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PubMed1975

Enzymatic repair of UV-irradiated DNA in vitro.

Excision repair of UV-damaged Bacillus subtilis transforming DNA has been carried out by a sequential enzyme system in vitro. Incision adjacent to the pyrimidine dimer in the DNA strand by correndonuclease II-initiated excision of the damage by the 5' in equilibrium 3'-directed exonuclease of the Micrococcus luteus DNA polymerase. Reinsertion of nucleotides into the gap in the strand by the DNA polymerase at 10 degrees C terminated in a single-strand break which was sealed by a polynucleotide ligase, thereby repairing the DNA strand. This restored biological activity to damaged DNA up to doses resulting in 60% inactivation of transforming activity. At higher doses, less repair was achieved, due to the development of double-strand breaks during the in vitro incision and excision steps.

Bacillus subtilis↗
PubMed1982

DNA glycosylases.

Various DNA glycosylases exist, which initiate the first step in base-excision repair. A summary of the kinetic and physical characteristics of three classes of DNA glycosylases are presented here. The first class discussed, include glycosylases which recognize alkylated DNA. Various data from enzymes derived from both prokaryotic and eukaryotic sources is discussed. The second class deals with a glycosylase that recognizes and initiates the excision of pyrimidine dimers in DNA. To date, this enzyme has only been uncovered from two sources, Micrococcus luteus and the T4 bacteriophage of E. coli. The third class consists of the most studied of the glycosylases, the uracil-DNA glycosylase enzymes. Various characteristics are presented for the uracil-DNA glycosylases derived from various sources. Recent information from our laboratory is presented implicating that herpes simplex virus may mediate a uracil-DNA glycosylase activity in productivity infected cells.

Chemical Phenomena↗
PubMed1979

Three additional genes involved in pyrimidine dimer removal in Saccharomyces cerevisiae: RAD7, RAD14 and MMS19.

The ability to remove ultraviolet (UV)-induced pyrimidine dimers from the nuclear DNA of yeast was examined in two radiation-sensitive (rad) mutants and one methyl methanesulfonate-sensitive (mms) mutant of the yeast Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by an endonuclease activity prepared from crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad7, rad14 and mms19 mutants were found to be defective in their ability to remove UV-induced dimers from nuclear DNA. All three mutants belong to the same epistatic group as the other mutants involved in excision-repair. All three mutants show enhanced UV-induced mutations. The rad14 mutant also shows epistatic interactions with genes in the other two UV repair pathways.

DNA Repair↗
PubMed1988

Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli.

Several hundred bacterial isolates were screened for bacteriolytic activity by growing them on agar medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells as the substrate. A Bacillus sp. producing the largest lytic zone was selected. A genomic bank of this selected bacterium was constructed in the multi-functional vector pTZ18R, with partial SauIIIA DNA fragments inserted at the SalI restriction site. Screening of 800 colonies of this bank for cell lysis gave 5 recombinants exhibiting lytic activity, as detected by analysis of extracts of sonicated Escherichia coli cells on denaturing polyacrylamide gels containing autoclaved, lyophilized M. lysodeikticus cells as the substrate. One clone (pBH2500), expressed in E. coli strain NM522, was found to code for a lytic enzyme corresponding, in molecular weight, to the 27 kDa Bacillus sp. hydrolase. This clone with an insertion of 2.5 kb was then subcloned as a 929 bp EcoRI-SauIIIA fragment in pTZ18R (pBH929) and showed higher cell lytic activity. A unique open reading frame for a protein of 251 amino acids, followed by a putative terminator sequence, was found after a consensus ribosome binding site. A putative leader sequence was identified in the first 37 amino acids. One truncated subclone (pBH703), corresponding to 196 out of 251 residues from the protein N-terminal end, still possessed lytic activity.

Amino Acid Sequence↗
PubMed1981

Rapid distinction between micrococci and staphylococci with furazolidone agars.

Furazolidone agar proved to be a suitable medium for separating strains of the genera Micrococcus and Staphylococcus. 720 isolates (including 24 type strains) of gram- and catalase-positive cocci were tested for growth on tryptone soya and peptone agar with the addition of 50 microgram/ml furazolidone. The results were compared with the classification obtained by the standard-O/F-test and by the test system of Schleifer and Kloos. For routine identification and separation of staphylococci from micrococci a peptone agar with 20 microgram furazolidone/ml is recommended.

Bacteriological Techniques↗
PubMed1988

Factors affecting the survival and growth of bacteria introduced into lake water.

The populations of Pseudomonas sp. B4, Escherichia coli, Klebsiella pneumoniae, Micrococcus flavus, and Rhizobium leguminosarum biovar phaseoli declined rapidly in lake water. The initially rapid decline of the two pseudomonads and R. phaseoli was followed by a period of slow loss of viability, but viable cells of the other species were not found after 10 days. The rapid initial phase of decline was not a result of Bdellovibrio spp., bacteriophages, or toxins in the water since Bdellovibrio spp. were not present and passage of the lake water through filters that should not have removed bacteriophages or soluble toxins led to the elimination of the rapid phase of decline. The addition of 250 micrograms of cycloheximide and 30 micrograms of nystatin per ml eliminated viable protozoa form the lake water, and the population of Pseudomonas sp. B4 did not fall and the decline of E. coli and K. pneumoniae was delayed or slowed under these conditions. Pseudomonas sp. L2 proliferated rapidly in lake water amended with glucose, phosphate, and NH4NO3, but its numbers subsequently fell abruptly; however, in water amended with cycloheximide and nystatin, which killed indigenous protozoa, the population density was higher and the fall in numbers was delayed. Of the nutrients, the chief response was to carbon, but when glucose was added, phosphorus and nitrogen stimulated growth further. Removing other bacteria by filtering the lake water before inoculation with Pseudomonas sp. L2 suggested that competition reduced the extent of response of the pseudomonad to added nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗
PubMed1979

Rate of drying and the survival of microorganisms.

The survival rate of cells of the genera Arthrobacter, Pseudomonas, Mycobacterium, Escherichia, Micrococcus and Saccharomyces when counted immediately after fast or slow drying (20 minutes and 24 hours, respectively) was rather similar. However, after prolonged periods of dry storage, the number of viable cells after slow drying was much higher as compared with the rapidly dried cells. Investigations with Escherichia coli demonstrated this phenomenon only when more than about 8 mg of water per 10(8) cells was available on a filter paper disc. In order to obtain optimum resistance to water loss the dessication period of 0.025 ml of suspension of E. coli must be longer than 13 hours.

Bacteria↗
PubMed1988

Characterization of 15 selected coccal bacteria isolated from Antarctic rock and soil samples from the McMurdo-Dry Valleys (South-Victoria Land).

Approximately 1500 cultures of microorganisms were isolated from rocks and soils of the Ross Desert (McMurdo-Dry Valleys). From these, 15 coccoid strains were chosen for more detailed investigation. They were characterized by morphological, physiological and chemotaxonomical properties. All isolates were Gram-positive, catalase-positive and nonmotile. Six strains showed red pigmentation and could be identified as members of the genera Micrococcus (M. roseus, M. agilis) or Deinococcus. In spite of their coccoid morphology, the remaining nine strains had to be associated with coryneform bacteria (Arthrobacter, Brevibacterium), because of their cell wall composition and G+C ratios. Most of the strains were psychrotrophic, but one strain was even obligately psychrophilic, with a temperature maximum below 20 degrees C. Red cocci had in vitro pH optima above 9.0 although they generally originated from acid samples. Most isolates showed a preference for sugar alcohols and organic acids, compounds which are commonly known to be released by lichens, molds and algae, the other components of the cryptoendolithic ecosystem. These properties indicate that our strains are autochthonous members of the natural Antarctic microbial population.

Antarctic Regions↗
PubMed1984

Murein hydrolase (N-acetyl-muramyl-L-alanine amidase) in human serum.

An enzyme was identified in human serum which unlike lysozyme cleaved the amide bond between N-acetyl-muramic acid and L-alanine of the peptide side chain of the rigid layer (murein) of Escherichia coli. The N-acetyl-muramyl-L-alanine amidase released all of the peptide side chains including those to which the lipoprotein is bound. A portion of the peptide side chains of the Micrococcus lysodeikticus murein was also hydrolysed from the polysaccharide chains. E. coli, M. lysodeikticus, Bacillus subtilis and Staphylococcus aureus were not killed by the amidase. Treatment of E. coli with EDTA or osmotic shock rendered the cells sensitive to the amidase and they were killed. Possible biological functions of the amidase are discussed. The enzyme was separated from lysozyme in human serum. Gel permeation chromatography indicated a molecular weight of the active enzyme of 82,000 while gel electrophoresis in the presence of sodium dodecyl sulfate revealed a molecular weight of 75,000. Thus, the enzyme probably consists of a single polypeptide chain. Incubation with neuraminidase rendered the amidase more basic suggesting the release of sialic acid residues. The modified glycoprotein disclosed an increased activity to murein. Enzyme activity was inhibited by p-chloromercuribenzene sulfonate and ethyleneglycol-bis(2-aminomethyl) tetraacetate (EGTA) at 1 and 0.2 mM concentration, respectively, whereas EDTA up to 5 mM was without effect. The amidase was also inactivated by agents that reduce disulfide bridges.

Amidohydrolases↗
PubMed1982

Utilization of phthalate esters by micrococci.

Several strains of Micrococcus have been isolated by enrichment with one of several phthalate esters as sole carbon source. They have been separated into four groups by their esterase content and nutritional characteristics. The catabolic potential for phthalate utilization found in these strains provides further support for designation of the four groups. Pathways for phthalate utilization by 4,5-dihydroxyphthalate and/or 3,4-dihydroxyphthalate and protocatechuate and/or 2,3-dihydroxybenzoate are outlined, which suggests that micrococci possess substantial potential for the catabolism of aromatic compounds.

Biodegradation, Environmental↗
PubMed1990

External eye flora as a nutrient source for Acanthamoeba.

Certain bacteria cause excystment of Acanthamoeba from cyst to trophozoite form and are then ingested by migrating trophozoites. We studied the response of Acanthamoeba cysts to inoculation on agar seeded with three types of commensal eye bacteria and Escherichia coli. Amoebae excysted on all bacteria tested, and the migration rate of Acanthamoeba trophozoites on each was compared. Acanthamoeba migrated with equal speed on E. coli and Staphylococcus epidermidis. Migration was observed, but was more slow on Micrococcus and Corynebacterium. Commensal bacteria on the eyelids, conjunctiva and tear film may have a role in pathogenesis of Acanthamoeba keratitis.

Acanthamoeba↗
PubMed1995

Comparative effects of gamma and microwave irradiation on the quality of black pepper.

Powdered black pepper from Egyptian markets, was irradiated with different recommended doses of gamma rays (5.0 and 10.0 kGy) and with microwaves for different periods (20, 40 and 75 s) to improve its hygienic quality. The most common bacterial isolates were of three genera Bacillus, Clostridium and Micrococcus (7.5 x 10(6)), whereas the predominant fungi (7.8 x 10(4)) were Aspergillus species, A. glaucus, A. flavus, A. niger and A. ochraceus. Doses of gamma irradiation used (5.0 and 10 kGy) were sufficient to decrease spore-forming bacteria (SFB) and to inhibit the fungal flora and coliforms which contaminated the black pepper powder. Microwave treatments for 40 s and 75 s were of the same effectiveness whereas treatment for 20 s was less so. GLC analysis proved the presence of 31 peaks, only 19 compounds were identified as monoterpene hydrocarbons (56.21%), the major one being beta-phellandrene and limonene. Sesquiterpenes were also present, mainly beta-caryollphyllene (3.69%) as well as oxygenated compounds such as terpenol, geraniol, Me-chavicol, eugenol and anisol. Gamma irradiation at 5 kGy and 10 kGy respectively decreased the numbers of identified compounds from 21 (86.58% concentration) in untreated pepper to 16 (59.22% concentration), 15 (54.06% concentration). In comparison, microwave treatments, particularly for 40 s and 75 s, increased the concentration of the same compounds. The results obtained indicate that microwave treatment, under these conditions, is a safe and suitable technique for decontamination of black pepper which does not result in a great loss of flavour compounds, as compared with recommended doses of gamma irradiation.

Aspergillus↗
PubMed1976

The inhibition of lysozyme by bile acids.

The effect of bile acids on the bacteriolytic activity of lysozyme towards Micrococcus lysodeikticus was studied in vitro. All bile acids tested inhibited lysozyme activity. Conjugated bile acids were better inhibitors than their unconjugated homologs and sulfation resulted in still stronger inhibition. A study of UV-difference spectra of bile acid-lysozyme mixtures suggests that bile acids distort the tertiary structure of the enzyme. The inhibition-concentration curves of micelle-forming bile acids were bell-shaped, and peak inhibition was apparently related to the critical micellar concentration. The inhibition-concentration curves of sulfated bile acids, which do not form micelles, are characterized by a plateau of maximal inhibition. A mechanism of lysozyme activation by bile acid micelles is proposed. Our results illustrate the complex interactions between antibacterial compounds in the gut. As bile acids are known to inhibit lipase activity as well, these studies suggest that bile acids may have an important influence on intestinal enzyme activity in general.

Bile Acids and Salts↗
PubMed1976

A method for automatic recording of serum lysozyme activity with the fragiligraph.

A quick and simple method for the estimation of lysozyme activity using the Fragiligraph, was described. Diminution of turbidity in a suspension of Micrococcus lysodeikticus produced by the addition of standard lysozyme (hen egg white) or serum sample, was continuously recorded for 5 min by the Fragiligraph. The normal mean serum lysozyme activity value obtained by this method is 6,80 mug/ml +/- 1.85.

Autoanalysis↗
PubMed1984

Bacterial growth in artificial capillary spaces.

In experiments on the influence of microscopic capillaries on the growth of Escherichia coli, Brevibacterium linens, Micrococcus flavus and Bacillus cereus, a distinct delay in growth was observed. The difference in counts was greatest after 8 h. Later a slight equilibration of counts was noted. With B. cereus, only slight or no spore formation was observed under microcapillary conditions.

Bacillus cereus↗
PubMed1987

Structure of 5S rRNA in actinomycetes and relatives and evolution of eubacteria.

The primary structure of 5S ribosomal RNA has been determined in five species belonging to the genus Mycobacterium and in Micrococcus luteus. The sequences of 5S RNAs from Actinomycetes and relatives point to the existence in this taxon of a bulge on the helix that joins the termini of the molecule. An attempt was made to reconstruct bacterial evolution from a sequence dissimilarity matrix based on 142 eubacterial 5S RNA sequences and corrected for multiple mutation. The algorithm is based on weighted pairwise clustering, and incorporates a correction for divergent mutation rates, as derived by comparison of sequence dissimilarities with an external reference group of eukaryotic 5S RNAs. The resulting tree is compared with the eubacterial phylogeny built on 16S rRNA catalog comparison. The bacteria for which the 5S RNA sequence is known form a number of clusters also discernible in the 16S rRNA phylogeny. However, the branching pattern leading to these clusters shows some notable discrepancies with the aforementioned phylogeny.

Bacteria↗
PubMed1992

Microorganisms associated with natural fermentation of Prosopis africana seeds for the production of okpiye.

Okpiye is a food condiment prepared by the fermentation of Prosopis africana seeds. The traditional process for the production and microbiological characteristics of the condiment were investigated. During laboratory fermentation that lasted 96 h, the mesquite seeds underwent a natural fermentation that was characterised by the growth of microorganisms to 10(6)-10(8) cfu/g. Several species of bacteria especially B. subtilis, B. licheniformis, B. megaterium, Staphylococcus epidermidis and Micrococcus spp were found to be the most actively involved organisms. However, significant contributions to the microbial ecology were made by Enterobacter cloacae and Klebsiella pneumoniae. Lactobacillus spp were present in low numbers towards the end of the fermentation. The presence of Proteus and Pseudomonas spp in traditional fermented samples demonstrate the variability which may exist in the microflora of individual fermented samples. Variations in the important microbial groups show that Bacillus spp were the most prevalent species and occurred until the end of fermentation. Temperature, pH and titratable acidity varied with time and were influenced by the metabolic activities of the microorganisms.

Bacillus↗
PubMed1995

Formation of polyhydroxylated isoflavones from the soybean seed isoflavones daidzein and glycitein by bacteria isolated from tempe.

Five tempe-derived bacterial strains identified as Micrococcus or Arthrobacter species were shown to transform the soybean isoflavones daidzein and glycitein to polyhydroxylated isoflavones by different hydroxylation reactions. All strains converted glycitein and daidzein to 6,7,4'-trihydroxyisoflavone (factor 2) and the latter substrate also to 7,8,4'-trihydroxyisoflavone. Three strains transformed daidzein to 7,8,3',4'-tetrahydroxyisoflavone and 6,7,3',4'-tetrahydroxyisoflavone. In addition, two strains formed 6,7,8,4'-tetrahydroxyisoflavone from daidzein. Conversion of glycitein by these two strains led to the formation of factor 2 and 6,7,3',4'-tetrahydroxyisoflavone. The structures of these transformation products were elucidated by spectroscopic techniques and chemical degradation.

Arthrobacter↗