No description available.

No description available.

Species of Arthrobacter luteus, also known as Oerskovia xanthineolytica, can utilise yeast cells as a growth substrate. This unusual ability is due to the secretion of a battery of hydrolytic enzymes which degrade the yeast cell wall and thus lyse the cells. Although many hydrolytic enzymes are important in the degradation of the yeast cell wall, the key activities are endo beta--l,3-glucanases. In order to characterise components of the yeast lytic system and the genetic organisation of this little-understood organism, a molecular cloning approach was adopted. Large clones expressing beta-1,3-glucanase were isolated from a library of A. luteus DNA constructed in the positive selection vector pKGW. By a combination of subcloning, restriction mapping and Southern analysis, it was determined that the clones contained virtually the same inserts. Additional subcloning, transposon mutagenesis, deletion mapping and nucleotide sequencing were used to identify at least one glucanase gene. The predicted protein product had a molecular weight of about 46 kD. When the gene was expressed in a number of in vivo and vitro systems including E. coli minicells and a Streptomyces coupled transcription/translation system, the protein observed had a similar molecular weight. Furthermore, when the protein was produced in E. coli and run on activity stained gels, the beta-glucanase activity co-migrated with the major glucanase of A. luteus. In addition the E. coli-produced glucanase had the ability to cause limited lysis of yeast.

A recombinant phage that produced a polypeptide possessing the characteristics of pneumolysin, the membrane damaging toxin of the pneumococcus, was isolated from a bank of pneumococcal sequences in ?gt10. Subclones carrying the pneumolysin gene in various plasmids were haemolytic regardless of the orientation of the insert. The nucleotide sequence of a 5 kb fragment carrying the pneumolysin gene was determined. An open reading frame 1413 bp long was identified that when translated encoded a polypeptide with 471 amino acids and a molecular weight 52.8 kD. The N-terminal amino acid sequence of the predicted protein was identical to that of native pneumolysin. A single cysteine residue was present at position 428 in the amino acid sequence. Comparison of the DNA and amino acid sequences of pneumolysin with streptolysin O (SLO) revealed extensive homology in the amino acid sequence. The longest region of identity was a sequence of 12 amino acids surrounding the unique cysteine. A hybrid gene consisting of the 5' region of the pneumolysin gene and the 3' end of the SLO gene was constructed. The fusion polypeptide was made in E. coli, but possessed a very low haemolytic activity. Using the technique of oligonucleotide-mediated site-directed mutagenesis, two mutant genes were constructed in which the cysteine codon was changed to either a glycine or serine codon. Modified toxins when purified from E. coli had a specific activity of about 1-2 % that of wild type pneumolysin.

No description available.

A conjugation system in Staphylococcus aureus has been investigated and shown to be determined, at least in part, by genes carried on plasmids. Conjugation required cell-to-cell contact but not calcium ions. The frequency of conjugation depended on the recipient used and on the incubation conditions. Two conjugative plasmids were mapped by restriction enzyme analysis but experiments to clone the conjugation-determining region were unsuccessful although separate regions specifying gentamicin resistance, ethidium bromide resistance and cadmium resistance were cloned. The gentamicin resistance determinant was probably part of Tn4001. Deletion of various sized pieces of DNA from one of the plasmids resulted in reduction of its ability to specify conjugation but no specific part of this plasmid could be implicated in the process. Further experiments led to the conclusion that this particular plasmid (p8325-4) is probably not self-transmissible but transferred by a phage-mediated system. Strains of Staphylococcus aureus produced a pheromone-like substance that elicited a clumping response in Streptococcus faecalis but no evidence was found for the involvement of staphylococcal conjugative plasmids in this. The conjugative plasmid, p8325-2, mobilized a small plasmid (pT181) but not a chromosomal gene. Insertion of transposon Tn551 was used to produce mutants of the conjugative plasmid p8325-2. Some twenty-six mutants were studied and the position of Tn551 in them mapped. There were preferred regions of insertion for Tn551 and twenty out of the twenty-six mutants had altered ability to conjugate. One showed a significantly higher frequency of conjugation and the other nineteen, all with substantially lower frequencies of conjugation, were mapped to two well-separated regions of the plasmid. Similarity between the locations of these putative regions and those reported for some other conjugative plasmids from staphylococci is striking and suggests a common origin.

No description available.

The detection of bacterial contamination of water, pharmaceutical products etc. is of great importance, and is most conveniently performed by the detection of bacterial ATP (Adenosine TriPhosphate) using the luciferin-luciferase bioluminescence system. This system uses unstable and expensive reagents, and emits transient light signals. In this study an FIA (Flow Injection Analysis) system was set up to monitor the light signal produced by the reaction. Using a luminometric detector (a liquid scintillation counter) with the FIA system, the reaction length, sample volume, flow rates, pH etc. were investigated.

No description available.

From Summary: A survey of collagenolytic aerobic bacteria from cured hides yielded three strains of Bacillus and eight of Achromobacter which degraded collagen at 0.4 M NaCl. Achromobacter sp. 2 was chosen for genetic studies due to its high collagenolytic activity and the lack of genetic information on Achromobacter. Four temperate bacteriophages specific for Achromobacter sp. 2 were isolated and their relationships studied. The phages caused lysogenic conversion resulting in the inability of lysogens to adsorb phage. Achromobacter sp. 2 was shown to be a cryptic lysogen as it was not immune to superinfection but had a very low rate of spontaneous induction which could be increased with mutagens. It is proposed that the cryptic lysogeny of this strain is maintained by a defective excision mechanism and the mode of prophage integration in the host chromosome. DNA extracted from phage α3a was used to transfect spheroplasts. The optimal conditions for the development of competence for transfection were determined. The presence of nuclease-attack on phage DNA under conditions of prolonged incubation of DNA and spheroplasts was proposed. A method for extracting Achromobacter DNA was devised which yielded purified, undegraded DNA, but it was not possible to transform Achromobacter sp. 2 with this DNA. The a phages were used to transduce a number of genetic markers into Achromobacter auxotrophs. The transduct ants had the ability to release the cryptic α3 prophage at a high rate while maintaining their sensitivity to homologous phage infection. It is proposed that this is due to complementation between the cryptic prophage and the residual phage functions in the transducing particles. The transductants segregated auxotrophs with a probability of 10⁻³ per cell per generation. It appears that an unusual system of generalised transduction is operating whereby the transducing particles contain both phage and bacterial DNA which is incorporated into the recipient genome by a single recombination event yielding unstable transductants. In a study on induction of Escherichia coli (λ), carcinogenic nitrosamines were shown to be inducers of phage development. This provides a screening system for potentially harmful nitrosamines.