bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and identification of eight microcystins from thirteen Oscillatoria agardhii strains and structure of a new microcystin
Authors: Luukkainen R , Sivonen K , Namikoshi M , Färdig M , Rinehart K L , Niemelä S I
Published: January 2020

Microcystins (cyclic heptapeptide hepatotoxins), isolated from 13 freshwater Oscillatoria agardhii strains from eight different Finnish lakes by high-performance liquid chromatography, were characterized by amino acid analysis, fast atom bombardment mass spectrometry (FABMS), and tandem FABMS (FABMS/collisionary-induced dissociation/MS). All strains produced two to five different microcystins. In total, eight different compounds, of which five were known microcystins, were isolated. The known compounds identified were [D-Asp3]MCYST (microcystin)-LR, [Dha7]MCYST-LR, [D-Asp3]MCYST-RR, [Dha7]MCYST-RR, and [D-Asp3,Dha7]MCYST-RR. This is the first time that isolation of these toxins from Oscillatoria spp., with the exception of [D-Asp3]MCYST-RR, has been reported. Three of the strains produced a new microcystin, and the structure was assigned as [D-Asp3,Mser7]MCYST-RR. The structures of two new microcystins, produced as minor components by one Oscillatoria strain, could not be determined because of the small amounts isolated from the cells. Four strains produced [Dha7]MCYST-RR as the main toxin, but [D-Asp3]MCYST-RR was clearly the most abundant and most frequently occurring toxin among these isolates of O. agardhii.
Simple solutions to false-positive staphylococcal enterotoxin assays with seafood tested with an enzyme-linked immunosorbent assay kit (TECRA)
Authors: Park C E , Akhtar M , Rayman M K
Published: January 2020

The TECRA kit, a commercial staphylococcal enterotoxin visual immunoassay kit, is an enzyme-linked immunosorbent assay system which utilizes polyvalent antisera against staphylococcal enterotoxin types A to E. The test is simple and rapid to perform (4 h) and has therefore been widely used for screening purposes. In this study, the TECRA kit produced a number of false-positive reactions with seafood; 25% of 218 samples of seven types of seafood gave false-positive results, particularly shellfish such as mussels (85%), clams (32%), oysters (23%), winkles (20%), and squid (13%). Some nonshellfish samples also gave false-positive results with the TECRA kit (smelt [20%] and trout [10%]). The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was: (i) heat labile, being completely inactivated by heating for 3 min at 70 degrees C, compared with 5% inactivation of true staphylococcal enterotoxins by the same heat treatment, (ii) in a selective reaction with normal rabbit or calf serum (nonspecific reactions were completely abolished by these sera, whereas staphylococcal enterotoxins were not affected), and (iii) incapable of binding to a copper-chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The false-positive reactions occurring with seafood were not associated with substances produced by microorganisms, since the bacterial isolates from the samples did not give positive results with the TECRA kit.(ABSTRACT TRUNCATED AT 250 WORDS)
Biodegradation of 4-nitrotoluene by Pseudomonas sp. strain 4NT
Authors: Haigler B E , Spain J C
Published: January 2020

A strain of Pseudomonas spp. was isolated from nitrobenzene-contaminated soil on 4-nitrotoluene as the sole source of carbon, nitrogen, and energy. The organism also grew on 4-nitrobenzaldehyde, and 4-nitrobenzoate. 4-Nitrobenzoate and ammonia were detected in the culture fluid of glucose-grown cells after induction with 4-nitrotoluene. Washed suspensions of 4-nitrotoluene- or 4-nitrobenzoate-grown cells oxidized 4-nitrotoluene, 4-nitrobenzaldehyde, 4-nitrobenzyl alcohol, and protocatechuate. Extracts from induced cells contained 4-nitrobenzaldehyde dehydrogenase, 4-nitrobenzyl alcohol dehydrogenase, and protocatechuate 4,5-dioxygenase activities. Under anaerobic conditions, cell extracts converted 4-nitrobenzoate or 4-hydroxylaminobenzoate to protocatechuate. Conversion of 4-nitrobenzoate to protocatechuate required NADPH. These results indicate that 4-nitrotoluene was degraded by an initial oxidation of the methyl group to form 4-nitrobenzyl alcohol, which was converted to 4-nitrobenzoate via 4-nitrobenzaldehyde. The 4-nitrobenzoate was reduced to 4-hydroxylaminobenzoate, which was converted to protocatechuate. A protocatechuate 4,5-dioxygenase catalyzed meta-ring fission of the protocatechuate. The detection of 4-nitrobenzaldehyde and 4-nitrobenzyl alcohol dehydrogenase and 4-nitrotoluene oxygenase activities in 4-nitrobenzoate-grown cells suggests that 4-nitrobenzoate is an inducer of the 4-nitrotoluene degradative pathway.
C
15
H
24
Volatile Compounds Unique to Aflatoxigenic Strains of
Aspergillus flavus
Authors: Zeringue H. J. , Bhatnagar D. , Cleveland T. E.
Published: January 2020

Headspace volatiles from eight strains of
Aspergillus flavus
(four aflatoxigenic strains and four nonaflatoxigenic strains), grown for 1, 2, 3, 4, 8, and 10 days in submerged cultures, were collected in Tenax GC traps. The traps were desorbed onto a 50-m gas-liquid chromatography capillary column by heat and gas purge from an external direct injector device. The column was interfaced with a mass spectrometer data acquisition system. Peaks were identified by comparing retention times and mass spectra with those obtained from authentic compounds and by using a computer-assisted mass spectral data base. Aflatoxigenic strains of
A. flavus
produced several C
15
H
24
compounds (e.g., alpha-gurjunene,
trans
-caryophyllene, and cadinene) which peaked in 3-day cultures and were not present in earlier (1- and 2-day) or later (8- and 10-day) cultures. None of these volatiles were detected in nonaflatoxigenic strains of
A. flavus
. There was an apparent correlation between the release of C
15
H
24
volatile compounds and the initiation of aflatoxin biosynthesis, and a correlation between decline of aflatoxin synthesis and the disappearance of the C
15
H
24
compounds unique to aflatoxigenic
A. flavus
also existed.
Microcosm and in situ field studies of enhanced biotransformation of trichloroethylene by phenol-utilizing microorganisms
Authors: Hopkins G D , Semprini L , McCarty P L
Published: January 2020

The ability of different aerobic groundwater microorganisms to cometabolically degrade trichloroethylene (TCE), 1,2-cis-dichloroethylene (c-DCE), and 1,2-trans-dichloroethylene (t-DCE) was evaluated both in groundwater-fed microcosms and in situ in a shallow aquifer. Microcosms amended with phenol or toulene were equally effective in removing c-DCE (> 90%) followed by TCE (60 to 70%), while the microcosm fed methane was most effective in removing t-DCE (> 90%). The microcosm fed ammonia was the least effective. None of the microcosms effectively degraded 1,1,1-trichloroethane. At the Moffett Field groundwater test site, in situ removal of c-DCE and TCE coincided with biostimulation through phenol and oxygen injection and utilization, with c-DCE removed more rapidly than TCE. Greater TCE and c-DCE removal was observed when the phenol concentration was increased. Over 90% removal of c-DCE and TCE was observed in the 2-m biostimulated zone. This compares with 40 to 50% removal of c-DCE and 15 to 25% removal of TCE achieved by methane-grown microorganisms previously evaluated in an adjacent in situ test zone. The in situ removal with phenol-grown microorganisms agrees qualitatively with the microcosm studies, with the rates and extents of removal ranked as follows: c-DCE > TCE > t-DCE. These studies demonstrate the potential for in situ TCE bioremediation using microorganisms grown on phenol.
Biodegradation of ortho-cresol by a mixed culture of nitrate-reducing bacteria growing on toluene
Authors: Flyvbjerg J , Jørgensen C , Arvin E , Jensen B K , Olsen S K
Published: January 2020

A mixed culture of nitrate-reducing bacteria degraded o-cresol in the presence of toulene as a primary growth substrate. No degradation of o-cresol was observed in the absence of toluene or when the culture grew on p-cresol and 2,4-dimethylphenol. In batch cultures, the degradation of o-cresol started after toluene was degraded to below 0.5 to 1.0 mg/liter but continued only for about 3 to 5 days after the depletion of toluene since the culture had a limited capacity for o-cresol degradation once toluene was depleted. The total amount of o-cresol degraded was proportional to the amount of toluene metabolized, with an average yield of 0.47 mg of o-cresol degraded per mg of toluene metabolized. Experiments with [ring-U-14C]o-cresol indicated that about 73% of the carbon from degraded o-cresol was mineralized to CO2 and about 23% was assimilated into biomass after the transient accumulation of unidentified water-soluble intermediates. A mathematical model based on a simplified Monod equation is used to describe the kinetics of o-cresol degradation. In this model, the biomass activity toward o-cresol is assumed to decay according to first-order kinetics once toluene is depleted. On the basis of nonlinear regression of the data, the maximum specific rate of o-cresol degradation was estimated to be 0.4 mg of o-cresol per mg of biomass protein per h, and the first-order decay constant for o-cresol-degrading biomass activity was estimated to be 0.15 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of Grazing by the Free-Living Soil Amoebae
Acanthamoeba castellanii, Acanthamoeba polyphaga
, and
Hartmannella vermiformis
on Various Bacteria
Authors: Weekers Peter H. H. , Bodelier Paul L. E. , Wijen John P. H. , Vogels Godfried D.
Published: January 2020

Cultures of 10 different bacteria were used to serve as food sources for axenically grown
Acanthamoeba castellanii, Acanthamoeba polyphaga
, and
Hartmannella vermiformis
. The nonpigmented enterobacteriaceae
Escherichia coli
K-12 and
Klebsiella aerogenes
appeared to be excellent feed to all three amoebae. Hardly any growth or ammonium production was observed in tests with
Chromatium vinosum
and
Serratia marcescens
, which share the presence of pigmented compounds. Distinct differences in net ammonium production were detected and were correlated to the amoebal growth yield. In general, growth of amoebae and ammonium production increased in the order
A. polyphaga, A. castellanii
, and
H. vermiformis
.
Brochocin-C, a new bacteriocin produced by Brochothrix campestris
Authors: Siragusa G R , Cutter C N
Published: January 2020

Brochotrix campestris ATCC 43754 produces a bacteriocin inhibitory towards Brochothrix thermosphacta, lactobacilli, Listeria spp., and other gram-positive bacteria. This antimicrobial agent is heat stable, sensitive to proteases, catalase insensitive, and free of organic acids. No phage particles were detected by transmission electron microscopy. Muramidase activity was not detected in the preparations. On the basis of established criteria, the antimicrobial agent was classified as a bacteriocin and named brochocin-C.
Enzymatic determination of itoic acid, a Bacillus subtilis siderophore, and 2,3-dihydroxybenzoic acid
Authors: Ito T
Published: January 2020

A specific enzymatic method to determine the amounts of itoic acid, a Bacillus subtilis siderophore, and 2,3-dihydroxybenzoic acid (2,3-DHBA) was devised. A sample was incubated first with hippurate hydrolase and then with 2,3-DHBA-3,4-dioxygenase. Itoic acid was estimated from the increase in A374. The incubation with the first enzyme was omitted for the determination of 2,3-DHBA.
Analysis of the Bradyrhizobium japonicum hemH gene and its expression in Escherichia coli
Authors: Frustaci J M , O'Brian M R
Published: January 2020

Complementation analysis showed that the Bradyrhizobium japonicum hemH gene was both necessary and sufficient to rescue mutant strains I110ek4 and I110bk2 in trans with respect to hemin auxotrophy, protoporphyrin accumulation, and the deficiency in ferrochelatase activity. The B. japonicum hemH gene was expressed in an Escherichia coli T7 expression system and yielded a 39-kDa protein, which was consistent with the predicted size of the deduced product. The overexpressed protein was purified and shown to contain ferrochelatase activity, thereby demonstrating that the hemH gene encodes ferrochelatase. When expressed from the lac promoter, the B. japonicum hemH gene was able to complement the enzyme activity of a ferrochelatase-defective E. coli mutant, and it also conferred hemin prototrophy on those cells. These latter findings confirm the identity of the hemH gene product and demonstrate that B. japonicum ferrochelatase can interact with the E. coli heme synthesis enzymes for heme formation in complemented cells.
Paracrystalline surface layers of dairy propionibacteria
Authors: Lortal S , Rouault A , Cesselin B , Sleytr U B
Published: January 2020

We examined 70 dairy propionibacteria and detected a crystalline surface layer (S-layer) in only 2 organisms (Propionibacterium freudenreichii CNRZ 722 and Propionibacterium jensenii CNRZ 87) by freeze-etching and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). Both S-layers exhibited oblique (p2) symmetry (a = 9.9 nm; b = 5.4 nm; gamma = 80 degrees) and completely covered the cell surface. Treatment for 15 min at the ambient temperature with 5 M guanidine hydrochloride or acidic conditions (250 mM ammonium acetate, pH 2.7) efficiently extracted the S-layer protein from intact cells of strain CNRZ 722, whereas treatment with 5 M guanidine hydrochloride at 100 degrees C for 15 min was necessary to isolate the S-layer protein of strain CNRZ 87. The precipitates obtained after dialysis of the extracting agents produced no regular patterns. The molecular masses of the two S-layer proteins, as estimated by SDS-PAGE, were 58.5 kDa for the strain CNRZ 722 and 67.3 kDa for the strain CNRZ 87. Mass spectrometry of the isolated S-layer protein of strain CNRZ 722 gave a molecular mass value close to the expected value (56,533 Da). The N-terminal sequences of the two purified S-layer proteins differed, as did their amino acid compositions, except that the same high hydrophobic amino acid content (52%) was observed.
Characterization of the methanotrophic bacterial community present in a trichloroethylene-contaminated subsurface groundwater site
Authors: Bowman J P , Jiménez L , Rosario I , Hazen T C , Sayler G S
Published: January 2020

Groundwater, contaminated with trichloroethylene (TCE) and tetrachloroethylene (PCE), was collected from 13 monitoring wells at Area M on the U.S. Department of Energy Savannah River Site near Aiken, S.C. Filtered groundwater samples were enriched with methane, leading to the isolation of 25 methanotrophic isolates. The phospholipid fatty acid profiles of all the isolates were dominated by 18:1 omega 8c (60 to 80%), a signature lipid for group II methanotrophs. Subsequent phenotypic testing showed that most of the strains were members of the genus Methylosinus and one isolate was a member of the genus Methylocystis. Most of the methanotroph isolates exhibited soluble methane monooxygenase (sMMO) activity. This was presumptively indicated by the naphthalene oxidation assay and confirmed by hybridization with a gene probe encoding the mmoB gene and by cell extract assays. TCE was degraded at various rates by most of the sMMO-producing isolates, whereas PCE was not degraded. Savannah River Area M and other groundwaters, pristine and polluted, were found to support sMMO activity when supplemented with nutrients and then inoculated with Methylosinus trichosporium OB3b. The maximal sMMO-specific activity obtained in the various groundwaters ranged from 41 to 67% compared with maximal rates obtained in copper-free nitrate mineral salts media. This study partially supports the hypothesis that stimulation of indigenous methanotrophic communities can be efficacious for removal of chlorinated aliphatic hydrocarbons from subsurface sites and that the removal can be mediated by sMMO.
Dominating Role of an Unusual Magnetotactic Bacterium in the Microaerobic Zone of a Freshwater Sediment
Authors: Spring Stefan , Amann Rudolf , Ludwig Wolfgang , Schleifer Karl-Heinz , van Gemerden Hans , Petersen Nikolai
Published: January 2020

A combination of polymerase chain reaction-assisted rRNA sequence retrieval and fluorescent oligonucleotide probing was used to identify in situ a hitherto unculturable, big, magnetotactic, rod-shaped organism in freshwater sediment samples collected from Lake Chiemsee. Tentatively named
“Magnetobacterium bavaricum,”
this bacterium is evolutionarily distant from all other phylogenetically characterized magnetotactic bacteria and contains unusually high numbers of magnetosomes (up to 1,000 magnetosomes per cell). The spatial distribution in the sediment was studied, and up to 7 × 10
5
active cells per cm
3
were found in the microaerobic zone. Considering its average volume (25.8 ± 4.1 μm
3
) and relative abundance (0.64 ± 0.17%),
“M. bavaricum”
may account for approximately 30% of the microbial biovolume and may therefore be a dominant fraction of the microbial community in this layer. Its microhabitat and its high content of sulfur globules and magnetosomes suggest that this organism has an iron-dependent way of energy conservation which depends on balanced gradients of oxygen and sulfide.
Effect of promoter modification on mosquitocidal cryIVB gene expression in Synechococcus sp. strain PCC 7942
Authors: Soltes-Rak E , Kushner D J , Williams D D , Coleman J R
Published: January 2020

The impact of promoter modification on the expression of the mosquitocidal Bacillus thuringiensis subsp. israelensis cryIVB gene when used to transform the cyanobacterium Synechococcus sp. strain PCC 7942 has been examined. Maximal transcript and protein abundances were achieved by the addition of the lacZ promoter upstream of the cryIVB sequence. Replacement of the endogenous corresponding Bacillus sequences with the Synechococcus petF1 promoter, ribosome binding site, and initiation codon also resulted in increased expression of the cryIVB gene relative to the expression obtained with the Bacillus promoter alone but decreased expression relative to the expression achieved with the tandem array of the Bacillus and lacZ promoters. Synechococcus cells carrying plasmids in which the expression of the cryIVB gene was regulated by either the lacZ or the petF1 promoter were readily consumed by first-instar Culex restuans larvae and proved to be toxic for these organisms.
Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts
Authors: Payment P , Franco E
Published: January 2020

To find the most suitable indicator of viral and parasitic contamination of drinking water, large-volume samples were collected and analyzed for the presence of pathogens (cultivable human enteric viruses, Giardia lamblia cysts, and Cryptosporidium oocysts) and potential indicators (somatic and male-specific coliphages, Clostridium perfringens). The samples were obtained from three water treatment plants by using conventional or better treatments (ozonation, biological filtration). All samples of river water contained the microorganisms sought, and only C. perfringens counts were correlated with human enteric viruses, cysts, or oocysts. For settled and filtered water samples, all indicators were statistically correlated with human enteric viruses but not with cysts or oocysts. By using multiple regression, the somatic coliphage counts were the only explanatory variable for the human enteric virus counts in settled water, while in filtered water samples it was C. perfringens counts. Finished water samples of 1,000 liters each were free of all microorganisms, except for a single sample that contained low levels of cysts and oocysts of undetermined viability. Three of nine finished water samples of 20,000 liters each revealed residual levels of somatic coliphages at 0.03, 0.10, and 0.26 per 100 liters. Measured virus removal was more than 4 to 5 log10, and cyst removal was more than 4 log10. Coliphage and C. perfringens counts suggested that the total removal and inactivation was more than 7 log10 viable microorganisms. C. perfringens counts appear to be the most suitable indicator for the inactivation and removal of viruses in drinking water treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of nutrients on exopolysaccharide production and surface properties of Aeromonas salmonicida
Authors: Bonet R , Simon-Pujol M D , Congregado F
Published: January 2020

Extracellular polysaccharide (EPS) and capsular polysaccharide (CPS) production by Aeromonas salmonicida A450 and the influence of the capsule on cell surface properties were studied. A. salmonicida did not produce CPS or EPS when glucose, phosphate, magnesium chloride, or trace mineral components were absent from the medium. The addition of yeast extract improved capsule production. Neither EPS nor CPS formation depended on the C/N ratio, although it appeared to be influenced by the level of carbon and nitrogen in the culture. Both EPS and CPS production started at the end of the logarithmic growth phase. The amounts of EPS and CPS produced were not influenced by temperature changes between 15 and 20 degrees C and was maximal from pH 7 to 7.5. Cell surface properties were strongly influenced by capsule production; high CPS production was associated with enhanced cell hydrophilicity and autoagglutination. The effect of CPS on cell surface properties was independent of the presence of the surface protein array (A-layer).
Stoichiometric interpretation of Escherichia coli glucose catabolism under various oxygenation rates
Authors: Varma A , Boesch B W , Palsson B O
Published: January 2020

Metabolic by-product secretion is commonly observed in oxygen-limited cultures. Oxygen limitations occur because of limits in the capacity of the respiratory system or because of the oxygenation limits of the cultivation method used. The latter restriction is of considerable practical importance since it results in a critical cell concentration above which oxygenation is insufficient, leading to by-product secretion. In this study we used a flux balance approach to determine optimal metabolic performance of Escherichia coli under variable oxygen limitations. This method uses linear optimization to find optimal metabolic flux patterns with respect to cell growth. Cell growth was defined as precursor requirements on the basis of a composition analysis. A growth-associated maintenance requirement of 23 mmol of ATP per g of biomass and a non-growth-associated maintenance value of 5.87 mmol at ATP per g (dry weight)-h were incorporated on the basis of a comparison with experimental data. From computations of optimal growth increased oxygen limitations were found to result in the secretion of acetate, formate, and ethanol in that order. Consistent with the experimental data in the literature, by-product secretion rates increased linearly with the growth rate. The computed optimal growth under increasing oxygen limitation revealed four critical growth rates at which changes in the by-product secretion pattern were observed. Concomitant with by-product secretion under oxygen limitations were changes in metabolic pathway utilization. The shifts in metabolism were characterized by changes in the metabolic values (computed as shadow prices) of the various redox carriers. The redox potential was thus identified as a likely trigger that leads to metabolic shifts.2+ ă
Kinetic Studies of Ammonia Monooxygenase Inhibition in
Nitrosomonas europaea
by Hydrocarbons and Halogenated Hydrocarbons in an Optimized Whole-Cell Assay
Authors: Keener William K. , Arp Daniel J.
Published: January 2020

The inhibitory effects of 15 hydrocarbons and halogenated hydrocarbons on NH
3
oxidation by ammonia monooxygenase (AMO) in intact cells of the nitrifying bacterium
Nitrosomonas europaea
were determined. Determination of AMO activity, measured as NO
2
-
production, required coupling of hydroxylamine oxidoreductase (HAO) activity with NH
3
-dependent NH
2
OH production by AMO. Hydrazine, an alternate substrate for HAO, was added to the reaction mixtures as a source of reductant for AMO. Most inhibitors exhibited competitive or noncompetitive inhibition patterns. The competitive character generally decreased (

K
i

E
/

K
i

ES
increased) as the molecular size of the inhibitors increased. For example, CH
4
and C
2
H
4
were competitive inhibitors of NH
3
oxidation, whereas the remaining alkanes (up to C
4
) and monohalogenated (Cl, Br, I) alkanes were noncompetitive. Oxidation of C
2
H
5
Br (noncompetitive) increased as the NH
4
+
concentration increased up to 40 mM, whereas oxidations of inhibitors with competitive character (

K
i

E
≪

K
i

ES
) were diminished at 40 mM NH
4
+
. Multichlorinated compounds produced nonlinear Lineweaver-Burk plots. Iodinated alkanes (CH
3
I, C
2
H
5
I) and C
2
Cl
4
were potent inhibitors of NH
3
oxidation. Maximum rates of NH
3
, C
2
H
4
, and C
2
H
6
oxidations were approximately equivalent, suggesting a common rate-determining step. These data support an active-site model for AMO consisting of an NH
3
-binding site and a second site that binds noncompetitive inhibitors, with oxidation occurring at either site.
Degradation of nitrobenzene by a Pseudomonas pseudoalcaligenes
Authors: Nishino S F , Spain J C
Published: January 2020

A Pseudomonas pseudoalcaligenes able to use nitrobenzene as the sole source of carbon, nitrogen, and energy was isolated from soil and groundwater contaminated with nitrobenzene. The range of aromatic substrates able to support growth was limited to nitrobenzene, hydroxylaminobenzene, and 2-aminophenol. Washed suspensions of nitrobenzene-grown cells removed nitrobenzene from culture fluids with the concomitant release of ammonia. Nitrobenzene, nitrosobenzene, hydroxylaminobenzene, and 2-aminophenol stimulated oxygen uptake in resting cells and in extracts of nitrobenzene-grown cells. Under aerobic and anaerobic conditions, crude extracts converted nitrobenzene to 2-aminophenol with oxidation of 2 mol of NADPH. Ring cleavage, which required ferrous iron, produced a transient yellow product with a maximum A380. In the presence of NAD, the product disappeared and NADH was produced. In the absence of NAD, the ring fission product was spontaneously converted to picolinic acid, which was not further metabolized. These results indicate that the catabolic pathway involves the reduction of nitrobenzene to nitrosobenzene and then to hydroxylaminobenzene; each of these steps requires 1 mol of NADPH. An enzyme-mediated Bamberger-like rearrangement converts hydroxylaminobenzene to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. The mechanism for release of ammonia and subsequent metabolism are under investigation.
Fate of enterohemorrhagic Escherichia coli O157:H7 in apple cider with and without preservatives
Authors: Zhao T , Doyle M P , Besser R E
Published: January 2020

A strain of enterohemorrhagic Escherichia coli serotype O157:H7 isolated from a patient in an apple cider-related outbreak was used to study the fate of E. coli O157:H7 in six different lots of unpasteurized apple cider. In addition, the efficacy of two preservatives, 0.1% sodium benzoate and 0.1% potassium sorbate, used separately and in combination was evaluated for antimicrobial effects on the bacterium. Studies were done at 8 or 25 degrees C with ciders having pH values of 3.6 to 4.0. The results revealed that E. coli O157:H7 populations increased slightly (ca. 1 log10 CFU/ml) and then remained stable for approximately 12 days in lots inoculated with an initial population of 10(5) E. coli O157:H7 organisms per ml and held at 8 degrees C. The bacterium survived from 10 to 31 days or 2 to 3 days at 8 or 25 degrees C, respectively, depending on the lot. Potassium sorbate had minimal effect on E. coli O157:H7 populations, with survivors detected for 15 to 20 days or 1 to 3 days at 8 or 25 degrees C, respectively. In contrast, survivors in cider containing sodium benzoate were detected for only 2 to 10 days or less than 1 to 2 days at 8 or 25 degrees C, respectively. The highest rates of inactivation occurred in the presence of a combination of 0.1% sodium benzoate and 0.1% potassium sorbate. The use of 0.1% sodium benzoate, an approved preservative used by some cider processors, will substantially increase the safety of apple cider in terms of E. coli O157:H7, in addition to suppressing the growth of yeasts and molds.
Characterization of Amylolytic Enzymes, Having Both α-1,4 and α-1,6 Hydrolytic Activity, from the Thermophilic Archaea
Pyrococcus furiosus
and
Thermococcus litoralis
Authors: Brown Stephen H. , Kelly Robert M.
Published: January 2020

Extracellular pullulanases were purified from cell-free culture supernatants of the marine thermophilic archaea
Thermococcus litoralis
(optimal growth temperature, 90°C) and
Pyrococcus furiosus
(optimal growth temperature, 98°C). The molecular mass of the
T. litoralis
enzyme was estimated at 119,000 Da by electrophoresis, while the
P. furiosus
enzyme exhibited a molecular mass of 110,000 Da under the same conditions. Both enzymes tested positive for bound sugar by the periodic acid-Schiff technique and are therefore glycoproteins. The thermoactivity and thermostability of both enzymes were enhanced in the presence of 5 mM Ca
2+
, and under these conditions, enzyme activity could be measured at temperatures of up to 130 to 140°C. The addition of Ca
2+
also affected substrate binding, as evidenced by a decrease in

K
m

for both enzymes when assayed in the presence of this metal. Each of these enzymes was able to hydrolyze, in addition to the α-1,6 linkages in pullulan, α-1,4 linkages in amylose and soluble starch. Neither enzyme possessed activity against maltohexaose or other smaller α-1,4-linked oligosaccharides. The enzymes from
T. litoralis
and
P. furiosus
appear to represent highly thermostable amylopullulanases, versions of which have been isolated from less-thermophilic organisms. The identification of these enzymes further defines the saccharide-metabolizing systems possessed by these two organisms.
MEL gene polymorphism in the genus Saccharomyces
Authors: Turakainen H , Aho S , Korhola M
Published: January 2020

In Saccharomyces spp. the ability to use melibiose depends on the presence of a MEL gene encoding alpha-galactosidase. We used two cloned MEL genes as probes to characterize the physical structure and chromosomal location of the MEL genes in several industrial and natural Mel+ strains of Saccharomyces cerevisiae, Saccharomyces pastorianus, and Saccharomyces bayanus. Electrokaryotyping showed that all of the S. pastorianus strains and most of the S. bayanus strains studied had one MEL locus. The MEL gene in S. bayanus strains was similar but not identical to the S. pastorianus MEL gene. Mel+ S. cerevisiae strains had one to seven loci containing MEL sequences. The MEL genes of these strains could be divided into two categories on the basis of hybridization to MEL1, one group exhibiting strong hybridization to MEL1 and the other group exhibiting weak hybridization to MEL1. In S. pastorianus and S. bayanus strains, the MEL gene was expressed as a single 1.5-kb transcript, and the expression was galactose inducible. In some S. cerevisiae strains, the MEL genes were expressed even without induction at fairly high levels. Expression was usually further induced by galactose. In two strains, CBS 5378 and CBS 4903, expression of the MEL genes was at the same level without induction as it was in most other strains with induction. In all S. cerevisiae strains, irrespective of the number of MEL genes, mRNA of only one size (1.6 kb) was observed.
Identification of
Vibrio splendidus
as a Member of the Planktonic Luminous Bacteria from the Persian Gulf and Kuwait Region with
luxA
Probes
Authors: Nealson K. H. , Wimpee B. , Wimpee C.
Published: January 2020

Hybridization probes specific for the
luxA
genes of four groups of luminous bacteria were used to screen luminous isolates obtained from the Persian Gulf, near Al Khiran, Kuwait Nine of these isolates were identified as
Vibrio harveyi
, a commonly encountered planktonic isolate, while three others showed no hybridization to any of the four probes (
V. harveyi, Vibrio fischeri, Photobacterium phosphoreum
, or
Photobacterium leiognathi
) under high-stringency conditions. Polymerase chain reaction amplification was used to prepare a
luxA
probe against one of these isolates, K-1, and this probe was screened under high-stringency conditions against a collection of DNAs from luminous bacteria; it was found to hybridize specifically to the DNA of the species
Vibrio splendidus
. A probe prepared against the type strain of
V. splendidus
(ATCC 33369) was tested against the collection of luminous bacterial DNA preparations and against the Kuwait isolates and was found to hybridize only against the type strain and the three unidentified Kuwait isolates. Extensive taxonomic analysis by standard methods confirmed the identification of the 13 isolates.
Degradation of trichloroethylene by Pseudomonas cepacia G4 and the constitutive mutant strain G4 5223 PR1 in aquifer microcosms
Authors: Krumme M L , Timmis K N , Dwyer D F
Published: January 2020

Pseudomonas cepacia G4 degrades trichloroethylene (TCE) via a degradation pathway for aromatic compounds which is induced by substrates such as phenol and tryptophan. P. cepacia G4 5223 PR1 (PR1) is a Tn5 insertion mutant which constitutively expresses the toluene ortho-monooxygenase responsible for TCE degradation. In groundwater microcosms, phenol-induced strain G4 and noninduced strain PR1 degraded TCE (20 and 50 microM) to nondetectable levels (< 0.1 microM) within 24 h at densities of 10(8) cells per ml; at lower densities, degradation of TCE was not observed after 48 h. In aquifer sediment microcosms, TCE was reduced from 60 to < 0.1 microM within 24 h at 5 x 10(8) PR1 organisms per g (wet weight) of sediment and from 60 to 26 microM over a period of 10 weeks at 5 x 10(7) PR1 organisms per g. Viable G4 and PR1 cells decreased from approximately 10(7) to 10(4) per g over the 10-week period.
Degradation of 2-Chloroethylvinylether by
Ancylobacter aquaticus
AD25 and AD27
Authors: van den Wijngaard Arjan J. , Prins Janke , Smal Arjan J. A. C. , Janssen Dick B.
Published: January 2020

Incubation of five different β-chloroethers with slurries prepared from brackish water sediment or activated sludge revealed that bis(2-chloroethyl)ether and 2-chloroethylvinylether (2-CVE) were biodegradable under aerobic conditions. After enrichment, two different cultures of
Ancylobacter aquaticus
that are capable of growth on 2-CVE were isolated. Both cultures were also able to grow on 1,2-dichloroethane. The cells contained a haloalkane dehalogenase that dehalogenated 2-CVE, 2-chloroethylmethylether, 2-bromoethylethylether, and epichlorohydrin. Experiments with cell extracts indicated that an alcohol dehydrogenase and an aldehyde dehydrogenase were also involved in the degradation of 2-CVE. This suggests that 2-CVE is metabolized via 2-hydroxyethylvinylether and vinyloxyacetaldehyde to vinyloxyacetic acid. Enzymatic ether cleavage was not detected. 2-CVE was also degraded by chemical ether cleavage, leading to the formation of 2-chloroethanol and acetaldehyde, both of which supported growth. We propose that
A. aquaticus
strains may be important for the detoxification and degradation of halogenated aliphatic compounds in the environment.
Glucose toxicity in Prevotella ruminicola: methylglyoxal accumulation and its effect on membrane physiology
Authors: Russell J B
Published: January 2020

When the ruminal bacterium prevotella ruminicola B(1)4-M was grown in a defined medium with an excess of glucose (3.6 mM ammonia and 50 mM glucose), the cells accumulated large amounts of cellular polysaccharide and the viable cell number decreased at least 1,000-fold. This decrease in viability was correlated with an accumulation of methylglyoxal in the supernatant (3 to 4 mM). Other genetically distinct strains of P. ruminicola produced methylglyoxal, but methylglyoxal production was not ubiquitous among the strains. When P. ruminicola B(1)4-M was grown in continuous culture (dilution rate, 0.1 h-1) with an excess of glucose, there was an oscillating pattern of growth and cell death which was correlated with the accumulation and washout of methylglyoxal from the culture vessel. Mutants which resisted an excess of glucose took up glucose at a slower rate and produced less methylglyoxal than the wild type. These mutants were, however, not stable. There was always a long lag time, and the mutants could only be maintained with a daily transfer schedule. When the mutants were transferred less frequently, methylglyoxal eventually accumulated and the cultures died. The mutants transported glucose at a threefold-slower rate than the wild type, and in each case the carrier had more than one binding site for glucose. Because glucose transport could not be driven by phosphoenolpyruvate or ATP, the glucose carrier of P. ruminicola is probably a proton symport system. When P. ruminicola B(1)4-M cultures were treated with 4 mM methylglyoxal, the delta psi decreased even though intracellular ATP concentrations were high.(ABSTRACT TRUNCATED AT 250 WORDS)
Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by
Trichoderma
Species
Authors: Serrano-Carreon L. , Hathout Y. , Bensoussan M. , Belin J.-M.
Published: January 2020

The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in
Trichoderma
species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of
Trichoderma harzianum
and
T. viride
with [U-
14
C]linoleic acid or [5-
14
C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [
14
C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [
14
C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-
14
C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in
Trichoderma
species.
Chemotaxis of
Azospirillum
Species to Aromatic Compounds
Authors: Lopez-de-Victoria Geralyne , Lovell Charles R.
Published: January 2020

Chemotaxis of
Azospirillum lipoferum
Sp 59b and
Azospirillum brasilense
Sp 7 and Sp CD to malate and to the aromatic substrates benzoate, protocatechuate, 4-hydroxybenzoate, and catechol was assayed by the capillary method and direct cell counts.
A. lipoferum
required induction by growth on 4-hydroxybenzoate for positive chemotaxis to this compound. Chemotaxis of
Azospirillum
spp. to all other substrates did not require induction. Maximum chemotactic responses for most aromatic compounds occurred at concentrations of 1 to 10 mM for
A. lipoferum
and 100 μM to 1 mM for
A. brasilense.
Threshold levels of these chemoattractants ranged from nanomolar to micromolar, with
A. brasilense
Sp CD showing the lowest threshold levels for the substrates tested. Benzoate was the strongest chemoattractant tested, with threshold concentrations in the nanomolar to picomolar range for all strains.
Azospirillum
spp. clearly have more sensitive chemosensory mechanisms for certain aromatic substrates than previously reported in some other soil bacteria. This sensitivity allows
Azospirillum
spp. to detect and respond to aromatic substrates at concentrations relevant to the soil and rhizosphere environments. The ability to detect such low concentrations of aromatic compounds in soils may confer advantages in survival and colonization of the rhizosphere by
Azospirillum
species.
Uncoupler-Resistant Glucose Uptake by the Thermophilic Glycolytic Anaerobe
Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum)
Authors: Cook Gregory M. , Janssen Peter H. , Morgan Hugh W.
Published: January 2020

The transport of glucose across the bacterial cell membrane of
Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum)
Rt8.B1 was governed by a permease which did not catalyze concomitant substrate transport and phosphorylation and thus was not a phosphoenolpyruvate-dependent phosphotransferase. Glucose uptake was carrier mediated, could not be driven by an artificial membrane potential (Δψ) in the presence or absence of sodium, and was not sensitive to inhibitors which dissipate the proton motive force (Δp; tetrachlorosalicylanilide,
N,N
-dicyclohexylcarboiimide, and 2,4-dinitrophenol), and no uptake of the nonmetabolizable analog 2-deoxyglucose could be demonstrated. The glucokinase apparent
K

m

for glucose (0.21 mM) was similar to the
K

t

(affinity constant) for glucose uptake (0.15 mM), suggesting that glucokinase controls the rate of glucose uptake. Inhibitors of ATP synthesis (iodoacetate and sodium fluoride) also inhibited glucose uptake, and this effect was due to a reduction in the level of ATP available to glucokinase for glucose phosphorylation. These results indicated that
T. thermosulfuricus
Rt8.B1 lacks a concentrative uptake system for glucose and that uptake is via facilitated diffusion, followed by ATP-dependent phosphorylation by glucokinase. In
T. thermosulfuricus
Rt8.B1, glucose is metabolized by the Embden-Meyerhof-Parnas pathway, which yields 2 mol of ATP (G. M. Cook, unpublished data). Since only 1 mol of ATP is used to transport 1 mol of glucose, the energetics of this system are therefore similar to those found in bacteria which possess a phosphotransferase.
Recombinational inactivation of the gene encoding nitrate reductase in Aspergillus parasiticus
Authors: Wu T S , Linz J E
Published: January 2020

Functional disruption of the gene encoding nitrate reductase (niaD) in Aspergillus parasiticus was conducted by two strategies, one-step gene replacement and the integrative disruption. Plasmid pPN-1, in which an internal DNA fragment of the niaD gene was replaced by a functional gene encoding orotidine monophosphate decarboxylase (pyrG), was constructed. Plasmid pPN-1 was introduced in linear form into A. parasiticus CS10 (ver-1 wh-1 pyrG) by transformation. Approximately 25% of the uridine prototrophic transformants (pyrG+) were chlorate resistant (Chlr), demonstrating their inability to utilize nitrate as a sole nitrogen source. The genetic block in nitrate utilization was confirmed to occur in the niaD gene by the absence of growth of the A. parasiticus CS10 transformants on medium containing nitrate as the sole nitrogen source and the ability to grow on several alternative nitrogen sources. Southern hybridization analysis of Chlr transformants demonstrated that the resident niaD locus was replaced by the nonfunctional allele in pPN-1. To generate an integrative disruption vector (pSKPYRG), an internal fragment of the niaD gene was subcloned into a plasmid containing the pyrG gene as a selectable marker. Circular pSKPYRG was transformed into A. parasiticus CS10. Chlr pyrG+ transformants were screened for nitrate utilization and by Southern hybridization analysis. Integrative disruption of the genomic niaD gene occurred in less than 2% of the transformants. Three gene replacement disruption transformants and two integrative disruption transformants were tested for mitotic stability after growth under nonselective conditions. All five transformants were found to stably retain the Chlr phenotype after growth on nonselective medium.(ABSTRACT TRUNCATED AT 250 WORDS)
Detection of the genes encoding botulinum neurotoxin types A to E by the polymerase chain reaction
Authors: Szabo E A , Pemberton J M , Desmarchelier P M
Published: January 2020

The polymerase chain reaction (PCR) was used as the basis for the development of highly sensitive and specific diagnostic tests for organisms harboring botulinum neurotoxin type A through E genes. Synthetic DNA primers were selected from nucleic acid sequence data for Clostridium botulinum neurotoxins. Individual components of the PCR for each serotype (serotypes A through E) were adjusted for optimal amplification of the target fragment. Each PCR assay was tested with organisms expressing each of the botulinum neurotoxin types (types A through G), Clostridium tetani, genetically related nontoxigenic organisms, and unrelated strains. Each assay was specific for the intended target. The PCR reliably identified multiple strains having the same neurotoxin type. The sensitivity of the test was determined with different concentrations of genomic DNA from strains producing each toxin type. As little as 10 fg of DNA (approximately three clostridial cells) was detected. C. botulinum neurotoxin types A, B, and E, which are most commonly associated with human botulism, could be amplified from crude DNA extracts, from vegetative cells, and from spore preparations. This suggests that there is great potential for the PCR in the identification and detection of botulinum neurotoxin-producing strains.
Metabolism of the plant toxins nitropropionic acid and nitropropanol by ruminal microorganisms
Authors: Anderson R C , Rasmussen M A , Allison M J
Published: January 2020

The nitro toxins 3-nitro-1-propionic acid (NPA) and 3-nitro-1-propanol (NPOH), which are found in many leguminous plants, are known to be detoxified by ruminal microorganisms. The rates of the detoxification reactions are critical to acquisition of tolerance to the plants by ruminant animals, but further information is needed about factors which influence reaction rates and about the nature of the detoxification reactions. We found that rates of disappearance of NPA and NPOH varied somewhat between samples of ruminal fluid but were usually about 0.4 and 0.1 mumol/ml of ruminal fluid per h, respectively, and that rates with threefold-concentrated cells from rumen fluid were correspondingly higher. We present evidence that ruminal microbes from both cattle and sheep reduce these nitro groups in situ, so that NPA is converted to bet-alanine and NPOH is converted to 3-amino-1-propanol. These products were identified by thin-layer chromatography and, as their dabsyl derivatives, separated by high-performance liquid chromatography. The product beta-alanine was itself metabolized by these mixed suspensions of rumen microbes, so its recovery was always less than what would be estimated from NPA loss, but as much as 87% of the NPOH lost from incubation mixtures was recovered as 3-amino-1-propanol. Addition of sulfide and ferrous ions to suspensions of ruminal microbes increased the rate of NPOH reduction about threefold, but rates of NPA reduction were not similarly increased. When incubations were under hydrogen gas instead of carbon dioxide, the addition of sulfide and ferrous ions led to even greater (five- to eightfold) increases in the rates of NPOH metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)