- Volume 134, Issue 8, 1988

Three homologous series of a-mycolic acids (dicyclopropanoyl acids, monocyclopropanoyl monoenoic acids and dienoic acids) from 16 rapidly growing and 14 slowly growing mycobacteria were separated by argentation thin-layer chromatography and analysed by gas chromatography/mass spectrometry of their trimethylsilyl ether derivatives. Mycobacterial species were separated into five groups. Strains of group A contained similar amounts of even and odd carbon-numbered dienoic acids, with a methyl branch on the odd acids and a C24-α-unit, as typified by Mycobacterium fortuitum and M. chitae. Group B strains possessed similar amounts of even carbon-numbered dicyclopropanoyl α-mycolic acids and odd carbon-numbered unsaturated acids with C22- and C24-α-units, as found in M. phlei and M. diernhoferi. Group C strains contained mainly even carbon-numbered dicyclopropanoyl acids with C22- and C24-a-units, as shown by M. vaccae and M. aurum. Group D strains possessed mainly odd carbon-numbered dienoic acids with a methyl branch and a C24-α-unit, as seen in M. triviale and M. nonchromogenicum. Group E strains had mainly even carbon-numbered dicyclopropanoyl acids with C24- or C26-α-units, as found in M. avium and M. tuberculosis. Many rapidly growing mycobacteria also produced α-mycolic acids which were shorter in the length of the main carbon chain but whose a-units were the same as those in α-mycolic acids from the same species. These α-mycolic acids had either one or two double bonds and showed variations in both their unsaturation and overall size, which may be useful in taxonomic studies.

Bacteroides fragilis NCDO 2217 produced three major proteases, P1, P2 and P3 of estimated molecular masses 73, 52 and 34 kDa respectively. Protease P1 weakly hydrolysed azocasein but strongly hydrolysed valyl-alanine p-nitroanilide (VAPNA), glycyl-proline p-nitroanilide (GPRPNA), and to a lesser extent leucine p-nitroanilide (LPNA), indicating it to be an exopeptidase. Proteases P2 and P3 hydrolysed only azocasein and LPNA. The high protease: arylamidase ratios of these enzymes indicated that they were probably endopepti-dases. Experiments with protease inhibitors suggested that P1 and P2 had characteristics of serine and metalloproteases respectively and that P3 was a cysteine protease. The proteolytic activity of whole cells was stimulated by divalent metal ions such as Mn2+, Ca2+ and Mg2+, but was strongly inhibited (about 95%) by Cu2+ and Zn2+. The temperature optimum for protein hydrolysis was 43 °C. Proteolysis was temperature sensitive, however (90% reduction at 60 °C) and was maximal at alkaline pH, with two broad peaks at pH 7·9 and pH 8·8. Cell fractionation showed that P1 was located intracellularly and in the periplasm, whereas P2 and P3 were largely associated with the outer membrane. Release of the membrane-bound proteases by treatment with 1 m-NaC1 suggested that ionic interactions were involved in the association of these enzymes with the membranes.

The cell membrane of Mycopfasma mobile was isolated by either ultrasonic or French press treatment of intact cells. The membrane fraction contained all of the cellular lipids, but only one-third of cellular proteins and had a density of 1·14 g ml−l. The soluble fraction contained the NADH dehydrogenase activity of the cells, as well as a protein with an apparent molecular mass of 55 kDa that was phosphorylated in the presence of ATP. Lipid analyses of M. mobile membranes revealed that membrane lipid could be labelled by radioactive glycerol, oleate and to a much higher extent by palmitate but not by acetic acid. The membrane lipid fraction was composed of 54% neutral and 46% polar lipid. The major constituents of the neutral lipid fraction were free fatty acid, free cholesterol and cholesterol esters (45,25 and 20%, respectively, of total neutral lipid fraction). The free cholesterol count was 13% (w/w) of total membrane lipids with a cholesterol:phospholipid molar ratio of about 0·9. Among the polar lipids, both phospho-and glycolipids were detected. The phospholipid fraction consisted of a major de novo-synthesized phosphatidylglycerol (63% of total phospholipids), plus exogenous phosphatidyl-choline and sphingomyelin incorporated in an unchanged form from the growth medium. The glycolipid fraction was dominated by a single glycolipid (~90% of total glycolipids) that was preferentially labelled by palmitic acid and showed a very high saturated:unsaturated fatty acids ratio.

Zymolyase released between 20 and 25% of the total protein from purified walls of yeast (Y) and mycelial (M) cells of Candida albicans. The material released contained 92% carbohydrate (86% mannose and 6% glucose) and 7:< protein. Over 85% of the carbohydrate was N-glycosidically linked to the protein and the rest (less than 15%) was linked O-glycosidically. Highly polydisperse, high molecular mass mannoproteins, resolved by electrophoresis as four defined bands in Y cells and two bands in M cells, had both types of sugar chains. A 34 kDa species found in both types of cells had a single 2·5 kDa N-glycosidically linked sugar chain and a 3 1·5 kDa protein moiety. Polydispersity in the high molecular mass mannoproteins was due to the N-linked sugar chains (mannan) with a molecular mass between 500 kDa and 20 kDa (average 100 kDa) in Y cells and between 400 kDa and 20 kDa (average 50 kDa) in M cells. Three mannoproteins of 34,30 and 29 kDa secreted by protoplasts were associated with the high molecular mass mannoproteins, suggesting that this type of interaction might be related to the regeneration of the cell wall.

Over 200 strains of actinomycetes, representing nine distinct genera, were screened directly for the ability to release reducing sugar from ball-milled wheat straw, using a microtitre plate assay system. Xylanase activity was detected in nearly all of the strains examined while activities against purified cellulosic substrates were less widespread and relatively low. Straw saccharification resulted from cooperative enzyme action and sugar yields were not simply correlated with substrate particle size. Straw-saccharifying activity was further characterized in selected strains comprising five representatives of the genera Thermomonospora and Streptomyces, one Micromonospora strain and the type strain of Microbispora bispora. Common features included optimal saccharification of straw in the pH range 6·0–9·0 and xylose and its oligomers as the principal products, although low concentrations of glucose were also detected. Optimal activity and increased stability at 70 °C was a feature of enzyme preparations from Thermomonospora and thermophilic Streptomyces strains. β-Xylosidase and β-glucosidase activities were largely intracellular, but significant amounts of extracellular β-xylosidase activity were also found in two strains. Other enzymes involved in straw saccharification include acetylesterase and arabinofuranosidase, and these activities were detected in all strains. Acetylesterase and arabinofuranosidase activities were largely extracellular, but in some strains significant amounts of intracellular activity were also detected.

The nitrogen-fixing symbiosis between Mimosa scabrella and Rhizobium Br 3454 was initiated by an infection sequence consisting of (a) surface colonization of roots; (b) penetration of mucigel and primary layers of radial walls of epidermal cells; (c) further penetration through primary wall layers and intercellular air spaces; (d) occasional intracellular penetration of epidermal and cortical cells, always occurring from a boundary between two neighbouring cells. Root hairs, when present, were never seen to be infected by the well-known infection thread mechanism, nor were infections seen to occur through wounds. This newly described method of infection may be common in legumes.

A repeating element of DNA has been isolated and sequenced from the genome of Bordetella pertussis. Restriction map analysis of this element shows single internal ClaI, SphI, BstEII and SalI sites. Over 40 DNA fragments are seen in ClaI digests of B. pertussis genomic DNA to which the repetitive DNA sequence hybridizes. Sequence analysis of the repeat reveals that it has properties consistent with bacterial insertion sequence (IS) elements. These properties include its length of 1053 bp, multiple copy number and presence of 28 bp of near-perfect inverted repeats at its termini. Unlike most IS elements, the presence of this element in the B. pertussis genome is not associated with a short duplication in the target DNA sequence. This repeating element is not found in the genomes of B. parapertussis or B. bronchiseptica. Analysis of a DNA fragment adjacent to one copy of the repetitive DNA sequence has identified a different repeating element which is found in nine copies in B. parapertussis and four copies in B. pertussis, suggesting that there may be other repeating DNA elements in the different Bordetella species. Computer analysis of the B. pertussis repetitive DNA element has revealed no significant nucleotide homology between it and any other bacterial transposable elements, suggesting that this repetitive sequence is specific for B. pertussis.

The maintenance of the plasmid vectors pTG201 and pTG206 (which both carry the Pseudomonas putida xylE gene) and pBH3 in Escherichia coli hosts was studied in free and immobilized continuous cultures. pTG201, containing the strong λPR promoter, was more quickly lost than plasmid pTG206, containing the tetracycline resistance gene promoter. The instability of pTG201 seems to be related to high expression of the cloned xylE gene. Fluctuations in the proportion of pTG201-containing cells were observed in the free system, suggesting the appearance of adaptive descendants (with and without plasmid) from the initial strains. The loss of plasmid vectors from E. coli cells and the fluctuations in the proportion of plasmid-containing cells could be prevented by immobilizing plasmid-containing bacteria in carrageenan gel beads.

We have isolated a set of strains with mutations (designated prs) that decrease secretion of α-amylase and have a pleiotropic effect on secretion of other exoproteins. The seven mutants were selected in a strain of Bacillus subtilis which overproduces α-amylase due to the presence of an α-amylase gene on a multicopy plasmid. The mutations were mapped to four different chromosomal loci. The phenotype of the mutants, especially their pleiotropic effects and the accumulation of α-amylase precursor, indicated that they have defects in the mechanism of protein export. Double mutants with certain pairwise combinations of mutations in different loci had additive effects on secretion, suggesting that these prs genes encode different components of the secretion pathway.

A non-specific deoxyribonuclease with a possible role in the restriction of some actinophages was detected in Streptomyces glaucescens ETHZ 22794. Production of this enzyme activity was influenced by the medium composition, indicating nutritional control of enzyme synthesis. Restriction was confirmed when phage adsorption and efficiency of plating in nuclease-productive and non-productive media were investigated, and also by analysis of a mutant which lacked exonucleolytic activity. In vivo escape from restriction in nuclease-productive media is mainly related to the ability of phages to adsorb in a growth phase earlier than that in which enzyme synthesis occurs.

A cloned EcoRI fragment from Legionella pneumophila, which includes 16s and 23s rRNA genes, was used to identify bacteria belonging to the genus Legionella by hybridization to a series of species specific restriction fragments. Examination of the type strains of 28 species of legionellae gave different band patterns in every case. When further isolates of these species were tested the patterns obtained were usually either identical, or very similar, to those of the respective type strains. Thirty-one coded isolates were examined and of these 29 were allocated to the correct species. The remaining strains (a non-Legionella and a L. pneumophila) could not be identified using this technique. The rRNA gene probe method should be of great value in the identification of legionellae, particularly for those species which are at present very difficult to distinguish serologically.

The plasmid pCS001, isolated from an enterotoxigenic strain of Escherichia coli, mediates expression of the CSl or CS2 and CS3 fimbrial adhesins in appropriate E. coli hosts. To characterize this further, HindIII-generated DNA fragments of this plasmid were cloned into the vector plasmid pBR322. A chimaera, called pCS200, which mediated expression of the CSl or CS2 fimbrial antigen but not of CS3 fimbrial antigen in appropriate host strains, was obtained. The DNA inserted into the vector sequences of plasmid pCS200 comprised HindIII fragments of 4.7 kbp and 0.8 kbp. Plasmid pCS200-carrying wild-type E. coli hosts of serotype O6:K15:H 16 that expressed the CS1 or CS2 antigen also caused mannose-resistant agglutination of bovine red blood cells, suggesting that functional fimbriae were present on the bacterial surface. As previously observed with strain K 12 recipients of CS-fimbriae-associated plasmids mobilized from wild-type enterotoxigenic E. coli, K 12 recipients of the chimaeric plasmid pCS200 did not express the CS1 or CS2 fimbrial antigen. An oligonucleotide probe, synthesized on the basis of the published N-terminal amino acid sequence of the CS2 fimbrial subunit, hybridized to plasmid pCS200, indicating that the gene for the structural subunit of this fimbria resided on the plasmid.

The effect of secreted virulence components of Bordetella pertussis on chemiluminescence (CL) of rabbit peritoneal neutrophils was determined with the chemotactic peptide N′-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) or intact B. pertussis as the stimulus. Pertussis toxin (PT) inhibited the response to fMLP in a dose-dependent manner, although only after the neutrophils had been exposed to the toxin for >15 min. Both filamentous haemagglutinin (FHA) and lipopolysaccharide (LPS) markedly enhanced the CL response to fMLP after ≥15 min incubation with the neutrophils. Similar effects to those of B. pertussis LPS were also seen with smooth and rough LPS from Salmonella minnesota. With the lowest dose of each component which elicited a maximal effect on CL, the inhibitory effect of PT overrode the enhancing effect of FHA and B. pertussis LPS. Pre-incubation of neutrophils with PT, FHA or B. pertussis LPS caused a slight reduction in the subsequent CL response to virulent B. pertussis Tohama. Virulent (phase I, or X-mode) organisms of B. pertussis 18334 and B. pertussis Tohama induced greater neutrophil CL than their avirulent (C-mode) derivatives. There appeared to be an inverse correlation between bacterial hydrophilicity and the ability to induce neutrophil CL: X-mode bacteria were significantly less hydrophilic than C-mode organisms. Three mutants, the adenylate cyclase (AC)- and haemolysin (HLY)-deficient B. pertussis BP348, the FHA-deficient B. pertussis BP353, and the PT-deficient B. pertussis BP357, generated similar levels of CL and had similar hydrophilicity values. The hydrophilicity value of the avirulent mutant B. pertussis BP347 (deficient in AC, HLY, FHA and PT) and the CL induced by this strain were similar to those of B. pertussis C-mode organisms. Thus, the interaction of B. pertussis with neutrophils appears to be complex, reflecting both the alteration of leucocyte function by secreted virulence components of the organism and, in the absence of opsonins, the surface properties of the bacterium.

Eight strains of Salmonella pullorum isolated from epidemiologically independent cases of pullorum disease (bacillary white diarrhoea) in young chickens possessed at least one large molecular mass plasmid in addition to smaller molecular mass plasmids. The 85 kb large plasmid, designated pBL001, of one of these strains was ‘tagged’ with an ampicillin resistance marker by the insertion of transposon Tn3. The plasmid was eliminated by passage in nutrient broth containing acridine orange. It was reintroduced into the strain from which it had been eliminated by mobilization using the F plasmid. Following oral inoculation of newly hatched Rhode Island Red chickens, the parent strain produced a high level of mortality (71%) with characteristic signs of pullorum disease. Following intramuscular inoculation of chickens of the same age, the bacterial LD50, was (log10 c.f.u.) 3·38 ± 0·43 (mean ± sem). The derivative lacking pBL001 produced no mortality or morbidity when inoculated orally and the bacterial LD50, value increased to (log10 c.f.u.) 5·54 ± 0·28. This increase was statistically significant (x 2 = 13·6, P < 0·01). Reintroduction of pBL001 restored virulence as gauged by oral inoculation of chickens (62% mortality) and by the intramuscular bacterial LD50value (log10, c.f.u. = 3·78 ± 0·25). These values were not significantly different to those produced by the parent strain (x 2 = 0·59, P= 0·4 and x 2=0·66, P =0·5, respectively). Following oral inoculation, the pBL001-cured derivative was less invasive than the parent strain and following intramuscular inoculation it persisted for a shorter period than the parent strain in the liver, spleen and the leg muscle into which it had been inoculated. In addition, the parent strain, but not the pBL001-cured derivative, localized in large numbers in the myocardium where it produced lesions typical of pullorum disease. Both the parent strain and the pBL001-cured derivative were serum resistant in the presence of rabbit serum and grew equally well in chick serum and broth.