bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotypic Characteristics and Virulence of
Vibrio anguillarum
-Related Organisms
Authors: Pazos F. , Santos Y. , Magari�os B. , Band�n I. , N��ez S. , Toranzo A. E.
Published: January 2020

The phenotypic, molecular, and virulence properties of 46
Vibrio anguillarum
-related (VAR) strains isolated from diseased fish and shellfish and from the environment were investigated. Twelve reference strains belonging to the 10 serotypes of
V. anguillarum
and the
Vibrio splendidus
type strain were included for comparison. Numerical taxonomy studies allowed us to group the isolates into four phena. The main phenotypic traits to differentiate VAR strains from
V. anguillarum
were fermentation of arabinose and mannitol, indole and Voges-Proskauer reactions, gelatin and casein hydrolysis, hemolytic activity, growth at 37 and 4�C, and resistance to ampicillin. Serological analysis confirmed that phena I and II were composed mainly of strains of
V. anguillarum
, while phena III and IV included VAR strains. Excluding the reference strains, the typeable isolates belonged to serotypes O3 (15 strains), O4 (3 strains), and O5 (2 strains) of
V. anguillarum.
The infectivity trials showed that only 9 of a total of 24 strains tested displayed virulence for rainbow trout. Virulent strains (50% lethal dose ranging from 10
2
to 10
6
cells) included
V. anguillarum
strains belonging to serotypes O1 (one strain), O2 (one strain), O3 (three isolates), and O4 (one isolate) and only three strains of the VAR group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of lipopolysaccharide and outer membrane proteins showed heterogeneity not only among the 10
V. anguillarum
serotypes but also within the VAR group. Immunoblot assays demonstrated a close relationship among
V. anguillarum
strains from the same serotype, while strains from different serotypes were not antigenically related. The VAR strains did not share antigenic components with the serotypes of
V. anguillarum
tested (serotypes O1 to O5). Plasmids were detected in only 19 of the total of 59 strains. The majority of the strains carrying plasmids were grouped within phenon IV, in which plasmid bands of 27 and 36 MDa were found in all the isolates. No correlation between the plasmid content of VAR microorganisms and their phenotypic or virulence characteristics was observed. From these results it can be concluded that VAR strains associated with disease should be included together with
V. anguillarum
in the formulation of vaccines against vibriosis.
A highly purified enrichment culture couples the reductive dechlorination of tetrachloroethene to growth
Authors: Holliger C , Schraa G , Stams A J , Zehnder A J
Published: January 2020

A microscopically pure enrichment culture of a gram-negative anaerobic bacterium, in the present article referred to as PER-K23, was isolated from an anaerobic packed-bed column in which tetrachloroethene (PCE) was reductively transformed to ethane via trichloroethene (TCE), cis-1,2-dichloroethene (cis-1,2-DCE), chloroethene, and ethene. PER-K23 catalyzes the dechlorination of PCE via TCE to cis-1,2-DCE and couples this reductive dechlorination to growth. H2 and formate were the only electron donors that supported growth with PCE or TCE as an electron acceptor. The culture did not grow in the absence of PCE or TCE. Neither O2, NO3-, NO2-, SO4(2-), SO3(2-), S2O3(2-), S, nor CO2 could replace PCE or TCE as an electron acceptor with H2 as an electron donor. Also, organic electron acceptors such as acetoin, acetol, dimethyl sulfoxide, fumarate, and trimethylamine N-oxide and chlorinated ethanes, DCEs, and chloroethene were not utilized. PER-K23 was not able to grow fermentatively on any of the organic compounds tested. Transferring the culture to a rich medium revealed that a contaminant was still present. Dechlorination was optimal between pH 6.8 and 7.6 and a temperature of 25 to 35 degrees C. H2 consumption was paralleled by chloride production, PCE degradation, cis-1,2-DCE formation, and growth of PER-K23. Electron balances showed that all electrons derived from H2 or formate consumption were recovered in dechlorination products and biomass. Exponential growth could be achieved only in gently shaken cultures.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of Polychlorinated Biphenyl Congener Concentration and Sediment Supplementation on Rates of Methanogenesis and 2,3,6-Trichlorobiphenyl Dechlorination in an Anaerobic Enrichment
Authors: Boyle Alfred W. , Blake Cheryl K. , Price W. Allen , May Harold D.
Published: January 2020

We have employed a method of enrichment that allows us to significantly increase the rate of reductive polychlorinated biphenyl (PCB) dechlorination. This method shortens the time required to investigate the effects that culture conditions have on dechlorination and provides an estimate of the potential activity of the PCB-dechlorinating anaerobes. The periodic supplementation of sterile sediment and PCB produced an enhanced, measurable, and sustained rate of dechlorination. We observed volumetric rates of the dechlorination of 2,3,6-trichlorobiphenyl (2,3,6-CB) to 2,6-dichlorobiphenyl (2,6-CB) of more than 300 μmol liter
-1
day
-1
when the cultures were supplemented daily. A calculation of this activity that is based on an estimate of the number of dechlorinating anaerobes present indicates that 1.13 pmol of 2,3,6-CB was dechlorinated to 2,6-CB day
-1
bacterial cell
-1
. This rate is similar to that of the reductive dechlorination of 3-chlorobenzoate by
Desulfomonile tiedjei.
Methanogenesis declined from 585.3 to 125.9 μmol of CH
4
liter
-1
day
-1
, while dechlorination increased from 8.2 to 346.0 μmol of 2,3,6-CB dechlorinated to 2,6-CB liter
-1
day
-1
.
Oxalate- and Glyoxylate-Dependent Growth and Acetogenesis by
Clostridium thermoaceticum
Authors: Daniel Steven L. , Drake Harold L.
Published: January 2020

The acetogenic bacterium
Clostridium thermoaceticum
ATCC 39073 grew at the expense of the two-carbon substrates oxalate and glyoxylate. Other two-carbon substrates (acetaldehyde, acetate, ethanol, ethylene glycol, glycolaldehyde, glycolate, and glyoxal) were not growth supportive. Growth increased linearly with increasing substrate concentrations up to 45 mM oxalate and glyoxylate, and supplemental CO
2
was not required for growth. Oxalate and glyoxylate yielded 4.9 and 9.4 g, respectively, of cell biomass (dry weight) per mol of substrate utilized. Acetate was the major reduced end product recovered from oxalate and glyoxylate cultures.
14
C labeling studies showed that oxalate was subject to decarboxylation, and product analysis indicated that oxalate was utilized by the following reaction: 4
-
OOC-COO
-
+ 5H
2
O → CH
3
COO
-
+ 6HCO
3
-
+ OH
-
. Oxalate- and glyoxylate-dependent growth produced lower acetate concentrations per unit of cell biomass synthesized than did H
2
-, CO-, methanol-, formate-,
O
-methyl-, or glucose-dependent growth. Protein profiles of oxalate-grown cells were dissimilar from protein profiles of glyoxylate-, CO-, or formate-grown cells, suggesting induction of new proteins for the utilization of oxalate.
C. thermoaceticum
DSM 2955 and
Clostridium thermoautotrophicum
JW 701/3 also grew at the expense of oxalate and glyoxylate. However, oxalate and glyoxylate did not support the growth of
C. thermoaceticum
OMD (a nonautotrophic strain) or six other species of acetogenic bacteria tested.
Corrinoid-Dependent Methyl Transfer Reactions Are Involved in Methanol and 3,4-Dimethoxybenzoate Metabolism by
Sporomusa ovata
Authors: Stupperich Erhard , Konle Ralph
Published: January 2020

Washed and air-oxidized proteins from
Sporomusa ovata
cleaved the C-O bond of methanol or methoxyaromatics and transferred the methyl to
dl
-tetrahydrofolate. The reactions strictly required a reductive activation by titanium citrate, catalytic amounts of ATP, and the addition of
dl
-tetrahydrofolate. Methylcorrinoid-containing proteins carried the methanol methyl, which was transferred to
dl
-tetrahydrofolate at a specific rate of 120 nmol h
-1
mg of protein
-1
. Tetrahydrofolate methylation diminished after the addition of 1-iodopropane or when the methyl donor methanol was replaced by 3,4-dimethoxybenzoate. However, whole
Sporomusa
cells utilize the methoxyl groups of 3,4-dimethoxybenzoate as a carbon source by a sequential O demethylation to 4-hydroxy-3-methoxybenzoate and 3,4-dihydroxybenzoate. The in vitro O demethylation of 3,4-[4-
methoxyl
-
14
C]dimethoxybenzoate proceeded via two distinct corrinoid-containing proteins to form 5-[
14
C]methyltetrahydrofolate at a specific rate of 200 nmol h
-1
mg of protein
-1
. Proteins from 3,4-dimethoxybenzoate-grown cells efficiently used methoxybenzoates with vicinal substituents only, but they were unable to activate methanol. These results emphasized that specific enzymes are involved in methanol activation as well as in the activation of various methoxybenzoates and that similar corrinoid-dependent methyl transfer pathways are employed in 5-methyl-tetrahydrofolate formation from these substrates. Methyl-tetrahydrofolate could be demethylated by a distinct methyl transferase. That enzyme activity was present in washed and air-oxidized cell extracts from methanol-grown cells and from 3,4-dimethoxybenzoate-grown cells. It used cob(I)alamin as the methyl acceptor in vitro, which was methylated at a rate of 48 nmol min
-1
mg of protein
-1
even when ATP was omitted from the assay mixture. This methyl-cob(III)alamin formation made possible a spectrophotometric quantification of the preceding methyl transfers from methanol or methoxybenzoates to
dl
-tetrahydrofolate.
Direct Quantification of Unadsorbed Viruses in Suspensions of Adsorbing Colloids with the Enzyme-Linked Immunosorbent Assay
Authors: Dornai D. , Mingelgrin U. , Frenkel H. , Bar-Joseph M.
Published: January 2020

Quantification of two plant viruses in suspensions of homoionic Ca-bentonite was conducted by applying to the enzyme-linked immunosorbent assay plates either the virus-clay mixture (direct method) or the supernatant obtained after the clay was allowed to settle (classic method). Both methods showed a similar dependence of free virus content on clay concentration. A higher content at equilibrium was measured for both viruses by the direct method. The advantage of using the direct over the classic method is discussed.
Purification and Properties of a Highly Active Organophosphorus Acid Anhydrolase from
Alteromonas undina
Authors: Cheng Tu-Chen , Harvey Steven P. , Stroup Adam N.
Published: January 2020

A highly active organophosphorus acid anhydrolase from
Alteromonas undina
was purified to homogeneity and found to be composed of a single polypeptide chain with a molecular weight of 53,000. With diisopropylfluorophosphate as a substrate, the purified enzyme has a specific activity of ∼575 μmol/min/mg of protein. The enzyme has optimum activity at pH 8.0 and 55�C and is stimulated by sulfhydryl reducing agents and manganese. It is capable of rapidly hydrolyzing a wide range of nerve agents and several chromogenic phosphinates.
Comparison and genomic sizing of Escherichia coli O157:H7 isolates by pulsed-field gel electrophoresis
Authors: Harsono K D , Kaspar C W , Luchansky J B
Published: January 2020

Genomic DNAs of Escherichia coli O157:H7 strains isolated from patients and food samples were analyzed by pulsed-field gel electrophoresis. The rare-cutting endonucleases SfiI and XbaI generated 6 and 10 distinct genomic profiles, respectively, for the 22 strains analyzed, indicating that this technique may find application for epidemiologic studies. Summation of XbaI fragments from five E. coli O157:H7 strains estimated the genomic length at ca. 4.7 Mb.
Evaluation of radioactive and nonradioactive gene probes and cell culture for detection of poliovirus in water samples
Authors: Moore N J , Margolin A B
Published: January 2020

Five nonradioactive probe assays were evaluated by using chemiluminescent and colormetric signals, along with two isotopic assays and cell culture, for the detection of poliovirus in concentrated water samples. In environmental samples, a 100% correlation existed between digoxigenin and single-stranded [32P]RNA probes. All probe assays detected more positive samples than the cell culture did.
Breakdown of N-terminally modified peptides and an isopeptide by rumen microorganisms
Authors: Wallace R J , Frumholtz P P , Walker N D
Published: January 2020

Treatment of Trypticase peptides with acetic anhydride, succinic anhydride, or maleic anhydride inhibited their breakdown to ammonia by rumen microorganisms by an average of 89% after 12 h of incubation in vitro. All three treatments gave similar protection. Acetylation also protected dipeptides containing lysine and methionine from degradation. However, more effective protection was obtained by linking lysine and methionine as N-epsilon-methionyl lysine.
Colonization of Crystalline Cellulose by
Clostridium cellulolyticum
ATCC 35319
Authors: Gelhaye E. , Gehin A. , Petitdemange H.
Published: January 2020

Cellulose colonization by
Clostridium cellulolyticum
was studied by using [

methyl-
3

H]thymidine incorporation. The colonization process indicated that a part of the bacterial population was released from cellulose to the liquid phase before binding and colonizing another adhesion site of the cellulose. We postulate that cellulose colonization occurs according to the following process: adhesion, colonization, release, and readhesion.
Growth phase-dependent regulation and membrane localization of SpaB, a protein involved in biosynthesis of the lantibiotic subtilin
Authors: Gutowski-Eckel Z , Klein C , Siegers K , Bohm K , Hammelmann M , Entian K D
Published: January 2020

The information responsible for biosynthesis of the lantibiotic subtilin is organized in an operon-like structure that starts with the spaB gene. The spaB gene encodes an open reading frame consisting of 1,030 amino acid residues, and it was calculated that a protein having a theoretical molecular mass of 120.5 kDa could be produced from this gene. This is consistent with the apparent molecular weight for SpaB of 115,000 which was estimated after sodium dodecyl sulfate-gel electrophoresis and identification with SpaB-specific antibodies. The SpaB protein is very similar to proteins EpiB and NisB, which were identified previously as being involved in epidermin and nisin biosynthesis. Upstream from SpaB a characteristic sigma A promoter sequence was identified. An immunoblot analysis revealed that SpaB expression was strongly regulated. No SpaB protein was detected in the early logarithmic growth phase, and maximum SpaB expression was observed in the early stationary growth phase. The expression of SpaB was strongly correlated with subtilin biosynthesis. Deletion mutations in either of two recently identified regulatory genes, spaR and spaK, which act as a "two-component" regulatory system necessary for growth phase-dependent induction of subtilin biosynthesis (C. Klein, C. Kaletta, and K. D. Entian, Appl. Environ. Microbiol. 59:296-303, 1993), also resulted in failure of SpaB expression. To investigate the intracellular localization of SpaB, vesicles of Bacillus subtilis were prepared. The SpaB protein cosedimented with the vesicle fraction and was released only after vigorous resuspension of the vesicles. Our results suggest that SpaB is membrane associated and that subtilin biosynthesis occurs at the cytoplasmic membrane of B. subtilis.
The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an agr-like regulatory system
Authors: Diep D B , Håvarstein L S , Nissen-Meyer J , Nes I F
Published: January 2020

Purification and amino acid sequencing of plantaricin A, a bacteriocin from Lactobacillus plantarum C11, revealed that maximum bacteriocin activity is associated with the complementary action of two almost-identical peptides, alpha and beta (J. Nissen-Meyer, A. G. Larsen, K. Sletten, M. Daeschel, and I. F. Nes, J. Gen. Microbiol. 139:1973-1978, 1993). A 5-kb chromosomal HindIII restriction fragment containing the structural gene of plantaricin A was cloned and sequenced. Only one gene encoding plantaricin A was found. The gene, termed plnA, encodes a 48-amino-acid precursor peptide, of which the 22 and 23 C-terminal amino acids correspond to the purified peptides. Northern (RNA) blot analysis demonstrated that a probe complementary to the coding strand of the plantaricin A gene hybridized to a 3.3-kb mRNA transcript. Further analysis of the 3.3-kb transcript demonstrated that it contains three additional open reading frames (plnB, plnC and plnD) downstream of plnA. The DNA sequences of plnB, plnC, and plnD revealed that their products closely resemble members of bacterial two-component signal transduction systems. The strongest homology was found to the accessory gene regulatory (agr) system, which controls expression of exoproteins during post-exponential growth in Staphylococcus aureus. The finding that plnABCD are transcribed from a common promoter suggests that the biological role played by the bacteriocin is somehow related to the regulatory function of the two-component system located on the same operon.
Inhibition of the lignin peroxidase of Phanerochaete chrysosporium by hydroxylamino-dinitrotoluene, an early intermediate in the degradation of 2,4,6-trinitrotoluene
Authors: Michels J , Gottschalk G
Published: January 2020

The ability of the white rot fungus Phanerochaete chrysosporium to mineralize 2,4,6-trinitrotoluene (TNT) was studied in the concentration range of 0.36 to 20.36 mg/liter. The initial rate of 14CO2 formation was 30% in 4 days at 0.36 mg of [14C]TNT per liter and decreased to 5% in 4 days at 20.36 mg of [14C]TNT per liter. Such a pronounced inhibition was not observed when a mixture of [14C]2-amino-4,6-dinitrotoluene and [14C]4-amino-2,6-dinitrotoluene was used as a substrate. 2-Hydroxylamino-4,6-dinitrotoluene and its isomer 4-hydroxylamino-2,6-dinitrotoluene were identified as the first detectable degradation products of TNT. Their transient accumulation correlated with the inhibition of TNT degradation and of the veratryl alcohol oxidase activity of lignin peroxidase. With purified lignin peroxidase H8, it could be shown that the two isomers of hydroxylamino-dinitrotoluene were oxidized by lignin peroxidase. The corresponding nitroso-dinitrotoluenes apparently were formed, as indicated by the formation of azoxy-tetranitrotoluenes.
Isolation of
Bacillus thuringiensis
from Stored Tobacco and
Lasioderma serricorne
(F.)
Authors: Kaelin Pascale , Morel Pascale , Gadani Ferruccio
Published: January 2020

Bacillus thuringiensis
was isolated from dried tobacco residues and dead tobacco beetles (
Lasioderma serricorne
(F.); Coleoptera: Anobiidae) collected in a large number of locations worldwide. Eighty-eight samples of stored tobacco were analyzed and yielded 78
B. thuringiensis
strains which were characterized on the basis of parasporal crystal morphology, sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles, and the results of an immunoblot analysis of the insecticidal crystal proteins. Flagellar antigen identification was used to differentiate selected isolates. Strains that produced rhomboidal crystals associated with the Coleoptera-specific pathotype (Cry III group) were the most abundant strains (59% of the isolates). Preliminary toxicity assays were performed with
L. serricorne
larvae, and the results suggested that activity is not restricted to isolates related to the Coleoptera-specific group. The results of our survey indicate that
B. thuringiensis
is part of the natural microflora in the stored-tobacco environment and that this special habitat represents a source of
B. thuringiensis
isolates that may be used to control stored-product pests.
Selective Desulfurization of Dibenzothiophene by
Rhodococcus erythropolis
D-1
Authors: Izumi Yoshikazu , Ohshiro Takashi , Ogino Hiroshi , Hine Yoshimitsu , Shimao Masayuki
Published: January 2020

A dibenzothiophene (DBT)-degrading bacterium,
Rhodococcus erythropolis
D-1, which utilized DBT as a sole source of sulfur, was isolated from soil. DBT was metabolized to 2-hydroxybiphenyl (2-HBP) by the strain, and 2-HBP was almost stoichiometrically accumulated as the dead-end metabolite of DBT degradation. DBT degradation by this strain was shown to proceed as DBT → DBT sulfone → 2-HBP. DBT at an initial concentration of 0.125 mM was completely degraded within 2 days of cultivation. DBT at up to 2.2 mM was rapidly degraded by resting cells within only 150 min. It was thought this strain had a higher DBT-desulfurizing ability than other microorganisms reported previously.
Isolation and characterization of soybean waste-degrading microorganisms and analysis of fertilizer effects of the degraded products
Authors: Kubo M , Okajima J , Hasumi F
Published: January 2020

Two microorganisms which could degrade soybean lees efficiently were isolated and identified as Bacillus circulans and B. stearothermophilus. These two strains secreted thermostable proteases into the medium and could digest soybean lees rapidly and completely at 50 degrees C. Initially, the soybean lees were degraded to proteins in approximately 20 h by these two strains, after which time the concentrations of peptides in the medium gradually increased. The degraded products from soybean lees contained abundant nitrogen compounds, such as peptides, amino acids, and amides. Approximately 10 times more fresh plant weight was obtained (in the case of Brassica campestris) when these degraded products were applied than when water was applied for 42 days. These stimulatory effects of the soybean lees products were almost equal to those of a chemically synthesized fertilizer.
Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2
Authors: Lin S C , Minton M A , Sharma M M , Georgiou G
Published: January 2020

Bacillus licheniformis JF-2 produces a very active biosurfactant under both aerobic and anaerobic conditions. We purified the surface-active compound to homogeneity by reverse-phase C18 high-performance liquid chromatography and showed that it is a lipopeptide with a molecular weight of 1,035. Amino acid analysis, fast atom mass and infrared spectroscopy, and, finally, 1H, 13C, and two-dimensional nuclear magnetic resonance demonstrated that the biosurfactant consists of a heterogeneous C15 fatty acid tail linked to a peptide moiety very similar to that of surfactin, a lipopeptide produced by Bacillus subtilis. Polyclonal antibodies were raised against surfactin and shown to exhibit identical reactivity towards purified JF-2 lipopeptide in competition enzyme-linked immunosorbent assays, thus providing further evidence for the structural similarity of these two compounds. Under optimal conditions, the B. licheniformis JF-2 biosurfactant exhibits a critical micelle concentration of 10 mg/liter and reduces the interfacial tension against decane to 6 x 10(-3) dyne/cm, which is one of the lowest interfacial tensions ever reported for a microbial surfactant.
A method for detection of aromatic metabolites at very low concentrations: application to detection of metabolites of anaerobic toluene degradation
Authors: Edwards E A , Edwards A M , Grbić-Galić D
Published: January 2020

Difficulties inherent in working with anaerobic microorganisms and mixed cultures have hampered efforts to detect and identify metabolites of anaerobic degradation of monoaromatic compounds. Isotope-trapping experiments and analysis using a high-performance liquid chromatograph equipped with a flow-through radioactivity detector were used to detect very low concentrations of metabolites. Data obtained by this method suggest that toluene was degraded via methyl hydroxylation by a mixed methanogenic culture.
Chemotaxis to oligopeptides by Pseudomonas aeruginosa
Authors: Kelly-Wintenberg K , Montie T C
Published: January 2020

A number of peptides were evaluated as chemoattractants for Pseudomonas aeruginosa. Several strains recognized tri-, tetra-, penta-, and hexapeptides in a capillary tube assay. Tripeptides altered at the carboxyl terminus were good attractants, whereas tripeptides altered at the amino terminus did not serve as chemoattractants. Methionine-containing peptides were relatively poor attractants. Arginine-containing peptides gave the best responses. Reduced responses to larger peptides suggest that porin penetration is required. No extracellular peptidase activity was detected. We conclude that oligopeptides are good attractants and that specificity for chemotactic recognition of oligopeptides exists.
Isolation of an insertion sequence (IS1051) from Xanthomonas campestris pv. dieffenbachiae with potential use for strain identification and characterization
Authors: Berthier Y , Thierry D , Lemattre M , Guesdon J L
Published: January 2020

A new insertion sequence was isolated from Xanthomonas campestris pv. dieffenbachiae. Sequence analysis showed that this element is 1,158 bp long and has 15-bp inverted repeat ends containing two mismatches. Comparison of this sequence with sequences in data bases revealed significant homology with Escherichia coli IS5. IS1051, which detected multiple restriction fragment length polymorphisms, was used as a probe to characterize strains from the pathovar dieffenbachiae.
Intracellular Concentrations of Coenzyme A and Its Derivatives from
Clostridium acetobutylicum
ATCC 824 and Their Roles in Enzyme Regulation
Authors: Boynton Zhuang L. , Bennett George N. , Rudolph Frederick B.
Published: January 2020

Intracellular levels of coenzyme A (CoA) and its derivatives involved in the metabolic pathways for
Clostridium acetobutylicum
ATCC 824 were analyzed by using reverse-phase high-performance liquid chromatography (HPLC). During the shift from the acidogenic to the solventogenic or stationary growth phase, the concentration of butyryl-CoA increased rapidly and the concentrations of free CoA and acetyl-CoA decreased. These changes were accompanied by a rapid increase of the solvent pathway enzyme activity and a decrease of the acid pathway enzyme activity. Assays with several non-solvent-producing mutant strains were also carried out. Upon entry of the mutant strains to the stationary phase, the butyryl-CoA concentrations for these mutant strains were comparable to those for the wild type even though the mutants were deficient in solvent-producing enzymes. Levels of acetoacetyl-CoA, β-hydroxy-butyryl-CoA, and crotonyl-CoA compounds in both wild-type and mutant extracts were below HPLC detection thresholds (<21 μM).
Isolation and properties of an extracellular beta-glucosidase from the polycentric rumen fungus Orpinomyces sp. strain PC-2
Authors: Chen H , Li X , Ljungdahl L G
Published: January 2020

An extracellular beta-glucosidase (EC 3.2.1.21) was purified from culture filtrate of the anaerobic rumen fungus Orpinomyces sp. strain PC-2 grown on 0.3% (wt vol-1) Avicel by using Q Sepharose anion-exchange chromatography, ammonium sulfate precipitation, chromatofocusing ion-exchange chromatography, and Superose 12 gel filtration. The enzyme is monomeric with a M(r) of 85,400, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, has a pI of 3.95, and contains about 8.5% (wt vol-1) carbohydrate. The N terminus appears to be blocked. The enzyme catalyzes the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (PNPG). The Km and Vmax values with cellobiose as the substrate at pH 6.0 and 40 degrees C are 0.25 mM and 27.1 mumol.min-1 x mg-1, respectively; with PNPG as the substrate, the corresponding values are of 0.35 mM and 27.7 mumol.min-1 x mg-1. Glucose (Ki = 8.75 mM, with PNPG as the substrate) and gluconolactone (Ki = 1.68 x 10(-2) and 2.57 mM, with PNPG and cellobiose as the substrates, respectively) are competitive inhibitors. Optimal activity with PNPG and cellobiose as the substrates is at pH 6.2 and 50 degrees C. The enzyme has high activity against sophorose (beta-1,2-glucobiose) and laminaribiose (beta-1,3-glucobiose) but has no activity against gentiobiose (beta-1,6-glucobiose). The activity of the beta-glucosidase is stimulated by Mg2+, Mn2+, Co2+, and Ni2+ and inhibited by Ag+, Fe2+, Cu2+, Hg2+, SDS, and p-chloromercuribenzoate.
Cloning and sequencing of the Thermoanaerobacterium saccharolyticum B6A-RI apu gene and purification and characterization of the amylopullulanase from Escherichia coli
Authors: Ramesh M V , Podkovyrov S M , Lowe S E , Zeikus J G
Published: January 2020

The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an M(r) of 142,000 +/- 2,000 and had same the catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase.
Methanotrophic Bacteria and Facilitated Transport of Pollutants in Aquifer Material
Authors: Jenkins Michael B. , Chen Jyh-Herng , Kadner Debra J. , Lion Leonard W.
Published: January 2020

In situ stimulation of methanotrophic bacteria has been considered as a methodology for aquifer remediation. Chlorinated aliphatic hydrocarbons such as trichloroethylene are fortuitously oxidized by the methane monooxygenase produced by methanotrophic bacteria. Experimental results are presented that indicate that both colloidal suspensions containing methanotrophic cells and the soluble extracellular polymers produced by methanotrophic cells have the potential to enhance the transport and removal of other environmental contaminants such as polynuclear aromatic hydrocarbons and transition metals in aquifer material. Three well-characterized methanotrophic bacteria were used in the experiments:
Methylomonas albus
BG8 (a type I methanotroph),
Methylosinus trichosporium
OB3b (a type II methanotroph), and
Methylocystis parvus
OBBP (a type II methanotroph). Isotherms were obtained for sorption of two radiolabeled pollutants, [
14
C] phenanthrene and
109
Cd, onto an aquifer sand in the presence and absence of washed cells and their extracellular polymer. Column transport experiments were performed with the washed methanotrophic cells and phenanthrene. The distribution coefficients for Cd with extracellular polymers were of the same order as that obtained with the aquifer sand, indicating that polymers from the methanotrophic bacteria could act to increase the transport of Cd in a porous medium. Polymer from BG8 significantly reduced the apparent distribution coefficient for Cd with an aquifer sand. [
14
C] phenanthrene also sorbed to extracellular polymer and to washed, suspended methanotrophic cells. The exopolymer of BG8 and OBBP significantly reduced the apparent distribution coefficient (

K
d

) for phenanthrene with aquifer sand. The distribution coefficients for phenanthrene with the methanotrophic cells were an order of magnitude greater than those previously reported for other heterotrophic bacteria. Cells of the methanotrophs also significantly reduced the apparent

K
d

for phenanthrene with an aquifer sand. The three strains of methanotrophs tested displayed mobility in a column of packed sand, and strain OBBP reduced the retardation coefficient of phenanthrene with an aquifer sand by 27%. These data indicate that both extracellular polymer and mobile cells of methanotrophic bacteria display a capacity to facilitate the mobility of pollutant metals and polynuclear aromatic hydrocarbons in aquifer material.
Ammonium and Nitrite Inhibition of Methane Oxidation by
Methylobacter albus
BG8 and
Methylosinus trichosporium
OB3b at Low Methane Concentrations
Authors: King Gary M. , Schnell Sylvia
Published: January 2020

Methane oxidation by pure cultures of the methanotrophs
Methylobacter albus
BG8 and
Methylosinus trichosporium
OB3b was inhibited by ammonium choride and sodium nitrite relative to that in cultures assayed in either nitrate-containing or nitrate-free medium.
M. albus
was generally more sensitive to ammonium and nitrite than
M. trichosporium
. Both species produced nitrite from ammonium; the concentrations of nitrite produced increased with increasing methane concentrations in the culture headspaces. Inhibition of methane oxidation by nitrite was inversely proportional to headspace methane concentrations, with only minimal effects observed at concentrations of>500 ppm in the presence of 250 μM nitrite. Inhibition increased with increasing ammonium at methane concentrations of 100 ppm. In the presence of 500 μM ammonium, inhibition increased initially with increasing methane concentrations from 1.7 to 100 ppm; the extent of inhibition decreased with methane concentrations of > 100 ppm. The results of this study provide new insights that explain some of the previously observed interactions among ammonium, nitrite, methane, and methane oxidation in soils and aquatic systems.
Monoclonal antibodies with a high degree of specificity for Listeria monocytogenes serotype 4b
Authors: Kathariou S , Mizumoto C , Allen R D , Fok A K , Benedict A A
Published: January 2020

Strains of Listeria monocytogenes serotype 4b account for a large fraction of sporadic listeriosis cases, as well as all major food-borne epidemics attributed to this pathogen. We have identified a set of three monoclonal antibodies which showed a high degree of specificity for strains of L. monocytogenes serotype 4b. Two of these antibodies (c74.33 and c74.180, isotypes immunoglobulin M [IgM] and IgG3, respectively) recognized all serotype 4b strains, whereas antibody c74.22 (isotype IgG1) failed to recognize certain epidemic-associated strains. The corresponding antigens were located on the surface of the bacteria and were expressed following bacterial growth in different media and over a wide range of temperatures (4, 22, and 37 degrees C). Heating L. monocytogenes cells at 80,90, or 100 degrees C abolished reactivity for c74.22 but not for c74.33 MAb. These MAbs were negative for all of the non-Listeria strains tested, including representatives of several gram-negative and gram-positive species. The surface antigen recognized by c74.22 appeared to be associated with the ability of the bacteria to enter (invade) mammalian cells in culture.
Antibody-direct epifluorescent filter technique for rapid, direct enumeration of Escherichia coli O157:H7 in beef
Authors: Tortorello M L , Stewart D S
Published: January 2020

Artificially inoculated Escherichia coli O157:H7 was directly enumerated in ground beef and beef exudate, without enrichment or selection, by the antibody-direct epifluorescent filter technique (Ab-DEFT). The total assay time of the Ab-DEFT was less than 1 h. The beef was homogenized, treated for 15 min with trypsin and Triton X-100, and passed through a 5-microns-pore-size prefilter and then through a 0.2-microns-pore-size black polycarbonate filter. The final filter was stained directly with fluorescein-labeled anti-O157 polyclonal antibody, rinsed, and examined by epifluorescence microscopy. The sensitivity of the Ab-DEFT was compared with that of a standard enrichment culture technique. Both methods reliably determined the presence of the pathogen in beef at 16 CFU/g. The Ab-DEFT was also useful for quantifying the pathogen and monitoring its growth in beef.
A Sensitive Method Using 4-Methylumbelliferyl-β-Cellobiose as a Substrate To Measure (1,4)-β-Glucanase Activity in Sediments
Authors: Boschker H. T. S. , Cappenberg T. E.
Published: January 2020

A sensitive method to measure (1,4)-β-glucanase activity in organic matter-rich sediments, using 4-methyl-umbelliferyl-β-cellobiose as a substrate, is described. β-Glucosidases, which were also able to hydrolyze this substrate, were inhibited with
d
-glucono-δ-lactone. The produced 4-methylumbelliferone was recovered quantitatively out of the sediment by an extraction with 80% ethanol. An inhibition experiment with known substrates or inhibitors suggested that at least 59% of the measured activity could be explained by enzymes of the exo-(1,4)-β-glucanase type and that the contribution of endo-(1,4)-β-glucanases was minor. Results of the inhibition experiment also suggested that the measured activity was of bacterial origin in the sediment used. First results of field measurements are given for the sediment from the reed bed of Lake Gooimeer.
Metabolism of Pyrene by the Basidiomycete
Crinipellis stipitaria
and Identification of Pyrenequinones and Their Hydroxylated Precursors in Strain JK375
Authors: Lambert Michael , Kremer Stefan , Sterner Olov , Anke Heidrun
Published: January 2020

The metabolism of pyrene, a polycyclic aromatic hydrocarbon, by submerged cultures of the basidiomycete
Crinipellis stipitaria
was studied. After incubation for 68 h at 25°C in a 20-liter fermentor with complex medium and 20 mg of pyrene per liter, five metabolites were detected. The compounds were isolated by preparative high-performance liquid chromatography on RP18 and DIOL gels. By UV, infrared, and
1
H nuclear magnetic resonance spectroscopy and mass spectrometry, 1-hydroxypyrene, 1,6-dihydroxypyrene, 1,8-dihydroxypyrene, 1,6-pyrenequinone, and 1,8-pyrenequinone were identified. 1,6- and 1,8-dihydroxypyrene were obtained from fungal cultures for the first time. The formation of these metabolites was confirmed by investigations with [4,5,9,10-
14
C]pyrene.
The NodD proteins of Rhizobium sp. strain BR816 differ in their interactions with coinducers and in their activities for nodulation of different host plants
Authors: van Rhijn P , Desair J , Vlassak K , Vanderleyden J
Published: January 2020

The early steps of symbiotic nodule formation by Rhizobium spp. on plants require coordinate expression of several nod gene operons, which is accomplished by the activating protein NodD. Rhizobium sp. strain BR816, isolated from Leucaena leucocephala, contains four nodD genes which differ in their interaction with flavonoids. Two of the four NodD proteins, namely, NodD1 and NodD2, obey the LysR rule regarding the need of a coinducer. NodD3 shows hardly any inducing activity, and NodD4 contains a high basal activity and no response to any of the flavonoids tested. Complementation experiments with the NGR234 nodD mutant by the different nodD genes of BR816, as well as the analysis of the nodulation phenotype of different nodD mutants of BR816, revealed that all the nodD genes of BR816 are functional, but differences can be noticed when different host plants are tested. Whereas the nodD2 and nodD4 genes of BR816 have a great impact on the nodulation of L. leucocephala, nodD3 and nodD4 appear to be important for the nodulation of Phaseolus vulgaris. It appears that NodD1 of BR816 can function as a transcriptional activator in bean nodulation but not in nodulation of L. leucocephala.
Induction of Carbon Starvation-Induced Proteins in
Vibrio vulnificus
Authors: Morton Darla S. , Oliver James D.
Published: January 2020

By using two-dimensional polyacrylamide gel electrophoresis of pulse-labelled proteins, carbon starvation-induced (Sti) proteins produced by
Vibrio vulnificus
were identified. At least 34 proteins were induced over a 26-h period of carbon starvation. Although the total rate of protein synthesis over the 26-h starvation period suggests that there is a dramatic decrease in total protein synthesis within the first hour of starvation, at least 23 of the Sti proteins were induced within the first 20 min of carbon depletion. Six temporal classes of proteins could be identified, with class A(ii) encompassing the largest (21 proteins) group. All of the proteins in this group could be characterized by one of two patterns of protein synthesis. The addition of chloramphenicol at sequential times throughout starvation revealed that proteins required for starvation survival are made within the first 4 h of starvation.
Occurrence and Role of Di-
myo
-Inositol-1,1′-Phosphate in
Methanococcus igneus
Authors: Ciulla Rose Anne , Burggraf Siegfried , Stetter Karl O. , Roberts Mary F.
Published: January 2020

Methanococcus igneus
, a hyperthermophilic marine methanogen (optimum growth temperature of 88°C) with a 25-min doubling time, synthesizes an unusual inositol phosphodiester which is present at high intracellular concentrations along with
l
-α-glutamate and β-glutamate. Identification of this compound as a dimeric inositol phosphodiester (di-
myo
-inositol-1,1′-phosphate) was provided by two-dimensional nuclear magnetic resonance methods. The intracellular levels of all three negatively charged solutes (
l
-α-glutamate, β-glutamate, and the inositol phosphodiester) increase with increasing levels of external NaCl, although the inositol compound shows much smaller increases with increasing NaCl levels than the glutamate isomers. The turnover of these solutes was examined by
13
CO
2
-pulse-CO
2
-chase experiments. The results indicated that both the β-glutamate and the inositol phosphodiester behaved as compatible solutes and were not efficiently metabolized by cells as was
l
-α-glutamate. At a fixed external NaCl concentration, lower ammonium levels increased the fraction of the inositol dimer present in extracts. The most pronounced changes in di-
myo
-inositol-1,1′-phosphate occurred as a function of cell growth temperature. While the organism grows over a relatively wide temperature range, the phosphodiester accumulated only when
M. igneus
was grown at temperatures of ≥80°C. Thus, this unusual compound is a non-nitrogen-containing osmolyte preferentially synthesized at high growth temperatures.
Detection of hepatitis A virus, rotavirus, and enterovirus in naturally contaminated shellfish and sediment by reverse transcription-seminested PCR
Authors: Le Guyader F , Dubois E , Menard D , Pommepuy M
Published: January 2020

A reverse transcription-PCR method was developed to detect enterovirus (EV), hepatitis A virus (HAV), and rotavirus (RV) RNAs in shellfish and sediment. The method was first tested under experimental conditions by using virus-spiked shellfish to evaluate assay sensitivity. The use of CC41 cellulose was found to be efficient for removing inhibitors of RV detection. For sediment samples, a Sephadex column was used to allow the detection of EV and HAV RNAs. The specificity of amplified products was controlled by hybridization with digoxigenin-labeled oligoprobes. The method was then applied to naturally contaminated shellfish and sediments. EV, HAV, and RV RNAs were detected in 22, 14, and 20% of the shellfish samples, respectively. No relationship between viral contamination and bacterial contamination was found. When viral RNAs (HAV or EV) were detected in sediments, they were also detected in shellfish.
Cloning and heterologous expression of a gene encoding an alkane-induced extracellular protein involved in alkane assimilation from Pseudomonas aeruginosa
Authors: Hardegger M , Koch A K , Ochsner U A , Fiechter A , Reiser J
Published: January 2020

Pseudomonas aeruginosa PG201 produces a 16-kDa extracellular protein in media containing n-hexadecane as a carbon source but not in media containing glycerol or glucose. This protein was purified, and the N-terminal amino acid sequence was determined. The amino acid composition of the protein was found to be very similar to that of the so-called protein-like activator for n-alkane oxidation (PA) from P. aeruginosa S7B1. This extracellular protein was previously characterized (K. Hisatsuka, T. Nakahara, Y. Minoda, and K. Yamada, Agric. Biol. Chem. 41:445-450, 1977) and found to stimulate the growth of P. aeruginosa on n-hexadecane and to possess emulsifying activity. To study the role(s) of the PA protein and to make it accessible for possible future applications, we have cloned the PA-encoding (pra) gene and determined its nucleotide sequence. This analysis revealed a protein-coding region of 162 amino acids, with the first 25 residues being reminiscent of those of a typical bacterial signal sequence. The pra gene was inactivated by insertional mutagenesis, and the resulting strain was found to lack extracellular PA protein and to be retarded in its growth in n-hexadecane-containing media. These results are consistent with the growth stimulatory role of the PA protein. The pra gene was expressed in Escherichia coli, and substantial amounts of the recombinant protein were found in the extracellular growth medium. The recombinant protein was purified by metal chelate affinity chromatography. The ability to produce secreted PA protein by E. coli provides a simple and safe means to analyze its function(s) in alkane assimilation in the future.
Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110
Authors: Varma A , Palsson B O
Published: January 2020

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)