- Volume 129, Issue 3, 1983

During axenic growth, microplasmodia of Physarum polycephalum secreted chitinase and β-N-acetylhexosaminidase. Chitin added to the medium was depolymerized to N-acetylglucosamine, but this was not used for growth. Spent culture medium contained three enzymes with activity towards 4-methylumbelliferyl-β-N-acetylglucosaminide and these were separated by chromatography on DEAE-cellulose. In previous work β-N-acetylhexosaminidases X and Y were identified. The latter form has now been resolved to yield a new form Z. The Y form can be readily distinguished from both X and Z by its lack of activity towards N,N′-diacetylchitobiose. Comparison of β-N-acetylhexosaminidases X and Z revealed differences in thermal stability, pH optima and K m values. 3,4-Dinitrophenyl-tetra-N-acetylchitotetraoside is hydrolysed by β-N-acetylhexosaminidase Z, but not by the X form of the enzyme.

A low molecular weight inhibitor of the thiol proteases papain and bromelain has been identified and partially purified from the culture filtrates of Aspergillus niger. Further studies have shown that this inhibitor reacts with compounds containing a free thiol group and as such it is capable of inhibiting other enzymes that contain a functional thiol group at their active site.

Extracts of Bacillus sphaericus NCTC 9602 catalysed the formation of meso-diaminopimelate from aspartic β-semialdehyde plus pyruvate, or from dihydrodipicolinate, even though no activities of tetrahydrodipicolinate acetylase (or succinylase) nor N-acetyl-(or N-succinyl-)ll-diaminopimelate deacylase nor diaminopimelate epimerase were found. However, meso-diaminopimelate d-dehydrogenase was present, and had very high activity at pH 7.5 in the direction of synthesis of meso-diaminopimelate from tetrahydrodipicolinate. A lysine-requiring mutant of B. sphaericus lacked diaminopimelate dehydrogenase, and this enzyme reappeared in a revertant that grew without lysine. Other lysine-requiring auxotrophs were defective in dihydrodipicolinate synthase or dihydrodipicolinate reductase or diaminopimelate decarboxylase, but had diaminopimelate dehydrogenase. Diaminopimelate dehydrogenase is not important in the assimilation of ammonia. Mutants that lack this enzyme or else cannot make one of its substrates (tetrahydrodipicolinate) still grow rapidly in minimal medium (plus 0.7 mm-l-lysine) containing ammonium chloride (36 mm) as the only major source of nitrogen. The wild-type grew with l-glutamine, but not with glutamate or lysine as sole source of nitrogen.

The effects of isomaltosaccharides of various molecular weights (isomaltose to dextran T2000) on glucan synthesis by a water-soluble glucan-synthesizing glucosyltransferase enzyme (GTase-S) and a water-insoluble glucan-synthesizing enzyme (GTase-I), both from Streptococcus mutans OMZ176, were examined. The activity of GTase-S was not affected by the addition of the isomaltosaccharides, but GTase-I was stimulated increasingly by isomaltosaccharides with degrees of polymerization more than 10. The GTase-I activity first increased and thereafter decreased slightly with increasing amounts of a soluble dextran. Maximal stimulation occurred at concentrations in the range 0·1 to 0·2 mg ml−1, when dextran T10 was used as a primer. The rate of glucan synthesis was highly enhanced by the combined action of GTase-S and GTase-I. The profile of the net activity of GTase-I in the presence of various amounts of GTase-S was similar to that of GTase-I in the presence of increasing amounts of an exogenous dextran. These results collectively suggest that soluble glucan produced by GTase-S from sucrose acts as an intrinsic primer for the glucan synthesis by GTase-I, indicating the contribution of autopriming in glucan synthesis by crude GTase of S. mutans.

Six penicillin-binding proteins (PBPs) were detected in clinical isolates of each one of three group D streptococci: Streptococcus bovis, S. faecalis and S. faecium. When examined in whole organisms, the PBPs of S. faecium, the most penicillin-resistant species of group D streptococci, generally had lower affinities for the antibiotic than those of S. faecalis (intermediate penicillin resistance), which in turn were of lower affinity than those of S. bovis (penicillin-sensitive): On the other hand, no quantitative correlation could be established between the binding of penicillin to any one PBP or group of PBPs, and the penicillin MIC value for the corresponding micro-organism. Examination of the amounts of antibiotic bound and the rates of binding to PBPs of equal numbers of protoplasts and whole bacteria of S. faecalis and S. faecium, indicated that there was no permeability barrier to benzylpenicillin in the cell walls of these species. The lower antibacterial effectiveness of cephalothin compared with ampicillin in group D streptococci was paralleled by the higher concentrations of cephalothin needed in competition assays to inhibit the lower molecular size PBPs of these bacteria.

Outer membrane fractions of Moraxella nonliquefaciens 7784 strains SC-c and N-b, isolated by extraction with lithium acetate, were analysed by SDS-PAGE. Three main proteins were found, of which one, with an apparent molecular weight of 19500, was undetectable in membranes isolated by lysis of lysozyme/EDTA spheroplasts. All three major proteins were heat modifiable.

Colicin E4 was produced in very large amounts when Escherichia coli K12 strain W3110 pColE4-CT9 was grown in the presence of 0.5 μg mitomycin C ml−1. The colicin was discharged from the producing cells when they lysed and was then degraded by a protease located on the surface of the producing cells. Colicins and proteolytic fragments derived from them could be concentrated from spent culture medium by filtration through Millipore membrane filters. Colicins are probably retained by these filters by hydrophobic interactions since binding was unaffected by changes in ionic conditions but-was completely inhibited in the presence of ionic or non-ionic detergents.

A characteristic mycobacterial wax, phthiocerol dimycocerosate, has been isolated from liver of armadillos experimentally infected with Mycobacterium leprae. The structure of this wax is generally similar to that produced by Mycobacterium tuberculosis, but the homologous phthiocerol and the mycocerosic acid components from M. leprae are significantly different from those of M. tuberculosis.

The effects were studied of increasing concentrations of bentonite clay on the interactions between the fungal pathogen Gaeumannomyces graminis var. tritici and two bacterial antagonists. Clay increased the growth rate of G. graminis; this increase was statistically significant, though small, and could have been due to an effect on water availability. The effectiveness of one of the bacterial culture nitrates in restricting the fungal growth was reduced by the clay, though antagonism was maintained in the presence of bacterial cells. The clay may adsorb some of the bacterial toxins, and lowered water availability may increase bacterial antagonism before it significantly reduces fungal growth. Antagonism by the other bacterium was not affected by clay.

Sapwood blocks of lime (Tilia vulgaris) and pine (Pinus sylvestris) placed to one side of growing colonies of several wood decay fungi markedly influenced their hyphal extension patterns, particularly by causing positive or negative tropic responses. A method was devised to measure and quantify these responses and analyse them statistically. Chemotropic responses were demonstrated by Chaetomium globosum, Trichoderma viride, Serpula lacrymans and Coniophora puteana in the presence of air-dried and heat-dried wood but not by Coriolus versicolor or Humicola grisea. The implications of chemotropism as a factor influencing the invasion and colonization of wood are discussed.

A sample of colonies from the Clarke-Carbon Co1E1-Escherichia coli DNA plasmid gene bank was screened by conjugation for complementation of the lipoamide dehydrogenase lesion of a deletion strain lacking all components of the pyruvate dehydrogenase complex, Δ(aroP aceE aceF lpd). Two Co1E1-lpd + hybrid plasmids were identified: pGS2 (Co1E1-ace lpd +; 24 kb) and pGS5 (Co1E1-lpd +; 14 kb). Enzymological studies confirmed that pGS2 expressed all the activities of the pyruvate dehydrogenase complex, whereas pGS5 expressed the lipoamide dehydrogenase and acetyltransferase activities (the latter from a ColE1 promoter). These and other plasmids were used to construct a 47-site (15 enzymes) restriction map for a 24·2 kb segment of bacterial DNA in the nadC-lpd region. A futher 13 sites (six enzymes) were defined in a 5·4 kb sub-segment containing the lpd gene. γ phage derivatives containing specific fragments were constructed and used in transduction studies which located the ace and lpd genes in a 7·78 kb sub-segment flanked by AccI and NruI sites.

DNA repair and genetic recombination have been studied in recF143 mutants of Escherichia coli in which expression of inducible, SOS repair activities was altered by additional mutations either in lexA or recA.recF143 and lexA3 appeared to act additively to increase sensitivity to UV irradiation and to reduce recombination proficiency. The recF defect was suppressed in strains carrying the tif-1 allele of recA but not in strains carrying mutations that increased synthesis of recA + protein or which directly inactivated lexA repressor. These and other data are interpreted to suggest that the recF defect is related to a reduced activity of recA protein. The implications of these findings are discussed in relation to the control of SOS activities and cell division during normal growth.

Bacillus subtilis strains possessing the trpE30 marker (splitting of the trpE locus and a non-tandem duplication of chromosome segment Ib: purB-tre) when transformed or transduced to tryptophan independence mainly give rise to haploid cells with the genetic structure of strain 168. However, among the Trp+ transformants or transductants about 10% are merodiploid carrying a non-tandem duplication of segment C (trpE-ilvA) while maintaining that of segment Ib. Linkage and segregation studies made it possible to determine their genetic structure, which can be represented by three different maps. In map a the copies of Ib are inverted repeats and one of them is flanked by two direct repeats of segment C; in map b two Ib-C segments are inverted repeats and in map c the copies of C are inverted repeats with one of them flanked by direct repeats of Ib. It is proposed that transition from map a to map b and then to map c, and vice versa, may occur by recombination between inverted repeats of either Ib or C. The merodiploids are unstable, recombination between direct repeats leading to haploid cells of 168-type structure. The models proposed for merodiploid formation call for fusion of two recipient chromosomes mediated by the donor segment and recombination between copies of a DNA sequence of the two chromosomes located in different regions. In the case of PBS-1 mediated transduction the greater length of the donor DNA segment makes it possible to obtain the merodiploids with a single recipient chromosome and this needs only a slight modification of the models. No trpE30 + merodiploids are found in transformation when the recipient carries a deletion of the SPβ prophage, or in transduction when both donor and recipient possess this deletion. These results indicate that the homologous sequences involved may be part of the SPβ prophage or that a sequence of bacterial DNA has a good homology with it.

Prime plasmids have been isolated from Methylophilus methylotrophus AS1 using the IncP-1 plasmid pMO172. Such plasmids can complement mutant function when transferred to appropriate strains of Pseudomonas aeruginosa PAO. From the range of particular functions complemented by each prime plasmid, a preliminary map of the M. methylotrophus AS1 genome with four groups of linked markers was obtained. Physical examination of two of these prime plasmids showed that each had acquired an additional DNA segment. Southern hybridization experiments showed there was sequence homology between the additional DNA segments and M. methylotrophus AS1 DNA, confirming that these prime plasmids carried a segment of the M. methylotrophus AS1 genome. Mapping by complementation may be applicable to other bacteria in which mutants suitable for selecting recombinants are not readily available.

The replication and incompatibility region of the IncFVI plasmid pSU502 has been isolated by in vitro DNA manipulation as part of a 12·6 kb plasmid, denominated pSU503. Plasmid pSU503 was strongly incompatible with its parental plasmid, pSU1, but was fully compatible with the haemolytic plasmids pSU316 (IncFIII/IV), pHly152 (IncI2) and pSU233 (Inc-pSU233). Furthermore, the 6.9 kb EcoRI fragment of pSU503 which carries the replication and incompatibility determinants of pSU1 did not show any detectable homology (<70 %) with any of the haemolysin-determining plasmids with which it is compatible. Thus, homologous haemolysin determinants have become linked to apparently unrelated replicons.