bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plant
Authors: Lindén T , Peetre J , Hahn-Hägerdal B
Published: January 2020

From a continuous spent sulfite liquor fermentation plant, two species of yeast were isolated, Saccharomyces cerevisiae and Pichia membranaefaciens. One of the isolates of S. cerevisiae, no. 3, was heavily flocculating and produced a higher ethanol yield from spent sulfite liquor than did commercial baker's yeast. The greatest difference between isolate 3 and baker's yeast was that of galactose fermentation, even when galactose utilization was induced, i.e., when they were grown in the presence of galactose, prior to fermentation. Without acetic acid present, both baker's yeast and isolate 3 fermented glucose and galactose sequentially. Galactose fermentation with baker's yeast was strongly inhibited by acetic acid at pH values below 6. Isolate 3 fermented galactose, glucose, and mannose without catabolite repression in the presence of acetic acid, even at pH 4.5. The xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) activities were determined in some of the isolates as well as in two strains of S. cerevisiae (ATCC 24860 and baker's yeast) and Pichia stipitis CBS 6054. The S. cerevisiae strains manifested xylose reductase activity that was 2 orders of magnitude less than the corresponding P. stipitis value of 890 nmol/min/mg of protein. The xylose dehydrogenase activity was 1 order of magnitude less than the corresponding activity of P. stipitis (330 nmol/min/mg of protein).
Effect of bacterial extracellular polymers on the saturated hydraulic conductivity of sand columns
Authors: Vandevivere P , Baveye P
Published: January 2020

Columns were packed with clean quartz sand, sterilized, and inoculated with different strains of bacteria, which multiplied within the sand at the expense of a continuous supply of fresh nutrient medium. The saturated hydraulic conductivity (HCsat) of the sand was monitored over time. Among the four bacterial strains tested, one formed a capsule, one produced slime layers, and two did not produce any detectable exopolymers. The last two strains were nonmucoid variants of the first two. Only one strain, the slime producer, had a large impact on the HCsat. The production of exopolymers had no effect on either cell multiplication within or movement through the sand columns. Therefore, the HCsat reduction observed with the slime producer was tentatively attributed to the obstruction of flow channels with slime. Compared with the results with Arthrobacter sp. strain AK19 used in a previous study, there was a 100-fold increase in detachment from the solid substratum and movement through the sand of the strains used in this study. All strains induced severe clogging when they colonized the inlet chamber of the columns. Under these conditions, the inlet end was covered by a confluent mat with an extremely low HCsat.
Chlorobenzene degradation by bacteria isolated from contaminated groundwater
Authors: Nishino S F , Spain J C , Belcher L A , Litchfield C D
Published: January 2020

Bacterial isolates were obtained from groundwater and soils contaminated with chlorobenzene (CB). The isolates were tested to determine whether the natural community could remove the groundwater contaminants. These isolates were identified and characterized as to their ability to grow on CB and related aromatic compounds. The complete consortium could mineralize approximately 54% of the CB within 7 days, with no accumulation of 3-chlorocatechol. Metabolic pathways were evaluated for several isolates. One phenotype was characterized by the ability to degrade CB by the modified ortho pathway. One strain also degraded p-dichlorobenzene by using the same pathway. Isolates exhibiting a second phenotype degraded p-cresol, benzene, and phenol by the classical ortho pathway and accumulated 3-chlorocatechol when grown in the presence of CB. Strains of the third phenotype grew on complex media in the presence of CB but did not transform any of the aromatic compounds tested. The results suggest that the indigenous microbial community at the contaminated site would be able to degrade CB if provided with the appropriate conditions.
Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels
Authors: Cancilla M R , Powell I B , Hillier A J , Davidson B E
Published: January 2020

Arbitrarily primed polymerase chain reaction, with incorporation of either radioactive or fluorescent labels, was used as a rapid and sensitive method for obtaining genomic fingerprints of strains of Lactococcus lactis. Closely related strains produced almost identical fingerprints. Fingerprints of other strains showed only some similarities.
Degradation of triglycerides by a pseudomonad isolated from milk: molecular analysis of a lipase-encoding gene and its expression in Escherichia coli
Authors: Johnson L A , Beacham I R , MacRae I C , Free M L
Published: January 2020

Psychrotrophic lipolytic bacteria represent a significant problem in the storage of refrigerated dairy products. A lipase-encoding gene has been cloned and characterized from a highly lipolytic strain of Pseudomonas. The nucleotide sequence of the gene predicts a polypeptide of M(r) 49,905, which was identified when the gene was expressed in Escherichia coli.
Demonstration of cel operon expression of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa at elevated temperatures refractory to their growth
Authors: Droffner M L , Yamamoto N
Published: January 2020

When Escherichia coli was incubated at the growth-refractory temperatures of 48 and 54 degrees C, expression of the cel operon was demonstrated by phospho-beta-glucosidase activity. This enzyme activity was also detected at the growth-refractory temperatures in Salmonella typhimurium and Pseudomonas aeruginosa. Thermotolerant and mesothermophilic mutants of E. coli, S. typhimurium, and P. aeruginosa, able to grow with generation times of 30 to 40 min at 48 and 54 degrees C, exhibited phospho-beta-glucosidase activity at their growth temperatures of 48 and 54 degrees C. Thus, the cel operon previously described as a cryptic operon in E. coli and S. typhimurium was found to be expressed at growth-refractory temperatures of the mesophilic parent and growth-permissive temperatures (48 and 54 degrees C) of the thermotolerant and mesothermophilic mutants.
Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes
Authors: Eardly B D , Young J P , Selander R K
Published: January 2020

Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far.
Cell-associated pectinolytic and cellulolytic enzymes in Rhizobium leguminosarum biovar trifolii
Authors: Mateos P F , Jimenez-Zurdo J I , Chen J , Squartini A S , Haack S K , Martinez-Molina E , Hubbell D H , Dazzo F B
Published: January 2020

The involvement of Rhizobium enzymes that degrade plant cell wall polymers has long been an unresolved question about the infection process in root nodule symbiosis. Here we report the production of enzymes from Rhizobium leguminosarum bv. trifolii that degrade carboxymethyl cellulose and polypectate model substrates with sensitive methods that reliably detect the enzyme activities: a double-layer plate assay, quantitation of reducing sugars with a bicinchoninate reagent, and activity gel electrophoresis-isoelectric focusing. Both enzyme activities were (i) produced commonly by diverse wild-type strains, (ii) cell bound with at least some of the activity associated with the cell envelope, and (iii) not changed appreciably by growth in the presence of the model substrates or a flavone that activates expression of nodulation (nod) genes on the resident symbiotic plasmid (pSym). Equivalent levels of carboxymethyl cellulase activity were found in wild-type strain ANU843 and its pSym-cured derivative, ANU845, consistent with previous results of Morales et al. (V. Morales, E. Martínez-Molina, and D. Hubbell, Plant Soil 80:407-415, 1984). However, polygalacturonase activity was lower in ANU845 and was not restored to wild-type levels in the recombinant derivative of pSym- ANU845 containing the common and host-specific nod genes within a 14-kb HindIII DNA fragment of pSym from ANU843 cloned on plasmid pRt032. Activity gel electrophoresis resolved three carboxymethyl cellulase isozymes of approximately 102, 56, and 33 kDa in cell extracts from ANU843. Isoelectric focusing activity gels revealed one ANU843 polygalacturonase isozyme with a pI of approximately 7.2. These studies show that R. leguminosarum bv. trifolii produces multiple enzymes that cleave glycosidic bonds in plant cell walls and that are cell bound.
Biodegradation of bisphenol A and other bisphenols by a gram-negative aerobic bacterium
Authors: Lobos J H , Leib T K , Su T M
Published: January 2020

A novel bacterium designated strain MV1 was isolated from a sludge enrichment taken from the wastewater treatment plant at a plastics manufacturing facility and shown to degrade 2,2-bis(4-hydroxyphenyl)propane (4,4'-isopropylidenediphenol or bisphenol A). Strain MV1 is a gram-negative, aerobic bacillus that grows on bisphenol A as a sole source of carbon and energy. Total carbon analysis for bisphenol A degradation demonstrated that 60% of the carbon was mineralized to CO2, 20% was associated with the bacterial cells, and 20% was converted to soluble organic compounds. Metabolic intermediates detected in the culture medium during growth on bisphenol A were identified as 4-hydroxybenzoic acid, 4-hydroxyacetophenone, 2,2-bis(4-hydroxyphenyl)-1-propanol, and 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Most of the bisphenol A degraded by strain MV1 is cleaved in some way to form 4-hydroxybenzoic acid and 4-hydroxyacetophenone, which are subsequently mineralized or assimilated into cell carbon. In addition, about 20% of the bisphenol A is hydroxylated to form 2,2-bis(4-hydroxyphenyl)-1-propanol, which is slowly biotransformed to 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Cells that were grown on bisphenol A degraded a variety of bisphenol alkanes, hydroxylated benzoic acids, and hydroxylated acetophenones during resting-cell assays. Transmission electron microscopy of cells grown on bisphenol A revealed lipid storage granules and intracytoplasmic membranes.
Specific and Quantitative Assessment of Naphthalene and Salicylate Bioavailability by Using a Bioluminescent Catabolic Reporter Bacterium
Authors: Heitzer Armin , Webb Oren F. , Thonnard Janeen E. , Sayler Gary S.
Published: January 2020

A bioassay was developed and standardized for the rapid, specific, and quantitative assessment of naphthalene and salicylate bioavailability by use of bioluminescence monitoring of catabolic gene expression. The bioluminescent reporter strain
Pseudomonas fluorescens
HK44, which carries a transcriptional
nahG-luxCDABE
fusion for naphthalene and salicylate catabolism, was used. The physiological state of the reporter cultures as well as the intrinsic regulatory properties of the naphthalene degradation operon must be taken into account to obtain a high specificity at low target substrate concentrations. Experiments have shown that the use of exponentially growing reporter cultures has advantages over the use of carbon-starved, resting cultures. In aqueous solutions for both substrates, naphthalene and salicylate, linear relationships between initial substrate concentration and bioluminescence response were found over concentration ranges of 1 to 2 orders of magnitude. Naphthalene could be detected at a concentration of 45 ppb. Studies conducted under defined conditions with extracts and slurries of experimentally contaminated sterile soils and identical uncontaminated soil controls demonstrated that this method can be used for specific and quantitative estimations of target pollutant presence and bioavailability in soil extracts and for specific and qualitative estimations of napthalene in soil slurries.
Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity
Authors: Heipieper H J , Diefenbach R , Keweloh H
Published: January 2020

A trans unsaturated fatty acid was found as a major constituent in the lipids of Pseudomonas putida P8. The fatty acid was identified as 9-trans-hexadecenoic acid by gas chromatography, argentation thin-layer chromatography, and infrared absorption spectrometry. Growing cells of P. putida P8 reacted to the presence of sublethal concentrations of phenol in the medium with changes in the fatty acid composition of the lipids, thereby increasing the degree of saturation. At phenol concentrations which completely inhibited the growth of P. putida, the cells were still able to increase the content of the trans unsaturated fatty acid and simultaneously to decrease the proportion of the corresponding 9-cis-hexadecenoic acid. This conversion of fatty acids was also induced by 4-chlorophenol in nongrowing cells in which the de novo synthesis of lipids had stopped, as shown by incorporation experiments with labeled acetate. The isomerization of the double bond in the presence of chloramphenicol indicates a constitutively operating enzyme system. The cis-to-trans modification of the fatty acids studied here apparently is a new way of adapting the membrane fluidity to the presence of phenols, thereby compensating for the elevation of membrane permeability induced by these toxic substances.
Heat susceptibility of aquatic mycobacteria
Authors: Schulze-Röbbecke R , Buchholtz K
Published: January 2020

An investigation was carried out to measure the heat susceptibility of opportunistic mycobacteria frequently isolated from domestic water supply systems. The study was conducted under standardized conditions designed to resemble those found in oligotrophic aquatic habitats. Strains of the following species were tested: Mycobacterium avium, M. chelonae, M. fortuitum, M. intracellulare, M. kansasii (two strains), M. marinum, M. phlei, M. scrofulaceum, and M. xenopi. Suspensions of the test strains were exposed to temperatures of 50, 55, 60, and 70 degrees C; samples were taken at defined intervals to determine the concentration of survivors. From these data, the decimal reduction times were calculated for each test strain and test temperature. The results indicate that M. kansasii is more susceptible to heat than Legionella pneumophila, whereas the heat susceptibilities of M. fortuitum, M. intracellulare, and M. marinum lie in the same order of magnitude as that of L. pneumophila. The strains of M. avium, M. chelonae, M. phlei, M. scrofulaceum, and M. xenopi were found to be more thermoresistant than L. pneumophila, with the highest resistance being found in M. xenopi. Thermal measures to control L. pneumophila may therefore not be sufficient to control the last five mycobacterial species in contaminated water systems.
Naphthalene degradation via salicylate and gentisate by Rhodococcus sp. strain B4
Authors: Grund E , Denecke B , Eichenlaub R
Published: January 2020

Rhodococcus sp. strain B4, isolated from a soil sample contaminated with polycyclic aromatic hydrocarbons, grows with naphthalene as the sole source of carbon and energy. Salicylate and gentisate were identified as intermediates in the catabolism of naphthalene. In contrast to the well-studied catabolic pathway encoded by the NAH7 plasmid of Pseudomonas putida, salicylate does not induce the genes of the naphthalene-degradative pathway in Rhodococcus sp. strain B4. The key enzymes of naphthalene degradation in Rhodococcus sp. strain B4 have unusual cofactor requirements. The 1,2-dihydroxynaphthalene oxygenase activity depends on NADH and the salicylate 5-hydroxylase requires NADPH, ATP, and coenzyme A.
Enumeration of Thiobacilli within pH-Neutral and Acidic Mine Tailings and Their Role in the Development of Secondary Mineral Soil
Authors: Southam G. , Beveridge T. J.
Published: January 2020

The Lemoine tailings of Chibougamau, Quebec, Canada, were deposited as a pH-neutral mineral conglomerate consisting of aluminum-silicates, iron-aluminum-silicates, pyrite, chalcopyrite, and sphalerite. These tailings are colonized by an active population of
Thiobacillus ferrooxidans
which is localized to an acid zone occupying 40% of the tailings' surface. This population peaked at 7 × 10
8
most probable number per gram of tailings during July and August 1990 and extended to a depth of 40 cm from the surface. Examination of samples over this depth profile by transmission electron microscopy and electron dispersive spectroscopy revealed a microbially mediated mineral transition from sulfides (below 40 cm) to chlorides and phosphates (at the surface). Silicate minerals were unaltered by microbial action. Transmission electron microscopy showed a tight association between
Thiobacillus
species and the sulfide minerals, which helps account for their prominence in tailings environments. Accurate enumeration of
T. ferrooxidans
from tailings required the disruption of their bonding to the mineral interface. Vortexing of a 10% aqueous suspension of the tailings material prior to most-probable-number analysis best facilitated this release. Even though heavy metals were highly mobile under acidic conditions at the Lemoine tailings, it was evident by transmission electron microscopy and electron dispersive spectroscopy that they were being immobilized as bona fide fine-grain minerals containing iron, copper, chlorine, phosphorus, and oxygen on bacterial surfaces and exopolymers. This biomineralization increased with increasing bacterial numbers and was most evident in the upper 3 cm of the acidic zone.
1,3-Propanediol:NAD+ oxidoreductases of Lactobacillus brevis and Lactobacillus buchneri
Authors: Veiga-da-Cunha M , Foster M A
Published: January 2020

In the cofermentation of glycerol with a sugar by Lactobacillus brevis and Lactobacillus buchneri, a 1,3-propanediol:NAD+ oxidoreductase provides an additional method of NADH disposal. The enzyme has been purified from both L. brevis B22 and L. buchneri B190 and found to have properties very similar to those reported for the enzyme from Klebsiella pneumoniae. The enzymes required Mn2+ and are probably octamers with a molecular mass of 350 kDa. Although not absolutely specific for 1,3-propanediol when tested as dehydrogenases, the enzymes have less than 10% activity with glycerol, ethanol, and 1,2-propanediol. These properties contrast sharply with those of a protein isolated from another Lactobacillus species (L. reuteri) that ferments glycerol with glucose and previously designated a 1,3-propanediol dehydrogenase.
Temperature and structural effects on transfer of double-stranded RNA among isolates of the chestnut blight fungus (Cryphonectria parasitica)
Authors: Friese C F , Allen M F , Martin R , van Alfen N K
Published: January 2020

Cryphonectria parasitica is a unique fungus which can serve as a model for understanding transfer of genes between eukaryotic microorganisms. We studied transfer of double-stranded RNA (dsRNA) between compatible and incompatible strains of C. parasitica to determine whether hyphal types or temperature could restrict that exchange. Hyphal connections between incompatible strains occurred at about 30% of the frequency of connections between compatible strains and differed morphologically. Gel electrophoresis and in situ hybridization confirmed that dsRNA was transferred through substrate hyphae but not through aerial hyphae. Freezing temperatures resulted in the loss of dsRNA from the new mycelium of the donor colony and stimulated the production of virulent pycnidiospores. These temperature and structural restrictions may help to explain the lack of spread of the dsRNA despite its presence in the field.
Evaluation of the Biolog automated microbial identification system
Authors: Klingler J M , Stowe R P , Obenhuber D C , Groves T O , Mishra S K , Pierson D L
Published: January 2020

Biolog's identification system was used to identify 39 American Type Culture Collection reference taxa and 45 gram-negative isolates from water samples. Of the reference strains, 98% were identified to genus level and 76% to species level within 4 to 24 h. Identification of some authentic strains of Enterobacter, Klebsiella, and Serratia was unreliable. A total of 93% of the water isolates were identified.
Identification of Brucella spp. by using the polymerase chain reaction
Authors: Herman L , De Ridder H
Published: January 2020

The application of two synthetic oligonucleotides as probes and as primers in the polymerase chain reaction is presented for a specific, sensitive, and quick identification of Brucella spp. The specific oligonucleotide sequences were chosen on the basis of a 16S rRNA sequence alignment between Brucella abortus and Agrobacterium tumefaciens.
Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum
Authors: Wood B E , Ingram L O
Published: January 2020

The Zymomonas mobilis genes for ethanol production have been integrated into the chromosome of Klebsiella oxytoca M5A1. The best of these constructs, strain P2, produced ethanol efficiently from cellobiose in addition to monomeric sugars. Utilization of cellobiose and cellotriose by this strain eliminated the requirement for external beta-glucosidase and reduced the amount of commercial cellulase needed to ferment Solka Floc SW40 (primarily crystalline cellulose). The addition of plasmids encoding endoglucanases from Clostridium thermocellum resulted in the intracellular accumulation of thermostable enzymes as coproducts with ethanol during fermentation. The best of these, strain P2(pCT603T) containing celD, was used to hydrolyze amorphous cellulose to cellobiose and produce ethanol in a two-stage process. Strain P2(pCT603T) was also tested in combination with commercial cellulases. Pretreatment of Solka Floc SW40 at 60 degrees C with endoglucanase D substantially reduced the amount of commercial cellulase required to ferment Solka Floc. The stimulatory effect of the endoglucanase D pretreatment may result from the hydrolysis of amorphous regions, exposing additional sites for attack by fungal cellulases. Since endoglucanase D functions as part of a complex in C. thermocellum, it is possible that this enzyme may complex with fungal enzymes or bind cellulose to produce a more open structure for hydrolysis.
Purification and characterization of two serine carboxypeptidases from Aspergillus niger and their use in C-terminal sequencing of proteins and peptide synthesis
Authors: Dal Degan F , Ribadeau-Dumas B , Breddam K
Published: January 2020

A procedure was developed to prepare in large amounts two carboxypeptidases, CPD-I and CPD-II, from Aspergillus niger. They were each shown to be serine proteases and single-chain monomers with molecular masses of ca. 81 kDa and containing 22% carbohydrates. Amino acid analysis, carbohydrate determination, and N-terminal sequencing (20 to 25 residues) were performed on each enzyme. CPD-I showed sequence homologies with malt carboxypeptidase II, while the N terminus of CPD-II was different from that of any known serine carboxypeptidase. Like carboxypeptidase Y from Saccharomyces cerevisiae and carboxypeptidase III from malt, CPD-II contained a free sulfhydryl group that could play a role in catalysis. Both A. niger enzymes had pH optima of about 4 and were unstable above pH 7. Their specificities for substrate positions P1 and P'1 were characterized by use of, as substrates, a series of N-blocked amino acid esters and dipeptides. Both enzymes were specific for Arg, Lys, and Phe in P1. CPD-I preferred hydrophobic residues in P'1, while CPD-II was highly specific for Arg and Lys in this position. Each displayed an original specificity when P1 and P'1 were considered together. The specificities were also studied by analyzing the time course of the release of amino acids from eight different peptides of various lengths. CPD-I and CPD-II appeared to be quite suitable for C-terminal sequence studies as well as for the synthesis of peptide bonds. The latter was studied with two peptide esters as aminolysis substrates and a series of amino acid amides as nucleophiles.(ABSTRACT TRUNCATED AT 250 WORDS)
Detection of Active Yeast Cells (
Saccharomyces cerevisiae
) in Frozen Dough Sections
Authors: Autio K. , Mattila-Sandholm T.
Published: January 2020

A new method based on fluorescence microscopy was developed to detect active yeast cells in cryosections of wheat dough. The sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) and counterstained with Evans blue. The active yeast cells in the sections appeared brilliant yellow and were readily distinguished from the red dough matrix. The dead cells allowed penetration of the Evans blue through the cell membrane, which interfered with the DAPI staining and caused the dead cells to blend into the red environment. The number of active yeast cells in fermenting dough sections containing different proportions of living and dead yeast cells correlated well with the gas-forming capability of the yeast in the dough but not with the results of the conventional plate count method. The new method allows the study of yeast activity not only during the different stages of frozen dough processing but also during the fermentation of doughs.
Enzymes Involved in Anaerobic Polyethylene Glycol Degradation by
Pelobacter venetianus
and
Bacteroides
Strain PG1
Authors: Frings Joachim , Schramm Edgar , Schink Bernhard
Published: January 2020

In extracts of polyethylene glycol (PEG)-grown cells of the strictly anaerobically fermenting bacterium
Pelobacter venetianus
, two different enzyme activities were detected, a diol dehydratase and a PEG-degrading enzyme which was characterized as a PEG acetaldehyde lyase. Both enzymes were oxygen sensitive and depended on a reductant, such as titanium citrate or sulfhydryl compounds, for optimal activity. The diol dehydratase was inhibited by various corrinoids (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, and methylcobalamin) by up to 37% at a concentration of 100 μM. Changes in ionic strength and the K
+
ion concentration had only limited effects on this enzyme activity; glycerol inhibited the enzyme by 95%. The PEG-degrading enzyme activity was stimulated by the same corrinoids by up to 80%, exhibited optimal activity in 0.75 M potassium phosphate buffer or in the presence of 4 M KCI, and was only slightly affected by glycerol. Both enzymes were located in the cytoplasmic space. Also, another PEG-degrading bacterium,
Bacteroides
strain PG1, contained a PEG acetaldehyde lyase activity analogous to the corresponding enzyme of
P. venetianus
but no diol dehydratase. Our results confirm that corrinoid-influenced PEG degradation analogous to a diol dehydratase reaction is a common strategy among several different strictly anaerobic PEG-degrading bacteria.
Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria
Authors: de Bruijn F J
Published: January 2020

The distribution of dispersed repetitive DNA (repetitive extragenic palindromic [REP] and enterobacterial repetitive intergenic consensus [ERIC]) sequences in the genomes of a number of gram-negative soil bacteria was examined by using conserved primers corresponding to REP and ERIC sequences and the polymerase chain reaction (PCR). The patterns of the resulting PCR products were analyzed on agarose gels and found to be highly specific for each strain. The REP and ERIC PCR patterns of a series of Rhizobium meliloti isolates, previously ordered in a phylogenetic tree based on allelic variations at 14 enzyme loci (B. D. Eardly, L. A. Materon, N. H. Smith, D. A. Johnson, M. D. Rumbaugh, and R. K. Selander, Appl. Environ. Microbiol. 56:187-194), were determined. Isolates which had been postulated to be closely related by multilocus enzyme electrophoresis also revealed similar REP and ERIC PCR patterns, suggesting that the REP and ERIC PCR method is useful for the identification and classification of bacterial strains.
Application of
13
C Nuclear Magnetic Resonance To Elucidate the Unexpected Biosynthesis of Erythritol by
Leuconostoc oenos
Authors: Veiga-Da-Cunha Maria , Firme Paula , Romão M. Vitória San , Santos Helena
Published: January 2020

Natural-abundance
13
C nuclear magnetic resonance (
13
C-NMR) revealed the production of erythritol and glycerol by nongrowing cells of
Leuconostoc oenos
metabolizing glucose. The ratio of erythritol to glycerol was strongly influenced by the aeration conditions of the medium. The elucidation of the metabolic pathway responsible for erythritol production was achieved by
13
C-NMR and
1
H-NMR spectroscopy using specifically
13
C-labelled
d
-glucose. The
1
H-NMR spectrum of the cell supernatant resulting from the metabolism of [2-
13
C]glucose showed that only 75% of the glucose supplied was metabolized heterofermentatively and that the remaining 25% was channelled to the production of erythritol. The synthesis of this polyol resulted from the reduction of the C-4 moiety of the intermediate fructose 6-phosphate. Oxygen has an inhibitory effect on the production of erythritol by
L. oenos.
Preaeration of a suspension of nongrowing cells of
L. oenos
resulted in 30% less erythritol and in 70% more glycerol formed during the anaerobic metabolism of glucose. The anaerobic production of erythritol from glucose was also found in growing cultures of
L. oenos
, although to a smaller extent.
Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction
Authors: Tsai Y L , Olson B H
Published: January 2020

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.
Oxidation of Nitrapyrin to 6-Chloropicolinic Acid by the Ammonia-Oxidizing Bacterium
Nitrosomonas europaea
Authors: Vannelli Todd , Hooper Alan B.
Published: January 2020

Suspensions of
Nitrosomonas europaea
catalyzed the oxidation of the commercial nitrification inhibitor nitrapyrin [2-chloro-6-(trichloromethyl)-pyridine]. Rapid oxidation of nitrapyrin (at a concentration of 10 μM) required the concomitant oxidation of ammonia, hydroxylamine, or hydrazine. The turnover rate was highest in the presence of 10 mM ammonia (0.8 nmol of nitrapyrin per min/mg of protein). The product of the reaction was 6-chloropicolinic acid. By the use of
18
O
2
, it was shown that one of the oxygens in 6-chloropicolinic acid came from diatomic oxygen and that the other came from water. Approximately 13% of the radioactivity of [2,6-
14
C]nitrapyrin was shown to bind to cells. Most (94%) of the latter was bound indiscriminately to membrane proteins. The nitrapyrin bound to membrane proteins may account for the observed inactivation of ammonia oxidation.
Characterization of bacteriocins from two strains of Bacillus thermoleovorans, a thermophilic hydrocarbon-utilizing species
Authors: Novotny J F , Perry J J
Published: January 2020

Bacillus thermoleovorans S-II and B. thermoleovorans NR-9 produce bacteriocins, and these bacteriocins are designated thermoleovorin-S2 and thermoleovorin-N9, respectively. The bacteriocins are effective against all but the producing strain of B. thermoleovorans, as well as being effective against Salmonella typhimurium, Branhamella catarrhalis, Streptococcus faecalis, and Thermus aquaticus. Thermoleovorins are produced during log-phase growth and are inhibitory to actively growing cells. The bacteriocins are proteinaceous in nature, being sensitive to selected proteases (protease type XI and pepsin). They are stable at pHs of 3 to 10. Thermoleovorin-S2 was more thermostable than thermoleovorin-N9 at 70 and 80 degrees C. Thermoleovorins-S2 and -N9 apparently act by binding to the susceptible organisms, resulting in lysis of the cell. Thermoleovorins-S2 has an estimated M(r) of 42,000, while thermoleovorin-N9 has a M(r) of 36,000.
Roles of manganese and organic acid chelators in regulating lignin degradation and biosynthesis of peroxidases by Phanerochaete chrysosporium
Authors: Perez J , Jeffries T W
Published: January 2020

We studied the effect of manganese and various organic chelators on the distribution, depolymerization, and mineralization of synthetic 14C-labeled lignins (DHP) in cultures of Phanerochaete chrysosporium. In the presence of high levels of manganese [Mn(II) or Mn(III)], along with a suitable chelator, lignin peroxidase (LiP) production was repressed and manganese peroxidase (MnP) production was stimulated. Even though partial lignin depolymerization was observed under these conditions, further depolymerization of the polymer to smaller compounds was more efficient when low levels of manganese were present. LiPs were prevalent under these latter conditions, but MnPs were also present. Mineralization was more efficient with low manganese. These studies indicate that MnP performs the initial steps of DHP depolymerization but that LiP is necessary for further degradation of the polymer to lower-molecular-weight products and mineralization. We also conclude that a soluble Mn(II)-Mn(III) organic acid complex is necessary to repress LiP.
Rates of Microbenthic and Meiobenthic Bacterivory in a Temperate Muddy Tidal Flat Community
Authors: Epstein Slava S. , Shiaris Michael P.
Published: January 2020

Rates of bacterivory in micro- and meiobenthic species were determined by an improved technique in a muddy tidal flat community in Boston Harbor, Mass. The predominant grazers of bacteria were identified, and their rates of grazing were measured in the top 1 cm of the sediment. Grazing rates were measured by a fluorescence-labeled bacteria (FLB) technique. A mixture of two
Enterococcus
spp. isolates and two isolates of
Escherichia coli
were prepared as FLB, and they were added to intact sediment cores by replacing the pore water in the upper centimeter of the core. A standard FLB procedure was modified by filtering sediment dilutions onto cellulose membrane filters and processing the filters to render them optically transparent while preserving the physical integrity of the micro- and meiobenthic organisms. Thus, it was possible, on the same microscopic field, to switch from light microscopy for identification of grazers to epifluorescence microscopy for counting FLB present in the gut contents of the same grazers. The majority of benthic organisms present in these sediments consumed FLB, but their consumption rates varied widely. Two ciliate species, a
Prorodon
sp. and a
Chlamidodon
sp., and a nematode, a
Metoncholaimus
sp., consumed fluorescence-labeled coliforms at the highest rates, 126 to 169 FLB per individual per h. Other ciliates and nematodes, as well as microflagellates and harpacticoid copepods, consumed fluorescence-labeled coliforms at lower rates, 1.2 to 26 FLB per individual per h. Foraminiferans and gastrotriches did not contain FLB. Some ciliate grazers discriminated between enterococci and coliforms, consuming the rod-shaped fluorescence-labeled coliforms at 74- to 155-fold-higher rates than did the coccus-shaped fluorescence-labeled enterococci. Other ciliates did not select between fluorescence-labeled enterococci and fluorescence-labeled coliforms. The high rates of bacterivory by some ciliates and nematodes indicated intensive grazing. However, at their low extant densities, the grazers consumed only a small portion of the bacterial standing stock. Major bacterial grazers, e.g., microflagellates, ciliates, and nematodes, could potentially consume, per day, only 0.2, 0.1, and 0.03%, respectively, of the bacterial standing stock (7.5 × 10
8
bacteria per cm
3
).
Utilization of 3-chloro-2-methylbenzoic acid by Pseudomonas cepacia MB2 through the meta fission pathway
Authors: Higson F K , Focht D D
Published: January 2020

Pseudomonas cepacia MB2 grew on 3-chloro-2-methylbenzoate as a sole carbon source by metabolism through the meta fission pathway with the subsequent liberation of chloride. meta pyrocatechase activity in cell extracts was induced strongly by 3-chloro-2-methylbenzoate, but not by nongrowth analogs 4- or 5-chloro-2-methylbenzoate. Although rapid turnover of metabolites precluded direct identification, a mutant strain MB2-G5 lacking meta pyrocatechase activity produced 4-chloro-3-methylcatechol when incubated with 3-chloro-2-methylbenzoate. The catecholic product, confirmed by nuclear magnetic resonance and mass spectral analyses, produced a transient meta fission product (lambda max = 391 nm) from cell extracts of the wild-type MB2 strain. Further confirmation of meta pyrocatechase activity was noted by conversion of 4-chlorocatechol to 2-hydroxy-5-chloromuconic semialdehyde, which was not further metabolized. In contrast to 3-chlorocatechol, which was not metabolized and is known to generate suicidal products, 4-chlorocatechols do not generate acyl halides. Thus, further metabolism of the ring fission products is governed in strain MB2 by their suitability as substrates for the hydrolase.
Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods
Authors: Park C E , Akhtar M , Rayman M K
Published: January 2020

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.
Relationship between Transport of Bacteria and Their Clogging Efficiency in Sand Columns
Authors: Vandevivere Philippe , Baveye Philippe
Published: January 2020

In an earlier article, we reported that, under conditions in which neither exopolymers nor bacterial mats were produced,
Arthrobacter
sp. strain AK19 was an effective plugging agent in sand columns, whereas the bacterial strain SLI
-
had no significant effect on the permeability of the medium. A laboratory experiment with sand columns was carried out to elucidate the causes of this difference in behavior. Measured values of the saturated hydraulic conductivity of the sand were explained in terms of biomass accumulation, which was estimated by solving a mass balance equation. The relationship between the saturated hydraulic conductivity and the biomass density within the sand was exponential, although two different exponential coefficients were needed to fit the data for biomass densities above or below 13 mg (wet weight) per cm
3
, suggesting that two different clogging mechanisms may be involved in different ranges of biomass densities. The experimental results suggest that the SLI
-
strain was a poor clogging agent partly because of its lower yield coefficient relative to the limiting nutrient (oxygen) and partly because 60% of the biomass produced in situ was washed out from the column, compared with only 1.2% in the case of
Arthrobacter
sp. strain AK19.
Novel Bacillus thuringiensis insecticidal crystal protein with a silent activity against coleopteran larvae
Authors: Lambert B , Höfte H , Annys K , Jansens S , Soetaert P , Peferoen M
Published: January 2020

A novel Bacillus thuringiensis crystal protein with a silent activity against the Colorado potato beetle is described. The crystal proteins are produced as bipyramidal crystals. These crystals contain a protein of 129 kDa with a trypsin-resistant core fragment of 72 kDa. Neither a spore-crystal mixture nor in vitro-solubilized crystals are toxic to any of several Lepidoptera and Coleoptera species tested. In contrast, a trypsin-treated solution containing the 72-kDa tryptic core fragment of the protoxin is highly toxic to Colorado potato beetle larvae. The crystal protein-encoding gene was cloned and sequenced. The inferred amino acid sequence of the putative toxic fragment has 37, 32, and 33% homology to the CryIIIA, CryIIIB, and CryIIID toxins, respectively. Interestingly, the 501 C-terminal amino acids show 41 to 48% amino acid identity with corresponding C-terminal amino acid sequences of other crystal proteins. Because of the toxicity of the fragment to the Colorado potato beetle and because of the distinct similarities of the toxic fragment with the other CryIII proteins, this gene was given a new subclass name (cryIIIC) within the CryIII class of coleopteran-active crystal proteins. CryIIIC represents the first example of a crystal protein with a silent activity towards coleopteran insect larvae. Natural CryIIIC crystals are not toxic. Toxicity is revealed only after an in vitro solubilization and activation step.
Ethanol Reassimilation and Ethanol Tolerance in
Pichia stipitis
CBS 6054 as Studied by
13
C Nuclear Magnetic Resonance Spectroscopy
Authors: Skoog Kerstin , Hahn-Hägerdal Bärbel , Degn Hans , Jacobsen Jens Peter , Jacobsen Hans Svankjaer
Published: January 2020

Ethanol reassimilation in
Pichia stipitis
CBS 6054 was studied by using continuous cultures, and the oxidation of [1-
13
C]ethanol was monitored by in vivo and in vitro
13
C nuclear magnetic resonance spectroscopy. Acetate was formed when ethanol was reassimilated. The ATP/ADP ratio and the carbon dioxide production decreased, whereas the malate dehydrogenase activity increased, in ethanol-reassimilating cells. The results are discussed in terms of the low ethanol tolerance in
P. stipitis
compared with that in
Saccharomyces cerevisiae
(S. W. Brown, S. G. Oliver, D. E. F. Harrison, and R. C. Righelato, Eur. J. Appl. Microbiol. Biotechnol. 11:151-155, 1981).
Effects of antibacterial agents on in vitro ovine ruminal biotransformation of the hepatotoxic pyrrolizidine alkaloid jacobine
Authors: Wachenheim D E , Blythe L L , Craig A M
Published: January 2020

Ingestion of pyrrolizidine alkaloids, naturally occurring plant toxins, causes illness and death in a number of animal species. Senecio jacobaea pyrrolizidine alkaloids cause significant economic losses due to livestock poisoning, particularly in the Pacific Northwest. Some sheep are resistant to pyrrolizidine alkaloid poisoning, because ovine ruminal biotransformation detoxifies free pyrrolizidine alkaloids in digesta. Antibacterial agents modify ruminal fermentation. Pretreatment with antibacterial agents may account for some animal variability in resistance to pyrrolizidine alkaloid toxicosis, and antibacterial agents can also be used for characterizing ruminal pyrrolizidine alkaloid-biotransforming microflora. The objective of this study was to evaluate the effects of antibacterial agents on biotransformation of a predominant S. jacobaea pyrrolizidine alkaloid, jacobine, in ovine ruminal contents. Ovine ruminal jacobine biotransformation was tested in vitro with 20 independent antibacterial agents. Low amounts of rifampin and erythromycin prevented jacobine biotransformation. Chlortetracycline, lasalocid, monensin, penicillin G, and tetracycline were slightly less effective at inhibiting jacobine biotransformation. Bacitracin, crystal violet, kanamycin, and neomycin were moderately inhibitory against jacobine biotransformation. Brilliant green, chloramphenicol, gramicidin, nalidixic acid, polymyxin B SO4, sodium azide, streptomycin, sulfisoxazole, and vancomycin had little to no effect on jacobine biotransformation. The antibiotics that were most effective at inhibiting biotransformation were those that are active against gram-positive bacteria. Therefore, gram-positive bacteria are most likely critical members of the jacobine-biotransforming consortia.