bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacterial Resistance to Ultrasonic Waves under Pressure at Nonlethal (Manosonication) and Lethal (Manothermosonication) Temperatures
Authors: Pagán R. , Mañas P. , Raso J. , Condón S.
Published: December 2019

The decimal reduction times of
Streptococcus faecium
,
Listeria monocytogenes
,
Salmonella enteritidis
, and
Aeromonas hydrophila
corresponding to heat treatment at 62°C were 7.1, 0.34, 0.024, and 0.0096 min, and those corresponding to manosonication treatment (40°C, 200 kPa, 117 μm) were 4.0, 1.5, 0.86, and 0.90 min, respectively. The manosonication decimal reduction times of the four species investigated decreased sixfold when the amplitude was increased from 62 to 150 μm and fivefold when the relative pressure was raised from 0 to 400 kPa. In
L. monocytogenes
,
S. enteritidis
, and
A. hydrophila
, the lethal effect of manothermosonication was the result of the addition of the lethal effects of heat and manosonication, whereas in
S. faecium
it was a synergistic effect.
Chlorine Inactivation of
Sphingomonas
Cells Attached to Goethite Particles in Drinking Water
Authors: Gauthier Vincent , Redercher Stéphane , Block Jean-Claude
Published: December 2019

Bacteria in drinking water, attached or not attached to goethite particles, were disinfected with chlorine. No additional protection was provided to the bacteria by their attachment to particles, and the limited efficiency of inactivation by chlorine was attributed to the presence of bacterial aggregates in both types of suspension.
In Situ, Real-Time Catabolic Gene Expression: Extraction and Characterization of Naphthalene Dioxygenase mRNA Transcripts from Groundwater
Authors: Wilson Mark S. , Bakermans Corien , Madsen Eugene L.
Published: December 2019

We developed procedures for isolating and characterizing in situ-transcribed mRNA from groundwater microorganisms catabolizing naphthalene at a coal tar waste-contaminated site. Groundwater was pumped through 0.22-μm-pore-size filters, which were then frozen in dry ice-ethanol. RNA was extracted from the frozen filters by boiling sodium dodecyl sulfate lysis and acidic phenol-chloroform extraction. Transcript characterization was performed with a series of PCR primers designed to amplify
nahAc
homologs. Several primer pairs were found to amplify
nahAc
homologs representing the entire diversity of the naphthalene-degrading genes. The environmental RNA extract was reverse transcribed, and the resultant mixture of cDNAs was amplified by PCR. A digoxigenin-labeled probe mixture was produced by PCR amplification of groundwater cDNA. This probe mixture hybridized under stringent conditions with the corresponding PCR products from naphthalene-degrading bacteria carrying a variety of
nahAc
homologs, indicating that diverse dioxygenase transcripts had been retrieved from groundwater. Diluted and undiluted cDNA preparations were independently amplified, and 28 of the resulting PCR products were cloned and sequenced. Sequence comparisons revealed two major groups related to the dioxygenase genes
ndoB
and
dntAc
, previously cloned from
Pseudomonas putida
NCIB 9816-4 and
Burkholderia
sp. strain DNT, respectively. A distinctive subgroup of sequences was found only in experiments performed with the undiluted cDNA preparation. To our knowledge, these results are the first to directly document in situ transcription of genes encoding naphthalene catabolism at a contaminated site by indigenous microorganisms. The retrieved sequences represent greater diversity than has been detected at the study site by culture-based approaches.
Ferric Iron Reduction by Bacteria Associated with the Roots of Freshwater and Marine Macrophytes
Authors: King G. M. , Garey Meredith A.
Published: December 2019

In vitro assays of washed, excised roots revealed maximum potential ferric iron reduction rates of >100 μmol g (dry weight)
−1
day
−1
for three freshwater macrophytes and rates between 15 and 83 μmol (dry weight)
−1
day
−1
for two marine species. The rates varied with root morphology but not consistently (fine root activity exceeded smooth root activity in some but not all cases). Sodium molybdate added at final concentrations of 0.2 to 20 mM did not inhibit iron reduction by roots of marine macrophytes (
Spartina alterniflora
and
Zostera marina
). Roots of a freshwater macrophyte,
Sparganium eurycarpum
, that were incubated with an analog of humic acid precursors, anthroquinone disulfate (AQDS), reduced freshly precipitated iron oxyhydroxide contained in dialysis bags that excluded solutes with molecular weights of >1,000; no reduction occurred in the absence of AQDS. Bacterial enrichment cultures and isolates from freshwater and marine roots used a variety of carbon and energy sources (e.g., acetate, ethanol, succinate, toluene, and yeast extract) and ferric oxyhydroxide, ferric citrate, uranate, and AQDS as terminal electron acceptors. The temperature optima for a freshwater isolate and a marine isolate were equivalent (approximately 32°C). However, iron reduction by the freshwater isolate decreased with increasing salinity, while reduction by the marine isolate displayed a relatively broad optimum salinity between 20 and 35 ppt. Our results suggest that by participating in an active iron cycle and perhaps by reducing humic acids, iron reducers in the rhizoplane of aquatic macrophytes limit organic availability to other heterotrophs (including methanogens) in the rhizosphere and bulk sediments.
Identification of Residues in Domain III of
Bacillus thuringiensis
Cry1Ac Toxin That Affect Binding and Toxicity
Authors: Lee Mi Kyong , You Taek H. , Gould Fred L. , Dean Donald H.
Published: December 2019

Alanine substitution mutations in the Cry1Ac domain III region, from amino acid residues 503 to 525, were constructed to study the functional role of domain III in the toxicity and receptor binding of the protein to
Lymantria dispar
,
Manduca sexta
, and
Heliothis virescens
. Five sets of alanine block mutants were generated at the residues
503
SS
504
,
506
NNI
508
,
509
QNR
511
,
522
ST
523
, and
524
ST
525
. Single alanine substitutions were made at the residues
509
Q,
510
N,
511
R, and
513
Y. All mutant proteins produced stable toxic fragments as judged by trypsin digestion, midgut enzyme digestion, and circular dichroism spectrum analysis. The mutations,
503
SS
504
-AA,
506
NNI
508
-AAA,
522
ST
523
-AA,
524
ST
525
-AA, and
510
N-A affected neither the protein’s toxicity nor its binding to brush border membrane vesicles (BBMV) prepared from these insects. Toward
L. dispar
and
M. sexta
, the
509
QNR
511
-AAA,
509
Q-A,
511
R-A, and
513
Y-A mutant toxins showed 4- to 10-fold reductions in binding affinities to BBMV, with 2- to 3-fold reductions in toxicity. Toward
H. virescens
, the
509
QNR
511
-AAA,
509
Q-A,
511
R-A, and
513
Y-mutant toxins showed 8- to 22-fold reductions in binding affinities, but only
509
QNR
511
-AAA and
511
R-A mutant toxins reduced toxicity by approximately three to four times. In the present study, greater loss in binding affinity relative to toxicity has been observed. These data suggest that the residues
509
Q,
511
R, and
513
Y in domain III might be only involved in initial binding to the receptor and that the initial binding step becomes rate limiting only when it is reduced more than fivefold.
Dimethylsulfoniopropionate and Methanethiol Are Important Precursors of Methionine and Protein-Sulfur in Marine Bacterioplankton
Authors: Kiene Ronald P. , Linn Laura J. , González José , Moran Mary Ann , Bruton Jody A.
Published: December 2019

Organic sulfur compounds are present in all aquatic systems, but their use as sources of sulfur for bacteria is generally not considered important because of the high sulfate concentrations in natural waters. This study investigated whether dimethylsulfoniopropionate (DMSP), an algal osmolyte that is abundant and rapidly cycled in seawater, is used as a source of sulfur by bacterioplankton. Natural populations of bacterioplankton from subtropical and temperate marine waters rapidly incorporated 15 to 40% of the sulfur from tracer-level additions of [35S]DMSP into a macromolecule fraction. Tests with proteinase K and chloramphenicol showed that the sulfur from DMSP was incorporated into proteins, and analysis of protein hydrolysis products by high-pressure liquid chromatography showed that methionine was the major labeled amino acid produced from [35S]DMSP. Bacterial strains isolated from coastal seawater and belonging to the α-subdivision of the divisionProteobacteriaincorporated DMSP sulfur into protein only if they were capable of degrading DMSP to methanethiol (MeSH), whereas MeSH was rapidly incorporated into macromolecules by all tested strains and by natural bacterioplankton. These findings indicate that the demethylation/demethiolation pathway of DMSP degradation is important for sulfur assimilation and that MeSH is a key intermediate in the pathway leading to protein sulfur. Incorporation of sulfur from DMSP and MeSH by natural populations was inhibited by nanomolar levels of other reduced sulfur compounds including sulfide, methionine, homocysteine, cysteine, and cystathionine. In addition, propargylglycine and vinylglycine were potent inhibitors of incorporation of sulfur from DMSP and MeSH, suggesting involvement of the enzyme cystathionine γ-synthetase in sulfur assimilation by natural populations. Experiments with [methyl-3H]MeSH and [35S]MeSH showed that the entire methiol group of MeSH was efficiently incorporated into methionine, a reaction consistent with activity of cystathionine γ-synthetase. Field data from the Gulf of Mexico indicated that natural turnover of DMSP supplied a major fraction of the sulfur required for bacterial growth in surface waters. Our study highlights a remarkable adaptation by marine bacteria: they exploit nanomolar levels of reduced sulfur in apparent preference to sulfate, which is present at 106- to 107-fold higher concentrations.
Diversity in Butane Monooxygenases among Butane-Grown Bacteria
Authors: Hamamura Natsuko , Storfa Ryan T. , Semprini Lewis , Arp Daniel J.
Published: December 2019

Butane monooxygenases of butane-grown
Pseudomonas butanovora
,
Mycobacterium vaccae
JOB5, and an environmental isolate, CF8, were compared at the physiological level. The presence of butane monooxygenases in these bacteria was indicated by the following results. (i) O
2
was required for butane degradation. (ii) 1-Butanol was produced during butane degradation. (iii) Acetylene inhibited both butane oxidation and 1-butanol production. The responses to the known monooxygenase inactivator, ethylene, and inhibitor, allyl thiourea (ATU), discriminated butane degradation among the three bacteria. Ethylene irreversibly inactivated butane oxidation by
P. butanovora
but not by
M. vaccae
or CF8. In contrast, butane oxidation by only CF8 was strongly inhibited by ATU. In all three strains of butane-grown bacteria, specific polypeptides were labeled in the presence of [
14
C]acetylene. The [
14
C]acetylene labeling patterns were different among the three bacteria. Exposure of lactate-grown CF8 and
P. butanovora
and glucose-grown
M. vaccae
to butane induced butane oxidation activity as well as the specific acetylene-binding polypeptides. Ammonia was oxidized by all three bacteria.
P. butanovora
oxidized ammonia to hydroxylamine, while CF8 and
M. vaccae
produced nitrite. All three bacteria oxidized ethylene to ethylene oxide. Methane oxidation was not detected by any of the bacteria. The results indicate the presence of three distinct butane monooxygenases in butane-grown
P. butanovora
,
M. vaccae
, and CF8.
Sulfonates as Terminal Electron Acceptors for Growth of Sulfite-Reducing Bacteria (Desulfitobacteriumspp.) and Sulfate-Reducing Bacteria: Effects of Inhibitors of Sulfidogenesis
Authors: Lie Thomas J. , Godchaux Walter , Leadbetter Edward R.
Published: December 2019

This study demonstrates the ability ofDesulfitobacteriumspp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction byDesulfitobacterium hafnienseresulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells ofDesulfitobacterium hafniense,Desulfitobacteriumsp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)—Desulfovibrio desulfuricansIC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135–149, 1998) andDesulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction byDesulfitobacterium dehalogenansandDesulfitobacteriumsp. strain PCE 1.Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB andDesulfitobacteriumspp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB andDesulfitobacterium hafniense.
Bacterial Production ofd-Erythroascorbic Acid andl-Ascorbic Acid through Functional Expression ofSaccharomyces cerevisiaed-Arabinono-1,4-Lactone Oxidase inEscherichia coli
Authors: Lee Byung-Hoon , Huh Won-Ki , Kim Seong-Tae , Lee Jung-Shin , Kang Sa-Ouk
Published: December 2019

d-Arabinono-1,4-lactone oxidase, which catalyzes the terminal step in the biosynthesis ofd-erythroascorbic acid inSaccharomyces cerevisiae, was functionally expressed inEscherichia coliinherently lacking the enzyme. The recombinantE. colistrain expressing the enzyme could overproduced-erythroascorbic acid andl-ascorbic acid when supplied withd-arabinono-1,4-lactone andl-galactono-1,4-lactone, respectively.
Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples
Authors: Miller D. N. , Bryant J. E. , Madsen E. L. , Ghiorse W. C.
Published: December 2019

We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.
Selection of a Highly Monensin-Resistant
Prevotella bryantii
Subpopulation with Altered Outer Membrane Characteristics
Authors: Callaway Todd R. , Russell James B.
Published: December 2019

Prevotella bryantii
cultures treated with monensin grew more slowly than untreated cultures, but only if the monensin concentration was greater than 1 μM. Cultures that were repeatedly transferred (eight transfers or 25 doublings) with monensin always grew rapidly, even at a 10 μM concentration. The amount of monensin needed to facilitate half-maximal potassium depletion (

K
d

) from monensin-selected cells was 16-fold greater than “unadapted” wild-type cultures (3,200 versus 200 nM). Cells taken from continuous culture had a

K
d

of 100 nM, and these inocula could not grow in batch culture when the monensin concentration was greater than 300 nM. Continuous cultures treated with monensin nearly washed out, but the surviving cells had a

K
d

of 1,300 nM. When wild-type cells were transferred in batch culture with 10 μM monensin, the

K
d

did not reach its maximum value (3,200 nM) until after eight transfers (25 doublings).

K
d

declined when monensin was removed, and it took eight transfers to reach the control value (200 nM). The most probable number of wild-type cells was 1,000-fold lower than of the monensin-selected cells, but calculations based on relative growth advantage and

K
d

indicated that the wild-type culture had 1 to 10% highly monensin-resistant cells. Cell pellets of wild-type cultures were more difficult to disperse than were monensin-selected cells, and water-soluble phenol extracts of monensin-selected cells had 1.8-fold more anthrone-reactive material than did the wild type. Wild-type cultures that were washed in Tris buffer (pH 8.0) released little alkaline phosphatase and were agglutinated by lysozyme. Monensin-selected cultures leaked ninefold more alkaline phosphatase and were not agglutinated by lysozyme. Wild-type colonies taken from high-dilution agar roll tubes retained the lysozyme agglutination phenotype even if transferred with monensin, and monensin-selected colonies were never agglutinated. These observations indicated that wild-type
P. bryantii
cultures had a subpopulation with different outer membrane characteristics and increased monensin resistance.
Biochemical and Genetic Analyses of Ferulic Acid Catabolism in
Pseudomonas
sp. Strain HR199
Authors: Overhage Jörg , Priefert Horst , Steinbüchel Alexander
Published: December 2019

The gene loci
fcs
, encoding feruloyl coenzyme A (feruloyl-CoA) synthetase,
ech
, encoding enoyl-CoA hydratase/aldolase, and
aat
, encoding β-ketothiolase, which are involved in the catabolism of ferulic acid and eugenol in
Pseudomonas
sp. strain HR199 (DSM7063), were localized on a DNA region covered by two
Eco
RI fragments (E230 and E94), which were recently cloned from a
Pseudomonas
sp. strain HR199 genomic library in the cosmid pVK100. The nucleotide sequences of parts of fragments E230 and E94 were determined, revealing the arrangement of the aforementioned genes. To confirm the function of the structural genes
fcs
and
ech
, they were cloned and expressed in
Escherichia coli
. Recombinant strains harboring both genes were able to transform ferulic acid to vanillin. The feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase activities of the
fcs
and
ech
gene products, respectively, were confirmed by photometric assays and by high-pressure liquid chromatography analysis. To prove the essential involvement of the
fcs
,
ech
, and
aat
genes in the catabolism of ferulic acid and eugenol in
Pseudomonas
sp. strain HR199, these genes were inactivated separately by the insertion of omega elements. The corresponding mutants
Pseudomonas
sp. strain HR
fcs
ΩGm and
Pseudomonas
sp. strain HR
ech
ΩKm were not able to grow on ferulic acid or on eugenol, whereas the mutant
Pseudomonas
sp. strain HR
aat
ΩKm exhibited a ferulic acid- and eugenol-positive phenotype like the wild type. In conclusion, the degradation pathway of eugenol via ferulic acid and the necessity of the activation of ferulic acid to the corresponding CoA ester was confirmed. The
aat
gene product was shown not to be involved in this catabolism, thus excluding a β-oxidation analogous degradation pathway for ferulic acid. Moreover, the function of the
ech
gene product as an enoyl-CoA hydratase/aldolase suggests that ferulic acid degradation in
Pseudomonas
sp. strain HR199 proceeds via a similar pathway to that recently described for
Pseudomonas fluorescens
AN103.
Flagellate Predation on a Bacterial Model Community: Interplay of Size-Selective Grazing, Specific Bacterial Cell Size, and Bacterial Community Composition
Authors: Hahn Martin W. , Höfle Manfred G.
Published: December 2019

The influence of grazing by the bacterivorous nanoflagellate
Ochromonas
sp. strain DS on the taxonomic and morphological structures of a complex bacterial community was studied in one-stage chemostat experiments. A bacterial community, consisting of at least 30 different strains, was fed with a complex carbon source under conditions of low growth rate (0.5 day
−1
when nongrazed) and low substrate concentration (9 mg liter
−1
). Before and after the introduction of the predator, the bacterial community composition was studied by in situ techniques (immunofluorescence microscopy and fluorescent in situ hybridization), as well as by cultivation on agar media. The cell sizes of nonspecifically stained and immunofluorescently labeled bacteria were measured by image analysis. Grazing by the flagellate caused a bidirectional change in the morphological structure of the community. Medium-size bacterial cells, which dominated the nongrazed community, were largely replaced by smaller cells, as well as by cells contained in large multicellular flocs. Cell morphological changes were combined with community taxonomic changes. After introduction of the flagellate, the dominating strains with medium-size cells were largely replaced by single-celled strains with smaller cells on the one hand and, on the other hand, by
Pseudomonas
sp. strain MWH1, which formed the large, floc-like forms. We assume that size-selective grazing was the major force controlling both the morphological and the taxonomic structures of the model community.
Microbial Desulfurization of Alkylated Dibenzothiophenes from a Hydrodesulfurized Middle Distillate by
Rhodococcus erythropolis
I-19
Authors: Folsom B. R. , Schieche D. R. , DiGrazia P. M. , Werner J. , Palmer S.
Published: December 2019

Rhodococcus erythropolis
I-19, containing multiple copies of key
dsz
genes, was used to desulfurize alkylated dibenzothiophenes (Cx-DBTs) found in a hydrodesulfurized middle-distillate petroleum (MD 1850). Initial desulfurization rates of dibenzothiophene (DBT) and MD 1850 by I-19 were 5.0 and 2.5 μmol g dry cell weight
−1
min
−1
, more than 25-fold higher than that for wild-type bacteria. According to sulfur K-edge X-ray absorption near-edge structure (XANES) analysis, thiophenic compounds accounted for >95% of the total sulfur found in MD 1850, predominantly Cx-DBTs and alkylated benzothiophenes. Extensive biodesulfurization resulted in a 67% reduction of total sulfur from 1,850 to 615 ppm S. XANES analysis of the 615-ppm material gave a sulfur distribution of 75% thiophenes, 11% sulfides, 2% sulfoxides, and 12% sulfones. I-19 preferentially desulfurized DBT and C1-DBTs, followed by the more highly alkylated Cx-DBTs. Shifting zero- to first-order (first-order) desulfurization rate kinetics were observed when MD 1850 was diluted with hexadecane. Apparent saturation rate constant (
K
0
) and half-saturation rate constant (
K
1
) values were calculated to be 2.8 μmol g dry cell weight
−1
min
−1
and 130 ppm, respectively. However, partial biocatalytic reduction of MD 1850 sulfur concentration followed by determination of initial rates with fresh biocatalyst led to a sigmoidal kinetic behavior. A competitive-substrate model suggested that the apparent
K
1
values for each group of Cx-DBTs increased with increasing alkylation. Overall desulfurization rate kinetics with I-19 were affected by the concentration and distribution of Cx-DBTs according to the number and/or lengths of alkyl groups attached to the basic ring structure.
The
ald
Gene, Encoding a Coenzyme A-Acylating Aldehyde Dehydrogenase, Distinguishes
Clostridium beijerinckii
and Two Other Solvent-Producing Clostridia from
Clostridium acetobutylicum
Authors: Toth Julianna , Ismaiel Adnan A. , Chen Jiann-Shin
Published: December 2019

The coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH) catalyzes a key reaction in the acetone- and butanol (solvent)-producing clostridia. It reduces acetyl-CoA and butyryl-CoA to the corresponding aldehydes, which are then reduced by alcohol dehydrogenase (ADH) to form ethanol and 1-butanol. The ALDH of
Clostridium beijerinckii
NRRL B593 was purified. It had no ADH activity, was NAD(H) specific, and was more active with butyraldehyde than with acetaldehyde. The N-terminal amino acid sequence of the purified ALDH was determined. The open reading frame preceding the
ctfA
gene (encoding a subunit of the solvent-forming CoA transferase) of
C. beijerinckii
NRRL B593 was identified as the structural gene (
ald
) for the ALDH. The
ald
gene encodes a polypeptide of 468 amino acid residues with a calculated
M
r
of 51,353. The position of the
ald
gene in
C. beijerinckii
NRRL B593 corresponded to that of the
aad/adhE
gene (encoding an aldehyde-alcohol dehydrogenase) of
Clostridium acetobutylicum
ATCC 824 and DSM 792. In Southern analyses, a probe derived from the
C. acetobutylicum aad/adhE
gene did not hybridize to restriction fragments of the genomic DNAs of
C. beijerinckii
and two other species of solvent-producing clostridia. In contrast, a probe derived from the
C. beijerinckii ald
gene hybridized to restriction fragments of the genomic DNA of three solvent-producing species but not to those of
C. acetobutylicum
, indicating a key difference among the solvent-producing clostridia. The amino acid sequence of the ALDH of
C. beijerinckii
NRRL B593 was most similar (41% identity) to those of the
eutE
gene products (CoA-acylating ALDHs) of
Salmonella typhimurium
and
Escherichia coli
, whereas it was about 26% identical to the ALDH domain of the aldehyde-alcohol dehydrogenases of
C. acetobutylicum
,
E. coli
,
Lactococcus lactis
, and amitochondriate protozoa. The predicted secondary structure of the
C. beijerinckii
ALDH suggests the presence of an atypical Rossmann fold for NAD
+
binding. A comparison of the proposed catalytic pockets of the CoA-dependent and CoA-independent ALDHs identified 6 amino acids that may contribute to interaction with CoA.
Comparative Phylogenetic Assignment of Environmental Sequences of Genes Encoding 16S rRNA and Numerically Abundant Culturable Bacteria from an Anoxic Rice Paddy Soil
Authors: Hengstmann Ulf , Chin Kuk-Jeong , Janssen Peter H. , Liesack Werner
Published: December 2019

We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042–5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 × 10
7
to 2.5 × 10
8
cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division
Verrucomicrobia
(strain PB90-1) and the
Cytophaga-Flavobacterium-Bacteroides
group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family
Bacillaceae
(strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.
Probiotics Shown To Change Bacterial Community Structure in the Avian Gastrointestinal Tract
Authors: Netherwood Trudy , Gilbert H. J. , Parker D. S. , O’Donnell A. G.
Published: December 2019

Culturing and molecular techniques were used to monitor changes in the bacterial flora of the avian gastrointestinal (GI) tract following introduction of genetically modified (GM) and unmodified probiotics. Community hybridization of amplified 16S ribosomal DNA demonstrated that the bacterial flora of the GI tract changed significantly in response to the probiotic treatments. The changes were not detected by culturing. Although both GM and non-GM strains of
Enterococcus faecium
NCIMB 11508 changed the bacterial flora of the chicken GI tract, they did so differently. Probing the community DNA with an
Enterococcus faecalis
-specific probe showed that the relative amount of
E. faecalis
in the total eubacterial population increased in the presence of the non-GM strain and decreased in the presence of the GM probiotic compared with the results obtained with an untreated control group.
Recovery in Embryonated Eggs of Viable but Nonculturable
Campylobacter jejuni
Cells and Maintenance of Ability To Adhere to HeLa Cells after Resuscitation
Authors: Cappelier J. M. , Minet J. , Magras C. , Colwell R. R. , Federighi M.
Published: December 2019

The existence of a viable but nonculturable (VBNC) state has been described for
Campylobacter jejuni
as it had been for a number pathogenic bacteria. Three
C. jejuni
human isolates were suspended in surface water and subsequently entered the VBNC state. After starvation for 30 days, VBNC cells were inoculated in the yolk sacs of embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs inoculated with VBNC
C. jejuni
cells. Recovered cells kept their adhesion properties.
Identification of a Transcriptional Activator (ChnR) and a 6-Oxohexanoate Dehydrogenase (ChnE) in the Cyclohexanol Catabolic Pathway in
Acinetobacter
sp. Strain NCIMB 9871 and Localization of the Genes That Encode Them
Authors: Iwaki Hiroaki , Hasegawa Yoshie , Teraoka Masahiro , Tokuyama Tai , Bergeron Hélène , Lau Peter C. K.
Published: December 2019

We identified
chnR
, a gene encoding an AraC-XylS type of transcriptional activator that regulates the expression of
chnB
, the structural gene for cyclohexanone monooxygenase (CHMO) in
Acinetobacter
sp. strain NCIMB 9871. The gene sequence of
chnE
, which encodes an NADP
+
-linked 6-oxohexanoate dehydrogenase, the enzyme catalyzing the fifth step of cyclohexanol degradation, was also determined. The gene arrangement is
chnB-chnE-chnR
. The predicted molecular masses of the three polypeptides were verified by radiolabeling by using the T7 expression system. Inducible expression of cloned
chnB
in
Escherichia coli
depended upon the presence of
chnR
. A transcriptional
chnB
::
lacZ
fusion experiment revealed that cyclohexanone induces
chnB
expression in
E. coli
, in which a 22-fold increase in activity was observed.
Genotypic Diversity among
Brevibacillus laterosporus
Strains
Authors: Zahner Viviane , Rabinovitch Leon , Suffys Phylip , Momen Hooman
Published: December 2019

In comparison with other entomopathogenic
Bacillus
species, the genome of
Brevibacillus laterosporus
is poorly characterized. The aim of this study was to examine genetic variability in
B. laterosporus
by using a range of typing methodologies. Strains of
B. laterosporus
were examined for variation in 13 chromosomal genes encoding enzymes by multilocus enzyme electrophoresis. Optimal conditions of pulsed-field gel electrophoresis and randomly amplified polymorphic DNA were established that allowed analysis of the genome of
B. laterosporus
. None of these techniques allowed the identification of a convenient molecular marker for entomopathogenic strains, although one specific primer amplified only DNA from almost all mosquitocidal strains.
Disruption of
TRI101
, the Gene Encoding Trichothecene 3-
O
-Acetyltransferase, from
Fusarium sporotrichioides
Authors: McCormick Susan P. , Alexander Nancy J. , Trapp Susan E. , Hohn Thomas M.
Published: December 2019

We screened a
Fusarium sporotrichioides
NRRL 3299 cDNA expression library in a toxin-sensitive
Saccharomyces cerevisiae
strain lacking a functional
PDR5
gene. Fourteen yeast transformants were identified as resistant to the trichothecene 4,15-diacetoxyscirpenol, and each carried a cDNA encoding the trichothecene 3-
O
-acetyltransferase that is the
F. sporotrichioides
homolog of the
Fusarium graminearum TRI101
gene. Mutants of
F. sporotrichioides
NRRL 3299 produced by disruption of
TRI101
were altered in their abilities to synthesize T-2 toxin and accumulated isotrichodermol and small amounts of 3,15-didecalonectrin and 3-decalonectrin, trichothecenes that are not observed in cultures of the parent strain. Our results indicate that TRI101 converts isotrichodermol to isotrichodermin and is required for the biosynthesis of T-2 toxin.
Osmoprotection ofEscherichia coliby Peptone Is Mediated by the Uptake and Accumulation of Free Proline but Not of Proline-Containing Peptides
Authors: Amezaga Maria-Rosario , Booth Ian R.
Published: December 2019

The effect of meat peptone type I (Sigma) on the growth ofEscherichia colicells under hyperosmotic stress has been investigated. Peptone is a complex mixture of peptides with a small content of free amino acids, which resembles nutrients found in natural environments. Our data showed that peptone enhances the growth ofE. colicells in high-osmolarity medium to levels higher than those achieved with the main compatible solute in bacteria, glycine betaine. The mechanism of osmoprotection by peptone comprises the uptake and accumulation of the compatible solute, proline. The main role of the peptides contained in peptone is the provision of nutrients rather than the intracellular accumulation of osmolytes. In contrast toListeria monocytogenes(M. R. Amezaga, I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth, Microbiology 141:41–49, 1995),E. colidoes not accumulate exogenous peptides for osmoprotection and peptides containing proline do not lead to the accumulation of proline as a compatible solute. In late-logarithmic-phase cultures ofE. coligrowing at high osmolarity plus peptone, proline becomes the limiting factor for growth, and the intracellular pools of proline are not maintained. This is a consequence of the low concentration of free proline in peptone, the catabolism of proline byE. coli, and the inability ofE. colito utilize proline-containing peptides as a source of compatible solutes. Our data highlight the role that natural components in food such as peptides play in undermining food preservation regimes, such as high osmolarity, and also that the specific mechanisms of osmoprotection by these compounds differ according to the organism.
Effects of Pulsed Electric Fields on Inactivation Kinetics of
Listeria innocua
Authors: Wouters Patrick C. , Dutreux Nicole , Smelt Jan P. P. M. , Lelieveld Huub L. M.
Published: December 2019

The effects of pulsed electric field (PEF) treatment and processing factors on the inactivation kinetics of
Listeria innocua
NCTC 11289 were investigated by using a pilot plant PEF unit with a flow rate of 200 liters/h. The electric field strength, pulse length, number of pulses, and inlet temperature were the most significant process factors influencing the inactivation kinetics. Product factors (pH and conductivity) also influenced the inactivation kinetics. In phosphate buffer at pH 4.0 and 0.5 S/m at 40°C, a 3.0-V/μm PEF treatment at an inlet temperature of 40°C resulted in ≥6.3 log inactivation of strain NCTC 11289 at 49.5°C. A synergistic effect between temperature and PEF inactivation was also observed. The inactivation obtained with PEF was compared to the inactivation obtained with heat. We found that heat inactivation was less effective than PEF inactivation under similar time and temperature conditions.
L. innocua
cells which were incubated for a prolonged time in the stationary phase were more resistant to the PEF treatment, indicating that the physiological state of the microorganism plays a role in inactivation by PEF. Sublethal injury of cells was observed after PEF treatment, and the injury was more severe when the level of treatment was increased. Overall, our results indicate that it may be possible to use PEF in future applications in order to produce safe products.
Molecular Analysis of the Microbial Diversity Present in the Colonic Wall, Colonic Lumen, and Cecal Lumen of a Pig
Authors: Pryde Susan E. , Richardson Anthony J. , Stewart Colin S. , Flint Harry J.
Published: December 2019

Random clones of 16S ribosomal DNA gene sequences were isolated after PCR amplification with eubacterial primers from total genomic DNA recovered from samples of the colonic lumen, colonic wall, and cecal lumen from a pig. Sequences were also obtained for cultures isolated anaerobically from the same colonic-wall sample. Phylogenetic analysis showed that many sequences were related to those of
Lactobacillus
or
Streptococcus
spp. or fell into clusters IX, XIVa, and XI of gram-positive bacteria. In addition, 59% of randomly cloned sequences showed less than 95% similarity to database entries or sequences from cultivated organisms. Cultivation bias is also suggested by the fact that the majority of isolates (54%) recovered from the colon wall by culturing were related to
Lactobacillus
and
Streptococcus
, whereas this group accounted for only one-third of the sequence variation for the same sample from random cloning. The remaining cultured isolates were mainly
Selenomonas
related. A higher proportion of
Lactobacillus reuteri
-related sequences than of
Lactobacillus acidophilus
- and
Lactobacillus amylovorus
-related sequences were present in the colonic-wall sample. Since the majority of bacterial ribosomal sequences recovered from the colon wall are less than 95% related to known organisms, the roles of many of the predominant wall-associated bacteria remain to be defined.
Linking Toluene Degradation with Specific Microbial Populations in Soil
Authors: Hanson Jessica R. , Macalady Jennifer L. , Harris David , Scow Kate M.
Published: December 2019

Phospholipid fatty acid (PLFA) analysis of a soil microbial community was coupled with
13
C isotope tracer analysis to measure the community’s response to addition of 35 μg of [
13
C]toluene ml of soil solution
−1
. After 119 h of incubation with toluene, 96% of the incorporated
13
C was detected in only 16 of the total 59 PLFAs (27%) extracted from the soil. Of the total
13
C-enriched PLFAs, 85% were identical to the PLFAs contained in a toluene-metabolizing bacterium isolated from the same soil. In contrast, the majority of the soil PLFAs (91%) became labeled when the same soil was incubated with [
13
C]glucose. Our study showed that coupling
13
C tracer analysis with PLFA analysis is an effective technique for distinguishing a specific microbial population involved in metabolism of a labeled substrate in complex environments such as soil.
Partitioning Effects during Terminal Carbon and Electron Flow in Sediments of a Low-Salinity Meltwater Pond near Bratina Island, McMurdo Ice Shelf, Antarctica
Authors: Mountfort Douglas O. , Kaspar Heinrich F. , Downes Malcolm , Asher Rodney A.
Published: December 2019

A study of anaerobic sediments below cyanobacterial mats of a low-salinity meltwater pond called Orange Pond on the McMurdo Ice Shelf at temperatures simulating those in the summer season (<5°C) revealed that both sulfate reduction and methane production were important terminal anaerobic processes. Addition of [2-14C]acetate to sediment samples resulted in the passage of label mainly to CO2. Acetate addition (0 to 27 mM) had little effect on methanogenesis (a 1.1-fold increase), and while the rate of acetate dissimilation was greater than the rate of methane production (6.4 nmol cm−3h−1compared to 2.5 to 6 nmol cm−3h−1), the portion of methane production attributed to acetate cleavage was <2%. Substantial increases in the methane production rate were observed with H2(2.4-fold), and H2uptake was totally accounted for by methane production under physiological conditions. Formate also stimulated methane production (twofold), presumably through H2release mediated through hydrogen lyase. Addition of sulfate up to 50-fold the natural levels in the sediment (interstitial concentration, ∼0.3 mM) did not substantially inhibit methanogenesis, but the process was inhibited by 50-fold chloride (36 mM). No net rate of methane oxidation was observed when sediments were incubated anaerobically, and denitrification rates were substantially lower than rates for sulfate reduction and methanogenesis. The results indicate that carbon flow from acetate is coupled mainly to sulfate reduction and that methane is largely generated from H2and CO2where chloride, but not sulfate, has a modulating role. Rates of methanogenesis at in situ temperatures were four- to fivefold less than maximal rates found at 20°C.
Visualization and Enumeration of Marine Planktonic Archaea and Bacteria by Using Polyribonucleotide Probes and Fluorescent In Situ Hybridization
Authors: DeLong Edward F. , Taylor Lance Trent , Marsh Terence L. , Preston Christina M.
Published: December 2019

Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4′,6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50–56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 10
5
/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or “ghosts,” as was suggested in a previous report.
Molecular Characterization of a Toluene-Degrading Methanogenic Consortium
Authors: Ficker Monica , Krastel Kirsten , Orlicky Stephen , Edwards Elizabeth
Published: December 2019

A toluene-degrading methanogenic consortium enriched from creosote-contaminated aquifer material was maintained on toluene as the sole carbon and energy source for 10 years. The species in the consortium were characterized by using a molecular approach. Total genomic DNA was isolated, and 16S rRNA genes were amplified by using PCR performed with kingdom-specific primers that were specific for 16S rRNA genes from either members of the kingdomBacteriaor members of the kingdomArchaea. A total of 90 eubacterial clones and 75 archaeal clones were grouped by performing a restriction fragment length polymorphism (RFLP) analysis. Six eubacterial sequences and two archaeal sequences were found in the greatest abundance (in six or more clones) based on the RFLP analysis. The relative abundance of each putative species was estimated by using fluorescent in situ hybridization (FISH), and the presence of putative species was determined qualitatively by performing slot blot hybridization with consortium DNA. Both archaeal species and two of the six eubacterial species were detected in the DNA and FISH hybridization experiments. A phylogenetic analysis of these four dominant organisms suggested that the two archaeal species are related to the generaMethanosaetaandMethanospirillum. One of the eubacterial species is related to the genusDesulfotomaculum, while the other is not related to any previously described genus. By elimination, we propose that the last organism probably initiates the attack on toluene.
Seasonal Enumeration of Fecal Coliform Bacteria from the Feces of Ring-Billed Gulls (
Larus delawarensis
) and Canada Geese (
Branta canadensis
)
Authors: Alderisio K. A. , DeLuca N.
Published: December 2019

Water suppliers have often implicated roosting birds for fecal contamination of their surface waters. Geese and gulls have been the primary targets of this blame although literature documenting the fecal coliform content of these birds is quite limited. To determine the actual fecal coliform concentrations of these birds, fecal samples from 249 ring-billed gulls and 236 Canada geese in Westchester County, N.Y., were analyzed over a 2-year period. Results indicate that gull feces contain a greater average concentration of fecal coliform bacteria per gram (3.68 × 10
8
) than do goose feces (1.53 × 10
4
); however, average fecal sample weights of the geese were more than 15 times higher than those of the gulls.