bluuebunny/crossref_metadata_2025 huggingface dataset:
Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora
Authors: Rafii F , Franklin W , Cerniglia C E
Published: January 2020

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.
Cloning and characterization of the thymidylate synthase gene from Lactococcus lactis subsp. lactis
Authors: Ross P , O'Gara F , Condon S
Published: January 2020

The thymidylate synthase (thyA) gene has been isolated from Lactococcus lactis subsp. lactis. The cloned gene was strongly expressed in Escherichia coli both in vivo and in vitro (maxicells and cell-free transcription and translation systems) and complemented E. coli thyA mutants. DNA-DNA hybridizations demonstrated that the thyA gene is encoded by the chromosome of L. lactis subsp. lactis. By sequential deletion of DNA outside the complementing region, the thyA gene was localized to a 1.1-kilobase DNA fragment. The nucleotide sequence of the lactococcal thyA gene was determined by the dideoxy-chain termination technique. The derived amino acid sequence indicated a protein size of 32,580 daltons, which is in good agreement with results obtained from maxicell and in vitro transcription and translation experiments. The primary sequence is homologous to 12 other thyA proteins from a variety of other organisms. Upstream from the structural gene, -10 and -35 promoter sequences which were almost canonical sigma-70 promoter sequences were identified, which may explain the strong expression of the thyA gene observed in E. coli. An A-T-rich sequence characteristic of gram-positive promoters was also noted adjacent to the -35 region. The thyA gene has potential as a marker for plasmid maintenance and selection in food systems.
Thermal ecology of Naegleria fowleri from a power plant cooling reservoir
Authors: Huizinga H W , McLaughlin G L
Published: January 2020

The pathogenic, free-living amoeba Naegleria fowleri is the causative agent of human primary amebic meningoencephalitis. N. fowleri has been isolated from thermally elevated aquatic environments worldwide, but temperature factors associated with occurrence of the amoeba remain undefined. In this study, a newly created cooling reservoir (Clinton Lake, Illinois) was surveyed for Naegleria spp. before and after thermal additions from a nuclear power plant. Water and sediment samples were collected from heated and unheated arms of the reservoir and analyzed for the presence of thermophilic Naegleria spp. and pathogenic N. fowleri. Amoebae were identified by morphology, in vitro cultivation, temperature tolerance, mouse pathogenicity assay, and DNA restriction fragment length analysis. N. fowleri was isolated from the thermally elevated arm but not from the ambient-temperature arm of the reservoir. The probability of isolating thermophilic Naegleria and pathogenic N. fowleri increased significantly with temperature. Repetitive DNA restriction fragment profiles of the N. fowleri Clinton Lake isolates and a known N. fowleri strain of human origin were homogeneous.
Melibiose is hydrolyzed exocellularly by an inducible exo-alpha-galactosidase in Azotobacter vinelandii
Authors: Wong T Y
Published: January 2020

Azotobacter vinelandii hydrolyzed melibiose exocellularly, leading to an accumulation of free glucose and galactose in the medium. This enzyme could also be induced by galactose, raffinose, and stachyose. The alpha-galactosidase activity could be detected quantitatively by using p-nitrophenyl-alpha-galactopyranoside as a substrate for intact cells. Chloramphenicol totally inhibited the induction of this enzyme. However, benzyl alcohol inhibited the secretion of this enzyme but did not inhibit the biosynthesis of the enzyme.
Replica plating of colonies from Listeria-selective agars to blood agar to improve the isolation of Listeria monocytogenes from foods
Authors: Cassiday P K , Graves L M , Swaminathan B
Published: January 2020

Bacterial colonies from Listeria-selective agars were replica plated to sheep blood agar to screen for beta-hemolysis. By using the replica plating method to test for the beta-hemolytic characteristic of all the colonies growing on Listeria-selective agars instead of picking 3 to 10 suspected colonies for further testing, we recovered Listeria monocytogenes from 59 of 142 Listeria-selective agar plates which contained colonies of hemolytic and nonhemolytic Listeria species and were negative when tested by conventional colony picks.
The unique stability of Vibrio proteolyticus neutral protease under alkaline conditions affords a selective step for purification and use in amino acid-coupling reactions
Authors: Durham D R
Published: January 2020

A procedure is described for the purification of a neutral protease from fermentation broths of Vibrio proteolyticus. The key feature of the purification scheme is the selective, irreversible inactivation of a contaminating exoenzyme, aminopeptidase, by alkali treatment, rather than removal of this enzyme by conventional chromatographic methods. Fermentation broths or concentrates were brought to pH 11.5 to 11.7 by Na2CO3-NaOH addition and incubated at 25 degrees C until aminopeptidase activity was diminished. The alkali treatment resulted in greater than 99% reduction of aminopeptidase activity with minimal loss of neutral protease activity. The neutral protease could be further purified to apparent homogeneity by QA-52 cellulose chromatography. The alkali treatment of fermentation concentrates was also useful for preparation of V. proteolyticus neutral protease to effect the coupling of N-protected aspartic acid and phenylalanine methyl ester for the production of N-aspartylphenylalanine methyl ester, a precursor for the sweetener aspartame.
Production of fumonisin B1 by Fusarium moniliforme NRRL 13616 in submerged culture
Authors: Jackson M A , Bennett G A
Published: January 2020

Fumonisin B1, a recently discovered mycotoxin, was synthesized by submerged cultures of Fusarium moniliforme NRRL 13616 grown for 29 days at 28 degrees C and 220 rpm in a basal salts medium (pH 5.0) supplemented with 90 g of glucose per liter and 3.5 g of ammonium sulfate per liter. Under these culture conditions, 74 +/- 23 micrograms of fumonisin B1 per ml was produced by 29-day-old F. moniliforme NRRL 13616 cultures. Fumonisin B1 was detected in liquid culture extracts by high-performance thin-layer chromatography. Fumonisin B1 was confirmed and quantitated by gas chromatography and gas chromatography-mass spectral analysis of the trimethylsilyl derivative. The use of a defined medium for producing fumonisin B1 in a submerged culture facilitates its isolation and provides an excellent method for conducting biosynthetic studies.
Incidence of Vibrio parahaemolyticus in U.S. coastal waters and oysters
Authors: DePaola A , Hopkins L H , Peeler J T , Wentz B , McPhearson R M
Published: January 2020

Oyster and seawater samples were collected seasonally from May 1984 through April 1985 from shellfish-growing areas in Washington, California, Texas, Louisiana, Alabama, Florida, South Carolina, Virginia, and Rhode Island which had been designated as approved or prohibited by the National Shellfish Sanitation Program. Fecal coliforms counts, aerobic plate counts, and Vibrio parahaemolyticus densities were determined for the samples. Mean V. parahaemolyticus density was more than 100 times greater in oysters than in water, whereas density of fecal coliforms was approximately 10 times higher in oysters. Seasonal and geographical distributions of V. parahaemolyticus were related to water temperature, with highest densities in samples collected in the spring and the summer along the Gulf coast. The synthetic DNA probe for thermostable direct hemolysin hybridized with 2 of 50 isolates, 1 of which was positive by the Kanagawa test.
Detection of Vibrio cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture methods
Authors: Huq A , Colwell R R , Rahman R , Ali A , Chowdhury M A , Parveen S , Sack D A , Russek-Cohen E
Published: January 2020

Vibrio cholerae O1 in plankton samples collected from ponds and rivers between February 1987 and January 1990 in Matlab, Bangladesh, was detected by the fluorescent-monoclonal antibody (FA) technique. Samples were collected at sites which were monitored fortnightly (fixed sites) as well as at sites that were part of a case-control study. FA results were compared with those obtained by conventional culture methods (CM). A total of 876 samples were collected; V. cholerae O1 was detected in 563 samples (64.27%) by the FA method and in 3 samples (0.34%) by CM. Of the fixed-site plankton samples, 439 (63.62%) were positive by FA and none were positive by CM. Of the 93 case sites sampled on the day after the occurrence of a case of cholera, 73 (78.49%) were positive for V. cholerae O1 by FA and 3 (3.2%) were positive by CM. In comparison, of the 93 first-day sample collections at control sites at the time a case of cholera occurred, only 51 (54.83%) were positive by FA and none were positive by CM. From the data, it is concluded that V. cholerae O1 is present throughout the year in the ponds and rivers of Bangladesh that were examined in this study and that V. cholerae can be detected by FA but not always by CM. The FA procedure was found to be very useful in detecting V. cholerae in plankton, with which it was associated and often occurred in large numbers in the nonculturable stage. Thus, studies investigating the significance of the role of environmental factors in the epidemiology of cholera can be performed effectively by using FA. Such studies are in progress.
Increased Bean (
Phaseolus vulgaris
L.) Nodulation Competitiveness of Genetically Modified
Rhizobium
Strains
Authors: Martinez-Romero Esperanza , Rosenblueth Monica
Published: January 2020

Rhizobium leguminosarum
bv. phaseoli strain collections harbor heterogeneous groups of bacteria in which two main types of strains may be distinguished, differing both in the symbiotic plasmid and in the chromosome. We have analyzed under laboratory conditions the competitive abilities of the different types of
Rhizobium
strains capable of nodulating
Phaseolus vulgaris
L. bean.
R. leguminosarum
bv. phaseoli type I strains (characterized by
nif
gene reiterations and a narrow host range) are more competitive than type II strains (that have a broad host range), and both types are more competitive than the promiscuous rhizobia isolated from other tropical legumes able to nodulate beans. Type I strains become even more competitive by the transfer of a non-Sym, 225-kilobase plasmid from type II strain CFN299. This plasmid has been previously shown to enhance the nodulation and nitrogen fixation capabilities of
Agrobacterium tumefaciens
transconjugants carrying the Sym plasmid of strain CFN299. Other type I
R. leguminosarum
bv. phaseoli transconjugants carrying two symbiotic plasmids (type I and type II) have been constructed. These strains have a diminished competitive ability. The increase of competitiveness obtained in some transconjugants seems to be a transient property.
Detection and analysis of two serotypes of ammonia-oxidizing bacteria in sewage plants by flow cytometry
Authors: Völsch A , Nader W F , Geiss H K , Nebe G , Birr C
Published: January 2020

Two different serotypes of the genus Nitrosomonas were isolated from samples of the sewage plant Heidelberg. These nitrifiers were enumerated in activated sludge of various other sewage plants after immunofluorescent labeling and staining with propidium iodide by flow cytometry. The concentrations of these serotypes of Nitrosomonas spp. were in the range of 0.1 to 2%. Also, a test for the determination of the activity of ammonia-oxidizing bacteria was developed. Nitrite-oxidizing bacteria were specifically inhibited with sodium chlorate, and the activity of ammonia-oxidizing bacteria could be calculated from the increase of nitrite. Concentrations and activities of ammonia oxidizers were measured for a period of 6 months in the sewage plant Heidelberg. With one exception, activities and concentrations of ammonia-oxidizing bacteria decreased and increased in parallel.
Restriction enzyme analysis of mitochondrial DNA of the Aspergillus flavus group: A. flavus, A. parasiticus, and A. nomius
Authors: Moody S F , Tyler B M
Published: January 2020

Mitochondrial DNA restriction fragment length polymorphisms were identified that clearly distinguish Aspergillus flavus, A. parasiticus, and A. nomius. Mitochondrial DNAs of A. flavus and A. parasiticus were found to be circular, and their size was estimated size to be 32 kilobases. A restriction map was constructed for the mitochondrial genome of an A. parasiticus isolate by using four restriction endonucleases. Four genes tested were found to have the same order as in the mitochondrial genome of A. nidulans. The mitochondrial genome of A. nomius was estimated to be 33 kilobases.
Detection of plasmid DNA from all Chlamydia trachomatis serovars with a two-step polymerase chain reaction
Authors: Welch D , Lee C H , Larsen S H
Published: January 2020

A polymerase chain reaction was used to amplify a 137-base-pair sequence of DNA from a Chlamydia trachomatis plasmid. Various parameters of the polymerase chain reaction were explored, and it was found that two short steps per reaction cycle were sufficient to achieve 10(12)-fold amplification in less than 1 h. By use of this procedure, 10(-18) g of a sequence of plasmid DNA, representing the amount of that sequence found in one C. trachomatis bacterium, was amplified to the point where it was clearly visible on an ethidium bromide-stained polyacrylamide gel under UV light. DNA from intact cells from each of the 15 serovars of C. trachomatis could also be amplified for visualization. With this procedure, the presence or absence of C. trachomatis DNA in a sample could be established in less than 1.5 h. The speed and extreme sensitivity of this detection procedure may make it a useful method for the detection of C. trachomatis, and similar techniques should be possible for any type of bacteria.
Effects of Culture Conditions on Poly(β-Hydroxybutyric Acid) Production by
Haloferax mediterranei
Authors: Lillo Jose Garcia , Rodriguez-Valera Francisco
Published: January 2020

The halobacterium
Haloferax mediterranei
accumulates poly(β-hydroxybutyrate) (PHB) as intracellular granules. The conditions for PHB production in batch and continuous cultures have been studied and optimized. Phosphate limitation is essential for PHB accumulation in large quantities. Glucose and starch are the best carbon sources. With 2% starch, 0.00375% KH
2
PO
4
, and 0.2% NH
4
Cl in batch culture, a production of ca. 6 g of PHB per liter was reached, being 60% of the total biomass dry weight, and giving a yield over the carbon source of 0.33 g/g. The PHB production in continuous cultures was stable over a 3-month period. Our results demonstrate that
H. mediterranei
is an interesting candidate for industrial production of biological polyesters.
Interrelationship of Xylanase Induction and Cellulase Induction of
Trichoderma longibrachiatum
Authors: Royer John C. , Nakas J. P.
Published: January 2020

Xylose oligomers rapidly induced xylanase activity of
Trichoderma longibrachiatum,
whereas induction was delayed in the presence of glucose. Cellobiose, cellopentaose, and xylobiose did not induce detectable levels of cellulase activity. However, mixtures of xylobiose with cellobiose or cellopentaose rapidly induced cellulase activity. In addition, mixtures of xylobiose with cellopentaose or cellobiose induced xylanase activity more effectively than xylobiose alone. Both xylanase and cellulase activity were detected after a lag period in the presence of lactose.
Selection and characterization of mutants of Phanerochaete chrysosporium exhibiting ligninolytic activity under nutrient-rich conditions
Authors: Tien M , Myer S B
Published: January 2020

Synthesis of the ligninolytic system of the wood-degrading fungus Phanerochaete chrysosporium is induced during secondary metabolism, brought about by nitrogen, carbon, or sulfur starvation. We describe here a strategy for selection of mutants which are ligninolytic (lignin----CO2) and overproduce lignin-degrading enzymes (ligninases) under nutrient-rich conditions (during primary metabolism). The strategy is based on using an adduct of lysine and a lignin model compound. Ligninase-dependent oxidation of this adduct releases free lysine, which complements the lysine requirements of a lysine auxotroph. Accordingly, a lysine auxotroph was mutagenized by UV irradiation and survivors were plated onto medium containing the adduct and high ammonia nitrogen. Four mutants which overproduce the ligninase isozymes were isolated by this procedure. Further characterization of one of the mutants, PSBL-1, indicated that the predominant isozymes produced are H1 (pI = 4.7) and H2 (pI = 4.4). The ligninase activity of PSBL-1, measured by veratryl alcohol oxidation, peaks on day 5 at over 1,000 U.liter-1. The mutant PSBL-1 was also able to degrade [14C]lignin to 14CO2, indicating that the complete ligninolytic system is deregulated.
Biodegradation of Halogenated Hydrocarbon Fumigants by Nitrifying Bacteria
Authors: Rasche Madeline E. , Hyman Michael R. , Arp Daniel J.
Published: January 2020

Three species of nitrifying bacteria were tested for the ability to degrade the halocarbon fumigants methyl bromide, 1,2-dichloropropane, and 1,2-dibromo-3-chloropropane. The soil nitrifiers
Nitrosomonas europaea
and
Nitrosolobus multiformis
degraded all three fumigants, while the marine nitrifier
Nitrosococcus oceanus
degraded only methyl bromide under the conditions tested. Inhibition of biodegradation by allylthiourea and acetylene, specific inhibitors of ammonia monooxygenase, suggests that ammonia monooxygenase is the enzyme which catalyzes fumigant degradation.
Tangential flow filtration and preliminary phylogenetic analysis of marine picoplankton
Authors: Giovannoni S J , DeLong E F , Schmidt T M , Pace N R
Published: January 2020

A procedure was developed for harvesting gram quantities of microbial biomass from oligotrophic waters, when mixed populations are present in low abundance. Picoplankton from Atlantic Ocean (Hydrostation S, Sargasso Sea) and Pacific Ocean (Aloha Station) sites were collected in a three-stage process: (i) collection of seawater through an intake covered with 10-microns-pore Nytex; (ii) concentration by a tangential flow filtration device equipped with 10 ft2 (0.929 m2) of 0.1-micron-pore fluorocarbon membrane; (iii) collection of cells from concentrate by centrifugation. The overall efficiency of picoplankton recovery was at least 37%. The cellular morphotypes recovered matched those of the original population. DNA was prepared from frozen cell pellets by enzymatic digestion, solvent extraction, and isopycnic centrifugation. As indicated by the binding of kingdom-specific hybridization probes to the purified DNA, the Sargasso Sea picoplankton in this collection were largely eubacteria.
Hydrophobicity of Bacillus and Clostridium spores
Authors: Wiencek K M , Klapes N A , Foegeding P M
Published: January 2020

The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography. Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells. The relative hydrophobicities determined by the two methods generally agreed. Sporulation media and conditions appeared to have little effect on spore hydrophobicity. However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity. The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces.
Effect of Sulfate and Organic Carbon Supplements on Reductive Dehalogenation of Chloroanilines in Anaerobic Aquifer Slurries
Authors: Kuhn Elmar P. , Townsend G. Todd , Suflita Joseph M.
Published: January 2020

When di-, tri-, and tetrachloroaniline were incubated in methanogenic groundwater slurries, they were reductively dehalogenated by the aquifer microbiota. 2,3,4-Trichloroaniline was metabolized by two pathways. Primary dehalogenation occurred at either the
meta
or
ortho
position of this substrate to form 2,4- and 3,4-dichloroaniline, respectively. The latter chemical could be stoichiometrically converted to 3-chloroaniline. 2,3,4,5-Tetrachloroaniline was degraded by the sequential removal of halogens from the
para
and then the
ortho
position to form 3,5-dichloroaniline. An additional pathway was observed with this substrate when the aquifer slurries were amended with butyrate. That is, halogens could be removed from both the
meta
and
ortho
positions of tetrachloroaniline. The amendment of sulfate to methanogenic aquifer slurries slowed the rate of 2,3,4,5-tetrachloroaniline degradation and increased the amount of substrate channeled through the additional pathway. The reported intermediates or end products are identified by their chromatographic mobility and mass-spectral profiles.
Xylanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum": overexpression of the gene in Escherichia coli and characterization of the gene product
Authors: Lüthi E , Jasmat N B , Bergquist P L
Published: January 2020

A xylanase encoded by the xynA gene of the extreme thermophile "Caldocellum saccharolyticum" was overexpressed in Escherichia coli by cloning the gene downstream from the temperature-inducible lambda pR and pL promoters of the expression vector pJLA602. Induction of up to 55 times was obtained by growing the cells at 42 degrees C, and the xylanase made up to 20% of the whole-cell protein content. The enzyme was located in the cytoplasmic fraction in E. coli. The temperature and pH optima were determined to be 70 degrees C and pH 5.5 to 6, respectively. The xylanase was stable for at least 72 h if incubated at 60 degrees C, with half-lives of 8 to 9 h at 70 degrees C and 2 to 3 min at 80 degrees C. The enzyme had high activity on xylan and ortho-nitrophenyl beta-D-xylopyranoside and some activity on carboxymethyl cellulose and para-nitrophenyl beta-D-cellobioside. The gene was probably expressed from its own promoter in E. coli. Translation of the xylanase overproduced in E. coli seemed to initiate at a GTG codon and not at an ATG codon as previously determined.
Effect of Agitation on Ligninase Activity and Ligninase Production by
Phanerochaete chrysosporium
Authors: Venkatadri Rajagopalan , Irvine Robert L.
Published: January 2020

The white rot fungus
Phanerochaete chrysosporium
produces extracellular ligninases as part of its idiophasic ligninolytic system. Agitation has been widely reported to suppress both ligninase production and lignin degradation. Results show that mechanical inactivation of ligninase is possibly the reason why ligninase accumulation is low or absent in agitated shake-flask cultures. Agitation seems to affect the catalytic activity of ligninase and has no apparent effect on either the rate of ligninase production or the physiology of
P. chrysosporium
. The detergents Tween 20, Tween 40, Tween 60, Tween 80, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) are able to protect both purified ligninase and extant ligninase in culture fluids (free of biomass) against mechanical inactivation due to agitation. Addition of Tween 80 at the end of primary growth to agitated shake flasks containing either pelleted or immobilized mycelial cultures results in production and maintenance of high levels of ligninase activity over several days under conditions of high agitation. Possible mechanisms by which the detergents could protect ligninase are discussed.
Adhesion of Cellulolytic Ruminal Bacteria to Barley Straw
Authors: Bhat Siva , Wallace R. J. , Ørskov E. R.
Published: January 2020

Adhesion of the cellulolytic ruminal bacteria
Ruminococcus flavefaciens
and
Fibrobacter succinogenes
to barley straw was measured by incubating bacterial suspensions with hammer-milled straw for 30 min, filtering the mixtures through sintered glass filters, and measuring the optical densities of the filtrates. Maximum adhesion of both species occurred at pH 6.0 and during mid- to late-exponential phase. Adhesion was saturable at 33 and 23 mg (dry weight) g of straw
−1
for
R. flavefaciens
and
F. succinogenes
, respectively. Methyl cellulose and carboxymethyl cellulose inhibited adhesion by 24 to 33%. Competition between species was determined by measuring characteristic cell-associated enzyme activities in filtrates of mixtures incubated with straw;
p
-nitrophenyl-β-
d
-lactopyranoside hydrolysis was used as a marker for
F. succinogenes
, while either β-xylosidase or carboxymethyl cellulase was used for
R. flavefaciens
, depending on the other species present.
R. flavefaciens
had no influence on
F. succinogenes
adhesion, and
F. succinogenes
had only a minor (<20%) effect on
R. flavefaciens
adhesion. The noncellulolytic ruminal bacteria
Bacteroides ruminicola
and
Selenomonas ruminantium
had no influence on adhesion of either cellulolytic species, although these organisms also adhered to the straw. We concluded that
R. flavefaciens
and
F. succinogenes
have separate, specific adhesion sites on barley straw that are not obscured by competition with non-cellulolytic species.
Enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus by the lactoperoxidase system
Authors: Kamau D N , Doores S , Pruitt K M
Published: January 2020

The lactoperoxidase system (LPS) enhanced thermal destruction of Listeria monocytogenes and Staphylococcus aureus. After LPS activation, biphasic survival curves were observed for L. monocytogenes at 57.8 degrees C and for S. aureus at 55.2 degrees C. The data were consistent with a model that assumed two bacterial populations differing in heat sensitivity. The more heat-sensitive fractions (93% of the L. monocytogenes, 92% of the S. aureus) were killed almost instantly. For these biphasic survival curves, D values were based on the much smaller, less-heat-sensitive fractions. For L. monocytogenes, the D52.2 degrees C values were 30.2 min (untreated milk) and 10.7 min (LPS activated); corresponding D55.2 degrees C values were 8.2 and 1.6 min; corresponding D57.8 degrees C values were 2.3 and 0.5 min. For S. aureus, the D52.2 degrees C values were 33.3 min (untreated milk) and 2.2 min (LPS activated), and the corresponding D55.2 degrees C values were 7.6 and 1.1 min, respectively. The most rapid killing of L. monocytogenes occurred when samples were heated soon after activation of the LPS. Activation of the LPS followed by heating can increase the margin of safety with respect to milkborne pathogens.
Stability of Integrated Plasmids in the Chromosome of
Lactococcus lactis
Authors: Leenhouts Kees J. , Kok Jan , Venema Gerard
Published: January 2020

Derivatives of plasmids pBR322, pUB110, pSC101, and pTB19, all containing an identical fragment of lactococcal chromosomal DNA, were integrated via a Campbell-like mechanism into the same chromosomal site of
Lactococcus lactis
MG1363, and the transformants were analyzed for the stability of the integrated plasmids. In all cases the erythromycin resistance gene of pE194 was used as a selectable marker. Transformants obtained by integration of the pBR322 derivatives contained a head-to-tail arrangement of several plasmid copies, which most likely was caused by integration of plasmid multimers. Single-copy integrations were obtained with the pSC101 and pTB19 derivatives. In all of these transformants no loss of the erythromycin gene was detected during growth for 100 generations in the absence of the antibiotic. In contrast, transformants containing integrated amplified plasmid copies of pUB110 derivatives were unstable under these conditions. Since pUB110 appeared to have replicative activity in
L. lactis
, we suggest that this activity destabilized the amplified structures in
L. lactis.
Insecticidal Activity of a Bacterial Crystal Protein Expressed by a Recombinant Baculovirus in Insect Cells
Authors: Martens John W. M. , Honée Guy , Zuidema Douwe , van Lent Jan W. M. , Visser Bert , Vlak Just M.
Published: January 2020

Baculoviruses are insect pathogens with a relatively slow speed of action, and this has limited their use as control agents of insect pests. Introduction into baculoviruses of genes which code for proteins interfering specifically with insect metabolism or metamorphosis, such as toxins, hormones, and enzymes, may enhance the pathogenicity of these viruses. The complete insecticidal crystal protein gene
cryIA(b)
of
Bacillus thuringiensis
subsp.
aizawai
7.21 was engineered into the nuclear polyhedrosis virus of
Autographa californica
(AcNPV) in place of the polyhedrin gene. In infected
Spodoptera frugiperda
cells, the
cryIA(b)
gene was expressed at a high level without interference with AcNPV production. The crystal protein was found in the cytoplasm of
S. frugiperda
cells, mainly as large crystals with an ultrastructure similar to that of
B. thuringiensis
crystals. Infected-cell extracts inhibited feeding of the large cabbage white
Pieris brassicae.
The toxicity of the crystal protein expressed by AcNPV recombinants was comparable with that of the crystal protein expressed by a corresponding
Escherichia coli
recombinant.
Binding of Colloidal MnO
2
by Extracellular Polysaccharides of
Pedomicrobium manganicum
Authors: Sly L. I. , Arunpairojana Vullapa , Dixon D. R.
Published: January 2020

The extracellular acidic polysaccharides of the manganese-oxidizing bacterium
Pedomicrobium manganicum
were able to bind preformed colloidal MnO
2
. The capacity of the cells to bind MnO
2
was pH dependent. Enhanced binding capacity below pH 5 suggests that ionic bonding forces are involved in the binding mechanism and that there is a charge reversal on the acidic polysaccharides between pH 5 and 4 that is due to increased protonation of carboxyl groups.
Regulation of Dissimilatory Fe(III) Reduction Activity in
Shewanella putrefaciens
Authors: Arnold Robert G. , Hoffmann Michael R. , DiChristina Thomas J. , Picardal Flynn W.
Published: January 2020

Under anaerobic conditions,
Shewanella putrefaciens
is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O
2
were presented simultaneously to batch cultures of
S. putrefaciens
indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN
−
resulted in a high rate of Fe(III) reduction in the presence of dissolved O
2
, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O
2
.
Preferential Osmolyte Accumulation: a Mechanism of Osmotic Stress Adaptation in Diazotrophic Bacteria
Authors: Madkour Magdy A. , Smith Linda Tombras , Smith Gary M.
Published: January 2020

A common cellular mechanism of osmotic-stress adaptation is the intracellular accumulation of organic solutes (osmolytes). We investigated the mechanism of osmotic adaptation in the diazotrophic bacteria
Azotobacter chroococcum, Azospirillum brasilense
, and
Klebsiella pneumoniae
, which are adversely affected by high osmotic strength (i.e., soil salinity and/or drought). We used natural-abundance
13
C nuclear magnetic resonance spectroscopy to identify all the osmolytes accumulating in these strains during osmotic stress generated by 0.5 M NaCl. Evidence is presented for the accumulation of trehalose and glutamate in
Azotobacter chroococcum
ZSM4, proline and glutamate in
Azospirillum brasilense
SHS6, and trehalose and proline in
K. pneumoniae.
Glycine betaine was accumulated in all strains grown in culture media containing yeast extract as the sole nitrogen source. Alternative nitrogen sources (e.g., NH
4
Cl or casamino acids) in the culture medium did not result in measurable glycine betaine accumulation. We suggest that the mechanism of osmotic adaptation in these organisms entails the accumulation of osmolytes in hyperosmotically stressed cells resulting from either enhanced uptake from the medium (of glycine betaine, proline, and glutamate) or increased net biosynthesis (of trehalose, proline, and glutamate) or both. The preferred osmolyte in
Azotobacter chroococcum
ZSM4 shifted from glutamate to trehalose as a consequence of a prolonged osmotic stress. Also, the dominant osmolyte in
Azospirillum brasilense
SHS6 shifted from glutamate to proline accumulation as the osmotic strength of the medium increased.
Direct Comparison of Phosphate Uptake by Adnate and Loosely Attached Microalgae within an Intact Biofilm Matrix
Authors: Burkholder JoAnn M. , Wetzel Robert G. , Klomparens Karen L.
Published: January 2020

We report a direct comparison of phosphate uptake by adnate and loosely attached microalgae in an intact biofilm matrix, with resolution at the level of individual cells. Track scanning electron microscope autoradiography enabled assay of [
33
P]phosphate uptake from the overlying water by adnate algae left undisturbed on mature leaves of the macrophyte
Potamogeton illinoensis
or on artificial plant mimics. The epiphyte communities developed in either phosphate-poor or moderately phosphate-enriched water, and they were assayed on both natural and artificial plants. All adnate taxa examined from both natural and artificial plants in both habitats took up significantly less radiolabel when assayed beneath the overlying matrix than when they were exposed to the water upon removal of the overstory material. Track scanning electron microscope autoradiography and track light microscope autoradiography were intercalibrated to enable comparison of [
33
P]phosphate uptake by adnate and loosely attached components of the epiphyte matrix. Loosely attached cells on substrata from both habitats took up significantly more radiolabel than did underlying adnate cells, indicating that access to phosphate supplies from the water depended on the position of microbial cells in the matrix. In this short-term assay, the adnate microalgae were relatively isolated from the water column nutrient source.
Thiobacillus ferrooxidans, a facultative hydrogen oxidizer
Authors: Drobner E , Huber H , Stetter K O
Published: January 2020

The type strain (ATCC 23270) and two other strains of Thiobacillus ferrooxidans were able to grow by hydrogen oxidation, a feature not recognized before. When cultivated on H2, a hydrogenase was induced and the strains were less extremely acidophilic than during growth on sulfidic ores. Cells of T. ferrooxidans grown on H2 and on ferrous iron showed 100% DNA homology. Hydrogen oxidation was not observed in eight other species of the genus Thiobacillus and in Leptospirillum ferrooxidans.
Preservation of Ruminal Bacterium Capsules by Using Lysine in the Electron Microscopy Fixative
Authors: Akin D. E. , Rigsby L. L.
Published: January 2020

Ruminal bacteria from axenic cultures of
Ruminococcus flavefaciens
FD1,
Butyrivibrio fibrisolvens
49, and bacterial types from the ruminal ecosystem that were fixed with 50 mM lysine (
l
-lysine hydrochloride) added to glutaraldehyde had better-preserved capsules and extracellular material than bacteria fixed without lysine.
Isolation and Enological Characterization of Malolactic Bacteria from the Vineyards of Northwestern Spain
Authors: Sieiro Carmen , Cansado José , Agrelo Dolores , Velázquez Jorge B. , Villa T. G.
Published: January 2020

Thirty-five strains of malolactic bacteria were isolated from grapes and alcoholic and malolactic fermentations in two vineyards from northwestern Spain. These belonged to six species of the genera
Lactobacillus
and
Leuconostoc.
The results of their partial enological characterization showed that 47.5% utilized more than 80% of the initial malic acid.
Method Designed To Detect Alginate-Degrading Bacteria
Authors: Kitamikado Manabu , Yamaguchi Kuniko , Tseng Chao-Huang , Okabe Bun'Ichi
Published: January 2020

A simple turbidimetric method was developed to detect alginate degradation. Bacteria were grown in alginate-containing media, and culture fluids were mixed with an acidic albumin solution. Failure to develop a white turbidity indicated an alginate degrader. The method showed alginate degradation by
Vibrio alginolyticus
ATCC 17749, in contrast to prior descriptions.
Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088
Authors: Muriana P M , Klaenhammer T R
Published: January 2020

Lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088 (NCK88), was purified and characterized. Lactacin F is heat stable, proteinaceous, and inhibitory to other lactobacilli as well as Enterococcus faecalis. The bacteriocin was isolated as a floating pellet from culture supernatants brought to 35 to 40% saturation with ammonium sulfate. Native lactacin F was sized at approximately 180 kDa by gel filtration. Column fractions having lactacin F activity were examined by electron microscopy and contained micelle-like globular particles. Purification by ammonium sulfate precipitation, gel filtration, and high-performance liquid chromatography resulted in a 474-fold increase in specific activity of lactacin F. The purified bacteriocin was identified as a 2.5-kDa peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The lactacin F peptide retained activity after extraction from SDS-PAGE gel slices, confirming the identity of the 2.5-kDa peptide. Variants of NCK88 that failed to exhibit lactacin F activity did not produce the 2.5-kDa band. Sequence analysis of purified lactacin F identified 25 N-terminal amino acids containing an arginine residue at the N terminus. Composition analysis indicates that lactacin F may contain as many as 56 amino acid residues.
Methylmercury Decomposition in Sediments and Bacterial Cultures: Involvement of Methanogens and Sulfate Reducers in Oxidative Demethylation
Authors: Oremland Ronald S. , Culbertson Charles W. , Winfrey Michael R.
Published: January 2020

Demethylation of monomethylmercury in freshwater and estuarine sediments and in bacterial cultures was investigated with
14
CH
3
HgI. Under anaerobiosis, results with inhibitors indicated partial involvement of both sulfate reducers and methanogens, the former dominating estuarine sediments, while both were active in freshwaters. Aerobes were the most significant demethylators in estuarine sediments, but were unimportant in freshwater sediments. Products of anaerobic demethylation were mainly
14
CO
2
as well as lesser amounts of
14
CH
4
. Acetogenic activity resulted in fixation of some
14
CO
2
produced from
14
CH
3
HgI into acetate. Aerobic demethylation in estuarine sediments produced only
14
CH
4
, while aerobic demethylation in freshwater sediments produced small amounts of both
14
CH
4
and
14
CO
2
. Two species of
Desulfovibrio
produced only traces of
14
CH
4
from
14
CH
3
HgI, while a culture of a methylotrophic methanogen formed traces of
14
CO
2
and
14
CH
4
when grown on trimethylamine in the presence of the
14
CH
3
HgI. These results indicate that both aerobes and anaerobes demethylate mercury in sediments, but that either group may dominate in a particular sediment type. Aerobic demethylation in the estuarine sediments appeared to proceed by the previously characterized organomercurial-lyase pathway, because methane was the sole product. However, aerobic demethylation in freshwater sediments as well as anaerobic demethylation in all sediments studied produced primarily carbon dioxide. This indicates the presence of an oxidative pathway, possibly one in which methylmercury serves as an analog of one-carbon substrates.
Isolation and characterization of zearalenone sulfate produced by Fusarium spp
Authors: Plasencia J , Mirocha C J
Published: January 2020

A water-soluble compound related to zearalenone was isolated from a culture of Fusarium graminearum 30 grown in rice. The structure of the novel metabolite was determined to be zearalenone-4-sulfate on the basis of fast-atom-bombardment mass spectrometry, proton nuclear magnetic resonance, UV spectroscopy, and by chemical and enzymatic reactions. Strains representing Fusarium equiseti, Fusarium sambucinum, and Fusarium roseum produced the sulfate conjugate as well. In the rat uterus enlargement bioassay, the metabolite or its hydrolysis product was found to retain the estrogenic activity characteristic of zearalenone. Natural occurrence of this novel metabolite might be significant because analytical methods devised for zearalenone in grain cannot detect the conjugate but the conjugate retains the biological properties of the mycotoxin when ingested by animals.
Cloning of the recA gene from a free-living leptospire and distribution of RecA-like protein among spirochetes
Authors: Stamm L V , Parrish E A , Gherardini F C
Published: January 2020

A recombinant plasmid carrying the recA gene of Leptospira biflexa serovar patoc was isolated from a cosmid library of genomic DNA by complementation of an Escherichia coli recA mutation. The cloned serovar patoc recA gene efficiently restored resistance to UV radiation and methyl methanesulfonate. Recombination proficiency was also restored, as measured by the formation of Lac+ recombinants from duplicated mutant lacZ genes. Additionally, the cloned recA gene increased the spontaneous and mitomycin C-induced production of lambda phage in lysogens of an E. coli recA mutant. The product of the cloned recA gene was identified in maxicells as a polypeptide with an Mr of 43,000. Antibodies prepared against the E. coli RecA protein cross-reacted with the serovar patoc RecA protein, indicating structural conservation. Southern hybridization data showed that the serovar patoc recA gene has diverged from the recA gene of L. interrogans, Leptonema illini, and E. coli. With the exception of the RecA protein of L. interrogans serovar hardjo, the RecA protein of the Leptospira serovars and L. illini were synthesized at elevated levels following treatment of cells with nalidixic acid. The level of detectable RecA correlated with previous studies demonstrating that free-living cells of L. biflexa serovars and L. illini were considerably more resistant to DNA-damaging agents than were those of parasitic L. interrogans serovars. RecA protein was not detected in cells of virulent Treponema pallidum or Borrelia burgdorferi.
Relationship between Cell Surface Properties and Transport of Bacteria through Soil
Authors: Gannon J. T. , Manilal V. B. , Alexander M.
Published: January 2020

A study was conducted to relate the properties of
Enterobacter, Pseudomonas, Bacillus, Achromobacter, Flavobacterium,
and
Arthrobacter
strains to their transport with water moving through soil. The bacteria differed markedly in their extent of transport; their hydrophobicity, as measured by adherence to
n
-octane and by hydrophobic-interaction chromatography; and their net surface electrostatic charge, as determined by electrostatic interaction chromatography and by measurements of the zeta potential. Transport of the 19 strains through Kendaia loam or their retention by this soil was not correlated with hydrophobicities or net surface charges of the cells or the presence of capsules. Among 10 strains tested, the presence of flagella was also not correlated with transport. Retention was statistically related to cell size, with bacteria shorter than 1.0 μm usually showing higher percentages of cells being transported through the soil. We suggest that more than one characteristic of bacterial cells determines whether the organisms are transported through soil with moving water.
Immobilization of bacteria and Saccharomyces cerevisiae in poly(tetrafluoroethylene) membranes
Authors: Hyde F W , Hunt G R , Errede L A
Published: January 2020

A novel method for immobilization of bacteria and Saccharomyces cerevisiae cells is described. Microorganisms may be entrapped in a matrix of poly(tetrafluoroethylene) (PTFE) fibrils. Cells are blended with an aqueous emulsion of PTFE stabilized with Triton X-100 surfactant to form a thick paste. The paste is calendered biaxially in a standard rubber mill. This process causes fibrillation of the PTFE and formation of the fibril matrix, which serves only to impart physical integrity to the composite microporous membrane. The cells trapped in the membrane were shown to be viable by incubation of the membrane on solid media and in broth culture. This bioactive membrane represents a new means of immobilization of cells for bioprocessing.
Effects of toxicity, aeration, and reductant supply on trichloroethylene transformation by a mixed methanotrophic culture
Authors: Alvarez-Cohen L , McCarty P L
Published: January 2020

The trichloroethylene (TCE) transformation rate and capacity of a mixed methanotrophic culture at room temperature were measured to determine the effects of time without methane (resting), use of an alternative energy source (formate), aeration, and toxicity of TCE and its transformation products. The initial specific TCE transformation rate of resting cells was 0.6 mg of TCE per mg of cells per day, and they had a finite TCE transformation capacity of 0.036 mg of TCE per mg of cells. Formate addition resulted in increased initial specific TCE transformation rates (2.1 mg/mg of cells per day) and elevated transformation capacity (0.073 mg of TCE per mg of cells). Significant declines in methane conversion rates following exposure to TCE were observed for both resting and formate-fed cells, suggesting toxic effects caused by TCE or its transformation products. TCE transformation and methane consumption rates of resting cells decreased with time much more rapidly when cells were shaken and aerated than when they remained dormant, suggesting that the transformation ability of methanotrophs is best preserved by storage under anoxic conditions.