- Volume 135, Issue 4, 1989

Lipopolysaccharide (LPS) was isolated and purified from Wolinella recta ATCC 33238 by the phenol-water procedure and RNAase treatment. The sugar components of the LPS were rhamnose, mannose, glucose, heptose, 2-keto-3-deoxyoctonate (KDO) (3-deoxy-d-mano-octulosonate) and glucosamine. The degraded polysaccharide prepared from LPS by mild acid hydrolysis was fractionated by Sephadex G-50 gel chromatography into three fractions: (1) a high-molecular-mass fraction, eluting just behind the void volume, consisting of a long chain of rhamnose (22 mols per 3 mols of heptose residue) with attached core oligosaccharide; (2) a core oligosaccharide containing heptose, glucose and KDO, substituted with a short side chain of rhamnose; (3) a low-molecular-mass fraction containing KDO and phosphate. The main fatty acids of the lipid A were C12:0′ C14:0′3-OH-C14:0 and 3-OH-C16:0. The biological activities of the LPS were similar to those of Salmonella typhimurium LPS in activation of the clotting enzyme of Limulus amoebocytes, the Schwartzman reaction and mitogenicity for murine lymphocytes, although all the biological activities of lipid A were lower than those of intact LPS.

Lipolysis by Lysobacter enzymogenes is due to the production of two major extracellular esterases. These were investigated using a turbidimetric assay with Tween 20 as the substrate. One esterase is cell-associated and found in the particulate fraction of cell-free extracts, the other esterase is secreted into the culture medium. In batch culture with 0·8% yeast extract as the medium the particulate esterase was produced mainly during the exponential growth phase and its yield was increased about twofold by the addition of olive oil and other substrates. The supernatant esterase was produced mainly after the exponetial growth phase and was induced six- to tenfold by olive oil. PAGE, combined with activity staining using Tween 20 as the substrate, indicated that the two enzymes have different electrophoretic mobilities. Intact cells expressed 87% of the particulate cell-associated esterase activity. The enzyme was released from cells or from the particulate fraction by incubation with 0·2% Zwittergent 3-14. Tween 20 (2%, v/v) or 1 mM-EDTA released little of the enzyme from whole cells. Separation of the inner and outer membranes of L. enzymogenes showed that the particulate esterase was localized in the outer membrane. Both esterases were very active with Tween 20 as the substrate, but they hydrolysed other compounds at very different rates. The relative activities with Tween 20, p-nitrophenyl palmitate, tributyrin and olive oil were 100, 3, 11 and 0 for the particulate esterase and 100, 73, 0 and 28 for the secreted esterase. In addition to these two extracellular esterases, L. enzymogenes contains a hydrolase in the cytoplasm which is most active with tributyrin.

The cellulase gene from Erwinia chrysanthemi coding for endoglucanase Z was subcloned into a broad-host-range plasmid pGSS33 in Escherichia coli. The recombinant pNB20 was transferred into Zymomonas mobilis ATCC 10988 by mobilization using the helper plasmid RP4. Plasmid pNB20 was stably maintained in E. coli and Z. mobilis hosts. The endoglucanase gene celZ was expressed efficiently and the level of expression was higher in Z. mobilis than in E. coli. The specific activity of the enzyme was comparable to that of the parent strain of Er. chrysanthemi. The proteins produced by Z. mobilis and Er. chrysanthemi presented identical immunological and electrophoretic properties. Biosynthesis of endoglucanase occurred during the exponential growth phase of Z. mobilis and about 35% of the enzyme was released into the medium in the absence of detectable cell lysis. The endoglucanase appeared to be located in the periplasmic space in Z. mobilis.

The surface ultrastructure of 21 strains of Bacteroides intermedius was investigated by electron microscopy. Rat monoclonal antibodies (mAbs) were used to define serogroups and to detect the location of surface antigens. All 21 isolates had capsules as demonstrated by the use of wet and dry Indian ink stains. Negative staining of whole cells with 1% (w/v) methylamine tungstate showed that all 21 isolates carried clumped peritrichous fibrils with strain dependent morphology, density and length (≤0·75 μm). Fibrils on 11 of 13 fresh clinical isolates were more conspicuously clumped and easily visible, whereas those on 6 of 8 laboratory strains were indistinct and were at the limits of the resolution of the negative staining technique. Staining with ruthenium red (RR), followed by thin sectioning, revealed a dense, amorphous RR staining layer (RRL), up to 24·8 ±3·0 nm thick, adjacent to the outer membrane on all of 15 strains examined. All isolates had a less dense RR staining matrix (RRM) extending away from the RRL. The structure of the RRM varied between strains. Four rat mAbs (37BI6.1, 38BI1, 39BI1.1 and 40BI3.2) were used to serogroup the 21 strains of B. intermedius. Immunonegative staining revealed that the mAbs were not directed against fibrils. Antigens recognized by mAb 37BI6.1 and mAb 39BI1.1 were located on the surfaces of cells, beneath fibrils, and on extracellular vesicles. mAb 38BI1 recognized an antigen which was most accessible on lysed cells, and non-specific binding of mAb 40BI3.2 to grids prevented its localization on the cell surface.

The spoIIA locus of Bacillus licheniformis was cloned into the phage vector 𝜙105J9; selection was based on the ability of the clone to complement mutations in both the first and the last of the spoIIA genes of B. subtilis. The B. licheniformis DNA was subcloned into M13 and sequenced; it includes three genes of identical lengths to those of B. subtilis. The average interspecies difference in nucleotide sequence is 24%; C occurs at the third position of codons 55% more often in the B. licheniformisthan in the B. subtilis sequence. The average difference in predicted amino acid sequence is 11%, but the distribution of these differences is far from random, and there are several long stretches of amino acid sequence that are identical in the two organisms. The distribution of non-conservative amino acid changes between the species is also strikingly non-random; no such changes are found in those regions in which missense mutations are known in B. subtilis. Each species has an open reading frame 5ʹ of the spoIIA gene, but the predicted amino acid sequence, and the distribution of differences between the two species in both nucleotides and predicted amino acids, suggest that these open reading frames are not expressed. Both species have regions 3ʹ of the open reading frames which resemble rhoindependent terminators of transcription, but that in B. licheniformis is longer and could form a more elaborate secondary structure than that in B. subtilis.

The DNA sequence of the region upstream from the amidase structural gene (amiE) of Pseudomonas aeruginosa indicates that amidase (EC 3.5.1.4) is transcribed from an Escherichia coli-like promoter located 150 bp before the amiE translation initiation codon. The sequence between the promoter and the coding sequence includes a single open reading frame followed by an E. coli-like rho-independent transcription terminator. A deletion within the presumed terminator region which disrupts the potential stem/loop formation leads to high constitutive amidase expression which is independent of the product of the regulator gene (amiR). It is proposed that the catabolic aliphatic amidase of P. aeruginosa is regulated by a transcription anti-termination mechanism. The magnoconstitutive mutant PAC433 has promoter and terminator sequences identical to the wild-type PAC1 but contains a single base pair change in the amiE gene ribosome-binding site.

A 26 bp DNA probe has been constructed with minimal degeneracy to the protein sequence for Clostridium perfringens enterotoxin. The probe has been hybridized against a 6–10 kb chromosomal bank from C. perfringens 8239, prepared as a HindIII partial digest in pHG165. From this survey a clone has been identified containing a 6·8 kb DNA insert with strong hybridization to the probe. Direct plasmid sequencing has identified a translational reading frame within this clone which correlates with the known protein sequence for the type A enterotoxin. DNA sequences 5′ to this open reading frame and containing the putative transcriptional control regions show areas of significant homology with regions upstream from the ATG codon of the tetanus toxin gene.

Two new temperate bacteriophages for Rhodobacter sphaeroides, designated ϕRsA and ϕRsD, were isolated from a stock of phage ϕRsGl using R. sphaeroides strain DSM 159–2 as the indicator strain. Electron-microscopic examination showed that phage ϕRsA belonged to group B and phage ϕRsD belonged to group A of Bradley’s morphological classification. Phage ϕRsA had a polyhedral head (50 nm in diameter) and a long, flexible, non-contractile tail (250 by 11 nm). Phage ϕRsD also had a polyhedral head (56 nm in diameter) and a sheathed contractile tail (160 by 18 nm). Both phages contained double-stranded DNA with cohesive ends. The size of the ϕRsA genome was about 40 kb and the G + C content was 72·4 mol%. The size of the ϕRsD genome was about 56 kb and the G + C content was approximately 72·6 mol%. Biotinylated DNA probes of phage ϕRsA and of phage ϕRsD hybridized to genomic DNA from R. sphaeroides strains Y and Si 4 but not to DNA from other sources. The host ranges of the two phages were determined using 26 R. sphaeroides strains and six strains of Rhodobacter capsulatus. Phage ϕRsA formed plaques on R. sphaeroides DSM 159–2 only, while phage ϕRsD plaqued on another six strains of R. sphaeroides but not on R. capsulatus.

Endo-1, 4-β-glucanase genes have been cloned from two strains of Ruminococcus albus recently isolated in this laboratory. Although the strains were phenotypically similar, cross-hybridization studies between them showed significant genetic differences, with only 20% of the genome forming DNA heteroduplexes. Heteroduplexes displayed an average dissociation temperature 9 °C lower than that of the homoduplex. Consistent with this, restriction maps of the two endoglucanase genes showed no similarity, and hybridization work using the endoglucanase genes as probes revealed that neither gene was present in the genome of the other isolate of R. albus. Comparative enzyme characterization showed differences between the enzymes in their response to temperature, pH and substrate preference.

The gene encoding a major protein antigen of Mycobacterium tuberculosis has been cloned and sequenced using oligonucleotide probes derived from the N-terminal sequence of the analogous protein from Mycobacterium bovis BCG. The gene analysis revealed a sequence encoding a protein of 99 amino acid residues, with a molecular mass of 10·7 kDa. Computer prediction suggests that the protein contains three T-cell-determined epitopes (of which one has been demonstrated experimentally) and three B-cell-determined epitopes. The protein sequence was homologous to two prokaryote heat-shock proteins and the gene possessed heat-shock-like promoter sequences upstream of the initiation codon. A hairpin loop identified in the upstream sequence may also be important in regulation of the gene.

The two linear, integrated mini-circle copies of Streptomyces coelicolor A3(2) were cloned in Escherichia coli and their positions on the S. coelicolor genetic map were determined. Mini-circle copy A is close to the argA locus, 20 kb upstream of the gyl operon. Mini-circle copy B is close to the cysD locus and is absent from S. coelicolor J1501, which has suffered a chromosomal deletion including mini-circle copy B and possibly associated with the pgl mutation. The mini-circle copies were not involved in any of the several previously identified physical interactions between the plasmid SCP1 and the S. coelicolor chromosome. A new insertion sequence was identified close to the right end of mini-circle copy B in S. coelicolor M145. The free mini-circle of S. coelicolor, when inserted into an attP-deleted derivative of phage ϕC31, actively integrated this phage into the chromosomes of S. coelicolor and S. lividans at preferred and secondary sites. The resulting prophages were stably inherited and remained physically intact. No precise excision of prophages from S. lividans lysogens carrying insertions at preferred or secondary integration sites was detected: instead, free phages were generated by imprecise excisions. These phages allowed the in vivo cloning of segments (> 3 kb in length) of the chromosomal DNA flanking preferred and secondary integration sites. Attempts to delete the preferred integration site by double homologous recombination with a clone carrying flanking DNA sequences and an antibiotic resistance gene were unsuccessful.

The plasmid-encoded β-lactamase genes of six strains of Staphylococcus aureus were cloned and shown to be expressed in Escherichia coli. The cloned genes were re-introduced into S. aureus via a shuttle vector, and expressed β-lactamase. However, clones containing only the small amount of DNA found necessary for expression of ampicillin resistance in E. coli did not express β-lactamase in S.aureus. Much larger pieces of DNA from the original plasmid were necessary to obtain expression in S.aureus. Some of the six strains of S.aureus synthesized β-lactamase constitutively and some released only a small proportion of the enzyme into the medium. Both these characteristics were maintained in the clones so it is concluded that they are features either of the gene itself or of the surrounding DNA. The cloned genes were sequenced and the putative amino acid sequences of the βlactamases were compared. There are several differences between the sequences and in particular one change in the N-terminal region, at a position believed to be especially important for export of proteins from the cell, is thought to have a key effect on whether or not the enzyme is found in the medium.

The segregational stability of two chimaeric plasmids has been examined in an isogenic series of haploid, diploid and tetraploid strains of Saccharomyces cerevisiae, constructed by transformation-associated spheroplast fusion. For the highly unstable, ARS-based plasmid YRp7M, a significant increase in its segregational stability was observed with increasing ploidy, while the relatively stable, 2µm-based plasmid pMA3a showed only a small increase in stability in strains of higher ploidy. The copy number of both pMA3a and the endogenous 2m plasmid increased in proportion with the host cell ploidy, while the copy number of TRp7M was increased in the higher ploidy strains but did not correlate with ploidy. These results suggest that the copy numbers of both the 2µm plasmid and a plasmid derived from it are controlled by a nuclear gene and that, in addition, there are 2µm sequences, other than those required for the FLP-mediated recombination system, that play a role in maintaining copy number.

Neisseria gonorrhoeae strains P9-2 (PenS) and KW2 (PenR) were grown in chemostats of non-ferrous design at constant growth rate, pH and dissolved oxygen tension. Iron limitation (µmax 0·1 h−1) was imposed by omitting iron salts from the defined medium and titrating increasing concentrations of the non-metabolizable iron chelators ovotransferrin and Desferal, to progressively decrease the growth yield. Metabolic activity during iron limitation was very high, with a q Glc which was 2-or 11-fold greater than during cystine-or glucose-limited growth, respectively. More aspartate and isoleucine were metabolized during cystine-limited growth, while more glutamate, proline and serine were metabolized during glucose-or iron-limited growth. Significant concentrations of alanine or valine were excreted during cystine-or glucose-limited growth, respectively. Iron-limited growth of an initial inoculum of non-piliated, transparent colony-forming (P−O−) gonococci resulted in the selection of 100% piliated bacteria. Initial inocula of P+O− gonococci retained this phenotype for over 100 generations. Iron-limited gonococci were extremely virulent in the guinea-pig subcutaneous chamber model and inocula of even 12 bacteria grew rapidly and persisted. By contrast, cystine-limited (iron-replete) gonococci retained piliation but did not survive in the chambers. Transition from iron-limited to glucose-limited growth resulted in marked loss of piliation but the bacteria remained virulent. Loss of virulence did not correlate with susceptibility to killing by normal human serum, nor with changes in the content or composition of lipooligosaccharide, which contained 2·9, 3·7, 4·3 and 4·8 kDa moieties. Additional proteins were detectable in Sarkosyl-purified outer membranes of iron-limited gonococci but several proteins with molecular masses similar to those described in the literature for iron-restricted gonococci were detectable in cystine-or glucose-limited bacteria.

Fourteen strains of Streptococcus mutans serotype c were examined for their cell-surface protein antigens in terms of hydrophobicity, M r and immunochemical specificities. Thirteen strains were hydrophobic, while strain GS-5 was markedly hydrophilic as compared to the other strains tested. Cell-surface protein antigens were then analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting. A protein antigen of M r 190000 (PAc) was found in cell extracts and culture supernatants of all the hydrophobic strains. Neither culture supernatant nor cell extract of strain GS-5 contained PAc. Strain GS-5, however, produced extracellularly a large amount of a protein of M r 155000 (PAGS-5) which reacted with rabbit anti-PAc serum. Immunodiffusion analysis showed that PAGS-5 lacked a part of the antigenic moieties in the PAc molecule. SDS-PAGE and radioimmunoassay showed a small amount of PAGS-5 on the cell surface of strain GS-5. These findings suggest that PAGS-5 may correspond to PAc which lacks a region participating in binding of PAc to the streptococcal cell.

The effects of the antibiotic validamycin A on the inositol content and hyphal branching of Rhizoctonia cerealis were investigated. Inositol was found associated with the cell wall and as a lipid extractable fraction composed of phospholipid, phosphosphingolipid and glycolipid. Validamycin A decreased the inositol content of the phospholipid fraction by 57% and the phosphosphingolipid fraction by 40%, but had no effect on the glycolipid fraction. The phosphatidylinositol (the only inositol-containing phospholipid detected) content of the biomass was reduced by 53 % in the presence of validamycin A and this decrease was compensated for by rises in the levels of phosphatidylserine and an unknown, minor phospholipid. Over the concentration range 1 to 5000 µm, validamycin A caused decreases in the colony radial growth rate, internode length (the distance between adjacent branches on a leading hypha) and inositol content of R. cerealis colonies, and a positive correlation was found between inositol content and internode length. Addition of inositol to the medium only slightly antagonized the effect of the antibiotic on mycelial morphology and did not restore phosphatidylinositol levels of the biomass to control values.

The regulation of the enzymes associated with one-carbon metabolism and the assimilation of nitrogen, together with the cellular composition of Hyphomicrobium X, were investigated. The effect of changing the methanol-carbon concentration with the NH+ 4-nitrogen concentration remaining constant (C: N ratio) in the medium during chemostat growth at a constant dilution rate was studied. As the medium changed from a C-limitation to a dual C- and N- and finally a N-limitation, the culture gradually passed through three definite growth phases. In response to these environmental conditions the cellular composition and the specific enzyme activity patterns changed. The C-content of the cells changed very little. The N- and protein-content was constant over C-limiting conditions, but under dual C- and N-limiting and N-limiting conditions an accumulation of poly-β-hydroxybutyrate (PHB) occurred and as a consequence the N-content and protein-content of the cells decreased. The enzyme associated with N-assimilation during C-limitation was an NADP+-dependent glutamate dehydrogenase which was replaced by the high affinity glutamine synthetase and glutamate synthase pathway immediately the NH+ 4-N concentration in the medium became limiting. Similarly the specific activity of methanol dehydrogenase, which was high during C-limiting conditions, dropped to a low level as the NH+ 4-N concentration decreased. Finally carbon balances were constructed throughout the experiment which showed that irrespective of the C:N ratio in the medium during C-limitation, the methanol-carbon was fluxed into biomass and CO2 only; during dual limitation the carbon was channelled into biomass, CO2 and PHB; and finally when the growth was in the presence of excess carbon no methanol-carbon was directed into over-metabolite production but, instead, the excess carbon was oxidized through the dissimilatory pathway.

The effect of growth conditions on the production of alginate by highly mucoid strains of Pseudomonas mendocina was investigated, using pH-controlled batch and continuous cultures. In batch culture, alginate was produced both during the exponential and stationary phases of growth. A polysaccharide concentration of 20 g 1−1 was obtained from medium containing glucose (50 g l−1). In steady-state continuous culture under nitrogen limitation (dilution rate, D = 0·05 h−1), a similar alginate concentration was attained; more than 60% of the utilized glucose was converted to alginate and the polysaccharide concentration was ten-fold the biomass concentration. Increasing the dilution rate above 0·1 h−1 decreased the proportion of glucose converted to alginate although the biomass was unchanged. The optimum dissolved oxygen tension (d.o.t.) for alginate production in nitrogen-limited continuous culture was approximately 40 mm Hg (1 mm Hg is approximately 133 Pa). The respiration rate and bacterial density were independent of d.o.t. above 10 mm Hg. Under oxygen limitation, no alginate was produced but acetate was excreted into the culture fluid. Variants producing little alginate arose during growth in continuous culture. Under carbon limitation, alginate production was substantially decreased but not eliminated.

The synthesis of the pyrimidine biosynthetic enzymes is repressed by the pyrimidine nucleotide end-products of the pathway. However, purine nucleotides also play a role. In this study, I have measured expression of the pyr genes (pyrA-E) in Salmonella typhimurium strains harbouring mutations that permit manipulation of the intracellular pools of both pyrimidine and purine nucleotides. The results identify the effectory purine compound as being a guanine nucleotide; it is probably GTP, but it may be GDP or GMP. The synthesis of carbamoylphosphate synthase, encoded by pyrA, and particularly dihydroorotase, encoded by pyrC, and dihydroorotate dehydrogenase, encoded by pyrD, is stimulated by the guanine nucleotide, while the synthesis of aspartate transcarbamoylase, encoded by pyrBI, and orotate phosphoribosyltransferase, encoded by pyrE, is inhibited by guanine nucleotides. The regulatory pattern of each pyr gene is discussed in relation to present knowledge on gene structure and regulatory mechanism.

The effect of light and dark incubation on the ultrastructure, pigments and lipids of five marine cyanobacteria was studied. All light-grown cyanobacteria contained in their lipid extracts the four major lipid classes characteristic of chloroplasts, namely monogalactosyldiacylglycerols, digalactosyldiacylglycerols, sulphoquinovosyldiacylglycerols, and phosphatidyl glycerols. However, the fatty acid patterns of the cyanobacterial total lipids and individual lipid classes were different from those of chloroplasts, and responded differently to dark incubation. In two cyanobacteria the fatty acid patterns of total lipids from light- and dark-incubated cultures were similar, but in the other three, dark incubation was associated with increased levels of oleic acid in the total lipids and individual lipid classes. The decrease of linolenic acid known to occur in lipids from photosynthetic eukaryotes in response to dark incubation was recorded only in Spirulina subsalsa. However, linolenic acid was not esterified in monogalactosyldiacylglycerols, as it is in chloroplasts. We also found the class of alcohol glycosides in three non-heterocystous cyanobacteria.

Plasma membranes were isolated from the acellular slime mould Physarum polycephalum by differential centrifugation and an aqueous two-phase polymer method. ATPase activity in the membrane fraction was optimal at pH 6·5 and was severely inhibited by vanadate but resistant to oligomycin. The protein components of the plasma membrane were analysed by polyacrylamide gel electrophoresis. Glycoproteins were located on Western blots by incubation of the sheets with lectin-peroxidase reagents. The results indicated that most of the membrane proteins were glycosylated. Pulse-chase experiments with [3H]glucosamine showed that when the plasmodia were induced to differentiate to macrocysts, turnover of the membrane glycoproteins occurred more rapidly than in growing plasmodia.

Five strains of Gram-negative denitrifying bacteria that used various ketones as sole carbon and energy sources were isolated from activated sludge from a municipal sewage plant. Three strains are related to the genus Pseudomonas; two non-motile species have not yet been affiliated. All strains grew well with ketones and fatty acids (C2 to C7), but sugars were seldom utilized. The physiology of anaerobic acetone degradation was studied with strain BunN, which was originally enriched with butanone. Bicarbonate was essential for growth with acetone under anaerobic and aerobic conditions, but not if acetate or 3-hydroxybutyrate were used as substrates. An apparent K s value of 5·6 mm-bicarbonate was determined for growth with acetone in batch culture. The molar growth yield was 24·8–29·8 g dry cell matter (mol acetone consumed)−1, with nitrate as the electron acceptor in batch culture; it varied slightly with the extent of poly-β-hydroxybutyric acid (PHB) formation. During growth with acetone, 14CO2 was incorporated mainly into the C-1 atom of the monomers of the storage polymer PHB. With 3-hydroxybutyrate as substrate, 14CO2 incorporation into PHB was negligible. The results provide evidence that acetone is channelled into the intermediary metabolism of this strain via carboxylation to acetoacetate.

The rRNA gene restriction patterns of 110 strains belonging to 12 staphylococcal species have been determined. The strains, isolated from various sources, were epidemiologically unrelated. Total DNA was cleaved with restriction enzymes HindIII and EcoRI, electrophoretically separated and probed with radiolabelled 16S rDNA from Bacillus subtilis inserted in a plasmid vector, pBR322. Fourty-four distinct Hindlll patterns and 44 distinct EcoRl patterns were observed. Strains belonging to different species had different patterns. Although distinct patterns were also observed within some species, a core of common bands could be discerned within each species or subspecies. Analysis of the patterns revealed two taxa in Staphylococcusxylosus which were not evident using phenotypic characteristics. Of 18 strains which were difficult to identify using phenotypic schemes, 15 showed patterns typical of known species. The three remaining atypical strains showed unusual patterns and may belong either to a known species, not included in the study, or to a new species. Since various patterns were observed within some species (e.g. S. aureus and S. epidermidis), rRNA gene restriction patterns may have epidemiological, as well as taxonomic interest.