bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism-Based Inactivation of Ammonia Monooxygenase in
Nitrosomonas europaea
by Allylsulfide
Authors: Juliette Lisa Y. , Hyman Michael R. , Arp Daniel J.
Published: January 2020

Allylsulfide caused an irreversible inactivation of ammonia monooxygenase (AMO) activity (ammonia-dependent O
2
uptake) in
Nitrosomonas europaea
. The hydroxylamine oxidoreductase activity (hydrazine-dependent O
2
uptake) of cells was unaffected by allylsulfide. Anaerobic conditions or the presence of allylthiourea, a reversible noncompetitive AMO inhibitor, protected AMO from inactivation by allylsulfide. Ammonia did not protect AMO from inactivation by allylsulfide but instead increased the rate of inactivation. The inactivation of AMO followed pseudo-first-order kinetics, but the observed rates did not saturate with increasing allylsulfide concentrations. The time course of recovery of AMO-dependent nitrite production after complete inactivation by allylsulfide required de novo protein synthesis. Incubation of cells with allylsulfide prevented the
14
C label from
14
C
2
H
2
(a suicide mechanism-based inactivator of AMO) from being incorporated into the 27-kDa polypeptide of AMO. Some compounds structurally related to allylsulfide were unable to inactivate AMO. We conclude that allylsulfide is a specific, mechanism-based inactivator of AMO in
N. europaea.
Characterization of enzymatic reduction of hexavalent chromium by Escherichia coli ATCC 33456
Authors: Shen H , Wang Y T
Published: January 2020

Chromium reduction by Escherichia coli ATCC 33456 quantitatively transferred hexavalent chromium, Cr(VI), to trivalent chromium, Cr(III). The reduced chromium was predominantly present in the external medium. Supernatant fluids of cell extract, obtained by centrifugation at 12,000 and 150,000 x g, showed almost the same Cr(VI) reduction activity, indicating that Cr(VI) reduction by E. coli ATCC 33456 was a largely soluble reductase activity. In studies with respiratory inhibitors, no inhibitory effects on aerobic and anaerobic Cr(VI) reduction were demonstrated by addition of cyanide, azide, and rotenone into both intact cell cultures and supernatant fluids of E. coli ATCC 33456. Although cytochromes b and d were identified in the membrane fraction of cell extracts, Cr(VI) was not reduced by the membrane fraction alone. The cytochrome difference spectra analysis also indicated that these cytochromes of the respiratory chain require the presence of the soluble Cr(VI) reductase to mediate electron transport to Cr(VI). Stimulation of Cr(VI) reduction by an uncoupler, 2,4-dinitrophenol, indicated that the respiratory-chain-linked electron transport to Cr(VI) was limited by the rate of dissipation of the proton motive force.
A new mechanism for the aerobic catabolism of dimethyl sulfide
Authors: Visscher P T , Taylor B F
Published: January 2020

Aerobic degradation of dimethyl sulfide (DMS), previously described for thiobacilli and hyphomicrobia, involves catabolism to sulfide via methanethiol (CH3SH). Methyl groups are sequentially eliminated as HCHO by incorporation of O2 catalyzed by DMS monooxygenase and methanethiol oxidase. H2O2 formed during CH3SH oxidation is destroyed by catalase. We recently isolated Thiobacillus strain ASN-1, which grows either aerobically or anaerobically with denitrification on DMS. Comparative experiments with Thiobacillus thioparus T5, which grows only aerobically on DMS, indicate a novel mechanism for aerobic DMS catabolism by Thiobacillus strain ASN-1. Evidence that both organisms initially attacked the methyl group, rather than the sulfur atom, in DMS was their conversion of ethyl methyl sulfide to ethanethiol. HCHO transiently accumulated during the aerobic use of DMS by T. thioparus but not with Thiobacillus strain ASN-1. Catalase levels in cells grown aerobically on DMS were about 100-fold lower in Thiobacillus strain ASN-1 than in T. thioparus T5, suggesting the absence of H2O2 formation during DMS catabolism. Also, aerobic growth of T. thioparus T5 on DMS was blocked by the catalase inhibitor 3-amino-1,2,4-triazole whereas that of Thiobacillus strain ASN-1 was not. Methyl butyl ether, but not CHCl3, blocked DMS catabolism by T. thioparus T5, presumably by inhibiting DMS monooxygenase and perhaps methanethiol oxidase. In contrast, DMS metabolism by Thiobacillus strain ASN-1 was unaffected by methyl butyl ether but inhibited by CHCl3. DMS catabolism by Thiobacillus strain ASN-1 probably involves methyl transfer to a cobalamin carrier and subsequent oxidation as folate-bound intermediates.
Role of the CryIVD polypeptide in the overall toxicity of Bacillus thuringiensis subsp. israelensis
Authors: Poncet S , Anello G , Delécluse A , Klier A , Rapoport G
Published: January 2020

The gene encoding the CryIVD protein of B. thuringiensis subsp. israelensis crystals was disrupted by in vivo recombination. The toxicity of the CryIVD protein-free inclusions was similar to that of the wild-type crystals on Anopheles stephensi larvae but was half the wild-type toxicity on Culex pipiens and Aedes aegypti larvae.
Acylated proteins in Borrelia hermsii, Borrelia parkeri, Borrelia anserina, and Borrelia coriaceae
Authors: Sambri V , Stefanelli C , Rossoni C , La Placa M , Cevenini R
Published: January 2020

Borrelia hermsii, Borrelia parkeri, Borrelia anserina, and Borrelia coriaceae produced several lipoproteins identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of bacteria grown in [3H]palmitate. Five major acylated proteins were demonstrated by sequential alkaline and acid hydrolysis. High-pressure liquid chromatography of isolated proteins confirmed that covalently bound radioactivity was represented by fatty acids.
Comparison of
Leuconostoc oenos
Strains by Pulsed-Field Gel Electrophoresis
Authors: Kelly W. J. , Huang C. M. , Asmundson R. V.
Published: January 2020

Pulsed-field gel electrophoresis of chromosomal DNA digested with
Not
I or
Sfi
I was used to differentiate individual strains of
Leuconostoc oenos. L. oenos
isolates with 13 different restriction digest patterns were detected in New Zealand wines undergoing malolactic fermentation. The average genome size was estimated to be 1,800 kb.
Nylon oligomer degradation gene, nylC, on plasmid pOAD2 from a Flavobacterium strain encodes endo-type 6-aminohexanoate oligomer hydrolase: purification and characterization of the nylC gene product
Authors: Kakudo S , Negoro S , Urabe I , Okada H
Published: January 2020

A new type of nylon oligomer degradation enzyme (EIII) was purified from an Escherichia coli clone harboring the EIII gene (nylC). This enzyme hydrolyzed the linear trimer, tetramer, and pentamer of 6-aminohexanoate by an endo-type reaction, and this specificity is different from that of the EI (nylA gene product) and EII (nylB gene product). Amino acid sequencing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified EIII demonstrated that the enzyme is made of two polypeptide chains arising from an internal cleavage between amino acid residues 266 and 267.
Nutritional aspects of cytotoxin production by Clostridium difficile
Authors: Osgood D P , Wood N P , Sperry J F
Published: January 2020

Arginine was the only amino acid used by Clostridium difficile that permitted cytotoxin synthesis in a peptone-based medium. Synthesis of cytotoxin was delayed when glucose was used as the substrate. Addition of rifampin or puromycin to cultures prior to release of cytotoxin inhibited the release of cytotoxin, suggesting that a protein essential for cytotoxin release is synthesized after cytotoxin is synthesized.
Polymerase chain reaction-restriction fragment length polymorphism analysis shows divergence among mer determinants from gram-negative soil bacteria indistinguishable by DNA-DNA hybridization
Authors: Osborn A M , Bruce K D , Strike P , Ritchie D A
Published: January 2020

Mercury resistant (Hgr) bacteria were isolated from four terrestrial sites: three containing high levels of mercury (sites T2, SE, and SO) and one uncontaminated site (SB). The frequencies of Hgr bacteria in the total cultivable populations were 0.05% (SB), 0.69% (SO), 4.8% (SE), and 25% (T2). Between 35 and 100% of the isolates from the four sites contained DNA sequences homologous to a DNA probe from the mercury resistance (mer) operon of the Tn501 Hgr determinant. The mer sequences of 10 Tn501-homologous Hgr determinants from each site were amplified by the polymerase chain reaction, with primers designed to consensus sequences of the mer determinants of Tn501, Tn21, and pMJ100, and were classified on the basis of the size of the amplified product and the restriction fragment length polymorphism pattern. Two main groups of amplification product were identified. The first, represented by the T2 and SB isolates and one SE isolate, gave an amplification product indistinguishable in size from that amplified from Tn501 (approximately 1,010 bp). The second group, represented by the SO isolates and the majority of the SE isolates, produced larger amplification products of 1,040 or 1,060 bp. Restriction fragment length polymorphism analysis revealed that each amplification product size group could be further subdivided into five subgroups.
Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction
Authors: Erb R W , Wagner-Döbler I
Published: January 2020

It was the aim of this study to specifically detect the DNA sequences for the bphC gene, the meta-cleavage enzyme of the aerobic catabolic pathway for biphenyl and polychlorinated biphenyl degradation, in aquatic sediments without prior cultivation of microorganisms by using extraction of total DNA, PCR amplification of bphC sequences, and detection with specific gene probes. The direct DNA extraction protocol used was modified to enhance lysis efficiency. Crude extracts of DNA were further purified by gel filtration, which yielded DNA that could be used for the PCR. PCR primers were designed for conserved regions of the bphC gene from a sequence alignment of five known sequences. The specificity of PCR amplification was verified by using digoxigenin-labeled DNA probes which were located internal to the amplified gene sequence. The detection limit for the bphC gene of Pseudomonas paucimobilis Q1 and Pseudomonas sp. strain LB400 was 100 cells per g (wet weight) or approximately five copies of the target sequence per PCR reaction mixture. In total-DNA extracts of aerobic top layers of sediment samples obtained from three different sampling sites along the Elbe River, which has a long history of anthropogenic pollution, Pseudomonas sp. strain LB 400-like sequences for the bphC gene were detected, but P. paucimobilis Q1 sequences were not detected. No bphC sequences were detected in an unpolluted lake sediment. A restriction analysis did not reveal any heterogeneity in the PCR product, and the possibility that sequences highly related to the bphC gene (namely, nahC and todE) were present was excluded.(ABSTRACT TRUNCATED AT 250 WORDS)
Distribution of Viruses and Dissolved DNA along a Coastal Trophic Gradient in the Northern Adriatic Sea
Authors: Weinbauer Markus G. , Fuks Dragica , Peduzzi Peter
Published: January 2020

The distribution of viral and other microbial abundances as well as the concentrations of dissolved DNA (D-DNA) along a trophic gradient in the northern Adriatic Sea were determined. Virus abundances, covering a range of 1.2 × 10
9
to 8.7 × 10
10
liter
-1
were on average 2.5-fold higher in eutrophic than in mesotrophic stations. A 2.5-fold enrichment was also measured for chlorophyll
a
concentrations, whereas the densities of bacteria and heterotrophic nanoflagellates were only approximately 1.5-fold higher. The frequency of bacteria containing mature phage increased linearly with bacterial abundance. Assuming that mature phage is only visible during the last 14 to 27% of the latent period (L. M. Proctor, A. Okubo, and J. A. Fuhrman, Microb. Ecol. 25:161-182, 1993), we estimated that between 3.5 and 7.3% of the bacterial population was infected at mesotrophic stations versus between 7.0 and 19.5% at eutrophic stations, indicating that the bacterial mortality due to viral lysis might increase with the degree of eutrophication. The frequency of bacteria with mature phage and the burst size varied significantly with the bacterial morphotype; rod-shape cells, the most abundant morphotype, showed low infection rates but a high burst size. Concentrations of D-DNA varied significantly with season but not with trophic conditions. The estimated percentage of viral DNA on total D-DNA concentrations averaged 17.1% (range, 0.7 to 88.3%). Some kind of interaction between heterotrophic nanoflagellates and viruses is proposed. We conclude (i) that the significance of viruses varies with changing trophic conditions and (ii) that viral activity may play a significant role in food web structure under changing trophic conditions.
Aerobic and anaerobic degradation of a range of alkyl sulfides by a denitrifying marine bacterium
Authors: Visscher P T , Taylor B F
Published: January 2020

A pure culture of a bacterium was obtained from a marine microbial mat by using an anoxic medium containing dimethyl sulfide (DMS) and nitrate. The isolate grew aerobically or anaerobically as a denitrifier on alkyl sulfides, including DMS, dimethyl disulfide, diethyl sulfide (DES), ethyl methyl sulfide, dipropyl sulfide, dibutyl sulfide, and dibutyl disulfide. Cells grown on an alkyl sulfide or disulfide also oxidized the corresponding thiols, namely, methanethiol, ethanethiol, propanethiol, or butanethiol. Alkyl sulfides were metabolized by induced or derepressed cells with oxygen, nitrate, or nitrite as electron acceptor. Cells grown on DMS immediately metabolized DMS, but there was a lag before DES was consumed; with DES-grown cells, DES was immediately used but DMS was used only after a lag. Chloramphenicol prevented the eventual use of DES by DMS-grown cells and DMS use by DES-grown cells, respectively, indicating separate enzymes for the metabolism of methyl and ethyl groups. Growth was rapid on formate, acetate, propionate, and butyrate but slow on methanol. The organism also grew chemolithotrophically on thiosulfate with a decrease in pH; growth required carbonate in the medium. Growth on sulfide was also carbonate dependent but slow. The isolate was identified as a Thiobacillus sp. and designated strain ASN-1. It may have utility for removing alkyl sulfides, and also nitrate, nitrite, and sulfide, from wastewaters.
Thermal Inactivation of
Aeromonas hydrophila
As Affected by Sodium Chloride and Ascorbic Acid
Authors: Stecchini Mara Lucia , Sarais Ileana , Giomo Andrea
Published: January 2020

The combined effects of sodium chloride (0, 1.0, 1.5, and 3.0%) and ascorbic acid (0, 1.0, and 2.0 mmol/liter) with mild heat (46°C) on the survival of
Aeromonas hydrophila
were evaluated. Because of the nonlinear nature of the survivor curves obtained, several equations yielding an
R
2
(coefficient of multiple determination) of ≅ 1 were tested. The equation that most closely fit the curvature of the observed data set was a hyperbolic function. Equation coefficients were combined to obtain a so-called death value. This value (46.67% explained variance) was calculated by extracting the larger eigenvalue and the relative eigenvector from the correlation matrix of the coefficients. the effects of the experimental factors on the death value were described by a quadratic response surface model. Results revealed that the death value was not influenced by the presence of ascorbic acid. However, increased mortality resulted from the interaction between sodium chloride and ascorbic acid.
DNA sequence variation and phylogenetic relationships among strains of Pseudomonas syringae pv. syringae inferred from restriction site maps and restriction fragment length polymorphism
Authors: Legard D E , Aquadro C F , Hunter J E
Published: January 2020

We evaluated the restriction fragment length polymorphism of genomic DNA among 53 strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Twenty-nine strains were isolated from beans, and the rest were isolated from 11 other hosts. Southern blots of DNA digested with EcoRI or HindIII were hybridized to two random probes from a cosmid library of P. syringae pv. syringae and a hrp (hypersensitive reaction and pathogenicity) cluster cloned from P. syringae pv. syringae. The size of hybridizing fragments was determined, and a similarity matrix was constructed by comparing strains on a pairwise basis for the presence or absence of fragments. The proportion of shared fragments was then used to estimate sequence divergence. Dendrograms were produced by using the unweighted pair group method with averages and the neighbor-joining method. For the hrp region, BamHI, EcoRI, EcoRV, and HindIII restriction sites were mapped for six representative bean strains and used to construct EcoRI and HindIII restriction maps for all 30 strains pathogenic on beans. Restriction mapping revealed the presence of a 3-kb insertion in nine bean strains and a probable second insertion or deletion event on the left-hand side of the hrp cluster that biased estimates of nucleotide sequence divergence from fragment comparisons. This demonstrated that the determination of phylogenetic relationships among bacteria by using restriction fragment length polymorphism data requires mapping restriction sites to remove the effect of insertion or deletion events on the analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Identification of endomycorrhizal fungi colonizing roots by fluorescent single-strand conformation polymorphism-polymerase chain reaction
Authors: Simon L , Lévesque R C , Lalonde M
Published: January 2020

A method to identify arbuscular endomycorrhizal fungi based on the amplification of portions of the nuclear gene coding for the small subunit rRNA is presented. By coupling the sensitivity of the polymerase chain reaction and the specificity afforded by taxon-specific primers, a variety of samples can be analyzed, including small amounts of colonized roots. Family-specific primers as well as generic primers are described and can be used to amplify small subunit rRNA fragments from endomycorrhizal fungi by polymerase chain reaction. The amplified products are then subjected to single-strand conformation polymorphism analysis to detect sequence differences. Among the advantages of this approach is the possibility of directly identifying the fungi inside field-collected roots, without having to rely on the fortuitous presence of spores. This technique should have obvious applications in the study of arbuscular endomycorrhizal fungi populations and allow closer examination of their host specificity.
Growth and Energy Generation by
Lactococcus lactis
subsp.
lactis
biovar diacetylactis during Citrate Metabolism
Authors: Hugenholtz Jeroen , Perdon Leo , Abee Tjakko
Published: January 2020

Growth of
Lactococcus lactis
subsp.
lactis
biovar diacetylactis was observed on media with citrate as the only energy source. At pH 5.6, steady state was achieved in a chemostat on a citrate-containing medium in the absence of a carbohydrate. Under these conditions, pyruvate, acetate, and some acetoin and butanediol were the main fermentation products. This indicated that energy was conserved in
L. lactis
subsp.
lactis
biovar diacetylactis during citrate metabolism and presumably during the conversion of citrate into pyruvate. The presumed energy-conserving step, decarboxylation of oxaloacetate, was studied in detail. Oxaloacetate decarboxylase was purified to homogeneity and characterized. The enzyme has a native molecular mass of approximately 300 kDa and consists of three subunits of 52, 34, and 12 kDa. The enzyme is apparently not sodium dependent and does not contain a biotin moiety, and it seems to be different from the energy-generating oxaloacetate decarboxylase from
Klebsiella pneumoniae.
Energy-depleted
L. lactis
subsp.
lactis
biovar diacetylactis cells generated a membrane potential and a pH gradient immediately upon addition of citrate, whereas ATP formation was slow and limited. In contrast, lactose energization resulted in rapid ATP formation and gradual generation of a proton motive force. These data were confirmed during studies on amino acid uptake. α-Aminoisobutyrate uptake was rapid but glutamate uptake was slow in citrate-energized cells, whereas lactose-energized cells showed the reverse tendency. These data suggest that, in
L. lactis
subsp.
lactis
bv. diacetylactis, a proton motive force could be generated during citrate metabolism as a result of electrogenic citrate uptake or citrate/product exchange together with proton consumption by the intracellular oxaloacetate decarboxylase.
An enzyme-linked immunosorbent assay-based isolation procedure for verotoxigenic Escherichia coli
Authors: Milley D G , Sekla L H
Published: January 2020

A colony enzyme-linked immunosorbent assay using the hydrophobic grid membrane filter format was developed for the isolation of verotoxigenic Escherichia coli from human and food samples. The method utilizes monoclonal antibodies directed against the verotoxins and is sensitive to all verotoxin 1- and/or 2-producing serotypes. E. coli that produced a minimum of 2 x 10(2) and 2 x 10(3) 50% cytotoxic doses per ml of verotoxins 1 and 2, respectively, were detectable. In a method comparison using human stool specimens, this procedure isolated 29% more E. coli O157 than did the standard sorbitol-MacConkey agar procedure, with no false-positive reactions. When applied to meat, 11 of 20 samples positive for verotoxin by polymyxin extraction yielded verotoxigenic E. coli of a variety of serotypes including O157:H7. Four false positives were noted. This procedure provides a sensitive means for the isolation of verotoxigenic E. coli and should facilitate recovery of those serotypes that are otherwise indistinguishable from nonpathogenic strains.
Heat Shock Induction of Manganese Peroxidase Gene Transcription in
Phanerochaete chrysosporium
Authors: Brown Julie A. , Li Dan , Alic Margaret , Gold Michael H.
Published: January 2020

The expression of manganese peroxidase (MnP) in nitrogen-limited cultures of
Phanerochaete chrysosporium
is regulated by heat shock at the level of gene transcription. Nitrogen limitation and manganous ion [Mn(II)] previously have been shown to regulate
mnp
gene transcription. Northern (RNA) blot analysis demonstrates that 45°C heat shock results in the accumulation of
mnp
mRNA, even in cells grown in the absence of Mn. Heat shock induces
mnp
gene transcription in 4- or 5-day-old cells, and
mnp
mRNA is detectable after 15 min at 45°C. Maximum accumulation of
mnp
mRNA is observed 1 to 2 h after transfer of cultures to 45°C. Two hours after heat shock-induced cultures grown in the absence of Mn are transferred back to 37°C,
mnp
mRNA is no longer detectable. Higher levels of
mnp
mRNA are obtained with simultaneous induction by Mn and heat shock than by either treatment alone. Neither MnP enzyme activity nor protein is detectable in heat-shocked cultures grown in the absence of Mn. However, higher MnP activity is found in the extracellular medium of cultures induced by both heat shock and Mn than in the medium of cultures induced by Mn alone. These results suggest that the putative heat shock elements found in the promoter region of the
mnp
genes are physiologically functional and that Mn may be required for a posttranscriptional step of MnP production under heat shock conditions.
A Novel Δ5-Desaturase-Defective Mutant of
Mortierella alpina
1S-4 and Its Dihomo-γ-Linolenic Acid Productivity
Authors: Jareonkitmongkol Saeree , Sakuradani Eiji , Shimizu Sakayu
Published: January 2020

A novel Δ5-desaturase-defective mutant was derived from an arachidonic acid-producing fungus,
Mortierella alpina
1S-4, after treating the parental spores with
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine. The mutant produced only a trace (about 1%) amount of arachidonic acid, and the ratio of dihomo-γ-linolenic acid (DGLA) to total fatty acids in each lipid class was markedly high, accounting for as much as 60% in phosphatidylcholine. Under submerged batch culture conditions, the mutant produced 2.4 g of DGLA per liter (43.3% of total fatty acids) when grown at 28°C for 7 days in a 5-liter jar fermentor. The other major (more that 1%) fatty acids were palmitic acid (21.2%), stearic acid (9.6%), oleic acid (14.3%), linoleic acid (4.4%), and γ-linolenic acid (5.8%). About 80 mol% of the DGLA produced was found in triacylglycerol.
Antimicrobial activity of a newly identified bacteriocin of Bacillus cereus
Authors: Naclerio G , Ricca E , Sacco M , De Felice M
Published: January 2020

A bacteriocin-producing Bacillus cereus strain was isolated. The bacteriocin, here called cerein, was shown to be active specifically against other B. cereus strains and inactive against all other bacterial species tested. Cerein was detected in the culture supernatants of stationary-phase cells, and its appearance was inhibited by induction of sporulation. The bacterial activity of cerein was insensitive to organic solvents and nonproteolytic enzymes, partially stable to heat, and active over a wide range of pH values. Direct detection of antimicrobial activity on sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of about 9 kDa.
Influence of Packaging and Processing Conditions on the Decontamination of Laboratory Biomedical Waste by Steam Sterilization
Authors: Ozanne Gérard , Huot Roger , Montpetit Claude
Published: January 2020

The conditions for optimal steam decontamination of polypropylene bags half loaded with laboratory biomedical waste were studied (276 bags were processed). Controls were single-closed bags without water added or incisions made in the top, standing freely in an autoclave set at 121°C. The average time required to reach 121°C at the load center was 46 min for controls. A significant increase in this time occurred following addition of water to bags without incisions (60 min), with double bagging (60 min), or when using vertical containers (82 min). A significant decrease occurred when bags were slashed (37 min) or processed at 123°C (32 min) or 132°C (19 min). Horizontal containers or addition of water to slashed bags had no significant effect.
Streptomyces subtilisin inhibitor-like proteins are distributed widely in streptomycetes
Authors: Taguchi S , Kikuchi H , Suzuki M , Kojima S , Terabe M , Miura K , Nakase T , Momose H
Published: January 2020

Streptomyces subtilisin inhibitor-like proteins were found to be distributed widely in streptomycetes by using the combination of the convenient, newly developed plate assay system and an established liquid culture assay. Almost all the strains formerly categorized as Streptoverticillium species produced proteins that exhibited inhibitory activity against both subtilisin BPN' and trypsin. N-terminal regions of three purified proteins showed high structural similarity to those of other previously reported SIL inhibitors.
Rapid glutamate decarboxylase assay for detection of Escherichia coli
Authors: Rice E W , Johnson C H , Dunnigan M E , Reasoner D J
Published: January 2020

A rapid test procedure for the enzyme glutamate decarboxylase was developed for detection of Escherichia coli. The assay procedure was able to confirm the presence of E. coli in enteric broth cultures with 95% specificity for both pure cultures and environmental samples. The procedure was capable of detecting survivors among chlorine-exposed cells.
Performance of anaerobic granules for degradation of pentachlorophenol
Authors: Wu W M , Bhatnagar L , Zeikus J G
Published: January 2020

Anaerobic granules degrading pentachlorophenol (PCP) with specific PCP removal activity up to 14.6 mg/g of volatile suspended solids per day were developed in a laboratory-scale anaerobic upflow sludge blanket reactor at 28 degrees C, by using a mixture of acetate, propionate, butyrate, and methanol as the carbon source. The reactor was able to treat synthetic wastewater containing 40 to 60 mg of PCP per liter at a volumetric loading rate of up to 90 mg/liter of reactor volume per day, with a hydraulic retention time of 10.8 to 15 h. PCP removal of more than 99% was achieved. Results of adsorption of PCP by granular biomass indicated that the PCP removal by the granules was due to biodegradation rather than adsorption. A radiotracer assay demonstrated that the PCP-degrading granules mineralized [14C]PCP to 14CH4 and 14CO2. Toxicity test results indicated that syntrophic propionate degraders and acetate-utilizing methanogens were more sensitive to PCP than syntrophic butyrate degraders. The PCP-degrading granules also exhibited a higher tolerance to the inhibition caused by PCP for methane production and degradation of acetate, propionate, and butyrate, compared with anaerobic granules unadapted to PCP.
Detection of Tn5-like sequences in kanamycin-resistant stream bacteria and environmental DNA
Authors: Leff L G , Dana J R , McArthur J V , Shimkets L J
Published: January 2020

Resistance to kanamycin and neomycin in the bacterial assemblage of a coastal plain stream was detected by growth of colonies on media containing antibiotics. Three of 184 kanamycin-resistant colonies hybridized with a probe containing the nptII gene from transposon Tn5; the nptII gene encodes the enzyme neomycin phosphotransferase and conveys resistance to kanamycin and neomycin. In one of these isolates, the homologous gene was cloned and shown to confer resistance to a kanamycin-sensitive Escherichia coli strain. Since enumeration of bacteria by acridine orange direct counts revealed that less than 0.2% of the bacteria present were cultivated, direct examination of environmental DNA was used to assess abundance of sequences that hybridize to the nptII gene. To examine the resistance potential of bacteria that were not cultured, total DNA was extracted from environmental samples and hybridized with specific probes. The relative amount of eubacterial DNA in each sample was determined by using a eubacterial specific rDNA probe. Then, the abundance of sequences that hybridize to the eubacterial neomycin phosphotransferase gene was determined by hybridization and expressed relative to the total eubacterial DNA in the assemblage. Relative gene abundance was significantly different among assemblages from different habitats (leaves, midchannel sediments, and bank sediments) but did not differ among stream sites.
Multiplex polymerase chain reaction for detection and differentiation of the microbial insecticide Bacillus thuringiensis
Authors: Bourque S N , Valéro J R , Mercier J , Lavoie M C , Levesque R C
Published: January 2020

A rapid identification of Bacillus thuringiensis strains was established by using multiplex polymerase chain reaction (PCR). Primers of high homology specific to regions within genes encoding three major classes of B. thuringiensis crystal proteins were used to generate a PCR product profile characteristic of each strain of B. thuringiensis subsp. kurstaki. Differentiation among these strains was made on the basis of the electrophoretic pattern of the PCR products. Known B. thuringiensis subsp. kurstaki strains as well as unidentified strains isolated from insect cadavers were analyzed by PCR. Small amounts of crude sample lysates were assayed in a two-step PCR containing five primers capable of distinguishing between the strains giving products of 1,500, 858, and 653 bp for the CryIA(a) CryIA(b), and CryIA(c) genes, respectively. The method can be applied to rapidly detect the strains of B. thuringiensis subsp. kurstaki in commercial formulations and in the field.
Overexpression of bacterial hemoglobin causes incorporation of pre-beta-lactamase into cytoplasmic inclusion bodies
Authors: Rinas U , Bailey J E
Published: January 2020

The expression of Vitreoscilla hemoglobin (VHb) in Escherichia coli JM101 (pRED2) causes the incorporation of the TEM beta-lactamase precursor into cytoplasmic inclusion bodies (IBs). Less pre-beta-lactamase is translocated and processed to its mature, periplasmic form in the strain coexpressing VHb than in the control strain E. coli JM101(pUC19) not expressing VHb. When cells are grown in a special fed-batch procedure, the formation of cytoplasmic IBs consisting of pre-beta-lactamase is also inducible in the control strain. Comparative microscopic and compositional analyses of IBs generated in E. coli JM101(pUC19) and JM101(pRED2) under identical growth conditions strongly suggest that pre-beta-lactamase and VHb coaggregate into common IBs in E. coli JM101 (pRED2).
On-Line Detection of Substrate Exhaustion by Using NAD(P)H Fluorescence
Authors: Kwong Simon C. W. , Randers Lisa , Rao Govind
Published: January 2020

In this study we examined the utility of NAD(P)H fluorescence for monitoring aerobic fermentations of the threonine auxotroph
Corynebacterium glutamicum
ATCC 14296. Instead of attempting complicated mathematical corrections for inner-filter effects, we found that it is possible to use the information contained in the on-line NAD(P)H fluorescence signal to assess culture metabolic activities during fermentation. The first derivative of the filtered fluorescence signal, which approximates the turnover rate of the NAD(P)H pool, can be used to precisely identify the temporal points of threonine and glucose exhaustion.
Amino Acid Requirements of Two Hyperthermophilic Archaeal Isolates from Deep-Sea Vents,
Desulfurococcus
Strain SY and
Pyrococcus
Strain GB-D
Authors: Hoaki Toshihiro , Wirsen Carl O. , Hanzawa Satoshi , Maruyama Tadashi , Jannasch Holger W.
Published: January 2020

Two sulfur-dependent hyperthermophilic archaea,
Desulfurococcus
strain SY and
Pyrococcus
strain GB-D, which were isolated from deep-sea hydrothermal vents, utilized free amino acids and peptides obtained from various molecular size fractions of yeast extract. It was found that 11 amino acids were essential for growth. The metabolic products were acetate,
i
-butyrate, and
i
-valerate.
In vivo labeling of Escherichia coli cell envelope proteins with N-hydroxysuccinimide esters of biotin
Authors: Bradburne J A , Godfrey P , Choi J H , Mathis J N
Published: January 2020

The primary amine coupling reagents succinimidyl-6-biotinamido-hexanoate (NHS-A-biotin) and sulfosuccinimidyl-6-biotinamido-hexanoate (NHS-LC-biotin) were tested for their ability to selectively label Escherichia coli cell envelope proteins in vivo. Probe localization was determined by examining membrane, periplasmic, and cytosolic protein fractions. Both hydrophobic NHS-A-biotin and hydrophilic NHS-LC-biotin were shown to preferentially label outer membrane, periplasmic, and inner membrane proteins. NHS-A- and NHS-LC-biotin were also shown to label a specific inner membrane marker protein (Tet-LacZ). Both probes, however, failed to label a cytosolic marker (the omega fragment of beta-galactosidase). The labeling procedure was also used to label E. coli cells grown in low-salt Luria broth medium supplemented with 0, 10, and 20% sucrose. Outer membrane protein A (OmpA) and OmpC were labeled by both NHS-A- and NHS-LC-biotin at all three sucrose concentrations. In contrast, OmpF was labeled by NHS-A-biotin but not by NHS-LC-biotin in media containing 0 and 10% sucrose. OmpF was not labeled by either NHS-A- or NHS-LC-biotin in E. coli cells grown in medium containing 20% sucrose. Coomassie-stained gels, however, revealed similar quantities of OmpF in E. coli cells grown at all three sucrose concentrations. These data indicate that there was a change in outer membrane structure due to increased osmolarity, which limits accessibility of NHS-A-biotin to OmpF.
Microbiological transformations of lipids: acyl-specific hydrolysis of lard by yeasts
Authors: el-Sharkawy S H , Dostal L , Rosazza J P
Published: January 2020

The fatty acid and positional specificities of Saccharomyces cerevisiae (UI-SACCH) and Schizosaccharomyces octosporus (NRRL Y-854) in the hydrolysis of lard were studied by using gas-liquid chromatography. Synthetic triglycerides were used to determine the positional specificities of the lipases of both organisms. Palmitic acid is specifically cleaved from all three triglyceride ester positions by S. cerevisiae, while S. octosporus was able to cleave stearic acid at either position 1 or position 3 of the glycerol moiety. Preparative scale fermentation with 200 g of lard per liter yielded 48.4 g of palmitic acid per liter with S. cerevisiae and 42 g of stearic acid per liter with S. octosporus. The free fatty acids produced by microbial transformation of lard were characterized spectrally (1H and C nuclear magnetic resonance and mass spectrometry) and chromatographically (thin-layer and gas chromatographies).
Glucose and carbon dioxide metabolism by Succinivibrio dextrinosolvens
Authors: O'Herrin S M , Kenealy W R
Published: January 2020

Growth rates and culture conditions affect the molar yields of catabolic end products and cells of Succinivibrio dextrinosolvens growing on glucose. When growth in chemostats occurred, a trend toward decreased succinate and acetate formation, increased lactate formation, and a higher yield of cells correlated with an increase in the growth rate. End product and cellular yields on defined medium indicate a high maintenance requirement for S. dextrinosolvens and are consistent with energy conservation steps during the formation of acetate and succinate. Simultaneous carbon dioxide consumption and production were determined from batch studies with NaH14CO3, and the amounts were used to calculate a fermentation balance. These data also indicated that CO2 consumption lags behind CO2 production early in the growth phase, becoming equivalent to it toward stationary phase. Significantly more CO2 was fixed by S. dextrinosolvens when the organism was cultured in chemostats sparged with CO2. Formate is in part derived from free CO2 in the medium, as shown by 13C nuclear magnetic resonance studies, and may be sensitive to CO2 availability. Nuclear magnetic resonance data are consistent with the carboxylation of a C3 intermediate of the Embden-Meyerhof-Parnas pathway of glycolysis to a C4 compound to eventually form succinate.
Antibody-magnetite method for selective concentration of Giardia lamblia cysts from water samples
Authors: Bifulco J M , Schaefer F W
Published: January 2020

An antibody-magnetite method was developed in order to selectively concentrate Giardia cysts from water samples. The indirect technique employed a mouse immunoglobulin G anti-Giardia antibody as a primary antibody and an anti-mouse immunoglobulin G antibody-coated magnetite particle as a secondary labeling reagent. The magnetically labeled cysts were then concentrated by high-gradient magnetic separation. Ninety percent of the seeded cysts were recovered from buffer when this method was employed. An average of 82% of the seeded cysts were recovered from water samples with various turbidities. Significantly higher cyst recoveries were obtained from water samples with turbidities below 600 nephelometric turbidity units.
ScrFI restriction-modification system of Lactococcus lactis subsp. cremoris UC503: cloning and characterization of two ScrFI methylase genes
Authors: Davis R , van der Lelie D , Mercenier A , Daly C , Fitzgerald G F
Published: January 2020

Two genes from the total genomic DNA of dairy starter culture Lactococcus lactis subsp. cremoris UC503, encoding ScrFI modification enzymes, have been cloned and expressed in Escherichia coli. No homology between the two methylase genes was detected, and inverse polymerase chain reaction of flanking chromosomal DNA indicated that both were linked on the Lactococcus genome. Neither clone encoded the cognate endonuclease. The DNA sequence of one of the methylase genes (encoded by pCI931M) was determined and consisted of an open reading frame 1,170 bp long, which could encode a protein of 389 amino acids (M(r), 44.5). The amino acid sequence contained the highly characteristic motifs of an m5C methylase. Extensive regions of homology were observed with the methylases of NlaX, EcoRII, and Dcm.
Identification of metabolites from the degradation of fluoranthene by Mycobacterium sp. strain PYR-1
Authors: Kelley I , Freeman J P , Evans F E , Cerniglia C E
Published: January 2020

Mycobacterium sp. strain PYR-1, previously shown to extensively mineralize high-molecular-weight polycyclic aromatic hydrocarbons in pure culture and in sediments, degrades fluoranthene to 9-fluorenone-1-carboxylic acid. In this study, 10 other fluoranthene metabolites were isolated from ethyl acetate extracts of the culture medium by thin-layer and high-performance liquid chromatographic methods. On the basis of comparisons with authentic compounds by UV spectrophotometry and thin-layer chromatography as well as gas chromatography-mass spectral and proton nuclear magnetic resonance spectral analyses, the metabolites were identified as 8-hydroxy-7-methoxyfluoranthene, 9-hydroxyfluorene, 9-fluorenone, 1-acenaphthenone, 9-hydroxy-1-fluorenecarboxylic acid, phthalic acid, 2-carboxybenzaldehyde, benzoic acid, phenylacetic acid, and adipic acid. Authentic 9-hydroxyfluorene and 9-fluorenone were metabolized by Mycobacterium sp. strain PYR-1. A pathway for the catabolism of fluoranthene by Mycobacterium sp. strain PYR-1 is proposed.
Survival of nonculturable Aeromonas salmonicida in lake water
Authors: Morgan J A , Rhodes G , Pickup R W
Published: January 2020

The survival of Aeromonas salmonicida subsp. salmonicida was investigated in sterile and untreated lake water. In sterile lake water (filtered and autoclaved), it was found that cells of A. salmonicida entered a nonculturable but viable condition. Viability was determined by flow cytometry with the dye rhodamine 123, which is taken up and maintained within cells with a membrane potential. For survival studies in untreated lake water, A. salmonicida was marked with the xylE gene by using the plasmid pLV1013. Marked cells were detected by growth on tryptone soy agar and tryptone soy agar supplemented with kanamycin. Cells were also detected by polymerase chain reaction DNA amplification of the xylE gene and a chromosomal DNA fragment specific for A. salmonicida (pLV1013). The results indicated that A. salmonicida entered a nonculturable condition in untreated lake water over a 21-day study. The viability of nonculturable cells could not be determined in mixed samples; however, the presence of nonculturable cells containing both chromosomal and plasmid DNA was confirmed.
Differential Responses of Nitrogen-Fixing Cyanobacteria to Salinity and Osmotic Stresses
Authors: Fernandes Tonina A. , Iyer Vimala , Apte Shree Kumar
Published: January 2020

Two nitrogen-fixing
Anabaena
strains were found to be differentially tolerant to salinity and osmotic stresses.
Anabaena torulosa
, a brackish-water, salt-tolerant strain, was relatively osmosensitive.
Anabaena
sp. strain L-31, a freshwater, salt-sensitive strain, on the other hand, displayed significant osmotolerance. Salinity and osmotic stresses affected nitrogenase activity differently. Nitrogen fixation in both of the strains was severely inhibited by the ionic, but not by the osmotic, component of salinity stress. Such differential sensitivity of diazotrophy to salinity-osmotic stresses was observed irrespective of the inherent tolerance of the two strains to salt-osmotic stress. Exogenously added ammonium conferred significant protection against salinity stress but was ineffective against osmotic stress. Salinity and osmotic stresses also affected stress-induced gene expression differently. Synthesis of several proteins was repressed by salinity stress but not by equivalent or higher osmotic stress. Salinity and osmotic stresses induced many common proteins. In addition, unique salt stress- or osmotic stress-specific proteins were also induced in both strains, indicating differential regulation of protein synthesis by the two stresses. These data show that cyanobacterial sensitivity and responses to salinity and osmotic stresses are distinct, independent phenomena.
Flow Cytometric Analysis of Marine Bacteria with Hoechst 33342
Authors: Monger Bruce C. , Landry Michael R.
Published: January 2020

We investigated the accuracy and precision of flow cytometric (FCM) estimates of bacterial abundances using 4′, 6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 (HO342, a bisbenzamide derivative) on paraformaldehyde-fixed seawater samples collected from two stations near Oahu, Hawaii. The accuracy of FCM estimates was assessed against direct counts by using epifluorescence microscopy. DAPI and HO342 differ in two aspects of their chemistry that make HO342 better suited for staining marine heterotrophic bacteria for FCM analysis. These differences are most important in studies of open-ocean ecosystems that require dual-beam FCM analysis to clearly separate heterotrophic bacterial populations from populations of photosynthetic
Prochlorococcus
spp. Bacterial populations were easier to distinguish from background fluorescence when stained with HO342 than when stained with DAPI, because HO342 has a higher relative fluorescence quantum yield. A substantially higher coefficient of variation of blue fluorescence, which was probably due to fluorescent complexes formed by DAPI with double-stranded RNA, was observed for DAPI-stained populations. FCM estimates averaged 2.0 and 12% higher than corresponding epifluorescence microscopy direct counts for HO342 and DAPI-stained samples, respectively. A paired-sample
t
test between FCM estimates and direct counts found no significant difference for HO342-stained samples but a significant difference for DAPI-stained samples. Coefficients of variation of replicate FCM abundance estimates ranged from 0.63 to 2.9% (average, 1.5%) for natural bacterial concentrations of 6 × 10
5
to 15 × 10
5
cells ml
-1
.
Direct extraction and purification of rRNA for ecological studies
Authors: Moran M A , Torsvik V L , Torsvik T , Hodson R E
Published: January 2020

Microgram quantities of rRNA were recovered from natural microbial communities in sediment, soil, and water with a lysozyme-hot phenol direct extraction method. Gel filtration with Sephadex G-75 spun columns readily removed humic-like contaminants without any measurable loss of rRNA and rendered RNA extracts of sufficient quality for molecular procedures.
Differences in the hybridization pattern of Bacillus subtilis genes coding for rDNA depend on the method of DNA preparation
Authors: Waterhouse R N , Glover L A
Published: January 2020

Three different methods of DNA isolation (organic deproteinization, potassium acetate deproteinization, and the use of cetyltrimethylammonium bromide) have been used to prepare DNA from Bacillus subtilis. Subsequent hybridization with an rDNA probe (DNA coding for rRNA) produces different patterns, which mirror those previously reported to indicate an rDNA deletion.
Chromosome Polymorphisms among Strains of
Hansenula polymorpha
(syn.
Pichia angusta
)
Authors: Marri Laura , Rossolini Gian Maria , Satta Giuseppe
Published: January 2020

Contour-clamped homogeneous electrophoresis and an embedded-agarose method of sample preparation were combined to carry out an analysis of the chromosome sets of nine strains of
Hansenula polymorpha
(syn.
Pichia angusta
). Chromosomal DNA molecules could be separated into a series of bands ranging, approximately, from 650 up to 2,200 kb in size. Polymorphism of the electrophoretic pattern was demonstrated among the strains investigated in this study. Cross-hybridization between
H. polymorpha
and
Saccharomyces cerevisiae
ribosomal DNA was also observed.