- Volume 141, Issue 7, 1995

Summary: The monoclonal antibody (mAb) B9E, which reacts with a cell wall surface determinant of Candida albicans serotype A, and a polyclonal monospecific antiserum against the antigen 6 (IF6) were used to investigate the expression of the antigens responsible for the serotype specificity in C. albicans under different growth conditions. By indirect immunofluorescence, both antibodies reacted with the cell wall surface of serotype A yeast cells and germ tubes grown in vitro but no reactivity was observed with serotype B yeast cells. In some cases, only a weak reactivity restricted to a zone close to the parent yeast cell was observed in serotype B germ tubes stained with mAb B9E. Both antibodies reacted strongly with yeast cells and germ tubes present in kidney abscesses from rabbits infected with both serotypes, but only serotype A yeast cells and germ tubes present in smears from patients with vulvovaginal candidiasis reacted with B9E and IF6 antibodies. The expression of antigens reactive with both antibodies was modulated by the pH of the environment in which the fungus was grown. Both antibodies showed a similar pattern of reactivity when studied with a spectrofluorometer. Serotype A yeast cells showed maximum reactivity when cells were grown on Sabouraud dextrose broth supplemented with yeast extract at pH 4.6. The lowest reactivity was observed in cells grown at pH 2.0. Conversely, the reactivity of serotype B yeast cells increased at alkaline pH values, the highest being in cells grown at pH values of 7.2 and 9.5. A precise use of the methods employed in studies on C. albicans serotype prevalence will be important to avoid the influence of pH on the expression of antigens conferring serotype specificity.

Summary: We have cloned and expressed the gp63 gene of Leishmania major in BCG to develop a recombinant vaccine against zoonotic cutaneous leishmaniasis. Two different expression systems were investigated. The first system consists of pAN, a Mycobacterium paratuberculosis promoter, which drives expression of ORF2, an open reading frame in IS900. This system allows the production of heterologous polypeptides as hybrids with the ORF2 gene product. The second expression system relies on the production of antigenic fragments as fusion proteins with the N-terminal region of Mycobacterium fortuitum β-lactamase. Both constructs resulted in the production of Gp63 in BCG. The ability of the two recombinant BCG strains to induce protective immunity against a challenge with L. major amastigotes was evaluated after vaccination of susceptible (BALB/c), and resistant (C57BL/6) mice. Recombinant BCG producing Gp63 as a hybrid protein with the N-terminal region of the β-lactamase elicited significant protection against a challenge with L. major in BALB/c-immunized mice.

Summary: Proteolytic enzyme activities of the wood-decaying basidiomycetes Serpula lacrymans and Coriolus versicolor, have been characterized using azocasein as substrate and by electrophoretic analysis with gelatin-containing polyacrylamide gels (gelatin-SDS-PAGE). In S. lacrymans, intracellular and extracellular azocaseinase activity was optimal at pH 5-6 and was inhibited by pepstatin A. Gelatin-SDS-PAGE revealed two highly active proteinases, S1 and S4 (apparent M r 65000 and 30000, respectively) and two less active enzymes, S2 and S3 (apparent M r 47000 and 43000, respectively). S1, the predominant intracellular proteinase, was present at all ages of the mycelium (tested up to 3 months). It is active over a broad pH range, with highest activity around neutral pH. As S1 was partially inhibited by 1,10-phenanthroline, the enzyme was considered to be a metalloproteinase although EDTA and phosphoramidon had no effect. A proteinase apparently identical to S1 was also detected in the medium of older cultures. S4 is a pepstatin-sensitive aspartic proteinase; its activity was highly pH-dependent and it was inactive in gelatin gels at pH 5-0 and above. S2 and S3 were identified as intracellular metalloproteinases, present in relatively young and growing cultures. They were distinct from S1 as they were inhibited by EDTA and phosphoramidon. During starvation-induced autolysis of S. lacrymans, proteinase S1 was the only enzyme present throughout (and the intracellular azocaseinase activity increased), which suggested a likely role of S1 in intra-hyphal protein mobilization. S4 is more likely to play a part in extracellular digestion of protein. The azocaseinase activities of cultures of C. versicolor were optimal at pH 7-0 (intracellular) and pH 5-6 (extracellular). Mycelial extracts gave one major band of proteinase activity in gelatin gels, C1 (apparent M r 62-64000). Since the activity was sensitive to inhibitors of both serine and metalloproteinases, there may have been overlapping bands due to enzymes of both types. Extracellular samples gave a more complex pattern, (five bands, C2-C6, M r 50000-100000). C2 and C4 are PMSF-sensitive proteinases, C5 and C6 are probably metalloproteinases, while C3, which was most active at pH 4-0, was unaffected by any of the inhibitors tested, including pepstatin A. No aspartic proteinase equivalent to S. lacrymans S4 appeared to be produced by C. versicolor. From the information gained about the intracellular or extracellular location of these enzymes, and the conditions under which they are active, an in vivo role may be tentatively ascribed to some of them.

Summary: To gain insight into the pathogenesis of tuberculosis, a molecular definition of the tubercle bacillus cell envelope, which is involved in the early stages of the infection, is required. The cell-surface-exposed material of the pathogen was isolated by mechanical means and chemically analysed. It was shown by scanning electron microscopy that the method used for extracting the surface-covering material preserves the integrity of the bacilli. Surprisingly, in view of the current opinion, only small amounts of lipids (1-6%) were present. Polysaccharides and proteins were the main components of the material. The polysaccharides were neutral and lipid-free D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 120, 13 and 4 kDa, respectively. Based on NMR spectroscopy and conventional chemical analyses, the major structural motifs of the purified polysaccharides were established as being identical to those of the polysaccharides we previously isolated from the culture filtrate of the tubercle bacillus. Immunocytochemical studies showed that these compounds were not only surface-located but were also present in the inner capsular compartment. The major protein constituents exhibited the same mobilities on SDS-PAGE as those of the culture filtrate of the tubercle bacillus and readily reacted with the monoclonal antibodies directed against these molecules. These proteins included the 19 and 38 kDa lipoproteins, the 30/31 kDa fibronectin-binding proteins and the 40 kDa L-alanine dehydrogenase. These findings suggest that the culture filtrate material represents part of the capsule which, in an in vivo context, could contribute to the electron transparent zone surrounding the tubercle bacillus. The 24 kDa (MPB/T64) protein was found to be a secreted protein, as it was detected almost exclusively in the culture filtrate. Taken together, the data give a new insight into the surface-exposed compounds of the tubercle bacillus and may explain part of the nature and limitation of the host immunity towards the pathogen.

Summary: The activity of the proline catabolic enzyme pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) was induced up to threehundredfold by the addition of three hundred proline to the growth medium of the Gram-positive bacterium Streptomyces coelicolor A3(2). Rifampicin, an inhibitor of RNA polymerase activity, abolished induction, implying that regulation was at the level of activation of gene transcription. The enzyme was purified and SDS-PAGE of the highly purified enzyme preparation revealed a single subunit with M r 68000. A single band of protein, which also stained for enzyme activity, was observed after native gel electrophoresis. The M r of the enzyme was estimated to be approximately 265000 by native gel electrophoresis and approximately 305000 by gel filtration, which indicated that the enzyme had a tetrameric quaternary structure. The apparent K m for pyrroline-5-carboxylate was 109 pm7-3 M, whilst that for NAD+ was 43-3+2-5 M. Product inhibition by NADH (apparent K i 0-6 mM) was observed. The observed V max was 22-0 pm 1 mol min−1 (mg protein)−1. Neither 1 nor 5 mM proline had any effect on enzyme activity, whilst glutamate was a very weak inhibitor.

Summary: The use of a novel monoclonal antibody (mAb) that reacts with (1,6)--glucan has permitted the study of the different covalent linkages between glucan and mannoproteins in the cell wall of Candida albicans. The mAb JRR1 was originally raised by immunization with Zymolyase extracts from C. albicans cell walls, but it soon became apparent that it reacted with a (1,6)--glucan epitope. By using this antibody, we show the existence of glucan-mannoprotein complexes between the (1,6)--glucan epitope recognized by the antibody and cell wall mannoproteins. The topology of the (1,6)--glucan in the cell wall of C. albicans has also been studied.

Summary: Substrains of Mycobacterium bovis BCG (BCG) have been divided into two major groups, high and low producers, on the basis of the amount of secretion of the MPB70 protein. The antigen is produced in high concentration by BCG Tokyo, Moreau, Russia and Sweden (high-producer substrains), whereas in BCG Pasteur, Copenhagen and Tice (low-producer substrains) it is detected at 1 % (w/w) or less of the concentration of BCG Tokyo. To investigate why this protein is secreted differently, the MPB70 genes of BCG Tokyo and Pasteur were cloned, sequenced and compared. The MPB70 genes in two substrains showed exactly the same sequence. Even the upstream and downstream regions of the MPB70 gene were identical. MPB70 gene expression was assessed by means of Northern hybridization analysis and reverse transcriptase polymerase chain reaction. The mRNA was clearly detected in BCG Tokyo, but at a very low level in BCG Pasteur. On the basis of these results, the difference in the secretion of the MPB70 protein between BCG Tokyo and Pasteur was attributed to differential transcription efficiencies.

Summary: The genes encoding the production of acidocin B, a bacteriocin produced by Lactobacillus acidophilus strain M46 which is active against Listeria monocytogenes, Clostridium sporogenes, Brochothrix thermosphacta, Lactobacillus fermentum and Lactobacillus delbrueckii subsp. bulgaricus, but inactive against most other Lactobacillus species, were previously localized on a 4 kb Xbal-HindIII fragment of plasmid pCV461. In the present work, DNA sequence analysis revealed the presence of three consecutive ORFs, which potentially code for hydrophobic peptides composed of 60, 91 and 114 amino acids, respectively, and a fourth ORF of opposite polarity which could potentially encode a peptide of 59 amino acids. The middle ORF (ORF-2; acdB) was identified as the gene encoding acidocin B by comparing the amino acid composition of highly purified acidocin B with the deduced amino acid sequence of ORF-2. Our results suggest that acidocin B is synthesized as a precursor which is processed at a site which conforms to the ' -3, -1' rules of von Heijne to yield active acidocin B (59 amino acids). The presence of an immunity-protein-encoding gene on the 4 kb Xbal-BamHl fragment was deduced from the capacity of a plasmid vector harbouring this fragment to confer immunity upon transformation of L. fermentum NCK127. One of the three non-assigned ORFs may encode this immunity protein.

Summary: Because of their potential usefulness as natural food preservatives, increased interest has focused on bacteriocins from lactic acid bacteria. Mesentericin Y105 is a small non-lantibiotic bacteriocin (class II) encoded within a 35 kb plasmid from Leuconostoc mesenteroides Y105 and it is active against Listeria monocytogenes. Using reverse genetic methodologies, an 8 kb Drall fragment has been cloned that contains the mesentericin Y105 structural gene, mesY, which encodes a precursor of the bacteriocin with a 24 amino acid N-terminal extension ending with a Gly-Gly motif upstream of the cleavage site, which is typical of class II bacteriocins. Four other putative genes are associated with mesY within two divergent putative operons. In addition to mesY, the first putative operon is predicted to encode a protein, similar to that encoded by ORF2 in the leucocin A operon, whose function remains to be elucidated. The second putative operon contains three ORFs, two of which, mesD and mesE, encode proteins that resemble ATP-dependent transporters and accessory factors, respectively. For three other class II bacteriocin systems (lactococcin A, pediocin PA-1, colicin V), these proteins have been shown to be involved in bacteriocin secretion independently of the general sec-dependent secretion pathway. The last putative gene (mesC) does not resemble any previously characterized gene. Results concerning the heterologous expression of the cloned mesY in Lactobacillus johnsonii NCK64 suggest that the maturation and secretion functions dedicated to lactacin F (another class II bacteriocin) are efficient for mesentericin Y105 as well. This characteristic may be of great interest in the development of industrial fermentation starters producing multiple bactericidal activities.

Summary: Most high-affinity systems for iron uptake in Gram-negative bacteria are thought to employ periplasmic-binding-protein-dependent transport. In Escherichia coli, FepB is a periplasmic protein required for uptake of iron complexed to its endogenously-synthesized siderophore enterobactin (Ent). Direct evidence that ferrienterobactin (FeEnt) binds to FepB is lacking because high background binding by FeEnt prevents use of the usual binding protein assays. Here the membrane localization vehicle LppOmpA [Francisco, J. A., Earhart, C. F. Georgiou, G. (1992). Proc Natl Acad Sci USA 89, 2713-2717] was employed to place FepB in the E. coli outer membrane. Plasmid pTX700 was constructed and shown to encode, under lac operator control, the ‘tribrid‘ protein LppOmpAFepB; the carboxy-terminal FepB portion lacks at most two amino acids of mature FepB. After short induction periods, most of the tribrid was in the outer membrane. A number of LppOmpAFepB species could be detected; some were degradation products and some may be related to the multiplicity of FepB forms previously observed in minicells and maxicells. Outer membrane harbouring the tribrid and lacking FepA, the normal outer membrane receptor for FeEnt, bound approximately four times more FeEnt than outer membrane from uninduced cells, from cells lacking pTX700 and from cells expressing only an LppOmpA ‘dibrid’. Similarly, whole UT5600(fepA)/pTX700 cells induced for tribrid synthesis bound FeEnt and this binding was not affected by energy poisons. The results demonstrated that FepB can bind FeEnt, thereby definitively placing FeEnt transport in the periplasmic permease category of transport systems, and that the LppOmpA localization vehicle can be used with periplasmic binding proteins.

Summary: Four major proteins are induced in Staphylococcus aureus in response to hyperosmotic shock caused by the presence of two different osmolytes, sucrose and NaCl. The gene encoding one of these proteins was isolated using a novel PCR procedure. The derived protein sequence shows extensive similarity to a subunit of alkyl hydroperoxide reductase (AhpC) from both Escherichia coli and Salmonella typhimurium. Exposure of S. aureus to varying concentrations of H2O2 did not result in the detectable induction of AhpC.

Summary: The mob locus of Escherichia coli encodes functions which catalyse the synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide, from molybdopterin and GTP. Reporter translational lac fusion mutations in the mobA gene have been constructed using λplacMu9 mutagenesis. The mob locus is expressed at very low levels under both aerobic and anaerobic growth conditions. Neither additions to the growth media (nitrate, tungstate or molybdate) nor secondary mutations at the moa, mob, mod, moe or mog loci affected the level of expression. Two transcription initiation sites and their associated promoter regions have been identified upstream of mobA. Both of the promoter regions show a poor match to the −35 and −10 consensus sequences for −70 promoters. A 2-2 kb chromosomal DNA fragment which complemented all available mob mutants has been sequenced. Two ORFs were identified, arranged as a single transcription unit. The encoded polypeptides have predicted molecular masses of 21642 Da and 19362 Da, respectively. The DNA has been subcloned into a T7 overexpression system and the predicted products identified. The mobA gene encodes protein FA, which has been purified to homogeneity and brings about the activation of inactive molybdoenzymes in cell extracts of mob mutants. The mobB gene encodes a polypeptide with a putative nucleotide binding site. All available mob mutations which have been selected for by their ability to grow anaerobically in the presence of chlorate are located in the mobA gene.

Summary: Twenty-three bacteria capable of growing with o-phthalate as sole carbon source were isolated from sewage sludge. One of these, named UCC61, was identified as a strain of Comamonas acidovorans and was selected for further study. UCC61 was found to carry a single plasmid (named pOPH1) of about 270 kbp in size. The phthalate-utilizing phenotype of UCC61 was unstable and could be lost either by complete curing of pOPH1, or by the deletion of a specific 70 kbp segment from pOPH1. This segment, termed the Pht element, was extensively restriction mapped (using four restriction endonucleases), and was found to be flanked by directly repeated sequences greater than 1-9 kbp in length. Data from comparative restriction analysis of pOPH1 and its deleted derivative were consistent with a deletion mechanism that involves homologous recombination between the direct repeats. UCC61 was found to catabolize o-phthalate via 4,5-oxygenation, dehydrogenation and decarboxylation to protocatechuate. These three activities were encoded within the Pht element, with the genes clustered near the right-hand terminus. Protocatechuate metabolism was chromosomally encoded, as also was the ability to catabolize the m- and p-isomers of phthalate.

Summary: The fliD genes of Salmonella typhimurium and Escherichia coli encode the filament-cap protein of the flagellar apparatus, which facilitates the polymerization of endogenous flagellin at the tips of the growing filaments. Previous sequence analysis of this operon in both organisms has revealed that the fliD gene constitutes an operon together with two additional genes, fliS and fliT. Based on the gene-disruption experiment in E. coli, both the fliS and fliT genes have been postulated to be necessary for flagellation. In the present study, we constructed S. typhimurium mutants in which either fliS or fliT on the chromosome was specifically disrupted. Both mutants were found to produce functional flagella, indicating that these genes are dispensable for motility development in S. typhimurium. However, flagellar filaments produced by the fliS mutant were much shorter than those produced by the wild-type strain. This indicates that the fliS mutation affects the elongation step of filament assembly. The excretion efficiency of flagellin was examined in the fliD-mutant background, where the exported flagellin molecules cannot assemble onto the hooks, resulting in their excretion into the culture media. We found that the amount of flagellin excreted was much reduced by the fliS mutation. Based on these results, we conclude that FliS facilitates the export of flagellin through the flagellum-specific export pathway.

Summary: Twenty-seven mutants defective in the biosynthesis of biotin were generated by N-methyl-N -nitro-N-nitrosoguanidine treatment of the obligate methylotrophic bacterium ‘Methylobacillus flagellatum’. The metabolic lesion in some of these mutants was determined in cross-feeding experiments using known Escherichia coli bio mutants. R-prime plasmids carrying various fragments of the ‘M. flagellatum’ chromosome were used for complementation of bio mutations in ‘M. flagellatum’ and E. coli. By complementation analysis and cross-feeding experiments, ‘M. flagellatum’ Bio- mutants were classified into five groups: bioA, bioB, bioD, bioH, and a group with unidentified bio mutations (bioF or/and bioC). Using R-prime complementation mapping and also Hfr-like mapping, most of the bio genes of ‘M. flagellatum’ identified in this study were localized on the ‘M. flagellatum’ chromosome.

Summary: The extreme thermophile Thermus sp. strain Rt41A produces an extracellular alkaline serine proteinase during growth. This enzyme is stable for more than 24 h at 70C and has a pH optimum of 8-0. The proteinase gene was identified using primers designed to amplify a region between two highly conserved amino acid motifs in subtilisin-like proteinases and the PCR product was used to identify a genomic fragment containing the gene. The amino acid sequence deduced from the Rt41A gene contained a region identical to that obtained by amino-terminal sequencing of purified Rt41A proteinase. Comparison of the entire derived peptide sequence with other subtilisin-like serine proteinases revealed significant homologies, especially with aqualysin I from Thermus aquaticus YT-1 and with exoprotease A from Vibrio alginolyticus. The Rt41A proteinase was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase as an aid for purification and to overcome difficulties experienced with other plasmid vectors which produced inactive protein. The enzyme is inactive as synthesized and activation was shown to be temperature-dependent, with shorter incubation times required at higher temperatures; removal of the hydrophobic signal peptide from the start of the gene reduced the time required for activation to less than a third of that required if the signal peptide was present.

Summary: The SpollAB protein of Bacillus subtilis is an anti-sigma factor that controls the release of -F in the prespore during sporulation. A missense mutation, spollAB22, in the N-terminal coding portion of spollAB was isolated previously on the basis of conferring increased -F activity on agar plates. We present the results of experiments further characterizing the phenotypic effects of the spollAB22 mutation. The mutation severely impairs spore formation on nutrient agar (NA) plates. Surprisingly, however, cultures induced to sporulate in liquid medium by the resuspension method showed little or no reduction in spore formation, and gene expression was more or less normal. The effects on sporulation on NA can be suppressed by mutations in the spollAC gene encoding -F. The implications for our understanding of the regulation of -F and -G activities, and the nature of the apparent medium dependence of sporulation in the mutant, are discussed.

Summary: High-level synthesis of exportable β-galactosidase (LacZ) fusion proteins in Bacillus subtilis results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants. We tested a plasmid-based, inducible lacZ fusion gene system for this purpose, but frequent mutations in cis. which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy. Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted. This led to the identification of a conditional B. subtilis secretion mutant after nitrosoguanidine mutagenesis. At 42°C, but not at 30°C, this mutant displayed extreme growth retardation when the LacZ fusion protein was produced, and was also defective in the secretion of subtilisin Carlsberg. The processing kinetics and secretion of a subtilisin Carlsberg-alkaline phosphatase fusion derivative were found to be defective specifically at the non-permissive temperature. The secretion defect was not linked to the secA/div locus.