Some Properties of Pentachloro-phenol-resistant Gram-negative Bacteria

The effects of derivatives containing Lys–Asp sequences on growth-hormone-mediated lipolysis were examined for fat cells isolated from rat epididymal adipose tissue. A dipeptide, Lys–Asp, had a weak but distinct ability to induce lipolysis and inhibit growth-hormone-mediated lipolysis. Among the derivatives tested, Lys(Z)–Asp(OEt)–QEt (6d), Lys(Z)–Asp(OC₄H₉)–OC₄H₉ (6e), Lys(Z)–Asp(OC₈H₁₇)–OC₈H₁₇ (6f), C₇H₁₅CO–Lys(Z)–Asp (4b), and C15H₃₁CO-Lys(Z)Asp (4c) had a fairly high lipolytic activity. The derivatives 6e, 6d and 4c inhibited growth-hormone-mediated lipolysis. A derivative, C₁₅H₃₁CO–Lys–Asp(OMe)–QMe (3c), had no lipolytic activity but strongly inhibited for growth-hormone-mediated lipolysis. It is suggested that charge groups in the Lys–Asp derivatives are responsible for lipolytic action and the hydrophobic hydrocarbon chains in the derivatives enhance the ability to induce lipolysis or inhibit the gorwthhormone-mediated lipolysis.     

Properties of the Glucose Transport System in Some Deep-Sea Bacteria

Many deep-sea bacteria are specifically adapted to flourish under the high hydrostatic pressures which exist in their natural environment. For better understanding of the physiology and biochemistry of these microorganisms, properties of the glucose transport systems in two barophilic isolates (PE-36, CNPT-3) and one psychrophilic marine bacterium (Vibrio marinus MP1) were studied. These bacteria use a phosphoenol-pyruvate:sugar phosphotransferase system (PTS) for glucose transport, similar to that found in many members of the Vibrionaceae and Enterobacteriaceae. The system was highly specific for glucose and its nonmetabolizable analog, methyl alpha-glucoside (a-MG), and exhibited little affinity for other sugars tested. The temperature optimum for glucose phosphorylation in vitro was approximately 20°C. Membrane-bound PTS components of deep-sea bacteria were capable of enzymatically cross-reacting with the soluble PTS enzymes of Salmonella typhimurium, indicating functional similarities between the PTS systems of these organisms. In CNPT-3 and V. marinus, increased pressure had an inhibitory effect on a-MG uptake, to the greatest extent in V. marinus. Relative to atmospheric pressure, increased pressure stimulated sugar uptake in the barophilic isolate PE-36 considerably. Increased hydrostatic pressure inhibited in vitro phosphoenolpyruvate-dependent a-MG phosphorylation catalyzed by crude extracts of V. marinus and PE-36 but enhanced this activity in crude extracts of the barophile CNPT-3. Both of the pressure-adapted barophilic bacteria were capable of a-MG uptake at higher pressures than was the nonbarophilic psychrophile, V. marinus.     

Properties of Alginate Lyases from Marine Bacteria

Alginate lyases (EC 4.2.2.3) from two marine bacteria were isolated and partially characterized. A cell-bound lyase from isolate A3 had a molecular weight of approximately 100,000 and cleaved mannuronate blocks, apparently in an exo manner. A lyase recovered from the culture medium of isolate W3 was soluble in saturated ammonium sulfate, cleaved guluronate blocks, apparently in an endo manner, and had a molecular weight of 35,000. The thiobarbiturate test and urea-polyacrylamide gel electrophoresis were used to determine substrate specificity and mode of substrate cleavage by the enzymes.     

Properties of Bacteria Isolated from Deep-Sea Sediments

Thirty-eight isolates were subjected to taxonomic analysis by computer. Of the 38 isolates, 31 were from sediment samples collected at depths from 9,400 to 10,400 meters in the Philippine and Marianas Trenches of the Pacific Ocean, and 7 cultures were from seawater samples collected at various depths from surface to 4,000 meters and from several locations in the Pacific Ocean. A total of 116 characteristics were determined for each isolate, coded, and transferred to punch cards. Similarity values were obtained by computer analysis, with the use of two recently developed computer programs. Five distinct phenetic clusters were observed from the numerical analyses. Four of the clusters were identified as species of the genus Pseudomonas, and one, as an aerogenic species of Aeromonas. Group IV was identified as pigmented Pseudomonas fluorescens, and the major cluster, consisting of groups I and II, which merged at a species level of similarity, was treated as a new species of Pseudomonas. The 38 strain data were compared with data for 132 marine and nonmarine strains previously subjected to computer taxonomic analysis. The barotolerant deep-sea strains, with the exception of the deep-sea P. fluorescens isolates, clustered separately from all other marine strains.     

Cellular Location and Some Properties of Proteolytic Enzymes of Rumen Bacteria

Several physical and chemical techniques were used to extract, and to identify the location of, proteolytic enzymes associated with mixed rumen bacteria. Most activity was removable by gentle physical methods such as shaking and brief blending, without cell disruption, indicating that it was associated with coat and capsular material. Proteases were present also in the cell envelope, corresponding to the inner membrane fraction of gram-negative bacteria, and intracellularly and were removable by detergent and French press treatment. Temperature and pH profiles were obtained for the coat enzymes, likely to be the most important in the digestion of food protein. Inhibition studies indicated that these proteases, and those of the whole bacterial fraction from rumen fluid, were predominantly of the cysteine protease type.     

Purification and Some Properties of Candida albicans DNA Polymerases

Abstract

DNA polymerases of Candida albicans were purified to near homogeneity. Three well distinguished peaks of DNA polymerase activity (Enzyme I, II and III respectively) were obtained bv DEAE-Sephacel chromatography. This purification step was followed by column chromatographies on Sepharose 6B and denatured DNA-cellulose. The enzymes molecular mass and biochemical properties, including their inhibition by aphidicolin, were studied. Molecular mass was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and was found to be 110 kDa for Enzyme I, 80 kDa for Enzyme II and 50 kDa for Enzyme III.     

Physiological Properties of Nitrogen-scavenging Bacteria from the Marine Environment

Nine selected strains of marine bacteria from the marine environment, although taxonomically heterogeneous in character, exhibited a capacity for prolonged growth on 'nitrogen-free' media; only one strain, a Klebsiella sp., fixed dinitrogen under any of a considerable range of test conditions. The non-nitrogen-fixing bacteria were able to scavenge low levels of nitrogenous materials, principally ammonia, from the atmosphere. Contrary to previous suggestions, this growth did not have an abnormally low cellular protein and nitrogen content. Some strains with apparently low intracellular protein contents, as determined on a cellular dry weight basis, showed accumulations of carbonaceous storage products which distorted the cytochemical analyses. Representative strains grown in chemostat culture showed a high affinity (i.e. low Ks values—0.35–0.52 μmol/1) for the NH₄+ ion, compared with Escherichia coli (1.75 μmol/1) and preliminary studies of their glutamine synthetases suggested that the affinities ( K_m values—0.25–0.29 μmol/1) of this enzyme for hydroxylamine were similar to the few values reported for other marine bacteria.     

Extremely Rapid Extraction of DNA from Bacteria and Yeasts

A very simple and rapid method for extracting genomic DNA from Gram-negative bacteria, Gram-positive bacteria and yeasts is presented. In this method, bacteria or yeasts are lysed directly by phenol and the supernatant is extracted with chloroform to remove traces of phenol. The supernatant contains DNA that is suitable for molecular analyses, such as PCR, restriction enzyme digestion and genomic library construction. This method is reproducible and simple for the routine DNA extraction from bacteria and yeasts.     

Interdomain Conjugal Transfer of DNA from Bacteria to Archaea

Escherichia coli transforms the methanogenic archaeon Methanococcus maripaludis at frequencies ranging from 0.2 × 10⁻⁶ to 2 × 10⁻⁶ per recipient cell. Transformation requires cell-to-cell contact, oriT, and tra functions, is insensitive to DNase I, and otherwise displays hallmarks of conjugation.Conjugal transfer of DNA involves a specific set of transfer (tra) functions that mediate the mobilization of DNA containing an origin of transfer (oriT) from a donor to a recipient in a process requiring cell-to-cell contact (9). While conjugation is often very efficient between members of a given species or genus, it can also occur at a lower efficiency between phylogenetically distant microorganisms with structurally distinct cell surfaces. Escherichia coli, for example, mediates conjugal transfer of DNA to such diverse bacterial recipients as cyanobacteria (23), spirochetes (14), and a variety of Gram-positive bacteria (17, 22); E. coli even mediates conjugal DNA transfer to members of the domain Eukarya, such as to Saccharomyces cerevisiae (6) and mammalian (20) cells. Because of its broad range of potential recipients, conjugation has proven to be a valuable genetic tool (11) and may be an important mechanism of horizontal gene transfer and a driver of genome evolution (7). Conjugation-like DNA transfer has also been demonstrated in members of the domain Archaea (5, 15). However, conjugation between Bacteria and Archaea has not been demonstrated, despite the observation that many whole-genome sequences of Archaea harbor DNA that appears to be of bacterial origin (7).To investigate whether conjugation can occur between Bacteria and Archaea, the RP4 (IncPα group) conjugal-transfer system was used to attempt to mobilize DNA from E. coli to the anaerobic, methanogenic archaeon Methanococcus maripaludis strain S2 (21). The RP4 system was selected because previous work demonstrated that this plasmid supports the transfer of DNA from E. coli to phylogenetically distant recipients, including yeast (3) and mammalian (20) cells. Additionally, E. coli has been shown to successfully conjugate with strictly anaerobic bacterial strains (22). M. maripaludis was chosen as a recipient because it has growth parameters similar to those of E. coli and has readily available selectable markers (1). For all the experiments described, M. maripaludis was grown in liquid or solid (excluding cysteine) McCas medium (12), supplemented with 2.5 μg/ml puromycin (Pur) where appropriate, using standard anaerobic techniques (2). All plating for conjugation experiments, except for determination of viable-E. coli cell counts, was performed in an anaerobic chamber (Coy, Grass Lake, MI) with an atmosphere of 5:5:90 H₂-CO₂-N₂. E. coli was grown in Difco LB medium (Becton-Dickinson, Sparks, MD) supplemented where appropriate with 50 μg/ml kanamycin sulfate (Kan) and ampicillin (Amp).To interrogate conjugal DNA transfer between E. coli and M. maripaludis, a set of vectors that either contained or lacked cis-acting sites required for mobilization by RP4 transfer functions were constructed (Table ​(Table1).1). Each of these vectors contained a Pur resistance (Pur^r) gene cassette (pac) (4) flanked by ∼0.5 kb DNA homologous to regions 5′ and 3′ of the M. maripaludis nrpR gene (nrpR::pac), which allows for selection by Pur in M. maripaludis and provides sites for homologous recombination into the nrpR locus of the M. maripaludis chromosome. This construct was selected because it has previously been used to transform M. maripaludis to Pur resistance by recombination into the nrpR locus using a polyethylene glycol (PEG)-mediated transformation protocol (10, 18). After it was demonstrated that plasmids of the appropriate genotypes support conjugation from donor strain E. coli S17-1, which contains the RP4 trans-acting transfer (tra) functions on the chromosome via an integrated RP4-2-Tc::Mu-Km::Tn7 cassette (16), to E. coli recipient cells (Table ​(Table1;1; see also the supplemental material), we investigated whether these same donor strains could support DNA transfer to M. maripaludis.

TABLE 1.

Transformation of M. maripaludis by E. coli

Biotechnological Potential of Some Cold-Adapted Bacteria Isolated from North-Western Himalaya

Microbiology - In the present study, cold-adapted bacteria were isolated from soil, water and glacial ice samples collected from various geographical locations within the north-western Himalayan...     

Isolation and Properties of New Photosynthetic Bacteria Isolated from Seawater

Three strains of purple nonsulfur photosynthetic bacteria were isolated from seawater. They showed the characteristic properties of Rhodobacter species. Their biochemical, physiological, and chemotaxonomical properties were different from those of previously known species.     

工程菌酯酶B1的纯化及性质研究

工程菌酯酶B1降解酶经硫酸铵分级沉淀、DEAE SepharoseCL 6B离子交换柱层析、SephadexG 1 5 0凝胶过滤 ,得到了分离纯化 ,SDS PAGE鉴定为单一组分。经 1 2 .5 %SDS PAGE法测得分子量约为 5 6KD ,提纯倍数为33.3,收率为 1 7.9%,比活力为 74 .7U/mg。该酶的最佳作用条件是 37℃ ,pH =7.0 ,该酶作用于α 乙酸萘酯的Km为1 .35× 1 0 -3 mM ,Vmax为 2 .7× 1 0 4μ/ml。酶在pH6～ 9范围内较稳定 ,重金属Cu2 对该酶具有明显的促进作用 ,而SDS对酶具有抑制作用 ,对有机磷农药对硫磷 (1 6 0 5 )的降解率在 90min内达 91 .5 %。     

Comparison of Antimicrobial Properties of Silver Nanoparticles Synthesized from Selected Bacteria

Green silver nanoparticle (AgNP) biosynthesis is facilitated by the enzyme mediated reduction of Ag ions by plants, fungi and bacteria. The antimicrobial activity of green AgNPs is useful to overcome the challenge of antimicrobial resistance. Antimicrobial properties of biosynthesized AgNPs depend on multiple factors including culture conditions and the microbial source. The antimicrobial activity of AgNPs biosynthesized by Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Acinetobacter baumannii (confirmed clinical isolate) were investigated in this study. Biosynthesis conditions (AgNO₃ concentration, pH, incubation temperature and incubation time) were optimized to obtain the maximum AgNP yield. Presence of AgNPs was confirmed by observing a characteristic UV–Visible absorbance peak in 420–435 nm range. AgNP biosynthesis was optimal at 0.4 g/L AgNO₃ concentration under alkaline conditions at 60–70 °C. The biosynthesized AgNPs showed higher stability compared to chemogenized AgNPs in the presence of electrolytes. AgNPs synthesized by P. aeruginosa were the most stable while NPs of S. aureus were the least stable. AgNPs synthesized by P. aeruginosa and S. aureus showed good antimicrobial potential against E. coli, P. aeruginosa, S. aureus, MRSA and Candida albicans. AgNPs synthesized by S. aureus had greater antimicrobial activity. The antimicrobial activity of NPs may vary depending on the size and the morphology of NPs.     

Catalytic Properties and Regulatory Diversity of Inorganic Pyrophosphatases from Photosynthetic Bacteria

Soluble inorganic pyrophosphatases of five species of nonsulfur purple bacteria were investigated in respect to reaction kinetics, regulatory behavior, and other characteristics. The enzymes appear to fall into two groups with correlated properties. The pyrophosphatases of Rhodopseudomonas capsulata and R. spheroides have molecular weights of approximately 60,000, are stabilized by Co(2+), and exhibit simple Michaelis-Menten reaction kinetics. On the other hand, the enzymes of R. palustris, R. gelatinosa, and Rhodospirillum rubrum are larger (molecular weight approximately 100,000), require Zn(2+) for maintenance of catalytic activity, and show complex reaction kinetics; these pyrophosphatases are activated by free Mg(2+) ions and, in the absence of the latter, are inhibited by 2-phosphoglyceric acid. The results described indicate the existence of alternative control patterns for regulation of intracellular turnover of phosphate, which is in part mediated by pyrophosphatases.     

Collagenolytic Activity of Some Marine Bacteria

Reconstituted, acid-extracted collagen was used to prepare a medium to screen proteolytic marine bacteria for their ability to elaborate collagenolytic enzymes. The medium was resistant to solubilization by trypsin, hyaluronidase, chondroitinase ABC, and various marine proteinases, but was readily hydrolyzed by commercial Clostridium collagenases. Eighty-seven marine isolates collected in the vicinity of Bermuda, Oahu (Hawaii), and Stone Harbor and Cape May, N. J., were screened. Approximately 44 per cent of the isolates were capable of elaborating enzymes that hydrolyzed reconstituted collagen gels. Several cultures produced collagenolytic enzymes only when grown in the presence of collagen or degradation products of collagen, and with very few exceptions the presence of collagen in the medium greatly enhanced collagenolytic enzyme production. The enzymes from a collagenolytic Bermuda marine isolate were studied in more detail to illustrate that the enzymes capable of hydrolyzing reconstituted collagen were separable from nonspecific proteinases by zone electrophoresis and that these enzymes were true collagenases by virtue of their ability to hydrolyze native bovine Achilles'tendon obtained from three different sources.     

Radiometric Detection of Some Food-Borne Bacteria

Studies on detection of bacteria by radiometric techniques have been concerned primarily with aerobic species in clinical specimens. The data presented here are related to detection of aerobic and anaerobic species that are of significance in foods, by measurement of (14)CO(2) evolved from the metabolism of (14)C-glucose. Salmonella typhimurium and Staphylococcus aureus were inoculated into tryptic soy broth containing 0.0139 muCi of (14)C glucose/ml of medium. Detection times ranged from 10 to 3 hr for inocula of 10(0) to 10(4) cells/ml of broth. Heat-shocked spores of Clostridium sporogenes or C. botulinum were incubated in tryptic soy broth supplemented with Thiotone and NaHCO(3). The medium was rendered anaerobic with N(2). Spores were detected when 0.0833 muCi of labeled glucose was available/ml of medium but not when 0.0139 muCi of glucose was present/ml. The spores required 3 to 4 hr longer for detection than did comparable numbers of aerobic vegetative cells. The results demonstrate the importance of availability of sufficient label in the media and the potential of the application of this technique for sterility testing of foods.     

Comparative Genomics of DNA Fragments from Six Antarctic Marine Planktonic Bacteria

Six environmental fosmid clones from Antarctic coastal water bacterioplankton were completely sequenced. The genome fragments harbored small-subunit rRNA genes that were between 85 and 91% similar to those of their nearest cultivated relatives. The six fragments span four phyla, including the Gemmatimonadetes, Proteobacteria (α and γ), Bacteroidetes, and high-G+C gram-positive bacteria. Gene-finding and annotation analyses identified 244 total open reading frames. Amino acid comparisons of 123 and 113 Antarctic bacterial amino acid sequences to mesophilic homologs from G+C-specific and SwissProt/UniProt databases, respectively, revealed widespread adaptation to the cold. The most significant changes in these Antarctic bacterial protein sequences included a reduction in salt-bridge-forming residues such as arginine, glutamic acid, and aspartic acid, reduced proline contents, and a reduction in stabilizing hydrophobic clusters. Stretches of disordered amino acids were significantly longer in the Antarctic sequences than in the mesophilic sequences. These characteristics were not specific to any one phylum, COG role category, or G+C content and imply that underlying genotypic and biochemical adaptations to the cold are inherent to life in the permanently subzero Antarctic waters.