bluuebunny/crossref_metadata_2025 huggingface dataset:
Symbiotic Role of the Viable but Nonculturable State of Vibrio fischeri in Hawaiian Coastal Seawater
Authors: Lee K , Ruby E G
Published: January 2020

To achieve functional bioluminescence, the developing light organ of newly hatched juveniles of the Hawaiian squid Euprymna scolopes must become colonized by luminous, symbiosis-competent Vibrio fischeri present in the ambient seawater. This benign infection occurs rapidly in animals placed in seawater from the host's natural habitat. Therefore, it was surprising that colony hybridization studies with a V. fischeri-specific luxA gene probe indicated the presence of only about 2 CFU of V. fischeri per ml of this infective seawater. To examine this paradox, we estimated the total concentration of V. fischeri cells present in seawater from the host's habitat in two additional ways. In the first approach, the total bacterial assemblage in samples of seawater was collected on polycarbonate membrane filters and used as a source of both a crude cell lysate and purified DNA. These preparations were then assayed by quantitative DNA-DNA hybridization with the luxA gene probe. The results suggested the presence of between 200 and 400 cells of V. fischeri per ml of natural seawater, a concentration more than 100 times that revealed by colony hybridization. In the second approach, we amplified V. fischeri-specific luxA sequences from microliter volumes of natural seawater by PCR. Most-probable-number analyses of the frequency of positive PCR results from cell lysates in these small volumes gave an estimate of the concentration of V. fischeri luxA gene targets of between 130 and 1,680 copies per ml. From these measurements, we conclude that in their natural seawater environment, the majority of V. fischeri cells become nonculturable while remaining viable and symbiotically infective. Experimental studies indicated that V. fischeri cells suspended in natural Hawaiian seawater enter such a state within a few days.
Regulation of manganese peroxidase gene transcription by hydrogen peroxide, chemical stress, and molecular oxygen
Authors: Li D , Alic M , Brown J A , Gold M H
Published: January 2020

The expression of manganese peroxidase (MnP) in nitrogen-limited cultures of the lignin-degrading fungus Phanerochaete chrysosporium is regulated at the level of gene transcription by H2O2 and various chemicals, including ethanol, sodium arsenite, and 2,4-dichlorophenol, as well as by Mn(II) and heat shock. Northern (RNA) blot analysis demonstrates that the addition of 1.0 mM H2O2 to 5-day-old cultures grown in the absence of Mn results in the appearance of mnp mRNA within 15 min. Higher levels of mnp mRNA are obtained with simultaneous induction by Mn and H2O2 than with H2O2 alone. Although neither MnP activity nor associated protein is detectable in H2O2-induced cultures grown in the absence of Mn, simultaneous induction with Mn and H2O2 results in a 1.6-fold increase in MnP activity compared with the MnP activity resulting from Mn induction alone. In the presence of Mn, purging of low-nitrogen cultures with 100% O2, in contrast to incubation under air, results in an increase in the accumulation of mnp mRNA and a 13-fold increase in MnP activity on day 5. However, in contrast to the effects of H2O2 and heat shock, O2 purging of Mn-deficient cultures results in negligible accumulation of mnp mRNA.
Specificity of reductive dehalogenation of substituted ortho-chlorophenols by Desulfitobacterium dehalogenans JW/IU-DC1
Authors: Utkin I , Dalton D D , Wiegel J
Published: January 2020

Resting cells of Desulfitobacterium dehalogenans JW/IU-DC1 growth with pyruvate and 3-chloro-4-hydroxyphenylacetate (3-Cl-4-OHPA) as the electron acceptor and inducer of dehalogenation reductively ortho-dehalogenate pentachlorophenol (PCP); tetrachlorophenols (TeCPs); the trichlorophenols 2,3,4-TCP, 2,3,6-TCP, and 2,4,6-TCP; the dichlorophenols 2,3-DCP, 2,4-DCP, and 2,6-DCP; 2,6-dichloro-4-R-phenols (2,6-DCl-4-RPs, where R is -H, -F, -Cl, -NO2, -CO2, or -COOCH3; 2-chloro-4-R-phenols (2-Cl-4-RPs, where R is -H, -F, -Cl, -Br, -NO2, -CO2-, -CH2CO2, or -COOCH3); and the bromophenols 2-BrP, 2,6-DBrP, and 2-Br-4ClP [corrected]. Monochlorophenols, the dichlorophenols 2,5-DCP, 3,4-DCP, and 3,5-DCP, the trichlorophenols 2,3,5-TCP, 2,4,5-TCP, and 3,4,5-TCP, and the fluorinated analog of 3-Cl-4-OHPA, 3-F-4-OHPA ("2-F-4-CH2CO2- P"), are not dehalogenated. A chlorine substituent in position 3 (meta), 4 (para), or 6 (second ortho) of the phenolic moiety facilitates ortho dehalogenation in position 2. Chlorine in the 5 (second meta) position has a negative effect on the dehalogenation rate or even prevents dechlorination in the 2 position. In general, 2,6-DCl-4-RPs are dechlorinated faster than the corresponding 2-Cl-4-RPs with the same substituent R in the 4 position. The highest dechlorination rate, however, was found for dechlorination of 2,3-DCP, with a maximal observed first-order rate constant of 19.4 h-1 g (dry weight) of biomass-1.(ABSTRACT TRUNCATED AT 250 WORDS)
A Simple and Rapid Method of Transformation of Streptomyces rimosus R6 and Other Streptomycetes by Electroporation
Authors: Pigac J , Schrempf H
Published: January 2020

Usually plasmid DNA is introduced into Streptomyces strains by polyethylene glycol-mediated transformation of protoplasts. However, many Streptomyces strains are only poorly or not at all transformable via protoplasts. Therefore, we have optimized the parameters critical for the application of electrotransformation of plasmid DNA into Streptomyces species. The most critical parameters evaluated for electrotransformation of the model strain Streptomyces rimosus R6 were the pretreatment of mycelia, buffer composition, and electric field strength. The electrocompetent mycelia were prepared from 24-h-old cultures, treated mildly with lysozyme, resuspended in sucrose-glycerol-polyethylene glycol buffer, and stored in aliquots at -70 deg C. The electric field strength of 10 kV/cm at 400 (Omega) and a capacitance of 25 (mu)F was applied. The method is simple and rapid, yielding transformant colonies in 48 to 72 h. Efficiencies of 10(sup5) to 10(sup6) transformants per (mu)g of plasmid DNA were reproducibly achieved for S. rimosus R6 and its mutants, and these numbers were 10(sup2) to 10(sup3) higher than those attained by polyethylene glycol-assisted transformation of protoplasts. In addition, we show that electroporation can be applied to other Streptomyces species, such as S. lividans 66, S. coelicolor A3(2), and an S. venezuelae strain. This last one could not be transformed by the standard protoplast procedure. Our data suggest that, because of the diversity of streptomycetes, the conditions have to be optimized for each strain.
Marine Vibrio species produce the volatile organic compound acetone
Authors: Nemecek-Marshall M , Wojciechowski C , Kuzma J , Silver G M , Fall R
Published: January 2020

While screening aerobic, heterotrophic marine bacteria for production of volatile organic compounds, we found that a group of isolates produced substantial amounts of acetone. Acetone production was confirmed by gas chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography. The major acetone producers were identified as nonclinical Vibrio species. Acetone production was maximal in the stationary phase of growth and was stimulated by addition of l-leucine but not the other common amino acids, suggesting that leucine degradation leads to acetone formation. Acetone production by marine vibrios may contribute to the dissolved organic carbon associated with phytoplankton, and some of the acetone produced may be volatilized to the atmosphere.
Genetic analysis of fumonisin production and virulence of Gibberella fujikuroi mating population A (Fusarium moniliforme) on maize (Zea mays) seedlings
Authors: Desjardins A E , Plattner R D , Nelsen T C , Leslie J F
Published: January 2020

The phytopathogenic fungus Gibberella fujikuroi mating population A (anamorph, Fusarium moniliforme) produces fumonisins, which are toxic to a wide range of plant and animal species. Previous studies of field strains have identified a genetic locus, designated fum1, that can determine whether fumonisins are produced. To test the relationship between fumonisin production and virulence on maize seedlings, a cross between a fum1+ field strain that had a high degree of virulence and a fum1- field strain that had a low degree of virulence was made, and ascospore progeny were scored for these traits. Although a range of virulence levels was recovered among the progeny, high levels of virulence were associated with production of fumonisins, and highly virulent, fumonisin-nonproducing progeny were not obtained. A survey of field strains did identify a rare fumonisin-nonproducing strain that was quite high in virulence. Also, the addition of purified fumonisin B1 to virulence assays did not replicate all of the seedling blight symptoms obtained with autoclaved culture material containing fumonisin. These results support the hypothesis that fumonisin plays a role in virulence but also indicate that fumonisin production is not necessary or sufficient for virulence on maize seedlings.
Molecular and Physiological Characterization of Pseudomonas syringae pv. tomato and Pseudomonas syringae pv. maculicola Strains That Produce the Phytotoxin Coronatine
Authors: Cuppels D A , Ainsworth T
Published: January 2020

The chlorosis-inducing phytotoxin coronatine is produced by several Pseudomonas syringae pathovars, including glycinea, morsprunorum, atropurpurea, and the closely related tomato and maculicola. To date, all coronatine-producing pv. glycinea, morsprunorum, and atropurpurea strains that have been examined carry the gene cluster that controls toxin production on a large plasmid. In the present study the genomic location of the coronatine gene cluster was determined for coronatine-producing strains of the pv. tomato-maculicola group by subjecting their genomic DNA to pulsed-field electrophoresis and Southern blot analysis with a hybridization probe from the coronatine gene cluster. The cluster was chromosomally borne in 10 of the 22 strains screened. These 10 strains infected both crucifers and tomatoes but could not use sorbitol as a sole source of carbon. The remaining 12 coronatine-producing strains had plasmid-borne toxin gene clusters and used sorbitol as a carbon source. Only one of these strains was pathogenic on both crucifers and tomatoes; the remainder infected just tomatoes. Restriction fragment length polymorphism analysis of the pv. tomato-maculicola coronatine gene clusters was performed with probes from P. syringae pv. tomato DC3000, a tomato and crucifer pathogen. Although the coronatine cluster appeared, in general, to be highly conserved across the pv. tomato-maculicola group, there were significant differences between plasmid-borne and chromosomally borne genes. The extensively studied coronatine cluster of pv. glycinea 4180 closely resembled the plasmid-borne clusters of the pv. tomato-maculicola group.
Heat-tolerant methanotrophic bacteria from the hot water effluent of a natural gas field
Authors: Bodrossy L , Murrell J C , Dalton H , Kalman M , Puskas L G , Kovacs K L
Published: January 2020

Methanotrophic bacteria were isolated from a natural environment potentially favorable to heat-tolerant methanotrophs. An improved colony plate assay was developed and used to identify putative methanotrophic colonies with high confidence. Fourteen new isolates were purified and partially characterized. These new isolates exhibit a DNA sequence homology of up to 97% with the conserved regions in the mmoX and mmoC genes of the soluble methane monooxygenase (MMO)-coding gene cluster of Methylococcus capsulatus Bath. The copper regulation of soluble MMO expression in the same isolates, however, differs from that of M. capsulatus Bath, as the new isolates can tolerate up to 0.8 microM copper without loss of MMO activity while a drastic reduction of MMO activity occurs already at 0.1 microM copper in M. capsulatus Bath. The isolates can be cultivated and utilized at elevated temperatures, and their copper- and heat-tolerant MMO activity makes these bacteria ideal candidates for future biotechnological use.
Molecular characterization of genes involved in the production of the bacteriocin leucocin A from Leuconostoc gelidum
Authors: van Belkum M J , Stiles M E
Published: January 2020

Leucocin A is a small heat-stable bacteriocin produced by Leuconostoc gelidum UAL187. A 2.9-kb fragment of plasmid DNA that contains the leucocin structural gene and a second open reading frame (ORF) in an operon was previously cloned (J. W. Hastings, M. Sailer, K. Johnson, K. L. Roy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 173:7491-7500, 1991). When a 1-kb DraI-HpaI fragment containing this operon was introduced into a bacteriocin-negative variant (UAL187-13), immunity but no leucocin production was detected. Leucocin production was observed when an 8-kb SacI-HindIII fragment of the leucocin plasmid was introduced into L. gelidum UAL187-13 and Lactococcus lactis IL1403. Nucleotide sequence analysis of this 8-kb fragment revealed the presence of three ORFs in an operon upstream of and on the strand opposite from the leucocin structural gene. The first ORF (lcaE) encodes a putative protein of 149 amino acids with no apparent function in leucocin A production. The second ORF (lcaC) contains 717 codons that encode a protein homologous to members of the HlyB family of ATP-binding cassette transporters. The third ORF (lcaD) contains 457 codons that encode a protein with marked similarity to LcnD, a protein essential for the expression of the lactococcal bacteriocin lactococcin A. Deletion mutations in lcaC and lcaD resulted in loss of leucocin production, indicating that LcaC and LcaD are involved in production and translocation of leucocin A. The secretion apparatus for lactococcin A did not complement mutations in the lcaCD genes to express leucocin A in L. lactis.(ABSTRACT TRUNCATED AT 250 WORDS)
Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying
Authors: Leslie S B , Israeli E , Lighthart B , Crowe J H , Crowe L M
Published: January 2020

The microorganisms Escherichia coli DH5 alpha and Bacillus thuringiensis HD-1 show an increased tolerance to freeze-drying when dried in the presence of the disaccharides trehalose and sucrose. When the bacteria were dried with 100 mM trehalose, 70% of the E. coli and 57% of the B. thuringiensis organisms survived, compared with 56 and 44%, respectively, when they were dried with sucrose. Only 8% of the E. coli and 14% of the B. thuringiensis organisms survived drying without the sugars. Fourier transform infrared spectroscopy was used to investigate the role of membrane phase transitions in the survival of the organisms during drying and rehydration. Both E. coli and B. thuringiensis showed an increase of 30 to 40 degrees C in the temperature of their phospholipid phase transition when dried without the sugars, while phase transition temperatures of those dried with the sugars remained near those of the hydrated cells. A Fourier transform infrared spectroscopy microscope made it possible to investigate the effects of drying on the protein structure in the intact cells. The amide II peak shifts from 1,543 cm-1 in the hydrated cells to about 1,533 cm-1 in the cells dried without sugar. There is no shift in the amide II peak when the cells are dried with trehalose or sucrose. We attribute the increased survival to the sugars' ability to lower the membrane phase transition temperature and to protect protein structure in the dry state.(ABSTRACT TRUNCATED AT 250 WORDS)
A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs
Authors: Pitulle C , Hedenstierna K O , Fox G E
Published: January 2020

Further improvements in technology for efficient monitoring of genetically engineered microorganisms (GEMs) in the environment are needed. Technology for monitoring rRNA is well established but has not generally been applicable to GEMs because of the lack of unique rRNA target sequences. In the work described herein, it is demonstrated that a deletion mutant of a plasmid-borne Vibrio proteolyticus 5S rRNA gene continues to accumulate to high levels in Escherichia coli although it is no longer incorporated into 70S ribosomes. This deletion construct was subsequently modified by mutagenesis to create a unique recognition site for the restriction endonuclease BstEII, into which new sequences could be readily inserted. Finally, a novel 17-nucleotide identifier sequence from Pennisetum purpureum was embedded into the construct to create an RNA identification cassette. The artificial identifier RNA, expressed from this cassette in vivo, accumulated in E. coli to levels comparable to those of wild-type 5S rRNA without being seriously detrimental to cell survival in laboratory experiments and without entering the ribosomes. These results demonstrate that artificial, stable RNAs containing sequence segments remarkably different from those present in any known rRNA can be designed and that neither the deleted sequence segment nor ribosome incorporation is essential for accumulation of an RNA product.
Effects of environmental parameters on the formation and turnover of acetate by forest soils
Authors: Kusel K , Drake H L
Published: January 2020

The capacity to form acetate from endogenous matter was a common property of diverse forest soils when incubated under anaerobic conditions. At 15 to 20(deg)C, acetate synthesis occurred without appreciable delay when forest soils were incubated as buffered suspensions or in microcosms at various percentages of their maximum water holding capacity. Rates for acetate formation with soil suspensions ranged from 35 to 220 (mu)g of acetate per g (dry weight) of soil per 24 h, and maximal acetate concentrations obtained in soil suspensions were two- to threefold greater than those obtained with soil microcosms at the average water holding capacity of the soil. Cellobiose degradation in soil suspensions yielded H(inf2) as a transient product. Under anaerobic conditions, supplemental H(inf2) and CO(inf2) were directed towards the acetogenic synthesis of acetate, and enrichments yielded a syringate-H(inf2)-consuming acetogenic consortium. At in situ temperatures, acetate was a relatively stable anaerobic end product; however, extended incubation periods induced acetoclastic methanogenesis and sulfate reduction. Higher mesophilic and thermophilic temperatures greatly enhanced the capacity of soils to form methane. Although methanogenic and sulfate-reducing activities under in situ-relevant conditions were negligible, these findings nonetheless demonstrated the occurrence of methanogens and sulfate-reducing bacteria in these aerated terrestrial soils. In contrast to the protracted stability of acetate under anaerobic conditions at 15 to 20(deg)C with unsupplemented soils, acetate formed by forest soils was rapidly consumed in the presence of oxygen and nitrate, and substrate-product stoichiometries indicated that acetate turnover was coupled to oxygen-dependent respiration and denitrification. The collective results suggest that acetate formed under anaerobic conditions might constitute a trophic link between anaerobic and aerobic processes in forest soils.
Substrate-dependent differential splicing of introns in the regions encoding the cellulose binding domains of two exocellobiohydrolase I-like genes in Phanerochaete chrysosporium
Authors: Birch P R , Sims P F , Broda P
Published: January 2020

Recently, we have shown differential splicing of an intron in the cbhI.2 gene of Phanerochaete chrysosporium ME446; this intron lies within the region of the gene encoding the cellulose binding domain (P.F.G. Sims, M. S. Soares-Felipe, Q. Wang, M.E. Gent, C. Tempelaars, and P. Broda, Mol. Microbiol. 12:209-216, 1994). Here, we show that such differential splicing occurs in the cbhI.1 gene of this fungus as well as in the cbhI.2 gene and that this phenomenon is substrate dependent. Avicel elicits the synthesis of both classes of mRNA transcripts from both of these genes. In contrast, carboxymethyl cellulose predominantly elicits the synthesis of fully spliced transcripts from both genes. Such differential splicing might allow this fungus to regulate the specificities of substrate binding for these cellulases.
Acid adaptation of Escherichia coli O157:H7 increases survival in acidic foods
Authors: Leyer G J , Wang L L , Johnson E A
Published: January 2020

Escherichia coli O157:H7 was adapted to acid by culturing for one to two doublings at pH 5.0. Acid-adapted cells had an increased resistance to lactic acid, survived better than nonadapted cells during a sausage fermentation, and showed enhanced survival in shredded dry salami (pH 5.0) and apple cider (pH 3.4). Acid adaptation is important for the survival of E. coli O157:H7 in acidic foods and should be considered a prerequisite for inocula used in food challenge studies.
Purification of Pyoverdines of Pseudomonas fluorescens 2-79 by Copper-Chelate Chromatography
Authors: Xiao R , Kisaalita W S
Published: January 2020

Three pyoverdines, Pf-A, Pf-B, and Pf-C, were purified with copper-chelate Sepharose and Sephadex G-15 columns from Pseudomonas fluorescens 2-79, and the yields (per 100 ml of culture supernatant) were 2.8, 21.6, and 3.2 mg, respectively. The absorption and fluorescence spectra of these pyoverdines were strongly pH dependent. Characteristic changes in the maximal absorbance wavelengths were observed when Fe(sup3+) or Cu(sup2+) was added. The addition of Cu(sup2+) shifted the pyoverdine Pf-B absorbance spectrum so that it exhibited a single peak at 410 nm but did not give rise to a new absorbance maximum at approximately 460 nm, which appeared when Fe(sup3+) was added. Fluorescence quenching experiments revealed that the forward reaction rate constant with pyoverdines was much higher with Cu(sup2+) (10(sup4) to 10(sup5) M(sup-1) s(sup-1)) than with Fe(sup3+) (10(sup2) M(sup-1) s(sup-1)). However, Cu(sup2+)-pyoverdine complexes were completely dissociated by EDTA at a low concentration (0.1 mM), while the level of Fe(sup3+)-pyoverdine complex dissociation at the same EDTA concentration was relatively low. The dissociation of Fe(sup3+)-pyoverdine complexes was EDTA concentration dependent. Formation of free pyoverdine was observed when the three types of Fe(sup3+)-pyoverdine complexes were incubated separately with P. fluorescens 2-79 cells, thus demonstrating that pyoverdines Pf-A, Pf-B, and Pf-C mediate iron transport.
Bioactivation of cysteine conjugates of 1-nitropyrene oxides by cysteine conjugate beta-lyase purified from Peptostreptococcus magnus
Authors: Kataoka K , Kinouchi T , Akimoto S , Ohnishi Y
Published: January 2020

To determine the role of cysteine conjugate beta-lyase (beta-lyase) in the metabolism of mutagenic nitropolycyclic aromatic hydrocarbons, we determined the effect of beta-lyase on the mutagenicities and DNA binding of cysteine conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), which are detoxified metabolites of the mutagenic compound 1-nitropyrene. We purified beta-lyase from Peptostreptococcus magnus GAI0663, since P. magnus is one of the constituents of the intestinal microflora and exhibits high levels of degrading activity with cysteine conjugates of 1-nitropyrene oxides (1-NP oxide-Cys). The activity of purified beta-lyase was optimal at pH 7.5 to 8.0, was completely inhibited by aminooxyacetic acid and hydroxylamine, and was eliminated by heating the enzyme at 55 degrees C for 5 min. The molecular weight of beta-lyase was 150,000, as determined by fast protein liquid chromatography. S-Arylcysteine conjugates were good substrates for this enzyme. As determined by the Salmonella mutagenicity test, 5 ng of beta-lyase protein increased the mutagenicity of the cysteine conjugate of 1-NP 9,10-oxide (10 nmol per plate) 4.5-fold in Salmonella typhimurium TA98 and 4.1-fold in strain TA100. However, beta-lyase had little effect on the cysteine conjugate of 1-NP 4,5-oxide (10 nmol per plate). Both conjugates exhibited only low levels of mutagenicity with nitroreductase-deficient strain TA98NR. In vitro binding of 1-NP oxide-Cys to calf thymus DNA was increased by adding purified beta-lyase or xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)
Bioconversion of Cellulose to Acetate with Pure Cultures of Ruminococcus albus and a Hydrogen-Using Acetogen
Authors: Miller T L , Wolin M J
Published: January 2020

Bioconversion of cellulose to acetate was accomplished with cocultures of two organisms. One was the cellulolytic species Ruminococcus albus. It ferments crystalline cellulose (Avicel) to acetate, ethanol, CO(inf2), and H(inf2). The other organism (HA) obtains energy for growth by using H(inf2) to reduce CO(inf2) to acetate. HA is a gram-negative coccobacillus that was isolated from horse feces. Coculture of R. albus with HA in batch or continuous culture alters the fermentation products formed from crystalline cellulose by the ruminococcus via interspecies H(inf2) transfer. The major product of the fermentation by R. albus and HA coculture is acetate. High concentrations of acetate (333 mM) were obtained when batch cocultures grown on 5% cellulose were neutralized with Ca(OH)(inf2). Continuous cocultures grown at retention times of 2 and 3.1 days produced 109 and 102 mM acetate, respectively, when fed 1% cellulose with utilization of 84% of the substrate.
Involvement of an extracellular H2O2-dependent ligninolytic activity of the white rot fungus Pleurotus ostreatus in the decolorization of Remazol brilliant blue R
Authors: Vyas B R , Molitoris H P
Published: January 2020

During solid-state fermentation of wheat straw, a natural lignocellulosic substrate, the white rot fungus Pleurotus ostreatus produced an extracellular H2O2-requiring Remazol brilliant blue R (RBBR)-decolorizing enzymatic activity along with manganese peroxidase, manganese-independent peroxidase, and phenol oxidase activities. The presence of RBBR was not essential for the production of RBBR-decolorizing enzymatic activity by P. ostreatus, because this activity was also produced in the absence of RBBR. This RBBR-decolorizing enzymatic activity in crude enzyme preparations of 14- and 20-day-old cultures exhibited an apparent Km for RBBR of 31 and 52 microM, respectively. The RBBR-decolorizing enzyme activity was maximal in the pH range 3.5 to 4.0. This activity was independent of manganese, and veratryl alcohol had no influence on it. Manganese peroxidase of P. ostreatus did not decolorize RBBR. This H2O2-dependent RBBR-decolorizing enzymatic activity behaved like an oxygenase possessing a catalytic metal center, perhaps heme, because it was inhibited by Na2S2O5, NaCN, NaN3, and depletion of dissolved oxygen. Na2S2O5 brought an early end to the reaction without interfering with the initial reaction rate of RBBR oxygenase. The activity was also inhibited by cysteine. Concentrations of H2O2 higher than 154 microM were observed to be inhibitory as well. Decolorization of RBBR by P. ostreatus is an oxidative process.
Comparative evaluation of modified m-FC and m-TEC media for membrane filter enumeration of Escherichia coli in water
Authors: Ciebin B W , Brodsky M H , Eddington R , Horsnell G , Choney A , Palmateer G , Ley A , Joshi R , Shears G
Published: January 2020

Two media used to detect fecal coliforms in water by membrane filtration, m-FC and m-TEC, were modified and supplemented with the chromogenic substrate 5-bromo-6-chloro-3-indoyl-beta-D-glucuronide (BCIG) and were compared for quantitative recovery of Escherichia coli. Student's t test of data from 181 water samples of sewage, rivers, lakes, and wells did not demonstrate any statistically significant differences (P = 0.05) in the enumeration of E. coli with these media. Target colonies were confirmed to be E. coli at rates of 98.6 and 97.3% by using FC-BCIG and TEC-BCIG media, respectively. Glucuronidase-negative isolates of E. coli were encountered at the same frequency (6.0%) on both media. This collaborative study demonstrated that either modified basal medium could be used successfully for detection of E. coli in various nontreated waters within 24 h.
Alternative Function of the Electron Transport System in Azotobacter vinelandii: Removal of Excess Reductant by the Cytochrome d Pathway
Authors: Liu J , Lee F , Lin C , Yao X , Davenport J W , Wong T
Published: January 2020

The N(inf2)-fixing bacterium Azotobacter vinelandii was grown in an O(inf2)-regulated chemostat with glucose or galactose as substrate. Increasing the O(inf2) partial pressure resulted in identical synthesis of the noncoupled cytochrome d terminal oxidase, which is consistent with the hypothesis that A. vinelandii uses high rates of respiration to protect the nitrogenase from oxygen. However, cell growth on glucose showed a lower yield of biomass, higher glycolytic rate, higher respiratory rate, and lower cytochrome o content than cell growth on galactose. Elemental analysis indicated no appreciable change in the C-to-N ratio of cell cultures, suggesting that the major composition of the cell was not influenced by the carbon source. A poor coordination of glucose and nitrogen metabolisms in A. vinelandii was suggested. The rapid hydrolysis of glucose resulted in carbonaceous accumulation in cells. Thus, Azotobacter species must induce a futile electron transport to protect cells from the high rates of glucose uptake and glycolysis.
Genetic and biochemical characterization of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H lysin
Authors: Vasala A , Välkkilä M , Caldentey J , Alatossava T
Published: January 2020

LL-H, a virulent phage of Lactobacillus delbrueckii subsp. lactis, produces a peptidoglycan-degrading enzyme, Mur, that is effective on L. delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus, and Pediococcus damnosus cell walls. In this study, the LL-H gene mur was cloned into Escherichia coli, its nucleotide sequence was determined, and the enzyme produced in E. coli was purified and biochemically characterized. Mur was purified 112-fold by means of ammonium sulfate precipitation and cation-exchange chromatography. The cell wall-hydrolyzing activity was found to be associated with a 34-kDa protein. The C-terminal domain of Mur is not essential for catalytic activity since it can be removed without destroying the lytic activity. The N-terminal sequence of the purified lysin was identical to that deduced from the nucleotide sequence, but the first methionine is absent from the mature protein. The N-terminal part of this 297-amino-acid protein had homology with several Chalaropsis-type lysozymes. Reduction of purified and Mur-digested L. delbrueckii cell wall material with labeled NaB3H4 indicated that the enzyme is a muramidase. The temperature optimum of purified Mur is between 30 and 40 degrees C, and the pH optimum is around 5.0. The LL-H lysin Mur is stable at temperatures below 60 degrees C.
Purification and properties of a novel thermostable galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces elviae CBS8119
Authors: Onishi N , Tanaka T
Published: January 2020

A thermostable beta-galactosidase which catalyzed the production of galacto-oligosaccharide from lactose was solubilized from a cell wall preparation of Sterigmatomyces elviae CBS8119. The enzyme was purified to homogeneity by means of chromatography on DEAE-Toyopearl, Butyl-Toyopearl, Chromatofocusing, and p-aminobenzyl 1-thio-beta-D-galactopyranoside agarose columns. The molecular weight of the purified enzyme was estimated to be about 170,000 by gel filtration with a Highload-Superdex 200pg column and 86,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its isoelectric point, determined by polyacrylamide gel electrofocusing, was 4.1. The optimal temperature for enzyme activity was 85 degrees C. It was stable at temperatures up to 80 degrees C for 1 h. The optimal pH range for the enzyme was 4.5 to 5.0, it was stable at pH 2.5 to 7.0, and its activity was inhibited by Hg2+. The Km values for o-nitrophenyl-beta-D-galactopyranoside and lactose were 9.5 and 2.4 mM, respectively, and the maximum velocities for these substrates were 96 and 240 mumol/min per mg of protein, respectively. In addition, this enzyme possessed a high level of transgalactosylation activity. Galacto-oligosaccharides, including tri- and tetrasaccharides, were produced with a yield, by weight, of 39% from 200-mg/ml lactose.
Purification and Characterization of Cystathionine (beta)-Lyase from Lactococcus lactis subsp. cremoris B78 and Its Possible Role in Flavor Development in Cheese
Authors: Alting A C , Engels W , van Schalkwijk S , Exterkate F A
Published: January 2020

An enzyme that degrades sulfur-containing amino acids was purified from Lactococcus lactis subsp. cremoris B78; this strain was isolated from a mixed-strain, mesophilic starter culture used for the production of Gouda cheese. The enzyme has features of a cystathionine (beta)-lyase (EC 4.4.1.8), a pyridoxal-5(prm1)-phosphate-dependent enzyme involved in the biosynthesis of methionine and catalyzing an (alpha),(beta)-elimination reaction. It is able to catalyze an (alpha),(gamma)-elimination reaction as well, which in the case of methionine, results in the production of methanethiol, a putative precursor of important flavor compounds in cheese. The native enzyme has a molecular mass of approximately 130 to 165 kDa and consists of four identical subunits of 35 to 40 kDa. The enzyme is relatively thermostable and has a pH optimum for activity around 8.0; it is still active under cheese-ripening conditions, viz., pH 5.2 to 5.4 and 4% (wt/vol) NaCl. A possible essential role of the enzyme in flavor development in cheese is suggested.
The protoxin composition of Bacillus thuringiensis insecticidal inclusions affects solubility and toxicity
Authors: Aronson A
Published: January 2020

Most Bacillus thuringiensis strains producing toxins active on lepidoptera contain several plasmid-encoded delta-endotoxin genes and package related protoxins into a single inclusion. It was previously found that in B. thuringiensis subsp. aizawai HD133, which produces an inclusion comprising the CryIAb, CryIC, and CryID protoxins, there is a spontaneous loss in about 1% of the cells of a 45-mDa plasmid containing the cryIAb gene. As a result, inclusions produced by the cured strain were less readily solubilized at pH 9.2 or 9.5 and had a decreased toxicity for Plodia interpunctella, despite the presence of the CryIC protoxin, which was active when solubilized. These results suggested that protoxin composition was a factor in inclusion solubility and toxicity and that the cryIAb gene, which is also present on an unstable plasmid in several other subspecies, may have a unique role in inclusion solubility and toxicity. Introduction of a cloned copy of this gene into the plasmid-cured derivative of B. thuringiensis subsp. aizawai HD133 resulted in an increase in the solubility at pH 9.2 of all of the inclusion proteins from less than 20% to greater than 45% and a lowering of the 50% lethal concentration (LC50, in micrograms [dry weight] per square centimeter) of inclusions for Spodoptera frugiperda from 35 to 10. These values are the same as those found with inclusions from B. thuringiensis subsp. aizawai HD133, and in all cases, the LC50 of the solubilized protoxins was 10. Transformants containing related cryIA genes produced inclusions which were more than 95% solubilized at pH 9.2 but also had LC50 of 10.(ABSTRACT TRUNCATED AT 250 WORDS)
Identification of prophage genes expressed in lysogens of the Lactococcus lactis bacteriophage BK5-T
Authors: Boyce J D , Davidson B E , Hillier A J
Published: January 2020

Bacteriophage BK5-T is a small isometric-headed temperate phage that infects Lactococcus lactis subsp. cremoris. Northern (RNA) analysis of mRNA produced by lysogenic strains containing BK5-T prophage revealed four major BK5-T transcripts that are 0.8, 1.3, 1.8, and 1.8 kb in size and enabled a transcription map of the prophage genome to be prepared. The position and size of each transcript corresponded closely to the position and size of open reading frames predicted from the nucleotide sequence of BK5-T. Analysis of the transcripts suggested that one of them was derived from the gene encoding the BK5-T integrase and another was from the gene encoding the BK5-T homolog of the lambda cI repressor. Computer analysis of the nucleotide sequence upstream of the BK5-T cI homolog predicted the presence of a pair of divergent promoters and three inverted repeat sequences, features characteristic of temperature-phage immunity regions. By analogy with lambda, the three inverted repeat sequences could be binding sites for cI or Cro homologs and the two divergent promoters could initiate transcription through the BK5-T equivalents of cI and cro.
Polymer production by Klebsiella pneumoniae 4-hydroxyphenylacetic acid hydroxylase genes cloned in Escherichia coli
Authors: Gibello A , Ferrer E , Sanz J , Martin M
Published: January 2020

The expression of Klebsiella pneumoniae hpaA and hpaH genes, which code for 4-hydroxyphenylacetic acid hydroxylase in Escherichia coli K-12 derivative strains, is associated with the production of a dark brown pigment in the cultures. This pigment has been identified as a polymer which shows several of the characteristics reported for microbial melanins and results from the oxidative activity of 4-hydroxyphenylacetic acid hydroxylase on some dihydroxylated compounds to form o-quinones. A dibenzoquinone is formed from the oxidation of different mono- or dihydroxylated aromatic compounds by the enzyme prior to polymerization. We report a hydroxylase activity, other than tyrosinase, that is associated with the synthesis of a bacterial melanin.
Rapid differentiation of bacterial species with multiple probes of different lengths in a single slot blot hybridization
Authors: Wang G C , Wang Y
Published: January 2020

We describe a highly efficient method for dot and slot blot hybridizations with multiple oligonucleotide probes for high throughput identification of organisms and studies of microbial community structures. Several probes with distinct specificities were designed to have the same melting temperature but unique lengths by adding different numbers of nonspecific nucleotides to one end. All of the probes were mixed, labelled with 32P, and hybridized to one piece of membrane on which genes coding for 16S rRNAs from different bacterial species had been immobilized. After hybridization, the bound probes were eluted and resolved on a denaturing polyacrylamide gel and the identities of the genes coding for 16S rRNAs were read from an autoradiograph of the gel. The results from the application of this technique to pure actinomycete cultures are reported here.
Use of Feedback-Resistant Threonine Dehydratases of Corynebacterium glutamicum To Increase Carbon Flux towards l-Isoleucine
Authors: Morbach S , Sahm H , Eggeling L
Published: January 2020

The biosynthesis of l-isoleucine proceeds via a highly regulated reaction sequence connected with l-lysine and l-threonine synthesis. Using defined genetic Corynebacterium glutamicum strains characterized by different fluxes through the homoserine dehydrogenase reaction, we analyzed the influence of four different ilvA alleles (encoding threonine dehydratase) in vectors with two different copy numbers on the total flux towards l-isoleucine. For this purpose, 18 different strains were constructed and analyzed. The result was that unlike ilvA in vectors with low copy numbers, ilvA in high-copy-number vectors increased the final l-isoleucine yield by about 20%. An additional 40% increase in l-isoleucine yield was obtained by the use of ilvA alleles encoding feedback-resistant threonine dehydratases. The strain with the highest yield was characterized by three hom(Fbr) copies encoding feedback-resistant homoserine dehydrogenase and ilvA(Fbr) encoding feedback-resistant threonine dehydratase on a multicopy plasmid. It accumulated 96 mM l-isoleucine, without any l-threonine as a by-product. The highest specific productivity was 0.052 g of l-isoleucine per g of biomass per h. This comparative flux analysis of isogenic strains showed that high levels of l-isoleucine formation from glucose can be achieved by the appropriate balance of homoserine dehydrogenase and threonine dehydratase activities in a strain background with feedback-resistant aspartate kinase. However, still-unknown limitations are present within the entire reaction sequence.
Isolation of Multiple Subspecies of Bacillus thuringiensis from a Population of the European Sunflower Moth, Homoeosoma nebulella
Authors: Itoua-Apoyolo C , Drif L , Vassal J M , Debarjac H , Bossy J P , Leclant F , Frutos R
Published: January 2020

Five subspecies of Bacillus thuringiensis were isolated from dead and diseased larvae obtained from a laboratory colony of the European sunflower moth, Homoeosoma nebulella. The subspecies isolated were B. thuringiensis subspp. thuringiensis (H 1a), kurstaki (H 3a3b3c), aizawai (H 7), morrisoni (H 8a8b), and thompsoni (H 12). Most isolates produced typical bipyramidal crystals, but the B. thuringiensis subsp. thuringiensis isolate produced spherical crystals and the B. thuringiensis subsp. thompsoni isolate produced a pyramidal crystal. Analysis of the parasporal crystals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the crystals from the B. thuringiensis subsp. kurstaki and aizawai isolates contained a protein of 138 kDa whereas those from B. thuringiensis subsp. morrisoni contained a protein of 145 kDa. The crystals from B. thuringiensis subsp. thuringiensis contained proteins of 125, 128, and 138 kDa, whereas those from B. thuringiensis subsp. thompsoni were the most unusual, containing proteins of 37 and 42 kDa. Bioassays of purified crystals conducted against second-instar larvae of H. nebulella showed that the isolates of B. thuringiensis subspp. aizawai, kurstaki, and thuringiensis were the most toxic, with 50% lethal concentrations (LC(inf50)s) of 0.15, 0.17, and 0.26 (mu)g/ml, respectively. The isolates of B. thuringiensis subspp. morrisoni and thompsoni had LC(inf50)s of 2.62 and 37.5 (mu)g/ml, respectively. These results show that a single insect species can simultaneously host and be affected by a variety of subspecies of B. thuringiensis producing different insecticidal proteins.
Sulfur-specific microbial desulfurization of sterically hindered analogs of dibenzothiophene
Authors: Lee M K , Senius J D , Grossman M J
Published: January 2020

Dibenzothiophenes (DBTs) bearing alkyl substitutions adjacent to the sulfur atom, such as 4,6-diethyldibenzothiophene (4,6-DEDBT), are referred to as sterically hindered with regard to access to the sulfur moiety. By using enrichment cultures with 4,6-DEDBT as the sole sulfur source, bacterial isolates which selectively remove sulfur from sterically hindered DBTs were obtained. The isolates were tentatively identified as Arthrobacter species. 4,6-DEDBT sulfone was shown to be an intermediate in the 4,6-DEDBT desulfurization pathway, and 2-hydroxy-3,3(prm1)-diethylbiphenyl (HDEBP) was identified as the sulfur-free end product.
Comparative Serology of the Marine Fish Pathogen Vibrio anguillarum
Authors: Grisez L , Ollevier F
Published: January 2020

The different serotyping systems, based on thermostable O antigens, reported for Vibrio anguillarum and V. ordalii were compared by quantitative agglutination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequent silver staining or Western blotting (immunoblotting) of purified lipopolysaccharide (LPS), using polyclonal rabbit antisera. The results demonstrate that 16 different serotypes within V. anguillarum (designated O1 to O16) can be distinguished. Each of these serotypes is characterized by a distinct polysaccharide banding pattern, as revealed by silver-stained gels of purified LPS. The comparative analysis allowed a complete alignment of the different serotypes for the first three serovars: O1, O2, and O3. Moreover, immunoblotting showed that strains belonging to each of these serotypes had the same LPS banding pattern independent of the origin of the strain. Serotype O2 contains different subtypes, O2a and O2b. While no differences were apparent between these subgroups in silver-stained gels, they could be separated by quantitative agglutination (titer determination) or immunoblotting. V. ordalii, the former biotype II of V. anguillarum, strongly reacts with anti-V. anguillarum O2a antiserum. Strains of the two species can be separated on the basis of different LPS profiles in the high-molecular-weight region of silver-stained gels of purified LPS. The silver-stained LPS profiles of the different serotypes of V. anguillarum that have been established are provided for further comparison in the future.
Halotolerance in Methanosarcina spp.: Role of N(sup(epsilon))-Acetyl-(beta)-Lysine, (alpha)-Glutamate, Glycine Betaine, and K(sup+) as Compatible Solutes for Osmotic Adaptation
Authors: Sowers K R , Gunsalus R P
Published: January 2020

The methanogenic Archaea, like the Bacteria and Eucarya, possess several osmoregulatory strategies that enable them to adapt to osmotic changes in their environment. The physiological responses of Methanosarcina species to different osmotic pressures were studied in extracellular osmolalities ranging from 0.3 to 2.0 osmol/kg. Regardless of the isolation source, the maximum rate of growth for species from freshwater, sewage, and marine sources occurred in extracellular osmolalities between 0.62 and 1.0 osmol/kg and decreased to minimal detectable growth as the solute concentration approached 2.0 osmol/kg. The steady-state water-accessible volume of Methanosarcina thermophila showed a disproportionate decrease of 30% between 0.3 and 0.6 osmol/kg and then a linear decrease of 22% as the solute concentration in the media increased from 0.6 to 2.0 osmol/kg. The total intracellular K(sup+) ion concentration in M. thermophila increased from 0.12 to 0.5 mol/kg as the medium osmolality was raised from 0.3 to 1.0 osmol/kg and then remained above 0.4 mol/kg as extracellular osmolality was increased to 2.0 osmol/kg. Concurrent with K(sup+) accumulation, M. thermophila synthesized and accumulated (alpha)-glutamate as the predominant intracellular osmoprotectant in media containing up to 1.0 osmol of solute per kg. At medium osmolalities greater than 1.0 osmol/kg, the (alpha)-glutamate concentration leveled off and the zwitterionic (beta)-amino acid N(sup(epsilon))-acetyl-(beta)-lysine was synthesized, accumulating to an intracellular concentration exceeding 1.1 osmol/kg at an osmolality of 2.0 osmol/kg. When glycine betaine was added to culture medium, it caused partial repression of de novo (alpha)-glutamate and N(sup(epsilon))-acetyl-(beta)-lysine synthesis and was accumulated by the cell as the predominant compatible solute. The distribution and concentration of compatible solutes in eight strains representing five Methanosarcina spp. were similar to those found in M. thermophila grown in extracellular osmolalities of 0.3 and 2.0 osmol/kg. Results of this study demonstrate that the mechanism of halotolerance in Methanosarcina spp. involves the regulation of K(sup+), (alpha)-glutamate, N(sup(epsilon))-acetyl-(beta)-lysine, and glycine betaine accumulation in response to the osmotic effects of extracellular solute.