- Volume 139, Issue 12, 1993

The gene encoding a periplasmic pH 2·5 acid phosphatase (appA) from Escherichia coli was placed under the control of an inducible promoter and integrated into the chromosome of Myxococcus xanthus. The majority of the AppA protein synthesized in M. xanthus accumulated in the periplasm whereas about 30% was secreted into the medium. The kinetics of AppA secretion through the ‘outer envelopes’ (i.e. periplasm + outer membrane) were followed after AppA induction. The results suggest that AppA crosses these envelopes by a mechanism involving diffusion and show that the periplasmic accumulation of AppA and envelope permeability are decreased by mutations that decrease the secretion of native proteins in M. xanthus.

A 5·4 kb HindIII fragment of Lactococcus lactis subsp. lactis was identified using a homologous dnaK probe generated by PCR and cloned in Escherichia coli. Upstream sequences were generated by inverse PCR. The two cloned fragments partially overlapped, and sequencing of 5915 bp revealed the presence of four open reading frames in the order orfl-grpE-dnaK-orf4. orf1 encodes a 39 kDa protein of unknown function which shows considerable sequence homology with the Orf39 and Orfa proteins of Bacillus subtilis and Clostridium acetobutylicum, respectively. The downstream ORFs showed high homology to the grpE and dnaK genes of other prokaryotes. The DnaK protein has a characteristic 24-amino-acid deletion exhibited by all the known DnaK proteins of Gram-positive species. In many bacteria the dnaK and dnaJ genes are found as part of the same operon. The L. lactis dnaK operon is unusual in that the dnaK gene is followed by a putative transcription terminator and a fourth large ORF which shares no homology with the dnaJ genes of other bacteria but has a small degree of homology with various membrane proteins. Vegetative promoter sequences are found upstream of both orf1 and orf4. A 12 bp inverted repeat is found upstream of the putative promoter of orf1 and an 8 bp inverted repeat is found between this promoter and the orf1 initiation codon. These repeats are thought to be involved in regulation of the heat-shock genes. The DnaK homologue is induced approximately 3-fold on heat shock at 42 °C.

An insertion sequence (IS) element of Brucella ovis, named IS6501, was isolated and its complete nucleotide sequence determined. IS6501 is 836 bp in length and occurs 20–35 times in the B. ovis genome and 5–15 times in other Brucella species. Analysis of the junctions at the sites of insertion revealed a small target site duplication of four bases and inverted repeats of 17 bp with one mismatch. IS6501 presents significant similarity (53·4%) with IS427 identified in Agrobacterium tumefaciens, suggesting a common ancestral sequence. A long ORF of 708 bp was identified encoding a protein with a predicted molecular mass of 26 kDa and sharing sequence identity with the hypothetical protein 1 of A. tumefaciens and with the transposase of Mycobacterium tuberculosis. IS6501 is present in all Brucella strains we have tested. Restriction fragment length polymorphism of reference and field strains of two species (B. melitensis and B. ovis) was studied using either pulsed field gel electrophoresis (PFGE) on Xbal-digested DNA or hybridization of EcoRI-digested DNA using IS6501 as a probe. The genome of B. melitensis biovar 3 contains about 10 IS copies per genome and field strains of the same species could not be distinguished either by IS hybridization or by XbaI (PFGE) restriction patterns. In contrast, the number of IS copies in the B. ovis genome is around 30 and the different field strains can be differentiated by both methods. As ISs have been shown to be implicated in chromosomal rearrangement, we propose that the chromosomal polymorphism revealed by PFGE and high copy number of IS6501 observed in B. ovis may be related to the presence of an active IS in this species.

A tryptophanase gene from Enterobacter aerogenes SM-18 was cloned and sequenced. The structural gene for tryptophanase, tnaA, consisted of 1389 bp encoding 462 amino acid residues, and its nucleotide sequence and deduced amino acid sequence showed significant homology to those of tnaA from Escherichia coli K12. A short open reading frame consisting of 31 amino acid residues was found upstream of tnaA, and it showed some similarity to the E. coli tnaC gene known to be a cis-acting regulatory element for transcription. A partial open reading frame homologous to the 5ʹ end of E. coli tnaB was observed at the 3ʹ-flanking region of tnaA. These genes may thus constitute an operon as in E. coli.

The immunosuppressants FK506 and cyclosporin A (CsA) bound to their receptors, FKBP12 or cyclophilin, inhibit the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, preventing T cell activation or, in yeast, recovery from α-mating factor arrest. Vegetative growth of yeast does not require calcineurin, and in strains sensitive to FK506 or CsA, growth is inhibited by concentrations of drug much higher than those required to inhibit T cell activation or recovery from mating factor arrest. We now describe the isolation of a mutant of Saccharomyces cerevisiae which is 100–1000-fold more sensitive to the growth inhibitory properties of these drugs. The mutation (fks1) also confers a slow growth phenotype which is partially suppressed by exogenously added Ca2+ and exacerbated by EGTA. Simultaneous disruption of the two genes (CNA1 and CNA2) encoding the alternative forms of the catalytic A subunit of calcineurin, or of the gene (CNB1) encoding the regulatory B subunit, is lethal in an fks1 mutant. Disruption of the gene encoding FKBP12 (FKB1) or the major, cytosolic cyclophilin (CPH1) in fks1 cells results in the loss of hypersensitivity to the relevant drug. Overexpression of CNA1 or CNA2, in conjunction with CNB1, results in a significant decrease in hypersensitivity to FK506 and CsA. The results show that the hypersensitivity of the fks1 mutant is due to the inhibition of calcineurin phosphatase activity by the receptor-drug complexes. The growth dependence of the mutant on the Ca2+/calcineurin signal pathway provides an important tool for studying in yeast certain aspects of immune suppression by these drugs.

The ability to aggregate human platelets was examined for five Lactobacillus rhamnosus strains and five Lactobacillus paracasei subsp. paracasei strains isolated from patients with infective endocarditis (IE), 25 laboratory isolates from the same two species, and 14 strains from five other oral species, namely Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus oris, Lactobacillus plantarum and Lactobacillus salivarius. Amongst the L. rhamnosus strains, platelets were aggregated by all five IE strains and 8/16 laboratory strains. For the L. paracasei subsp. paracasei strains, the respective numbers were 2/5 and 2/9. Aggregation also occurred with 11/14 strains of the other five species; each species was represented. The optimal ratio of bacteria to platelets for aggregation was approximately 1:1, and there was considerable variation in the lag phase that preceded aggregation, depending on the source of the platelets. Overall, the lag phase varied between 0·25±01·and 20·4±3·2 min and the percentage aggregation ranged between 70±2·6 and 104±13·5%. Confirmation that aggregation was being observed came from studies with five strains on the inhibitory effects of EDTA, dipyridamole, apyrase, imipramine, acetylsalicylic acid and quinacrine. Inhibition of aggregation by L. rhamnosus strains by the peptide arginine-glycine-aspartic acid-serine (RGDS) further indicated a role for fibronectin and/or fibrinogen. Pronase treatment of cells for 1 h and extraction of bacterial surface components with 0·1 m-Tris/HCl (pH 8·5) at 37 °C for 1 h stopped aggregation in 8/9 IE strains. Extracted surface proteins (200 μg) completely inhibited platelet aggregation by 8/9 of the homologous strains. A comparison of the platelets from four donors showed that an increase in the lag phase by 50% required 11·5, 14·8, 33·3 or 115 μg of extract, indicating a variability in donor response to aggregation by lactobacilli. The data indicate that platelet aggregation by lactobacilli may be an important contributory factor in IE. The potential to cause IE is present in the general population of oral lactobacilli and due regard should be taken when strain selection is made for probiotic purposes.

This study was undertaken to search for the presence of immunoglobulin G (IgG)-binding proteins in Streptococcus suis, an important swine pathogen. Whole bacterial cells were incubated with human or pig IgG conjugated to gold particles and examined by transmission electron microscopy. Cells of some S. suis strains were labelled as were cells of the positive control strain, Staphylococcus aureus Cowan I. Binding of pig and human IgG to five different bacterial species of group D streptococci, to reference strains representing the 29 capsular types of S. suis, and to 12 S. suis capsular type 2 strains was then examined using Western blotting. All strains interacted with pig and human IgG, although the binding profiles were slightly different. A 52 kDa protein was observed in all capsular types of S. suis. This protein, absent in other group D streptococcal species, was observed in all capsular type 2 isolates originating from diseased or clinically healthy pigs, and was shown to bind human IgG-Fc fragments. The IgG-binding activity was also observed in the culture supernatant and was sensitive to proteolysis.

Peptidoglycan (PG) has been isolated from some species of spirochaetes, including Leptospira interrogans. Although leptospiral PG has been chemically characterized, no study has been carried out on its potential biological activity. Since PG of Treponema and Borrelia is biologically active both in vivo and in vitro, we investigated the capacity of a leptospiral PG preparation to induce relevant biological effects. PG extracted from L. interrogans strain Teramo was mitogenic at 0·1 μg ml−1 for human peripheral blood mononuclear cells (PBMC) since it increased the PBMC fraction positive for Ki-67, an antigen expressed by human proliferating cells; at 4 μg ml−1, PG was able to induce complement consumption and to stimulate leucocyte phagocytosis and the metabolic burst of resting as well as phagocytosing leucocytes. These findings indicate that Leptospira PG may play a role in modulating the immunocompetent cell functions and suggest that PG can contribute to the host response during Leptospira infection.

Four proteins from Candida albicans extracts have been isolated by ATP affinity chromatography. These proteins were found to be at elevated levels in extracts of cells raised from 25 °C to 37 °C, but were present at low levels in cells grown at 25 °C. The molecular masses of the proteins (38–42 kDa, 66–68 kDa, 70–72 kDa and 74–76 kDa) correspond to the published sizes of C. albicans heat-shock proteins. Three of the four proteins were recognized by the sera of patients with oral and/or oesophageal C. albicans infections, with the 70–72 kDa protein reacting in all cases tested. Binding of antibodies to two of the other proteins (38–42 kDa and 74–76 kDa) differed from patient to patient. IgA antibodies were the dominant immunoglobulin class in these mucosal C. albicans infections. The IgA antibody titre may be of diagnostic value and seemed to be correlated to the severity of infections, with a higher level in oesophageal infections compared to oral infections. Antibody binding to these proteins was specific as the sera did not show the same enhanced recognition with bacterial or HeLa cell heat-shock proteins.

Different exocellular extracts were isolated by concentrating the supernatants of yeast- and mycelial-phase Candida albicans cultures incubated in a synthetic medium. The only difference between the extracts obtained from the two phases was the presence in those obtained from mycelial cultures of a polysaccharide-rich, high-molecular-mass component, migrating in SDS-polyacrylamide gels at a position that would correspond to proteins with molecular masses of 245–265 kDa. The electrophoretic band patterns obtained before and after concanavalin A-Sepharose 4B affinity column treatments confirmed that the 245–265 kDa band was the only one of mannoprotein nature. The extract obtained from 24 h mycelial-phase culture (EA) was selected as the exocellular antigen for this work. The dry weight of EA obtained from 1 litre of culture medium was 30 mg; it contained 53% carbohydrate (18·3% glucose and 21·7% mannose measured by gas-liquid chromatography) and 10% protein. Rabbit antisera against EA were absorbed with yeast-phase organisms and used to stain Western blots of gels loaded with EAs. These antisera clearly recognized bands in the 21, 33 and 44 kDa areas. The antiserum obtained was employed to develop a double-antibody enzyme-linked immunosorbent assay for measuring EA concentrations in a culture medium. Most of the EA was released during the exponential phase of growth.

Macrophage (MΦ) chemiluminescence (CL) induced by interaction with the two types of colonial variants of Mycobacterium intracellulare was studied. A smooth, opaque and dome-shaped (SmD) colonial variant triggered more intense MΦ CL than did a smooth, transparent and flat colonial variant (SmT). MΦ CL-inducing activity of the SmD variant was reduced by heating or by treatments with either Pronase P, some endoglycosidases or Tween 80, thereby indicating that the SmD variant possesses MΦ CL-inducing substance(s) having peptide, sugar and/or lipid-like moieties. Treatment of the SmD variant organism with some endoglycosidases, such as cellulase, pectinase, dextranase or α-amylase decreased its MΦ CL-inducing ability. On the other hand, MΦ CL-inducing activity of the SmT variant was not affected by any of above treatments except that it was slightly increased by Pronase P treatment and reduced by α-amylase and dextranase.

Synthetic oligonucleotide primers derived from a sequence of the thermostable direct haemolysin (tdh) gene were used in a polymerase chain reaction (PCR) amplification technique to detect this gene in strains of Vibrio parahaemolyticus. A total of 36 TDH-producing, and 89 TDH-negative Vibrio parahaemolyticus strains and 46 other vibrios and enteric pathogens were studied. In all, 36 strains of Vibrio parahaemolyticus from which the tdh gene could be successfully amplified by PCR were found to be TDH-positive in TDH haemolysin assay. No amplification products were obtained from Vibrio parahaemolyticus strains that were TDH-negative in the haemolysin assay or from other vibrios and enteric pathogens, with the exception of two strains. The PCR results were consistent with DNA hybridization tests. The detection limit for the tdh gene by PCR amplification was 40 pg of total DNA, or broth culture containing 1000 viable cells. Amplification products were confirmed by restriction enzyme digestion and Southern blot hybridization. The PCR method could detect the tdh sequences in stool samples from patients with gastroenteritis caused by V. parahaemolyticus. This PCR protocol clearly identified TDH-producing strains of V. parahaemolyticus and provides an alternative to conventional methods for TDH detection by research laboratories, clinical laboratories, regulatory agencies, and the seafood industry.

A series of thermosensitive cell growth mutants of Enterococcus hirae have been isolated. Most of these mutants elongate and some show reduced autolytic activity when incubated at the non-permissive temperature (42 °C) in comparison to the wild-type incubated at the same temperature. When mutants were incubated for longer than 15 min at 42 °C and were then shifted to 30 °C, a lag proportional to the time of preincubation at 42 °C was observed before division, indicating that a certain time is necessary to restore normal levels of an active molecule(s) needed for septum formation and division. The addition of wild-type muramidase-1 permitted the immediate formation of septa and a single cell division; further addition of the enzyme stimulated the cells to divide once again. The other E. hirae autolytic enzyme, peptidoglycan-hydrolase-2, which is found in the culture medium, seemed to be involved in separation of daughter cells but may also take over the function of muramidase-1. A key role of both enzymes in septum formation and division is postulated.

A temperature-sensitive mutation in a new Escherichia coli gene, located at 62·5 min on the linkage map and designated lytF, resulted in bacteriolysis at the restrictive temperature. Temperature sensitivity and lytF-mediated lysis were simultaneously suppressed by either of two previously described unlinked mutations designated smhA1 and smhB1. The smhA1 and smhB1 alleles were originally isolated as specific extragenic suppressors of temperature-sensitive mutations in three other genes known as murH (99 min), lytD (13 min) and lytE (25 min) which conferred lysis phenotypes indistinguishable from that of the lytF mutation. The murH, lytD and lytE genes have been proposed to be related on the bases of phenotypic similarities and the specificities of their extragenic suppressors. It is now further proposed that lytF belongs to this group. The isolation of new alleles of smhA and smhB as extragenic suppressors of lytF further supports this proposal.

In Corynebacterium glutamicum peptide uptake increases the internal concentration of amino acids and thus triggers amino acid secretion. The peptide uptake system is stimulated by a factor of two in cells grown on pure peptone medium in comparison to peptone media with additional carbon sources. Uptake depends on the proton-motive force and shows a broad substrate spectrum. Peptide uptake is characterized by a K m of about 230 μm and a V max of 12 nmol min−1 (mg dry wt)−1 for the peptide lysyl-alanine (Lys-Ala). Lysine secretion in the wild-type of C. glutamicum does not show Michaelis-Menten-type kinetics as reported for the producing strains DG 52–5 and MH 20–22B. The secretion of lysine depends on the composition of the medium in which the cells were grown prior to the initiation of secretion by peptide uptake. The lack of secretion activity when the cells are shifted to peptone medium in the presence of chloramphenicol indicates that protein synthesis is necessary for this regulatory process.

Growth assays reveal that Rhodococcus rhodochrous IGTS8 uses a wide range of organosulphur compounds as the sole source of sulphur, yet none of the compounds serve as carbon sources. Compounds that are utilized include thiophenes, sulphides, disulphides, mercaptans, sulphoxides, and sulphones. A convenient spectrophotometric assay (Gibbs assay), based on the chromogenic reaction of 2,6-dichloroquinone-4-chloroimide with aromatic hydroxyl groups, was developed and used in conjunction with GC/MS analyses to examine the kinetics of dibenzothiophene metabolism by axenic and mixed cell cultures of Rhodococcus rhodochrous IGTS8. The desulphurization trait is expressed at increasing levels during the exponential phase of growth and then declines in stationary-phase cells. Mixtures of streptomycin-resistant Rhodococcus rhodochrous IGTS8 and Enterobacter cloacae (an organism incapable of cleaving carbon-sulphur bonds in relevant test compounds) were prepared in ratios that varied over six orders of magnitude. Growth studies revealed that E. cloacae was able to gain access to sulphur liberated from organosulphur compounds by IGTS8; however, cell-to-cell contact appears to be required. These experiments also indicate that the desulphurization activity, on a per cell basis, is higher in mixed cultures than in axenic cultures.