- Volume 133, Issue 8, 1987

SUMMARY: A mutant of Candida albicans ATCC 10261 was isolated that was defective in the production of β-N-acetylglucosaminidase (chitobiase). The mutant grew normally in minimal medium supplemented with either glucose or N-acetyl-D-glucosamine (GlcNAc) as carbon and energy source, and the cells formed germ-tubes at 37°C when induced to do so with GlcNAc. However, unlike the wild-type parent strain, the mutant strain did not utilize N, N'-diacetylchitobiose for growth. The mutant and parent strains had similar growth rates on glucose or GlcNAc, similar rates of uptake of these sugars and similar rates of 14C-labelled amino acid incorporation. The chitobiase mutant did, however, contain 53-85% more chitin than the wild-type strain. No reversion of the mutant phenotype was observed following induction of mitotic recombination with UV light, suggesting that the mutant allele (chi) was carried homozygously in the chitobiase-deficient mutant. Although the chitobiase-deficient mutant was pathogenic, it was not as virulent as the wild-type strain.

SUMMARY: Cell-envelope fractions were isolated from the rapidly growing saprophyte Mycobacterium smegmatis following growth in glycerol/asparagine medium under both iron-limited (0·02 μg Fe ml-1) and iron-sufficient (2·0 to 4·0 μg Fe ml-1) conditions. Examination of these preparations by SDS-PAGE demonstrated the production of at least four additional proteins when iron was limiting. These iron-regulated envelope proteins (IREPs) were ascribed apparent molecular masses of 180 kDa (protein I), 84 kDa (protein II), 29 kDa (protein III) and 25 kDa (protein IV). All four proteins were present in both cell-wall and membrane preparations but spheroplast preparations were devoid of the 29 kDa protein. Attempts at labelling the proteins with 55FeCl3 or 55Fe-exochelin, the siderophore for iron uptake, were unsuccessful, though this was attributed to the denatured state of the proteins following electrophoresis. Antibodies were raised to each of the four proteins; the one raised to protein III inhibited exochelin-mediated iron uptake into iron-deficiently grown cells by 70% but was ineffective against iron uptake into iron-sufficiently grown cells. As exochelin is taken up into both types of cells by a similar process, protein III may not be a simple receptor for iron uptake though the results imply some function connected with this process. The role of the other IREPs is less certain.

SUMMARY: From the highly chloramphenicol-resistant cytophaga-like bacterium Flavobacterium CB60, which can both acetylate chloramphenicol and degrade it in co-metabolism, the chloramphenicol acetyltransferase (CAT) was purified to homogeneity and characterized. The purification included fractional precipitation with ammonium sulphate and two affinity chromatography steps, eluting CAT the first time with 5 mM-chloramphenicol and the second time with a linear gradient (0–10 mM) of chloramphenicol. The purification was 3979-fold. Properties of this CAT were investigated and compared with CATs from other bacteria. Although CAT from Flavobacterium CB60 shares some properties with the enzymes from Escherichia coli and other Gram-negative bacteria - especially with CATII and CATIII - it has distinct properties like extreme heat lability and the inability to produce diacetylchloramphenicol, so that it might be regarded as a new variant.

SUMMARY: A new exopolysaccharide, secreted in addition to cellulose, has been isolated from the culture medium of Acetobacter xylinum NRRL B42. This polysaccharide, for which the name acetan is proposed, contains glucose, mannose, glucuronic acid and rhamnose in a molar ratio of 4:1:1:1. On the basis of methylation, thin-layer, paper and gas-liquid chromatography, paper electrophoresis and mass spectrometry studies of the degradation products obtained by total and partial hydrolysis and acetolysis of acetan, the following structure is proposed for its repeating unit:

Since our previous work with this strain demonstrated the in vitro synthesis of a lipid-linked heptasaccharide with the same structure, the possibility of acetan being the result of its polymerization is discussed. One to two O-acetyl residues per repeating unit are also present in positions not yet determined.

SUMMARY: A bacterium capable of growth on 4-ethylphenol was isolated from soil and identified as Pseudomonas putida. Intact cells grown on 4-ethylphenol rapidly oxidized 4-hydroxyaceto-phenone as well as growth substrate and the bacterium was also capable of growth on 4-hydroxyacetophenone. The initial enzymes for 4-ethylphenol catabolism were still present, although at lower activities, in succinate-grown cells which oxidized 4-ethylphenol to 4-hydroxyacetophenone. Extracts of 4-ethylphenol-grown cells oxidized 4-hydroxyacetophenone when provided with NADPH. When this activity was partially purified a stoichiometry of 1 μmol O2 consumed per μmol of substrate was observed with the production of hydroquinone as required for a monooxygenase producing 4-hydroxyphernyl acetate followed by hydrolysis by an esterase. Cell extracts contained esterase activity and hydrolysed 4-hydroxyphenyl acetate to yield hydroquinone. Intact cells converted the analogue, acetophenone, into phenol. Hydroquinone served as the ring-fission substrate and was cleaved by an O2-requiring reaction. The enzymes of the proposed pathway were induced by growth on 4-ethylphenol or 4-hydroxyacetophenone.

SUMMARY: Acholeplasma laidlawii possesses a biochemical pathway for tyrosine and phenylalanine biosynthesis, while Mycoplasma iowae and Mycoplasma gallinarum do not. The detection of 7-phospho-2-dehydro-3-deoxy-d-arabino-heptonate (DAHP) synthase (EC 4.1.2.15), dehydro-shikimate reductase (EC 1.1.1.25) and 3-enol-pyruvoylshikimate-5-phosphate synthase (EC 2.5.1.19) activities in cell-free extracts established the presence in A. laidlawii of a functional shikimate pathway. l-Phenylalanine synthesis occurs solely through the phenylpyruvate route via prephenate dehydratase (EC 4.2.1.51), no arogenate dehydratase activity being found. Although arogenate dehydrogenase was detected, l-tyrosine synthesis appears to occur mainly through the 4-hydroxyphenylpyruvate route, via prephenate dehydrogenase (EC 1.3.1.12), which utilized NAD+ as a preferred coenzyme substrate. l-Tyrosine was found to be the key regulatory molecule governing aromatic biosynthesis. DAHP synthase was feedback inhibited by l-tyrosine, but not by l-phenylalanine or l-tryptophan; l-tyrosine was a potent feedback inhibitor of prephenate dehydrogenase and an allosteric activator of prephenate dehydratase. Chorismate mutase (EC 5.4.99.5) was sensitive to product inhibition by prephenate. Prephenate dehydratase was feedback inhibited by l-phenylalanine. It was also activated by hydrophobic amino acids (l-valine, l-isoleucine and l-methionine), similar to results previously found in a number of other genera that share the Gram-positive line of phylogenetic descent. Aromatic-pathway-encoded cistrons present in saprophytic large-genome mycoplasmas may have been eliminated in the parasitic small-genome mycoplasmas.

SUMMARY: Carboxymethylcellulase (endo-1,4-β-glucanase; EC 3.2.1.4) was purified from the culture filtrate of Bacillus subtilis AU-1 by (NH4)2SO4 precipitation, Avicel affinity chromatography, DEAE Sephadex G-75 chromatography and Sulphopropyl Sephadex C-50 chromatography. The enzyme was purified 36-fold and had an M r of 23000 as determined by gel filtration on a Sephadex G-75 column. The pH optimum of the purified enzyme was 5·5; the enzyme was stable at 65°C. Activity of the purified enzyme was significantly reduced by Cu2+, Pb2+, Sn2+, Ag+, Hg2+ and Fe2+, but was increased by 139·5% in the presence of Co2+. Inhibition studies indicated that the purified enzyme was either a metalloprotein or required certain metal ions for activation/stabilization; that iron was not a prosthetic group of the enzyme; that a tryptophanyl group was not involved in enzyme action; and that reduced thiol groups were required for enzyme activity and involved in the active site of the enzyme. The K m of the purified enzyme for carboxymethylcellulose was 4 mg ml-1, and the V max for carboxymethylcellulose hydrolysis was 0·42 mg d-glucose min-1 (mg protein)-1.

SUMMARY: Aerobic cells of the yeast Schizosaccharomyces pombe accumulated 2-deoxy-d-glucose (2-DOG) and glucosamine, but were unable to accumulate 3-O-methyl-d-glucose, 6-deoxy-d-glucose, d-xylose and d-arabinose. Uptake of all sugars tested displayed saturation kinetics; however, only 2-DOG, glucosamine and d-glucose exhibited mutual competitive inhibition of uptake and the phenomenon of exchange transport. Thus, they share a common ‘glucose transport system’. Uncouplers inhibited sugar accumulation or induced sugar outflow from preloaded cells. The uptake of sugars of the glucose transport system was pH dependent and was accompanied by a stoicheiometric cotransport of H+. Since millimolar concentrations of the lipophilic cation tetraphenylphosphonium (which depolarizes the membrane potential) prevented sugar accumulation, the transport was electrogenic. Thus, the glucose carrier is a H+-symporter. Accumulation of sugars was inhibited by the plasma membrane ATPase inhibitors Dio-9 and N, N’-dicyclohexylcarbodiimide. Anaerobic cells of S. pombe were virtually unable to transport 2-DOG and glucosamine. However, when energized by glucose, the accumulation of both sugars was completely restored. This anaerobic transport was also catalysed by the glucose carrier.

SUMMARY: Recombinant plasmids carrying the amidase genes of Pseudomonas aeruginosa were used to study the genetic control of amidase synthesis in Escherichia coli and Pseudomonas aeruginosa. The amidase regulator gene, amiR, was found to lie about 2 kbp downstream from the structural gene, amiE. Using plasmids with in vitro-constructed deletions, and plasmids containing sub-cloned DNA fragments, the amiR gene was located within a l kbp ClaI-XhoI DNA fragment. The structural and regulator genes were shown to be transcribed in the same direction. Deletion of DNA sequences between the two genes resulted in increased synthesis of amidase in both E. coli and P. aeruginosa. The intervening sequences showed no repressing effect when tested in trans. The results suggested that the amiR gene could be transcribed from more than one promoter.

SUMMARY: A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae M5al and Escherichia coli K12 has been developed. The method, which uses a freeze-thaw cycle in the presence of CaCl2 to facilitate DNA uptake, is substantially more efficient for K. pneumoniae M5al than the conventional transformation procedure for E. coli. The simplicity and speed of the method makes it very attractive for routine transformation of K. pneumoniae M5al and E. coli K12.

SUMMARY: Azotobacter vinelandii OP which had been naturally induced to competence by growth in iron-and molybdenum-limited medium was transformed with the broad-host-range cloning vector pKT210. However, the transformation frequency at nearly saturating levels of DNA was 1000-fold lower for pKT210 than for a single chromosomal DNA marker (nif +). Plasmid- and chromosomal-DNA-mediated transformation events were competitive, magnesium-dependent, 42°C-sensitive processes specific to double-stranded DNA, suggesting a common mechanism of DNA binding and uptake. The low frequency of plasmid transformation was not related to restriction of transforming DNA or to the growth period allowed for phenotypic expression. Covalently-closed-circular and open-circular forms of pKT210 transformed cells equally well whereas EcoRI- or HindIII-linearized pKT210 transformed cells with two to three times greater efficiency. Genetic transformation was enhanced 10- to 50-fold when pKT210 contained an insert fragment of A. vinelandii nif DNA, indicating that A. vinelandii possessed a homology-facilitated transformation system. However, all transformants failed to maintain the plasmid-encoded antibiotic resistance determinants, and extrachromosomal plasmid DNA was not recovered from these cells. Flush-ended pKT210 was not active in transformation; however, competent cells were transformed to Nif+ by HindII -digested plasmid DNA containing the cloned A. vinelandii nif-10 marker.

SUMMARY: The ability of Pseudomonas putida mt-2 strain UCC2 to grow with aniline, m- and p-toluidine and m-toluate (the Tdn+ phenotype) is plasmid encoded. Strain UCC2 contains two plasmids of which pUCC2 is a deleted derivative of the TOL plasmid pWW0 (Worsey & Williams, 1975) and can be lost from the strain with no effect on the Tdn+ phenotype. The second plasmid in strain UCC2, pTDN1, harbours genes involved in the Tdn+ phenotype and is conjugative. The catechol 2,3-dioxygenase (C230) structural gene resident on pTDN1 has been cloned into the broad host range vector pKT231. The relative specific activity towards substituted catechols of C23O expressed in the cloned fragment in Escherichia coli is similar to that expressed in P. putida strain UCC2.

SUMMARY: The Bacillus subtilis spoOF gene was cloned on a 6·3 kbp BglII fragment. The effect on sporulation of amplification of the spoOF region was examined. Sporulation was inhibited to less than 5% of that of the parental strain when as few as four copies of the spoOF region were present. Subclones, constructed in autonomous or integrative vectors, were used to demonstrate that the region responsible for the copy-number-dependent asporogeny corresponded closely with the spoOF structural gene. A possible mechanism for this effect is discussed.

SUMMARY: In Bacillus subtilis the α, β, γ and δ components comprise 80–90% of the total acid-soluble spore proteins (ASSPs). Sequence analysis demonstrates that α and β share 32 of their first 36 amino acids and are closely related to the A and C ASSPs of Bacillus megaterium spores, confirming the results of analysis of their cloned genes. Despite the difference in apparent size of γ and δ, they have identical N-terminal sequences (37 residues). Unless γ and δ derive from very recently duplicated genes, it appears that γ is derived from δ, either in vivo or during isolation. Although the sequenced regions of γ and δ have no homology to α and β, outside of the previously recognized pentapeptide recognition sequence for the spore endopeptidase, they share 10 and 15 residue peptides flanking this sequence with ASSP B of B. megaterium, but in reverse order. At least two groups of ASSPs have, therefore, been conserved between B. subtilis and B. megaterium: the multigene ACαβ family and the Bγ(δ) group. Sequence conservation in each group implies selection for functions in addition-to storage. Both the α and β components of B. subtilis ASSPs and their mRNAs are located in the forespore compartment of cells at t 5·5 of sporulation, the time of most rapid ASSP synthesis. The sizes of these transcripts (250–350 bp) and their ability to direct the in vitro synthesis of ASSPs of mature size, indicate that genes for these ASSPs are monocistronic, consistent with dispersed map location. Synthesis of ASSPs is, therefore, coordinately controlled by selective transcription in the forespore.

SUMMARY: Plasmid pHH502-1 shows incompatibility only towards members of the IncP group, but has a narrower host range than typical members of that group. In contrast to other IncP plasmids its replication was not affected by a high-copy-number plasmid carrying the replication origin (oriV) of IncP plasmid RK2. Southern blotting of pHH502-1 revealed homology to oriV, consistent with its incompatibility phenotype, but no homology to trfA, the essential replication gene of RK2. Thus it is probable that pHH502-1 does not possess a functional IncP replication system, accounting for its restricted host range. A restriction map of pHH502-1 was constructed and the mercury-resistance determinant was localized to Tn735, which also carries the trimethoprim-resistance determinant and is related to Tn21. The presence of a korB-like function on pHH502-1 was also demonstrated.

SUMMARY: We have investigated the immunity to E colicins conferred by three group A klebicin plasmids. pP5a, which encodes klebicin Al-P5, like pClo-DF13, confers immunity to colicin E6 on Escherichia coli K12, whilst pP5b and pP3, which encode klebicins A2-P5 and A3-P3 respectively, both confer immunity to colicin E3. We have determined the restriction endonuclease and functional maps of the three group A klebicin plasmids. By sub-cloning and transposon mutagenesis we have investigated the relationship between the klebicin immunity and the E colicin immunity conferred by these plasmids. The colicin E6 and the klebicin A1 immunity are encoded by a single gene present on pP5a. The colicin E3 and the klebicin A2 immunity are encoded by a single gene present on pP5b. The colicin E3 and the klebicin A3 immunity are encoded by separate genes present on pP3. Recombinant pML8412, which is derived from the ColE6-CT14 plasmid and encodes colicin E6 immunity, confers klebicin Al-P5 immunity upon Klebsiella pneumoniae UNF5023. Recombinant pKC23, which is derived from the ColE3-CA38 plasmid and confers colicin E3 immunity, confers immunity to klebicin A2-P5, but not to klebicin A3-P3.