bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Nitrate Availability and the Presence of Glyceria maxima on the Composition and Activity of the Dissimilatory Nitrate-Reducing Bacterial Community
Authors: Nijburg J W , Coolen M , Gerards S , Gunnewiek P , Laanbroek H J
Published: January 2020

The effects of nitrate availability and the presence of Glyceria maxima on the composition and activity of the dissimilatory nitrate-reducing bacterial community were studied in the laboratory. Four different concentrations of NO(inf3)(sup-), 0, 533, 1434, and 2,905 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), were added to pots containing freshwater sediment, and the pots were then incubated for a period of 69 days. Upon harvest, NH(inf4)(sup+) was not detectable in sediment that received 0 or 533 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1). Nitrate concentrations in these pots ranged from 0 to 8 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1) at harvest. In pots that received 1,434 or 2,905 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), final concentrations varied between 10 and 48 (mu)g of NH(inf4)(sup+)-N g of dry sediment(sup-1) and between 200 and 1,600 (mu)g of NO(inf3)(sup-)-N g of dry sediment(sup-1), respectively. Higher input levels of NO(inf3)(sup-) resulted in increased numbers of potential nitrate-reducing bacteria and higher potential nitrate-reducing activity in the rhizosphere. In sediment samples from the rhizosphere, the contribution of denitrification to the potential nitrate-reducing capacity varied from 8% under NO(inf3)(sup-)-limiting conditions to 58% when NO(inf3)(sup-) was in ample supply. In bulk sediment with excess NO(inf3)(sup-), this percentage was 44%. The nitrate-reducing community consisted almost entirely of NO(inf2)(sup-)-accumulating or NH(inf4)(sup+)-producing gram-positive species when NO(inf3)(sup-) was not added to the sediment. The addition of NO(inf3)(sup-) resulted in an increase of denitrifying Pseudomonas and Moraxella strains. The factor controlling the composition of the nitrate-reducing community when NO(inf3)(sup-) is limited is the presence of G. maxima. In sediment with excess NO(inf3)(sup-), nitrate availability determines the composition of the nitrate-reducing community.
Multiplication of different Legionella species in Mono Mac 6 cells and in Acanthamoeba castellanii
Authors: Neumeister B , Schöniger S , Faigle M , Eichner M , Dietz K
Published: January 2020

Survival and distribution of legionellae in the environment are assumed to be associated with their multiplication in amoebae, whereas the ability to multiply in macrophages is usually regarded to correspond to pathogenicity. Since most investigations focused on Legionella pneumophila serogroup 1, we examined the intracellular multiplication of different Legionella species in Mono Mac 6 cells, which express phenotypic and functional features of mature monocytes, and in Acanthamoeba castellanii, an environmental host of Legionella spp. According to the bacterial doubling time in Mono Mac 6 cells and in A. castellanii, seven clusters of legionellae could be defined which could be split further with regard to finer differences. L. longbeachae serogroup 1, L. jordanis, and L. anisa were not able to multiply in either A. castellanii or Mono Mac 6 cells and are members of the first cluster. L. dumoffi did not multiply in Mono Mac 6 cells but showed a delayed multiplication in A. castellanii 72 h after infection and is the only member of the second cluster. L. steigerwaltii, L. gormanii, L. pneumophila serogroup 6 ATCC 33215, L. bozemanii, and L. micdadei showed a stable bacterial count in Mono Mac 6 cells after infection but a decreasing count in amoebae. They can be regarded as members of the third cluster. As the only member of the fourth cluster, L. oakridgensis was able to multiply slight in Mono Mac 6 cells but was killed within amoebae. A strain of L. pneumophila serogroup 1 Philadelphia obtained after 30 passages on SMH agar and a strain of L. pneumophila serogroup 1 Philadelphia obtained after intraperitoneal growth in guinea pigs are members of the fifth cluster, which showed multiplication in Mono Mac 6 cells but a decrease of bacterial counts in A. castellanii. The sixth cluster is characterized by intracellular multiplication in both host cell systems and consists of several strains of L. pneumophila serogroup 1 Philadelphia, a strain of L. pneumophila serogroup 2, and a fresh clinical isolate of L. pneumophila serogroup 6. Members of the seventh cluster are a strain of agar-adapted L. pneumophila serogroup 1 Bellingham and a strain of L. pneumophila serogroup 1 Bellingham which was passaged fewer than three times on BCYE alpha agar after inoculation and intraperitoneal growth in guinea pigs. In comparison to members of the sixth cluster, both strains showed a slightly enhanced multiplication in Mono Mac 6 cells but a reduced multiplication in amoebae. From our investigations, we could demonstrate a correlation between prevalence of a given Legionella species and their intracellular multiplication in Mono Mac 6 cells. Multiplication of members of the genus Legionella in A. castellanii seems to be dependent on mechanisms different from those in monocytes.
Molecular detection of streptomycin-producing streptomycetes in Brazilian soils
Authors: Huddleston A S , Cresswell N , Neves M C , Beringer J E , Baumberg S , Thomas D I , Wellington E M
Published: January 2020

Actinomycetes were isolated from soybean rhizosphere soil collected as two field sites in Brazil. All the isolates were identified as Streptomyces species and were screened for streptomycin production and the presence of two genes, strA and strB1, known to be involved in streptomycin biosynthesis in Streptomyces griseus. Antibiotic resistance profiles were determined for 53 isolates from cultivated and uncultivated sites, and approximately half the strains were streptomycin resistance. Clustering by the unweighted pair group method with averages indicated the presence of two major clusters, with the majority of resistant strains from cultivated sites being placed in cluster 1. Only representatives from this cluster contained strA. Streptomycetes containing strA and strB1 were phenotypically diverse, and only half could be assigned to known species. Sequence comparison of 16S rRNA and trpBA (tryptophan synthetase) genes revealed that streptomycin- producing streptomycetes were phylogenetically diverse. It appeared that a population of streptomycetes had colonized the rhizosphere and that a proportion of these were capable of streptomycin production.
Variation in microbial community structure in two boreal peatlands as determined by analysis of phospholipid Fatty Acid profiles
Authors: Sundh I , Nilsson M , Borga P
Published: January 2020

Analyses of phospholipid fatty acids (PLFAs) were used to assess variation in community structure and total microbial biomass in two boreal peatlands in Sweden. The total PLFA concentration in peat ranged from 0.16 to 7.0 nmol g of wet peat(sup-1) (median, 0.70 nmol g of wet peat(sup-1)). Principal-component analysis of PLFA data revealed that the degree of depth-related variation in PLFA composition was high among peatland habitats, with general differences between wet sites, with water tables within a few centimeters of the moss surface, and dry sites, with water tables >10 cm below the moss surface. However, variation in PLFA composition over the growing season was negligible. In the principal-component analyses, most PLFAs were determined to be parts of clusters of covarying fatty acids, suggesting that they originated in the same functional groups of microorganisms. Major clusters were formed by monounsaturated (typical of gram-negative eubacteria), terminally branched (gram-positive or anaerobic gram-negative eubacteria), methyl-branched and branched unsaturated (sulfate-reducing bacteria and/or actinomycetes), (omega)8 monounsaturated (methane-oxidizing bacteria), and polyunsaturated (eucaryotes) PLFAs. Within the clusters, PLFAs had rather distinct concentration-depth distributions. For example, PLFAs from sulfate-reducing bacteria and/or actinomycetes and those from methane-oxidizing bacteria had maximum concentrations slightly below and at the average water table depth, respectively.
Survival and phospholipid Fatty Acid profiles of surface and subsurface bacteria in natural sediment microcosms
Authors: Kieft T L , Wilch E , O'connor K , Ringelberg D B , White D C
Published: January 2020

Although starvation survival has been characterized for many bacteria, few subsurface bacteria have been tested, and few if any have been tested in natural subsurface porous media. We hypothesized that subsurface bacteria may be uniquely adapted for long-term survival in situ. We further hypothesized that subsurface conditions (sediment type and moisture content) would influence microbial survival. We compared starvation survival capabilities of surface and subsurface strains of Pseudomonas fluorescens and a novel Arthrobacter sp. in microcosms composed of natural sediments. Bacteria were incubated for up to 64 weeks under saturated and unsaturated conditions in sterilized microcosms containing either a silty sand paleosol (buried soil) or a sandy silt nonpaleosol sediment. Direct counts, plate counts, and cell sizes were measured. Membrane phospholipid fatty acid (PLFA) profiles were quantified to determine temporal patterns of PLFA stress signatures and differences in PLFAs among strains and treatments. The Arthrobacter strains survived better than the P. fluorescens strains; however, differences in survival between surface and subsurface strains of each genus were not significant. Bacteria survived better in the paleosol than in the nonpaleosol and survived better under saturated conditions than under unsaturated conditions. Cell volumes of all strains decreased; however, sediment type and moisture did not influence rates of miniaturization. Both P. fluorescens strains showed PLFA stress signatures typical for gram-negative bacteria: increased ratios of saturated to unsaturated fatty acids, increased ratios of trans- to cis-monoenoic fatty acids, and increased ratios of cyclopropyl to monoenoic precursor fatty acids. The Arthrobacter strains showed few changes in PLFAs. Environmental conditions strongly influenced PLFA profiles.
Influence of water chlorination on the counting of bacteria with DAPI (4',6-diamidino-2-phenylindole)
Authors: Saby S , Sibille I , Mathieu L , Paquin J L , Block J C
Published: January 2020

Counting bacteria in drinking water samples by the epifluorescence technique after 4',6-diamidino-2-phenylindole (DAPI) staining is complicated by the fact that bacterial fluorescence varies with exposure of the cells to sodium hypochlorite. An Escherichia coli laboratory-grown suspension treated with sodium hypochlorite (5 to 15 mg of chlorine liter-1) for 90 min was highly fluorescent after DAPI staining probably due to cell membrane permeation and better and DAPI diffusion. At chlorine concentrations greater than 25 mg liter-1, DAPI-stained bacteria had only a low fluorescence. Stronger chlorine doses altered the DNA structure, preventing the DAPI from complexing with the DNA. When calf thymus DNA was exposed to sodium hypochlorite (from 15 to 50 mg of chlorine liter-1 for 90 min), the DNA lost the ability to complex with DAPI. Exposure to monochloramine did not have a similar effect. Treatment of drinking water with sodium hypochlorite (about 0.5 mg of chlorine liter-1) caused a significant increase in the percentage of poorly fluorescent bacteria, from 5% in unchlorinated waters (40 samples), to 35 to 39% in chlorinated waters (40 samples). The presence of the poorly fluorescent bacteria could explain the underestimation of the real number of bacteria after DAPI staining. Microscopic counting of both poorly and highly fluorescent bacteria is essential under these conditions to obtain the total number of bacteria. A similar effect of chlorination on acridine orange-stained bacteria was observed in treated drinking waters. The presence of the poorly fluorescent bacteria after DAPI staining could be interpreted as a sign of dead cells.
On-farm monitoring of mouse-invasive Salmonella enterica serovar enteritidis and a model for its association with the production of contaminated eggs
Authors: Guard-Petter J , Henzler D J , Rahman M M , Carlson R W
Published: January 2020

Mice (Mus musculus) captured in henhouses were assessed for the presence of salmonellae in spleens. Of 621 and 526 spleens cultured during the first and second years of collection, 25.0 and 17.9%, respectively, were positive for Salmonella enterica serovar Enteritidis. Contaminated eggs were cultured from nine houses during the first year of sampling, and for eight of these houses, serovar Enteritidis was recovered from the spleens of mice. Rank sum statistical analysis of positive mouse spleens indicated that three overlapping bacterial populations were present. This pattern of infection was repeated when lipopolysaccharide (LPS) variants were used to infect chicks, and the worst infections were associated with isolates producing high-molecular-weight (HMW) LPS. Mouse isolates were capable of producing unprecedented amounts of HMW LPS as indicated by compositional analysis of six isolates that swarmed across 2% agar, which is a type of bacterial migration dependent upon production of HMW LPS. It is suggested that serovar Enteritidis cultured from the spleens of mice caught on farms will detect strains that are enhanced in their ability to contaminate eggs, in part because they are able to produce HMW LPS.
Sporulation of Cyclospora sp. oocysts
Authors: Smith H V , Paton C A , Mitambo M M , Girdwood R W
Published: January 2020

Cyclospora sp. oocysts sporulated maximally at 22 and 30 degrees C for 14 days retarded sporulation. Up to 12% of human- and baboon-derived oocysts previously stored at 4 degrees C for 1 to 2 months sporulated when stored for 6 to 7 days at 30 degrees C.
Isolation and characterization of two new homoacetogenic hydrogen-utilizing bacteria from the human intestinal tract that are closely related to Clostridium coccoides
Authors: Kamlage B , Gruhl B , Blaut M
Published: January 2020

Two gram-positive, strictly anoxic, coccoid- to rod-shaped strains of bacteria, Clostridium coccoides 1410 and C. coccoides 3110, were isolated from human feces on the typical homoacetogenic substrates formate plus H2 plus CO2 (strain 1410) and vanillate plus H2 plus CO2 (strain 3110) in the presence of 2-bromoethanesulfonate to inhibit methanogenesis. On the basis of 16S rRNA sequencing, DNA-DNA hybridization, and physiological and morphological parameters, both isolates are closely related to C. coccoides DSM 935T. The G+C contents of the DNA were 46.1 and 46.2 mol% for C. coccoides 1410 and C. coccoides 3110, respectively. Cytochromes could not be detected. Formate was degraded exclusively to acetate, whereas vanillate was O-demethylated, resulting in acetate and 3,4-dihydroxybenzoate, the latter being further decarboxylated to catechol. In the presence of organic substrates, H2 was cometabolized to acetate, but both strains failed to grow autotrophically. Lactose, lactulose, sorbitol, glucose, and various other carbohydrates supported growth as well. Untypical of homoacetogens, glucose and sorbitol were fermented not exclusively to acetate; instead, considerable amounts of succinate and D-lactate were produced. H2 was evolved from carbohydrates only in negligible traces. Acetogenesis from formate plus H2 plus CO2 or vanillate plus H2 plus CO2 was constitutive, whereas utilization of carbohydrates was inducible. Hydrogenase, CO dehydrogenase, formate dehydrogenase, and all of the tetrahydrofolic acid-dependent, C1 compound-converting enzymes of the acetyl-coenzyme A pathway of homoacetogenesis were present in cell extracts.
Molecular epidemiological survey of rotaviruses in sewage by reverse transcriptase seminested PCR and restriction fragment length polymorphism assay
Authors: Dubois E , Le Guyader F , Haugarreau L , Kopecka H , Cormier M , Pommepuy M
Published: January 2020

Rotavirus double-stranded RNA was detected directly in sewage treatment plant samples over a 1-year period by reverse transcription followed by PCR amplification of the VP7 gene and Southern blot hybridization. The presence of naturally occurring rotaviruses was demonstrated in 42% of raw sewage samples and in 67% of treated effluent samples. Amplified viral sequences were analyzed by restriction enzymes. Ten different restriction profiles were characterized, most of which were found in treated effluent samples. A mixture of restriction profiles was observed in 75% of contaminated effluent samples. The profiles were compared with those obtained from human rotavirus isolates involved in infections in children from the same area (six different profiles were detected). Five identical viral sequences were detected in both environmental and clinical samples. Restriction profiles were also compared to profiles from known genomic sequences of human and animal viruses. Both human and animal origins of rotavirus contamination of water seemed likely.
Hydrogenases in Nostoc sp. Strain PCC 73102, a Strain Lacking a Bidirectional Enzyme
Authors: Tamagnini P , Troshina O , Oxelfelt F , Salema R , Lindblad P
Published: January 2020

The present study was carried out in order to examine and characterize the bidirectional hydrogenase in the cyanobacterium Nostoc sp. strain PCC 73102. Southern hybridizations with the probes Av1 and Av3 (hoxY and hoxH, bidirectional hydrogenase small and large subunits, respectively) revealed the occurrence of corresponding sequences in Anabaena variabilis (control), Anabaena sp. strain PCC 7120, and Nostoc muscorum but not in Nostoc sp. strain PCC 73102. As a control, hybridizations with the probe hup2 (hupL, uptake hydrogenase large subunit) demonstrated the presence of a corresponding gene in all the cyanobacteria tested, including Nostoc sp. strain PCC 73102. Moreover, with three different growth media, a bidirectional enzyme that was functional in vivo was observed in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis, whereas Nostoc sp. strain PCC 73102 consistently lacked any detectable in vivo activity. Similar results were obtained when assaying for the presence of an enzyme that is functional in vitro. Native polyacrylamide gel electrophoresis followed by in situ hydrogenase activity staining was used to demonstrate the presence or absence of a functional enzyme. Again, bands corresponding to hydrogenase activity were observed for N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis but not for Nostoc sp. strain PCC 73102. In conclusion, we were unable to detect a bidirectional hydrogenase in Nostoc sp. strain PCC 73102 with specific physiological and molecular techniques. The same techniques clearly showed the presence of an inducible bidirectional enzyme and corresponding structural genes in N. muscorum, Anabaena sp. strain PCC 7120, and A. variabilis. Hence, Nostoc sp. strain PCC 73102 seems to be an unusual cyanobacterium and an interesting candidate for future biotechnological applications.
Production of Fumonisin B(inf1) and Moniliformin by Gibberella fujikuroi from Rice from Various Geographic Areas
Authors: Desjardins A E , Plattner R D , Nelson P E
Published: January 2020

Gibberella fujikuroi strains isolated from rice in the United States, Asia, and other geographic areas were tested for sexual fertility with members of mating population D and for production of fumonisin B(inf1) and moniliformin in culture. Of the 59 field strains tested, 32 (54%) were able to cross with tester strains of mating population D, but only a few ascospores were produced in most of these crosses. Thirty-four strains produced more than 10 (mu)g of fumonisin B(inf1) per g, but only three strains produced more than 1000 (mu)g/g. Twenty-five strains produced more than 100 (mu)g of moniliformin per g, and 15 produced more than 1,000 (mu)g/g. Seven field strains produced both fumonisin B(inf1) and moniliformin, but none of these strains produced a high level of fumonisin B(inf1) (>1,000 (mu)g/g). However, a genetic cross between a strain that produced fumonisin B(inf1) but no moniliformin and a strain that produced moniliformin but no fumonisin B(inf1) yielded progeny that produced high levels of both toxins. Strains of G. fujikuroi isolated from rice infected with bakanae disease are similar to strains of mating population D isolated from maize in their ability to produce both fumonisins and moniliformin. This finding suggests a potential for contamination of rice with both fumonisins and moniliformin.
Toluene Elicits a Carbon Starvation Response in Pseudomonas putida mt-2 Containing the TOL Plasmid pWW0
Authors: Vercellone-Smith P , Herson D S
Published: January 2020

Pseudomonas putida mt-2(pWWO) exhibited a carbon starvation response in the presence of toluene, a utilizable carbon source. When growth-supporting (4-mg/liter), inhibitory (130-mg/liter), and lethal (267-mg/ liter) levels of toluene were provided as the sole carbon source, P. putida responded by rapidly inhibiting protein synthesis and by producing 26 new proteins, 22 of which overlapped with those induced by carbon starvation. P. putida produced the same proteins when cultures were starved by depleting their carbon source or were downshifted into a carbon-free medium. Carbon supplementation of toluene-exposed cells suppressed the production of the toluene-induced proteins. The level of toluene provided as the sole carbon source influenced the length of time that this response was observed. Following 1.5 to 3 h in a basal salts medium with 4 mg of toluene per liter, protein synthesis increased, the production of the majority of the toluene-induced proteins ceased, and the cells began to grow. In cells provided with 130 mg of toluene per liter, protein synthesis remained inhibited over a 6.5-h experimental period. At this concentration, the production of 15 toluene-induced proteins was prolonged, with nine still detectable in the profiles at 6.5 h. In cells provided with 267 mg of toluene per liter, there was a rapid loss of viability and the toluene-induced proteins were detected prior to death. In cells provided with 4 mg of toluene per liter, the carbon starvation response is transient and likely reflects a period of induction and/or adaptation prior to growth on toluene. At the toluene concentrations which inhibit growth, P. putida exhibits a prolonged starvation response despite the presence of an excess of a utilizable carbon source.
Natural transformation and availability of transforming DNA to Acinetobacter calcoaceticus in soil microcosms
Authors: Nielsen K M , van Weerelt M D , Berg T N , Bones A M , Hagler A N , van Elsas J D
Published: January 2020

A small microcosm, based on optimized in vitro transformation conditions, was used to study the ecological factors affecting the transformation of Acinetobacter calcoaceticus BD413 in soil. The transforming DNA used was A. calcoaceticus homologous chromosomal DNA with an inserted gene cassette containing a kanamycin resistance gene, nptII. The effects of soil type (silt loam or loamy sand), bacterial cell density, time of residence of A. calcoaceticus or of DNA in soil before transformation, transformation period, and nutrient input were investigated. There were clear inhibitory effects of the soil matrix on transformation and DNA availability. A. calcoaceticus cells reached stationary phase and lost the ability to be transformed shortly after introduction into sterile soil. The use of an initially small number of A. calcoaceticus cells and nutrients, resulting in bacterial growth, enhanced transformation frequencies within a limited period. The availability of introduced DNA for transformation of A. calcoaceticus cells disappeared within a few hours in soil. Differences in transformation frequencies between soils were found; A. calcoaceticus cells were transformed at a higher rate and for a longer period in a silt loam than in a loamy sand. Physical separation of DNA and A. calcoaceticus cells had a negative effect on transformation. Transformation was also detected in nonsterile soil microcosms, albeit only in the presence of added nutrients and at a reduced frequency. These results suggest that chromosomal DNA released into soil rapidly becomes unavailable for transformation of A. calcoaceticus. In addition, strain BD413 quickly loses the ability to receive, stabilize, and/or express exogenous DNA after introduction into soil.
Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52
Authors: Fiorella P D , Spain J C
Published: January 2020

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.
Stereospecific oxidation of (R)- and (S)-1-indanol by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4
Authors: Lee K , Resnick S M , Gibson D T
Published: January 2020

A recombinant Escherichia coli strain which expresses naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized (S)-1-indanol to trans-(1S,3S)-indan-1,3-diol (95.5%) and (R)-3-hydroxy-1-indanone (4.5%). The same cells oxidized (R)-1-indanol to cis-1,3-indandiol (71%), (R)-3-hydroxy-1-indanone (18.2%), and cis-1,2,3-indantriol (10.8%). Purified NDO oxidized (S)-1-indenol to both syn- and anti-2,3-dihydroxy-1-indanol.
Molecular cloning and analysis of the ptsHI operon in Lactobacillus sake
Authors: Stentz R , Lauret R , Ehrlich S D , Morel-Deville F , Zagorec M
Published: January 2020

The ptsH and ptsI genes of Lactobacillus sake, encoding the general enzymes of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS), were cloned and sequenced. HPr (88 amino acids), encoded by ptsH, and enzyme I (574 amino acids), encoded by ptsI, are homologous to the corresponding known enzymes of other bacteria. Nucleotide sequence and mRNA analysis showed that the two genes are cotranscribed in a large transcript encoding both HPr and enzyme I. The transcription of ptsHI was shown to be independent of the carbon source. Four ptsI mutants were constructed by single-crossover recombination. For all mutants, growth on PTS carbohydrates was abolished. Surprisingly, the growth rates of mutants on ribose and arabinose, two carbohydrates which are not transported by the PTS, were accelerated. This unexpected phenotype suggests that the PTS negatively controls ribose and arabinose utilization in L. sake by a mechanism different from the regulation involving HPr described for other gram-positive bacteria.
In situ analysis of denitrifying toluene- and m-xylene-degrading bacteria in a diesel fuel-contaminated laboratory aquifer column
Authors: Hess A , Zarda B , Hahn D , Häner A , Stax D , Höhener P , Zeyer J
Published: January 2020

A diesel fuel-contaminated aquifer was bioremediated in situ by the injection of oxidants (O2 and NO3-) and nutrients in order to stimulate microbial activity. After 3.5 years of remediation, an aquifer sample was excavated and the material was used (i) to isolate bacterial strains able to grow on selected hydrocarbons under denitrifying conditions and (ii) to construct a laboratory aquifer column in order to simulate the aerobic and denitrifying remediation processes. Five bacterial strains isolated from the aquifer sample were able to grow on toluene (strains T2 to T4, T6, and T10), and nine bacterial strains grew on toluene and m-xylene (strains M3 to M7 and M9 to M12). Strains T2 to T4, T6, and T10 were cocci, and strains M3 to M7 and M9 to M12 were rods. The morphological and physiological differences were also reflected in small sequence variabilities in domain III of the 23S rRNA and in the 16S rRNA. Comparative sequence analyses of the 16S rRNA of one isolate (T3 and M3) of each group revealed a close phylogenetic relationship for both groups of isolates to organisms of the genus Azoarcus. Two 16S rRNA-targeted oligonucleotide probes (Azo644 and Azo1251) targeting the experimental isolates, bacteria of the Azoarcus tolulyticus group, and Azoarcus evansii were used to investigate the significance of hydrocarbon-degrading Azoarcus spp. in the laboratory aquifer column. The number of bacteria in the column determined after DAPI (4',6-diamidino-2-phenylindole) staining was 5.8 x 10(8) to 1.1 x 10(9) cells g of aquifer material-1. About 1% (in the anaerobic zone of the column) to 2% (in the aerobic zone of the column) of these bacteria were detectable by using a combination of probes Azo644 and Azo1251, demonstrating that hydrocarbon-degrading Azoarcus spp. are significant members of the indigenous microbiota. More than 90% of the total number of bacteria were detectable by using probes targeting higher phylogenetic groups. Approximately 80% of these bacteria belonged to the beta subdivision of the class Proteobacteria (beta-Proteobacteria), and 10 to 16% belonged to the gamma-Proteobacteria. Bacteria of the alpha-Proteobacteria were present in high numbers (10%) only in the aerobic zone of the column.
Activity of H(+)-ATPase in ruminal bacteria with special reference to acid tolerance
Authors: Miwa T , Esaki H , Umemori J , Hino T
Published: January 2020

Batch culture experiments showed that permeabilized cells and membranes of Ruminococcus albus and Fibrobacter succinogenes, acid-intolerant celluloytic bacteria, have only one-fourth to one-fifth as much H(+)-ATPase as Megasphaera elsdenii and Streptococcus bovis, which are relatively acid tolerant. Even in the cells grown in continuous culture at pH 7.0, the acid-intolerant bacteria contained less than half as much H(+)-ATPase as the acid-tolerant bacteria. The amounts of H(+)-ATPase in the acid-tolerant bacteria were increased by more than twofold when the cells were grown at the lowest pH permitting growth, whereas little increase was observed in the case of the acid-intolerant bacteria. These results indicate that the acid-intolerant bacteria not only contain smaller amounts of H(+)-ATPase at neutral pH but also have a lower capacity to enhance the level of H(+)-ATPase in response to low pH than the acid-tolerant bacteria. In addition, the H(+)-ATPases of the acid-intolerant bacteria were more sensitive to low pH than those of the acid-tolerant bacteria, although the optimal pHs were similar.
Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation
Authors: Muñoz C , Guillén F , Martínez A T , Martínez M J
Published: January 2020

Two laccase isoenzymes produced by Pleurotus eryngii were purified to electrophoretic homogeneity (42- and 43-fold) with an overall yield of 56.3%. Laccases I and II from this fungus are monomeric glycoproteins with 7 and 1% carbohydrate content, molecular masses (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 65 and 61 kDa, and pIs of 4.1 and 4.2, respectively. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for laccase I was reached at 65 degrees C and pH 4, and that for laccase II was reached at 55 degrees C and pH 3.5. Both isoenzymes are stable at high pH, retaining 60 to 70% activity after 24 h from pH 8 to 12. Their amino acid compositions and N-terminal sequences were determined, the latter strongly differing from those of laccases of other basidiomycetes. Antibodies against laccase I reacted with laccase II, as well as with laccases from Pleurotus ostreatus, Pleurotus pulmonarius, and Pleurotus floridanus. Different hydroxy- and methoxy-substituted phenols and aromatic amines were oxidized by the two laccase isoenzymes from P. eryngii, and the influence of the nature, number, and disposition of aromatic-ring substituents on kinetic constants is discussed. Although both isoenzymes presented similar substrate affinities, the maximum rates of reactions catalyzed by laccase I were higher than those of laccase II. In reactions with hydroquinones, semiquinones produced by laccase isoenzymes were in part converted into quinones via autoxidation. The superoxide anion radical produced in the latter reaction dismutated, producing hydrogen peroxide. In the presence of manganous ion, the superoxide union was reduced to hydrogen peroxide with the concomitant production of manganic ion. These results confirmed that laccase in the presence of hydroquinones can participate in the production of both reduced oxygen species and manganic ions.
Cloning and functional expression in Escherichia coli of the gene encoding the di- and tripeptide transport protein of Lactobacillus helveticus
Authors: Nakajima H , Hagting A , Kunji E R , Poolman B , Konings W N
Published: January 2020

The gene encoding the di- and tripeptide transport protein (DtpT) of Lactobacillus helveticus (DtpTLH) was cloned with the aid of the inverse PCR technique and used to complement the dipeptide transport-deficient and proline-auxotrophic Escherichia coli E1772. Functional expression of the peptide transporter was shown by the uptake of prolyl-[14C] alanine in whole cells and membrane vesicles. Peptide transport via DtpT in membrane vesicles is driven by the proton motive force. The system has specificity for di- and tripeptides but not for amino acids or tetrapeptides. The dtpTLH gene consists of 1,491 bp, which translates into a 497-amino-acid polypeptide. DtpTLH shows 34% identity to the di- and tripeptide transport protein of Lactococcus lactis and is also homologous to various peptide transporters of eukaryotic origin, but the similarity between these proteins is confined mainly to the N-terminal halves.
Cloning, sequencing, and overexpression of the Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase (pckA) gene
Authors: Laivenieks M , Vieille C , Zeikus J G
Published: January 2020

The phosphoenolpyruvate (PEP) carboxykinase-encoding gene from the anaerobic, CO2-fixing, succinate-producing bacterium Anaerobiospirillum succiniciproducens was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a 532-residue polypeptide with a calculated molecular mass of 58.7 kDa. The sequence of the A. succiniciproducens PEP carboxykinase was similar to those of all known ATP/ADP-dependent PEP carboxykinases. In particular, the A. succiniciproducens enzyme was 67.3% identical and 79.2% similar to the E. coli enzyme. The A. succiniciproducens pckA transcription start site was determined, and putative promoter regions were identified. The recombinant enzyme was overexpressed in E. coli. The purified enzyme was indiscernible from the native enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had the same activity as the native enzyme.
Natural horizontal transfer of a naphthalene dioxygenase gene between bacteria native to a coal tar-contaminated field site
Authors: Herrick J B , Stuart-Keil K G , Ghiorse W C , Madsen E L
Published: January 2020

Horizontal transfer of genes responsible for pollutant biodegradation may play a key role in the evolution of bacterial populations and the adaptation of microbial communities to environmental contaminants. However, field evidence for horizontal gene transfer between microorganisms has traditionally been very difficult to obtain. In this study, the sequences of the 16S rRNA and naphthalene dioxygenase iron-sulfur protein (nahAc) genes of nine naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site, as well as a naphthalene-degrading bacterium from a contaminated site in Washington state and two archetypal naphthalene-degrading strains, were compared. Seven strains from the study site had a single nahAc allele, whereas the 16S rRNA gene sequences of the strains differed by as much as 7.9%. No nahAc alleles from the site were identical to those of the archetypal strains, although the predominant allele was closely related to that of Pseudomonas putida NCIB 9816-4, isolated in the British Isles. However, one site-derived nahAc allele was identical to that of the Washington state strain. Lack of phylogenetic congruence of the nahAc and 16S rRNA genes indicates that relatively recent in situ horizontal transfer of the nahAc gene has occurred, possibly as a direct or indirect consequence of pollutant contamination. Alkaline lysis plasmid preparations and pulsed-field gel electrophoresis have revealed the presence of plasmids ranging in size from 70 to 88 kb in all site isolates. Southern hybridizations with a 407-bp nahAc probe have suggested that the nahAc gene is plasmid borne in all the site isolates but one, a strain isolated from subsurface sediment 400 m upstream from the source of the other site isolates. In this strain and in the naphthalene-degrading strain from Washington state, nahAc appears to be chromosomally located. In addition, one site isolate may carry nahAc on both chromosome and plasmid. Within the group of bacteria with identical nahAc sequences the Southern hybridizations showed that the gene was distributed between plasmids of different sizes and a chromosome. This suggests that plasmid modification after transfer may have been effected by transposons. Horizontal transfer of catabolic genes may play a significant role in the acclimation of microbial communities to pollutants.
Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration
Authors: Presser K A , Ratkowsky D A , Ross T
Published: January 2020

The growth rate responses of Escherichia coli M23 (a nonpathogenic strain) to suboptimal pH and lactic acid concentration were determined. Growth rates were measured turbidimetrically at 20 degrees C in the range of pH 2.71 to 8.45. The total concentration of lactic acid was fixed at specific values, and the pH was varied by the addition of a strong acid (hydrochloric) or base (sodium hydroxide) to enable the determination of undissociated and dissociated lactic acid concentrations under each condition. In the absence of lactic acid, E. coli grew at pH 4.0 but not at pH 3.7 and was unable to grow in the presence of > or = 8.32 mM undissociated lactic acid. Growth rate was linearly related to hydrogen ion concentration in the absence of lactic acid. In the range 0 to 100 mM lactic acid, growth rate was also linearly related to undissociated lactic acid concentration. A mathematical model to describe these observations was developed based on a Bĕlehrádek-like model for the effects of water activity and temperature. This model was expanded to describe the effects of pH and lactic acid by the inclusion of novel terms for the inhibition due to the presence of hydrogen ions, undissociated lactic acid, and dissociated lactic acid species. Preliminary data obtained for 200 and 500 mM total lactic acid concentrations show that the response to very high lactic acid concentrations was less well described by the model. However, for 0 to 100 mM lactic acid, the model described well the qualitative and quantitative features of the response.
Effect of bile on Vibrio parahaemolyticus
Authors: Pace J L , Chai T J , Rossi H A , Jiang X
Published: January 2020

Many enteric pathogens are thought to enter a viable but nonculturable state when deprived of nutrients. Virulent strains of the enteric pathogen Vibrio parahaemolyticus are rarely isolated from their low-nutrient aquatic environments, possibly due to their nonculturability. Host factors such as bile may trigger release from dormancy and increase virulence in these strains. In this study, the addition of bile or the bile acid deoxycholic acid to estuarine water-cultured bacteria led to an increase in the direct viable count and colony counts among the virulent strains. This effect was not demonstrated in the nonvirulent strains, and it was reversed by extraction of bile acids with cholestyramine. Bile-treated V. parahaemolyticus had lower levels of intracellular calcium than untreated cells, and this effect coincided with an increase in the number of metabolically active cells. Chelation of intracellular calcium with BAPTA/AM (R. Y. Tsien, Biochemistry 19:2396-2402, 1980) produced similar results. Addition of bile to V. parahaemolyticus cultures in laboratory medium enhanced factors associated with virulence such as Congo red binding, bacterial capsule size, and adherence to epithelial cells. These results suggest that a bile acid-containing environment such as that found in the human host favors growth of virulent strains of V. parahaemolyticus and that bile acids enhance the expression of virulence factors. These effects seem to be mediated by a decrease in intracellular calcium.
An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton
Authors: Scanlan D J , Silman N J , Donald K M , Wilson W H , Carr N G , Joint I , Mann N H
Published: January 2020

In the marine cyanobacterium Synechococcus sp. strain WH7803, PstS is a 32-kDa cell wall-associated phosphate-binding protein specifically synthesized under conditions of restricted inorganic phosphate (P1) availability (D. J. Scanlan, N. H. Mann, and N. G. Carr, Mol. Microbiol. 10:181-191, 1993). We have assessed its use as a potential diagnostic marker for the P status of photosynthetic picoplankton. Expression of PstS in Synechococcus sp. strain WH7803 was observed when the P1 concentration fell below 50 nM, demonstrating that the protein is induced at concentrations of P1 typical of oligotrophic conditions. PstS expression could be specifically detected by use of standard Western blotting (immunoblotting) techniques in natural mesocosm samples under conditions in which the N/P ratio was artificially manipulated to force P depletion. In addition, we have developed an immunofluorescence assay that can detect PstS expression in single Synechococcus cells both in laboratory cultures and natural samples. We show that antibodies raised against PstS cross-react with P-depleted Prochlorococcus cells, extending the use of these antibodies to both major groups of prokaryotic photosynthetic picoplankton. Furthermore, DNA sequencing of a Prochlorococcus pstS homolog demonstrated high amino acid sequence identity (77%) with the marine Synechococcus sp. strain WH7803 protein, including those residues in Escherichia coli PstS known to be directly involved in phosphate binding.
Impact of nutrient composition on a degradative biofilm community
Authors: Moller S , Korber D R , Wolfaardt G M , Molin S , Caldwell D E
Published: January 2020

A microbial community was cultivated in flow cells with 2,4,6-trichlorobenzoic acid (2,4,6-TCB) as sole carbon and energy source and was examined with scanning confocal laser microscopy and fluorescent molecular probes. The biofilm community which developed under these conditions exhibited a characteristic architecture, including a basal cell layer and conspicuous mounds of bacterial cells and polymer (approximately 20 to 30 (mu)m high and 25 to 40 (mu)m in diameter) occurring at 20- to 200-(mu)m intervals. When biofilms grown on 2,4,6-TCB were shifted to a labile, nonchlorinated carbon source (Trypticase soy broth), the biofilms underwent an architectural change which included the loss of mound structures and the formation of a more homogeneous biofilm. Neutrally charged fluorescent dextrans, which upon hydration become cationic, were observed to bind to mounds, as well as to the basal cell layer, in 14-day biofilms. In contrast, polyanionic dextrans bound only to the basal cell layer, indicating that this material incorporated sites with both positive and negative charge. The results from this study indicate that nutrient composition has a significant impact on both the architecture and the physicochemistry of degradative biofilm communities.
Effects of aerobic and microaerobic conditions on anaerobic ammonium-oxidizing (anammox) sludge
Authors: Strous M , Van Gerven E , Kuenen J G , Jetten M
Published: January 2020

The anaerobic ammonium oxidation (Anammox) process is a promising novel option for removing nitrogen from wastewater. In this study it was shown that the Anammox process was inhibited reversibly by the presence of oxygen. Furthermore, aerobic nitrifiers were shown not to play an important role in the Anammox process.
Development of a radioactive protein A-based assay for analysis of surface protein expression in gram-positive bacteria
Authors: Coyle T C , Franke C A , Hruby D E
Published: January 2020

This paper describes an immunochemical method which uses radioactive protein A for the detection and analysis of streptococcal M6 protein epitopes on the surface of recombinant Streptococcus gordonii. With this assay, recombinant S. gordonii cells expressing a portion of the M6 protein on their surfaces show a 75-fold increase in bound radioactivity over cells of the control S. gordonii parental strain. Furthermore, use of the assay to monitor the amount of M6 protein present on the surface of the S. gordonii recombinant during growth in culture demonstrated that expression is highest at late log phase, with the protein being sloughed off during stationary phase. This simple assay allows analysis of surface protein without any protein purification or sophisticated instrumentation. As such, it should be broadly applicable to following the expression of most surface-accessible bacterial proteins.
Ennoblement of Stainless Steel by the Manganese-Depositing Bacterium Leptothrix discophora
Authors: Dickinson W H , Caccavo F , Olesen B , Lewandowski Z
Published: January 2020

The noble shift in open-circuit potential exhibited by microbially colonized stainless steel (ennoblement) was investigated by examining the relationship among surface colonization, manganese deposition, and open-circuit potential for stainless steel coupons exposed to batch cultures of the manganese-depositing bacterium Leptothrix discophora. Open-circuit potential shifted from -100 to +330 mV(infSCE) as a biofilm containing 75 nmol of MnO(infx) cm(sup-2) formed on the coupon surface but changed little further with continued MnO(infx) deposition up to 270 nmol cm(sup-2). Increased open-circuit potential corresponded to decreasing Mn(II) concentration in solution and to increased MnO(infx) accumulation and attached cell density on the coupon surfaces. MnO(infx) deposition was attributable to biological activity, and Mn(II) was observed to enhance cell attachment. The experimental results support a mechanism of ennoblement in which open-circuit potential is fixed near +350 mV(infSCE) by the cathodic activity of biomineralized MnO(infx).