- Volume 128, Issue 10, 1982

Fructose-1,6-bisphosphatase (EC 3.1.3.11) has been partially purified from methanol- and ethanol-grown Hansenula polymorpha. The enzyme was specific for fructose 1,6-bisphosphate as a substrate and did not react with sedoheptulose 1,7-bisphosphate. The enzyme purified from ethanol-grown cells resembled that from methanol-grown cells in all properties investigated, namely, temperature stability, substrate affinity, pH-dependent inhibition by AMP, and pH-dependent stimulation of activity by EDTA. It is concluded that, although the metabolic role of fructose-1,6-bisphosphatase during growth on methanol and ethanol is completely different, growth on these substrates probably involves the same enzyme.

The enzymes of N-acetyl-d-glucosamine (GlcNAc) metabolism, GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase were found to be inducible in Candida albicans. The pattern of induction for these enzymes was the same under conditions of germ-tube formation (37 °C) and where yeast cells metabolized GlcNAc with no change in morphology (28 °C); this indicates that these enzymes are not control points in the dimorphic development of C. albicans. During induction there was a 40- and 25-fold increase in specific activity for the deacetylase and the deaminase, respectively, and the maximum specific activity corresponded to the time when all the GlcNAc had been metabolized. The presence of lomofungin (an inhibitor of transcription) or trichodermin (an inhibitor of translation) in cell suspensions of C. albicans containing GlcNAc prevented the increase in specific activity of these enzymes. 2-Deoxyglucose inhibited germ-tube formation, partially inhibited the induction of the deacetylase (43%) and the deaminase (60%), but did not affect the growth of C. albicans on either Glc or GlcNAc. GlcN-6-phosphate was a competitive inhibitor of the deacetylase with a K i of 1·45 mm while the other product of the reaction, acetate, did not inhibit the enzyme. The K m value for GlcN-6-phosphate on GlcN-6-phosphate deaminase was 0·24 mm. Incubation of starved yeast cells with GlcNAc produced a four-fold increase in the specific activity of UDP-GlcNAc-pyrophosphorylase at either 28 °C or 37 °C.

The thermophilic fungus Talaromyces emersonii when grown on cellulose-containing medium produces three extracellular forms of β-glucosidase, I(M r 135000), II (M r, 100000), III (M r, 45700), and one intracellular form, β-glucosidase IV (M r, 57600). With the exception of β-glucosidase II which has yet to be isolated in quantities sufficient for characterization each has been shown to be a glycoprotein and to exist as a single polypeptide. Each of the purified enzyme preparations catalysed cellobiose hydrolysis but only β-glucosidase IV, at the concentrations of substrates used, also catalysed glucosyl transfer to cellobiose.

The generic distribution of nitrate-respiring bacteria in tidal mudflats of six Scottish east-coast river estuaries has been determined. The predominant bacteria involved were identified as Aeromonas/Vibrio spp., together with lower numbers of enterobacteria. Pseudomonads accounted for only a small proportion of the nitrate-respiring populations. Samples of sediment from all the estuaries produced nitrite and ammonia after 7 d incubation at 15 °C with added nitrate. Studies with the isolate Vibrio V48 and strains of Vibrio parahaemolyticus (NCMB 1903 and NCMB 2047) demonstrated that when these bacteria were grown anaerobically under conditions of glucose limitation, nitrite was the primary product of nitrate respiration, whereas ammonia was excreted into the medium when nitrogen was limiting. Nitrate reduction was accompanied by the synthesis of a particulate nitrate reductase and under N-limitation a nitrite reductase was also induced.

All seven species of aquatic hyphomycetes tested produced both polygalacturonases and pectin lyases. The polygalacturonases were constitutive, whereas the pectin lyases were induced on pectic substrates at pH 6.5 and above. Some species could grow on pectic substrates at both pH 5 and pH 7; other species at pH 7 only. Tricladium splendens grew as well on a polypectate substrate as it did on glucose. This fungus elaborated three endo-polygalacturonase isoenzymes, as did Articulospora tetracladia. Tetrachaetum elegans secreted a presumed exo-pectin lyase and a pectin esterase, and Mycocentrospora angulata an endo-pectin lyase and a pectin esterase. The four species macerated alder-leaf strips totally within 9 to 12 d at stream pH 7, utilizing predominantly pectin lyases and pectin esterases. These enzymes are stimulated by Ca2+, and this may explain why plant decay proceeds most rapidly in calcium-rich streams of pH 6.5 and above. The frequency with which the four species of aquatic hyphomycetes were observed on experimental leaf packs placed in a stream does not appear to be related to their pectolytic capability.

A total of 15 bacterial species were screened for the presence of the Escherichia coli K12 insertion sequences IS1, IS2 and IS3 by Southern blotting and DNA hybridization. The only positive signals obtained were for IS1 in Serratia marcescens, where the sequence was present on a plasmid, and a weak hybridization to IS3 in Erwinia carotovora. This shows that the capacity for transposition alone is not sufficient to allow the spread of sequences through a wide range of bacterial species.

Strain FC1, a wild-type isolate of the phytopathogenic fungus Fusarium oxysporum, is resistant to nifuroxime, a nitrofuran derivative. Mutants sensitive to this compound arise at high frequency by vegetative segregation. Resistance to nifuroxime is inherited as an extranuclear marker in crosses between a nifuroxime-sensitive variant and the wild-type. Nifuroxime-sensitive strains are also unable to express an exopolygalacturonase activity present in the wild-type. For one strain tested, it was shown that the ability to parasitize a susceptible tomato plant variety was also lost. Loss of nifuroxime resistance was concomitant with the loss of a 46·7 kb circular DNA molecule.

Mutants affected in production of alkaline extracellular protease (xpr mutants) have been characterized. The alkaline protease produced by the xpr mutants, except for that of the structural gene mutant, was indistinguishable from the wild-type protease on the basis of isoelectric focusing in polyacrylamide gels, SDS-PAGE and thermal stability. Some revertants of xpr mutants were temperature sensitive for protease production, but the thermal stability of the revertant protease was not altered. The xpr mutants grew as rapidly as the wild-type with glutamic acid, leucine or urea as sole nitrogen source. The pleiotropic xpr mutants which produced lowered levels of extracellular RNAase also produced less extracellular acid proteases (4 to 40% of wild-type). These results suggest that the pleiotropic xpr mutants may affect secretion. However, production of phosphatase(s) (which was secreted but located primarily in the cell envelope) was much less affected in these mutants (65 to 100% of wild-type). A possible explanation for these results is that at least one step (component) of the secretion pathway for the extracellular proteases and RNAases is not shared by the phosphatase(s).

Pseudomonas sp. NCIB 9816 contains two plasmids: pWW60, an IncP9 plasmid of 87 kb encoding genes for the catabolism of naphthalene, and pWW61, a cryptic plasmid of about 65 kb. The ability to degrade naphthalene was transferred at low frequency by conjugation from strain NCIB 9816 into a plasmid-free strain of Pseudomonas putida, PaW340. A transconjugant, PaW701, containing the naphthalene plasmid pWW60-1, metabolized naphthalene and salicylate via the ortho pathway. 2-Methylnaphthalene was not a growth substrate but was partly metabolized with accumulation of a brown compound in the medium (λ max = 440 nm). Spontaneous mutants of PaW701 with the ability to grow on 2-methylnaphthalene arose at a frequency of about 10−5. These fell into two groups. Group A mutants had no detectable salicylate hydroxylase activity and accumulated salicylate from naphthalene in culture supernatants: they appeared to grow on the pyruvate released from oxidation of the first ring of both substrates. Their plasmids all contained a 16·7 kb insert in different sites within a small, limited region of the plasmid. Group B mutants used a meta pathway for catabolism of naphthalene and 2-methylnaphthalene. Their plasmids had undergone a small deletion of from 1·2 to 1·6 kb in a region of the plasmid close to the sites of the insertions in the group A mutants.

The genetics of Pachysolen tannophilus, a yeast able to ferment xylose to alcohol, was investigated. This yeast is a strongly homothallic organism in which the haplophase is predominant. Typically, diploidy is a consequence of fusion of two mitotic products within a specialized conjugant cell. The diploid nucleus proceeds directly to meiosis and the production of a four-spored ascus. A technique for isolating both homozygous and heterozygous diploids has been devised. The species is amenable to tetrad analysis, and is well-suited to the study of pentose fermentation in yeast.

The uptake of the labelled precursors, thymidine, deoxyadenosine and adenine, into the nuclear and mitochondrial DNA of the cellular slime mould, Dictyostelium discoideum, at various stages of development was studied. Labelling of the different species of DNA, nuclear main-band, mitochondrial, and satellite, was analysed by CsC1 gradients using the AT-specific drug netropsin to enhance the density resolution. Constitutive and gamma ray modified uptake patterns were obtained. During early development, through late aggregation, constitutive uptake of thymidine and deoxyadenosine was exclusively into DNA at the density of mitochondrial and nuclear satellite I DNA, believed to be primarily due to mitochondrial DNA labelling. Labelled adenine was not incorporated into any DNA before early culmination. During the period of early culmination uptake of all three of these precursors into the main-band nuclear DNA increased dramatically, to considerably exceed uptake into the mitochondrial DNA. The molecular basis for these constitutive uptake patterns during development is not understood, but they appear to involve developmentally associated periods of DNA replication in some or all of the cells, possibly accompanied by changes in precursor transport and/or pool sizes that are different for the mitochondrial and nuclear DNA metabolic pathways. Early in development gamma rays had little effect on the constitutive uptake of precursor into the mitochondrial DNA but induced new dose-dependent uptake of thymidine and deoxyadenosine into the nuclear DNA. This unscheduled nuclear DNA synthesis may be a manifestation of gamma ray induced repair replication. At the early culmination stage gamma rays severely depressed the constitutive uptake of thymidine, deoxyadenosine and adenine into the nuclear DNA, to a level of about 10% at 20 krad. Higher doses induced a slight increase over this level. These changes may be due to the inhibition of the constitutive semiconservative replication by low gamma ray doses with the superposition of some induced repair replication.

Phages C-2 and J were isolated from sewage. Phage C-2 was filamentous and formed plaques on Salmonella typhimurium strains carrying various C plasmids. It also plated on Proteus mirabilis and Serratia marcescens strains carrying particular C plasmids, but failed to form plaques on lines of Escherichia coli K12 strains harbouring most of these plasmids, although in all cases, phage multiplication on the strains was demonstrated. No phage increase occurred in any strain which lacked a C plasmid or contained plasmids of other incompatibility groups. The phage was sensitive to chloroform and, unlike other filamentous bacterial viruses, adsorbed to shafts of conjugative pili. It had a disc-like structure at the end which attached to the pilus. Phage C-2 had a buoyant density of 1·30 g cm-3 and a single-stranded circular DNA genome of 3·0 MDal. Phage J had an hexagonal head with an inter-apical distance of 40 nm and a short non-contractile tail. It was resistant to chloroform and diethyl ether. The phage formed plaques or propagated on E. coli strains harbouring some IncC plasmids and all IncJ and IncD plasmids tested. The phage did not form plaques but propagated on P. mirabilis and Ser. marcescens strains carrying these plasmids. It did not plate or propagate on S. typhimurium strains harbouring the plasmids. The plaques were very hazy and variable in size. The phage attached sparsely, at a site which appeared to be located at the base of the tail, to sides of conjugative pili.

We have evaluated 66 β-lactamase-producing (β-Lac+) clinical isolates of Haemophilus influenzae for the presence of plasmid DNA, ability to conjugate with an H. influenzae type b recipient, and stability of the β-Lac+ phenotype. Of the 66 strains, 23 (35%) contained a plasmid demonstrable by either of two methods of cleared cell lysate preparation. The molecular weight of 17 of the plasmids was approximately 30 × 106. Four strains carried a plasmid of mol.wt 3 × 106 and single strains carried plasmids of mol. wt 36 × 106 or 50 × 106. Of 23 plasmids that were unstable in vitro, 10 (43%) had molecular weights of 30 × 106 and coded only for β-lactamase. Stability of the β-Lac+ phenotype was observed in all 43 strains in which plasmid DNA was not demonstrated by physical methods. When β-Lac+,‘plasmid-free’ strains were conjugated with strain Eagan (H. influenzae type b), ampicillin-resistant transconjugants were isolated in 21 out of 43 matings (the lower limit of detection was 10−7 per donor cell per 30 min mating). With few exceptions, homologous (type b × type b) crosses with β-Lac+, plasmid-containing donors were more efficient (frequency 10−2–10-3) than heterologous (untypable × type b) matings (frequency 10−6–10−7). β-Lac+,‘plasmid-free’ strains were inefficient donors (frequency 10−6–10−7) irrespective of serotype, but all transconjugants from these crosses were efficient donors (frequency 10−1–10−4) and contained a plasmid of mol. wt 30 × 106 that was unstable in 9 cases out of 26 (35%). The increase in transferability and phenotypic instability of plasmid-bearing β-Lac+ strains reinforces the concept of a chromosomal locus for β-lactamase genes in‘plasmid-free’ β-Lac+ strains. Carriage of β-lactamase genes on 30 × 106 mol. wt R plasmids aids transferability at the expense of genetic stability.

A synthetic derivative of muramoyl dipeptide, 6-O-stearoyl-N-acetylmuramoyl-l-alanyl-d-iso-glutamine [L18-MDP(A)], showed a protective effect against bacteraemic and non-bacteraemic pneumonia caused by Pseudomonas aeruginosa in immunosuppressed guinea pigs. In about half of the animals treated with the compound before infection, death from bacteraemic pneumonia produced by intratracheal inoculation of P. aeruginosa was delayed for 7 d, although all of the animals infected without prior treatment with the compound died within 4 d of infection. Multiplication of the organisms in the lung was also suppressed for at least 10 d by treatment with the compound when the animals inhaled an aerosol of P. aeruginosa. In contrast, in untreated animals the numbers of bacteria in the lung gradually increased from 106 to 109 c.f.u. g−1, and a few animals in which the organism increased to 109 c.f.u. g−1 had died by 6 and 10 d after infection. In both healthy and immunosuppressed animals, the accumulation of polymorphonuclear leukocytes (PMNs) in a subcutaneous air-pouch injected with heat-killed organisms was augmented by subcutaneous treatment with L18-MDP(A) 1 d before bacterial injection. The phagocytic activity of peritoneal PMNs was also increased by treatment with this compound. The augmentation of protective mechanisms against pseudomonas pneumonia by L18-MDP(A) may be attributed at least partly to the increased chemotactic and phagocytic activity of PMNs.

Enterochelin, the iron chelator produced by a number of pathogenic enterobacteria, appears to be an essential metabolite for multiplication within the host, where it transports iron from the host iron-binding proteins to the bacteria. Previous work showed that complexes of enterochelin containing either scandium (Sc3+) or indium (In3+) exerted a bacteriostatic effect on Klebsiella pneumoniae in serum, whilst the Sc3+ complex exerted a significant therapeutic effect on mice infected with K. pneumoniae. These observations have now been extended to a number of pathogenic serotypes of Escherichia coli including those carrying either the K1 antigen or the ColV plasmid. The Sc3+ and In3+ complexes each exert a bacteriostatic effect on these organisms growing in either whole serum or media containing an iron-binding protein. Evidence is presented that the Sc3+ complex may act as a competitive inhibitor of the Fe3+ complex. In contrast to their effects on K. pneumoniae, sideramines other than enterochelin fail to reverse the bacteriostatic effect of the Sc3+ complex of enterochelin in E. coli, suggesting that the complex produces a more profound derangement of metabolism in this organism. The Sc3+ complex exerts a significant therapeutic effect on E. coli infections in mice although the In3+ complex is less active.