bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of genetically engineered gallidermin and epidermin analogs
Authors: Ottenwälder B , Kupke T , Brecht S , Gnau V , Metzger J , Jung G , Götz F
Published: January 2020

Gallidermin (Gdm) and epidermin (Epi) are highly homologous tetracyclic polypeptide antibiotics that are ribosomally synthesized by a Staphylococcus gallinarum strain and a Staphylococcus epidermidis strain, respectively. These antibiotics are secreted into media and are distinguished by the presence of the unusual amino acids lanthionine, 3-methyllanthionine, didehydrobutyrine, and S-(2-aminovinyl)-D-cysteine, which are formed by posttranslational modification. To study the substrate specificities of the modifying enzymes and to obtain variants that exhibit altered or new biological activities, we changed certain amino acids by performing site-specific mutagenesis with the Gdm and Epi structural genes (gdmA and epiA, respectively). S. epidermidis Tü3298/EMS6, an epiA mutant of the Epi-producing strain, was used as the expression host. This mutant synthesized Epi, Gdm, or analogs of these antibiotics when the appropriate genes were introduced on a plasmid. No Epi or Gdm analogs were isolated from the supernatant when (i) hydroxyamino acids involved in thioether amino acid formation were replaced by nonhydroxyamino acids (S3N and S19A); (ii) C residues involved in thioether bridging were deleted (delta C21, C22 and delta C22); or (iii) a ring amino acid was replaced by an amino acid having a completely different character (G10E and Y20G). A strong decrease in production was observed when S residues involved in thioether amino acid formation were replaced by T residues (S16T and S19T). A number of conservative changes at positions 6, 12, and 14 on the Gdm backbone were tolerated and led to analogs that had altered biological properties, such as enhanced antimicrobial activity (L6V) or a remarkable resistance to proteolytic degradation (A12L and Dhb14P). The T14S substitution led to simultaneous production of two Gdm species formed by incomplete posttranslational modification (dehydration) of the S-14 residue. The fully modified Dhb14Dha analog exhibited antimicrobial activity similar to that of Gdm, whereas the Dhb14S analog was less active. Both peptides were more sensitive to tryptic cleavage than Gdm was.
Differential secretion of isoforms of Serratia marcescens extracellular nuclease
Authors: Suh Y , Alpaugh M , Krause K L , Benedik M J
Published: January 2020

Extracellular secretion of the Serratia marcescens nuclease occurs in a two-step process: (i) rapidly to the periplasm via a signal sequence-dependent pathway and then (ii) slowly to the extracellular growth medium without cell lysis. There are two major isoforms of the nuclease in the culture supernatant of S. marcescens. We have isolated, purified, and determined the sequences of both isoforms. The first isoform, the mature nuclease (Sm2), is the result of signal sequence processing. The second isoform (Sm1) has three additional amino acids missing from the N terminus of the mature nuclease. Sm1 starts to appear extracellularly only during prolonged growth of a culture (16 to 48 h), probably because of cell lysis. However, pulse-chase experiments show that it is made early with Sm2 but is not secreted efficiently.
Sequence analysis of the Lactococcus lactis temperate bacteriophage BK5-T and demonstration that the phage DNA has cohesive ends
Authors: Boyce J D , Davidson B E , Hillier A J
Published: January 2020

The Lactococcus lactis temperate bacteriophage BK5-T is a type phage in the lactococcal phage classification (A. W. Jarvis, G. F. Fitzgerald, M. Mata, A. Mercenier, H. Neve, I. B. Powell, C. Ronda, M. Saxelin, and M. Teuber, Intervirology 32:2-9, 1991). The nucleotide sequence of 18,935 bp of the genome of BK5-T was determined and analyzed for the presence of open reading frames and other structural features. Thirty-two open reading frames longer than 60 codons were identified, and these appeared to be grouped into at least seven transcriptional units. A search of the nucleotide sequence for restriction sites identified a small number of discrepancies with the previously published physical map of the BK5-T genome (G. Lakshmidevi, B. E. Davidson, and A. J. Hillier, Appl. Environ. Microbiol. 54:1039-1045, 1988). Subsequent analysis of restriction digests of BK5-T DNA which were heated prior to electrophoresis indicated that BK5-T DNA was not terminally redundant as previously reported but contained cohesive ends.
A meta cleavage pathway for 4-chlorobenzoate, an intermediate in the metabolism of 4-chlorobiphenyl by Pseudomonas cepacia P166
Authors: Arensdorf J J , Focht D D
Published: January 2020

Bacterial degradation of biphenyl and polychlorinated biphenyls proceeds by a well-studied pathway which produces benzoate and 2-hydroxypent-2,4-dienoate (or, in the case of polychlorinated biphenyls, the chlorinated derivatives of these compounds). Pseudomonas cepacia P166 utilizes 4-chlorobiphenyl for growth and produces 4-chlorobenzoate as a central intermediate. In this study we found that strain P166 further transforms 4-chlorobenzoate to 4-chlorocatechol, which is mineralized by a meta cleavage pathway. Key metabolites which we identified include the meta cleavage product (5-chloro-2-hydroxymuconic semialdehyde), 5-chloro-2-hydroxymuconate, 5-chloro-2-oxopent-4-enoate, 5-chloro-4-hydroxy-2-oxopentanoate, and chloroacetate. Chloroacetate accumulated transiently, and slow but stoichiometric dehalogenation was observed.
A critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria
Authors: Glickmann E , Dessaux Y
Published: January 2020

We examined the sensitivity and the specificity of three versions of the Salkowski colorimetric technique. Two of these allowed the detection of indoleacetic acid (IAA) over a low range of concentrations (0.5 to 20 (mu)g/ml), while the third permitted the detection of IAA over a range of higher concentrations (5 to 200 (mu)g/ml). Overall, the three formulations reacted not only with auxin (IAA) but also with indolepyruvic acid and indoleacetamide. Therefore, these techniques appear to be specific for IAA, indolepyruvic acid, and indoleacetamide rather than for IAA alone.
Comparative Study of Substrates and Inhibitors of Azospirillum lipoferum and Pyricularia oryzae Laccases
Authors: Faure D , Bouillant M , Bally R
Published: January 2020

Azospirillum lipoferum and Pyricularia oryzae laccases were compared, using several substrates and inhibitors. Sixteen phenolic or nonphenolic compounds were found to be substrates of both fungal and bacterial laccases. In the presence of different phenol oxidase inhibitors, P. oryzae and A. lipoferum laccase activities had similar properties.
Purification and Properties of Extracellular Amylase from the Hyperthermophilic Archaeon Thermococcus profundus DT5432
Authors: Chung Y C , Kobayashi T , Kanai H , Akiba T , Kudo T
Published: January 2020

A hyperthermophilic archaeon, Thermococcus profundus DT5432, produced extracellular thermostable amylases. One of the amylases (amylase S) was purified to homogeneity by ammonium sulfate precipitation, DEAE-Toyopearl chromatography, and gel filtration on Superdex 200HR. The molecular weight of the enzyme was estimated to be 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 5.5 to 6.0 and was stable in the range of pH 5.9 to 9.8. The optimum temperature for the activity was 80(deg)C. Half-life of the enzyme was 3 h at 80(deg)C and 15 min at 90(deg)C. Thermostability of the enzyme was enhanced in the presence of 5 mM Ca(sup2+) or 0.5% soluble starch at temperatures above 80(deg)C. The enzyme activity was inhibited in the presence of 5 mM iodoacetic acid or 1 mM N-bromosuccinimide, suggesting that cysteine and tryptophan residues play an important role in the catalytic action. The amylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen to produce maltose and maltotriose of (alpha)-configuration as the main products. Smaller amounts of larger maltooligosaccharides were also produced with a trace amount of glucose. Pullulan; (alpha)-, (beta)-, and (gamma)-cyclodextrins; maltose; and maltotriose were not hydrolyzed.
Insecticidal Activity of the Toxins from Bacillus thuringiensis subspecies kurstaki and tenebrionis Adsorbed and Bound on Pure and Soil Clays
Authors: Tapp H , Stotzky G
Published: January 2020

The release of transgenic plants and microorganisms expressing truncated genes from various subspecies of Bacillus thuringiensis that encode active insecticidal toxins rather than inactive protoxins could result in the accumulation of these active proteins in soil, especially when bound on clays and other soil particles. Toxins from B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. tenebrionis, either free or adsorbed at equilibrium or bound on pure clay minerals (montmorillonite or kaolinite) or on the clay size fraction of soil, were toxic to larvae of the tobacco hornworm (Manduca sexta) and the Colorado potato beetle (Leptinotarsa decemlineata), respectively. The 50% lethal concentrations (LC(inf50)) of free toxins from B. thuringiensis subsp. kurstaki were higher than those of both bound and adsorbed complexes of these toxins with clays, indicating that adsorption and binding of these toxins on clays increase their toxicity in diet bioassays. The LC(inf50) of the toxin from B. thuringiensis subsp. tenebrionis that was either free or adsorbed on montmorillonite were comparable, whereas the toxin bound on this clay had higher LC(inf50) and the toxin bound on kaolinite had lower LC(inf50) than when adsorbed on this clay. Results obtained with the clay size fraction separated from unamended soil or soil amended with montmorillonite or kaolinite were similar to those obtained with the respective pure clay minerals. Therefore, insecticidal activity of these toxins is retained and sometimes enhanced by adsorption and binding on clays.
Activation of plasma membrane H(+)-ATPase and expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cells grown at supraoptimal temperatures
Authors: Viegas C A , Sebastião P B , Nunes A G , Sá-Correia I
Published: January 2020

During exponential growth at temperatures of 30 to 39 degrees C, the specific activity of H(+)-ATPase in the plasma membrane of Saccharomyces cerevisiae (assayed at the standard temperature 30 degrees C) increased with increases in growth temperature. In addition, the optimal temperature for in vitro activity of this ATPase was 42 degrees C. Therefore, the maximum values of ATPase activity were expected to occur in cells that grew within the supraoptimal range of temperatures. Activation induced by supraoptimal temperatures was not the result of increased synthesis of this membrane enzyme. When the growth temperature increased from 30 to 40 degrees C, expression of the essential PMA1 gene, monitored either by the level of PMA1 mRNA or the beta-galactosidase activity of the lacZ-PMA1 fusion, was reduced. Consistently, quantitative immunoassays showed that the ATPase content in the plasma membrane decreased. Like ATPase activity, the efficiency of the PMA2 promoter increased with increases in growth temperature in cells that had been grown at 30 to 39 degrees C, but its level of expression was several hundred-fold lower than that of PMA1. These results suggest that the major PMA1 ATPase is activated at supraoptimal temperatures.
Minimal Amino Acid Requirements of the Hyperthermophilic Archaeon Pyrococcus abyssi, Isolated from Deep-Sea Hydrothermal Vents
Authors: Watrin L , Martin-Jezequel V , Prieur D
Published: January 2020

Vol. 61, no. 3, p. 1138, column 1, line 4 from bottom: "(A(inf600) = 0.1 = 8 x 10(sup8) cells ml(sup-1))" should read "(A(inf600) = 0.1 = 1.8 x 10(sup8) cells ml(sup-1))." [This corrects the article on p. 1138 in vol. 61.].
Effects of Waterborne Copper, Cyanide, Ammonia, and Nitrite on Stress Parameters and Changes in Susceptibility to Saprolegniosis in Rainbow Trout (Oncorhynchus mykiss)
Authors: Carballo M , Munoz M J , Cuellar M , Tarazona J V
Published: January 2020

The effects of toxic exposures on the susceptibility of rainbow trout (Oncorhynchus mykiss) to saprolegniosis were evaluated. Fish were exposed to sublethal concentrations of copper (0.25 mg/liter), cyanide (0.07 mg/liter), ammonia (0.5 mg/liter), and nitrite (0.24 mg/liter) for 24 h. After exposure, the fish were challenged by Saprolegnia parasitica (3.6 x 10(sup6) zoospores per liter) for 10 min. Cortisol and cholesterol were used to indicate stress response. Similar increases of cortisol were found for the four tested chemicals. All fish with cortisol levels higher than 370 ng/ml developed the disease, while only 24% of the fish with cortisol levels lower than 370 ng/ml were infected. Cholesterol levels remained unchanged after toxic exposure. Increased susceptibilities to the pathogen were observed for ammonia (71%), copper (57%), nitrite (50%), and cyanide (33%). The increases in susceptibility as a result of cyanide and nitrite exposure could be explained by the stress response. For copper and ammonia, the combination of two different effects, the stress response and specific impairments of the defense mechanism of trout against saprolegniosis, should be considered.
Identification of grass-associated and toluene-degrading diazotrophs, Azoarcus spp., by analyses of partial 16S ribosomal DNA sequences
Authors: Hurek T , Reinhold-Hurek B
Published: January 2020

The genus Azoarcus includes nitrogen-fixing, grass-associated strains as well as denitrying toluene degraders. In order to identify and group members of the genus Azoarcus, phylogenetic analysis based on partial sequences of 16S rRNA genes (16S rDNAs) is proposed. 16S rRNA-targeted PCR using specific primers to exclude amplification in the majority of other members of the beta subclass of the class Proteobacteria was combined with direct sequencing of the PCR products. Tree inference from comparisons of 446-bp rDNA fragments yielded similar results for the three known Azoarcus spp. sequences and for analysis of the complete 16S rDNA sequence. These three species formed a phylogenetically coherent group with representatives of two other Azoarcus species which were subjected to 16S rRNA sequencing in this study. This group was related to Rhodocyclus purpureus and Thauera selenatis. New isolates and also sequences of so far uncultured bacteria from roots of Kallar grass were assigned to the genus Azoarcus as well. Also, strains degrading monoaromatic hydrocarbons anaerobically in the presence of nitrate clustered within this genus, albeit not with grass-associated isolates. All representative members of the five species harboring rhizospheric bacteria were able to form N2O from nitrate and showed anaerobic growth on malic acid with nitrate but not on toluene. In order to visualize different Azoarcus spp. by whole-cell in situ hybridizations, we generated 16S rRNA-targeted, fluorescent probes by in vitro transcription directly from PCR products which spanned the variable region V2. Hybridization was species specific for Azoarcus communis and Azoarcus indigens.(ABSTRACT TRUNCATED AT 250 WORDS)
Gene cloning, sequence analysis, purification, and secretion by Escherichia coli of an extracellular lipase from Serratia marcescens
Authors: Li X , Tetling S , Winkler U K , Jaeger K E , Benedik M J
Published: January 2020

The gene encoding extracellular lipase of Serratia marcescens has been identified from a phage lambda genomic library. Formation of orange-red fluorescent plaques on rhodamine B-triolein plates was used to identify phages carrying the lipase gene. A 2.8-kb SalI fragment was subcloned into a plasmid, and lipase was expressed in Escherichia coli. Extracellular lipase was detected in the presence of the secretion plasmid pGSD6 carrying the genes prtD, -E, and -F, which guide the secretion of protease from Erwinia chrysanthemi. Determination of the nucleotide sequence of the entire cloned fragment revealed an open reading frame coding for a 613-amino-acid protein with a predicted M(r) of 64,800. Analysis of the amino acid sequence revealed significant homology (around 70%) to lipases of Pseudomonas fluorescens strains. The lipase-specific consensus sequence G-X1-S-X2-G resided in the amino-terminal part of the protein, and carboxyl-terminal consensus sequences were an L-X-G-G-B-G-B-B-X repeat motif and a so-called aspartate box, respectively, which are both found in proteins secreted by the class I secretion pathway. Lipase was purified from the supernatant of a culture carrying a lipase expression vector, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an M(r) of 64,000 for the purified protein. Our results suggest that the lipase of S. marcescens belongs to the group of extracellular enzyme proteins secreted by the class I secretion pathway.
3-Methylindole (skatole) and indole production by mixed populations of pig fecal bacteria
Authors: Jensen M T , Cox R P , Jensen B B
Published: January 2020

Pig fecal slurries converted added L-tryptophan either to indole without detectable intermediates or to 3-methylindole (skatole) via indole-3-acetate. The initial rate of production of 3-methylindole was greatest at pH 6.5 and less at pH 5.0 and 8.0; the initial rates of indole production were similar at pH 6.5 and 8.0. More than 80% of the tryptophan added was converted to 3-methylindole at pH 5.0; at pH 8.0 85% was converted to indole. Both pathways had similar Km values for tryptophan and similar maximum rates. Indole-3-carbinol and indole-3-acetonitrile completely inhibited the production of 3-methylindole from indole-3-acetate but had no effect on the reactions involving L-tryptophan.
Stereoselective production of (+)-trans-chrysanthemic acid by a microbial esterase: cloning, nucleotide sequence, and overexpression of the esterase gene of Arthrobacter globiformis in Escherichia coli
Authors: Nishizawa M , Shimizu M , Ohkawa H , Kanaoka M
Published: January 2020

The gene coding for a novel esterase which stereoselectively hydrolyzes the (+)-trans (1R,3R) stereoisomer of ethyl chrysanthemate was cloned from Arthrobacter globiformis SC-6-98-28 and overexpressed in Escherichia coli. The cellular content of the active enzyme reached 33% of the total soluble protein in the recombinant E. coli JM105 cells and 5.6 g/liter of culture by high-density cell cultivation. The hydrolytic activity of the recombinant E. coli cells for ethyl chrysanthemate reached 605 mumol of chrysanthemic acid per min per g of dry cells, which is approximately 2,500-fold higher than that of A. globiformis cells. The stereoselective hydrolysis by the recombinant E. coli cells was efficient at substrate concentrations of up to 40% by removing the produced chrysanthemic acid by ultrafiltration. The (+)-trans-chrysanthemic acid produced had 100% optical purity. The amino acid sequence of the esterase was found to be similar to that of several class C beta-lactamases, D,D-carboxypeptidase, D-aminopeptidase, 6-aminohexanoate-dimer hydrolase, and Pseudomonas esterase. The sequence comparison also suggested that the Ser-X-X-Lys motif in the esterase was at the active site of the enzyme.
Evaluation of molecular typing techniques to assign genetic diversity among Saccharomyces cerevisiae strains
Authors: Baleiras Couto M M , Eijsma B , Hofstra H , Huis in't Veld J H , van der Vossen J M
Published: January 2020

Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite oligonucleotide primers (GAC)5 and (GTG)5 enabled discrimination between the strains tested. Additionally, restriction enzyme analysis, with TaqI and MseI, of PCR-amplified fragments from the complete internal transcribed spacer and nontranscribed spacer, both present in the rRNA-encoding gene cluster, proved to be suitable for generating intraspecies-specific patterns. Random amplified polymorphic DNA with primers 24 and OPA-11 and PCR fingerprinting with primer (GTG)5 appeared to generate the highest degree of diversity. However, the results indicated that there was no single PCR-mediated typing technique enabling discrimination on the strain level. Discrimination of each individual strain was nevertheless possible by combining the results obtained with all typing techniques.
A glycerol-3-phosphate dehydrogenase-deficient mutant of Saccharomyces cerevisiae expressing the heterologous XYL1 gene
Authors: Lidén G , Walfridsson M , Ansell R , Anderlund M , Adler L , Hahn-Hägerdal B
Published: January 2020

The gene XYL1, encoding a xylose reductase, from Pichia stipitis was transformed into a mutant of Saccharomyces cerevisiae incapable of glycerol production because of deletion of the genes GPD1 and GPD2. The transformed strain was capable of anaerobic glucose conversion in the presence of added xylose, indicating that the xylose reductase reaction can fulfill the role of the glycerol-3-phosphate dehydrogenase reaction as a redox sink. The specific xylitol production rate obtained was 0.38 g g-1 h-1.
Population analysis in a denitrifying sand filter: conventional and in situ identification of Paracoccus spp. in methanol-fed biofilms
Authors: Neef A , Zaglauer A , Meier H , Amann R , Lemmer H , Schleifer K H
Published: January 2020

The microbial community of a denitrifying sand filter in a municipal wastewater treatment plant was examined by conventional and molecular techniques to identify the bacteria actively involved in the removal of nitrate. In this system, denitrification is carried out as the last step of water treatment by biofilms growing on quartz grains with methanol as a supplemented carbon source. The biofilms are quite irregular, having a median thickness of 13 to 20 microns. Fatty acid analysis of 56 denitrifying isolates indicated the occurrence of Paracoccus spp. in the sand filter. 16S rRNA-targeted probes were designed for this genus and the species cluster Paracoccus denitrificans-Paracoccus versutus and tested for specificity by whole-cell hybridization. Stringency requirements for the probes were adjusted by use of a formamide concentration gradient to achieve complete discrimination of even highly similar target sequences. Whole-cell hybridization confirmed that members of the genus Paracoccus were abundant among the isolates. Twenty-seven of the 56 isolates hybridized with the genus-specific probes. In situ hybridization identified dense aggregates of paracocci in detached biofilms. Probes complementary to the type strains of P. denitrificans and P. versutus did not hybridize to cells in the biofilms, suggesting the presence of a new Paracoccus species in the sand filter. Analysis using confocal laser scanning microscopy detected spherical aggregates of morphologically identical cells exhibiting a uniform fluorescence. Cell quantification was performed after thorough disruption of the biofilms and filtration onto polycarbonate filters. An average of 3.5% of total cell counts corresponded to a Paracoccus sp., whereas in a parallel sand filter with no supplemented methanol, and no measurable denitrification, only very few paracocci (0.07% of cells stained with 4',6-diamidino-2-phenylindole) could be detected. Hyphomicrobium spp. constituted approximately 2% of all cells in the denitrifying unit and could not be detected in the regular sand filter. This clear link between in situ abundance and denitrification suggests an active participation of paracocci and hyphomicrobia in the process. Possible selective advantages favoring the paracocci in this habitat are discussed.
Phospholipid Fatty Acid Composition and Heavy Metal Tolerance of Soil Microbial Communities along Two Heavy Metal-Polluted Gradients in Coniferous Forests
Authors: Pennanen T , Frostegard A , Fritze H , Baath E
Published: January 2020

The effects of long-term heavy metal deposition on microbial community structure and the level of bacterial community tolerance were studied along two different gradients in Scandinavian coniferous forest soils. One was near the Harjavalta smelter in Finland, and one was at Ronnskar in Sweden. Phospholipid fatty acid (PLFA) analysis revealed a gradual change in soil microbial communities along both pollution gradients, and most of the individual PLFAs changed similarly to metal pollution at both sites. The relative quantities of the PLFAs br18:0, br17:0, i16:0, and i16:1 increased with increasing heavy metal concentration, while those of 20:4 and 18:2(omega)6, which is a predominant PLFA in many fungi, decreased. The fungal part of the microbial biomass was found to be more sensitive to heavy metals. This resulted in a decreased fungal/bacterial biomass ratio along the pollution gradient towards the smelters. The thymidine incorporation technique was used to study the heavy metal tolerance of the bacteria. The bacterial community at the Harjavalta smelter, exposed mainly to Cu deposition, exhibited an increased tolerance to Cu but not to Cd, Ni, and Zn. At the Ronnskar smelter the deposition consisting of a mixture of metals increased the bacterial community tolerance to all tested metals. Both the PLFA pattern and the bacterial community tolerance were affected at lower soil metal concentrations than were bacterial counts and bacterial activities. At Harjavalta the increased Cu tolerance of the bacteria and the change in the PLFA pattern of the microbial community were found at the same soil Cu concentrations. This indicated that the altered PLFA pattern was at least partly due to an altered, more metal-tolerant bacterial community. At Ronnskar, where the PLFA data varied more, a correlation between bacterial community tolerance and an altered PLFA pattern was found up to 10 to 15 km from the smelter. Farther away changes in the PLFA pattern could not be explained by an increased community tolerance to metals.
Occurrence of urease-positive Vibrio parahaemolyticus in Kanagawa, Japan, with specific reference to presence of thermostable direct hemolysin (TDH) and the TDH-related-hemolysin genes
Authors: Osawa R , Okitsu T , Morozumi H , Yamai S
Published: January 2020

A total of 132 strains of V. parahaemolyticus isolated from patients and from the suspected causal food items of past food poisoning cases occurring in Kanagawa Prefecture, Japan, were examined for the ability to hydrolyze urea, with specific reference to the presence of the thermostable direct hemolysin gene (tdh) and the gene for thermostable direct hemolysin-related hemolysin (trh). Ten strains belonging to five different O-antigen serotypes were positive for urea hydrolysis (UH+), and four of these strains did not carry tdh. A total of 106 strains carried tdh, but less than 6% of them were UH+, whereas all trh-carrying strains were UH+. The evidence suggests that urea hydrolysis is not a reliable marker for identifying tdh-carrying V. parahaemolyticus strains in Japan (the Pacific Northeast) but may be a marker for trh-carrying strains.
Detection and serogroup differentiation of Salmonella spp. in food within 30 hours by enrichment-immunoassay with a T6 monoclonal antibody capture enzyme-linked immunosorbent assay
Authors: Ng S P , Tsui C O , Roberts D , Chau P Y , Ng M H
Published: January 2020

We previously described an antigen capture enzyme-linked immunosorbent assay which makes use of monoclonal antibody T6, which recognizes an epitope on the outer core polysaccharide of Salmonella lipopolysaccharide molecules that is common to almost all Salmonella serovars. In this paper, we show that this assay can detect between 10(5) and 10(7) Salmonella cells per ml even in the presence of excess Escherichia coli. A total of 153 of 154 (99%) serogroup A to E strains and 51 of 78 (71%) serogroup F to 67 strains were reactive as determined by this assay. This corresponds to a detection rate of approximately 98% of all salmonellae known to affect humans. None of the 65 strains of non-Salmonella bacteria tested positive. Taking advantage of the O-factor polysaccharides also present on the antigen captured by the immobilized T6 antibody, we showed that 136 of 154 Salmonella serogroup A to E strains (88%) were correctly differentiated according to their serogroups by use of enzyme conjugates of a panel of O-factor-specific monoclonal antibodies. We evaluated this assay for the detection and serogroup differentiation of salmonellae directly from enrichment cultures of simulated food, eggs, pork, and infant formula milk. All 26 samples which had been contaminated with Salmonella spp. were detected by T6 (100% sensitivity), with only one false-positive result from 101 samples not contaminated by Salmonella spp. (99% specificity). The detection time was substantially reduced to between 17 and 29 h, depending on the enrichment methods used. Since there were no false-negative results, we concluded that this enrichment-immunoassay method can afford rapid screening for Salmonella spp. in food samples.
Phytoplasma-specific PCR primers based on sequences of the 16S-23S rRNA spacer region
Authors: Smart C D , Schneider B , Blomquist C L , Guerra L J , Harrison N A , Ahrens U , Lorenz K H , Seemüller E , Kirkpatrick B C
Published: January 2020

In order to develop a diagnostic tool to identify phytoplasmas and classify them according to their phylogenetic group, we took advantage of the sequence diversity of the 16S-23S intergenic spacer regions (SRs) of phytoplasmas. Ten PCR primers were developed from the SR sequences and were shown to amplify in a group-specific fashion. For some groups of phytoplasmas, such as elm yellows, ash yellows, and pear decline, the SR primer was paired with a specific primer from within the 16S rRNA gene. Each of these primer pairs was specific for a specific phytoplasma group, and they did not produce PCR products of the correct size from any other phytoplasma group. One primer was designed to anneal within the conserved tRNA(Ile) and, when paired with a universal primer, amplified all phytoplasmas tested. None of the primers produced PCR amplification products of the correct size from healthy plant DNA. These primers can serve as effective tools for identifying particular phytoplasmas in field samples.
Genetic relationships among strains of Xanthomonas fragariae based on random amplified polymorphic DNA PCR, repetitive extragenic palindromic PCR, and enterobacterial repetitive intergenic consensus PCR data and generation of multiplexed PCR primers useful for the identification of this phytopathogen
Authors: Pooler M R , Ritchie D F , Hartung J S
Published: January 2020

Genetic relationships among 25 isolates of Xanthomonas fragariae from diverse geographic regions were determined by three PCR methods that rely on different amplification priming strategies: random amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) PCR, and enterobacterial repetitive intergenic consensus (ERIC) PCR. The results of these assays are mutually consistent and indicate that pathogenic strains are very closely related to each other. RAPD, ERIC, and REP PCR assays identified nine, four, and two genotypes, respectively, within X. fragariae isolates. A single nonpathogenic isolate of X. fragariae was not distinguishable by these methods. The results of the PCR assays were also fully confirmed by physiological tests. There was no correlation between DNA amplification product patterns and geographic sites of isolation, suggesting that this bacterium has spread largely through exchange of infected plant germ plasm. Sequences identified through the RAPD assays were used to develop three primer pairs for standard PCR assays to identify X. fragariae. In addition, we developed a stringent multiplexed PCR assay to identify X. fragariae by simultaneously using the three independently derived sets of primers specific for pathogenic strains of the bacteria.
Effects of lactobacilli on yeast-catalyzed ethanol fermentations
Authors: Narendranath N V , Hynes S H , Thomas K C , Ingledew W M
Published: January 2020

Normal-gravity (22 to 24 degrees Plato) wheat mashes were inoculated with five industrially important strains of lactobacilli at approximately 10(5), approximately 10(6), approximately 10(7), approximately 10(8), and approximately 10(9) CFU/ml in order to study the effects of the lactobacilli on yeast growth and ethanol productivity. Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus #3, Lactobacillus rhamnosus, and Lactobacillus fermentum were used. Controls with yeast cells but no bacterial inoculation and additional treatments with bacteria alone inoculated at approximately 10(7) CFU/ml of mash were included. Decreased ethanol yields were due to the diversion of carbohydrates for bacterial growth and the production of lactic acid. As higher numbers of the bacteria were produced (depending on the strain), 1 to 1.5% (wt/vol) lactic acid resulted in the case of homofermentative organisms. L. fermentum, a heterofermentative organism, produced only 0.5% (wt/vol) lactic acid. When L. plantarum, L. rhamnosus, and L. fermentum were inoculated at approximately 10(6) CFU/ml, an approximately 2% decrease in the final ethanol concentration was observed. Smaller initial numbers (only 10(5) CFU/ml) of L. paracasei or Lactobacillus #3 were sufficient to cause more than 2% decreases in the final ethanol concentrations measured compared to the control. Such effects after an inoculation of only 10(5) CFU/ml may have been due to the higher tolerance to ethanol of the latter two bacteria, to the more rapid adaptation (shorter lag phase) of these two industrial organisms to fermentation conditions, and/or to their more rapid growth and metabolism. When up to 10(9) CFU of bacteria/ml was present in mash, approximately 3.8 to 7.6% reductions in ethanol concentration occurred depending on the strain. Production of lactic acid and a suspected competition with yeast cells for essential growth factors in the fermenting medium were the major reasons for reductions in yeast growth and final ethanol yield when lactic acid bacteria were present.
Phylogenetic diversity of Archaea in sediment samples from a coastal salt marsh
Authors: Munson M A , Nedwell D B , Embley T M
Published: January 2020

The Archaea present in salt marsh sediment samples from a tidal creek and from an adjacent area of vegetative marshland, both of which showed active methanogenesis and sulfate reduction, were sampled by using 16S rRNA gene libraries created with Archaea-specific primers. None of the sequences were the same as reference sequences from cultured taxa, although some were closely related to sequences from methanogens previously isolated from marine sediments. A wide range of Euryarchaeota sequences were recovered, but no sequences from Methanococcus, Methanobacterium, or the Crenarchaeota were recovered. Clusters of closely related sequences were common and generally contained sequences from both sites, suggesting that some related organisms were present in both samples. Recovery of sequences closely related to those of methanogens such as Methanococcoides and Methanolobus, which can use substrates other than hydrogen, provides support for published hypotheses that such methanogens are probably important in sulfate-rich sediments and identifies some likely candidates. Sequences closely related to those of methanogens such as Methanoculleus and Methanogenium, which are capable of using hydrogen, were also discovered, in agreement with previous inhibitor and process measurements suggesting that these taxa are present at low levels of activity. More surprisingly, we recovered a variety of sequences closely related to those from different halophilic Archaea and a cluster of divergent sequences specifically related to the marine group II archaeal sequences recently shown by PCR and probing to have a cosmopolitan distribution in marine samples.
Partial purification and characterization of manganese-oxidizing factors of Pseudomonas fluorescens GB-1
Authors: Okazaki M , Sugita T , Shimizu M , Ohode Y , Iwamoto K , de Vrind-de Jong E W , de Vrind J P , Corstjens P L
Published: January 2020

The Mn(2+)-oxidizing bacterium Pseudomonas fluorescens GB-1 deposits Mn oxide around the cell. During growth of a culture, the Mn(2+)-oxidizing activity of the cells first appeared in the early stationary growth phase. It depended on the O2 concentration in the culture during the late logarithmic growth phase. Maximal activity was observed at an oxygen concentration of 26% saturation. The activity could be recovered in cell extracts and was proportional to the protein concentration in the cell extracts. The specific activity was increased 125-fold by ammonium sulfate precipitation followed by reversed-phase and gel filtration column chromatographies. The activity of the partly purified Mn(2+)-oxidizing preparation had a pH optimum of circa 7 and a temperature optimum of 35 degrees C and was lost by heating. The Mn(2+)-oxidizing activity was sensitive to NaN3 and HgCl2. It was inhibited by KCN, EDTA, Tris, and o-phenanthroline. Although most data indicated the involvement of protein in Mn2+ oxidation, the activity was slightly stimulated by sodium dodecyl sulfate at a low concentration and by treatment with pronase and V8 protease. By polyacrylamide gel electrophoresis, two Mn(2+)-oxidizing factors with estimated molecular weights of 180,000 and 250,000 were detected.
Cellobiose dehydrogenase from Phanerochaete chrysosporium is encoded by two allelic variants
Authors: Li B , Nagalla S R , Renganathan V
Published: January 2020

The complete nucleotide sequences of two alleles of cellobiose dehydrogenase, cdh-1 (3,627 bp) and cdh-2 (3,623 bp), from Phanerochaete chrysosporium OGC101 are reported. The nucleotide sequences of cdh-1 and cdh-2 exhibit 97% similarity. A total of eighty-six point mutations between cdh-1 and cdh-2 are observed. Both alleles have 14 exons, and the introns are located at exactly the same positions. The translation products of these alleles have identical amino acid sequences. Restriction fragment length polymorphism analyses of homokaryotic derivatives show segregation of the CDH alleles.
Quantification of 2,4-Diacetylphloroglucinol Produced by Fluorescent Pseudomonas spp. In Vitro and in the Rhizosphere of Wheat
Authors: Bonsall R F , Weller D M , Thomashow L S
Published: January 2020

The broad-spectrum antibiotic 2,4-diacetylphloroglucinol (Phl) is a major determinant in the biological control of a wide range of plant diseases by fluorescent Pseudomonas spp. A protocol was developed to readily isolate and quantify Phl from broth and agar cultures and from the rhizosphere environment of plants. Extraction with ethyl acetate at an acidic pH was suitable for both in vitro and in situ sources of Phl. For soil samples, the addition of an initial extraction step with 80% acetone at an acidic pH was highly effective in eliminating polar organic soil components, such as humic and fulvic acids, which can interfere with Phl detection by high-performance liquid chromotography. The efficiency of Phl recovery from soil by a single extraction averaged 54.6%, and a second extraction added another 6.1%. These yields were substantially greater than those achieved by several standard protocols commonly used to extract polar phenolic compounds from soil. For the first time Phl was isolated from the rhizosphere environment in raw soil. Following application of Pseudomonas fluorescens Q2-87 and the Phl-overproducing strain Q2-87(pPHL5122) to the seeds of wheat, 2.1 and 2.4 (mu)g of Phl/g of root plus rhizosphere soil, respectively, were isolated from wheat grown in a Ritzville silt loam; 0.47 and 1.3 (mu)g of Phl/g of root plus rhizosphere soil, respectively, were isolated from wheat grown in a Shano silt loam. However, when the amount of Phl was calculated on the basis of cell density, Q2-87(pPHL5122) produced seven and six times more antibiotic than Q2-87 in Ritzville silt loam, and Shano silt loam, respectively.
Population structure of microbial communities associated with two deep, anaerobic, alkaline aquifers
Authors: Fry N K , Fredrickson J K , Fishbain S , Wagner M , Stahl D A
Published: January 2020

Microbial communities of two deep (1,270 and 316 m) alkaline (pH 9.94 and 8.05), anaerobic (Eh, -137 and -27 mV) aquifers were characterized by rRNA-based analyses. Both aquifers, the Grande Ronde (GR) and Priest rapids (PR) formations, are located within the Columbia River Basalt Group in south-central Washington, and sulfidogenesis and methanogenesis characterize the GR and PR formations, respectively. RNA was extracted from microorganisms collected from groundwater by ultrafiltration through hollow-fiber membranes and hybridized to taxon-specific oligonucleotide probes. Of the three domains, Bacteria dominated both communities, making up to 92.0 and 64.4% of the total rRNA from the GR and PR formations, respectively. Eucarya comprised 5.7 and 14.4%, and Archaea comprised 1.8% and 2.5%, respectively. The gram-positive target group was found in both aquifers, 11.7% in GR and 7.6% in PR. Two probes were used to target sulfate- and/or metal-reducing bacteria within the delta subclass of Proteobacteria. The Desulfobacter groups was present (0.3%) only in the high-sulfate groundwater (GR). However, comparable hybridization to a probe selective for the desulfovibrios and some metal-reducing bacteria was found in both aquifers, 2.5 and 2.9% from the GR and PR formations, respectively. Selective PCR amplification and sequencing of the desulfovibrio/metal-reducing group revealed a predominance of desulfovibrios in both systems (17 of 20 clones), suggesting that their environmental distribution is not restricted by sulfate availability.
The acquisition of indigenous plasmids by a genetically marked pseudomonad population colonizing the sugar beet phytosphere is related to local environmental conditions
Authors: Lilley A K , Bailey M J
Published: January 2020

The transfer of naturally occurring conjugative plasmids from the indigenous microflora to a genetically modified population of bacteria colonizing the phytospheres of plants has been observed. The marked strain (Pseudomonas fluorescens SBW25EeZY6KX) was introduced as a seed dressing to sugar beets (Beta vulgaris var. Amethyst) as part of a field experiment to assess the ecology and genetic stability of deliberately released bacterial inocula. The sustained populations of the introduced strain, which colonized the phytosphere, were assessed throughout the growing season for the acquisition of plasmids conferring mercury resistance (Hg(supr)). Transconjugants were isolated only from root and leaf samples collected within a narrow temporal window coincident with the midseason maturation of the crop. Conjugal-transfer events were recorded during this defined period in two separate field release experiments conducted over consecutive years. On one occasion seven of nine individual plants sampled supported transconjugant P. fluorescens SBW25EeZY6KX, demonstrating that conjugative gene transfer between bacterial populations in the phytosphere may be a common event under specific environmental conditions. The plasmids acquired in situ by the colonizing inocula were identified as natural variants of restriction digest pattern group I, III, or IV plasmids from five genetically distinct groups of large, conjugative mercury resistance plasmids known to persist in the phytospheres of sugar beets at the field site. These data demonstrate not only that gene transfer may be a common event but also that the genetic and phenotypic stability of inocula released into the natural environment cannot be predicted.
Accumulation of Trichothecenes in Liquid Cultures of a Fusarium sporotrichioides Mutant Lacking a Functional Trichothecene C-15 Hydroxylase
Authors: McCormick S P , Hohn T M
Published: January 2020

A mutant strain of Fusarium sporotrichioides NRRL 3299 produced by disruption of Tri11, a gene encoding a cytochrome P-450 monooxygenase, was shown to be altered in its ability to biosynthesize T-2 toxin. This mutant strain produced four trichothecenes that were not observed in cultures of the parent strain. The compounds were identified as isotrichodermin, 8-hydroxyisotrichodermin, 8-hydroxyisotrichodermol, and 3,4,8-trihydroxytricothecene on the basis of their nuclear magnetic resonance and mass spectra. This is the first report of these 8-hydroxytrichothecenes as metabolites of F. sporotrichioides. The accumulation of isotrichodermin and the results of whole-cell feeding experiments with a Tri11(sup-) strain confirm that oxygenation of C-15 is blocked.
Potential Role of the Eastern Oyster, Crassostrea virginica, in the Epidemiology of Cryptosporidium parvum
Authors: Fayer R , Farley C A , Lewis E J , Trout J M , Graczyk T K
Published: January 2020

Oysters were placed in an aquarium containing artificial seawater, and Cryptosporidium parvum oocysts were added. Oocysts were later found in the gill washings, hemocytes, and gut contents of the oysters. Hemocytes containing oocysts were intubated into four mice. C. parvum stages developed in the ileal epithelia of all of the mice, indicating that the oocysts in the hemocytes remained infective.
Bacterial population dynamics in a meromictic lake
Authors: Tuomi P , Torsvik T , Heldal M , Bratbak G
Published: January 2020

Polyclonal antibodies against nine different bacteria isolated from Lake Saelenvannet in western Norway were produced, and the population dynamics of these strains in the lake were monitored through two spring seasons by immunofluorescence staining. The total counts of bacteria varied over time and space from 1.5 x 10(6) to 1.5 x 10(7) cells ml-1. The counts of specific bacteria were in the range of 10(3) to 10(4) cells ml-1 or less; in sum, they generally made up less than 1% of the bacterial community. Some populations showed significant changes in abundance, with blooms lasting 1 to 3 weeks. The rate of change (increase and decrease) in abundance during blooms was estimated to be 0.2 to 0.6 day-1. The average virus-to-bacteria ratio was 50, and there was a significant correlation between the abundances of virus and bacteria. Both protozoan grazing and lytic virus infection were assessed as possible mechanisms driving the variations in bacterial population density.
Occurrence of a Highly Heat-Sensitive Spore Subpopulation of Bacillus coagulans STCC 4522 and Its Conversion to a More Heat-Stable Form
Authors: Palop A , Sala F J , Condon S
Published: January 2020

The profile of the survival curves, at different heating temperatures, of B. coagulans STCC 4522 sporulated at 52(deg)C has been studied, focusing on the early moments of treatment. A highly heat-sensitive spore subpopulation that includes more than 90% of the total spore population has been found. This heat-sensitive spore fraction was inactivated after 2 s of treatment at 111(deg)C. Its heat resistance was as much as 200-fold lower than that of the heat-resistant spore fraction (D(inf111(deg)C) of 0.01 min for the heat-sensitive spore fraction compared with D(inf111(deg)C) of 2 min for the heat-resistant fraction). The shape of the survival curve at 108.5(deg)C was modified after a sublethal heat shock at 80(deg)C for 3.5 h, resulting in a straight-line survival curve. The temperature of treatment also influenced the shape of the survival curves. The conversion of the highly heat-sensitive spore subpopulation to a more heat-stable form is discussed.