A fourth metalloprotease gene in Erwinia chrysanthemi

Erwinia chrysanthemi, a Gram-negative phytopathogenic bacterium, was previously shown to secrete 3 related extracellular metalloproteases, A, B and C via a specific signal-peptide-independent pathway. A new gene (prtG) encoding a fourth, 52-kDa metalloprotease was identified on the same recombinant cosmid (pEW1) that carries the genes for the previously described proteases (prtA, prtB and prtC), for the specific secretion factors (prtD, prtE and prtF) and for a protease inhibitor (inh) cloned from E. chrysanthemi B374. The predicted sequence of PrtG was similar to those of PrtA, PrtB and PrtC, its secretion required PrtD, PrtE and PrtF; its secretion signal was located at the C terminus but its proteolytic activity was distinct from that of the 3 other proteases. Results presented here suggest that prtG could be the first gene of an operon that includes inh, prtD, prtE and prtF.     

Regulation of nisin biosynthesis by continuous cultures and by resting cell of Lactococcus lactis subsp. lactis

Nisin production by Lactococcus lactis subsp. lactis has been investigated using lactose as carbon source. Whether or not continuous cultures were lactose-limited, maximum nisin titre was observed at an intermediate mu value with a sharp peak of activity between 0.2 and 0.3/h. The maximum specific growth rate obtained in the medium used was 0.6/h and the maximum titre of nisin at mu = 0.25/h (160 AU/ml) was about nine-fold higher as compared with activity obtained at a dilution rate of 0.05/h or 0.4/h. With a constant dilution rate of 0.25/h and varying initial lactose concentrations from 3 to 40 g/l, there is an increase in nisin biosynthesis with increasing lactose concentration correlated with higher rates of sugar consumption. A Ymax value of 0.2 g bacterial dry weight and a maintenance coefficient of 124 mg lactose/g bacterial dry weight/h were determined. Lactose consumption increased from 1 to 3.28 g of lactose/g (dry wt) of cell mass/h and the nisin titre from 12.5 to 164.2 AU/ml. At higher values, nisin production declined. This implies that biosynthesis of nisin is regulated by a system of repression and derepression. Addition of lanthionine and beta-methyllanthionine precursors to the medium decreased the nisin titre when either threonine, threonine-cysteine, or cysteine-serine-threonine was added at the optimal dilution rate of 0.25/h; however, simultaneous addition of serine and cysteine elicited a slight increase in nisin activity. Studies with resting cells confirm that the biosynthesis of nisin is tightly regulated, since the production rate can be 5.6-fold higher than in cells grown in continuous culture. In addition, cell-adhered nisin appears to play a role in the production of the enzyme: low levels of cell-adhered nisin elicited high production rates, whereas high levels were not associated with nisin biosynthesis. In addition to pH, magnesium sulphate and lactose concentrations, nitrogen sources were also able to interfere in cell-adherence nisin.     

Intracellular movements of Rickettsia conorii adn R. typhi based on actin polymerization

Human vascular endothelial, Vero and human embryonic lung cells infected with rickettsiae for 24 h or 48 h were labelled for polymerized actin with NBD-phallacidin. Between 20 and 68% of the intracellular Rickettsia conorii had an actin tail of between 0.33 and 15 microns, with the longest tails being observed in Vero cells. In the case of R. typhi less than 1% of the organisms had actin tails and these were considerably shorter than those of R. conorii. These findings provide new information concerning the different cytopathic effects observed with the two rickettsial species.     

Characterization of a linear extrachromosomal DNA element (pBL1) isolated after interspecific mating between Streptomyces bambergiensis and S. lividans

Streptomyces bambergiensis S712 harbours a giant linear plasmid PSB1 of 640 kb. After mating with the plasmidless S. lividans strain TK64, conjugants carrying a smaller extrachromosomal DNA element, pBL1, were identified. pBL1 is a 43-kb linear DNA molecule bound to a protein which protects it from attack by both 3'- and 5'-exonucleases. The absence of this protein drastically reduces the transforming efficiency of pBL1. pBL1 shares homology with linear plasmids and chromosomal DNA from S. bambergiensis strains.     

Nucleotide sequence of the cellulase gene celF of Clostridium thermocellum

The nucleotide sequence of the celF gene of Clostridium thermocellum was determined. The open reading frame extended over 2217 bp. The encoded 739-aa polypeptide, CelF, with a M_w = 82,015, was an endoglucanase with activity against carboxymethylcellulose. The N terminus showed a typical signal peptide, and a cleavage site after Ala-27 was predicted. From residues 28 to 470, the sequence of CelF was related to the catalytic domains of type E₂ endoglucanases, with a strong homology to the endoglucanases CelZ of Clostridium stercorarium and CenB of Cellulomonas fimi. The catalytic region was followed by a 134-aa segment also present in C. stercorarium CelZ and in C. fimi CenB, and belonging to the family of non-catalytic, presumably cellulose-binding domains first identified in Bacillus subtilis endoglucanase. A 21-aa segment rich in Pro/Thr/Ser residues separated the putative cellulose-binding region from the COOH-terminal region, which contained two conserved stretches of 24 amino acids closely similar to those previously described in endoglucanases CelA, CelB, CelD, CelE, CelH and CelX, and xylanase XynZ of C. thermocellum.     

Molecular typing of thermotolerant species of Campylobacter with ribosomal RNA gene patterns

Ribosomal RNA gene restriction patterns (ribopatterns) of 72 strains representing Campylobacter jejuni (subspecies jejuni and doylei), C. coli, C. upsaliensis and C. lari including the urealytic (UPTC) biovar were determined using four common restriction endonucleases (HaeIII, HindIII, PstI and PvuII). The relative effectiveness of these enzymes for general molecular typing of the thermotolerant campylobacters was assessed. Ribotypes (HaeIII) were defined on the basis of computer-assisted numerical analysis. For C. jejuni, C. coli and C. lari, the HaeIII ribopatterns provided a high level of typability and discrimination, with patterns that were reproducible and easy to code for numerical analysis. There was evidence of diversity within three of the HaeIII types, and PstI ribopatterns proved the most reliable for detecting such differences. C. upsaliensis also could be ribotyped with HaeIII, but HindIII, PvuII and PstI were less satisfactory for this species. For such campylobacters, the HindIII ribopatterns generally were complex and difficult to compare, and the PvuII profiles provided the least discrimination. We conclude that the choice of restriction endonuclease is of critical importance when applying ribotyping to different species of Campylobacter. HaeIII ribopatterns were the most effective means of typing strains of different thermotolerant species of campylobacters and, when combined with PstI ribopatterns, offered a highly discriminatory basis for molecular typing.     

Listeria monocytogenes infection of Caco-2 cells: role of growth temperature

The aim of the present study was to evaluate the role of temperature in the virulence of Listeria monocytogenes, a Gram-positive facultative intracellular food-borne pathogen. The capacity of bacteria grown at 37, 25 and 4°C to develop haemolytic activity, to enter the Caco-2 enterocyte-like cell line and to multiply intracellularly was investigated. We demonstrated that L. monocytogenes penetration was not significantly influenced by the growth temperature of cultures and that bacteria grown at low temperature were capable of synthesizing internalin and, during the infection process, of restoring the haemolytic phenotype which is normally lacking in the extracellular environment at 4 and 25°C.It can be concluded that L. monocytogenes, frequently present in numerous environmental sources and also in refrigerated food products, produces at low temperature, the virulence factors necessary to invade intestinal cells.     

Cloning and nucleotide sequence analysis of immunodominant heat-shock protein of Yersinia enterocolitica

Yersinia enterocolitica, a highly antigenic 60-kDa protein, designated cross-reacting protein antigen (CRPA), is a member of the chaperonin-60 family of molecular chaperons. The gene encoding CRPA was cloned, expressed and sequenced. A partial library from Y. enterocolitica O:3 genomic DNA was constructed in vector pUC19 and was screened by the immunoreactivity to monoclonal antibody 1A4, which has specificity for a species-specific epitope on the CRPA molecule. The crpA gene region consists of a putative two-cistron operon encoding proteins of 549 and 97 amino acids. The operon structure was led by a consensus heat-shock promoter sequence. Homology searches using the derived amino acid sequence have revealed that CRPA is 88.2% identical to GroEL of Escherichia coli. CRPB, another protein encoded by the operon, shows extensive sequence homology, 91.8% identical to GroES of E. coli which is a member of chaperonin-10.     

Distribution of antibody titres against phenolic glycolipids from Mycobacterium tuberculosis in the sera from tuberculosis patients and healthy controls

Sera from tuberculosis (TB) patients and healthy controls were tested by ELISA for their antibody titres against the two major phenolic glycolipids (PGLs) of Mycobacterium tuberculosis, PGL-tbO (a 1:3 mixture of PGL-tb1 and its analogue whose phthiocerol moiety is phenolphthiotriol A) and PGL-tbK. Both PGL-tbs were shown to be specific to M. tuberculosis, and the profiles of serum anti-PGL-tbK titres revealed that PGL-tbK, like PGL-tb1, was fairly widely distributed among strains of M. tuberculosis. Even when these two PGL-tbs were used, however, the rate of ELISA-positives was not very high among TB patients, which is probably explained by the nature of the disease. Moreover, a considerable number of sera from healthy controls, especially from younger age groups, had high anti-PGL-tb titres, which implies that environmental exposure to M. tuberculosis is much higher than has been estimated from the actual TB cases. The ELISA system using these species-specific PGL-tb antigens may be useful for the survey of TB infection, since it gives more direct information on TB infection than the PPD skin test.     

Characterization of smooth Brucella lipopolysaccharides and polysaccharides by monoclonal antibodies

Two mouse monoclonal antibodies (mAb) generated to the M antigen of Brucella melitensis 16M were analyzed. Binding profiles of both monoclonals were established by a competitive enzyme-linked immunosorbent assay using chemically defined lipopolysaccharides, O polysaccharides and native hapten polysaccharides from B. melitensis 16M, B. abortus 544 and Yersinia enterocolitica O:9. Using this assay, significant differences in the reactivity of both antibodies were found with A and M antigens from Brucella spp. and the O polysaccharide from Y. enterocolitica O:9. These findings are consistent with the simultaneous expression of A and M epitopes on the lipopolysaccharide of all smooth Brucella strains. Quantitatively similar inhibitory powers were established for the native hapten and O polysaccharide from B. melitensis 16M. However, different behaviour was observed between both antigenic preparations obtained from B. abortus 544. The use of lipopolysaccharide-M-specific mAb in different serological tests instead of polyclonal antisera may be of great practical use for minimizing the risk of the appearance of cross-serological reactions between smooth Brucella strains and Y. enterocolitica O:9.     

Fate of the major outer membrane protein P.IA in early and late events of gonococcal infection of epithelial cells

We investigated the fate of the major outer membrane protein of Neisseria gonorrhoeae, P.IA, during gonococcal infection of Chang conjunctiva epithelial cells by using immunoelectron microscopy. Probing of P.IA epitopes with mono- and polyclonal antibodies revealed variable, fixation-dependent P.IA epitope exposure in the gonococci used as an inoculum in the infection experiments. Detection of invariable exposed P.IA epitopes in cryosections of infected epithelial cells with a polyclonal antiserum revealed unaltered P.IA exposure on the bacterial membranes during early attachment of the bacteria to the eukaryotic cells. Upon entry of the bacteria into the host cells, however, labelling was extended to membraneous structures that intercalated between the bacteria and the host cell surface, and, occasionally, to the host cell plasma membrane. The latter observation is consistent with the suggested active role of P.I. in the uptake process (as shown in 1985 by E.C. Gotschlich). Once inside the epithelial cells, both morphologically intact and disintegrating bacteria could be distinguished. The disintegration of the bacteria was accompanied by a loss of P.IA immunoreactivity.     

Phylogenetic analysis of the putative phosphorylation domain in the pyruvate kinase of Bacillus stearothermophilus

All sequenced phosphoenolpyruvate synthases (PPS), pyruvate:phosphate dikinases (PPDK) and enzymes I (EI) of the phosphoenolpyruvate:sugar phosphotransferase system comprise the PEP family. Linked to the C terminus of the sequenced pyruvate kinase from Bacillus stearothermophilus (PKBst) is a domain that is homologous to the putative phosphorylation domains of PEP family enzymes. We report sequence and phylogenetic analyses that lead to the following conclusions: (1) the phosphorylation domain of PKBst was derived from a PPS, late in the evolutionary process, after the divergence of PPSs from PPDKs and EIs; (2) this domain is probably functional in phosphoryl transfer; (3) the C-terminal phosphorylation domain in PKBst probably defines a compact domain in all PEP family proteins that is linked to other domains in these proteins via flexible linkers.     

Expression of P32 protein of goatpox virus in Pichia pastoris and its potential use as a diagnostic antigen in ELISA

The present study was undertaken to express goatpox virus (GTPV) P32 protein in Pichia pastoris and evaluate its potential use as a diagnostic antigen in ELISA. The amplified P32 gene of GTPV was cloned into pPICZαA vector and characterized by PCR, restriction enzyme digestion and sequencing. The characterized linear recombinant plasmids were transformed in Pichia host GSII5 strain by electroporation and the zeocin resistant Pichia transformant containing P32 gene was selected and confirmed by PCR. The expression of P32 protein in Pichia was induced with 0.5% methanol at 30 °C. The optimum expression was observed at 72 h post-induction and the yield was 100 mg/L of culture. The expressed protein was precipitated with polyethylene glycol and analyzed by SDS-PAGE and Western blot using GTPV specific serum and GTPV-P32 protein specific monoclonal antibody. Further, the protein precipitated with acetone was evaluated as diagnostic antigen in indirect ELISA in order to replace the whole GTPV. The standardized P32 protein based indirect ELISA had relative specificity and sensitivity of 84.2% and 94.2–100%, respectively when compared with serum neutralization test and whole virus based indirect ELISA. This study showed a potential of the yeast expressed GTPV-P32 protein as safe antigen in ELISA for seroepidemiological study of the capripox infection in sheep and goats, in India as well as capripox enzootic countries.     

Oxidative damage to DNA, p53 gene expression and p53 protein level in the process of aging in rat brain

Levels of 8-oxo2'dG (HPLC), p53 mRNA (PCR) and p53 protein (Western Blot) were estimated in four structures of rat brain, including grey matter (GM) of cerebral cortex, cerebral white matter (WM), cerebellum (C) and medulla oblongata (MO) of control (3.0-3.5-month-old) rats, 12- and 24-month-old rats. The level of oxidative DNA was statistically significantly higher in C of 24-month-old animals. Expression of p53 gene increased in C and also in the all other investigated brain parts, while the protein level of p53 was enhanced only in GM of 24-month-old rats. These data indicated that DNA oxidative damage and p53 gene expression increased significantly in aged brain. The higher expression of p53 gene in aged brain may suggest the activation of DNA repair processes.     

Comparison of the class-1 outer membrane protein from B:15:P1.16 Neisseria meningitidis strains isolated from patients previously immunized with a serogroup B outer membrane protein vaccine in Norway

A previous report of a large, double blind, efficacy trial of an experimental Group B meningococcal outer membrane protein vaccine carried out in Norwegian Teenagers, showed a protection rate of 57%. Previous studies had demonstrated the occurrence of mutations in the class-1 outer membrane protein which alter its immunological properties. The occurrence of new mutations might compromise the efficacy of a vaccine and explain the occurrence of any vaccine failures. The porA gene, which encodes expression of the class 1 protein, was sequenced in all isolates from vaccine failures and compared to that of the vaccinating strain H44/76 (B:15:P1.7,16). The porA DNA and deduced amino acid sequences were all identical to that of the vaccinating strain except for that of one isolate which had a sequence identical to strains previously reported in Norway and England with a 'masked P1.7' epitope. The absence of new mutations in the trial was encouraging for the further development of outer membrane protein vaccines.     

Expression of retinoblastoma protein and P16 proteins in classic Hodgkin lymphoma: relationship with expression of p53 and presence of Epstein-Barr virus in the regulation of cell growth and death

Deregulation of several genes involved in cell cycle control has been reported in classic Hodgkin lymphoma (cHL). This study aimed to investigate the expression of tumor suppressor proteins (P16(INK4A), retinoblastoma protein, and p53) in cHL in relation to the proliferation and apoptosis of Hodgkin/Reed-Sternberg (H/RS) cells, correlating with the status of Epstein-Barr virus (EBV). A total of 66 cHL cases and 10 nonneoplastic reactive lymphoid tissues were retrieved from the archives. Immunohistochemistry technique was used for the detection of protein expression. Presence of EBV infection was detected by EBV early RNA in situ hybridization. p16(INK4A) gene deletion status was assessed by fluorescence in situ hybridization technique. Expression of P16(INK4A) was observed in 49.2% of the cases, whereas positive retinoblastoma protein and p53 expressions in the H/RS cells were detected in 89.1% and 81.5% of the cases, respectively. Epstein-Barr virus positivity was detected in 53.0% of the cases. Proliferation marker, Ki-67 expression, was observed in 86.7% of the cases. There was no significant correlation between the expression of the various tumor suppressor proteins and Ki-67. Retinoblastoma protein and p53 were also not associated with the presence of EBV. An inverse relationship was observed between the expression of P16(INK4A) and the presence of EBV. There were no significant homozygous or hemizygous deletions of the p16(INK4A) gene. However, an aberrant copy number of chromosome 9 with the loss of one or more p16(INK4A) loci was detected in all cases assessable by fluorescence in situ hybridization. Loss of function of one or more tumor suppressor proteins may be involved in defective cell regulation of H/RS cells. Epstein-Barr virus may have a role in inhibiting P16(INK4A) expression, thus resulting in a perturbed p16(INK4A)-Rb cell cycle checkpoint.