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PubMed2005

Effects of orally administered tetracycline on the intestinal community structure of chickens and on tet determinant carriage by commensal bacteria and Campylobacter jejuni.

There is a growing concern that antibiotic usage in animal production has selected for resistant food-borne bacteria. Since tetracyclines are common therapeutic antibiotics used in poultry production, we sought to evaluate the effects of oral administration on the resistance of poultry commensal bacteria and the intestinal bacterial community structure. The diversity indices calculated from terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA amplicons did not indicate significant changes in the cecal bacterial community in response to oxytetracycline. To evaluate its effects on cultivable commensals, Enterococcus spp., Escherichia coli, and Campylobacter spp. were isolated from the cecal droppings of broiler chickens. Enterococcus spp. and E. coli expressed tetracycline MICs of >8 microg/ml and harbored a variety of tet resistance determinants regardless of the tetracycline exposure history of the birds. The enterococcal isolates possessed tetM (61%), tetL (25.4%), and tetK (1.3%), as well as tetO (52.5%), the determinant known to confer a tetracycline resistance phenotype in Campylobacter jejuni. E. coli isolates harbored tetA (32.2%) or tetB (30.5%). Tetracycline MICs remained at <2 microg/ml for Campylobacter isolates before and after tetracycline treatment of the chickens, even though isolates expressing MICs of >16 mug/ml were commonly cultured from flocks that did not receive oxytetracycline. The results imply that complex ecological and genetic factors contribute to the prevalence of antibiotic resistance arising from resistance gene transfer in the production environment.

Animals↗
PubMed1982

Effects of oil and gas well-drilling fluids on the biomass and community structure of microbiota that colonize sands in running seawater.

Well-drilling fluid and a number of the known components (barite, clay, Aldacide, Surflo, and Dowicide, were tested for effects on the biomass and community structure of the microbiota that colonize marine sands exposed for eight weeks to running ambient seawater. Shading the microbiota from light depressed the microflora without a significant effect on the biomass, while well-drilling fluids layered on the surface or mixed with the sand significantly increased a component of the bacteria and the microfauna as reflected in changes in the fatty acid composition. There were some shading effects from the surface layering of well-drilling fluids as reflected in the fatty acids from the microflora when compared to the sands mixed with well-drilling fluids. Barite had essentially no effect on the biomass or community structure while clays increased nearly all of the biomass indicators for the bacteria as well as the microfauna; the clay overlay mirrors the effect of the drilling fluids. Aldacide shifted the bacterial composition, depressing the proportions of microbes containing the cyclopropane fatty acids and the anaerobic pathways of desaturation. Concentrations of 1 and 15 microgram/L increased the bacterial biomass as reflected in the total lipid (16:0) and extractable lipid phosphate coupled with a decrease in the total microeukaryotes. Surflo increased the biomass and shifted the bacterial community structure at concentrations between 4 and 800 microgram/L. The lowest level also stimulated the microfauna. Dowicide at 100 microgram/L increased the bacteria forming cis-vaccenic acid and the microfauna similar to low concentrations of Surflo.

Fatty Acids↗
PubMed2001

Structure of proline 3-hydroxylase. Evolution of the family of 2-oxoglutarate dependent oxygenases.

Iron (II)/2-oxoglutarate (2-OG)-dependent oxygenases catalyse oxidative reactions in a range of metabolic processes including the hydroxylation of proline and lysine residues during the post-translational modification of collagen. 2-OG oxygenases commonly require ascorbate for full activity. In the vitamin C deficient disease, scurvy, reduced activity of 2-OG oxygenases results in impaired formation of collagen. Here we report the crystal structure of bacterial proline 3-hydroxylase from Streptomyces sp., an enzyme which hydroxylates proline at position 3, the first of a 2-OG oxygenase catalysing oxidation of a free alpha-amino acid. Structures were obtained for the enzyme in the absence of iron (to 2.3A resolution, R=20.2%, Rfree=25.3%) and that complexed to iron (II) (to 2.4A resolution, R=19.8%, Rfree=22.6%). The structure contains conserved motifs present in other 2-OG oxygenases including a 'jelly roll' beta strand core and residues binding iron and 2-oxoglutarate, consistent with divergent evolution within the extended family. The structure differs significantly from many other 2-OG oxygenases in possessing a discrete C-terminal helical domain. Analysis of the structure suggests a model for proline binding and a mechanism for uncoupling of proline and 2-OG turnover.

Amino Acid Sequence↗
PubMed1990

Copper-zinc superoxide dismutase of Caulobacter crescentus: cloning, sequencing, and mapping of the gene and periplasmic location of the enzyme.

Although widely found in the cytoplasm of eucaryotes, the copper-zinc form of superoxide dismutase (CuZnSOD) has been identified in only a small number of bacterial species. One species is the freshwater bacterium Caulobacter crescentus, which also contains an SOD with iron as the metal cofactor (FeSOD). To investigate the function of this CuZnSOD and its structural relationship to the eucaryotic CuZnSODs, the gene encoding CuZnSOD (sodC) of C. crescentus CB15 was cloned and sequenced. By hybridization to pulsed-field electrophoresis gels, sodC was mapped near cysE in the C. crescentus chromosome. Through analysis of spheroplasts, the two SODs of C. crescentus were shown to be differently localized, CuZnSOD in the periplasm and FeSOD in the cytoplasm. In its natural habitat, C. crescentus is frequently associated with blue-green algae (cyanobacteria). The oxygen evolved by these photosynthetic algae may create an extracellular oxidative stress against which the periplasmic CuZnSOD may defend more effectively than the cytoplasmic FeSOD. Amino acid sequence alignments of C. crescentus CuZnSOD with eucaryotic CuZnSODs and with CuZnSOD of Photobacterium leiognathi (the only other bacterium from which CuZnSOD has been isolated and sequenced) suggest similar supersecondary structures for bacterial and eucaryotic CuZnSODs but reveal four novel substitutions in C. crescentus CuZnSOD: a phenylalanine critical to intrasubunit hydrophobic bonding replaced by alanine, a histidine ligand of zinc replaced by aspartate, and substitutions of two other previously invariant residues that stabilize zinc or both copper and zinc. These amino acid substitutions in C. crescentus CuZnSOD may have implications for its catalysis and stability.

Amino Acid Sequence↗
PubMed2005

Bacterial community shift along a subsurface geothermal water stream in a Japanese gold mine.

Change of bacterial community occurring along a hot water stream in the Hishikari gold mine, Japan, was investigated by applying a combination of various culture-independent techniques. The stream, which is derived from a subsurface anaerobic aquifer containing plentiful CO2, CH4, H2, and NH4+, emerges in a mine tunnel 320 m below the surface providing nutrients for a lush microbial community that extends to a distance of approximately 7 m in the absence of sunlight-irradiation. Over this distance, the temperature decreases from 69 degrees C to 55 degrees C, and the oxidation-reduction potential increases from -130 mV to +59 mV. In the hot upper reaches of the stream, the dominant phylotypes were: 1) a deeply branching lineage of thermophilic methane-oxidizing gamma-Proteobacteria, and 2) a thermophilic hydrogen- and sulfur-oxidizing Sulfurihydrogenibium sp. In contrast, the prevailing phylotypes in the middle and lower parts of the stream were closely related to ammonia-oxidizing Nitrosomonas and nitrite-oxidizing Nitrospira spp.. Changes in the microbial metabolic potential estimated by competitive PCR analysis of genes encoding the enzymes, particulate methane monooxygenase (pmoA), ammonia monooxygenase (amoA), and putative nitrite oxidoreductase (norB), also substantiated the community shift indicated by 16S rRNA gene analysis. The diversity of putative norB lineages was assessed for the first time in the hot water environment. Estimation of dominant phylotypes by whole-cell fluorescent in situ hybridization and changes in inorganic nitrogen compounds such as decreasing ammonium and increasing nitrite and nitrate in the mat-interstitial water along the stream were consistent with the observed transition of the bacterial community structure in the stream.

Ammonia↗
PubMed2005

High molecular weight DNA recovery from soils prerequisite for biotechnological metagenomic library construction.

Soil is a complex environment considered as one of the main reservoirs of microbial diversity. However, the inability to cultivate most soil bacteria hampered fundamental attempts to determine the diversity of the prokaryotic world and limited its industrial exploitation. In the last 20 years, new methods have been developed to overcome these limitations based on the direct extraction of DNA from bacteria in their natural environment. In addition to fundamental research, the cloning of the extracted DNA for the development of metagenomic DNA clone libraries offers possibilities to discover novel bio-molecules through the expression of genes from uncultivated bacteria in surrogate bacterial hosts. However, such objectives require adapting DNA extraction methods and cloning strategies in order that entire gene clusters encoding biosynthetic pathway for secondary metabolites can be cloned. In this paper, we report that the size of DNA fragments extracted from soil varied in a range between less than 100 kb and more than 400 kb depending on the soil. The relatively limited size of DNA fragments extracted from some soil was not only due to mechanical, chemical or enzymatic shearing of the DNA during the extraction process but partly to the microbial growth status. Stimulating bacteria in situ by providing nutrients to the soil improved the size of extracted DNA, but it modified the bacterial community structure.

Bacteria↗
PubMed2002

Down-regulation of pili and capsule of Neisseria meningitidis upon contact with epithelial cells is mediated by CrgA regulatory protein.

The initial attachment of Neisseria meningitidis to the target cell surface appears to be largely pilus depend-ent in capsulated bacteria. Intimate adhesion subsequently occurs to permit colonization. We recently reported that insertional inactivation of the crgA gene, which encodes a transcriptional regulator belonging to the LysR family, decreased meningococcal adhesion to epithelial cells and abolished intimate adhesion. In this report, we analyse expression of the pilE and sia genes, which are involved in the biosynthesis of pili and capsule respectively, during bacteria-host cell interactions. Western blotting, transcriptional fusion and reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression of these genes was downregulated during intimate adhesion. DNA-binding assays, footprinting and RT-PCR analysis indicated that this downregulation was directly mediated by the CrgA protein. The pilE and sia promoters were found to have a CrgA binding motif in common. These results strongly suggest that N. meningitidis displays an adaptive response upon cell contact. CrgA may play a central regulatory role in meningococcal adhesion, particularly in switching from initial to intimate adhesion by downregulating the bacterial surface structures that hinder this adhesion.

Bacterial Adhesion↗
PubMed1970

Fine structure of the motile cells and flagella in a member of the Actinoplanaceae (Actinomycetales).

The motile cells (sporangiospores) of an undescribed member of the Actinoplanaceae are studied by electron microscopy as shadowed, negatively stained, and sectioned preparations. The rod-shaped spores exhibit a typically bacterial internal structure. However, a single tubular structure (rhapidosome) is positioned just inside the site of flagellar attachment of each spore and is oriented perpendicular to the direction of the flagella. Flagella arise from basal dises and pass through the plasma membrane and the two-layered cell wall to become associated with other flagella to function as a posteriorly directed unit. Each flagellum consists of a helical band or ribbon which dissociates into 5 or 6 subfibrils.

Actinomycetales↗
PubMed1979

Hemagglutination and adhesiveness of toxigenic Escherichia coli isolated from humans.

Toxigenic strains of Escherichia coli isolated from humans were studied for adherence to human buccal mucosal epithelial cells. The E. coli strains were labeled with 3H-amino acids or fluorescein isothiocyanate. Toxigenic E. coli strains varied in their ability to adhere in the presence of mannose. Of 32 toxigenic strains examined, 52% bound to the buccal cells, whereas none of 8 control strains did so (Mann-Whitney U test, P =0.007). The control strains were nontoxigenic E. coli isolates from humans, enterotoxigenic E. coli isolates from animals, and E. coli K-12 containing the K88 or K99 plasmid; these strains exhibited only background-level adherence in this assay. Among the toxigenic E. coli strains that bound to human buccal mucosal cells, there was no correlation with mannose-resistant hemagglutination (MR-HA) of guinea pig and human erythrocytes. Screening 32 strains, we found the following phenotypes: (i) MR-HA+, buccal adherent; (ii) MR-HA+, buccal nonadherent; (iii) MR-HA-, buccal adherent. Presumably the third group represents strains with another type(s) of surface attachment components not involved in the MR-HA reaction. Our findings indicate that a number of bacterial surface structures can function in MR-HA and buccal adherence.

Agglutinins↗
PubMed2006

Molecular analysis of bacteria in asymptomatic and symptomatic endodontic infections.

The purpose of the present study was to use terminal restriction fragment length polymorphism analysis and the 16S rRNA gene clone library to investigate the diversity of the microbiota associated with asymptomatic and symptomatic endodontic infections and to compare the bacterial community structure in these two clinical conditions. Samples were taken from asymptomatic endodontic infections associated with chronic periradicular lesions and from symptomatic infections clinically diagnosed as acute abscesses. 16S rRNA genes from DNA isolated from clinical samples were used to construct clone libraries or were subjected to terminal restriction fragment length polymorphism analysis. Sequence analysis of 186 clones revealed 42 taxa; 23 (55%) were uncultivated phylotypes, of which seven were unique to endodontic infections. Clone sequencing and terminal restriction fragment length polymorphism analysis revealed that the most commonly detected taxa were Fusobacterium nucleatum (including terminal restriction fragment types 1 and 2), Peptostreptococcus micros/Peptostreptococcus sp. oral clone AJ062/BS044/FG014, Prevotella species, Dialister species, Mogibacterium species, Lachnospiraceae oral clone 55A-34, Filifactor alocis, Megasphaera sp. oral clone CS025/BS073, and Veillonella sp. oral clone BP1-85/Veillonella dispar/V. parvula. Bacteroides-like sp. oral clone X083/Bacteroidales oral clone MCE7_20 and Dialister sp. oral clone BS016/MCE7_134 were detected only in asymptomatic teeth. On the other hand, F. nucleatum terminal restriction fragment type 2, Prevotella intermedia, Dialister pneumosintes, and some phylotypes were exclusively detected in symptomatic samples. Bacterial profiles of symptomatic endodontic infections generated by terminal restriction fragment length polymorphism analysis were clearly different from those of asymptomatic infections. Overall, the average number of terminal restriction fragments in symptomatic samples was significantly larger than in asymptomatic samples. Molecular analysis of the microbiota associated with symptomatic or asymptomatic endodontic infections indicates that the endodontic bacterial diversity is greater than previously described by culture methods and that the structure of the microbiota differ significantly between asymptomatic and symptomatic infections.

Adolescent↗
PubMed1996

The 1.8 A resolution structure of hevamine, a plant chitinase/lysozyme, and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18.

The three-dimensional structure of hevamine, a plant enzyme with chitinase and lysozyme activity, has been refined at 1.8 A resolution to an R-factor of 14.9% and a free R-factor of 19.6%. The final model consists of all 273 amino acid residues and 206 ordered water molecules. Two non-proline cis-peptides were identified, involving Phe32 and Trp255, both of which are implicated in substrate binding. Other glycosyl hydrolase family 18 proteins with known three-dimensional structure are bacterial chitinase A, endo-beta-N-acetylglucosaminidase F1, endo-beta-N-acetylglucosaminidase H, and the two plant proteins concanavalin B and narbonin, which have no known enzymatic activity. All these structures contain a (beta alpha)8 barrel fold, with the two family 18 consensus regions roughly corresponding to the third and fourth barrel strands. This confirms the grouping of these proteins into family 18, which was only based on weak and local sequence similarity. The substrate specificity of the enzymes is determined by the loops following the barrel strands that form the substrate binding site. All enzymes have an aspartic acid and a glutamic acid residue in positions identical with Asp 125 and the catalytic Glu127 of hevamine. The lack of chitinase activity of concanavalin B and narbonin can be explained by the absence of one of these carboxylate groups, and by differences in the loops that form the substrate-binding cleft in hevamine.

Amino Acid Sequence↗
PubMed1993

Genetic structure of Neisseria gonorrhoeae populations: a non-clonal pathogen.

Reproduction by binary fission generates a clonal genetic structure in bacterial populations in the absence of a high rate of recombination. The extent of recombination in natural populations of Neisseria gonorrhoeae was determined from an analysis of electrophoretically demonstrable allelic variation at structural genes encoding nine enzyme loci in 227 worldwide isolates. No significant linkage disequilibrium was evident in the population, indicating that recombination must be frequent, relative to binary fission. The genetic structure of N. gonorrhoeae was compared with that of Bacillus subtilis from an earlier study. Linkage disequilibrium was less extreme in the N. gonorrhoeae population than in the local population of B. subtilis, in which only modest clonal structure was evident. Thus, N. gonorrhoeae, unlike pathogens so far examined, has a non-clonal population structure. As expected in a freely recombining population, no correlation was found between electrophoretic genotype and serovar or auxotype.

Genetic Linkage↗
PubMedDate not supplied

Effect of Cry3Bb transgenic corn and tefluthrin on the soil microbial community: biomass, activity, and diversity.

Transgenic Bt corn expressing the Cry3Bb insecticidal protein active against corn rootworm (CRW) (Diabrotica spp.; Coleoptera: Chrysomelidae) was released for commercial use in 2003 and is expected to be widely adopted. Yet, the direct and indirect risks to soil microorganisms of growing this CRW-resistant Bt corn versus applying insecticides to control the rootworm have not been assessed under field conditions. The effects of CRW Bt corn and the insecticide tefluthrin [2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1RS)-cis-3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate] on soil microbial biomass, activity (N mineralization potential, short-term nitrification rate, and soil respiration), and bacterial community structure as determined by terminal restriction fragment length polymorphism (T-RFLP) analysis were assessed over two seasons in a field experiment. Bt corn had no deleterious effects on microbial activity or bacterial community measures compared with the non-transgenic isoline. The T-RFLP analysis indicated that amplifiable bacterial species composition and relative abundance differed substantially between years, but did not differ between rhizosphere and bulk soils. The application of tefluthrin also had no effect on any microbial measure except decreased soil respiration observed in tefluthrin-treated plots compared with Bt and non-transgenic isoline (NoBt) plots in 2002. Our results indicate that the release of CRW Bt corn poses little threat to the ecology of the soil microbial community based on parameters measured in this study.

Animals↗
PubMed1981

Conformational transitions in Pf3 and their implications for the structure and assembly of filamentous bacterial viruses.

Laser Raman and circular dichroism spectra of filamentous bacteriophage Pf3 show that its coat protein is predominantly alpha-helical, similar to the subunits of bacteriophages Pf1 and fd. Unlike Pf1 and fd, however, the subunits of Pf3 are converted to beta-sheet structures by raising the temperature, the transition temperature depending upon phage and NaCl concentrations. On cooling, the beta structure reverts to an alpha structure the same as or similar to the native structure. On further heating it converts irreversibly to a second alpha-helical form different from the original one. The spectra also show that aromatic amino acid residues of Pf3 undergo dramatic changes in molecular environment during the alpha leads to beta transition. Similar transitions are observed to take place in the filamentous bacteriophage Xf.

Bacteriophages↗
PubMed2005

Cell selectivity and mechanism of action of antimicrobial model peptides containing peptoid residues.

To develop a useful method for designing cell-selective antimicrobial peptides and to investigate the effect of incorporating peptoid residues into an alpha-helical model peptide on structure, function, and mode of action, we synthesized a series of model peptides incorporating Nala (Ala-peptoid) into different positions of an amphipathic alpha-helical model peptide (KLW). Incorporation of one or two Nala residues into the hydrophobic helix face of KLW was more effective at disrupting the alpha-helical structure and bacterial cell selectivity than incorporation into the hydrophilic helix face or hydrophobic/hydrophilic interface. Tryptophan fluorescence studies of peptide interaction with model membranes indicated that the cell selectivity of KLW-L9-a and KLW-L9,13-a is closely correlated with their selective interactions with negatively charged phospholipids. KLW-L9,13-a, which has two Nala residues in its hydrophobic helix face, showed a random structure in membrane-mimicking conditions. KLW-L9,13-a exhibited the highest selectivity toward bacterial cells, showing no hemolytic activity and no or less cytotoxicity compared with other peptides against four mammalian cell lines. Unlike other model peptides, KLW-L9,13-a caused no or little membrane depolarization in Staphylococcus aureus or lipid flip-flop in negatively charged vesicles. In addition, KLW-L9,13-a caused very little fluorescent dye leakage from negatively charged vesicles. Furthermore, confocal laser-scanning microscopy and DNA-binding assays showed that KLW-L9,13-a probably exerts its antibacterial action by penetrating the bacterial membrane and binding to cytoplasmic compounds (e.g., DNA), resulting in cell death. Collectively, our results demonstrate that the incorporation of two Nala residues into the central position of the hydrophobic helix face of noncell-selective alpha-helical peptides is a promising strategy for the rational design of intracellular, cell-selective antimicrobial peptides.

Amino Acid Sequence↗
PubMed1993

Eubacterium yurii subspecies margaretiae is resistant to nonopsonic phagocytic ingestion.

We have previously shown that strains of Eubacterium yurii are hydrophobic, as compared with human polymorphonuclear leukocytes (PMNs), possibly because of a crystalline surface layer (S-layer) covering the cell envelope of this potential endo-perio pathogen. The aim of the present study was to investigate the phagocytic ingestion by PMNs of the three E. yurii subspecies, with special attention to bacterial surface structures and hydrophobicity. Type strains of subspp. margaretiae, yurii, and schtitka, together with three clinical isolates from necrotic root canals, were studied. All strains were hydrophobic when tested by a two-phase partition method. E. yurii subspp. margaretiae strains ATCC43715T, ES4C, and ES14B-8E were resistant to PMN ingestion in the absence of opsonins, whereas strains of the two other subspecies were readily ingested. The presence of a resistant strain (subsp. margaretiae ATCC43715T) did not inhibit the ingestion of a sensitive strain (subsp. schtitka ATCC43716T). Ingestion of E. yurii subsp. margaretiae strains required opsonization by normal human serum or specific antibodies. Electron microscopy revealed an S-layer in all strains and fimbria-like structures in the subspp. margaretiae and yurii strains. The antiserum prepared against the S-protein of E. yurii subsp. margaretiae ATCC43715T showed only slight cross-reactivity with other E. yurii strains and indicated the presence of strain-specific rather than species- or subspecies-specific antigens in the S-protein of E. yurii subsp. margaretiae ATCC43715T. The results suggest that the mere presence of the S-layer or fimbria-like structures cannot explain the susceptibility to ingestion by the PMNs.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkanes↗
PubMed2003

Protistan grazing analysis by flow cytometry using prey labeled by in vivo expression of fluorescent proteins.

Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.

Animals↗