- Volume 130, Issue 6, 1984

Chitinase activities were detected in the supernatant and microsomal fractions from homogenized mycelium of surface-grown cultures of Mucor mucedo. Most properties of the two preparations were very similar, e.g. pH optima were 5·65 and 5·55, respectively, and K m values were 16·7 and 19·2 mg chitin ml−1, respectively. Neither activity had a cationic requirement, but both were inhibited by Cu2+Some properties differed. On freezing, the supernatant activity remained constant over 4 d and then gradually decreased, whereas microsomal activity increased, suggesting that endogenous activation was occurring. Prolonged incubations at 30 °C also allowed endogenous activation of microsomal activity, which could be prevented by incubation with phenylmethanesulphonyl fluoride. This enzyme activity could also be activated by treatment of the microsomes with proteases such as trypsin. Inactivation occurred after treatment with phospholipases, suggesting that microsomal chitinase requires phospholipid-enzyme interactions for activity. Thus the microsomal activity behaved as a membrane-bound zymogen.

β-Glucosidase activity in Cellulomonas fimi is strictly intracellular. Cell-free extracts contain at least two β-glucosidases. One of the enzymes is constitutive and it hydrolyses p -nitrophenyl-β-d-glucoside (PNPG) but not cellobiose. A second enzyme is induced about fourfold by growth on Avicel, and about sevenfold by growth on cellobiose; it hydrolyses both PNPG and cellobiose.

Purified walls from Saccharomyces cerevisiae were treated chemically to release intrinsic mannoproteins. Boiling in 2% SDS gave the best results, although treatment in 6 M-urea at room temperature also released significant amounts of mannoprotein radioactivity. Triton X-100, sodium deoxycholate and EDTA were poor solubilizers. Electrophoretic patterns of SDS- or urea-released mannoproteins in SDS-acrylamide gels indicated a great heterogeneity of molecular species, with more than 60 bands. Zymolyase, a glucan-digesting complex, released about half of the mannoproteins, but these species showed an altered mobility on SDS-acrylamide gels and had a lowered capacity for precipitation by ethanol. Action of the enzyme on isolated walls was favoured by dithiothreitol, as is the case with whole cells, and repeated treatments with SDS and Zymolyase released all of the mannoproteins from the wall. Solubilizing treatments other than SDS had a differential effect on recently or formerly incorporated mannoproteins in the wall. The results suggest an asymmetrical arrangement of molecules in the envelope and point to dynamic changes inside the wall as it thickens as a result of cell aging.

An endodeoxyribonuclease specific to apurinic sites in DNA was purified 12000-fold from vegetative cells of the anaerobic thermophilic bacterium, Desulfotomaculum nigrificans strain IFO 13698. The enzyme specifically hydrolyses phosphodiester bonds of apurinic double-stranded DNA, without action on normal, alkylated, or single-stranded depurinated DNA. The endodeoxyribonuclease has a molecular weight of about 18500 and a sedimentation value of 2·2S. The enzyme has an optimum pH of 7·5–8·0 and an optimum temperature of 55 °C. The half-life of purified enzyme is 55 min at 60 °C but it can be heated at 60 °C for at least 60 min without loss of activity in the presence of BSA. The purified enzyme has an absolute requirement for Mg2+ or Mn2+, and is inhibited by EDTA. Enzyme activity is completely inhibited by 0·5 m-NaCl or 1 mm-p-chloromercuribenzoate. All these properties differ from those of apurinic/apyrimidinic endodeoxyribonucleases isolated from Bacillus subtilis, Bacillus stearothermophilus. and Escherichia coli.

A sensitive immunoassay developed previously was used to isolate a series of Escherichia coli clones expressing cellulase genes from Cellulomonas fimi. The clones fell into three groups. Clones in the first group contained plasmids with a 6·6 kb insert of C. fimi DNA, were strongly antigenic, and contained low levels of CM-cellulase activity. Those in the second group contained plasmids with a 5·0 kb insert, were weakly antigenic, and contained high levels of CM-cellulase activity. Those in the third group contained plasmids with a 5·6 kb insert, were weakly antigenic, and contained low levels of CM-cellulase activity. Restriction analysis showed that the three groups carried different cellulase genes. All of these CM-cellulase activities were exported to the periplasm in E. coli. but with different efficiencies. These activities represented important components of the C. fimi cellulase complex. Their properties indicated that they were different from each other and that they probably had complementary actions.

A method has been developed to isolate protoplasts from dermatophytes using Novozym 234. A simple technique of flotation in MgSO4 has been adapted to separate protoplasts from incubation mixture. Electron microscopic studies confirmed the absence of cell wall material on these protoplasts. The recovery of DNA from protoplasts was higher than from mycelia.

The removal of divalent cations from suspensions of zoospores of Phytophthora cinnamomi decreased cell viability. When zoospores were exposed to combinations of cations, the degree of zoospore encystment was reduced to the mean level of that stimulated by the two cations acting separately. When Ca2+ was present in mM concentrations, any damaging effect of the second cation was reduced. In the absence of added K+ and Ca2+, the ionophores valinomycin and A23187 induced zoospore encystment within minutes. The addition of Ca2+ to zoospores treated with A23187 induced germination of the cysts. Strontium at 25 mM was as effective as Ca2+ in stimulating germination. The calmodulin-inhibitory drugs trifluoperazine, imipramine hydrochloride and dibucaine reduced zoospore viability and, with the exception of dibucaine, were ineffective at inducing encystment.

Two different replication determinants were found on an antibiotic resistance plasmid, pTB19, from a thermophilic bacillus. One replication determinant (designated RepA) was functional only in Bacillus subtilis. whereas the other (designated RepB) functioned in both B. subtilis and Bacillus stearothermophilus. A deletion plasmid, pTB90, carrying the RepB derived from pTB19 coincidentally contained the specific 1·0 MDal Eco RI fragment of a cryptic plasmid pBSO2 from B. stearothermophilus. The presence of this 1·0 MDal Eco RI fragment in various deletion plasmids from pTB90 increased transformation frequencies for B. stearothermophilus 103 to 104 times and lowered plasmid copy numbers in the host strain to about one-tenth of those found for plasmids lacking this fragment.

The acidic proteins of six different mycoplasma serotypes causing bovine or caprine pleuro-pneumonia were compared by two-dimensional gel electrophoresis of extracts of 35S-labelled cells. The organisms investigated were Mycoplasma mycoides subsp. mycoides (PG1), M. mycoides subsp. mycoides (Y-goat), M. mycoides subsp. capri (PG3), M. capricolum (California kid), the unclassified bovine serogroup 7 of Leach (PG50) and the F38-like group (F38). The results suggested a close relationship between M. capricolum and F38 and a similarly close relationship between the different M. mycoides subspecies, whereas the two M. mycoides subspecies appeared to be quite distant from M. capricolum and F38. The representative strain of the bovine serogroup 7 of Leach was equally distant from F38, M. capricolum and the three strains of M. mycoides. Strikingly, all six mycoplasma strains apparently shared six proteins in the two-dimensional gels. In Escherichia coli minicells, DNA from strain PG50 cloned in the vector pBR325 gave rise to incorporation of radioactive label into proteins which were identified as mycoplasma proteins by two-dimensional electrophoresis and immuno-precipitation.

Eleven transfer-derepressed plasmids from incompatibility groups I1, I5, B, K and Z were constructed using the dnaG3 mutant Escherichia coli strain BW86. All were found to determine thin flexible and thick rigid pili constitutively. Immune electron microscopy was used to relate thick and thin pilus serotypes with incompatibility grouping. Mutant plasmids that determined only thick pili constitutively transferred efficiently on an agar surface but not in a liquid, whereas plasmids with both kinds of pili transferred equally well in both environments. A mutant of the Incl2 plasmid R721 determined thin pili constitutively, and thick pili at a repressed level, as indicated by electron microscopy. Experiments with this indicated that thin pili were apparently not involved directly in conjugation but were only used to stabilize mating aggregates.

Genes involved in the biosynthesis of a fluorescent pigment by Pseudomonas syringae pv. syringae (P. s. syringae) JL2000 were investigated. A genomic library of this strain was constructed using the broad host range cosmid vector, pLAFRl. Nonfluorescent (Flu−) mutants of JL2000, defective in the biosynthesis of a fluorescent pigment, were obtained after ethyl methanesulphonate mutagenesis. Individual recombinant plasmids from the genomic library were introduced into Flu− mutants. Of a total of 146 Flu− mutants, 36 were restored to fluorescence following matings with individual recombinant colonies in the genomic library. Four separate fluorescence restoration groups, each comprised of 5 to 11 Flu− mutants restored to fluorescence by one of four structurally distinct recombinant plasmids, were identified. Whereas the 36 Flu− mutant strains differed in their abilities to grow on an iron-deficient medium, wild-type P. s. syringae strain JL2000 and all Flu+ transconjugants from these 36 crosses grew on an iron-deficient medium. These results indicate that at least four genes or gene clusters are involved in the production of a fluorescent pigment of P. s. syringae strain JL2000. These genes, with few exceptions, also control the ability of strain JL 2000 to grow under iron-limiting conditions.

The properties of the tryptophanase constitutive mutation tnaR3 have been investigated. It has previously been reported that mutants carrying tnaR3 grow poorly on medium that selects for constitutive expression of tryptophanase. Our results now show that this poor growth can be explained by the inability of tryptophanase to catalyse the synthesis of L-tryptophan from indole, pyruvate and ammonia at a rate sufficient to allow normal growth. Improved media for the characterization of tryptophanase constitutive mutants are described. The mutation tnaR3 rendered tryptophanase synthesis constitutive (at a differential rate that at 37 C is 30% of the fully induced wild-type) and not further inducible. Diploid studies showed that tnaR3 is cis-dominant, but no evidence was found for any effect in trans. In addition to rendering tryptophanase synthesis constitutive, tnaR3 affects the response of tryptophanase synthesis to different growth temperatures.

The S1 homothallic wild-type strain of Nectria haematococca produces grey-purplish colonies on PDA medium, due to synthesis of fusarubin and related naphthazarins. Seventeen yellow, beige or white mutants were isolated. On the basis of recombination tests they were divided into six distinct chromosomal regions. Four of them, designated befB. befC. belB and belC. may be regarded as regulatory genes which uncouple the processes of ageing and pigmentation that are normally associated in the wild-type. The two others, alyA and yelJ-beiW. control the biosynthesis of naphthazarins. Mutations in alyA were found epistatic to yelJ and beiW mutations, indicating that the alyA locus acts on an earlier biosynthetic step than the yelJ -beiW region. The latter was shown to comprise two closely linked genes, yielding yellow or beige colours when mutated. Analysis of polar mutations suggests that they form a single unit of transcription. One of these two genes probably controls a late oxidation step in naphthazarin biosynthesis.

Protoplasts from hyphae of Saprolegnia monoica. collected from lytic medium after different times of incubation, represented a sequential fractionation of mycelium from apical to distal zones. This method gives the opportunity to study the intrahyphal localization of cell wall glucan synthases in relation to apical growth. (l→4)-β-Glucan synthases were mainly found in early protoplasts (i.e. from apical zones) while the highest (l→3)-β-glucan synthase activities were recovered in late protoplasts (i.e. from the subapical zones). Early protoplasts also exhibited the highest rate of cell wall synthesis during regeneration. The distribution of the enzymes along the fungal hyphae might provide a clue to the mechanisms of cell wall polysaccharide deposition during apical growth.

From a λgtWES library of the chromosome of Bacillus subtilis. phages carrying DNA from the hisA and ilv-leu regions were isolated. They were identified by their ability to form complementing plaques on hisB. iluC or leuB mutants of Escherichia coli K12 under selective conditions and in the presence of a helper phage. The his phages complemented E. coli his A, B or D mutations and could transform seven mutations in the hisA region of the B. subtilis chromosome; each carried a single Eco RI insert of about 8·2 kb. Phages complementing E. coli ilvC or leuB mutations and carrying the equivalent B. subtilis genes ilvC and leuC transformed a range of mutations in the B. subtilis ilv-leu region. The distribution of genetic markers carried by the phages suggests that the entire ilv -leu cluster from azlA through leuD is covered in the collection of phages obtained and is carried in three Eco RI restriction fragments of approximately 6·7, 4·7 and 2·85 kb.

The non-conjugative plasmids pAS9, RSF1010, pBR325 and pML21 were mobilized from Escherichia coli into Rhodopseudomonas sphaeroides by conjugative plasmids of the P-1 incompatibility group or their derivatives. The transfer of plasmids pAS9 and RSF1010 was rec, A -independent and both plasmids could be maintained in the autonomous state in R. sphaeroides cells. In contrast, the transfer of the plasmids pML21 and pBR325 based on the ColEl replicon was dependent on the recA status of donor strains. Replication and maintenance of these plasmids occurred as a result of the production of cointegrates formed by recombination between the mobilized and mobilizing plasmids.