bluuebunny/crossref_metadata_2025 huggingface dataset:
Estimation of Nitrifying Bacterial Activities by Measuring Oxygen Uptake in the Presence of the Metabolic Inhibitors Allylthiourea and Azide
Authors: Ginestet Philippe , Audic Jean-Marc , Urbain Vincent , Block Jean-Claude
Published: December 2019

The effects of two metabolic inhibitors on an enriched nitrifying biomass during incubation for short periods of time were investigated by determining respirometric measurements. Allylthiourea (86 μM) and azide (24 μM) were shown to be strong, selective inhibitors of ammonia and nitrite oxidation, respectively. Consequently, a differential respirometry method for estimating nitrifying and heterotrophic bacterial activities within a mixed biomass is proposed.
Seasonal Abundance of Lysogenic Bacteria in a Subtropical Estuary
Authors: Cochran Pamela K. , Paul John H.
Published: December 2019

Seasonal changes in the abundance of inducible lysogenic bacteria in a eutrophic estuarine environment were investigated over a 13-month period. Biweekly water samples were collected from Tampa Bay, Fla., and examined for prophage induction by mitomycin C treatment. At the conclusion of the study, we determined that 52.2% of the samples displayed prophage induction, as indicated by significant increases in viral direct counts compared with uninduced controls. Samples that displayed prophage induction occurred during the warmer months (February through October), when surface water temperatures were above 19°C, and no induction was observed in November, December, or January. This study presents clear evidence that there is seasonal variation in the number of inducible lysogenic bacteria in an estuarine environment.
Analysis of Molecular Size Distributions of Cellulose Molecules during Hydrolysis of Cellulose by Recombinant
Cellulomonas fimi
β-1,4-Glucanases
Authors: Stålbrand Henrik , Mansfield Shawn D. , Saddler John N. , Kilburn Douglas G. , Warren R. Antony J. , Gilkes Neil R.
Published: December 2019

Four β-1,4-glucanases (cellulases) of the cellulolytic bacterium
Cellulomonas fimi
were purified from
Escherichia coli
cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of β-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.
Abundance in Sewage of Bacteriophages That InfectEscherichia coliO157:H7 and That Carry the Shiga Toxin 2 Gene
Authors: Muniesa Maite , Jofre Juan
Published: December 2019

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such asEscherichia coliO157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negativeE. coliO157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious forE. coliO157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infectingE. coliO157:H7.
Cloning of
Phanerochaete chrysosporium leu2
by Complementation of Bacterial Auxotrophs and Transformation of Fungal Auxotrophs
Authors: Schick Zapanta Laura , Hattori Takefumi , Rzetskaya Magarita , Tien Ming
Published: December 2019

A
Phanerochaete chrysosporium
cDNA library was constructed in an expression vector that allows expression in both
Escherichia coli
and
Saccharomyces cerevisiae
. This expression vector, λYES, contains the
lacZ
promoter for expression in
E. coli
and the
GAL1
promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the β-isopropylmalate dehydrogenase from
P. chrysosporium
was characterized further. The genomic clone (g
leu2
) was subsequently isolated and was used successfully as a selectable marker to transform
P. chrysosporium
auxotrophs for LEU2. Protoplasts for transformation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous
P. chrysosporium
transformation procedures. The method described here allows other genes to be isolated from
P. chrysosporium
for use as selectable markers.
Microbial Community Composition of Wadden Sea Sediments as Revealed by Fluorescence In Situ Hybridization
Authors: Llobet-Brossa Enric , Rosselló-Mora Ramon , Amann Rudolf
Published: December 2019

The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides. A large fraction (up to 73%) of the DAPI (4′,6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes. Nearly 45% of the total cells could be further identified as belonging to known phyla. Members of the
Cytophaga-Flavobacterium
cluster were most abundant in all layers, followed by the sulfate-reducing bacteria.
Plasposons: Modular Self-Cloning Minitransposon Derivatives for Rapid Genetic Analysis of Gram-Negative Bacterial Genomes
Authors: Dennis Jonathan J. , Zylstra Gerben J.
Published: December 2019

A series of modular mini-transposon derivatives which permit the rapid cloning and mapping of the DNA flanking the minitransposon’s site of insertion has been developed. The basic plasposon, named Tn
Mod
, consists of the Tn
5
inverted repeats, a conditional origin of replication, rare restriction endonuclease multiple cloning sites, and exchangeable antibiotic resistance cassettes. The broad host range and low target DNA sequence specificity of the Tn
5
transposase, in combination with the flexibility afforded by the modular arrangement of Tn
Mod
, result in a versatile tool for the mapping of insertional mutations and the rapid recovery of clones from gram-negative bacteria.
Use of Luciferase Genes as Biosensors To Study Bacterial Physiology in the Digestive Tract
Authors: Corthier G. , Delorme C. , Ehrlich S. D. , Renault P.
Published: December 2019

A method based on the use of the bacterial luciferase genes was developed in order to investigate
Lactococcus lactis
gene expression in the mouse digestive tract. Germfree mice were monoassociated with different strains containing transcriptional fusions of promoters with the luciferase genes. Our results demonstrate that this method is readily applicable to the study of promoter strength and physiology of bacteria in the digestive tract.
Importance of Extracellular Polymeric Substances fromThiobacillus ferrooxidansfor Bioleaching
Authors: Gehrke Tilman , Telegdi Judit , Thierry Dominique , Sand Wolfgang
Published: December 2019

Leaching bacteria such asThiobacillus ferrooxidansattach to pyrite or sulfur by means of extracellular polymeric substances (EPS) (lipopolysaccharides). The primary attachment to pyrite at pH 2 is mediated by exopolymer-complexed iron(III) ions in an electrochemical interaction with the negatively charged pyrite surface. EPS from sulfur cells possess increased hydrophobic properties and do not attach to pyrite, indicating adaptability to the substrate or substratum.
Gene Transfer by Transduction in the Marine Environment
Authors: Jiang Sunny C. , Paul John H.
Published: December 2019

To determine the potential for bacteriophage-mediated gene transfer in the marine environment, we established transduction systems by using marine phage host isolates. Plasmid pQSR50, which contains transposon Tn
5
and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. Both marine bacterial isolates and concentrated natural bacterial communities were used as recipients in transduction studies. Transductants were detected by a gene probe complementary to the neomycin phosphotransferase (
npt
II) gene in Tn
5
. The transduction frequencies ranged from 1.33 × 10
−7
to 5.13 × 10
−9
transductants/PFU in studies performed with the bacterial isolates. With the mixed bacterial communities, putative transductants were detected in two of the six experiments performed. These putative transductants were confirmed and separated from indigenous antibiotic-resistant bacteria by colony hybridization probed with the
npt
II probe and by PCR amplification performed with two sets of primers specific for pQSR50. The frequencies of plasmid transduction in the mixed bacterial communities ranged from 1.58 × 10
−8
to 3.7 × 10
−8
transductants/PFU. Estimates of the transduction rate obtained by using a numerical model suggested that up to 1.3 × 10
14
transduction events per year could occur in the Tampa Bay Estuary. The results of this study suggest that transduction could be an important mechanism for horizontal gene transfer in the marine environment.
A Glutathione
S
-Transferase with Activity towards
cis
-1,2-Dichloroepoxyethane Is Involved in Isoprene Utilization by
Rhodococcus
sp. Strain AD45
Authors: van Hylckama Vlieg Johan E. T. , Kingma Jaap , van den Wijngaard Arjan J. , Janssen Dick B.
Published: December 2019

Rhodococcus
sp. strain AD45 was isolated from an enrichment culture on isoprene (2-methyl-1,3-butadiene). Isoprene-grown cells of strain AD45 oxidized isoprene to 3,4-epoxy-3-methyl-1-butene,
cis
-1,2-dichloroethene to
cis
-1,2-dichloroepoxyethane, and
trans
-1,2-dichloroethene to
trans
-1,2-dichloroepoxyethane. Isoprene-grown cells also degraded
cis
-1,2-dichloroepoxyethane and
trans
-1,2-dichloroepoxyethane. All organic chlorine was liberated as chloride during degradation of
cis
-1,2-dichloroepoxyethane. A glutathione (GSH)-dependent activity towards 3,4-epoxy-3-methyl-1-butene, epoxypropane,
cis
-1,2-dichloroepoxyethane, and
trans
-1,2-dichloroepoxyethane was detected in cell extracts of cultures grown on isoprene and 3,4-epoxy-3-methyl-1-butene. The epoxide-degrading activity of strain AD45 was irreversibly lost upon incubation of cells with 1,2-epoxyhexane. A conjugate of GSH and 1,2-epoxyhexane was detected in cell extracts of cells exposed to 1,2-epoxyhexane, indicating that GSH is the physiological cofactor of the epoxide-transforming activity. The results indicate that a GSH
S
-transferase is involved in the metabolism of isoprene and that the enzyme can detoxify reactive epoxides produced by monooxygenation of chlorinated ethenes.
Acyl Transfer Activity of an Amidase from
Rhodococcus
sp. Strain R312: Formation of a Wide Range of Hydroxamic Acids
Authors: Fournand David , Bigey Frederic , Arnaud Alain
Published: December 2019

The enantioselective amidase from
Rhodococcus
sp. strain R312 was produced in
Escherichia coli
and was purified in one chromatographic step. This enzyme was shown to catalyze the acyl transfer reaction to hydroxylamine from a wide range of amides. The optimum working pH values were 7 with neutral amides and 8 with α-aminoamides. The reaction occurred according to a Ping Pong Bi Bi mechanism. The kinetic constants demonstrated that the presence of a hydrophobic moiety in the carbon side chain considerably decreased the
K

m
amide

values (e.g.,
K

m
amide

= 0.1 mM for butyramide, isobutyramide, valeramide, pivalamide, hexanoamide, and benzamide). Moreover, very high turnover numbers (
k
cat
) were obtained with linear aliphatic amides (e.g.,
k
cat
= 333 s
−1
with hexanoamide), whereas branched-side-chain-, aromatic cycle- or heterocycle-containing amides were sterically hindered. Carboxylic acids, α-amino acids, and methyl esters were not acyl donors or were very bad acyl donors. Only amides and hydroxamic acids, both of which contained amide bonds, were determined to be efficient acyl donors. On the other hand, the highest affinities of the acyl-enzyme complexes for hydroxylamine were obtained with short, polar or unsaturated amides as acyl donors (e.g.,
K

m

NH
2
OH

= 20, 25, and 5 mM for acetyl-, alanyl-, and acryloyl-enzyme complexes, respectively). No acyl acceptors except water and hydroxylamine were found. Finally, the purified amidase was shown to be
l
-enantioselective towards α-hydroxy- and α-aminoamides.
Detection ofEhrlichia risticii, the Agent of Potomac Horse Fever, in Freshwater Stream Snails (Pleuroceridae:Jugaspp.) from Northern California
Authors: Barlough Jeffrey E. , Reubel Gerhard H. , Madigan John E. , Vredevoe Larisa K. , Miller Paul E. , Rikihisa Yasuko
Published: December 2019

Ehrlichia DNA was identified by nested PCR in operculate snails (Pleuroceridae:Jugaspp.) collected from stream water in a northern California pasture in which Potomac horse fever (PHF) is enzootic. Sequencing of PCR-amplified DNA from a suite of genes (the 16S rRNA,groESLheat shock operon, 51-kDa major antigen genes) indicated that the source organism closely resembledEhrlichia risticii, the causative agent of PHF. The minimum percentage ofJugaspp. harboring the organism in the population studied was 3.5% (2 of 57 snails). No ehrlichia DNA was found in tissues of 123 lymnaeid, physid, and planorbid snails collected at the same site. These data suggest that pleurocerid stream snails may play a role in the life cycle ofE. risticiiin northern California.
Strain IMB-1, a Novel Bacterium for the Removal of Methyl Bromide in Fumigated Agricultural Soils
Authors: Connell Hancock Tracy L. , Costello Andria M. , Lidstrom Mary E. , Oremland Ronald S.
Published: December 2019

A facultatively methylotrophic bacterium, strain IMB-1, that has been isolated from agricultural soil grows on methyl bromide (MeBr), methyl iodide, methyl chloride, and methylated amines, as well as on glucose, pyruvate, or acetate. Phylogenetic analysis of its 16S rRNA gene sequence indicates that strain IMB-1 classes in the alpha subgroup of the class
Proteobacteria
and is closely related to members of the genus
Rhizobium
. The ability of strain IMB-1 to oxidize MeBr to CO
2
is constitutive in cells regardless of the growth substrate. Addition of cell suspensions of strain IMB-1 to soils greatly accelerates the oxidation of MeBr, as does pretreatment of soils with low concentrations of methyl iodide. These results suggest that soil treatment strategies can be devised whereby bacteria can effectively consume MeBr during field fumigations, which would diminish or eliminate the outward flux of MeBr to the atmosphere.
Spatial and Temporal Deposition of
Hyphomonas
Strain VP-6 Capsules Involved in Biofilm Formation
Authors: Langille Stephen E. , Weiner Ronald M.
Published: December 2019

Hyphomonas
strain VP-6 is a prosthecate bacterium isolated from the Guayamas vent region and is a member of a genus of primary and common colonizers of marine surfaces. It adheres to solid substrata as a first step in biofilm formation. Fine-structure microscopy and the use of specific stains and lectins reveal that it synthesizes two different extracellular polymeric substances (EPS). One is a temporally synthesized, polar holdfast EPS, and the other is a capsular EPS that is present during the complete life cycle and surrounds the entire cell, including the prosthecum. The timing and location of
Hyphomonas
strain VP-6 EPS elaboration correlate with adhesion to surfaces, suggesting that the EPS serves not only as the biofilm matrix but also as a primary adhesin. The temporality and polarity of VP-6 EPS expression substantially differ from those properties of
Hyphomonas
strain MHS-3 EPS expression.
Volatile Compounds Originating from Mixed Microbial Cultures on Building Materials under Various Humidity Conditions
Authors: Korpi Anne , Pasanen Anna-Liisa , Pasanen Pertti
Published: December 2019

We examined growth of mixed microbial cultures (13 fungal species and one actinomycete species) and production of volatile compounds (VOCs) in typical building materials in outside walls, separating walls, and bathroom floors at various relative humidities (RHs) of air. Air samples from incubation chambers were adsorbed on Tenax TA and dinitrophenylhydrazine cartridges and were analyzed by thermal desorption-gas chromatography and high-performance liquid chromatography, respectively. Metabolic activity was measured by determining CO
2
production, and microbial concentrations were determined by a dilution plate method. At 80 to 82% RH, CO
2
production did not indicate that microbial activity occurred, and only 10% of the spores germinated, while slight increases in the concentrations of some VOCs were detected. All of the parameters showed that microbial activity occurred at 90 to 99% RH. The microbiological analyses revealed weak microbial growth even under drying conditions (32 to 33% RH). The main VOCs produced on the building materials studied were 3-methyl-1-butanol, 1-pentanol, 1-hexanol, and 1-octen-3-ol. In some cases fungal growth decreased aldehyde emissions. We found that various VOCs accompany microbial activity but that no single VOC is a reliable indicator of biocontamination in building materials.
Effect of Tungstate on Nitrate Reduction by the Hyperthermophilic Archaeon
Pyrobaculum aerophilum
Authors: Afshar Sepideh , Kim Christopher , Monbouquette Harold G. , Schröder Imke
Published: December 2019

Pyrobaculum aerophilum
, a hyperthermophilic archaeon, can respire either with low amounts of oxygen or anaerobically with nitrate as the electron acceptor. Under anaerobic growth conditions, nitrate is reduced via the denitrification pathway to molecular nitrogen. This study demonstrates that
P. aerophilum
requires the metal oxyanion WO
4
2−
for its anaerobic growth on yeast extract, peptone, and nitrate as carbon and energy sources. The addition of 1 μM MoO
4
2−
did not replace WO
4
2−
for the growth of
P. aerophilum
. However, cell growth was completely inhibited by the addition of 100 μM MoO
4
2−
to the culture medium. At lower tungstate concentrations (0.3 μM and less), nitrite was accumulated in the culture medium. The accumulation of nitrite was abolished at higher WO
4
2−
concentrations (<0.7 μM). High-temperature enzyme assays for the nitrate, nitrite, and nitric oxide reductases were performed. The majority of all three denitrification pathway enzyme activities was localized to the cytoplasmic membrane, suggesting their involvement in the energy metabolism of the cell. While nitrite and nitric oxide specific activities were relatively constant at different tungstate concentrations, the activity of nitrate reductase was decreased fourfold at WO
4
2−
levels of 0.7 μM or higher. The high specific activity of the nitrate reductase enzyme observed at low WO
4
2−
levels (0.3 μM or less) coincided with the accumulation of nitrite in the culture medium. This study documents the first example of the effect of tungstate on the denitrification process of an extremely thermophilic archaeon. We demonstrate here that nitrate reductase synthesis in
P. aerophilum
occurs in the presence of high concentrations of tungstate.
Randomly Amplified Polymorphic DNA Analysis of Starved and Viable but Nonculturable
Vibrio vulnificus
Cells
Authors: Warner Jennifer M. , Oliver James D.
Published: December 2019

Vibrio vulnificus
is an estuarine bacterium capable of causing a rapidly fatal infection in humans. Because of the low nutrient levels and temperature fluctuations found in the organism’s natural habitat, the starvation state and viable but nonculturable (VBNC) state are of particular interest. A randomly amplified polymorphic DNA (RAPD) PCR protocol was developed previously for the detection of
V. vulnificus
strains grown in rich media and has been applied to starved and VBNC cells of
V. vulnificus
in the present study. As cells were subjected to starvation in artificial seawater, changes in the RAPD profile were detected as early as 15 min into the starvation period. Most noticeable was a uniform loss of RAPD amplification products. By 4 h of starvation, the cells were undetectable by the RAPD method. Cells that had been starved for up to 1 year again became detectable by the RAPD method when nutrients were added to the starvation microcosm. The same loss of signal, but at a lower rate, was also seen as cells entered the VBNC state. VBNC cells were resuscitated by a temperature upshift and were once again detectable by the RAPD method. The addition of chloramphenicol prevented the RAPD signal from being lost in both the starvation and VBNC states. This suggests that DNA binding proteins produced during starvation and entrance into the VBNC state may be responsible for the inability of the RAPD method to amplify
V. vulnificus
DNA in these states.
Comparison of C
18
-Carboxypropylbetaine and Glass Bead DNA Extraction Methods for Detection of
Mycobacterium bovis
in Bovine Milk Samples and Analysis of Samples by PCR
Authors: Cornejo Brandon J. , Sahagún-Ruiz Alfredo , Suárez-Güemes Francisco , Thornton Charles G. , Ficht Thomas A. , Adams L. Garry
Published: December 2019

The purpose of this prospective study was to compare two different milk preparation methods to assay for the presence of
Mycobacterium bovis
by PCR. Detection by a C
18
-carboxypropylbetaine (CB-18)-based sample processing method was compared to extraction of DNA from milk with glass beads. Samples from 17 skin test-positive cattle were analyzed. Following CB-18 processing and glass bead extraction, the sensitivity of IS
6110
-based PCR was 94.1 and 58.8%, respectively (
P
< 0.025). Because CB-18 processing will permit the proficient use of PCR for diagnosis and surveillance of bovine tuberculosis, it will contribute to the more efficient detection and control of tuberculosis.
Isolation ofMycobacterium paratuberculosisfrom Milk by Immunomagnetic Separation
Authors: Grant Irene R. , Ball Hywel J. , Rowe Michael T.
Published: December 2019

An immunomagnetic separation (IMS) technique was developed to facilitate selective isolation ofMycobacterium paratuberculosiscells from milk. Rabbit polyclonal antibodies against radiation-killed intactM. paratuberculosiscells were produced and used to coat sheep anti-rabbit immunoglobulin G (IgG) type M-280 Dynabeads. The rabbit anti-M. paratuberculosisIgG-coated beads (IMB) reacted strongly with laboratory strains ofM. paratuberculosisas determined by slide agglutination, and microscopic examination confirmed thatM. paratuberculosiscells attached to the IMB. The IMB were found to have a maximum binding capacity of 104to 105CFU ofM. paratuberculosis. Studies showed that IMS selectively recoveredM. paratuberculosisfrom inoculated milk containing as few as 10 CFU ofM. paratuberculosisper ml when 10 μl of IMB (ca. 106beads) was added to 1 ml of milk and the preparation was incubated for 30 min at room temperature with gentle agitation. Larger volumes of milk (10 and 50 ml) were centrifuged and resuspended in 1 ml of phosphate-buffered saline–0.05% Tween 20 prior to IMS in order to increase the sensitivity of the method. Currently, primary isolation ofM. paratuberculosisfrom a milk sample relies on chemical decontamination, followed by culturing on Herrold’s egg yolk medium, which must be incubated at 37°C for up to 18 weeks. The potential value of our IMS method is as an aid for rapid detection ofM. paratuberculosisin milk when it is used in conjunction with end point detection methods, such as IS900PCR or an enzyme-linked immunosorbent assay.
Aerobic Growth on Nitroglycerin as the Sole Carbon, Nitrogen, and Energy Source by a Mixed Bacterial Culture
Authors: Accashian John V. , Vinopal Robert T. , Kim Byung-Joon , Smets Barth F.
Published: December 2019

Nitroglycerin (glycerol trinitrate [GTN]), an explosive and vasodilatory compound, was metabolized by mixed microbial cultures from aeration tank sludge previously exposed to GTN. Aerobic enrichment cultures removed GTN rapidly in the absence of a supplemental carbon source. Complete denitration of GTN, provided as the sole C and N source, was observed in aerobic batch cultures and proceeded stepwise via the dinitrate and mononitrate isomers, with successive steps occurring at lower rates. The denitration of all glycerol nitrate esters was found to be concomitant, and 1,2-glycerol dinitrate (1,2-GDN) and 2-glycerol mononitrate (2-GMN) were the primary GDN and GMN isomers observed. Denitration of GTN resulted in release of primarily nitrite-N, indicating a reductive denitration mechanism. Biomass growth at the expense of GTN was verified by optical density and plate count measurements. The kinetics of GTN biotransformation were 10-fold faster than reported for complete GTN denitration under anaerobic conditions. A maximum specific growth rate of 0.048 ± 0.005 h−1(mean ± standard deviation) was estimated for the mixed culture at 25°C. Evidence of GTN toxicity was observed at GTN concentrations above 0.3 mM. To our knowledge, this is the first report of complete denitration of GTN used as a primary growth substrate by a bacterial culture under aerobic conditions.
Biodegradation of Atrazine byAgrobacterium radiobacterJ14a and Use of This Strain in Bioremediation of Contaminated Soil
Authors: Struthers J. K. , Jayachandran K. , Moorman T. B.
Published: December 2019

We examined the ability of a soil bacterium,Agrobacterium radiobacterJ14a, to degrade the herbicide atrazine under a variety of cultural conditions, and we used this bacterium to increase the biodegradation of atrazine in soils from agricultural chemical distribution sites. J14a cells grown in nitrogen-free medium with citrate and sucrose as carbon sources mineralized 94% of 50 μg of [14C-U-ring]atrazine ml−1in 72 h with a concurrent increase in the population size from 7.9 × 105to 5.0 × 107cells ml−1. Under these conditions cells mineralized the [ethyl-14C]atrazine and incorporated approximately 30% of the14C into the J14a biomass. Cells grown in medium without additional carbon and nitrogen sources degraded atrazine, but the cell numbers did not increase. Metabolites produced by J14a during atrazine degradation include hydroxyatrazine, deethylatrazine, and deethyl-hydroxyatrazine. The addition of 105J14a cells g−1into soil with a low indigenous population of atrazine degraders treated with 50 and 200 μg of atrazine g−1soil resulted in two to five times higher mineralization than in the noninoculated soil. Sucrose addition did not result in significantly faster mineralization rates or shorten degradation lag times. However, J14a introduction (105cells g−1) into another soil with a larger indigenous atrazine-mineralizing population reduced the atrazine degradation lag times below those in noninoculated treatments but did not generally increase total atrazine mineralization.
Genotypic and Phenotypic Responses of a Riverine Microbial Community to Polycyclic Aromatic Hydrocarbon Contamination
Authors: Langworthy Donald E. , Stapleton Raymond D. , Sayler Gary S. , Findlay Robert H.
Published: December 2019

The phenotypic and genotypic adaptation of a freshwater sedimentary microbial community to elevated (22 to 217 μg g [dry weight] of sediment
−1
) levels of polycyclic aromatic hydrocarbons (PAHs) was determined by using an integrated biomolecular approach. Central to the approach was the use of phospholipid fatty acid (PLFA) profiles to characterize the microbial community structure and nucleic acid analysis to quantify the frequency of degradative genes. The study site was the Little Scioto River, a highly impacted, channelized riverine system located in central Ohio. This study site is a unique lotic system, with all sampling stations having similar flow and sediment characteristics both upstream and downstream from the source of contamination. These characteristics allowed for the specific analysis of PAH impact on the microbial community. PAH concentrations in impacted sediments ranged from 22 to 217 μg g (dry weight) of sediment
−1
, while PAH concentrations in ambient sediments ranged from below detection levels to 1.5 μg g (dry weight) of sediment
−1
. Total microbial biomass measured by phospholipid phosphate (PLP) analysis ranged from 95 to 345 nmol of PLP g (dry weight) of sediment
−1
. Nucleic acid analysis showed the presence of PAH-degradative genes at all sites, although observed frequencies were typically higher at contaminated sites. Principal component analysis of PLFA profiles indicated that moderate to high PAH concentrations altered microbial community structure and that seasonal changes were comparable in magnitude to the effects of PAH pollution. These data indicate that this community responded to PAH contamination at both the phenotypic and the genotypic level.
Genetic Diversity of the Biofilm Covering
Montacuta ferruginosa
(Mollusca, Bivalvia) as Evaluated by Denaturing Gradient Gel Electrophoresis Analysis and Cloning of PCR-Amplified Gene Fragments Coding for 16S rRNA
Authors: Gillan David C. , Speksnijder Arjen G. C. L. , Zwart Gabriel , De Ridder Chantal
Published: December 2019

The shell of the bivalve
Montacuta ferruginosa
, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain
Bacteria
. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera
Pseudoalteromonas
,
Colwellia
, and
Oceanospirillum
(members of the γ-
Proteobacteria
lineage), as well as
Flexibacter maritimus
(a member of the
Cytophaga-Flavobacter-Bacteroides
lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.
Biofilms on Indwelling Urethral Catheters Produce Quorum-Sensing Signal Molecules In Situ and In Vitro
Authors: Stickler David J. , Morris Nicola S. , McLean Robert J. C. , Fuqua Clay
Published: December 2019

Acylated homoserine lactones (AHLs) are chemical signals that mediate population density-dependent (quorum-sensing) gene expression in numerous gram-negative bacteria. In this study, gram-negative bacilli isolated from catheters were screened for AHL production by a cross-feeding assay utilizing an AHL-responsive
Agrobacterium tumefaciens
reporter strain. Positive reactions were obtained from 14 isolates of
Pseudomonas aeruginosa
; negative or weakly positive reactions were recorded for isolates of five other species.
P. aeruginosa
biofilms were then produced on catheters in a physical model of the bladder. Sections of colonized all-silicone catheters gave positive reactions for the quorum-sensing signal molecules as did sections that had been cleaned of biofilm and autoclaved. Control sections of unused catheters were negative in the tests. Sections from four of nine catheters that had been freshly removed from patients gave positive reactions for AHLs. Cleaned autoclaved sections of three of these catheters also gave strongly positive reactions for AHLs. These results demonstrate that AHLs are produced by biofilms as they develop on the catheters both in vitro in the model and in vivo in the patient’s bladder. They represent the first demonstration of AHL production by biofilms in a clinical setting.
Genetically Modified Ruminal Bacteria Protect Sheep from Fluoroacetate Poisoning
Authors: Gregg Keith , Hamdorf Brenton , Henderson Kerrin , Kopecny Jan , Wong Cheryl
Published: December 2019

Four strains of
Butyrivibrio fibrisolvens
, transformed with a gene encoding fluoroacetate dehalogenase, maintained a combined population of 10
6
to 10
7
cells ml
−1
in the rumens of test sheep. Five inoculated sheep showed markedly reduced toxicological symptoms after fluoroacetate poisoning when behavioral, physiological, and histological effects were compared with those of five uninoculated control sheep.
Pseudomonassp. Strain 273, an Aerobic α,ω-DichloroalkaneDegrading Bacterium
Authors: Wischnak Catrin , Löffler Frank E. , Li Jieran , Urbance John W. , Müller Rudolf
Published: December 2019

A gram-negative, aerobic bacterium was isolated from soil; this bacterium grew in 50% (vol/vol) suspensions of 1,10-dichlorodecane (1,10-DCD) as the sole source of carbon and energy. Phenotypic and small-subunit ribosomal RNA characterizations identified the organism, designated strain 273, as a member of the genusPseudomonas. After induction with 1,10-DCD,Pseudomonassp. strain 273 released stoichiometric amounts of chloride from C5to C12α,ω-dichloroalkanes in the presence of oxygen. No dehalogenation occurred under anaerobic conditions. The best substrates for dehalogenation and growth were C9to C12chloroalkanes. The isolate also grew with nonhalogenated aliphatic compounds, and decane-grown cells dechlorinated 1,10-DCD without a lag phase. In addition, cells grown on decane dechlorinated 1,10-DCD in the presence of chloramphenicol, indicating that the 1,10-DCD-dechlorinating enzyme system was also induced by decane. Other known alkane-degradingPseudomonasspecies did not grow with 1,10-DCD as a carbon source. Dechlorination of 1,10-DCD was demonstrated in cell extracts ofPseudomonassp. strain 273. Cell-free activity was strictly oxygen dependent, and NADH stimulated dechlorination, whereas EDTA had an inhibitory effect.
Chemostat Production of Plantaricin C By
Lactobacillus plantarum
LL441
Authors: Bárcena J. M. Bruno , Siñeriz F. , González de Llano D. , Rodríguez Ana , Suárez Juan E.
Published: December 2019

Plantaricin C, a bacteriocin synthesized by
Lactobacillus plantarum
LL441, was optimally produced in chemostats kept at pH 5.0, 30°C, 150 rpm, and a dilution rate of 0.05 h
−1
when glucose was used as carbon source and a dilution rate of 0.10 to 0.12 h
−1
when sucrose or fructose was used instead. Production was abolished at high dilution rates, i.e., when the cells grew rapidly in all carbon sources.
Prevalence of the
Rhizobium etli
-Like Allele in Genes Coding for 16S rRNA among the Indigenous Rhizobial Populations Found Associated with Wild Beans from the Southern Andes in Argentina
Authors: Aguilar O. Mario , López María Verónica , Riccillo Pablo M. , González Ramón A. , Pagano Marcela , Grasso Daniel H. , Pühler Alfred , Favelukes Gabriel
Published: December 2019

A collection of rhizobial isolates from nodules of wild beans,
Phaseolus vulgaris
var. aborigineus, found growing in virgin lands in 17 geographically separate sites in northwest Argentina was characterized on the basis of host range, growth, hybridization to a
nifH
probe, analysis of genes coding for 16S rRNA (16S rDNA), DNA fingerprinting, and plasmid profiles. Nodules in field-collected wild bean plants were largely dominated by rhizobia carrying the 16S rDNA allele of
Rhizobium etli
. A similar prevalence of the
R. etli
allele was observed among rhizobia trapped from nearby soil. Intragroup diversity of wild bean isolates with either
R. etli
-like or
Rhizobium leguminosarum
bv. phaseoli-like alleles was generally found across northwest Argentina. The predominance of the
R. etli
allele suggests that in this center of origin of
P. vulgaris
the coevolution of
Rhizobium
spp. and primitive beans has resulted in this preferential symbiotic association.
Fate of
Escherichia coli
O157:H7 on Fresh-Cut Apple Tissue and Its Potential for Transmission by Fruit Flies
Authors: Janisiewicz W. J. , Conway W. S. , Brown M. W. , Sapers G. M. , Fratamico P. , Buchanan R. L.
Published: December 2019

Pathogenic
Escherichia coli
O157:H7, as well as nonpathogenic strains ATCC 11775 and ATCC 23716, grew exponentially in wounds on Golden Delicious apple fruit. The exponential growth occurred over a longer time period on fruit inoculated with a lower concentration of the bacterium than on fruit inoculated with a higher concentration. The bacterium reached the maximum population supported in the wounds regardless of the initial inoculum concentrations. Populations of
E. coli
O157:H7 in various concentrations of sterilized apple juice and unsterilized cider declined over time and declined more quickly in diluted juice and cider. The decline was greater in the unsterilized cider than in juice, which may have resulted from the interaction of
E. coli
O157:H7 with natural populations of yeasts that increased with time. Experiments on the transmission of
E. coli
by fruit flies, collected from a compost pile of decaying apples and peaches, were conducted with strain F-11775, a fluorescent transformant of nonpathogenic
E. coli
ATCC 11775. Fruit flies were easily contaminated externally and internally with
E. coli
F-11775 after contact with the bacterium source. The flies transmitted this bacterium to uncontaminated apple wounds, resulting in a high incidence of contaminated wounds. Populations of the bacterium in apple wounds increased significantly during the first 48 h after transmission. Further studies under commercial conditions are necessary to confirm these findings.
Identification of Enzymes and Quantification of Metabolic Fluxes in the Wild Type and in a Recombinant
Aspergillus oryzae
Strain
Authors: Pedersen Henrik , Carlsen Morten , Nielsen Jens
Published: December 2019

Two α-amylase-producing strains of
Aspergillus oryzae
, a wild-type strain and a recombinant containing additional copies of the α-amylase gene, were characterized with respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition, and metabolic fluxes through the central metabolism during glucose-limited chemostat cultivations. Citrate synthase and isocitrate dehydrogenase (NAD) activities were found only in the mitochondria, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase (NADP) activities were found only in the cytosol, and isocitrate dehydrogenase (NADP), glutamate oxaloacetate transaminase, malate dehydrogenase, and glutamate dehydrogenase (NAD) activities were found in both the mitochondria and the cytosol. The measured biomass components and ash could account for 95% (wt/wt) of the biomass. The protein and RNA contents increased linearly with increasing specific growth rate, but the carbohydrate and chitin contents decreased. A metabolic model consisting of 69 fluxes and 59 intracellular metabolites was used to calculate the metabolic fluxes through the central metabolism at several specific growth rates, with ammonia or nitrate as the nitrogen source. The flux through the pentose phosphate pathway increased with increasing specific growth rate. The fluxes through the pentose phosphate pathway were 15 to 26% higher for the recombinant strain than for the wild-type strain.
Bacterial Adhesion to Soil Contaminants in the Presence of Surfactants
Authors: Stelmack Patricia L. , Gray Murray R. , Pickard Michael A.
Published: December 2019

It has been proposed that addition of surfactants to contaminated soil enhances the solubility of target compounds; however, surfactants may simultaneously reduce the adhesion of bacteria to hydrophobic surfaces. If the latter mechanism is important for the biodegradation of virtually insoluble contaminants, then the use of surfactants may not be beneficial. The adhesion of a
Mycobacterium
strain and a
Pseudomonas
strain, isolated from a creosote-contaminated soil, to the surfaces of highly viscous non-aqueous-phase liquids (NAPLs) was measured. The NAPLs were organic material extracted from soils from two creosote-contaminated sites and two petroleum-contaminated sites. Cells suspended in media with and without surfactant were placed in test tubes coated with an NAPL, and the percentages of cells that adhered to the surface of the NAPL in the presence and absence of surfactant were compared by measuring optical density. Test tubes without NAPLs were used as controls. The presence of either Triton X-100 or Dowfax 8390 at a concentration that was one-half the critical micelle concentration (CMC) inhibited adhesion of both species of bacteria to the NAPLs. Both surfactants, when added at concentrations that were one-half the CMCs to test tubes containing previously adhered bacteria, also promoted the removal of the cells from the surfaces of the NAPL-coated test tubes. Neither surfactant was toxic to the bacteria. Further investigation showed that a low concentration of surfactant also inhibited the growth of both species on anthracene, indicating that the presence of a surfactant resulted in a reduction in the uptake of the solid carbon source.
Bacterial Filament Formation, a Defense Mechanism against Flagellate Grazing, Is Growth Rate Controlled in Bacteria of Different Phyla
Authors: Hahn Martin W. , Moore Edward R. B. , Höfle Manfred G.
Published: December 2019

A facultatively filamentous bacterium was isolated from eutrophic lake water and was identified asFlectobacillussp. strain MWH38 (a member of theCytophaga-Flavobacterium-Bacteroidesphylum) by comparative 16S rRNA gene sequence analysis. Filament formation byFlectobacillussp. strain MWH38 and filament formation byFlectobacillus major, the closest known relative of strain MWH38, were studied in chemostat cultures under grazing pressure by the bacterivorous flagellateOchromonassp. strain DS and without predation at several growth rates. The results clearly demonstrated that filament formation by the two flectobacilli is growth rate controlled and thus independent of the presence of a predator. However, flagellate grazing positively influenced bacterial growth rates by decreasing bacterial biomass and thus indirectly stimulated filament formation. The results of investigations of cell elongation and filament formation byComamonas acidovoransPX54 (a member of the β subclass of the classProteobacteria) supported the recent proposal that in this species the mechanism of filament formation is growth rate controlled. The finding that the grazing defense mechanism consisting of filament formation is growth rate controlled in the flectobacilli investigated andC. acidovoransPX54 (i.e., in bacteria belonging to divergent evolutionary phyla) may indicate that this mechanism is a phylogenetically widely distributed defense strategy against grazing.
Homologs of Aflatoxin Biosynthesis Genes and Sequence of
aflR
in
Aspergillus oryzae
and
Aspergillus sojae
Authors: Watson Adrian J. , Fuller Linda J. , Jeenes David J. , Archer David B.
Published: December 2019

The presence, but not expression, of homologs of three structural genes and a regulatory gene necessary for aflatoxin biosynthesis in
Aspergillus parasiticus
and
A. flavus
was shown for
A. oryzae
and
A. sojae
. Homologs of the regulatory gene
aflR
were cloned and sequenced from
A. oryzae
and
A. sojae
.
StableEscherichia coli-Clostridium acetobutylicumShuttle Vector for Secretion of Murine Tumor Necrosis Factor Alpha
Authors: Theys J. , Nuyts S. , Landuyt W. , Van Mellaert L. , Dillen C. , Böhringer M. , Dürre P. , Lambin P. , Anné J.
Published: December 2019

Recombinant plasmids were constructed to secrete mouse tumor necrosis factor alpha (mTNF-α) fromClostridium acetobutylicum. The shuttle plasmids contained the clostridial endo-β1,4-glucanase (eglA) promoter and signal sequence that was fused in frame to the mTNF-α cDNA. The construction was first tested inEscherichia coliand then introduced inC. acetobutylicumDSM792 by electroporation. Controls confirmed the presence and stability of the recombinant plasmids in this organism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an in vitro cytotoxic assay were used to monitor expression and secretion of mTNF-α during growth. Significant levels of biologically active mTNF-α were measured in both lysates and supernatants. The present report deals with investigations on the elaboration of a gene transfer system for cancer treatment using anaerobic bacteria.
Localization and In Situ Activities of Homoacetogenic Bacteria in the Highly Compartmentalized Hindgut of Soil-Feeding Higher Termites (
Cubitermes
spp.)
Authors: Tholen Anne , Brune Andreas
Published: December 2019

Methanogenesis and homoacetogenesis occur simultaneously in the hindguts of almost all termites, but the reasons for the apparent predominance of methanogenesis over homoacetogenesis in the hindgut of the humivorous species is not known. We found that in gut homogenates of soil-feeding
Cubitermes
spp., methanogens outcompete homoacetogens for endogenous reductant. The rates of methanogenesis were always significantly higher than those of reductive acetogenesis, whereas the stimulation of acetogenesis by the addition of exogenous H
2
or formate was more pronounced than that of methanogenesis. In a companion paper, we reported that the anterior gut regions of
Cubitermes
spp. accumulated hydrogen to high partial pressures, whereas H
2
was always below the detection limit (<100 Pa) in the posterior hindgut, and that all hindgut compartments turned into efficient H
2
sinks when external H
2
was provided (D. Schmitt-Wagner and A. Brune, Appl. Environ. Microbiol. 65:4490–4496, 1999). Using a microinjection technique, we found that only the posterior gut sections P3/4a and P4b, which harbored methanogenic activities, formed labeled acetate from H
14
CO
3
−
. Enumeration of methanogenic and homoacetogenic populations in the different gut sections confirmed the coexistence of both metabolic groups in the same compartments. However, the in situ rates of acetogenesis were strongly hydrogen limited; in the P4b section, no activity was detected unless external H
2
was added. Endogenous rates of reductive acetogenesis in isolated guts were about 10-fold lower than the in vivo rates of methanogenesis, but were almost equal when exogenous H
2
was supplied. We conclude that the homoacetogenic populations in the posterior hindgut are supported by either substrates other than H
2
or by a cross-epithelial H
2
transfer from the anterior gut regions, which may create microniches favorable for H
2
-dependent acetogenesis.