants were able to cross with a matA3 gadA + tester strain. These ‘class I’ revertants failed to cross with one another or with similar revertants of two matA2 gadA mutants; they appear all to lack a single gene function (npfC +) that is necessary in plasmodium development. Other revertants failed to cross with the matA3 gadA + tester. These ‘class II’ revertants also failed to cross with one another, but showed several different patterns of crossing when mixed with other strains. The behaviour of some of the class II revertants suggests that they lack a further gene function (npfB +) necessary in plasmodium development. The gadA mutations may affect a cis-acting regulator of npfB +.