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The biphenyl- and 4-chlorobiphenyl-catabolic transposon Tn4371, a member of a new family of genomic islands related to IncP and Ti plasmids.

The nucleotide sequence of the biphenyl catabolic transposon Tn4371 has been completed and analyzed. It confirmed that the element has a mosaic structure made of several building blocks. In addition to previously identified genes coding for a tyrosine recombinase related to phage integrases and for biphenyl degradation enzymes very similar to those of Achromobacter georgiopolitanum KKS102, Tn4371 carries many plasmid-related genes involved in replication, partition, and other, as-yet-unknown, plasmid functions. One gene cluster contains most of the genes required to express a type IV secretion-mating pair formation apparatus coupled with a TraG ATPase, all of which are related to those found on IncP and Ti plasmids. Orthologues of all Tn4371 plasmid-related genes and of the tyrosine recombinase gene were found, with a very similar organization, in the chromosome of Ralstonia solanacearum and on the yet-to-be-determined genomic sequences of Erwinia chrysanthemi and Azotobacter vinelandii. In each of these chromosomal segments, conserved segments were separated by different groups of genes, which also differed from the Tn4371 bph genes. The conserved blocks of genes were also identified, in at least two copies, in the chromosome of Ralstonia metallidurans CH34. Tn4371 thus appears to represent a new family of potentially mobile genomic islands with a broad host range since they reside in a wide range of soil proteobacteria, including plant pathogens.

Base Sequence↗

Exposure of sink drain microcosms to triclosan: population dynamics and antimicrobial susceptibility.

Recent concern that the increased use of triclosan (TCS) in consumer products may contribute to the emergence of antibiotic resistance has led us to examine the effects of TCS dosing on domestic-drain biofilm microcosms. TCS-containing domestic detergent (TCSD) markedly lowered biofouling at 50% (wt/vol) but was poorly effective at use levels. Long-term microcosms were established and stabilized for 6 months before one was subjected to successive 3-month exposures to TCSD at sublethal concentrations (0.2 and 0.4% [wt/vol]). Culturable bacteria were identified by 16S rDNA sequence analysis, and their susceptibilities to four biocides and six antibiotics were determined. Microcosms harbored ca. 10 log(10) CFU/g of biofilm, representing at least 27 species, mainly gamma proteobacteria, and maintained dynamic stability. Viable cell counts were largely unaffected by TCSD exposure, but species diversity was decreased, as corroborated by denaturing gradient gel electrophoresis analysis. TCS susceptibilities ranged widely within bacterial groups, and TCS-tolerant strains (including aeromonads, pseudomonads, stenotrophomonads, and Alcaligenes spp.) were isolated before and after TCSD exposure. Several TCS-tolerant bacteria related to Achromobacter xylosoxidans became clonally expanded during dosing. TCSD addition did not significantly affect the community profiles of susceptibility to the test biocides or antibiotics. Several microcosm isolates, as well as reference bacteria, caused clearing of particulate TCS in solid media. Incubations of consortia and isolates with particulate TCS in liquid led to putative TCS degradation by the consortia and TCS solubilization by the reference strains. Our results support the view that low-level exposure of environmental microcosms to TCS does not affect antimicrobial susceptibility and that TCS is degradable by common domestic biofilms.

Bacteria↗

Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creosote-contaminated soil.

Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the alpha-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the gamma-Proteobacteria group (genus Xanthomonas), the alpha-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the gamma-Proteobacteria group (genus Xathomonas), the beta-Proteobacteria group (genera Alcaligenes and Achromobacter), and the alpha-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.

Bacteria↗

Application of replica plating and computer analysis for rapid identification of bacteria in some foods. II. Analysis of microbial flora in irradiated Dover sole (Microstomus pacificus).

This investigation was carried out to determine the nature of the microbial flora shifts in dover sole as a result of irradiation and storage at 6 C. The relationship was determined between the microorganisms which initially survive irradiation and those making up the final spoilage flora. A total of 2,723 isolates were examined by use of the replica-plating and computer analysis method. The spoilage of the unirradiated control samples during storage at 6 C was almost entirely due to the growth of Pseudomonas. This group, which occupied 25% of the fresh flora, grew to nearly 100% in 2 days of storage. In contrast, irradiation doses of 0.1, 0.2, 0.3, and 0.4 Mrad favored the growth of Achromobacter and yeasts. The Micrococcus, which survived radiation, did not grow at 6 C. At 0.5 Mrad, spoilage of fish samples was due entirely to the growth of yeasts.

Alcaligenes↗

Bacteria responsible for the retting of Brazilian flax.

Twenty-two species of bacteria were isolated from Linum usitatissimum stored for retting. Achromobacter parvulus, Clostridium beijerinckii, C. saprogenes, C. saccharoacetoperbutylicum, C. perenne, and Pseudomonas aeruginosa and its achromogenic variety are retting agents. The last species mentioned performs the retting in only 72 hr. This is the first time A. parvulus has been shown to be a retting agent.

Bacteria↗

Comparative effects of chlortetracycline, freezing, and gamma radiation on microbial population of ocean perch.

The microbial populations in chlortetracycline (CTC)-treated (50, 100, 200, and 500 ppm), frozen (-15 C), and irradiated (0.1 Mrad) ocean perch (Sebastodes alutus) were compared. The control sample spoiled at 7 C, primarily because of the growth of Pseudomonas. Irradiation changed this to Achromobacter-dominated spoilage. Freezing or CTC treatment altered the spoilage pattern very little. CTC was particularly effective against ultraviolet fluorescent Pseudomonas species at the higher concentrations. Freezing and CTC were not effective against "coryneforms."

Alcaligenes↗

Selection of microbial population in chlortetracycline-treated ocean perch (Sebastodes alutus).

Microorganisms isolated from chlortetracycline (CTC)-treated ocean perch were individually tested to determine their relative sensitivity to CTC by use of CTC-impregnated paper discs. The CTC sensitivity varied among isolates obtained from untreated fish. The proportion of CTC-resistant species increased with higher CTC concentration and the length of storage at 7 C. Among individual generic groups, "coryneforms" and yeasts were more resistant to CTC than other groups. Pseudomonas, Achromobacter, Flavobacterium, Bacillus, and Lactobacillus all contained species either resistant or sensitive to CTC. More CTC-resistant species in these genera accumulated with the increased CTC concentrations and with the length of storage in the presence of CTC.

Alcaligenes↗

Properties of Pseudomonas enalia, a marine bacterium pathogenic for the invertebrate Crassostrea gigas (Thunberg).

Bacteriological investigations of dead and dying oysters in populations of Crassostrea gigas grown in Hood Canal, Oyster Bay, and Willapa Bay, Washington, were undertaken. Living, and presumably normal, oysters within the same sample set were also examined. Results indicated that the natural flora of Crassostrea gigas (Thunberg) is composed of organisms representing the genera Pseudomonas, Achromobacter, Flavobacterium, and Vibrio. Pollution indicator organisms such as Escherichia coli were not found. The flora of dead or dying oysters included a somewhat greater incidence of Pseudomonas sp.; a seawater-requiring organism isolated on several occasions from oyster gapers which had been collected from different geographical areas was identified as P. enalia. A description of the organism has been provided, and the characteristics are listed to facilitate identification by other workers encountering the organism in future studies of a similar kind. The seawater requirement exhibited by P. enalia was deduced to be a requirement for sodium chloride for growth of the organism. Experiments to determine the pathogenicity of Pseudomonas enalia were performed by use of experimentally infected animals maintained in aerated seawater tanks. Death of C. gigas occurred when the animal body tissue was injected with viable bacterial cell suspension. Results of histological studies of the normal and infected oyster tissue suggest that bacterial invasion of the tissue occurred.

Alcaligenes↗

Roles of microorganisms in the deterioration of rockfish.

Seven species of microorganisms indigenous to fish were inoculated into sterile fish muscle, obtained from Sebastodes alutus and Sebastalobus alascanus, and the production of total volatile acids (TVA) and total volatile bases (TVB) was determined during storage at 1 C. No production of TVA and TVB was noted in sterile fish, fish inoculated with Pseudomonas type III species (PIII-985), or fish inoculated with Flavobacterium (F-1070). The inactivity of PIII-985 was due to its low maximal growth level, whereas the inactivity of F-1070 was due to its slow growth rate. Pseudomonas type I species (PI-950), Pseudomonas type II species (PII-952), and two Achromobacter (Acinetobacter) species (A-981 and A-70) grew and actively produced TVA and TVB. TVB levels increased steadily even after the microbial number reached a maximum, whereas TVA levels declined toward the end of the storage period. It appeared that a minimal microbial growth level of 10(8) cells/g was needed before detectable levels of TVA or TVB could be produced. Regardless of genera, microorganisms which grew well in fish were able to produce TVA and TVB.

Alcaligenes↗

Effect of nitrofurans and chlortetracycline on microorganisms associated with shrimp.

Nitrofuran AF-2 displayed greater inhibitory effect than did nitrofuran Z when a mixed bacterial culture, including several proteolytic bacteria, isolated from shrimp was subjected to these compounds in vitro. Nitrofuran Z exhibited greater bactericidal properties than did chlortetracycline in all cultures used. Only 10 mug of nitrofuran AF-2 per ml was sufficient to inhibit the growth of mixed bacteria in nutrient broth, whereas 50 mug of nitrofuran Z per ml was necessary to accomplish the same inhibition. A 50-mug amount of chlortetracycline per ml displayed about the same inhibitory effect as either 10 mug of AF-2 per ml or 20 mug of Z per ml. The isolated proteolytic bacteria showed greater suppression of growth when subjected to AF-2 than when subjected to Z; however, both nitrofurans were effective in preventing growth. The addition of either 1 mug of AF-2 per ml or 5 mug of Z per ml to nutrient broth inhibited the growth of Achromobacter aquarmarinus, whereas chlortetracycline was less effective, requiring about 20 mug to suppress growth to the same degree.

Alcaligenes↗

Almond harvesting, processing, and microbial flora.

This survey was set up on a statistical sampling plan to determine the microbial quality of almonds as they are received at the processing plant. The total aerobic bacterial count and yeast and mold count distribution were skewed by a few high counts compared with the majority of relatively low counts. Hard shell varieties of almonds had lower counts than did soft shell, and almonds with complete shells had lower counts than shelled almonds. Almonds harvested onto canvas had lower counts than those harvested by knocking onto the ground. Nuts with the least amounts of foreign material mixed into the sample had the lowest counts, as did nuts with the least amount of insect damage. Coliforms, Escherichia coli, and Streptococcus were isolated from the nuts, and their presence was correlated with soil contamination. When almonds are stored, the total plate count, the Streptococcus count, and the E. coli count after an initial drop remain nearly constant for more than 3 months. In addition to the indicator organisms, several genera of bacteria were isolated including Bacillus, Xanthomonas, Achromobacter, Pseudomonas, Micrococcus or Staphylococcus, and Brevibacterium.

Bacteria↗

Characterization of radiation-resistant vegetative bacteria in beef.

Ground beef contains numerous microorganisms of various types. The commonly recognized bacteria are associated with current problems of spoilage. Irradiation, however, contributes a new factor through selective destruction of the microflora. The residual microorganisms surviving a nonsterilizing dose are predominantly gram-negative coccobacilli. Various classifications have been given, e.g., Moraxella, Acinetobacter, Achromobacter, etc. For a more detailed study of these radiation-resistant bacteria occurring in ground beef, an enrichment procedure was used for isolation. By means of morphological and biochemical tests, most of the isolates were found to be Moraxella, based on current classifications. The range of growth temperatures was from 2 to 50 C. These bacteria were relatively heat sensitive, e.g., D10 of 5.4 min at 70 C or less. The radiation resistance ranged from D10 values of 273 to 2,039 krad. Thus, some were more resistant than any presently recognized spores. A reference culture of Moraxella osloensis was irradiated under conditions comparable to the enrichment procedure used with the ground beef. The only apparent changes were in morphology and penicillin sensitivity. However, after a few subcultures these bacteria reverted to the characteristics of the parent strain. Thus, it is apparent that these isolates are a part of the normal flora of ground beef and not aberrant forms arising from the irradiation procedure. The significance, if any, of these bacteria is not presently recognized.

Dose-Response Relationship, Radiation↗

Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients.

Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescent-phase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methyl-accepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and IlvC), secretion (TatB and plasmid Achromobacter secretion [PAS] factor), transcriptional activation (IlvY and HlyU), and the activity of a putative lipoprotein (YaeC). In addition, one identified open reading frame encoded a hypothetical protein. Isogenic mutants of the 12 in vivo-expressed (ive) genes were constructed and tested for cytotoxicity. Cytotoxic activity of the mutant strains, as measured by lactate dehydrogenase release from HeLa cells, was nearly abolished in pyrH, purH, and hlyU mutants. The intraperitoneal 50% lethal dose in mice increased by ca. 10- to 50-fold in these three mutants. PyrH and PurH seem to be essential for in vivo growth. HlyU appears to be one of the master regulators of in vivo virulence expression. The successful identification of ive genes responsible for the in vivo bacterial virulence, as done in the present study, demonstrates the usefulness of IVIAT for the detection of new virulence genes.

Animals↗

Polyphasic taxonomy of the genus vibrio: numerical taxonomy of Vibrio cholerae, Vibrio parahaemolyticus, and related Vibrio species.

A set of 86 bacterial cultures, including 30 strains of Vibrio cholerae, 35 strains of V. parahaemolyticus, and 21 representative strains of Pseudomonas, Spirillum, Achromobacter, Arthrobacter, and marine Vibrio species were tested for a total of 200 characteristics. Morphological, physiological, and biochemical characteristics were included in the analysis. Overall deoxyribonucleic acid (DNA) base compositions and ultrastructure, under the electron microscope, were also examined. The taxonomic data were analyzed by computer by using numerical taxonomy programs designed to sort and cluster strains related phenetically. The V. cholerae strains formed an homogeneous cluster, sharing overall S values of >/=75%. Two strains, V. cholerae NCTC 30 and NCTC 8042, did not fall into the V. cholerae species group when tested by the hypothetical median organism calculation. No separation of "classic" V. cholerae, El Tor vibrios, and nonagglutinable vibrios was observed. These all fell into a single, relatively homogeneous, V. cholerae species cluster. V. parahaemolyticus strains, excepting 5144, 5146, and 5162, designated members of the species V. alginolyticus, clustered at S >/=80%. Characteristics uniformly present in all the Vibrio species examined are given, as are also characteristics and frequency of occurrence for V. cholerae and V. parahaemolyticus. The clusters formed in the numerical taxonomy analyses revealed similar overall DNA base compositions, with the range for the Vibrio species of 40 to 48% guanine plus cytosine. Generic level of relationship of V. cholerae and V. parahaemolyticus is considered dubious. Intra- and intergroup relationships obtained from the numerical taxonomy studies showed highly significant correlation with DNA/DNA reassociation data.

Alcaligenes↗

Occurrence of taurine: -ketoglutarate aminotransferase in bacterial extracts.

High activity of taurine:alpha-ketoglutarate aminotransferase was found exclusively in cell-free extracts of Achromobacter superficialis and A. polymorph. The former was chosen for characterization of the enzymatic reaction. The enzyme activity was enhanced by addition of beta-alanine to the growth medium. The product from alpha-ketoglutarate was identified as l-glutamate. Another product has been isolated, purified, and identified as sulfoacetaldehyde (2-oxoethanesulfonate), a deamination product from taurine, by comparison between the 2,4-dinitrophenylhydrazones of the synthetic and enzymatic products on the basis of studies by paper chromatography, by visible, infrared, and nuclear magnetic resonance spectrophotometries, and by elemental analysis. This enzymatic transamination was found to proceed stoichiometrically and reversibly as follows: NH(2).CH(2).CH(2).SO(3)H + HOOC.CH(2).CH(2).CO.COOH right harpoon over left harpoon OHC.CH(2).SO(3)H + HOOC.CH(2).CH(2).CH(NH(2)).COOH.

Alanine↗

Nitric oxide-dependent proton translocation in various denitrifiers.

Respiration of NO resulted in transient proton translocation in anaerobically grown cells of four physiologically diverse denitrifiers. Paracoccus denitrificans, Rhodopseudomonas sphaeroides subsp. denitrificans, "Achromobacter cycloclastes," and Rhizobium japonicum gave, respectively, H+/NO ratios of 3.65, 4.96, 1.94, and 1.12. Antimycin A completely inhibited NO-dependent proton translocation in P. denitrificans and severely restricted translocation in the R. sphaeroides strain. Proton uptake during NO respiration with antimycin A-inhibited cells supplied with an artificial electron source provided evidence for the periplasmic consumption of protons. Values obtained were consistent with the expected ratios of 0.5 mol of H+/mol of NO for reduction of NO to N2O and 1.0 mol of H+/mol of NO for reduction of NO to N2. These data are consistent with the presence of a unique NO reductase found only in anaerobically grown denitrifying cells.

Alcaligenes↗

Molecular relationship of chromosomal genes encoding biphenyl/polychlorinated biphenyl catabolism: some soil bacteria possess a highly conserved bph operon.

All the genes we examined that encoded biphenyl/polychlorinated biphenyl (PCB) degradation were chromosomal, unlike many other degradation-encoding genes, which are plasmid borne. The molecular relationship of genes coding for biphenyl/PCB catabolism in various biphenyl/PCB-degrading Pseudomonas, Achromobacter, Alcaligenes, Moraxella, and Arthrobacter strains was investigated. Among 15 strains tested, 5 Pseudomonas strains and one Alcaligenes strain possessed the bphABC gene cluster on the XhoI 7.2-kilobase fragment corresponding to that of Pseudomonas pseudoalcaligenes KF707. More importantly, the restriction profiles of these XhoI 7.2-kilobase fragments containing bphABC genes were very similar, if not identical, despite the dissimilarity of the flanking chromosomal regions. Three other strains also possessed bphABC genes homologous with those of KF707, and five other strains showed weak or no significant genetic homology with bphABC of KF707. The immunological cross-reactivity of 2,3-dihydroxybiphenyl dioxygenases from various strains corresponded well to the DNA homology. On the other hand, the bphC gene of another PCB-degrading strain, Pseudomonas paucimobilis Q1, lacked genetic as well as immunological homology with any of the other 15 biphenyl/PCB degraders tested. The existence of the nearly identical chromosomal genes among various strains may suggest that a segment containing the bphABC genes has a mechanism for transferring the gene from one strain to another.

Bacteria↗

Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG.

Agrobacterium tumefaciens virulence genes are induced by plant signals through the VirA-VirG two-component regulatory system. The VirA protein is a membrane-spanning sensor molecule that possesses an autophosphorylating activity, and the VirG protein is a sequence-specific DNA-binding protein. In this report, we demonstrate that the VirG protein is phosphorylated by the VirA protein and that the phosphate is directly transferred from the phosphorylated VirA molecule (phosphohistidine) to the VirG protein. The chemical stability of the phospho-VirG bond suggested that the VirG protein was phosphorylated at the aspartate and/or glutamate residue. The phosphorylated VirG protein was reduced with tritiated sodium borohydride and subjected to proteolytic digestion with the Achromobacter protease I enzyme. The resulting peptide fragments were separated by C8 reversed-phase high-pressure liquid chromatography, and the tritium-labeled peptide was sequenced. Amino acid sequence data showed that the aspartate residue at position 52 was the only site phosphorylated. Changing this aspartate into asparagine resulted in a nonphosphorylatable and biologically nonfunctional gene product. As a control, a randomly chosen aspartate was changed into an asparagine (position 72), and no effect on its phosphorylation or biological activity was observed. Unlike its homologs, including CheA-CheY, EnvZ-OmpR, and NtrB-NtrC, the phospho-VirG molecule was very stable in vitro. The possible implications of these observations and the function of VirG phosphorylation in vir gene activation are discussed.

Aspartic Acid↗