bluuebunny/crossref_metadata_2025 huggingface dataset:
Degradative capacities and 16S rRNA-targeted whole-cell hybridization of sulfate-reducing bacteria in an anaerobic enrichment culture utilizing alkylbenzenes from crude oil
Authors: Rabus R , Fukui M , Wilkes H , Widdle F
Published: January 2020

A mesophilic sulfate-reducing enrichment culture growing anaerobically on crude oil was used as a model system to study which nutritional types of sulfate-reducing bacteria may develop on original petroleum constituents in oil wells, tanks, and pipelines. Chemical analysis of oil hydrocarbons during growth revealed depletion of toluene and o-xylene within 1 month and of m-xylene, o-ethyltoluene, m-ethyltoluene, m-propyltoluene, and m-isopropyltoluene within approximately 2 months. In anaerobic counting series, the highest numbers of CFU (6 x 10(6) to 8 x 10(6) CFU ml-1) were obtained with toluene and benzoate. Almost the same numbers were obtained with lactate, a substrate often used for detection of the vibrio-shaped, incompletely oxidizing Desulfovibrio sp. In the present study, however, lactate yielded mostly colonies of oval to rod-shaped, completely oxidizing, sulfate-reducing bacteria which were able to grow slowly on toluene or crude oil. Desulfovibrio species were detected only at low numbers (3 x 10(5) CFU ml-1). In agreement with this finding, a fluorescently labeled, 16S rRNA-targeted oligonucleotide probe described in the literature as specific for members of the Desulfovibrionaceae (suggested family) hybridized only with a small portion (< 5%) of the cells in the enrichment culture. These results are consistent with the observation that known Desulfovibrio species do not utilize aromatic hydrocarbons, the predominant substrates in the enrichment culture. All known sulfate-reducing bacteria which utilize aromatic compounds belong to a separate branch, the Desulfobacteriaceae (suggested family). Most members of this family are complete oxidizers. For specific hybridization with members of this branch, the probe had to be modified by a nucleotide exchange. Indeed, this modified probe hybridized with more than 95% of the cells in the enrichment culture. The results show that completely oxidizing, alkylbenzene-utilizing sulfate-reducing bacteria rather than Desulfovibrio species have to be considered in attempts to understand the microbiology of sulfide production in oil wells, tanks, and pipelines when no electron donors other than the indigenous oil constituents are available.
Measurement of minimum substrate concentration (Smin) in a recycling fermentor and its prediction from the kinetic parameters of Pseudomonas strain B13 from batch and chemostat cultures
Authors: Tros M E , Bosma T N , Schraa G , Zehnder A J
Published: January 2020

The minimum substrate concentration required for growth, Smin, was measured for Pseudomonas sp. strain B13 with 3-chlorobenzoate (3CB) and acetate in a recycling fermentor. The substrates were provided alone or in a mixture. Smin values predicted with kinetic parameters from resting-cell batches and chemostat cultures differed clearly from the values measured in the recycling fermentor. When 3CB and acetate were fed as single substrates, the measured Smin values were higher than the individual Smin values in the mixture. The Smin in the mixture reflected the relative energy contributions of the two substrates in the fermentor feed. The energy-based maintenance coefficients during zero growth in the recycling fermentor were comparable for all influent compositions (mean +/- standard deviation, 0.34 +/- 0.07 J mg [dry weight]-1 h-1). Maintenance coefficient values for acetate were significantly higher in chemostat experiments than in recycling-fermentor experiments. 3CB maintenance coefficients were comparable in both experimental systems. The parameters for 3CB consumption kinetics varied remarkably with the experimental growth conditions in batch, chemostat, and recycling-fermentor environments. The results demonstrate that the determination of kinetic parameters in the laboratory for prediction of microbial activity in complex natural systems should be done under conditions which best mimic the system under consideration.
Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin
Authors: de Ruyter P G , Kuipers O P , de Vos W M
Published: January 2020

The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes. In the nisin-producing L. lactis strain NZ9700, the specific beta-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, beta-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no beta-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more beta-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The beta-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.
Estimation of the abundance of an uncultured soil bacterial strain by a competitive quantitative PCR method
Authors: Lee S Y , Bollinger J , Bezdicek D , Ogram A
Published: January 2020

Strain EA25 was identified in a clone library of bacterial 16S rRNA gene sequences that had been amplified from DNA extracted from soil collected in eastern Washington State. EA25 was subsequently shown to be related to members of the genera Planctomyces and Chlamydia and most closely related (93% similarity) to strain MC18, a strain identified in an Australian soil sample (W. Liesack and E. Stackebrandt, J. Bacteriol. 174:5072-5078, 1992). A competitive quantitative PCR method developed by Zachar et al. (V. Zachar, R.A. Thomas, and A.S. Goustin, Nucleic Acids Res. 21:2017-2018, 1993) was used to estimate the abundance of this uncultured strain in soil. An estimation of the abundance of EA25 was based on the number of copies of the sequence in the DNA extracted and the efficiency of the DNA extraction. In addition, amplification rates of Escherichia coli DNAs added to soil were shown to be similar to those of DNAs from laboratory cultures of E. coli. The number of EA25 16S rRNA genes was estimated to be 2.17 x 10(8) copies per g of soil, suggesting that strains similar to EA25 and the similar Australian strain could be widely distributed and present in significant numbers in soils from temperate regions. This represents the first enumeration of 16S rDNA copies from an uncultured strain in soil.
Complete Sucrose Metabolism Requires Fructose Phosphotransferase Activity in Corynebacterium glutamicum To Ensure Phosphorylation of Liberated Fructose
Authors: Dominguez H , Lindley N D
Published: January 2020

Sucrose uptake by Corynebacterium glutamicum involves a phosphoenolpyruvate-dependent sucrose phosphotransferase (PTS), but in the absence of fructokinase, further metabolism of the liberated fructose requires efflux of the fructose and reassimilation via the fructose PTS. Mutant strains lacking detectable fructose-transporting PTS activity accumulated fructose extracellularly but consumed sucrose at rates comparable to those of the wild-type strain.
Prevalence of Lyme disease spirochetes in Ixodes persulcatus and wild rodents in far eastern Russia
Authors: Sato Y , Miyamoto K , Iwaki A , Masuzawa T , Yanagihara Y , Korenberg E I , Gorelova N B , Volkov V I , Ivanov L I , Liberova R N
Published: January 2020

Borrelia spirochetes were isolated from the adult ixodid tick (Ixodes persulcatus) in three areas of far eastern Russia, namely, Khabarovsk, Vladivostok, and Yuzhno-Sakhalinsk. Borrelia infective rates of ticks in those areas were 24.5, 41.4, and 25.1%, respectively (total rate was 26.6%). Spirochetes were also isolated from the tissues of small mammals captured at Khabarovsk (infective rate was 20.8%). Samples were classified by rRNA gene restriction fragment length polymorphism (RFLP) analysis. The isolated spirochetes from ticks were classified mainly RFLP ribotype group IV (B. garinii), followed by groups II (B. garinii), III (B. afzelii), and V (B. garinii), showing that B. garinii is a dominant species among them. Both B. garinii and B. afzelii were also found in rodents, and multiple infections with those two species were observed in some rodents. B. burgdorferi sensu stricto (group I) was not isolated from either ticks or rodents.
Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat
Authors: Santegoeds C M , Nold S C , Ward D M
Published: January 2020

Previous studies investigating microbial diversity in the Octopus Spring cyanobacterial mat community (Yellowstone National Park) have shown a discrepancy between bacterial populations observed by molecular retrieval and cultivation techniques. To investigate how selective enrichment culture techniques affect species composition, we used denaturing gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S rRNA gene fragments to monitor the populations contained within enrichment cultures of aerobic chemoorganotrophic bacteria from the ca. 50 degrees C region of the mat community. By varying the degree of dilution of the inoculum, medium composition, and enrichment conditions and duration and by analyzing the cultures by DGGE, we detected 14 unique 16S rRNA sequence types. These corresponded to alpha-, beta-, gamma-, and delta-proteobacteria, Thermus relatives, and gram-positive bacteria with high G + C ratio and, at the highest inoculum dilutions, Chloroflexus aurantiacus relatives, which were estimated to still be approximately 300 times less abundant than cells of the mat primary producer, Synechococcus spp. Only three of these populations were previously cultivated on solidified medium after similar enrichment. Only two of these population have 16S rRNA sequences which were previously cloned directly from the mat. These results reveal a diversity of bacterial populations in enrichment culture which were not detected by either molecular retrieval or strain purification techniques.
Evaluation of test media for routine monitoring of Escherichia coli in nonpotable waters
Authors: Gaudet I D , Florence L Z , Coleman R N
Published: January 2020

Six test media, m-TEC, m-TEC with 4-methylumbelliferyl-beta-D-glucuronide (MUG), lauryl tryptose agar (LTA) with MUG, LTA with 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Glue), EC medium with MUG, and lauryl tryptose broth with MUG, were evaluated for their usefulness in enumerating Escherichia coli in nonpotable waters on a routine basis. The media were chosen for their case of interpretation of target colonies, ability to allow enumeration at low and high concentrations, and ability to inhibit nontarget microorganisms. The recoveries on the test media were compared with those on three reference media, R2A, m-FC, and m-Endo, by analysis of spiked samples of filter-sterilized waters. The test media were then further tested for their ability to differentiate nontarget but closely related microorganisms. Statistical analysis indicated that the best recoveries were obtained with lauryl tryptose agar with added MUG and X-Gluc. The media were then tested with surface waters that could be expected to have high levels of total and fecal coliforms along with Escherichia coli.
Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR
Authors: Bourne D G , Jones G J , Blakeley R L , Jones A , Negri A P , Riddles P
Published: January 2020

An isolated bacterium, identified as a new Sphingomonas species, was demonstrated to contain a novel enzymatic pathway which acted on microcystin LR, the most common cyanobacterial cyclic peptide toxin. Degradation of microcystin LR was mediated by at least three intracellular hydrolytic enzymes. The use of classic protease inhibitors allowed (i) the classification of these enzymes into general protease families and (ii) the in vitro accumulation of otherwise transient microcystin LR degradation products. The initial site of hydrolytic cleavage of the parent cyclic peptide by an enzyme that we designate microcystinase is at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg peptide bond. Two intermediates of microcystin LR enzymatic degradation have been identified; one is linearized (acyclo-) microcystin LR, NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-Leu-beta-methylas partate-Arg-OH, and the other is the tetrapeptide NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-OH. The intermediate degradation products were less active than the parent cyclic peptide; the observed 50% inhibitory concentrations for crude chicken brain protein phosphatase were 0.6 nM for microcystin LR, 95 nM for linear LR, and 12 nM for the tetrapeptide. These linear peptides were nontoxic to mice at doses up to 250 micrograms/kg. Ring opening of the potent hepatotoxin microcystin LR by bacterial microcystinase effectively renders the compound nontoxic by dramatically reducing the interaction with the target protein phosphatase.
Enzymes of Poly-(beta)-Hydroxybutyrate Metabolism in Soybean and Chickpea Bacteroids
Authors: Kim S A , Copeland L
Published: January 2020

The enzymatic capacity for metabolism of poly-(beta)-hydroxybutyrate (PHB) has been examined in nitrogen-fixing symbioses of soybean (Glycine max L.) plants, which may accumulate substantial amounts of PHB, and chickpea (Cicer arietinum L.) plants, which contain little or no PHB. In the free-living state, both Bradyrhizobium japonicum CB 1809 and Rhizobium sp. (Cicer) CC 1192, which form nodules on soybean and chickpea plants, respectively, produced substantial amounts of PHB. To obtain information on why chickpea bacteroids do not accumulate PHB, the specific activities of enzymes of PHB metabolism (3-ketothiolase, acetoacetyl-coenzyme A reductase, PHB depolymerase, and 3-hydroxybutyrate dehydrogenase), the tricarboxylic acid cycle (malate dehydrogenase, citrate synthase, and isocitrate dehydrogenase), and related reactions (malic enzyme, pyruvate dehydrogenase, and glutamate:2-oxoglutarate transaminase) were compared in extracts from chickpea and soybean bacteroids and the respective free-living bacteria. Significant differences were noted between soybean and chickpea bacteroids and between the bacteroid and free-living forms of Rhizobium sp. (Cicer) CC 1192, with respect to the capacity for some of these reactions. It is suggested that a greater potential for oxidizing malate to oxaloacetate in chickpea bacteroids may be a factor that favors the utilization of acetyl-coenzyme A in the tricarboxylic acid cycle over PHB synthesis.
Nitrogen removal by tubular gel containing Nitrosomonas europaea and Paracoccus denitrificans
Authors: Uemoto H , Saiki H
Published: January 2020

A new bioreactor for the removal of nitrogen from wastewater is described which consists of a tubular polymeric gel containing Nitrosomonas europaea and Paracoccus denitrificans. The outer surface of the tube is in aerobic contact with wastewater containing ammonia, while the inside of the tube is in anaerobic contact with ethanol flowing through the tube. N. europaea oxidizes ammonia to nitrite in the gel, and then P. denitrificans reduces the nitrite to nitrogen gas in the same gel. This concept would be effective for simplifying nitrogen removal systems requiring aerobic and anaerobic operations.
Ex vivo protocol for testing virus survival on human skin: experiments with herpesvirus 2
Authors: Graham M L , Springthorpe V S , Sattar S A
Published: January 2020

We report an ex vivo method, which uses pieces of human skin excised during routine plastic surgery, for testing survival of hazardous pathogens. Using this procedure, we compared the survival of human herpesvirus 2 on human skin and on metal disks. At the physiological skin temperature of 32 degrees C, the half-life of the virus on skin was 1.44 h while on metal disks it was 0.36 h. Even at ambient temperature (22 degrees C), the virus lost infectivity faster (half-life = 0.96 h) on metal disks than on the skin at 32 degrees C. The method described could be used to assess the survival of other human pathogens on skin and to evaluate the germicidal activity of handwashing agents and other topicals.
PCR amplification of the fimA gene sequence of Salmonella typhimurium, a specific method for detection of Salmonella spp
Authors: Cohen H J , Mechanda S M , Lin W
Published: January 2020

The goal of this study was to evaluate the suitability of the fimA gene amplification by PCR as a specific method for detection of Salmonella strains. Salmonella typhimurium and other pathogenic members of the family Enterobacteriaceae produce morphologically and antigenically related, thin, aggregative, type 1 fimbriae. A single gene, fimA, encodes the major fimbrial unit. In order to obtain higher specificity, we have selected a series of primers internal to the fimA gene sequence and have developed a PCR method for detecting Salmonella strains. A collection of 376 strains of Salmonella comprising over 80 serovars, isolated from animals and humans in Canada, have been used to evaluate this PCR method. Forty non-Salmonella strains were also tested by the same procedure. Cultures were screened by inoculating a single colony of bacteria directly into a PCR mixture containing a pair of primers specific for the fimA gene. The specific PCR product is an 85-bp fragment which was visualized by polyacrylamide gel electrophoresis and ethidium bromide staining. All Salmonella strains gave positive results by the PCR. Feed and milk samples contaminated by Salmonella strains were also detected by this procedure. The detection of all Salmonella strains tested and the failure to amplify the fragment from non-Salmonella strains confirm that the fimA gene contains sequences unique to Salmonella strains and demonstrate that this gene is a suitable PCR target for detection of Salmonella strains in food samples.
Degradation of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans by the white rot fungus Phanerochaete sordida YK-624
Authors: Takada S , Nakamura M , Matsueda T , Kondo R , Sakai K
Published: January 2020

A method for the degradation of dioxins by white rot fungi was developed. Degradation of a mixture of 10 kinds of tetra- to octachlorodibenzo-p-dioxins (polychlorinated dibenzo-p-dioxins [PCDDs]) and tetra- to octachlorodibenzofurans (polychlorinated dibenzofurans [PCDFs]), which were chlorinated at 2-, 3-, 7-, and 8-positions of the molecules, by the white rot fungus Phanerochaete sordida YK-624 was studied in a stationary low-nitrogen medium. The percent degradation values of PCDDs and PCDFs were approximately 40 (tetra-chloro-) to 76% (hexachloro-) and 45 (tetrachloro-) to 70% (hexachloro-), respectively. Metabolites of 2,3,7,8-tetra- and octaCDD formed by P. sordida YK-624 included 4,5-dichlorocatechol and tetrachlorocatechol, respectively. These results suggest that white rot fungus is able to substantially degrade both PCDDs and PCDFs. This is the first report of the degradation of highly chlorinated PCDDs and PCDFs by a microorganism.
Ochratoxin production by Aspergillus species
Authors: Varga J , Kevei E , Rinyu E , Téren J , Kozakiewicz Z
Published: January 2020

Ochratoxin production was tested in 172 strains representing species in sections Fumigati, Circumdati, Candidi, and Wentii of the genus Aspergillus by an immunochemical method using a monoclonal antibody preparation against ochratoxin A. Ochratoxin A was detected in Aspergillus ochraceus, A. alliaceus, A. sclerotiorum, A. sulphureus, A. albertensis, A. auricomus, and A. wentii strains. This is the first report of production of ochratoxins in the latter three species. Ochratoxin production by these species was confirmed by high-performance thin-layer chromatography and by high-performance liquid chromatography. The chemical methods also indicated the production of ochratoxin B by all of the Aspergillus strains mentioned above.
Analysis of cumene (isopropylbenzene) degradation genes from Pseudomonas fluorescens IP01
Authors: Habe H , Kasuga K , Nojiri H , Yamane H , Omori T
Published: January 2020

We obtained the DNA fragments encoding 2-hydroxy-6-oxo-7-methylocta-2,4-dienoic acid (HOMODA) hydrolase in the cumene (isopropylbenzene) degrader Pseudomonas fluorescens strain IP01 via PCR using two synthesized oligonucleotides corresponding to the conserved regions within known meta-cleavage compound hydrolases. Following colony hybridization using the amplified DNA as a probe, a 4.5-kb HindIII fragment was isolated from P. fluorescens IP01. After determining the nucleotide sequence of this fragment, three open reading frames (ORF11 [cumH], ORF12 [cumD], and ORF13) were identified. The deduced amino acid sequence of ORF12 showed homology with meta-cleavage compound hydrolases encoded by the tod, dmp, xyl, and bph operons. Although the product of ORF12 was found to exhibit HOMODA and 2-hydroxy-6-oxohepta-2,4-dienoic acid (HOHDA) hydrolase activities, it did not exhibit 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase activity. The deduced amino acid sequence of ORF11 showed 40.4% homology with the sequence of todX in Pseudomonas putida F1 (Y. Wang, M. Ralings, D. T. Gibson, D. Labbé, H. Bergeron, R. Brousseau, and P. C. K. Lau, Mol. Gen. Genet. 246:570-579, 1995). The nucleotide sequence of ORF13 and its flanking region showed strong homology (91.0%) with IS52 from Pseudomonas savastanoi (Y. Yamada, P.-D. Lee, and T. Kosuge, Proc. Natl. Acad. Sci. USA 83:8263-8267, 1982). By characterization of cumH and cumD, the entire cum gene cluster from the cumene-degrader P. fluorescens IP01 (cumA1A2A3A4BCEGFHD) has been identified.
Morphological and metabolic responses to starvation by the dissimilatory metal-reducing bacterium Shewanella alga BrY
Authors: Caccavo F , Ramsing N B , Costerton J W
Published: January 2020

The response of the dissimilatory metal-reducing bacterium Shewanella alga BrY to carbon and nitrogen starvation was examined. Starvation resulted in a gradual decrease in the mean cell volume from 0.48 to 0.2 micron 3 and a dramatic decrease in Fe(III) reductase activity. Growth of starved cultures was initiated with O2, ferric oxyhydroxide, Co(III)-EDTA, or Fe(III)-bearing subsurface materials as the sole electron acceptor. Microbially reduced subsurface materials reduced CrO(4)2-. Starvation of dissimilatory metal-reducing bacteria may provide a means of delivering this metabolism to contaminated subsurface environments for in situ bioremediation.
The endosymbiont (Buchnera sp.) of the aphid Diuraphis noxia contains plasmids consisting of trpEG and tandem repeats of trpEG pseudogenes
Authors: Lai C Y , Baumann P , Moran N
Published: January 2020

Most aphids are dependent for their survival on prokaryotic endosymbionts assigned to the genus Buchnera. Among the functions of Buchnera species is the synthesis of tryptophan, which is required by the aphid host. In Buchnera species from the aphid Diuraphis noxia, the genes for anthranilate synthase (trpEG) were found on a plasmid which consisted of seven tandem repeats of a 3.2-kb unit and one 2.6-kb unit which differed in containing a 0.6-kb deletion. One of the 3.2-kb units contained open reading frames corresponding to trpEG; the remaining units contained trpEG pseudogenes (psi). The nucleotide sequence upstream of trpE contained a region that has characteristics of an origin of replication (ori). Relative to trpB (a chromosomal gene), there were about two copies of the trpEG-containing plasmid. Comparisons of the nucleotide sequences of the 3.2-kb units containing trpEG and psi trpEG indicated that most changes occurred in a 700-nucleotide segment that included the region upstream of trpE and the portion of this gene coding for the N terminus. The consequence of these changes was the silencing of trpEG by inactivation of the putative promoter region and premature termination of the TrpE peptide. In contrast, the nucleotide sequence of the segment corresponding to ori was conserved in the units containing trpEG and psi trpEG. We offer a number of speculations on the evolutionary pressure in this lineage which resulted in the silencing of most of trpEG while still retaining the regions resembling ori.
Bacteriocin Typing of Burkholderia (Pseudomonas) solanacearum Race 1 of the French West Indies and Correlation with Genomic Variation of the Pathogen
Authors: Frey P , Smith J J , Albar L , Prior P , Saddler G S , Trigalet-Demery D , Trigalet A
Published: January 2020

Burkholderia solanacearum race 1 isolates indigenous to the French West Indies were characterized by bacteriocin typing and two genomic fingerprinting methods: pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction endonucleases (RC-PFGE) and PCR with primers corresponding to repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and BOX elements (collectively known as rep-PCR). The survey comprised 24 reference strains and 65 isolates obtained from a field trial in Guadeloupe in 1993. Comparison of the data identified RC-PFGE as the most discriminatory method, delineating 17 pulsed-field gel profile types. rep-PCR and bacteriocin typing identified nine rep-PCR profile types and nine bacteriocin groups. Independent determination of similarity coefficients and clustering of RC-PFGE and rep-PCR data identified six groups common to both sets of data that correlated to biovar and bacteriocin groups. Further study of bacteriocin production in planta gave results consistent with in vitro bacteriocin typing. It was observed that spontaneous bacteriocin-resistant mutants exhibited a cross-resistance to other bacteriocins as identified by the typing scheme and that such mutants possessed a selective advantage for growth over isogenic nonmutants in the presence of a bacteriocin. The results are significant in the search for biological control of disease by nonpathogenic mutants of the wild-type organism.
Comparative sporicidal effects of liquid chemical agents
Authors: Sagripanti J L , Bonifacino A
Published: January 2020

We compared the effectiveness of glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid, cupric ascorbate (plus a sublethal amount of hydrogen peroxide), sodium hypochlorite, and phenol to inactivate Bacillus subtilis spores under various conditions. Each chemical agent was distinctly affected by pH, storage time after activation, dilution, and temperature. Only three of the preparations (hypochlorite, peracetic acid, and cupric ascorbate) studied here inactivated more than 99.9% of the spore load after a 30-min incubation at 20 degrees C at concentrations generally used to decontaminate medical devices. Under similar conditions, glutaraldehyde inactivated approximately 90%, and hydrogen peroxide, formaldehyde, and phenol produced little killing of spores in suspension. By kinetic analysis at different temperatures, we calculated the rate of spore inactivation (k) and the activation energy of spore killing (delta E) for each chemical agent. Rates of spore inactivation had a similar delta E value of approximately 20 kcal/mol (ca.83.68 kJ/mol) for every substance tested. The variation among k values allowed a quantitative comparison of liquid germicidal agents.
Characterization, distribution, and localization of ISRl2, and insertion sequence element isolated from Rhizobium leguminosarum bv. viciae
Authors: Mazurier S I , Rigottier-Gois L , Amarger N
Published: January 2020

An insertion sequence (IS) element, ISR12, from Rhizobium leguminosarum bv. viciae strain MSDJ4184 was isolated by insertional inactivation of the sacRB gene of pSUP104-sac, which allows positive selection. ISRl2 is 932 bp long, is flanked by 17-bp imperfect terminal inverted repeats, and generated a 3-bp target site duplication. ISRl2 was found to be 63 to 77% homologous to insertion elements of the IS5 group of the IS4 superfamily. A probe incorporating a full-length copy of ISRl2 was used to screen genomic DNAs from a collection of strains and from two field populations of R. leguminosarum to detect and estimate the copy numbers of homologous sequences. Among the collection of 63 strains representing the different species and genera of members of the family Rhizobiaceae, homology to ISRl2 was found within strains belonging to Sinorhizobium meliloti and S. fredii; within four of the six recognized Rhizobium species. R. leguminosarum, R. tropici, R. etli, and R. galegae; and within Rhizobium sp. (Phaseolus) genomic species 2. The apparent copy numbers of ISRl2 varied from one to eight. Among 139 isolates of R. leguminosarum from two field populations, homology to ISRl2 was detected in 91% of the isolates from one site and in 17% from the other. Analysis of the 95 isolates that hybridize to ISRl2 revealed a total of 20 distinct hybridization patterns composed of one to three bands. Probing blots of Eckhardt gels showed that sequences with homology to ISRl2 may be found on plasmids or the chromosome. Analysis of their genomic distribution demonstrated relationships and diversity among the R. leguminosarum isolates tested.
Use of radiolabelled thymidine and leucine to estimate bacterial production in soils from continental antarctica
Authors: Tibbles B J , Harris J M
Published: January 2020

Tritiated thymidine incorporation (TTI) into DNA was used to examine bacterial production in two soil types from the Robertskollen group of nunataks in northwestern Dronning Maud Land, providing the first estimates of bacterial production in soil habitats on the Antarctic continent. Although estimates of bacterial productivity in soils near to bird nests (344 (plusmn) 422 ng of C g [dry weight](sup-1) h(sup-1)) were higher than those for soils from beneath mosses (175 (plusmn) 90 ng of C g [dry weight](sup-1) h(sup-1); measured by TTI at 10(deg)C), these differences were not significant because of patchiness of bacterial activity (P > 0.05). TTI- and [(sup14)C]leucine ([(sup14)C]Leu)-derived estimates of bacterial production were similar when incubations of 3 h were used, although incubations as short as 1 h were sufficient for measurable uptake of radiolabel. Dual-label incorporation of [(sup3)H]thymidine ([(sup3)H]TdR) into DNA and [(sup14)C]Leu into protein indicated that TTI did not reflect bacterial production of in situ assemblages when incubations were longer than 3 h. Isotope dilution analysis indicated that dilution of the specific activity of exogenously supplied [(sup3)H]TdR by de novo synthesis of TdR precursor could be limited by additions of [(sup3)H]TdR at a concentration of 1 nmol per ca. 115 mg of soil. TTI exhibited a psychrotrophic response to variation in temperature, with a temperature optimum of ca. 15(deg)C and a Q(inf10) value for 0 to 10(deg)C of 2.41.
Identification of genetic heterogeneity in the Cryptosporidium parvum ribosomal repeat
Authors: Carraway M , Tzipori S , Widmer G
Published: January 2020

Oocysts of the protozoan parasite Cryptosporidium parvum are found in most surface waters and can contaminate municipal water supplies, as demonstrated by recent outbreaks of cryptosporidiosis. A method capable of fingerprinting C. parvum isolates from the environment would facilitate the study of epidemiology and transmission cycles and aid in the implementation of preventive measures to reduce water contamination by oocytes. In this study, we report polymorphism in C. parvum isolates on the basis of analysis of random amplified polymorphic DNA and nucleotide sequences in a region of the 18S rRNA and the internal transcribed spacer 1. Isolate-specific primers for these two regions were designed, and PCR tests capable of discriminating between isolates were developed. In both PCR assays, the five C. parvum isolates analyzed segregated into two subgroups. One group consisted of isolates that originated directly from human patients, and the other group had various host origins and had been propagated in laboratory animals. These results demonstrate the feasibility of distinguishing C. parvum isolates by sequence-specific PCR tests.
Food-grade cloning and expression system for Lactococcus lactis
Authors: Platteeuw C , van Alen-Boerrigter I , van Schalkwijk S , de Vos W M
Published: January 2020

A versatile set of cloning and expression vectors has been developed for application in self-cloning and other genetic modifications of Lactococcus lactis. The expression vectors were equipped with the controlled and strong lacA promoter of the lactococcal lactose operon. In addition, the transcriptional terminator of the aminopeptidase N gene, pepN, was inserted, which in some cases increased the genetic stabilities of the vectors and the cloned DNA. The small, 0.3-kb lacF gene encoding the soluble carrier enzyme IIALac was used as a dominant selection marker in the plasmid-free L. lactis strain NZ3000 carrying an in-frame deletion of the chromosomal lacF gene. Lactose-utilizing transformants were easily selected on lactose indicator plates at high frequencies and showed a copy number of approximately 50 plasmids per cell. All vectors were stably maintained in the lacF strain NZ3000 when grown on lactose, and only the high-level expression vectors showed some instability when their host was grown on glucose-containing medium. The application potentials of the expression vectors carrying the lacF marker were determined by cloning of the promoterless Escherichia coli gusA reporter gene under control of the lacA promoter followed by analysis of its expression. While in one of the vectors this resulted in a promoter-down mutation in the -10 region of the lacA promoter, in other vectors high-level and controlled expression of the gusA gene was observed.
Characterization of a restriction-modification system of the thermotolerant methylotroph Bacillus methanolicus
Authors: Cue D , Lam H , Hanson R S , Flickinger M C
Published: January 2020

We report the isolation of a restriction endonuclease, BmeTI, an isoschizomer of BclI, that recognizes the DNA sequence 5' TGATCA 3'. We also report that BmeTI sites are modified to TGm6ATCA. These findings provide the basis for devising strategies to prevent BmeTI restriction of any DNA introduced into Bacillus methanolicus.
Development of a Defined Minimal Medium for the Growth of Edwardsiella ictaluri
Authors: Collins L A , Thune R L
Published: January 2020

In this report, a complete defined medium and a minimally defined medium are described for Edwardsiella ictaluri. The complete defined medium consists of 46 individual components, including a basal salt solution, glucose, magnesium sulfate, iron sulfate, six trace metals, four nucleotides, 10 vitamins, and 19 amino acids. This medium supports growth in broth and on solid media. Optimal growth at 30(deg)C was obtained at pH 7.0, and at an osmolality of 390 mosmol/kg of H(inf2)O, with a glucose concentration of 4 g/liter. The defined minimal medium reduces the 46 components of the complete medium to eight essential components, including the basal salt solution, glucose, magnesium sulfate, pantothenic acid, and niacinamide. In addition, specific amino acids that depend on the specific requirements of the individual strains of E. ictaluri are added.
A computer analysis of primer and probe hybridization potential with bacterial small-subunit rRNA sequences
Authors: Brunk C F , Avaniss-Aghajani E , Brunk C A
Published: January 2020

Analysis of restriction fragment length polymorphism of bacterial small-subunit (SSU) rRNA sequences represents a potential means for characterizing complex bacterial populations such as those found in natural environments. In order to estimate the resolution potential of this approach, we have examined the SSU rRNA sequences in the Ribosomal Database Project bank using a computer algorithm which simulates hybridization between DNA sequences. Simulated hybridizations between a primer or probe sequence and an SSU rRNA sequence yield a value for each potential hybridization. This algorithm has been used to evaluate sites for PCR primers and hybridization probes used for classifying SSU rRNA sequences. Our analysis indicates that length variation in terminal restriction fragments of PCR products from the SSU rRNA sequences can identify a wide spectrum of bacteria. We also observe that the majority of restriction fragment length variation is the result of insertions and deletions rather than restriction site polymorphisms. This approach is also used to evaluate the relative efficiency and specificity of a number of published hybridization probes.
Assessment of indoor climate in an apartment by use of a fungal index
Authors: Abe K , Nagao Y , Nakada T , Sakuma S
Published: January 2020

Indoor climate was assessed in an apartment in Isehara City, Kanagawa Prefecture, Japan, by use of a fungal index. The index represents the environmental (climate) capacity to allow fungal growth; it is determined by measuring the growth rate of a biosensor fungus, Eurotium herbariorum J-183. Differences in climate among various parts of the apartment (microclimate) and its changes could be clarified by using the index. The index in the entire apartment was high in summer, low in winter, and intermediate in spring and autumn. According to the part of the apartment, the index was high in water-associated areas and cool areas. This high fungal index in cool areas was caused by the air at the same absolute humidity showing an increase in the relative humidity with a decrease in temperature. Fungal contamination rapidly progressed in areas with a high fungal index in this apartment. A correlation was observed between the fungal index and fungal contamination. Therefore, areas susceptible to fungal contamination can be estimated by use of the fungal index.
Identification of Aeromonas hydrophila hybridization group 1 by PCR assays
Authors: Cascón A , Anguita J , Hernanz C , Sánchez M , Fernández M , Naharro G
Published: January 2020

Synthetic oligonucleotide primers of 24 and 23 bases were used in a PCR assay to amplify a sequence of the lip gene, which encodes a thermostable extracellular lipase of Aeromonas hydrophila. A DNA fragment of approximately 760 bp was amplified from both sources, i.e., lysed A. hydrophila cells and isolated DNA. The amplified sequence was detected in ethidium bromide-stained agarose gels or by Southern blot analysis with an internal HindIII-BamHI 356-bp fragment as a hybridization probe. With A. hydrophila cells, the sensitivity of the PCR assay was < 10 CFU, and with the isolated target, the lower detection limit was 0.89 pg of DNA. Primer specificity for A. hydrophila was determined by the PCR assay with cells of 50 strains of bacteria, including most of the 14 currently recognized DNA hybridization groups of Aeromonas spp. as well as other human and environmental Aeromonas isolates. Detection of A. hydrophila by PCR amplification of DNA has great potential for rapid identification of this bacterium because it has proved to be highly specific.
Aflatoxin variability in pistachios
Authors: Mahoney N E , Rodriguez S B
Published: January 2020

Pistachio fruit components, including hulls (mesocarps and epicarps), seed coats (testas), and kernels (seeds), all contribute to variable aflatoxin content in pistachios. Fresh pistachio kernels were individually inoculated with Aspergillus flavus and incubated 7 or 10 days. Hulled, shelled kernels were either left intact or wounded prior to inoculation. Wounded kernels, with or without the seed coat, were readily colonized by A. flavus and after 10 days of incubation contained 37 times more aflatoxin than similarly treated unwounded kernels. The aflatoxin levels in the individual wounded pistachios were highly variable. Neither fungal colonization nor aflatoxin was detected in intact kernels without seed coats. Intact kernels with seed coats had limited fungal colonization and low aflatoxin concentrations compared with their wounded counterparts. Despite substantial fungal colonization of wounded hulls, aflatoxin was not detected in hulls. Aflatoxin levels were significantly lower in wounded kernels with hulls than in kernels of hulled pistachios. Both the seed coat and a water-soluble extract of hulls suppressed aflatoxin production by A. flavus.
Hydrophobic and electrostatic cell surface properties of Cryptosporidium parvum
Authors: Drozd C , Schwartzbrod J
Published: January 2020

Microbial adhesion to hydrocarbons and microelectrophoresis were investigated in order to characterize the surface properties of Cryptosporidium parvum. Oocysts exhibited low removal rates by octane (only 20% on average), suggesting that the Cryptosporidium sp. does not demonstrate marked hydrophobic properties. A zeta potential close to -25 mV at pH 6 to 6.5 in deionized water was observed for the parasite. Measurements of hydrophobicity and zeta potential were performed as a function of pH and ionic strength or conductivity. Hydrophobicity maxima were observed at extreme pH values, with 40% of adhesion of oocysts to octane. It also appeared that ionic strength (estimated by conductivity) could influence the hydrophobic properties of oocysts. Cryptosporidium oocysts showed a pH-dependent surface charge, with zeta potentials becoming less negative as pH was reduced, starting at -35 mV for alkaline pH and reaching 0 at isoelectric points for pH 2.5. On the other hand, variation of surface charge with respect to conductivity of the suspension tested in this work was quite small. The knowledge of hydrophobic properties and surface charge of the parasite provides information useful in, for example, the choice of various flocculation treatments, membrane filters, and cleaning agents in connection with oocyst recovery.
Responses to Stress and Nutrient Availability by the Marine Ultramicrobacterium Sphingomonas sp. Strain RB2256
Authors: Eguchi M , Nishikawa T , Macdonald K , Cavicchioli R , Gottschal J C , Kjelleberg S
Published: January 2020

Sphingomonas sp. strain RB2256 was isolated from Resurrection Bay in Alaska and possibly represents the dominant bacterial species in some oligotrophic marine environments. Strain RB2256 has a high-affinity nutrient uptake system when growing under nutrient-limiting conditions, and growing cells are very small (<0.08 (mu)m(sup3)). These characteristics indicate that RB2256 is highly evolved for withstanding nutrient limitations and grazing pressure by heterotrophic nanoflagellates. In this study, strain RB2256 was subjected to nutrient starvation and other stresses (high temperature, ethanol, and hydrogen peroxide). It was found that growing cells were remarkably resistant, being able to survive at a temperature of 56(deg)C, in 25 mM hydrogen peroxide, or in 20% ethanol. In addition, growing cells were generally as resistant as starved cells. The fact that vegetative cells of this strain are inherently resistant to such high levels of stress-inducing agents indicates that they possess stress resistance mechanisms which are different from those of other nondifferentiating bacteria. Only minor changes in cell volume (0.03 to 0.07 (mu)m(sup3)) and maximum specific growth rate (0.13 to 0.16 h(sup-1)) were obtained for cells growing in media with different organic carbon concentrations (0.8 to 800 mg of C per liter). Furthermore, when glucose-limited, chemostat-grown cultures or multiple-nutrient-starved batch cultures were suddenly subjected to excess glucose, maximum growth rates were reached immediately. This immediate response to nutrient upshift suggests that the protein-synthesizing machinery is constitutively regulated. In total, these results are strong evidence that strain RB2256 possesses novel physiological and molecular strategies that allow it to predominant in natural seawater.
Cloning of a cDNA encoding cellobiose dehydrogenase, a hemoflavoenzyme from Phanerochaete chrysosporium
Authors: Li B , Nagalla S R , Renganathan V
Published: January 2020

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by cellulose-degrading cultures of the wood-degrading basidiomycete Phanerochaete chrysosporium. CDH contains one flavin adenine dinucleotide (FAD) and one heme b per molecule, and it oxidizes cellobiose to cellobionolactone. In this report, a 2.4-kb cDNA encoding CDH was isolated by screening an expression library of P. chrysosporium OGC101 with a CDH-specific polyclonal antibody. The cDNA encodes a 755-amino-acid protein with a predicted mass of 80,115 Da. Sequence analysis suggests that the heme domain is located at the N terminus and that the falvin domain is located at the C terminus. The flavin domain shows a beta 1-alpha A-beta 2 motif for FAD binding and has high sequence similarity to several FAD-dependent enzymes. Little sequence similarity to hemoflavoenzymes is found. CDH binds to cellulose similarly to cellulases. However, little sequence similarity is observed with the conserved cellulose-binding sequences of cellulases. This suggests that CDH might possess a specific sequence for cellulose binding which is different from that of cellulases. Northern (RNA) blot analysis of total RNA from cellulose-, glucose-, and cellobiose-grown P. chrysosporium indicated that CDH mRNA is produced only in cellulose-grown cells. This suggests that CDH expression is regulated at the transcriptional level by either cellulose or one of its degradation products. Southern blot analysis suggests the presence of only a single gene for CDH in P. chrysosporium OGC101.
Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA
Authors: Kuo W S , Chak K F
Published: January 2020

Two pairs of universal oligonucleotide primers were designed to probe the most conserved regions of all known cryI-type gene sequences so that the amplified PCR fragments of the DNA template from Bacillus thuringiensis strains may contain all possible cryI-type gene sequences. The restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified fragments revealed that 14 distinct cry-type genes have been identified from 20 B. thuringiensis strains. Those cry-type genes included cryIA(a), cryIA(a), cryIA(b), cryIA(b), cryIA(c), cryIB, cryIC, cryIC, cryIC(b), cryID, cryIE, cryIF, cryIF, and cryIII (a dagger at the end of a gene designation indicates a novel cry-type gene determined by restriction mapping or DNA sequences). Among them, the sequences of cryIA(a), cryIA(b), cryIB, cryIC, cryIF, and cryIII were found to be different from the corresponding published cry gene sequences. Interestingly, five cry-type genes [cryIA(a)-, cryIB-, cryIC-, cryIC(b)-, and cryIF-type genes] and seven cry-type genes [cryIA(a)-, cryIA(b)-, cryIB-, cryIC-, cryIC(b)-, cryIF-, and cryIII-type genes] have been detected from B. thuringiensis subsp. morrisoni HD-12 and B. thuringiensis subsp. wuhanensis, respectively. Therefore, the PCR-RFLP typing system is a facile method to detect both known and novel cry genes existing in B. thuringiensis strains.