bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122
Authors: Kita K , Ishimaru K , Teraoka M , Yanase H , Kato N
Published: January 2020

Alcaligenes faecalis AE122 that used poly(3-hydroxybutyrate) (PHB) as a sole source of carbon was newly isolated from a coastal seawater sample. The strain required seawater for growth on PHB as well as in a nutrient broth, in which seawater could be replaced by an appropriate concentration of NaCl. PHB depolymerase was purified to homogeneity from the culture supernatant of A. faecalis AE122 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme consisted of a monomer subunit with a molecular mass of 95.5 kDa. The N-terminal amino acid sequence was GAWQNNLAGGFNKV. The dimeric and trimeric esters of 3-hydroxybutyrate were the main hydrolysis products of the purified enzyme. The enzyme was most active at pH 9.0 and 55 degrees C and was inhibited by phenylmethylsulfonyl fluoride. Several cations in seawater greatly enhanced the enzyme activity.
Cellodextrin efflux by the cellulolytic ruminal bacterium Fibrobacter succinogenes and its potential role in the growth of nonadherent bacteria
Authors: Wells J E , Russell J B , Shi Y , Weimer P J
Published: January 2020

When glucose or cellobiose was provided as an energy source for Fibrobacter succinogenes, there was a transient accumulation (as much as 0.4 mM hexose equivalent) of cellobiose or cellotriose, respectively, in the growth medium. Nongrowing cell suspensions converted cellobiose to cellotriose and longer-chain cellodextrins, and in this case the total cellodextrin concentration was as much as 20 mM (hexose equivalent). Because cell extracts of glucose- or cellobiose-grown cells cleaved cellobioise and cellotriose by phosphate-dependent reactions and glucose 1-phosphate was an end product, it appeared that cellodextrins were being produced by a reversible phosphorylase reaction. This conclusion was supported by the observation that the ratio of cellodextrins to cellodextrins with one greater hexose [n/(n + 1)] was approximately 4, a value similar to the equilibrium constant (Keq) of cellobiose phosphorylase (J. K. Alexander, J. Bacteriol. 81:903-910, 1961). When F. succinogenes was grown in a cellobiose-limited chemostat, cellobiose and cellotriose could both be detected, and the ratio of cellotriose to cellobiose was approximately 1 to 4. On the basis of these results, cellodextrin production is an equilibrium (mass action) function and not just an artifact of energy-rich cultural conditions. Cellodextrins could not be detected in low-dilution-rate, cellulose-limited continuous cultures, but these cultures had a large number of nonadherent cells. Because the nonadherent cells had a large reserve of polysaccharide and were observed at all stages of cell division, it appeared that they were utilizing cellodextrins as an energy source for growth.(ABSTRACT TRUNCATED AT 250 WORDS)
Survival of Azospirillum brasilense in the Bulk Soil and Rhizosphere of 23 Soil Types
Authors: Bashan Y , Puente M E , Rodriguez-Mendoza M N , Toledo G , Holguin G , Ferrera-Cerrato R , Pedrin S
Published: January 2020

The survival of Azospirillum brasilense Cd and Sp-245 in the rhizosphere of wheat and tomato plants and in 23 types of plant-free sterilized soils obtained from a wide range of environments in Israel and Mexico was evaluated. Large numbers of A. brasilense cells were detected in all the rhizospheres tested, regardless of soil type, bacterial strain, the origin of the soil, or the amount of rainfall each soil type received prior to sampling. Survival of A. brasilense in soils without plants differed from that in the rhizosphere and was mainly related to the geographical origin of the soil. In Israeli soils from arid, semiarid, or mountain regions, viability of A. brasilense rapidly declined or populations completely disappeared below detectable levels within 35 days after inoculation. In contrast, populations in the arid soils of Baja California Sur, Mexico, remained stable or even increased during the 45-day period after inoculation. In soils from Central Mexico, viability slowly decreased with time. In all soils, percentages of clay, nitrogen, organic matter, and water-holding capacity were positively correlated with bacterial viability. High percentages of CaCO(inf3) and fine or rough sand had a highly negative effect on viability. The percentage of silt, pH, the percentage of phosphorus or potassium, electrical conductivity, and C/N ratio had no apparent effect on bacterial viability in the soil. Fifteen days after removal of inoculated plants, the remaining bacterial population in the three soil types tested began to decline sharply, reaching undetectable levels 90 days after inoculation. After plant removal, percolating the soils with water almost eliminated the A. brasilense population. Viability of A. brasilense in two artificial soils containing the same major soil components as the natural soils from Israel did was almost identical to that in the natural soils. We conclude that A. brasilense is a rhizosphere colonizer which survives poorly in most soils for prolonged periods of time; that outside the rhizosphere, seven abiotic parameters control the survival of this bacterium in the soil; and that disturbance of the soil (percolation with water or plant removal) directly and rapidly affects the population levels.
Surface phenotypic characteristics and virulence of Spanish isolates of Aeromonas salmonicida after passage through fish
Authors: Fernández A I , Pérez M J , Rodríguez L A , Nieto T P
Published: January 2020

Eleven strains of Aeromonas salmonicida were passaged twice by intraperitoneal injection through rainbow trout and reisolated from the kidney of moribund fish. The surface characteristics and virulence of the strains changed following passage through fish. None of the in vitro tests used could effectively predict the in vivo virulence.
Biodegradation of Poly-(beta)-Hydroxyalkanoates in a Lake Sediment Sample Increases Bacterial Sulfate Reduction
Authors: Urmeneta J , Mas-Castella J , Guerrero R
Published: January 2020

The addition of a poly-(beta)-hydroxyalkanoate (PHA) to incubation bottles containing anaerobic sediment from Lake Ciso (Banyoles, Catalonia, northeast Spain) was correlated with an increase in sulfide production. The concentration of PHA diminished to 51 to 99% of the initial amount. Sodium molybdate (1 mM) decreased the rate of PHA degradation and inhibited sulfide production, thus indicating that PHAs serve as carbon and electron sources for sulfate reduction.
Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1
Authors: Masai E , Yamada A , Healy J M , Hatta T , Kimbara K , Fukuda M , Yano K
Published: January 2020

Rhodococcus sp. strain RHA1 is a gram-positive polychlorinated biphenyl (PCB) degrader which can degrade 10 ppm of PCB48 (equivalent to Aroclor1248), including tri-, tetra-, and pentachlorobiphenyls, in a few days. We isolated the 7.6-kb EcoRI-BamHI fragment carrying the biphenyl catabolic genes of RHA1 and determined their nucleotide sequence. On the basis of deduced amino acid sequence homology, we identified six bph genes, bphA1A2A3A4, bphB, and bphC, that are responsible for the initial three steps of biphenyl degradation. The order of bph genes in RHA1 is bphA1A2A3A4-bphC-bphB. This gene order differs from that of other PCB degraders reported previously. The amino acid sequences deduced from the RHA1 bph genes have a higher degree of homology with the tod genes from Pseudomonas putida F1 (49 to 79%) than with the bph genes of Pseudomonas sp. strains KF707 and KKS102 (30 to 65%). In Escherichia coli, bphA gene activity was not observed even when expression vectors were used. The activities of bphB and bphC, however, were confirmed by observing the transformation of biphenyl to a meta-cleavage compound with the aid of benzene dioxygenase activity that complemented the bphA gene activity (S. Irie, S. Doi, T. Yorifuji, M. Takagi, and K. Yano, J. Bacteriol. 169:5174-5179, 1987). The expected products of the cloned bph genes, except bphA3, were observed in E. coli in an in vitro transcription-translation system. Insertion mutations of bphA1 and bphC of Rhodococcus sp. strain RHA1 were constructed by gene replacement with cloned gene fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
Fate of Salmonella montevideo on and in raw tomatoes as affected by temperature and treatment with chlorine
Authors: Zhuang R Y , Beuchat L R , Angulo F J
Published: January 2020

A study was undertaken to determine the survival patterns of Salmonella montevideo G4639 on and in tomatoes during storage and the efficacy of chlorine treatment on inactivation of the pathogen. The population of S. montevideo on the surfaces of inoculated tomatoes stored at 10 degrees C did not change significantly (P < 0.05) throughout an 18-day storage period. Significant increases in population occurred within 7 days and within 1 day when tomatoes were stored at 20 and 30 degrees C, respectively. A significantly higher number of cells was taken up by the core tissue of tomatoes tempered at 25 degrees C when the tomatoes were dipped in a suspension at 10 degrees C compared with the number taken up when the tomatoes were dipped in cell suspensions tempered at 25 or 37 degrees C. Populations remained constant throughout subsequent storage for 8 days at 10 degrees C, regardless of the temperature differential between tomatoes and the dip suspension. Storage of tomatoes at 20 degrees C, however, resulted in significant increases in populations of S. montevideo. Populations of the pathogen on the surfaces and in the core tissues of tomatoes were significantly reduced by dipping for 2 min in a solution containing 60 or 110 ppm (60 or 110 micrograms/ml) chlorine, respectively; however, treatment in solution containing 320 ppm chlorine did not result in complete inactivation. Populations of S. montevideo remained unchanged in chopped tomatoes stored at 5 degrees C for 216 h (9 days) but increased significantly after storage for 96 or 22 h at 20 or 30 degrees C, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Investigations related to the epidemic strain involved in the French listeriosis outbreak in 1992
Authors: Jacquet C , Catimel B , Brosch R , Buchrieser C , Dehaumont P , Goulet V , Lepoutre A , Veit P , Rocourt J
Published: January 2020

Two hundred seventy-nine cases of human listeriosis (92 pregnancy-related cases and 187 non-pregnancy-related cases) caused by a serovar 4b and phagovar 2389:2425:3274:2671:47:108:340 strain were identified in France between March and December 1992. Epidemiological investigations included a case-control study (not described here) and microbiological analyses of foods. Results of the case-control study and characterization of food isolates identified pork tongue in jelly, a ready-to-eat meat product, as the major vehicle of this outbreak, and to a lesser extent, delicatessen products contaminated secondarily during handling in food stores. As far as serotyping, phage typing, DNA macrorestriction pattern analysis (obtained by pulsed-field gel electrophoresis [PFGE]), and ribotyping are concerned, this epidemic strain is phenotypically and genomically closely related to strains responsible for major outbreaks of listeriosis previously observed in Europe and North America. The epidemic strain sensu stricto as defined by PFGE (2/1/3) displayed the same serovar, phagovar, ribovar, and ApaI and NotI PFGE patterns as the epidemic strains from outbreaks in Switzerland, California, and Denmark, but it consistently showed differences in the SmaI PFGE profile. This information greatly contributed to the identification of the major food vehicle (pork tongue in jelly) and further allowed exclusion of other foods (cheese) as possible sources of this major listeriosis epidemic.
Unraveling the extent of diversity within the order Planctomycetales
Authors: Ward N , Rainey F A , Stackebrandt E , Schlesner H
Published: January 2020

The phylogenetic positions of 22 isolates that morphologically resemble members of the family Planctomycetaceae were determined by sequence analysis of genes coding for 16S rRNA. While nine and eight isolates could be assigned to the genera Planctomyces and Pirellula, respectively, three strains grouped near Isosphaera pallida and one strain was closely related to Gemmata obscuriglobus. No isolate was found to be related to a previously described species of any of the four genera at the species level. Morphological characters and sequence idiosyncrasies of genes coding for 16S rRNA of the isolates generally correlated with features described for the four genera to which the isolates could be assigned. One strain stands phylogenetically isolated and may be representative of a novel genus of the family. Comparison with environmental clone sequences representing planctomycetes in soil and water revealed that three of the novel isolates were related to one clone of soil origin, but no close relationships between clones and the other new strains were found. The study reveals that the biodiversity of planctomycetes is significantly greater than was previously determined.
PCR analysis of the viral complex associated with La France disease of Agaricus bisporus
Authors: Romaine C P , Schlagnhaufer B
Published: January 2020

Reverse transcription PCR analysis was used to investigate the involvement of two RNA-genome viruses, La France isometric virus (LIV) and mushroom bacilliform virus (MBV), in the etiology of La France disease of the cultivated mushroom Agaricus bisporus. Reverse transcription PCR amplification of sequences targeted to the genomes of LIV and MBV, with a sensitivity of detection of < 10 fg of viral RNA, showed diseased mushrooms to be either singly infected by LIV or doubly infected by LIV and MBV. Of 70 geographically diverse diseased mushroom isolates, 100% were infected by LIV, whereas almost 60% of these isolates were coinfected by MBV. Of 58 mushroom isolates determined to be free of infection by LIV, 3 were found to be infected by MBV. This represents the first documented report of the independent replication of these two viruses. Our data support the hypothesis that La France disease is associated with infection by two autonomously replicating viruses in which LIV is the primary causal agent and MBV, although possibly pathogenic and capable of modulating symptoms, is not required for pathogenesis.
Effect of oxytetracycline-medicated feed on antibiotic resistance of gram-negative bacteria in catfish ponds
Authors: DePaola A , Peeler J T , Rodrick G E
Published: January 2020

The effect of oxytetracycline-medicated feeds on antibiotic resistance in gram-negative bacteria from fish intestines and water in catfish ponds was investigated. In experiments in the fall and spring, using ponds with no previous history of antibiotic usage, percentages of tetracycline-resistant bacteria in catfish intestines obtained from medicated ponds increased significantly after 10 days of treatment. In the fall, resistance of the intestinal and aquatic bacteria returned to pretreatment levels within 21 days after treatment. In the spring, resistance declined after treatment but remained higher than pretreatment levels for at least 21 days in intestinal bacteria and for 5 months in aquatic bacteria. Plesiomonas shigelloides, Aeromonas hydrophila, and Citrobacter freundii were isolated frequently in both spring and fall; Escherichia coli, Klebsiella pneumoniae, Edwardsiella tarda, and Enterobacter spp. were isolated primarily in the spring. Oxytetracycline treatment did not affect the distribution of bacterial species in the fall but may have accelerated a shift toward greater prevalence of members of the family Enterobacteriaceae in the spring. Multiple antibiotic resistance did not appear to be elicited by oxytetracycline treatment.
A serine (threonine) protein kinase confers fungicide resistance in the phytopathogenic fungus Ustilago maydis
Authors: Orth A B , Rzhetskaya M , Pell E J , Tien M
Published: January 2020

A mutant of Ustilago maydis (VR43) with single-gene resistance to the dicarboximide fungicide vinclozolin was previously isolated and characterized. A genomic library was constructed, and an 8.7-kb resistance-conferring fragment was isolated by sib selection. Sequencing this fragment, we identified an 1,218-bp open reading frame, which, if disrupted by deletion, no longer confers resistance. Analyses of the data in GenBank demonstrated a high degree of homology between the product of the 1,218-bp open reading frame, referred to as the adr-1 gene, and Ser (Thr) protein kinases.
Overproduction, purification, and biochemical characterization of a xylanase (Xys1) from Streptomyces halstedii JM8
Authors: Ruiz-Arribas A , Fernández-Abalos J M , Sánchez P , Garda A L , Santamariá R I
Published: January 2020

Streptomyces halstedii JM8, isolated from straw, produces and secretes into the culture supernatant at least two proteins with hydrolytic activity towards xylan. The cloning of a DNA fragment of this microorganism in several Streptomyces strains permitted us to overproduce both proteins. N-terminal sequence analyses, immunoblot assays, and time course overproduction experiments allowed us to ensure that both xylanases were encoded by the same gene and that the smallest form (35 kDa) originated from the large one (45 kDa) by proteolytic cleavage on the C terminus. The production of both forms was studied in different strains carrying the gene in a multicopy plasmid. The best production was obtained with Streptomyces parvulus transformed with the plasmid pJM9, a pIJ702 derivative, which yielded 144 U/ml. Both forms of the xylanase were purified with a fast-performance liquid chromatography system and characterized biochemically. The optimal pH and temperature, for both, were 6.3 and 60 degrees C, respectively, in 7.5-min assays. Both proteins were highly stable in a wide range of pHs (4 to 10) and temperatures (4 to 50 degrees C); nevertheless, after 1-h incubations, both enzymes lost most of their activity at temperatures over 55 to 60 degrees C. Endoxylanolytic activity was demonstrated in both enzymes, but no beta-xylosidase activity was detected.
Role of fadR and atoC(Con) mutations in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli
Authors: Rhie H G , Dennis D
Published: January 2020

Recombinant Escherichia coli fadR atoC(Con) mutants containing the polyhydroxyalkanoate (PHA) biosynthesis genes from Alcaligenes eutrophus are able to incorporate significant levels of 3-hydroxyvalerate (3HV) into the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]. We have used E. coli fadR (FadR is a negative regulator of fatty acid oxidation) and E. coli atoC(Con) (AtoC is a positive regulator of fatty acid uptake) mutants to demonstrate that either one of these mutations alone can facilitate copolymer synthesis but that 3HV levels in single mutant strains are much lower than in the fadR atoC(Con) strain. E. coli atoC(Con) mutants were used alone and in conjunction with atoA and atoD mutants to determine that the function of the atoC(Con) mutation is to increase the uptake of propionate and that this uptake is mediated, at least in part, by atoD+. Similarly, E. coli fadR mutants were used alone and in conjunction with fadA, fadB, and fadL mutants to show that the effect of the fadR mutation is dependent on fadB+ and fadA+ gene products. Strains that were mutant in the fadB or fadA locus were unable to complement a PHA biosynthesis pathway that was mutant at the phaA locus (thiolase), but a strain containing a fadR mutation and which was fadA+ fadB+ was able to complement the phaA mutation and incorporated 3HV into P(3HB-co-3HV) to a level of 29 mol%.
Modelling Bacterial Growth of Lactobacillus curvatus as a Function of Acidity and Temperature
Authors: Wijtzes T , de Wit J C , In Huis  , Van't R , Zwietering M H
Published: January 2020

Models that describe the effect of acidity, temperature, and the combined effect of these variables on the growth parameters of Lactobacillus curvatus are developed and validated. Growth parameters (lag time, specific growth rate, and maximum population density) were calculated from growth data at different temperature-acidity combinations. Experiments were set up to assess the quantitative effects of temperature and acidity on the growth parameters rather than for parameter estimation solely. The effect of acidity is monitored at several constant temperature values. Models are set up and fitted to the data. The same procedure is used at constant acidity values to model the effect of temperature. For lag time, specific growth rate, and maximum population density, the effect of temperature could be multiplied with the effect of acidity to obtain combinatory models that describe the effect of both controlling factors on the growth parameters. Lag time measurements showed large deviations, and therefore the lag time models developed can only be used to estimate the order of magnitude of lag time.
The Termite Gut Microflora as an Oxygen Sink: Microelectrode Determination of Oxygen and pH Gradients in Guts of Lower and Higher Termites
Authors: Brune A , Emerson D , Breznak J A
Published: January 2020

Clark-type oxygen microelectrodes and glass pH microelectrodes, each with a tip diameter of <=10 (mu)m, were used to obtain high-resolution profiles of oxygen concentrations and pH values in isolated termite guts. Radial oxygen profiles showed that oxygen penetrated into the peripheral hindgut contents up to about 150 to 200 (mu)m below the epithelial surface in both the lower termite Reticulitermes flavipes (Kollar) and the higher termite Nasutitermes lujae (Wasmann). Only the central portions (comprising less than 40% of the total volume) of the microbe-packed, enlarged hindgut compartments ("paunches") were completely anoxic, indicating that some members of the hindgut microbiota constitute a significant oxygen sink. From the slopes of the oxygen gradients, we estimated that the entire paunches (gut tissue plus resident microbiota) of R. flavipes and N. lujae accounted for 21 and 13%, respectively, of the respiratory activity of the intact animals. Axial oxygen profiles also confirmed that in general, only the paunches were anoxic in their centers, whereas midguts and posterior hindgut regions contained significant amounts of oxygen (up to about 50 and 30% air saturation, respectively). A remarkable exception to this was the posterior portion of an anterior segment (the P1 segment) of the hindgut of N. lujae, which was completely anoxic despite its small diameter ((apprx=)250 (mu)m). Axial pH profiles of the guts of Nasutitermes nigriceps (Haldeman) and Microcerotermes parvus (Haviland) revealed that there were extreme shifts as we moved posteriorly from the midgut proper (pH (apprx=)7) to the P1 segment of the hindgut (pH >10) and then to the P3 segment (paunch; pH (apprx=)7). The latter transition occurred at the short enteric valve (P2 segment) and within a distance of less than 500 (mu)m. In contrast, R. flavipes, which lacks a readily distinguishable P1 segment, did not possess a markedly alkaline region, and the pH around the midgut-hindgut junction was circumneutral. The oxic status of the peripheral hindgut lumen and its substantial oxygen consumption, together with previous reports of large numbers of aerobic and facultatively anaerobic bacteria in the hindgut microflora, challenge the notion that termite hindguts are a purely anoxic environment and, together with the steep axial pH gradients in higher termites, refine our concept of this tiny microbial habitat.
Biochemical Characterization and Agglutinating Properties of Xenorhabdus nematophilus F1 Fimbriae
Authors: Moureaux N , Karjalainen T , Givaudan A , Bourlioux P , Boemare N
Published: January 2020

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with nematodes of the family Steinernematidae, occur spontaneously in two phases. Only the phase I variants of Xenorhabdus nematophilus F1 expressed fimbriae when the bacteria were grown on a solid medium (nutrient agar; 24 and 48 h of growth). These appendages were purified and characterized. They were rigid, with a diameter of 6.4 (plusmn) 0.3 nm, and were composed of 16-kDa pilin subunits. The latter were synthesized and assembled during the first 24 h of growth. Phase II variants of X. nematophilus did not possess fimbriae and apparently did not synthesize pilin. Phase I variants of X. nematophilus have an agglutinating activity with sheep, rabbit, and human erythrocytes and with hemocytes of the insect Galleria mellonella. The purified fimbriae agglutinated sheep and rabbit erythrocytes. The hemagglutination by bacteria and purified fimbriae was mannose resistant and was inhibited by porcine gastric mucin and N-acetyl-lactosamine. The last sugar seems to be a specific inhibitor of hemagglutination by X. nematophilus.
Microbial formation of dimethyl sulfide in anoxic sphagnum peat
Authors: Kiene R P , Hines M E
Published: January 2020

Peat bogs dominated by Sphagnum spp. have relatively high areal rates of dimethyl sulfide (DMS) emission to the atmosphere. DMS was produced in anoxic slurries of Sphagnum peat with a linear time course and with an average rate of 40.4 (range, 22.0 to 68.6) nmol per liter of slurry (middot) day(sup-1) observed in nine batches of slurry. Methanethiol (MeSH) was produced at roughly similar rates over the typical 4- to 8-day incubations. DMS and MeSH production in these acidic (pH 4.2 to 4.6) peats were biological, as they were stopped completely by autoclaving and inhibited strongly by addition of antibiotics and 500 (mu)M chloroform. Endogenous DMS production may be due to the degradation of S-methyl-methionine, dimethyl sulfoxide, or methoxyaromatic compounds (e.g., syringic acid), each of which stimulated DMS formation when added at 5 to 10 (mu)M concentrations. However, on the basis of the high rates of thiol (MeSH and ethanethiol) methylation activity that we observed and the availability of endogenous MeSH, we suggest that methylation of MeSH is the major pathway leading to DMS formation in anaerobic peat. Solid-phase adsorption of MeSH plays a key role in its availability for biomethylation reactions. Additions of acetate (1.5 mM) or compounds which could cause acetate to accumulate (e.g., glucose, alanine, and 2-bromoethanesulfonate) suppressed DMS formation. It is likely that acetogenic bacteria are involved in DMS formation, but our data are insufficient to allow firm conclusions about the metabolic pathways or organisms involved. Our observations are the first which point to the methylation of MeSH as the major mechanism for endogenous DMS production in any environment. The rates of net DMS production observed are sufficient to explain the relatively high fluxes of DMS emitted to the atmosphere from Sphagnum sp.-dominated wetlands.
DNA fingerprinting by pulsed-field gel electrophoresis to investigate a nosocomial pneumonia caused by Legionella bozemanii serogroup 1
Authors: L�ck P C , Helbig J H , Hagedorn H J , Ehret W
Published: January 2020

We typed 18 isolates of Legionella bozemanii obtained from clinical and environmental sources by pulsed-field gel electrophoresis. Each of the unrelated strains showed individual restriction patterns of the genomic DNA when either the SfiI or NotI restriction enzyme was used. One strain isolated from a patient with nosocomial legionellosis and two strains from the corresponding hospital water supply were indistinguishable, arguing for a transmission of L. bozemanii from the water supply to the patient. In conclusion, macrorestriction analysis is a valuable tool for studies of the molecular epidemiology of L. bozemanii.
Genetic marking of Lactococcus lactis shows its survival in the human gastrointestinal tract
Authors: Klijn N , Weerkamp A H , de Vos W M
Published: January 2020

A human feeding study was performed with Lactococcus lactis TC165.5, which is genetically marked by insertion of the sucrose-nisin conjugative transposon Tn5276 and chromosomal resistance to rifampin and streptomycin. The fate of strain TC165.5 and its nucleic acids was monitored by conventional plating methods and by molecular detection techniques based on specific PCR amplification of the nisin (nisA) gene from DNA extracted from human feces. A method was developed for the efficient extraction of microbial DNA from human feces. The results show that a fraction of viable cells of L. lactis TC165.5 survived passage through the human gastrointestinal tract. Only cells that passed within 3 days of ingestion could be recovered from the feces of the volunteers, and they accounted for approximately 1% of the total number of cells consumed. The presence of nisA in DNA extracted from feces could be detected up to 4 days, when viable cells were no longer present.
Enterobacterial repetitive intergenic consensus sequences and the PCR to generate fingerprints of genomic DNAs from Vibrio cholerae O1, O139, and non-O1 strains
Authors: Rivera I G , Chowdhury M A , Huq A , Jacobs D , Martins M T , Colwell R R
Published: January 2020

Enterobacterial repetitive intergenic consensus (ERIC) sequence polymorphism was studied in Vibrio Cholerae strains isolated before and after the cholera epidemic in Brazil (in 1991), along with epidemic strains from Peru, Mexico, and India, by PCR. A total of 17 fingerprint patterns (FPs) were detected in the V. cholerae strains examined; 96.7% of the toxigenic V. cholerae O1 strains and 100% of the O139 serogroup strains were found to belong to the same FP group comprising four fragments (FP1). The nontoxigenic V. cholerae O1 also yielded four fragments but constituted a different FP group (FP2). A total of 15 different patterns were observed among the V. cholerae non-O1 strains. Two patterns were observed most frequently for V. cholerae non-01 strains, 25% of which have FP3, with five fragments, and 16.7% of which have FP4, with two fragments. Three fragments, 1.75, 0.79, and 0.5 kb, were found to be common to both toxigenic and nontoxigenic V. cholerae O1 strains as well as to group FP3, containing V. cholerae non-O1 strains. Two fragments of group FP3, 1.3 and 1.0 kb, were present in FP1 and FP2 respectively. The 0.5-kb fragment was common to all strains and serogroups of V. cholerae analyzed. It is concluded from the results of this study, based on DNA FPs of environmental isolates, that it is possible to detect an emerging virulent strain in a cholera-endemic region. ERIC-PCR constitutes a powerful tool for determination of the virulence potential of V. cholerae O1 strains isolated in surveillance programs and for molecular epidemiological investigations.
Microbial diversity in uranium mine waste heaps
Authors: Schippers A , Hallmann R , Wentzien S , Sand W
Published: January 2020

Two different uranium mine waste heaps near Ronneburg, Thuringia, Germany, which contain the remains of the activity of the former uranium-mining Soviet-East German company Wismut AG, were analyzed for the occurrence of lithotrophic and chemoorganotropic leach bacteria. A total of 162 ore samples were taken up to a depth of 5 m. Cell counts of ferrous iron-, sulfur-, sulfur compound-, ammonia-, and nitrite-oxidizing bacteria were determined quantitatively by the most-probable-number technique. Sulfate-, nitrate-, ferric iron-, and manganese-reducing bacteria were also detected. In addition, the metabolic activity of sulfur- and iron-oxidizing bacteria was measured by microcalorimetry. Generally, all microorganisms mentioned above were detectable in the heaps. Aerobic and anaerobic microorganisms thrived up to a depth of 1.5 to 2 m. Up to 99% of Thiobacillus ferrooxidans cells, the dominant leaching bacteria, occurred to this depth. Their numbers correlated with the microbial activity measurements. Samples below 1.5 to 2 m exhibited reduced oxygen concentrations and reduced cell counts for all microorganisms.
Characterization of rhizosphere colonization by luminescent Enterobacter cloacae at the population and single-cell levels
Authors: Rattray E A , Prosser J I , Glover L A , Killham K
Published: January 2020

A bioluminescence marker system was used to characterized colonization of the rhizosphere by a bacterial inoculum, both in terms of population activity and at the single-cell level. Plasmid pQF70/44, which contains luxAB genes under the control of a strong constitutive phage promoter, was introduced into the rhizobacterium and model biocontrol agent Enterobacter cloacae. Light output from the lux-modified strain was detected by luminometry of samples from growing cultures of E. cloacae and from inoculated soil and wheat root samples. The minimum detection limits for fully active cells under optimum conditions were 90 and 445 cells g-1 for liquid culture and soil, respectively. The metabolic activities of the lux-marked population of E. cloacae, characterized by luminometry, contrasted in rhizosphere and nonrhizosphere soil. Cells in the rhizosphere were active, and there was a linear relationship between light output and cell concentration. The activity of cells in nonrhizosphere coil could not be detected unless the soil was supplied with substrate. Novel use of a charge-coupled device is reported for the spatial characterization of rhizosphere colonization by E. cloacae (pQF70/44) at the single-cell and population levels. Used macroscopically, the charge-coupled device identified differences in colonization due to competition from indigenous soil organisms. The lux-marked bacterium was able to colonize all depths of roots in the absence of competition but was restricted tot he spermosphere in the presence of competition (nonsterile soil).(ABSTRACT TRUNCATED AT 250 WORDS)
Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR
Authors: Atmar R L , Neill F H , Romalde J L , Le Guyader F , Woodley C M , Metcalf T G , Estes M K
Published: January 2020

A method for the detection of Norwalk virus and hepatitis A virus from shellfish tissues by PCR was developed. Virus was added to the stomach and hepatopancreatic tissues of oysters or hard-shell clams, and viral nucleic acids were purified by a modification of a previously described method (R.L. Atmar, T.G. Metcalf, F.H. Neill, and M.K. Estes, Appl. Environ. Microbiol. 59:631-635, 1993). The new method had the following advantages compared with the previously described method: (i) more rapid sample processing; (ii) increased test sensitivity; (iii) decreased sample-associated interference with reverse transcription-PCR; and (iv) use of chloroform-butanol in place of the chlorofluorocarbon trichlorotrifluoroethane. In addition, internal standards for both Norwalk virus and hepatitis A virus were made which demonstrated when inhibitors to reverse transcription-PCR were present and allowed quantitation of the viral nucleic acids present in samples. This assay can be used to investigate shellfish-associated gastroenteritis outbreaks and to study factors involved in virus persistence in shellfish.
Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples
Authors: Langendijk P S , Schut F , Jansen G J , Raangs G C , Kamphuis G R , Wilkinson M H , Welling G W
Published: January 2020

Three 16S rRNA hybridization probes were developed and tested for genus-specific detection of Bifidobacterium species in the human fecal flora. Variable regions V2, V4, and V8 of the 16S rRNA contained sequences unique to this genus and proved applicable as target sites for oligodeoxynucleotide probes. Determination of the genus specificity of the oligonucleotides was performed by whole-cell hybridization with fluorescein isothiocyanate-labelled probes. To this end, cells were fixed on glass slides, hybridized with the probes, and monitored by videomicroscopy. In combination with image analysis, this allowed quantification of the fluorescence per cell and objective evaluation of hybridization experiments. One of the probes developed was used to determine the population of Bifidobacterium spp. in human fecal samples. A comparison was made with results obtained by cultural methods for enumeration. Since both methods gave similar population estimates, it was concluded that all bifidobacteria in feces were culturable. However, since the total culturable counts were only a fraction of the total microscopic counts, the contribution of bifidobacteria to the total intestinal microflora was overestimated by almost 10-fold when cultural methods were used as the sole method for enumeration.
Salmonella enteritidis phage type 4 isolates more tolerant of heat, acid, or hydrogen peroxide also survive longer on surfaces
Authors: Humphrey T J , Slater E , McAlpine K , Rowbury R J , Gilbert R J
Published: January 2020

In a comparative study of different Salmonella enteritidis phage type 4 isolates we found that those isolates with enhanced heat tolerance also survived better than isolates that were heat sensitive either at pH 2.6, in 10 mM H2O2, or on surfaces. Culture to the stationary phase increased the heat tolerance of all isolates and the acid and H2O2 tolerance of heat-tolerant isolates. With heat-sensitive isolates, however, extended culture had no impact on survival in H2O2 and only a marginal impact on acid tolerance. The growth phase had no appreciable impact on the surface survival of any of the isolates.
Aqueous two-phase polymer systems as tools for the study of a recombinant surface-expressed Escherichia coli hemagglutinin
Authors: Lutwyche P , Norris-Jones R , Brooks D E
Published: January 2020

The surface expression of an integral membrane hemagglutinin, HRA1, cloned from Escherichia coli O9: H10:K99 in heterologous E. coli strains was studied by utilizing a variety of polyethylene glycol-dextran and dextran-Ficoll aqueous two-phase polymer systems. Bacteria containing plasmids that encoded the hemagglutinin were found to partition differently from both the host bacteria lacking the plasmid and the original hemagglutinating strain in several of these systems. By using molecular biological techniques, the origin of the partition difference was unambiguously correlated to the expression of HRA1, providing evidence independent of the agglutination phenotype that the protein was accessible to the surrounding milieu. It was demonstrated by using bacterial partition in charge-sensitive systems that the agglutination event was not likely to be due to the presence of a nonspecific positively charged surface protein, as HRA1-expressing clones showed no less affinity for the relatively positive polyethylene glycol-rich upper phase than did control bacteria. This work demonstrates the utility of aqueous polymer two-phase systems for the study of surface-expressed recombinant proteins, due to the sensitivity of the systems and the presence of excellent controls (the host bacteria before plasmid introduction). In cloning and expression studies of surface-associated proteins, two-phase aqueous polymer systems could be used as an alternative to antibody production for the monitoring of surface expression, and these systems may give valuable information on the surface exposure of the protein.
Reduction of Nitrated Diphenylamine Derivatives under Anaerobic Conditions
Authors: Drzyzga O , Schmidt A , Blotevogel K
Published: January 2020

2-Nitrodiphenylamine, 4-nitrodiphenylamine, and 2,4-dinitrodiphenylamine were anaerobically metabolized in sediment-water batch enrichments inoculated with mud from the German North Sea coast. The first intermediate in 2,4-dinitrodiphenylamine degradation was 2-amino-4-nitrodiphenylamine, which appeared in large (nearly stoichiometric) amounts before being completely reduced to 2,4-diaminodiphenylamine. Of the second theoretically expected metabolite, 4-amino-2-nitrodiphenylamine, only traces were detected by gas chromatographic-mass spectrometric analysis in highly concentrated extracts. In addition, low levels of 4-nitrodiphenylamine, which may be the product of ortho deamination of intermediately produced 2-amino-4-nitrodiphenylamine, were observed. 2-Nitrodiphenylamine and 4-nitrodiphenylamine were primarily reduced to 2-aminodiphenylamine and 4-aminodiphenylamine, respectively. Diphenylamine was never detected in any experiment as a theoretically possible intermediate. Results from studies with dense cell suspensions of anaerobic, aromatic-compound-mineralizing bacteria confirmed the transformation reactions, which were carried out by microorganisms indigenous to the anaerobic coastal water sediment.
Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay
Authors: Jacobsen C S
Published: January 2020

A magnetic capture-hybridization PCR technique (MCH-PCR) was developed to eliminate the inhibitory effect of humic acids and other contaminants in PCRs targeting specific soil DNA. A single-stranded DNA probe, which was complementary to an internal part of the target gene, was used to coat magnetic beads. After hybridization in a suspension of soil DNA, magnetic extraction of the beads separated the hybrid DNA from all other soil DNA, humic acids, and other interfering soil components. The MCH was followed by PCR amplification of the specific target DNA. In barley rhizosphere soil, detection of a lux gene inserted in a Pseudomonas fluorescens strain could be demonstrated in nonsterile soil samples (0.5 mg). This corresponded to a detection of fewer than 40 bacterial cells per cm of barley root. The MCH-PCR technique greatly improves the current protocols for PCR detection of specific microorganisms or genes in soil because specific target DNA sequences from very small soil samples can be extracted and determined.
Cloning, characterization, and expression of a gene region from Pseudomonas sp. strain ADP involved in the dechlorination of atrazine
Authors: de Souza M L , Wackett L P , Boundy-Mills K L , Mandelbaum R T , Sadowsky M J
Published: January 2020

We previously identified a Pseudomonas sp. strain, ADP, which rapidly metabolized atrazine in liquid culture, agar plates, and soils (R. T. Mandelbaum, D. L. Allan, L. P. Wackett, Appl. Environ. Microbiol. 61:1451-1457, 1995). In this study, we report the cloning and partial characterization of a gene region from Pseudomonas sp. strain ADP that encodes atrazine degradation activity. A 22-kb EcoRI genomic DNA fragment, designated pMD1, was shown to encode atrazine dechlorination activity in Escherichia coli DH5 alpha. Atrazine degradation was demonstrated by a zone-clearing assay on agar medium containing crystalline atrazine and by chromatographic methods. A gene conferring the atrazine-clearing phenotype was subsequently subcloned as a 1.9-kb AvaI fragment in pACYC184, designated pMD4, and was expressed in E. coli. This result and random Tn5 mutagenesis established that the 1.9-kb AvaI fragment was essential for atrazine dechlorination. High-pressure liquid and thin-layer chromatographic analyses were used to rigorously establish that E. coli containing pMD4 degraded atrazine and accumulated hydroxyatrazine. Hydroxyatrazine was detected only transiently in E. coli containing pMD1. This is consistent with the idea that hydroxyatrazine is the first metabolite in atrazine degradation by Pseudomonas sp. strain ADP. A 0.6-kb ApaI-PstI fragment from pMD4, containing the putative atrazine chlorohydrolase gene, hybridized to DNA from atrazine-degrading bacteria isolated in Switzerland and Louisiana.(ABSTRACT TRUNCATED AT 250 WORDS)
Efficacy of ozonated water against various food-related microorganisms
Authors: Restaino L , Frampton E W , Hemphill J B , Palnikar P
Published: January 2020

The antimicrobial effects of ozonated water in a recirculating concurrent reactor were evaluated against four gram-positive and four gram-negative bacteria, two yeasts, and spores of Aspergillus niger. More than 5 log units each of Salmonella typhimurium and Escherichia coli cells were killed instantaneously in ozonated water with or without addition of 20 ppm of soluble starch (SS). In ozonated water, death rates among the gram-negative bacteria--S. typhimurium, E. coli, Pseudomonas aeruginosa, and Yersinia enterocolitica--were not significantly different (P > 0.05). Among gram-positive bacteria, Listeria monocytogenes was significantly P < 0.05) more sensitive than either Staphylococcus aureus or Enterococcus faecalis. In the presence of organic material, death rates of S. aureus compared with L. monocytogenes and E. coli compared with S. typhimurium in ozonated water were not significantly (P > 0.05) affected by SS addition but were significantly reduced (P < 0.05) by addition of 20 ppm of bovine serum albumin (BSA). More than 4.5 log units each of Candida albicans and Zygosaccharomyces bailii cells were killed instantaneously in ozonated water, whereas less than 1 log unit of Aspergillus niger spores was killed after a 5-min exposure. The average ozone output levels in the deionized water (0.188 mg/ml) or water with SS (0.198 mg/ml) did not differ significantly (P < 0.05) but were significantly lower in water containing BSA (0.149 mg/ml).
Morphological variation of new Thermoplasma acidophilum isolates from Japanese hot springs
Authors: Yasuda M , Oyaizu H , Yamagishi A , Oshima T
Published: January 2020

We isolated 12 strains of Thermoplasma acidophilum from hot springs in Hakone, Japan. T. acidophilum strains showed morphological variation in the crystal-like structure in the cell and the fibrous structure on the cell surface. Two strains tested were sensitive to novobiocin. However, a novobiocin-resistant mutant was obtained by spontaneous mutation.
Biodiversity of gas vacuolate bacteria from Antarctic sea ice and water
Authors: Gosink J J , Staley J T
Published: January 2020

Psychrophilic, gas vacuolate, heterotrophic bacteria indigenous to sea ice communities in Antarctica have been isolated. Phylogenetic analysis of representative members of these bacteria shows that they belong to the alpha, beta, and gamma Proteobacteria and the Flavobacteria-Cytophaga group. This is the first report of gas vacuolate bacteria from the beta Proteobacteria and the Flavobacteria-Cytophaga groups.
Regulation of Proteolytic Enzyme Activity in Lactococcus lactis
Authors: Meijer W , Marugg J D , Hugenholtz J
Published: January 2020

Two different Lactococcus lactis host strains, L. lactis subsp. lactis MG1363 and L. lactis subsp. cremoris SK1128, both containing plasmid pNZ521, which encodes the extracellular serine proteinase (PrtP) from strain SK110, were used to study the medium and growth-rate-dependent activity of three different enzymes involved in the proteolytic system of lactococci. The activity levels of PrtP and both the intracellular aminopeptidase PepN and the X-prolyl-dipeptidyl aminopeptidase PepXP were studied during batch and continuous cultivation. In both strains, the PrtP activity level was regulated by the peptide content of the medium. The highest activity level was found during growth in milk, and the lowest level was found during growth in the peptide-rich laboratory medium M17. Regulation of the intracellular peptidase activity appeared to be a strain-dependent phenomenon. In cells of strain MG1363, the activity levels of PepN and PepXP were regulated in a similar way to that observed for PrtP. In cells of strain SK1128, the levels of both peptidases were not significantly influenced by the peptide content of the medium. The presence of specific concentrations of the dipeptide prolylleucine could mimic the low activity levels of the regulated proteolytic enzymes, even to the activity level found on M17 medium. The effect of the presence of the dipeptide prolylleucine in the medium on the activity level of the regulated proteolytic enzymes was confirmed at fixed growth rates in chemostat cultures.