- Volume 137, Issue 8, 1991

The structure–activity relationships of different nikkomycins were studied to evaluate the structural requirements for a potent chitin synthase inhibitor. We investigated the transport of the nikkomycins via the peptide transport system of the yeast Yarrowia lipolytica and determined the kinetic parameters for nikkomycin Z uptake [K m = 24 μM, V max = 2·2 nmol min−1 (mg dry wt)−1]. We demonstrated that the β-methyl group of the N-terminal amino acid of dipeptide nikkomycins protects the molecule against peptidase activity in crude cell-extracts of different fungi. Furthermore, the relationship between inhibition constants for chitin synthase, transport of the nikkomycins via the peptide transport system, susceptibility to degradation by cellular proteases and whole-cell activity of the nikkomycins are discussed.

The production of pectinase was studied in Neurospora crassa, using the hyperproducer mutant exo-1, which synthesized and secreted five to six times more enzyme than the wild-type. Polygalacturonase, pectin lyase and pectate lyase were induced by pectin, and this induction was glucose-repressible. Polygalacturonase was induced by galactose four times more efficiently than by pectin; in contrast the activity of lyases was not affected by galactose. The inducing effect of galactose on polygalacturonase was not glucose-repressible. Extracellular pectinases were separated by ion exchange chromatography. Pectate and pectin lyases eluted into three main fractions containing both activities; polygalacturonase eluted as a single, symmetrical peak, apparently free of other protein contaminants, and was purified 56-fold. The purified polygalacturonase was a monomeric glycoprotein (38% carbohydrate content) of apparent molecular mass 36·6–37.0 kDa (Sephadex G-100 and urea-SDS-PAGE, respectively). The enzyme hydrolysed predominantly polypectate. Pectin was also hydrolysed, but at 7% of the rate for polypectate. K m and V max for polypectate hydrolysis were 5·0 mg ml−1 and 357 μmol min−1 (mg protein)−1, respectively. Temperature and pH optima were 45°C and 6·0, respectively. The purified polygalacturonase reduced the viscosity of a sodium polypectate solution by 50%, with an increase of 7% in reducing sugar groups. The products of hydrolysis at initial reaction times consisted of oligogalacturonates without detectable monomer. Thus, the purified Neurospora crassa enzyme was classified as an endopolygalacturonase [poly(1,4-α-d-galacturonide) glycanohydrolase; EC 3.2.1.15].

Rhodococcus rhodochrous strain CTM co-metabolized 2-methylaniline and some of its chlorinated isomers in the presence of ethanol as additional carbon source. Degradation of 2-methylaniline proceeded via 3-methylcatechol, which was metabolized mainly by meta-cleavage. In the case of 3-chloro-2-methylaniline, however, only a small proportion (about 10%) was subjected to meta-cleavage; the chlorinated meta-cleavage product was accumulated in the culture fluid as a dead-end metabolite. In contrast, 4-chloro-2-methylaniline was degraded via ortho-cleavage exclusively. Enzyme assays showed the presence of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase as inducible enzymes in strain CTM. Extended cultivation of strain CTM with 2-methylaniline and 3-chloro-2-methylaniline yielded mutants, including R. rhodochrous strain CTM2, that had lost catechol 2,3-dioxygenase activity; these mutants degraded the aromatic amines exclusively via the ortho-cleavage pathway. DNA hybridization experiments using a gene probe revealed the loss of the catechol 2,3-dioxygenase gene from strain CTM2.

Rhodococcus rhodochrous strain CTM degrades 2-methylaniline mainly via the meta-cleavage pathway. Conversion of the metabolite 3-methylcatechol was catalysed by an M r 156 000 catechol 2,3-dioxygenase (C23OI) comprising four identical subunits of M r 39000. The corresponding gene was detected by using an oligonucleotide as a gene probe. This oligonucleotide was synthesized on the basis of a partial amino acid sequence obtained from the purified enzyme from R. rhodochrous. The structural gene of C23OI was located on a 3.5 kb Bg/II restriction fragment of plasmid pTCl. On the same restriction fragment the gene for a second catechol 2,3-dioxygenase, designated C23OII. was found. This gene coded for the synthesis of the M r 40000 polypeptide of the M r 158000 tetrameric C23OII. More precise mapping of the structural genes showed that the C23OI gene was located on a 1.2 kb Bg/II SmaI fragment and the C23OII gene on the adjacent 1.15 kb SmaI fragment. Comprehensive substrate range analysis showed that C23OII accepted all the substrates that C23OI did, but additionally cleaved 2,3-dihydroxybiphenyl and catechols derived from phenylcarboxylic acids. C23OI exhibited highest activity towards methylcatechols, whereas C23OII cleaved unsubstituted catechol preferentially.

A chitosanase was purified from the intercellular fluid extract of chemically stressed barley (Hordeum vulgare) leaves. Purification was achieved by preparative PAGE involving separation under native conditions at pH 4.3 (Reisfeld system) followed by denaturing PAGE in the presence of SDS. One basic chitosanase with a molecular mass of 19 kDa could hydrolyse chitosan and glycol chitosan of crustacean origin in addition to fungal chitosan isolated after treatment of Mucor mucedo cells with sodium hydroxide followed by extraction in acetic acid. This enzyme did not exhibit activity towards β-1,4-polymers such as chitin, cellulose or peptidoglycan (from Micrococcus luteus) after SDS-PAGE. The chitosanase was conjugated to colloidal gold at pH 9·5 and applied to fungal tissue sections or to isolated glycol chitosan, chitosan, cellulose or chitin. The chitosanase-gold complex reacted intensely with glycol chitosan and chitosan but did not bind to chitin or cellulose. The enzyme-gold complex also labelled chitosan isolated from M. mucedo. However, it did not react with M. mucedo cells in tissue sections. Labelling with the chitosanase-gold complex was easily detected over cell walls of Fusarium oxysporum f. sp. radicis-lycopersici spores and hyphae as well as over cell walls of Ophiostoma ulmi, Aspergillus niger and Colletotrichum lindemuthianum. No binding was observed with Pythium ultimum.

Survival of Bacillus subtilis strain 168 containing plasmid pAB224, which carries a gene for tetracycline resistance, was studied in mushroom compost under mesophilic and thermophilic conditions. Stable populations of B. subtilis were maintained as spores in both sterile and fresh mushroom compost incubated at 37°C. At 65°C, the introduced B. subtilis populations declined during incubation but spores were still detectable after 28 d. Survival at the higher temperature was greater in fresh than in sterile compost. There was no apparent loss of plasmid pAB224 or plasmid-determined phenotype from the introduced B. subtilis population at either incubation temperature. The frequency of tetracycline resistance in the indigenous Bacillus population was very low (10-10), but some tetracycline-resistant isolates contained plasmid DNA. Four plasmid DNA profiles were found associated with five Bacillus phenotypes, and some evidence for homology with pAB224 was found. However, pAB224 was found to be a suitable marker for release studies because it was easily recovered, readily distinguished from indigenous plasmids on agarose gels, and was maintained in compost-grown B. subtilis 168 in the absence of any selective pressure.

By use of a functional assay for activity, an autolysin structural gene was cloned on a 3 kb EcoRI DNA fragment from a λgt11 expression library of Bacillus subtilis 168 trpC2 genomic DNA. Sequencing of the fragment showed five open reading frames, the central one of which encoded the lytic enzyme as found by subclone activity mapping and its homology to a recently sequenced autolysin gene. The protein had a deduced sequence of 272 amino acids and a molecular mass of 29957 Da. When expressed in Escherichia coli DH5α, the protein was processed to a 21 kDa form, as estimated by renaturing SDS-PAGE. The autolysin was an N-acetylmuramyl-L-alanine amidase and its activity was MgCI2-dependent (20 mM optimum) and LiC1-sensitive. The enzyme could bind to and hydrolyse a wide range of peptidoglycan substrates isolated from Gram-positive bacteria; the binding was also MgC12-dependent. Initial mapping experiments located the autolysin gene near aroD on the B. subtilis 168 chromosome.

Enterotoxigenic Escherichia coli (ETEC) strains of nineteen serogroups which produced colonization factors (coli-surface-associated antigens CS5, CS6, CS7 and CS17, colonization factor antigen CFA/III and putative colonization factors PCFO159:H4, PCFO166 and PCFO9) were tested for hybridization with a DNA probe containing the cfaD sequence that regulates expression of CFA/I. Strong colony hybridization, similar to that with the CFA/I-positive control strain H10407, occurred with ETEC strains of serogroups O27, O159 and O169 which produced CS6 antigen, and with all the strains which produced PCFO166 fimbriae. Weak colony hybridization, compared to the control strain, was found with ETEC producing CS5 fimbriae with CS6 antigen, CFA/III fimbriae with CS6 antigen, CS7 fimbriae or PCFO159:H4 fimbriae. CS6-antigen-positive strains of serogroups O79, O89 and O148 and all the CS17-antigen-positive and PCFO9-fimbriae-positive strains were negative in colony hybridization tests with the cfaD probe. Plasmid DNA of nine ETEC strains and their colonization-factor-negative derivatives was tested for hybridization with the cfaD probe and with ST and LT oligonucleotide probes. The sequences that hybridized with the cfaD probe were on the plasmids which coded for enterotoxin production. Fifteen strains were transformed with NTP513, a recombinant plasmid which contains the CFA/I region 1 fimbrial subunit operon but lacks a functional cfaD sequence, in order to determine whether DNA in any of these strains could substitute for the cfaD sequence in the regulation of production of CFA/I fimbriae. Transformants of five strains which produced the colonization factors CS6, PCFO166, CS5 + CS6, CS7 and PCFO9, and of one strain which was a colonization-factor-negative derivative of the CS5,CS6-producing strain E17018, gave good production of CFA/I fimbriae comparable to the CFA/I-positive control strain H10407. Transformants of two strains, producing PCFO159 fimbriae and CS17 antigen, respectively, gave weak CFA/I production. Transformants of one strain producing CS6 antigen and of six colonization-factor-negative derivatives did not produce CFA/I fimbriae. These results showed that plasmids in seven of eight types of colonization-factor-positive strains contained gene sequences which could substitute functionally for the cfaD sequence. Only two of these strains had gene sequences that hybridized strongly with the cfaD probe.

Each of five monoclonal antibodies (mAbs) prepared against the type 1 fimbriae of Actinomyces viscosus T14V reacted with a 54 kDa cloned protein previously identified as a fimbrial subunit. This purified protein completely inhibited the reaction of a specific anti-type-1-fimbria rabbit antibody with A. viscosus whole cells. Maximum values for the number of antibody molecules bound per bacterial cell ranged from 7 × 103 to 1·2 × 104 for the different 125I-labelled mAbs and was approximately 7 × 104 for 125I-labelled rabbit IgG or Fab against either type 1 fimbriae or the 54 kDa cloned protein. Although the different mAbs, either individually or as a mixture, failed to inhibit the type-1-fimbria-mediated adherence of A. viscosus T14V to saliva-treated hydroxyapatite, each rabbit antibody gave 50% inhibition of adherence when approximately 5 × 104 molecules of IgG were bound per cell. However, binding of each corresponding rabbit Fab had no significant effect on bacterial attachment unless much higher concentrations were used. These findings suggest that antibodies directed solely against the 54 kDa fimbrial subunit do not react with the putative receptor binding sites of A. viscosus T14V type 1 fimbriae. Instead, inhibition of attachment by the polyclonal antibodies may depend on an indirect effect of antibody binding that prevents the fimbria-receptor interaction.

The unicellular cyanobacterium Gloeothece sp. PCC 6909 was grown diazotrophically in continuous culture. Various light-dark regimes and dilution rates were applied. Nitrogenase activity (acetylene reduction), oxygen-production and -consumption, and glycogen were measured over 24 h periods covering one complete light-dark cycle. Gloeothece fixed nitrogen predominantly in the light, concomitant with photosynthetic oxygen evolution. This result was found irrespective of the imposed light-dark regime or dilution rate. It is concluded that temporal separation of photosynthesis and nitrogen fixation is not obligatory in Gloeothece.

Using a variety of techniques the distribution of the glutathione-regulated KefC K+-transport system among bacterial species was investigated. The presence of similar systems in a number of Gram-negative bacteria was demonstrated. In contrast, the system appeared to be absent from most Gram-positive bacteria tested with the exception of Staphylococcus aureus. Using the cloned Escherichia coli kefC gene as a probe for Southern hybridization it was shown that only limited DNA sequence homology exists with other bacteria, even when closely related members of the enteric group were examined.

Hyphal growth and secretion of proteins in Aspergillus niger were studied using a new method of culturing the fungus between perforated membranes which allows visualization of both parameters. At the colony level the sites of occurrence of growth and general protein secretion were correlated. In 4-d-old colonies both growth and secretion were localized at the periphery of the colony, whereas in a 5-d-old colony growth and secretion also occurred in a more central zone of the colony where conidiophore differentiation was observed. However, in both cases glucoamylase secretion was mainly detected at the periphery of the colonies. At the hyphal level immunogold labelling showed glucoamylase secretion at the tips of leading hyphae only. Microautoradiography after labelling with N-acetylglucosamine showed that these hyphae were probably all growing. Glucoamlyase secretion could not be demonstrated immediately after a temperature shock which stopped growth. These results indicate that glucoamylase secretion is located at the tips of growing hyphae only.