bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro analysis of the role of glucose oxidase from Talaromyces flavus in biocontrol of the plant pathogen Verticillium dahliae
Authors: Stosz S K , Fravel D R , Roberts D P
Published: January 2020

Culture filtrates from Talaromyces flavus grown on glucose contained high levels of glucose oxidase activity, while culture filtrates from T. flavus grown on xylan contained negligible glucose oxidase activity. Culture filtrates from T-flavus grown on both media contained complex protein profiles. However, only culture filtrates from T. flavus grown on glucose inhibited germination of microsclerotia of Verticillium dahliae in in vitro inhibition assays. A polyclonal antiserum preparation, pABGO-1, raised against purified glucose oxidase from T. flavus was highly specific for glucose oxidase. Only one protein band in culture filtrates (from glucose medium), migrating at 71 kDa, was detected in Western blots (immunoblots) with this antiserum. This band comigrated with purified glucose oxidase. No bands were detected in culture filtrates from the xylan medium. Glucose oxidase was removed via immunoprecipitation from culture filtrates of T. flavus grown in glucose medium, resulting in filtrates which no longer inhibited in vitro microsclerotial germination. When glucose oxidase-depleted filtrates were amended with purified glucose oxidase from T. flavus, the ability to kill microsclerotia in vitro was restored to original levels. We conclude that glucose oxidase is the only protein in culture filtrates of T. flavus responsible for inhibition of germination of microsclerotia of V. dahliae.
Monoclonal antibodies for detection of the H7 antigen of Escherichia coli
Authors: He Y , Keen J E , Westerman R B , Littledike E T , Kwang J
Published: January 2020

Two murine monoclonal antibodies (MAbs) (2B7 and 46E9-9) reactive with the H7 flagellar antigen of Escherichia coli were produced and characterized. A total of 217 E. coli strains (48 O157:H7, 4 O157:NM, 23 O157:non-H7, 22 H7:non-O157, and 120 non-O157:nonH7), 17 Salmonella serovars, and 29 other gram-negative bacteria were used to evaluate the reactivities of the two MAbs by indirect enzyme-linked immunosorbent assay (ELISA). Both MAbs reacted strongly with all E. coli strains possessing the H7 antigen and with H23- and H24-positive E. coli strains. Indirect ELISA MAb specificity was confirmed by inhibition ELISA and by Western blotting (immunoblotting), using partially purified flagellins from E. coli O157:H7 and other E. coli strains. On a Western blot, MAb 46E9-9 was more reactive against H7 flagellin of E. coli O157:H7 than against H7 flagellin of E. coli O1:K1:H7. Competition ELISA suggested that MAbs 2B7 and 46E9-9 reacted with closely related H7 epitopes. When the ELISA reactivities of the MAbs and two commercially available polyclonal anti-H7 antisera were compared, both polyclonal antisera and MAbs reacted strongly with E. coli H7 bacteria. However, the polyclonal antisera cross-reacted strongly both with non-H7 E. coli and with many non-E. coli bacteria. The polyclonal antisera also reacted strongly with H23 and H24 E. coli isolates. The data suggest the need to define serotype-specific epitopes among H7, H23, and H24 E. coli flagella. The anti-H7 MAbs described in this report have the potential to serve as high-quality diagnostic reagents, used either alone or in combination with O157-specific MAbs, to identify or detect E. coli O157:H7 in food products or in human and veterinary clinical specimens.
Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase
Authors: McDaniels A E , Rice E W , Reyes A L , Johnson C H , Haugland R A , Stelma G N
Published: January 2020

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.
Comparison of the Efficacies of Chloromethane, Methionine, and S-Adenosylmethionine as Methyl Precursors in the Biosynthesis of Veratryl Alcohol and Related Compounds in Phanerochaete chrysosporium
Authors: Harper D B , McRoberts W C , Kennedy J T
Published: January 2020

The effect on veratryl alcohol production of supplementing cultures of the lignin-degrading fungus Phanerochaete chrysosporium with different methyl-(sup2)H(inf3)-labelled methyl precursors has been investigated. Both chloromethane (CH(inf3)Cl) and l-methionine caused earlier initiation of veratryl alcohol biosynthesis, but S-adenosyl-l-methionine (SAM) retarded the formation of the compound. A high level of C(sup2)H(inf3) incorporation into both the 3- and 4-O-methyl groups of veratryl alcohol occurred when either l-[methyl-(sup2)H(inf3)]methionine or C(sup2)H(inf3)Cl was present, but no significant labelling was detected when S-adenosyl-l-[methyl-(sup2)H(inf3)]methionine was added. Incorporation of C(sup2)H(inf3) from C(sup2)H(inf3)Cl was strongly antagonized by the presence of unlabelled l-methionine; conversely, incorporation of C(sup2)H(inf3) from l-[methyl-(sup2)H(inf3)]methionine was reduced by CH(inf3)Cl. These results suggest that l-methionine is converted either directly or via an intermediate to CH(inf3)Cl, which is utilized as a methyl donor in veratryl alcohol biosynthesis. SAM is not an intermediate in the conversion of l-methionine to CH(inf3)Cl. In an attempt to identify the substrates for O methylation in the metabolic transformation of benzoic acid to veratryl alcohol, the relative activities of the SAM- and CH(inf3)Cl-dependent methylating systems on several possible intermediates were compared in whole mycelia by using isotopic techniques. 4-Hydroxybenzoic acid was a much better substrate for the CH(inf3)Cl-dependent methylation system than for the SAM-dependent system. The CH(inf3)Cl-dependent system also had significantly increased activities toward both isovanillic acid and vanillyl alcohol compared with the SAM-dependent system. On the basis of these results, it is proposed that the conversion of benzoic acid to veratryl alcohol involves para hydroxylation, methylation of 4-hydroxybenzoic acid, meta hydroxylation of 4-methoxybenzoic acid to form isovanillic acid, and methylation of isovanillic acid to yield veratric acid.
Prevalence of enterohemorrhagic Escherichia coli in raw and treated municipal sewage
Authors: Grant S B , Pendroy C P , Mayer C L , Bellin J K , Palmer C J
Published: January 2020

Municipal sewage was screened for DNA encoding Shiga-like Toxin (SLT) II, a key protein involved in the virulence of enterohemorrhagic Escherichia coli. PCR analysis of sewage concentrates showed that DNA encoding SLT II was present in a single sample of untreated sewage and absent in all other samples tested (n = 6). Thermotolerant E. coli cultured from the sewage (n = 1,520) also tested negative for SLT II by colony hybridization.
Attachment of Agrobacterium tumefaciens B6 and A. radiobacter K84 to Tomato Root Tips
Authors: Penalver R , Serra M T , Duran-Vila N , Lopez M M
Published: January 2020

Agrobacterium tumefaciens B6 and the avirulent Agrobacterium radiobacter strain K84 attached to in vitro-cultured tomato root tips, but the binding of strain B6 to root tips was greater than the binding of strain K84. Strain K84 was not able to block the attachment of A. tumefaciens B6 to in vitro-cultured tomato root tips.
A selective and differential medium for Vibrio harveyi
Authors: Harris L , Owens L , Smith S
Published: January 2020

A new medium, termed Vibrio harveyi agar, has been developed for the isolation and enumeration of V. harveyi. It is possible to differentiate V. harveyi colonies from the colonies of strains representing 15 other Vibrio species with this medium. This medium has been shown to inhibit the growth of two strains of marine Pseudomonas spp. and two strains of marine Flavobacterium spp. but to allow the growth of Photobacterium strains. Colonies displaying typical V. harveyi morphology were isolated from the larval rearing water of a commercial prawn hatchery with V. harveyi agar as a primary isolation medium and were positively identified, by conventional tests, as V. harveyi. This agar displays great potential as a primary isolation medium and offers significant advantages over thiosulfate-citrate-bile salts-sucrose agar as a medium for differentiating V. harveyi from other marine and estuarine Vibrio species.
Penetration of Sulfate Reducers through a Porous North Sea Oil Reservoir
Authors: Beeder J , Nilsen R K , Thorstenson T , Torsvik T
Published: January 2020

The presence of mesophilic benzoate-degrading sulfate-reducing bacteria in the water systems of three Norwegian oil platforms was investigated. Strain 4502 was isolated from the injection water system, and specific antibodies were produced against this isolate. It was present in the injection water system during a period of 3 years, but not in the in situ reservoir water. Later it was found in water samples collected from the oil field production system. This showed that strain 4502 had penetrated the reservoir together with the injection water and eventually reached the production well.
Degradation of Chlorophenols by Alcaligenes eutrophus JMP134(pJP4) in Bleached Kraft Mill Effluent
Authors: Valenzuela J , Bumann U , Cespedes R , Padilla L , Gonzalez B
Published: January 2020

The ability of Alcaligenes eutrophus JMP134(pJP4) to degrade 2,4-dichlorophenoxyacetic acid, 2,4,6-trichlorophenol, and other chlorophenols in a bleached kraft mill effluent was studied. The efficiency of degradation and the survival of strain JMP134 and indigenous microorganisms in short-term batch or long-term semicontinuous incubations performed in microcosms were assessed. After 6 days of incubation, 2,4-dichlorophenoxyacetate (400 ppm) or 2,4,6-trichlorophenol (40 to 100 ppm) were extensively degraded (70 to 100%). In short-term batch incubations, indigenous microorganisms were unable to degrade such of compounds. Degradation of 2,4,6-trichlorophenol by strain JMP134 was significantly lower at 200 to 400 ppm of compound. This strain was also able to degrade 2,4-dichlorophenoxyacetate, 2,4,6-trichlorophenol, 4-chlorophenol, and 2,4,5-trichlorophenol when bleached Kraft mill effluent was amended with mixtures of these compounds. On the other hand, the chlorophenol concentration and the indigenous microorganisms inhibited the growth and survival of the strain in short-term incubations. In long-term (>1-month) incubations, strain JMP134 was unable to maintain a large, stable population, although extensive 2,4,6-trichlorophenol degradation was still observed. The latter is probably due to acclimation of the indigenous microorganisms to degrade 2,4,6-trichlorophenol. Acclimation was observed only in long-term, semicontinuous microcosms.
Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon
Authors: Fuerst J A , Gwilliam H G , Lindsay M , Lichanska A , Belcher C , Vickers J E , Hugenholtz P
Published: January 2020

Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae. The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented. A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria. Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group. Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species. A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology.
Characterization of Di-myo-Inositol-1,1(prm1)-Phosphate in the Hyperthermophilic Bacterium Thermotoga maritima
Authors: Ramakrishnan V , Verhagen M , Adams M
Published: January 2020

Di-myo-inositol-1,1(prm1)-phosphate (DIP) is present at a significant concentration ((symbl)160 nmol/mg of protein) in the cytoplasm of the hyperthermophilic bacterium Thermotoga maritima. The concentration of DIP was independent of the pH of the growth medium or the cell growth phase but increased with increasing concentrations of NaCl in the growth medium, reaching a maximum ((symbl)450 nmol/mg of protein) at 0.4 to 0.6 M NaCl. A large-scale purification procedure for DIP which yields approximately 18 g of DIP per kg of cells (wet weight) is described. Purified DIP was stable at 90(deg)C for at least 5 h. The presence of DIP (50 mM) did not increase the stability at 90(deg)C of pure forms of the hydrogenase or pyruvate ferredoxin oxidoreductase of T. maritima, suggesting that DIP is not a general thermoprotectant.
Temporal Transcriptional Pattern of Three Melanin Biosynthesis Genes, PKS1, SCD1, and THR1, in Appressorium-Differentiating and Nondifferentiating Conidia of Colletotrichum lagenarium
Authors: Takano Y , Kubo Y , Kuroda I , Furusawa I
Published: January 2020

A phytopathogenic fungus, Colletotrichum lagenarium, produces melanized appressoria that display temperature-sensitive differentiation. Conidia incubated in water at 24(deg)C germinated, and germ tubes differentiated into melanized appressoria. On the other hand, conidia incubated in water at 32(deg)C germinated and produced germ tubes that elongated without appressorium differentiation. Conidia in 0.1% yeast extract solution at 32(deg)C germinated and developed into vegetative hyphae. In this study, we investigated the temporal transcriptional pattern of cloned melanin biosynthesis genes, PKS1, SCD1, and THR1, in these differentiating and nondifferentiating conidia. During appressorium differentiation, de novo transcripts of the three melanin biosynthesis genes accumulated 1 to 2 h after the start of conidial incubation at 24(deg)C, and the amount of transcripts began to decrease at 6 h. In conidia germinating in water at 32(deg)C, the transcriptional pattern of the PKS1, SCD1, and THR1 genes was similar to that of these genes in appressorium-forming conidia, although no appressoria were formed. However, in conidia in 0.1% yeast extract solution at 32(deg)C, transcripts of the three melanin biosynthesis genes hardly accumulated.
Statistical modeling of methane production from landfill samples
Authors: Gurijala K R , Sa P , Robinson J A
Published: January 2020

Multiple-regression analysis was conducted to evaluate the simultaneous effects of 10 environmental factors on the rate of methane production (MR) from 38 municipal solid-waste (MSW) samples collected from the Fresh Kills landfill, which is the world's largest landfill. The analyses showed that volatile solids (VS), moisture content (MO), sulfate (SO(inf4)(sup2-)), and the cellulose-to-lignin ratio (CLR) were significantly associated with MR from refuse. The remaining six factors did not show any significant effect on MR in the presence of the four significant factors. With the consideration of all possible linear, square, and cross-product terms of the four significant variables, a second-order statistical model was developed. This model incorporated linear terms of MO, VS, SO(inf4)(sup2-), and CLR, a square term of VS (VS(sup2)), and two cross-product terms, MO x CLR and VS x CLR. This model explained 95.85% of the total variability in MR as indicated by the coefficient of determination (R(sup2) value) and predicted 87% of the observed MR. Furthermore, the t statistics and their P values of least-squares parameter estimates and the coefficients of partial determination (R values) indicated that MO contributed the most (R = 0.7832, t = 7.60, and P = 0.0001), followed by VS, SO(inf4)(sup2-), VS(sup2), MO x CLR, and VS x CLR in that order, and that CLR contributed the least (R = 0.4050, t = -3.30, and P = 0.0045) to MR. The SO(inf4)(sup2-), VS(sup2), MO x CLR, and CLR showed an inhibitory effect on MR. The final fitted model captured the trends in the data by explaining vast majority of variation in MR and successfully predicted most of the observed MR. However, more analyses with data from other landfills around the world are needed to develop a generalized model to accurately predict MSW methanogenesis.
Cloning and sequence analysis of a novel hemolysin gene (vllY) from Vibrio vulnificus
Authors: Chang T M , Chuang Y C , Su J H , Chang M C
Published: January 2020

A gene (vllY) encoding a novel hemolysin of Vibrio vulnificus CKM-1 has been cloned and sequenced. When the vllY gene was expressed in minicells, a unique peptide of approximately 40 kDa was identified. Subcellular fractionation of Escherichia coli cells carrying the vllY gene indicated that the VllY protein was distributed in both the cytoplasmic and the periplasmic fractions, with the notable ability to appear in the latter compartment. Nucleotide sequence analysis predicted a single open reading frame of 1,071 bp encoding a 357-amino acid polypeptide with an estimated pI of 5.02. The deduced amino acid sequence of VllY showed high similarity to the sequence of legiolysin, responsible for hemolysis, pigment production, and fluorescence in Legionella pneumophila. The enzyme also exhibited sequence homology to the MelA protein sequence of Shewanella colwelliana and the sequences of 4-hydroxyphenylpyruvate dioxygenase family proteins from various organisms. PCR screening and Southern blotting of V. vulnificus strains revealed that all of the 41 V. vulnificus clinical isolates contained vllY-like genes.
Dehalogenation and biodegradation of brominated phenols and benzoic acids under iron-reducing, sulfidogenic, and methanogenic conditions
Authors: Monserrate E , Häggblom M M
Published: January 2020

The anaerobic biodegradation of monobrominated phenols and benzoic acids by microorganisms enriched from marine and estuarine sediments was determined in the presence of different electron acceptors [i.e., Fe(III), SO4(2-), or HCO3-]. Under all conditions tested, the bromophenol isomers were utilized without a lengthy lag period whereas the bromobenzoate isomers were utilized only after a lag period of 23 to 64 days. 2-Bromophenol was debrominated to phenol, with the subsequent utilization of phenol under all three reducing conditions. Debromination of 3-bromophenol and 4-bromophenol was also observed under sulfidogenic and methanogenic conditions but not under iron-reducing conditions. In the bromobenzoate-degrading cultures, no intermediates were observed under any of the conditions tested. Debromination rates were higher under methanogenic conditions than under sulfate-reducing or iron-reducing conditions. The stoichiometric reduction of sulfate or Fe(III) and the utilization of bromophenols and phenol indicated that biodegradation was coupled to sulfate or iron reduction, respectively. The production of phenol as a transient intermediate demonstrates that reductive dehalogenation is the initial step in the biodegradation of bromophenols under iron- and sulfate-reducing conditions.
Gene sequence and expression of an analog of proliferating cell nuclear antigen (PCNA) in the alga Tetraselmis chui and detection of the encoded protein with anti-rat PCNA monoclonal antibody
Authors: Cheng L C , Hwang S P , Chang J
Published: January 2020

To identify a phytoplankton cell cycle marker detected by a monoclonal antibody against mammalian proliferating cell nuclear antigen (PCNA) (S. Lin, J. Chang, and E. J. Carpenter, J. Phycol. 30:449-456, 1994), a PCNA gene fragment was isolated by reverse transcription-PCR from the marine unicellular alga Tetraselmis chui Butcher (Prasinophyceae). The gene fragment was 616 bp in length and contained an open reading frame of 205 amino acids. The deduced amino acid sequence showed 80 and 88% similarity to human and rice PCNA, respectively. Southern hybridization indicated that the isolated gene fragment was part of the T. chui genome, with up to three copies in each haploid nucleus. Northern hybridization was used to detect a PCNA mRNA with a size of 1.2 kb from an exponentially growing algal culture. The T. chui gene fragment has been cloned into an expression vector, and a fusion protein was subsequently generated. Anti-rat PCNA simultaneously recognized the PCNA fusion protein and a single 33-kDa band in T.chui total protein extract. Our results indicate that T. chui PCNA is highly similar to its mammalian counterpart and that anti-rat PCNA is a good tool for detecting phytoplankton PCNA in general.
Escherichia coli in settled-dust and air samples collected in residential environments in Mexico City
Authors: Rosas I , Salinas E , Yela A , Calva E , Eslava C , Cravioto A
Published: January 2020

Escherichia coli, an important indicator of the presence of fecal material, was isolated from indoor and outdoor environments in Mexico City. The heterogeneity of E. coli was represented by 89 serotypes, most of them coming from settled-dust indoor samples; 21% of them presented antibiotic multiresistance. The numbers of plasmids were higher among the antibiotic-resistant strains. The results of this study suggest that intestinal infections produced by environmental strains could be of more epidemiological impact than previously thought.
Visualization of specific gene expression in individual Salmonella typhimurium cells by in situ PCR
Authors: Tolker-Nielsen T , Holmstrøm K , Molin S
Published: January 2020

An in situ PCR protocol by which we can monitor the presence or absence of lac mRNA in individual cells of a Salmonella typhimurium F' lac+ strain has been developed. In this protocol, fixed cells are permeabilized with lysozyme and subjected to a seminested reverse transcriptase PCR using reporter molecule-labeled primers, and subsequently, intracellular reporter molecules are detected microscopically at the individual-cell level by use of a horseradish peroxidase-conjugated antifluorescein antibody assay. In order to determine the sensitivity of the in situ PCR assay, the ability to detect lac mRNA in suboptimally isopropyl-beta-D-thiogalactopyranoside-induced cells was investigated. By use of a single-cell beta-galactosidase assay, it was confirmed that homogeneous suboptimally induced cultures of S. typhimurium F' lacY cells could be established, and the number of functional lac mRNAs in individual cells was estimated from standard population level beta-galactosidase assays. Cells estimated to contain a single lac mRNA were detected as containing lac mRNA by the in situ PCR method. Conclusively, we demonstrate the potential of in situ PCR for detection of even poorly expressed mRNA in individual bacterial cells.
Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test
Authors: Fach P , Popoff M R
Published: January 2020

A duplex PCR procedure was evaluated for the detection of Clostridium perfringens in food and biological samples and for the identification of enterotoxigenic strains. This method uses two sets of primers which amplify in the same reaction two different DNA fragments simultaneously: the 283-bp C. perfringens phospholipase C gene fragment and the 426-bp enterotoxin gene fragment. Internal primers within the two primer sets confirmed the specificity of the method by DNA-DNA hybridization with the PCR products. No cross-reaction was observed with other Clostridium species or with other bacteria routinely found in food. The detection level was approximately 10(5) C. perfringens cells per g of stool or food sample. When overnight enrichment culture was used, 10 C. perfringens cells per g was detected in 57 artificially contaminated food samples. The duplex PCR is a rapid, sensitive, and reliable method for the detection and identification of enterotoxigenic C. perfringens strains in food samples. A slide latex agglutination test was also evaluated as a rapid, simple technique for the detection of C. perfringens enterotoxin in stool samples.
Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR
Authors: Klein P G , Juneja V K
Published: January 2020

Detection of pathogens in contaminated food products by PCR can result in false-positive data due to the amplification of DNA from nonviable cells. A new method based on reverse transcription-PCR (RT-PCR) amplification of mRNA for the specific detection of viable Listeria monocytogenes was developed. The expression of three L. monocytogenes genes, iap, hly, and prfA, was examined to determine a suitable target for amplification of RT-PCR. Total RNA from L. monocytogenes was isolated, and following DNase treatment, the RNA was amplified by both RT-PCR and PCR with primers specific for the three genes. Amplicon detection was accomplished by Southern hybridization to digoxigenin-labeled gene probes. The levels of expression of these three genes differed markedly, and the results indicated that the iap gene would provide a good target for development of a specific method for detection of viable L. monocytogenes based on RT-PCR amplification. After a 1-h enrichment, the 371-bp iap-specific product was detected with a sensitivity of ca. 10 to 15 CFU/ml from pure culture. Detection of the 713-bp hly-specific amplicon was ca. 4,000 times less sensitive after 1 h, whereas detection of the 508-bp prfA product showed the lowest level of sensitivity, with detection not observed until after a 5-h enrichment period. The amplification of the iap mRNA was specific for L. monocytogenes. Overall, the assay could be completed in ca. 54 h. The use of RT-PCR amplification for the detection of viable L. monocytogenes was validated in artificially contaminated cooked ground beef. Following a 2-h enrichment incubation, the iap-specific amplification product could be detected in a cooked meat sample that was originally inoculated with ca. 3 CFU/g. These results support the usefulness of RT-PCR amplification of mRNA as a sensitive method for the specific detection of viable L. monocytogenes and indicate that this method may prove useful in the detection of this pathogen in ready-to-eat, refrigerated meat products.
Development of a diagnostic DNA probe for xanthomonads causing bacterial spot of peppers and tomatoes
Authors: Kuflu K M , Cuppels D A
Published: January 2020

Xanthomonas vesicatoria and Xanthomonas axonopodis pv. vesicatoria, causal agents for bacterial spot of tomatoes and peppers, are difficult to distinguish from other xanthomonads found on field-grown plants. A genomic subtraction technique with subtracter DNA from nonpathogenic epiphytic xanthomonads was used to enrich for sequences that could serve as diagnostic probes for these pathogens. A 1.75-kb PstI-NotI fragment (KK1750) that preferentially hybridized to X. vesicatoria DNA and X. axonopodis pv. vesicatoria DNA was identified and cloned into pBluescriptII KS+. It hybridized to 46 (89%) of the 52 geographically diverse bacterial spot-causing xanthomonad (bsx) strains included in this study. The six probe-negative strains were genotypically and pathologically distinct from the other bsx strains studied. Two of these strains, DC91-1 and DC91-2, resembled X. campestris pv. raphani in that they also infected radish plants. X. vesicatoria strains gave stronger hybridization signals than did most X. axonopodis pv. vesicatoria strains. In a survey of 110 non-bsx plant-associated bacteria, including 44 nonvesicatoria phytopathogenic xanthomonads and 43 epiphytic xanthomonad strains, only 8 were probe positive, but the responses were weak. Further testing revealed that one of these strains was actually a tomato pathogen. Pulsed-field gel electrophoresis and Southern blot analysis of 46 bsx strains indicated that KK1750 sequences could be either plasmid-borne (10.9%), chromosome-borne (43.4%), or present on both replicons (45.7%). KK1750, unique in its ability to hybridize to both X. axonopodis pv. vesicatoria and X. vesicatoria strains, should facilitate disease diagnosis for these important plant pathogens.
Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR
Authors: Takagi M , Nishioka M , Kakihara H , Kitabayashi M , Inoue H , Kawakami B , Oka M , Imanaka T
Published: January 2020

The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.
Comparison of PCR and plaque assay for detection and enumeration of coliphage in polluted marine waters
Authors: Rose J B , Zhou X , Griffin D W , Paul J H
Published: January 2020

A total of 68 marine samples from various sites impacted by sewage and storm waters were analyzed by both the plaque assay and a reverse transcriptase (RT) PCR technique for F(sup+)-specific coliphage. The coliphage levels detected by the plaque assay averaged 1.90 x 10(sup4) PFU/100.0 ml. Using a most probable number (MPN) PCR approach, the levels averaged 2.40 x 10(sup6) MPN-PCR units/100.0 ml. Two samples were positive by RT-PCR but negative by plaque assay, and 12 samples were positive by plaque assay but negative by RT-PCR (levels lower than 11.00 PFU/100.0 ml). The host system used for the plaque assay may detect somatic coliphage in addition to the F(sup+)-specific coliphage. When it is used as an indicator of pollution, contamination may be missed with more restrictive systems. The difference in results may be due to the sensitivity, specificity, or inhibition of RT-PCR in marine samples. This study provides information on quantifying PCR results by an MPN method and insights into interpretation of PCR data for detection of viruses in marine environments.
Detection of Activity Responsible for Induction of the Agrobacterium tumefaciens Virulence Genes in Bacteriological Agar
Authors: Loubens I , Chilton W S , Dion P
Published: January 2020

Agrobacterium tumefaciens C58 grown on acidic medium containing glucose and solidified with bacteriological agar expressed a virB::lacZ fusion. No expression of this fusion was observed on a similar medium which was solidified with purified agarose. The fraction from bacteriological agar which was responsible for vir gene induction was extracted with methanol and partially purified by preparative thin-layer chromatography.
A relative of the broad-host-range plasmid RSF1010 detected in Erwinia amylovora
Authors: Palmer E L , Teviotdale B L , Jones A L
Published: January 2020

Streptomycin- and sulfonamide-resistant Erwinia amylovora CA3R from California contained an 8.7-kb plasmid, pEa8.7, with a sulII-strA-strB resistance region; furthermore, PCR, sequencing, hybridization, and restriction analyses showed that pEa8.7 was closely related or identical to broad-host-range plasmid RSF1010. Although RSF1010 has been found in a variety of bacteria, this is the first report of its presence in plant pathogenic bacteria.
Conjugative plasmids isolated from bacteria in marine environments show various degrees of homology to each other and are not closely related to well-characterized plasmids
Authors: Dahlberg C , Linberg C , Torsvik V L , Hermansson M
Published: January 2020

Mercury resistance plasmids were exogenously isolated, i.e., recovered after transfer to a model recipient bacterium, from marine air-water interface, bulk water, and biofilm communities during incubation in artificial seawater without added nutrients. Ninety-five plasmids from different environments were classified by restriction endonuclease digestion, and 12 different structural plasmid groups were revealed. The plasmid types isolated from different habitats and from different sampling occasions showed little similarity to each other based on their restriction endonuclease patterns, indicating high variation and possibly a low transfer between microhabitats and/or a different composition of the microbial communities at different sites and times. With another approach in which probes derived from one of the isolated plasmids and a mercury resistance (mer) probe from Tn501 were used, similarities between plasmids from several different groups were found. The plasmids were further tested for their incompatibility by use of the collection of inc/rep probes (B/O, com9, FI, FII, HI1, HI2, I1, L/M, N, P, Q, U, W, Y) described by Couturier et al. (M. F. Couturier, P. Bex, L. Bergquist, and W. K. Maas, Microbiol. Rev. 52:375-395, 1988). Hybridizations did not reveal any identity between the 12 plasmid groups and any of the inc/rep probes tested. The results indicate that plasmids isolated from different marine habitats have replication and/or incompatibility systems that are different from the well-characterized plasmids that are commonly used in plasmid biology. This shows the need for the use of more relevant plasmids in studies of plasmid activity in the environment and development of new inc/rep probes for their characterization.
The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations
Authors: Rotthauwe J H , Witzel K P , Liesack W
Published: January 2020

The naturally occurring genetic heterogeneity of autotrophic ammonia-oxidizing populations belonging to the beta subclass of the Proteobacteria was studied by using a newly developed PCR-based assay targeting a partial stretch of the gene which encodes the active-site polypeptide of ammonia monooxygenase (amoA). The PCR yielded a specific 491-bp fragment with all of the nitrifiers tested, but not with the homologous stretch of the particulate methane monooxygenase, a key enzyme of methane-oxidizing bacteria. The assay also specifically detected amoA in DNA extracted from various aquatic and terrestrial environments. The resulting PCR products retrieved from rice roots, activated sludge, a freshwater sample, and an enrichment culture were used for the generation of amoA gene libraries. No false positives were detected in a set of 47 randomly selected clone sequences that were analyzed further. The majority of the environmental sequences retrieved from rice roots and activated sludge grouped within the phylogenetic radiation defined by cultured strains of the genera Nitrosomonas and Nitrosospira. The comparative analysis identified members of both of these genera in activated sludge; however, only Nitrosospira-like sequences with very similar amino acid patterns were found on rice roots. Further differentiation of these molecular isolates was clearly possible on the nucleic acid level due to the accumulation of synonymous mutations, suggesting that several closely related but distinct Nitrosospira-like populations are the main colonizers of the rhizosphere of rice. Each of the amoA gene libraries obtained from the freshwater sample and the enrichment culture was dominated by a novel lineage that shared a branch with the Nitrosospira cluster but could not be assigned to any of the known pure cultures. Our data suggest that amoA represents a very powerful molecular tool for analyzing indigenous ammonia-oxidizing communities due to (i) its specificity, (ii) its fine-scale resolution of closely related populations, and (iii) the fact that a functional trait rather than a phylogenetic trait is detected.
Carboxylation as an initial reaction in the anaerobic metabolism of naphthalene and phenanthrene by sulfidogenic consortia
Authors: Zhang X , Young L Y
Published: January 2020

The anaerobic biodegradation of naphthalene (NAP) and phenanthrene (PHE) was investigated by using sediment collected from the Arthur Kill in New York/New Jersey harbor. The initial cultures were composed of 10% sediment and 90% mineral medium containing 20 mM sulfate. Complete loss of NAP and PHE (150 to 200 muM) was observed after 150 days of incubation. Upon refeeding, NAP and PHE were utilized within 14 days. The utilization of both compounds was inhibited in the presence of 20 mM molybdate. [14C]NAP and [14C]PHE were mineralized to 14CO2. The activities could be maintained and propagated by subculturing in mineral medium. In the presence of halogenated analogs, 2-naphthoate was detected in NAP-utilizing enrichments. The mass spectrum of the derivatized 2-napththoate from the enrichment supplemented with both [13C]bicarbonate and NAP indicates the incorporation of 13CO2 into NAP. In the PHE-utilizing enrichment, a metabolite was detected by both high-pressure liquid chromatography and gas chromatography-mass spectrometry analyses. The molecular ion and fragmentation pattern of its mass spectrum indicate that it was phenanthrenecarboxylic acid. The results obtained with [13C] bicarbonate indicate that 13CO2 was incorporated into PHE. It appears, therefore, that carboxylation is an initial key reaction for the anaerobic metabolism and NAP and PHE. To our knowledge, this is the first report providing evidence for intermediates of PAH degradation under anaerobic conditions.
Expression of Barley Endopeptidase B in Trichoderma reesei
Authors: Saarelainen R , Mantyla A , Nevalainen H , Suominen P
Published: January 2020

The gene for barley endopeptidase B (EPB) has been expressed in the filamentous fungus Trichoderma reesei from the cbh1 promoter. The EPB signal sequence allowed secretion of over 90% of the recombinant protein. Yields reached about 500 mg of immunoreactive protein per liter and exceeded values for any other protein derived from a higher eukaryotic organism produced in T. reesei.
Conditions for natural transformation of Ralstonia solanacearum
Authors: Bertolla F , Van Gijsegem F , Nesme X , Simonet P
Published: January 2020

The development of competence allowing natural transformation of Ralstonia solanacearum was found to occur during exponential growth and not in response to any excreted factors. Linear DNAs were effectively integrated by recombination requiring a minimum of 50 bp of homologous DNA. Therefore, DNA from other genera and species were ineffective.
Inactivation of Helicobacter pylori by chlorination
Authors: Johnson C H , Rice E W , Reasoner D J
Published: January 2020

Three strains of Helicobacter pylori were studied to determine their resistance to chlorination. The organisms were readily inactivated by free chlorine and should therefore be controlled by disinfection practices normally employed in the treatment of drinking water.
One-step purification of nisin A by immunoaffinity chromatography
Authors: Suárez A M , Azcona J I , Rodríguez J M , Sanz B , Hernández P E
Published: January 2020

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method.
Aerobic and anaerobic metabolism of 6,10,14-trimethylpentadecan-2-one by a denitrifying bacterium isolated from marine sediments
Authors: Rontani J F , Gilewicz M J , Michotey V D , Zheng T L , Bonin P C , Bertrand J C
Published: January 2020

This report describes the metabolism of 6,10,14-trimethylpentadecan-2-one by a denitrifying bacterium (Marinobacter sp. strain CAB) isolated from marine sediments. Under aerobic and denitrifying conditions, this strain efficiently degraded this ubiquitous isoprenoid ketone. Several bacterial metabolites, 4,8,12-trimethyl-tridecan-1-ol, 4,8,12-trimethyltridecanal, 4,8,12-trimethyltridecanoic acid, Z-3,7-dimethylocten-2-oic acid, Z-3,7,11-trimethyldodecen-2-oic acid, and 6,10,14-trimethylpentadecan-2-ol, were formally identified, and different pathways were proposed to explain the formation of such isoprenoid compounds.
Intersporal Genetic Variation of Gigaspora margarita, a Vesicular Arbuscular Mycorrhizal Fungus, Revealed by M13 Minisatellite-Primed PCR
Authors: Zeze A , Sulistyowati E , Ophel-Keller K , Barker S , Smith S
Published: January 2020

Spores of vesicular arbuscular mycorrhizal (VAM) fungi contain thousands of nuclei. In order to understand the karyotic structure of a VAM fungus spore, the genetic variation of the first generation of spores from a VAM fungus (Gigaspora margarita) was examined. Spores originating from both single- and multispore inoculations of the species G. margarita were analyzed by M13 minisatellite-primed PCR. In both cases, different fingerprints were obtained from individual spores with few spores exhibiting similar fingerprints. These results can be explained only by a heterokaryotic status of the nuclear population within a spore.
The ldh phylogeny for environmental isolates of Lactococcus lactis is consistent with rRNA genotypes but not with phenotypes
Authors: Urbach E , Daniels B , Salama M S , Sandine W E , Giovannoni S J
Published: January 2020

Lactate dehydrogenase (ldh) gene sequences, levels of 16S rRNA group-specific probe binding, and phenotypic characteristics were compared for 45 environmental isolates and four commercial starter strains of Lactococcus lactis to identify evolutionary groups best suited to cheddar cheese manufacture, ldh sequences from the environmental isolates showed high similarity to those from two groups of L. lactis used for industrial fermentations, L. lactis subsp. cremoris and subsp. lactis. Within each phylogenetically defined subspecies, ldh sequence similarities were greater than 99.1%. Strains with phenotypic traits formerly diagnostic for both subspecies were found in each ldh similarity group, but only strains belonging to L. lactis subsp. cremoris by both the newer, genetic and the older, superseded phenotypic criteria were judged potentially suitable for the commercial production of cheddar cheese. Identical evolutionary relationships were inferred from ldh sequences and from binding of subspecies-specific, 16S rRNA-directed oligonucleotide probes. However, groups defined according to these chromosomal traits bore no relationship to patterns of arginine deamination, carbon substrate utilization, or bacteriophage sensitivity, which may be encoded by cryptic genes or sexually transmissible genetic elements. Fourteen new L. lactis subsp. cremoris isolates were identified as suitable candidates for cheddar cheese manufacture, and 10 of these were completely resistant to three different batteries of commercial bacteriophages known to reduce starter activity.
Supply-Side Analysis of Growth of Bacillus subtilis on Glucose-Citrate Medium: Feasible Network Alternatives and Yield Optimality
Authors: Lee J , Goel A , Ataai M M , Domach M M
Published: January 2020

Our prior work revealed that compared to the case for glucose metabolism, increased carbon yield and nil acid formation result when Bacillus subtilis grows on glucose medium containing citrate. To scrutinize further how citrate addition may alter metabolic flux regulation and the degree that the observed carbon yield corresponds to the maximal value, experimental (by least-squares analysis) and optimal (by linear programming) fluxes and yields were contrasted. Networks with differing reaction routes, directionality constraints, and transhydrogenase activities were examined. To attain an elevated carbon yield, citrate-glucose utilization need not alleviate any stoichiometric constraints that can sometimes interfere with the attainment of network objectives. Rather, the high carbon yield and nil acid formation attained may be linked to restriction of glycolytic capacity, particularly at the level of pyruvate kinase, which is consistent with a hypothesized effect of coupled metal-citrate uptake. Allowing for malic enzyme activity, hexose monophosphate pathway cycling, and transhydrogenase activity may also lead to the flux distributions underlying the high carbon yield observed. Finally, the observed carbon yield corresponded well to the maximum yield provided by all the network alternatives examined. Collectively, these results suggest that (i) the observed carbon yield is essentially equal to the maximal values associated with plausible networks and (ii), as suggested by others, nonoptimal flux regulation may contribute significantly to apparent cellular maintenance requirements.
Mutational analysis of the feedback sites of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli
Authors: Kikuchi Y , Tsujimoto K , Kurahashi O
Published: January 2020

In Escherichia coli, aroF, aroG, and aroH encode 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isozymes that are feedback inhibited by tyrosine, phenylalanine, and tryptophan, respectively. In vitro chemical mutagenesis of the cloned aroG gene was used to identify residues and regions of the polypeptide essential for phenylalanine feedback inhibition.