- Volume 136, Issue 2, 1990

[U-14C]Phenylalanine was injected into spring barley plants during 140 d growth. Autoradiography of the plants revealed that, apart from high concentrations around the injection sites, radioactivity was evenly distributed throughout the plants. [14C]Lignin lignocellulose was prepared from plants cropped at 30, 58, 82 and 140 d after sowing, using organic and aqueous solvents followed by enzymic hydrolysis with commercial protease and polysaccharidase preparations. Weight loss due to solvent and enzymic treatments was greatest in the youngest plants, resulting in a preparation of [14C]lignocellulose of specific activity 2-8-fold higher than preparations from more mature plants. When subjected to crude preparations of extracellular protein from either Phanerochaete chrysosporium or Streptomyces cyaneus the substrate derived from the youngest plants (30 d) was particularly susceptible to solubilization of lignocellulose by polysaccharidases. Substrates prepared from plants after 58 d growth (i.e. fully grown) showed significant levels of lignocellulose solubilization that were not due to the action of polysaccharidases. When used in similar assays the oldest crops (82 and 140 d) were solubilized at very low levels. The desirability of using fully grown plants as substrates in lignin solubilization and biodegradation studies is clearly demonstrated.

Valine dehydrogenase (VDH) from Streptomyces coelicolor A3(2) was purified from cell-free extracts to apparent homogeneity. The enzyme had an M r 41000 in denaturing conditions and an M r 70000 by gel filtration chromatography, indicating that it is composed of two identical subunits. It oxidized l-valine and l-α-aminobutyric acid efficiently, l-isoleucine and l-leucine less efficiently, and did not act on d-valine. It required NAD+ as cofactor and could not use NADP+. Maximum dehydrogenase activity with valine was at pH 10.5 and the maximum reductive amination activity with 2-oxoisovaleric acid and NH4Cl was at pH 9. The enzyme exhibited substrate inhibition in the forward direction and a kinetic pattern with NAD+ that was consistent with a sequential ordered mechanism with non-competitive inhibition by valine. The following Michaelis constants were calculated from these data: l-valine, 10·0 mm; NAD+, 0·17 mm; 2-oxoisovalerate, 0·6 mm; and NADH, 0·093 mm In minimal medium, VDH activity was repressed in the presence of glucose and NH4 +, or glycerol and NH4 + or asparagine, and was induced by d- and l-valine. The time required for full induction was about 24 h and the level of induction was 2- to 23-fold.

A monoclonal antibody (mAb), designated 15D8, was produced from BALB/c splenocytes of mice injected with Escherichia coli flagella. ELISA of motile cells, non-motile cells and partially purified flagellin proteins showed that the mAb reacted specifically with flagella of E. coli and with other members of the family Enterobacteriaceae. Western immunoblot analyses of enterobacterial flagella or cell extracts demonstrated that the antibody reacted with a single protein species in the extracts which was identical in size to purified flagellin. The antigenic determinant for this antibody appears to be surface exposed and linear in configuration, since the antibody reacted with native flagella and flagella which had been denatured. This antibody was also used to demonstrate that although the flagella proteins are heterogeneous in size, at least one epitope is highly conserved.

A rapid and efficient method of transforming Corynebacterium glutamicum by electroporation is described. A number of factors are important in determining the level of transformation obtained. Cells grown in the presence of glycine and isonicotinic acid hydrazide and harvested in early exponential growth phase were much easier to transform. The recovery medium on which transformants were isolated also had a significant effect on the number of transformants obtained because cells were osmotically or electrochemically sensitive following electroporation. Transformation efficiencies of up to 5 × 105 transformants per μg plasmid DNA with homologously derived DNA and 2 × 103 transformants per μg of DNA derived from Escherichia coli were obtained.

A 6·5 kb DNA fragment containing a chloramphenicol-resistance gene of Streptomyces venezuelae ISP5230 was cloned in Streptomyces lividans M252 using the high-copy-number plasmid vector pIJ702. The gene was located within a 2·4 kb KpnI-SstI fragment of the cloned DNA and encoded an enzyme (chloramphenicol hydrolase) that catalysed removal of the dichloroacetyl moiety from the antibiotic. The deacylated product, p-nitrophenylserinol, was metabolized to p-nitrobenzyl alcohol and other compounds by enzymes present in S. lividans M252. Examination of the genomic DNA from several sources using the cloned 6·5 kb SstI fragment from S. venezuelae ISP5230 as a probe showed a hybridizing region in the DNA from S. venezuelae 13s but none in the DNA from another chloramphenicol producer, Streptomyces phaeochromogenes NRRLB 3559. The resistance phenotype was not expressed when the 6·5 kb SstI fragment or a subfragment was subcloned behind the lac-promoter of plasmid pTZ18R in Escherichia coli.

The structural gene (glnA) encoding the ammonia-assimilation enzyme glutamine synthetase (GS) has been cloned from the obligate methanotroph Methylococcus capsulatus (Bath). Complementation of Escherichia coli glnA mutants was demonstrated. In vitro expression analysis revealed that the cloned glnA gene coded for a polypeptide of apparent M r 60000, as determined by PAGE. Expression of the M. capsulatus (Bath) glnA gene in E. coli was regulated by nitrogen levels in an Ntr+ but not an Ntr− background. The nucleotide sequence of the M. capsulatus (Bath) glnA gene and flanking sequences was determined. This gene, of 1407 bp, encoded a polypeptide of M r 51717 containing 468 amino acids. The 5′ leader region contained three putative promoters. Promoters P1 and P3 resembled the canonical - 10 - 35 E. coli-type promoter. Promoter P2, which was located between P1 and P3, resembled the NtrA-dependent promoters of enteric organisms. A potential NtrC-binding site was also determined, flanking the Pribnow box at P1. Comparisons of nucleotide-derived amino acid sequences of GS enzymes from prokaryotes and eukaryotes with GS from M. capsulatus are made.

The aroC genes from Salmonella typhi and Escherichia coli, encoding 5-enolpyruvylshikimate-3-phosphate phospholyase (chorismate synthase) were cloned in E. coli and their DNA sequences were determined. The aroC gene from S. typhi was isolated from a cosmid gene bank by complementation of an E. coli aroC mutant. The corresponding E. coli gene was isolated from a pBR322 gene bank by colony hybridization using DNA encoding the aroC gene from S. typhi as a hybridization probe. Analysis of the nucleotide sequence revealed that both genes have an open reading frame capable of encoding proteins comprising 361 amino acids. The calculated molecular mass of the protein from S. typhi is 39108 Da while that of the protein from E. coli is 39138 Da. Homology is particularly strong between the coding regions of the genes: 95% when protein sequences are compared, and 83% when DNA sequences are examined. Use of a deletion variant of the E. coli aroC gene demonstrates that the C-terminal 36 amino acids are not essential for the correct folding or functional activity of the chorismate synthase enzyme.

Lipopolysaccharides (LPS) were extracted from whole cells of seven strains of Bacteroides gingivalis - 381, ATCC 33277, BH18/10, OMZ314, OMZ409, 6/26 and HW24D-1 - by the phenol/water procedure, and purified by treatment with nuclease and by repeated ultracentrifugation. These LPS were composed of hexoses, hexosamines, fatty acids, phosphorus and phosphorylated 2-keto-3-deoxyoctonate (KDO). The major components of the lipid portion of these LPS were hexadecanoic, 3-hydroxyhexadecanoic, branched 3-hydroxypentadecanoic and branched 3-hydroxyheptadecanoic acids. All the LPS preparations induced marked mitogenic and in vitro polyclonal B cell activation responses in spleen cells from both C3H/HeN and C3H/HeJ mice, exhibited no definitive preparatory activity in the local Shwartzman reaction in rabbits, but were active in the chromogenic Limulus amoebocyte lysate test. A monoclonal antibody (mAb) raised against the LPS from B. gingivalis strain 6/26 reacted with LPS from all other B. gingivalis strains tested. Other mAbs raised against LPS from B. gingivalis strains 381 and 6/26 reacted with the LPS from strains 381, ATCC 33277, BH18/10 and 6/26 (these strains were termed LPS serogroup I), as revealed by ELISA and immunodiffusion. The LPS from these strains except for 6/26 showed almost indentical patterns in SDS-PAGE stained with ammoniacal silver. A mAb raised against the LPS from B. gingivalis HW24D-1 reacted with the LPS from strains OMZ314, HW24D-1 and OMZ409 (LPS serogroup II). These LPS, except OMZ409, exhibited very similar profiles in SDS-PAGE. These results indicate that there are at least two different antigenic groups present among LPS from B. gingivalis strains, as well as a common, species-specific antigen.

A protective glycolipid antigen (PAg) was extracted from Leptospira interrogans serovar canicola with chloroform/methanol/water (1:2:0.8, by vol.) and partially purified by silica gel column chromatography. The PAg elicited a protective response in hamsters and in cyclophosphamide-treated mice subsequently challenged with homologous Leptospira. The PAg band was detected as a single smear-like band, corresponding to a protein of 23–30 kDa, by silver-staining in SDS-PAGE. In immunoblots, this band reacted with a monoclonal antibody, A5, which agglutinated serovar canicola and recognized a serovar-specific antigen. Furthermore, the PAg did not migrate on silica gel TLC, but was detected at the origin as a ninhydrin- and naphthol-positive spot. This suggests that PAg is a hydrophilic molecule with a carbohydrate chain that contains amino groups, possibly as amino sugars.

Proteolytic activities, active against casein and insulin are present in cell-free extracts of the psychrotrophic bacterium Arthrobacter globiformis S155. These activities were compared (i) following growth of the bacterium at different temperatures (10, 20 and 32C) and (ii) after a temperature shift from 10 to 32C. Both activities (measured at 20C) were greater in cells grown at 32C and after a temperature shift. Chloramphenicol prevented the increases in activities. The proteolysis of casein was stimulated by ATP; the stimulation was greater following a temperature shift. In addition, proteolysis of casein (measured at 20C) was activated by pre-incubation at temperatures between 45 and 60C; the proportion of proteolytic activity activated by such treatment also increased with growth temperature. Comparison by two-dimensional electrophoresis of the polypeptides synthesized at 10 and 32C suggests that A. globiformis S155 produces proteins specific to both temperatures (13 species were either exclusive to or present in increased amounts during growth at 10C, while at least 21 polypeptides were preferentially synthesized at 32C). A temperature shift from 10 to 32C promoted the synthesis of at least 16 polypeptides, many with M r values similar to those of heat shock proteins synthesized at higher temperatures in mesophiles. A temperature up-shift of A. globiformis S155 from 10 to 20C did not provoke the synthesis of new proteins; heat shock proteins were produced only at 28C and higher. The similarity in kinetics of the appearance of heat shock proteins and the increase in proteolytic activities suggest that in A. globiformis S155 some heat shock proteins might also possess protein catabolic function.

Yeast cells of Candida albicans which had been attached to polylysine-coated microscope slides were induced to form buds or germ tubes in the presence of external electrical fields. The sites of budding and germ tube formation and the growth of germ tubes and hyphal branches were polarized preferentially towards the cathode. Buds were not converted to pseudohyphae or germ tubes by the field and the field had no effect on the positioning of nuclei or septa in the yeast cell or germ tube. Buds were less polarized than germ tubes at any given applied voltage. The polarization of buds reached a peak at an electrical field of 12 m V per cell. Polarization of germ tubes was biphasic, increasing rapidly with increasing field strengths up to 5 mV per cell, and then more slowly in stronger fields. An electrical field was only required for a fraction of the time taken for germ tubes to start to form, so cells retained a memory of experiencing an electrical field which influenced the selection of sites of evagination. Increasing the electrical field delayed the time of germ tube evagination and inhibited the rate of germ tube extension. Unlike previous findings with other filamentous fungi, germ tubes grew unidirectionally towards the cathode for extended periods and did not deviate to a perpendicular orientation. This result suggests that the septal pore of the filamentous form may have high electrical resistance and would act as an effective barrier to solute transport between intercalary compartments.

A dual-laser flow cytometer was used to analyse different species of bacteria for the molar percentage of guanine-plus-cytosine (% G + C) without the need for DNA extraction or purification. Ethanol-fixed bacterial cells were stained with a combination of DNA-specific fluorochromes, Hoechst 33258 and chromomycin A3, which bind to AT- and GC-rich regions of DNA, respectively. A linear relationship (r = 0·99) was demonstrated between the log of the ratio of chromomycin A3 to Hoechst 33258 fluorescence and the log of the % G + C as determined by thermal denaturation (T m) or buoyant density centrifugation (Bd) methods. Linearity was maintained for all bacterial species tested over the range of 28–67% G + C. A standard curve was constructed using five strains whose % G + C had been determined by other methods. From the equation describing this line, the % G + C values of nine other strains with known DNA base composition, together with the five strains used to construct the curve, were calculated using the chromomycin A3 to Hoechst 33258 ratio and were in agreement with values obtained by T m, Bd or HPLC. The reproducibility of flow cytometric analysis (mean error 0·7% G + C) compared well with the reproducibility of other methods. Mixtures containing two species were also analysed. Two cell populations could be discerned in mixtures containing two species which differed in base composition by as little as 4% G + C. Dual-laser flow cytometric analysis of stained bacteria is a rapid, simple and accurate method for determining the % G + C of bacterial DNA and can be used to distinguish populations of bacteria with differing % G + C content.

Ninety-one strains of Bacillus sphaericus, including representatives of all the established DNA homology groups, related round-spored and oval-spored species, and six strains pathogenic for mosquito larvae, were examined for 155 characters. Numerical analyses (Jaccard coefficient/average linkage clustering) based on the 88 variable features revealed 14 clusters at the 79% similarity level that contained more than one strain and 17 single member clusters. All insect pathogenic strains were recovered in a single cluster and the classification was in accord with an established classification based on DNA sequence homology. Two frequency matrices for probabilistic identification were constructed and tested. A comprehensive matrix comprising 14 mesophilic, round-spored taxa and 27 tests gave good results for identification of hypothetical median organisms, cluster overlap and identifications of representative strains (based on data generated in the classification study). Reference strains for the 14 taxa and eight additional insect pathogenic strains were examined for the 27 tests and were correctly identified with high scores using this matrix. A second matrix comprising seven taxa and 13 tests also performed well in the theoretical evaluation and correctly identified the reference strains and insect pathogenic strains.