bluuebunny/crossref_metadata_2025 huggingface dataset:
Monitoring Methanotrophic Bacteria in Hybrid Anaerobic-Aerobic Reactors with PCR and a Catabolic Gene Probe
Authors: Miguez Carlos B. , Shen Chun F. , Bourque Denis , Guiot Serge R. , Groleau Denis
Published: December 2019

We attempted to mimic in small upflow anaerobic sludge bed (UASB) bioreactors the metabolic association found in nature between methanogens and methanotrophs. UASB bioreactors were inoculated with pure cultures of methanotrophs, and the bioreactors were operated by using continuous low-level oxygenation in order to favor growth and/or survival of methanotrophs. Unlike the reactors in other similar studies, the hybrid anaerobic-aerobic bioreactors which we used were operated synchronously, not sequentially. Here, emphasis was placed on monitoring various methanotrophic populations by using classical methods and also a PCR amplification assay based on themmoXgene fragment of the soluble methane monooxygenase (sMMO). The following results were obtained: (i) under the conditions used,Methylosinus sporiumappeared to survive better thanMethylosinus trichosporium; (ii) the PCR method which we used could detect as few as about 2,000 sMMO gene-containing methanotrophs per g (wet weight) of granular sludge; (iii) inoculation of the bioreactors with pure cultures of methanotrophs contributed greatly to increases in the sMMO-containing population (although the sMMO-containing population decreased gradually with time, at the end of an experiment it was always at least 2 logs larger than the initial population before inoculation); (iv) in general, there was a good correlation between populations with the sMMO gene and populations that exhibited sMMO activity; and (v) inoculation with sMMO-positive cultures helped increase significantly the proportion of sMMO-positive methanotrophs in reactors, even after several weeks of operation under various regimes. At some point, anaerobic-aerobic bioreactors like those described here might be used for biodegradation of various chlorinated pollutants.
Anaerobic versus Aerobic Degradation of Dimethyl Sulfide and Methanethiol in Anoxic Freshwater Sediments
Authors: Lomans Bart P. , Op den Camp Huub J. M. , Pol Arjan , Vogels Godfried D.
Published: December 2019

Degradation of dimethyl sulfide and methanethiol in slurries prepared from sediments of minerotrophic peatland ditches were studied under various conditions. Maximal aerobic dimethyl sulfide-degrading capacities (4.95 nmol per ml of sediment slurry · h−1), measured in bottles shaken under an air atmosphere, were 10-fold higher than the maximal anaerobic degrading capacities determined from bottles shaken under N2or H2atmosphere (0.37 and 0.32 nmol per ml of sediment slurry · h−1, respectively). Incubations under experimental conditions which mimic the in situ conditions (i.e., not shaken and with an air headspace), however, revealed that aerobic degradation of dimethyl sulfide and methanethiol in freshwater sediments is low due to oxygen limitation. Inhibition studies with bromoethanesulfonic acid and sodium tungstate demonstrated that the degradation of dimethyl sulfide and methanethiol in these incubations originated mainly from methanogenic activity. Prolonged incubation under a H2atmosphere resulted in lower dimethyl sulfide degradation rates. Kinetic analysis of the data resulted in apparentKmvalues (6 to 8 μM) for aerobic dimethyl sulfide degradation which are comparable to those reported forThiobacillusspp.,Hyphomicrobiumspp., and other methylotrophs. ApparentKmvalues determined for anaerobic degradation of dimethyl sulfide (3 to 8 μM) were of the same order of magnitude. The low apparentKmvalues obtained explain the low dimethyl sulfide and methanethiol concentrations in freshwater sediments that we reported previously. Our observations point to methanogenesis as the major mechanism of dimethyl sulfide and methanethiol consumption in freshwater sediments.
Influence of Light Intensity on Methanotrophic Bacterial Activity in Petit Saut Reservoir, French Guiana
Authors: Dumestre J. F. , Guézennec J. , Galy-Lacaux C. , Delmas R. , Richard S. , Labroue L.
Published: December 2019

One year after impoundment in January 1994, methanotrophic bacteria in Petit Saut Reservoir (French Guiana) were active at the oxic-anoxic interface. This activity was revealed by the sudden extinction of diffusive methane emission (600 metric tons of CH
4
· day
−1
for the whole lake surface area, i.e., 360 km
2
). Lifting of inhibition was suspected. After reviewing the potential inhibitors of this physiological guild (O
2
, NH
4
+
, sulfides) and considering the similarities with nitrifiers, we suggest that sunlight influenced the methanotrophic bacteria. On the basis of phospholipid analysis, only a type II methanotrophic community was identified in the lake. Both growth and methanotrophic activity of an enriched culture, obtained in the laboratory, were largely inhibited by illumination over 150 microeinsteins · m
−2
· s
−1
. These results were confirmed on a pure culture of
Methylosinus trichosporium
OB3B. In situ conditions showed that water transparency was quite stable in 1994 and 1995 and that the oxycline moved steadily deeper until January 1995. Considering the mean illumination profile during this period, we showed that removal of methanotrophic growth inhibition could only occur below a 2-m depth. The oxycline reached this level in October 1994, allowing methanotrophic bacteria to develop and to consume the entire methane emission 4 months later.
Production of Medium-Chain-Length Poly(3-Hydroxyalkanoates) from Gluconate by RecombinantEscherichia coli
Authors: Klinke Stefan , Ren Qun , Witholt Bernard , Kessler Birgit
Published: December 2019

It was shown recently that recombinantEscherichia coli, defective in the β-oxidation cycle and harboring a medium-chain-length (MCL) poly(3-hydroxyalkanoate) (PHA) polymerase-encoding gene ofPseudomonas, is able to produce MCL PHA from fatty acids but not from sugars or gluconate (S. Langenbach, B. H. A. Rehm, and A. Steinbüchel, FEMS Microbiol. Lett. 150:303–309, 1997; Q. Ren, Ph.D. thesis, ETH Zürich, Zürich, Switzerland, 1997). In this study, we report the formation of MCL PHA from gluconate by recombinantE. coli. By introduction of genes coding for an MCL PHA polymerase and the cytosolic thioesterase I (′thioesterase I) intoE. coliJMU193, we were able to engineer a pathway for the synthesis of MCL PHA from gluconate. We used two expression systems, i.e., thebadpromoter andalkpromoter, for the ′thioesterase I- and PHA polymerase-encoding genes, respectively, which enabled us to modulate their expression independently over a range of inducer concentrations, which resulted in a maximum MCL PHA accumulation of 2.3% of cell dry weight from gluconate. We found that the amount of PHA and the ′thioesterase I activity are directly correlated. Moreover, the polymer accumulated in the recombinantE. coliconsisted mainly of 3-hydroxyoctanoate monomers. On the basis of our data, we propose an MCL PHA biosynthesis pathway scheme for recombinantE. coliJMU193, harboring PHA polymerase and ′thioesterase I, when grown on gluconate, which involves both de novo fatty acid synthesis and β-oxidation.
Isolation and Characterization of a
Mycobacterium
Species Capable of Degrading Three- and Four-Ring Aromatic and Aliphatic Hydrocarbons
Authors: Churchill Sharon A. , Harper Jennifer P. , Churchill Perry F.
Published: December 2019

Mycobacterium
sp. strain CH1 was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated freshwater sediments and identified by analysis of 16S rDNA sequences. Strain CH1 was capable of mineralizing three- and four-ring PAHs including phenanthrene, pyrene, and fluoranthene. In addition, strain CH1 could utilize phenanthrene or pyrene as a sole carbon and energy source. A lag phase of at least 3 days was observed during pyrene mineralization. This lag phase decreased to less than 1 day when strain CH1 was grown in the presence of phenanthrene or fluoranthene. Strain CH1 also was capable of using a wide range of alkanes as sole carbon and energy sources. No DNA hybridization was detected with the
nahAc
gene probe, indicating that enzymes involved in PAH metabolism are not related to the well-characterized naphthalene dioxygenase gene. DNA hybridization was not detected when the
alkB
gene from
Pseudomonas oleovorans
was used under high-stringency conditions. However, there was slight but detectable hybridization under low-stringency conditions. This suggests a distant relationship between genes involved in alkane oxidation.
Production and Distribution of Endoglucanase, Cellobiohydrolase, and β-Glucosidase Components of the Cellulolytic System of
Volvariella volvacea
, the Edible Straw Mushroom
Authors: Cai Yi Jin , Chapman Sandra J. , Buswell John A. , Chang Shu-ting
Published: December 2019

The edible straw mushroom,
Volvariella volvacea
, produces a multicomponent enzyme system consisting of endo-1,4-β-glucanase, cellobiohydrolase, and β-glucosidase for the conversion of cellulose to glucose. The highest levels of endoglucanase and cellobiohydrolase were recorded in cultures containing microcrystalline cellulose (Avicel) or filter paper, while lower but detectable levels of activity were also produced on carboxymethyl cellulose, cotton wool, xylitol, or salicin. Biochemical analyses of different culture fractions in cultures exhibiting peak enzyme production revealed that most of the endoglucase was present either in the culture filtrate (45.8% of the total) or associated with the insoluble pellet fraction remaining after centrifugation of homogenized mycelia (32.6%). Cellobiohydrolase exhibited a similar distribution pattern, with 58.9% of the total enzyme present in culture filtrates and 31.0% associated with the pellet fraction. Conversely, most β-glucosidase activity (63.9% of the total) was present in extracts of fungal mycelia whereas only 9.4% was detected in culture filtrates. The endoglucanase and β-glucosidase distribution patterns were confirmed by confocal laser scanning microscopy combined with immunolabelling. Endoglucanase was shown to be largely cell wall associated or located extracellularly, with the highest concentrations being present in a region 1 to 2 μm wide immediately adjacent to the outer surface of (and possibly including) the hyphal wall and extending 60 to 70 μm from the hyphal tip. Immunofluorescence patterns indicated little if any intracellular endoglucanase. Most β-glucosidase was located intracellularly in the apical area extending 60 to 70 μm below the hyphal tip, although enzyme was also evident in the extracellular region extending approximately 15 μm all around the hyphal tip and trailing back along the length of the hypha. The regions of the hypha located some distance from the apical region appeared to be devoid of intracellular β-glucosidase, and the enzyme appears to be associated almost exclusively with, or located on the outside surface of, the hyphal wall.
Leaching of Pyrite by Acidophilic Heterotrophic Iron-Oxidizing Bacteria in Pure and Mixed Cultures
Authors: Bacelar-Nicolau Paula , Johnson D. Barrie
Published: December 2019

Seven strains of heterotrophic iron-oxidizing acidophilic bacteria were examined to determine their abilities to promote oxidative dissolution of pyrite (FeS
2
) when they were grown in pure cultures and in mixed cultures with sulfur-oxidizing
Thiobacillus
spp. Only one of the isolates (strain T-24) oxidized pyrite when it was grown in pyrite-basal salts medium. However, when pyrite-containing cultures were supplemented with 0.02% (wt/vol) yeast extract, most of the isolates oxidized pyrite, and one (strain T-24) promoted rates of mineral dissolution similar to the rates observed with the iron-oxidizing autotroph
Thiobacillus ferrooxidans
. Pyrite oxidation by another isolate (strain T-21) occurred in cultures containing between 0.005 and 0.05% (wt/vol) yeast extract but was completely inhibited in cultures containing 0.5% yeast extract. Ferrous iron was also needed for mineral dissolution by the iron-oxidizing heterotrophs, indicating that these organisms oxidize pyrite via the “indirect” mechanism. Mixed cultures of three isolates (strains T-21, T-23, and T-24) and the sulfur-oxidizing autotroph
Thiobacillus thiooxidans
promoted pyrite dissolution; since neither strains T-21 and T-23 nor
T. thiooxidans
could oxidize this mineral in yeast extract-free media, this was a novel example of bacterial synergism. Mixed cultures of strains T-21 and T-23 and the sulfur-oxidizing mixotroph
Thiobacillus acidophilus
also oxidized pyrite but to a lesser extent than did mixed cultures containing
T. thiooxidans
. Pyrite leaching by strain T-23 grown in an organic compound-rich medium and incubated either shaken or unshaken was also assessed. The potential environmental significance of iron-oxidizing heterotrophs in accelerating pyrite oxidation is discussed.
Characteristics of Garden Dormice That Contribute to Their Capacity as Reservoirs for Lyme Disease Spirochetes
Authors: Matuschka Franz-Rainer , Allgöwer Rainer , Spielman Andrew , Richter Dania
Published: December 2019

To describe the contribution of garden dormice to the epizootiology of Lyme disease, we compared their reservoir capacity for these pathogens to that of other sympatric hosts. Garden dormice are trapped most abundantly during early spring and again during midsummer, when their offspring forage. They are closely associated with moist forests. Garden dormice serve as hosts to nymphal ticks far more frequently than do other small mammals. Spirochetal infection is most prevalent in dormice, and many more larval ticks acquire infection in the course of feeding on these than on other rodents in the study site. Mature dormice appear to contribute more infections to the vector population than juveniles do. Replete larval ticks generally detach while their dormouse hosts remain within their nests. The population of garden dormice contributes five- to sevenfold more infections to the vector population than the mouse population does. Their competence, nymphal feeding density, and preference for a tick-permissive habitat combine to favor garden dormice over other putative reservoir hosts of Lyme disease spirochetes.
Characterization of Two Novel Propachlor Degradation Pathways in Two Species of Soil Bacteria
Authors: Martin Margarita , Mengs Gerardo , Allende Jose Luis , Fernandez Javier , Alonso Ramon , Ferrer Estrella
Published: December 2019

Propachlor (2-chloro-
N
-isopropylacetanilide) is an acetamide herbicide used in preemergence. In this study, we isolated and characterized a soil bacterium,
Acinetobacter
strain BEM2, that was able to utilize this herbicide as the sole and limiting carbon source. Identification of the intermediates of propachlor degradation by this strain and characterization of new metabolites in the degradation of propachlor by a previously reported strain of
Pseudomonas
(PEM1) support two different propachlor degradation pathways. Washed-cell suspensions of strain PEM1 with propachlor accumulated
N
-isopropylacetanilide, acetanilide, acetamide, and catechol.
Pseudomonas
strain PEM1 grew on propachlor with a generation time of 3.4 h and a

K
s

of 0.17 ± 0.04 mM.
Acinetobacter
strain BEM2 grew on propachlor with a generation time of 3.1 h and a

K
s

of 0.3 ± 0.07 mM. Incubations with strain BEM2 resulted in accumulation of
N
-isopropylacetanilide,
N
-isopropylaniline, isopropylamine, and catechol. Both degradative pathways were inducible, and the principal product of the carbon atoms in the propachlor ring was carbon dioxide. These results and biodegradation experiments with the identified metabolites indicate that metabolism of propachlor by
Pseudomonas
sp. strain PEM1 proceeds through a different pathway from metabolism by
Acinetobacter
sp. strain BEM2.
A Novel Chromogenic Ester Agar Medium for Detection of Salmonellae
Authors: Cooke Venitia M. , Miles R. J. , Price R. G. , Richardson A. C.
Published: December 2019

A novel agar medium, chromogenic
Salmonella
esterase (CSE) agar, for the differentiation of salmonellae is described. The agar contains peptones and nutrient extracts together with the following (grams per liter unless otherwise specified): 4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)-vinyl]-quinolinium-1-(propan-3-yl carboxylic acid) bromide (SLPA-octanoate; bromide form), 0.3223; lactose, 14.65; trisodium citrate dihydrate, 0.5; Tween 20, 3.0; ethyl 4-dimethylaminobenzoate, 0.035% (wt/vol), novobiocin, 70 mg liter
−1
. The key component of the medium is SLPA-octanoate, a newly synthesized ester formed from a C
8
fatty acid and a phenolic chromophore. In CSE agar, the ester is hydrolyzed by
Salmonella
spp. to yield a brightly colored phenol which remains tightly bound within colonies. After 24 h of incubation at 37 or 42°C, colonies of typical
Salmonella
spp. were burgundy colored on a transparent yellow background, whereas non-
Salmonella
spp. were white, cream, yellow or transparent. CSE agar was evaluated by using a panel of strains including a high proportion of
Salmonella
and non-
Salmonella
strains giving atypical reactions on other differential agars. The sensitivity (93.1%) of CSE agar for non-
typhi
salmonellae compared favorably with those of Rambach (82.8%), xylose-lysine-deoxycholate (XLD; 91.4%), Hektoen-enteric (89.7%), and SM ID (91.4%) agars. The specificity (93.9%) was also comparable to those of other
Salmonella
media (SM ID agar, 95.9%; Rambach agar, 91.8%; XLD agar, 91.8%; Hektoen-enteric agar, 87.8%). Strains of
Citrobacter freundii
and
Proteus
spp. giving false-positive reactions with other media gave a negative color reaction on CSE agar. CSE agar enabled the detection of >30
Salmonella
serotypes, including
agona
,
anatum
,
enteritidis
,
hadar
,
heidelberg
,
infantis
,
montevideo
,
thompson
,
typhimurium
, and
virchow
, which accounted for 91.8% of the salmonella isolates recorded by the Public Health Laboratory Service (Colindale, London, England) for 1997.
Isolation of
Deinococcus
Species from Commercial Oyster Extract
Authors: Chan W. F. , O’Toole D. K.
Published: December 2019

Deinococci with radiation resistance greater than that of
Deinococcus radiophilus
(ATCC 27603) were isolated from three commercial oyster extracts stored at 4, 20, and 30°C. During storage the number of other bacteria declined and deinococci became the predominant group in the microflora, particularly at 20°C, although at 30°C the number of deinococci as well rapidly declined. The results suggest that the natural habitat of deinococci is an aerobic environment containing a slightly elevated saline content, soluble protein, and low sugar levels.
Detection of Shiga-Like Toxin (

stx
1

and

stx
2

), Intimin (
eaeA
), and Enterohemorrhagic
Escherichia coli
(EHEC) Hemolysin (EHEC
hlyA
) Genes in Animal Feces by Multiplex PCR
Authors: Fagan Peter K. , Hornitzky Michael A. , Bettelheim Karl A. , Djordjevic Steven P.
Published: December 2019

A multiplex PCR was developed for the rapid detection of genes encoding Shiga toxins 1 and 2 (

stx
1

and

stx
2

), intimin (
eaeA
), and enterohemolysin A (
hlyA
) in 444 fecal samples derived from healthy and clinically affected cattle, sheep, pigs, and goats. The method involved non-solvent-based extraction of nucleic acid from an aliquot of an overnight culture of feces in EC (modified) broth. The detection limit of the assay for both fecal samples and pure cultures was between 18 and 37 genome equivalents.

stx
1

and
hlyA
were the most commonly encountered virulence factors.
A Comparative Study of Methods To Validate Formaldehyde Decontamination of Biological Safety Cabinets
Authors: Munro Kerry , Lanser Janice , Flower Robert
Published: December 2019

Methods of validation of formaldehyde decontamination of biological safety cabinets were compared. Decontamination of metal strips inoculated with
Mycobacterium bovis
, poliovirus, or
Bacillus
spp. spores was compared with the results obtained with three biological indicators. Conditions for successful decontamination, particularly relative humidity, were defined. The Attest 1291 biological indicator was the only biological indicator which was an aid in the detection of gross decontamination failure.
High-Affinity Methane Oxidation by a Soil Enrichment Culture Containing a Type II Methanotroph
Authors: Dunfield Peter F. , Liesack Werner , Henckel Thilo , Knowles Roger , Conrad Ralf
Published: December 2019

Methanotrophic bacteria in an organic soil were enriched on gaseous mixing ratios of <275 parts per million of volume (ppmv) of methane (CH
4
). After 4 years of growth and periodic dilution (>10
20
times the initial soil inoculum), a mixed culture was obtained which displayed an apparent half-saturation constant [

K
m(app)

] for CH
4
of 56 to 186 nM (40 to 132 ppmv). This value was the same as that measured in the soil itself and about 1 order of magnitude lower than reported values for pure cultures of methane oxidizers. However, the

K
m(app)

increased when the culture was transferred to higher mixing ratios of CH
4
(1,000 ppmv, or 1%). Denaturing gradient gel electrophoresis of the enrichment grown on <275 ppmv of CH
4
revealed a single gene product of
pmoA
, which codes for a subunit of particulate methane monooxygenase. This suggested that only one methanotroph species was present. This organism was isolated from a sample of the enrichment culture grown on 1% CH
4
and phylogenetically positioned based on its 16S rRNA,
pmoA
, and
mxaF
gene sequences as a type II strain of the
Methylocystis/Methylosinus
group. A coculture of this strain with a
Variovorax
sp., when grown on <275 ppmv of CH
4
, had a

K
m(app)

(129 to 188 nM) similar to that of the initial enrichment culture. The data suggest that the affinity of methanotrophic bacteria for CH
4
varies with growth conditions and that the oxidation of atmospheric CH
4
observed in this soil is carried out by type II methanotrophic bacteria which are similar to characterized species.
Polynucleotide Probes That Target a Hypervariable Region of 16S rRNA Genes To Identify Bacterial Isolates Corresponding to Bands of Community Fingerprints
Authors: Heuer Holger , Hartung Kathrin , Wieland Gabriele , Kramer Ina , Smalla Kornelia
Published: December 2019

Temperature gradient gel electrophoresis (TGGE) is well suited for fingerprinting bacterial communities by separating PCR-amplified fragments of 16S rRNA genes (16S ribosomal DNA [rDNA]). A strategy was developed and was generally applicable for linking 16S rDNA from community fingerprints to pure culture isolates from the same habitat. For this, digoxigenin-labeled polynucleotide probes were generated by PCR, using bands excised from TGGE community fingerprints as a template, and applied in hybridizations with dot blotted 16S rDNA amplified from bacterial isolates. Within 16S rDNA, the hypervariable V6 region, corresponding to positions 984 to 1047 (
Escherichia coli
16S rDNA sequence), which is a subset of the region used for TGGE (positions 968 to 1401), best met the criteria of high phylogenetic variability, required for sufficient probe specificity, and closely flanking conserved priming sites for amplification. Removal of flanking conserved bases was necessary to enable the differentiation of closely related species. This was achieved by 5′ exonuclease digestion, terminated by phosphorothioate bonds which were synthesized into the primers. The remaining complementary strand was removed by single-strand-specific digestion. Standard hybridization with truncated probes allowed differentiation of bacteria which differed by only two bases within the probe target site and 1.2% within the complete 16S rDNA. However, a truncated probe, derived from an excised TGGE band of a rhizosphere community, hybridized with three phylogenetically related isolates with identical V6 sequences. Only one of the isolates comigrated with the excised band in TGGE, which was shown to be due to identical sequences, demonstrating the utility of a combined TGGE and V6 probe approach.
Sensitivity, Specificity, and Predictive Values of Three
Salmonella
Rapid Detection Kits Using Fresh and Frozen Poultry Environmental Samples versus Those of Standard Plating
Authors: Peplow Melissa O. , Correa-Prisant Maria , Stebbins Martha E. , Jones Frank , Davies Peter
Published: December 2019

To reduce human exposure to
Salmonella
spp. in poultry products, broiler chicken flocks have been tested by culture methods. Since the standard techniques may take 3 to 5 days, rapid detection methods have been developed. In this study we tested the performance of three rapid tests originally developed for food samples by using environmental samples obtained from poultry houses. These rapid tests were Reveal, an enzyme-linked immunosorbent assay from Neogen Corp.; BIND, a bacterial ice nucleation detection method from Idetek Corp.; and a filter monitor method from Future Medical Technologies, Inc. For the standard culture, brilliant green with novabiocin and xylose–lysine–tergitol-4 agar were used for presumptive identification, and identities were confirmed by using poly-O antisera. Environmental samples were collected from farms belonging to an integrated poultry company prior to chick placement and 1 week before slaughter. Sensitivities, specificities, and predictive values with 95% confidence intervals were calculated. Statistical differences were determined by using McNemar’s chi square test. The sensitivities of the different tests were not stable, varying widely between sample times, and were affected by freezing of the samples. All of the rapid tests had low sensitivities, which led to many false-negative results. All tests were able to detect
Salmonella
spp. at a concentration of 10 CFU/ml in at least one of four trials. The BIND and Reveal tests were simple to use with multiple samples and reduced laboratory time by up to 1 day. Based on our results, we do not recommend that any of these rapid tests, in their present state of development, be utilized with environmental samples collected with drag swabs.
Isolation from Estuarine Sediments of a
Desulfovibrio
Strain Which Can Grow on Lactate Coupled to the Reductive Dehalogenation of 2,4,6-Tribromophenol
Authors: Boyle Alfred W. , Phelps Craig D. , Young L. Y.
Published: December 2019

Strain TBP-1, an anaerobic bacterium capable of reductively dehalogenating 2,4,6-tribromophenol to phenol, was isolated from estuarine sediments of the Arthur Kill in the New York/New Jersey harbor. It is a gram-negative, motile, vibrio-shaped, obligate anaerobe which grows on lactate, pyruvate, hydrogen, and fumarate when provided sulfate as an electron acceptor. The organism accumulates acetate when grown on lactate and sulfate, contains desulfoviridin, and will not grow in the absence of NaCl. It will not utilize acetate, succinate, propionate, or butyrate for growth via sulfate reduction. When supplied with lactate as an electron donor, strain TBP-1 will utilize sulfate, sulfite, sulfur, and thiosulfate for growth but not nitrate, fumarate, or acrylate. This organism debrominates 2-, 4-, 2,4-, 2,6-, and 2,4,6-bromophenol but not 3- or 2,3-bromophenol or monobrominated benzoates. It will not dehalogenate monochlorinated, fluorinated, or iodinated phenols or chlorinated benzoates. Together with its physiological characteristics, its 16S rRNA gene sequence places it in the genus
Desulfovibrio
. The average growth yield of strain TBP-1 grown on a defined medium supplemented with lactate and 2,4,6-bromophenol is 3.71 mg of protein/mmol of phenol produced, and the yield was 1.42 mg of protein/mmol of phenol produced when 4-bromophenol was the electron acceptor. Average growth yields (milligrams of protein per millimole of electrons utilized) for
Desulfovibrio
sp. strain TBP-1 grown with 2,4,6-bromophenol, 4-bromophenol, or sulfate are 0.62, 0.71, and 1.07, respectively. Growth did not occur when either lactate or 2,4,6-bromophenol was omitted from the growth medium. These results indicate that
Desulfovibrio
sp. strain TBP-1 is capable of growth via halorespiration.
Fusarium subglutinans
f. sp.
pini
Represents a Distinct Mating Population in the
Gibberella fujikuroi
Species Complex
Authors: Britz H. , Coutinho T. A. , Wingfield M. J. , Marasas W. F. O. , Gordon T. R. , Leslie J. F.
Published: December 2019

Fusarium
strains in the
Gibberella fujikuroi
species complex cause diseases on a variety of economically important plants. One of these diseases, pitch canker of
Pinus
spp., is caused by strains identified as
Fusarium subglutinans
f. sp.
pini
. Fertile crosses were detected between
F. subglutinans
f. sp.
pini
strains from South Africa, California, and Florida.
F. subglutinans
f. sp.
pini
strains were not cross-fertile with the standard tester strains of six of the seven other mating populations of
G. fujikuroi
. Sporadic perithecia with ascospores were obtained in two crosses with the mating population B tester strains. These perithecia were homothallic, and the ascospores derived from these perithecia were vegetatively compatible with the mating population B tester strain parent. We concluded that fertile
F. subglutinans
f. sp.
pini
isolates represent a new mating population (mating population H) of
G. fujikuroi
and that they belong to a unique biological species in a distinct taxon.
Dissimilatory Reduction of Fe(III) and Other Electron Acceptors by a
Thermus
Isolate
Authors: Kieft T. L. , Fredrickson J. K. , Onstott T. C. , Gorby Y. A. , Kostandarithes H. M. , Bailey T. J. , Kennedy D. W. , Li S. W. , Plymale A. E. , Spadoni C. M. , Gray M. S.
Published: December 2019

A thermophilic bacterium that can use O
2
, NO
3
−
, Fe(III), and S
0
as terminal electron acceptors for growth was isolated from groundwater sampled at a 3.2-km depth in a South African gold mine. This organism, designated SA-01, clustered most closely with members of the genus
Thermus
, as determined by 16S rRNA gene (rDNA) sequence analysis. The 16S rDNA sequence of SA-01 was >98% similar to that of
Thermus
strain NMX2 A.1, which was previously isolated by other investigators from a thermal spring in New Mexico. Strain NMX2 A.1 was also able to reduce Fe(III) and other electron acceptors. Neither SA-01 nor NMX2 A.1 grew fermentatively, i.e., addition of an external electron acceptor was required for anaerobic growth.
Thermus
strain SA-01 reduced soluble Fe(III) complexed with citrate or nitrilotriacetic acid (NTA); however, it could reduce only relatively small quantities (0.5 mM) of hydrous ferric oxide except when the humic acid analog 2,6-anthraquinone disulfonate was added as an electron shuttle, in which case 10 mM Fe(III) was reduced. Fe(III)-NTA was reduced quantitatively to Fe(II); reduction of Fe(III)-NTA was coupled to the oxidation of lactate and supported growth through three consecutive transfers. Suspensions of
Thermus
strain SA-01 cells also reduced Mn(IV), Co(III)-EDTA, Cr(VI), and U(VI). Mn(IV)-oxide was reduced in the presence of either lactate or H
2
. Both strains were also able to mineralize NTA to CO
2
and to couple its oxidation to Fe(III) reduction and growth. The optimum temperature for growth and Fe(III) reduction by
Thermus
strains SA-01 and NMX2 A.1 is approximately 65°C; their optimum pH is 6.5 to 7.0. This is the first report of a
Thermus
sp. being able to couple the oxidation of organic compounds to the reduction of Fe, Mn, or S.
Morphological and Compositional Changes in a Planktonic Bacterial Community in Response to Enhanced Protozoan Grazing
Authors: Jürgens Klaus , Pernthaler Jakob , Schalla Sven , Amann Rudolf
Published: December 2019

We analyzed changes in bacterioplankton morphology and composition during enhanced protozoan grazing by image analysis and fluorescent in situ hybridization with group-specific rRNA-targeted oligonucleotide probes. Enclosure experiments were conducted in a small, fishless freshwater pond which was dominated by the cladoceranDaphnia magna. The removal of metazooplankton enhanced protozoan grazing pressure and triggered a microbial succession from fast-growing small bacteria to larger grazing-resistant morphotypes. These were mainly different types of filamentous bacteria which correlated in biomass with the population development of heterotrophic nanoflagellates (HNF). Small bacterial rods and cocci, which showed increased proportion after removal ofDaphniaand doubling times of 6 to 11 h, belonged nearly exclusively to the beta subdivision of the classProteobacteriaand theCytophaga-Flavobacteriumcluster. The majority of this newly produced bacterial biomass was rapidly consumed by HNF. In contrast, the proportion of bacteria belonging to the gamma and alpha subdivisions of theProteobacteriaincreased throughout the experiment. The alpha subdivision consisted mainly of rods that were 3 to 6 μm in length, which probably exceeded the size range of bacteria edible by protozoa. Initially, these organisms accounted for less than 1% of total bacteria, but after 72 h they became the predominant group of the bacterial assemblage. Other types of grazing-resistant, filamentous bacteria were also found within the beta subdivision ofProteobacteriaand theCytophaga-Flavobacteriumcluster. We conclude that the predation regimen is a major structuring force for the bacterial community composition in this system. Protozoan grazing resulted in shifts of the morphological as well as the taxonomic composition of the bacterial assemblage. Grazing-resistant filamentous bacteria can develop within different phylogenetic groups of bacteria, and formerly underepresented taxa might become a dominant group when protozoan predation is the major selective pressure.
Kinetics of Sulfate and Hydrogen Uptake by the Thermophilic Sulfate-Reducing Bacteria
Thermodesulfobacterium
sp. Strain JSP and
Thermodesulfovibrio
sp. Strain R1Ha3
Authors: Sonne-Hansen Jacob , Westermann Peter , Ahring Birgitte K.
Published: December 2019

Half-saturation constants (

K
m

), maximum uptake rates (
V
max
), and threshold concentrations for sulfate and hydrogen were determined for two thermophilic sulfate-reducing bacteria (SRB) in an incubation system without headspace.

K
m

values determined for the thermophilic SRB were similar to the constants described for mesophilic SRB isolated from environments with low sulfate concentrations.
In Situ Analysis of Phototrophic Sulfur Bacteria in the Chemocline of Meromictic Lake Cadagno (Switzerland)
Authors: Tonolla Mauro , Demarta Antonella , Peduzzi Raffaele , Hahn Dittmar
Published: December 2019

Comparative sequence analysis of a 16S rRNA gene clone library from the chemocline of the meromictic Lake Cadagno (Switzerland) revealed the presence of a diverse number of phototrophic sulfur bacteria. Sequences resembled those of rRNA of type strains
Chromatium okenii
DSM169 and
Amoebobacter purpureus
DSM4197, as well as those of four bacteria forming a tight cluster with
A. purpureus
DSM4197 and
Lamprocystis roseopersicina
DSM229. In situ hybridization with fluorescent (Cy3 labeled) oligonucleotide probes indicated that all large-celled phototrophic sulfur bacteria in the chemocline of Lake Cadagno were represented by
C. okenii
DSM169, while small-celled phototrophic sulfur bacteria consisted of four major populations with different distribution profiles in the chemocline indicating different ecophysiological adaptations.
Steady-State Nitrogen Isotope Effects of N
2
and N
2
O Production in
Paracoccus denitrificans
Authors: Barford Carol C. , Montoya Joseph P. , Altabet Mark A. , Mitchell Ralph
Published: December 2019

Nitrogen stable-isotope compositions (δ
15
N) can help track denitrification and N
2
O production in the environment, as can knowledge of the isotopic discrimination, or isotope effect, inherent to denitrification. However, the isotope effects associated with denitrification as a function of dissolved-oxygen concentration and their influence on the isotopic composition of N
2
O are not known. We developed a simple steady-state reactor to allow the measurement of denitrification isotope effects in
Paracoccus denitrificans
. With [dO
2
] between 0 and 1.2 μM, the N stable-isotope effects of NO
3
−
and N
2
O reduction were constant at 28.6‰ ± 1.9‰ and 12.9‰ ± 2.6‰, respectively (mean ± standard error,
n
= 5). This estimate of the isotope effect of N
2
O reduction is the first in an axenic denitrifying culture and places the δ
15
N of denitrification-produced N
2
O midway between those of the nitrogenous oxide substrates and the product N
2
in steady-state systems. Application of both isotope effects to N
2
O cycling studies is discussed.
Effectiveness of Chemometric Techniques in Discrimination of
Lactobacillus helveticus
Biotypes from Natural Dairy Starter Cultures on the Basis of Phenotypic Characteristics
Authors: Gatti Monica , Contarini Giovanna , Neviani Erasmo
Published: December 2019

Lactobacillus helveticus
is the dominant organism in natural starter cultures used for the production of typical Italian cheeses. In this study, 74
L. helveticus
strains, isolated from grana and provolone cheese natural whey starters, were distinguished with respect to their origin by using both cell wall protein profiles and chemometric evaluation of some phenotypic parameters, such as the ability to acidify cultures and the presence of nonspecific proteolytic and peptidase activities. Cell wall protein patterns allowed
L. helveticus
strains to be distinguished with respect to their source of isolation. Among the different phenotypes studied, no single specific parameter permitted the two groups of strains to be separated. A good discrimination between the two groups of
L. helveticus
species was obtained by multivariate statistical techniques, which permitted the extraction of all of the discriminating information retained in the phenotypic activities. Associations between strain phenotype expression and dairy environmental ecosystem source are discussed.
Cloning and Nucleotide Sequence Analysis of
gyrB
of
Bacillus cereus
,
B. thuringiensis
,
B. mycoides
, and
B. anthracis
and Their Application to the Detection of
B. cereus
in Rice
Authors: Yamada Shoichi , Ohashi Eiji , Agata Norio , Venkateswaran Kasthuri
Published: December 2019

As 16S rRNA sequence analysis has proven inadequate for the differentiation of
Bacillus cereus
from closely related species, we employed the gyrase B gene (
gyrB
) as a molecular diagnostic marker. The
gyrB
genes of
B. cereus
JCM 2152
T
,
Bacillus thuringiensis
IAM 12077
T
,
Bacillus mycoides
ATCC 6462
T
, and
Bacillus anthracis
Pasteur #2H were cloned and sequenced. Oligonucleotide PCR primer sets were designed from within
gyrB
sequences of the respective bacteria for the specific amplification and differentiation of
B. cereus
,
B. thuringiensis
, and
B. anthracis
. The results from the amplification of
gyrB
sequences correlated well with results obtained with the 16S rDNA-based hybridization study but not with the results of their phenotypic characterization. Some of the reference strains of both
B. cereus
(three serovars) and
B. thuringiensis
(two serovars) were not positive in PCR amplification assays with
gyrB
primers. However, complete sequencing of 1.2-kb
gyrB
fragments of these reference strains showed that these serovars had, in fact, lower homology than their originally designated species. We developed and tested a procedure for the specific detection of the target organism in boiled rice that entailed 15 h of preenrichment followed by PCR amplification of the
B. cereus
-specific fragment. This method enabled us to detect an initial inoculum of 0.24 CFU of
B. cereus
cells per g of boiled rice food homogenate without extracting DNA. However, a simple two-step filtration step is required to remove PCR inhibitory substances.
Surface-Grafted, Environmentally Sensitive Polymers for Biofilm Release
Authors: Ista Linnea K. , Pérez-Luna Víctor H. , López Gabriel P.
Published: December 2019

Controlling bacterial biofouling is desirable for almost every human enterprise in which solid surfaces are introduced into nonsterile aqueous environments. One approach that is used to decrease contamination of manufactured devices by microorganisms is using materials that easily slough off accumulated material (i.e., fouling release surfaces). The compounds currently used for this purpose rely on low surface energy to inhibit strong attachment of organisms. In this study, we examined the possible use of environmentally responsive (or “smart”) polymers as a new class of fouling release agents; a surface-grafted thermally responsive polymer, poly(
N
-isopropylacrylamide) (PNIPAAM), was used as a model compound. PNIPAAM is known to have a lower critical solubility temperature of ∼32°C (i.e., it is insoluble in water at temperatures above 32°C and is soluble at temperatures below 32°C). Under experimental conditions, >90% of cultured microorganisms (
Staphylococcus epidermidis
,
Halomonas marina
) and naturally occurring marine microorganisms that attached to grafted PNIPAAM surfaces during 2-, 18-, 36-, and 72-h incubations were removed when the hydration state of the polymer was changed from a wettability that was favorable for attachment to a wettability that was less favorable. Of particular significance is the observation that an organism known to attach in the greatest numbers to hydrophobic substrata (i.e.,
H. marina
) was removed when transition of PNIPAAM to a more hydrated state occurred, whereas an organism that attaches in the greatest numbers to hydrophilic substrata (i.e.,
S. epidermidis
) was removed when the opposite transition occurred. Neither solvated nor desolvated PNIPAAM exhibited intrinsic fouling release properties, indicating that the phase transition was the important factor in removal of organisms. Based on our observations of the behavior of this model system, we suggest that environmentally responsive polymers represent a new approach for controlling biofouling release.
Detection of Small Numbers ofCampylobacter jejuniandCampylobacter coliCells in Environmental Water, Sewage, and Food Samples by a Seminested PCR Assay
Authors: Waage Astrid S. , Vardund Traute , Lund Vidar , Kapperud Georg
Published: December 2019

A rapid and sensitive assay was developed for detection of small numbers ofCampylobacter jejuniandCampylobacter colicells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the twoCampylobacterflagellin genes,flaAandflaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU ofC. jejuniper 100 ml in water samples containing a background flora consisting of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as ≤3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection ofC. jejuniorC. coliin drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms.
Effects of Surfactant Mixtures, Including Corexit 9527, on Bacterial Oxidation of Acetate and Alkanes in Crude Oil
Authors: Bruheim Per , Bredholt Harald , Eimhjellen Kjell
Published: December 2019

Mixtures of nonionic and anionic surfactants, including Corexit 9527, were tested to determine their effects on bacterial oxidation of acetate and alkanes in crude oil by cells pregrown on these substrates. Corexit 9527 inhibited oxidation of the alkanes in crude oil byAcinetobacter calcoaceticusATCC 31012, while Span 80, a Corexit 9527 constituent, markedly increased the oil oxidation rate. Another Corexit 9527 constituent, the negatively charged dioctyl sulfosuccinate (AOT), strongly reduced the oxidation rate. The combination of Span 80 and AOT increased the rate, but not as much as Span 80 alone increased it, which tentatively explained the negative effect of Corexit 9527. The results of acetate uptake and oxidation experiments indicated that the nonionic surfactants interacted with the acetate uptake system while the anionic surfactant interacted with the oxidation system of the bacteria. The overall effect of Corexit 9527 on alkane oxidation byA. calcoaceticusATCC 31012 thus seems to be the sum of the independent effects of the individual surfactants in the surfactant mixture. WhenRhodococcussp. strain 094 was used, the alkane oxidation rate decreased to almost zero in the presence of a mixture of Tergitol 15-S-7 and AOT even though the Tergitol 15-S-7 surfactant increased the alkane oxidation rate and AOT did not affect it. This indicated that there was synergism between the two surfactants rather than an additive effect like that observed forA. calcoaceticusATCC 31012.
Analysis and Dynamics of the Chromosomal Complements of Wild Sparkling-Wine Yeast Strains
Authors: Nadal Dolors , Carro David , Fernández-Larrea Juan , Piña Benjamin
Published: December 2019

We isolatedSaccharomyces cerevisiaeyeast strains that are able to carry out the second fermentation of sparkling wine from spontaneously fermenting musts in El Penedès (Spain) by specifically designed selection protocols. All of them (26 strains) showed one of two very similar mitochondrial DNA (mtDNA) restriction patterns, whereas their karyotypes differed. These strains showed high rates of karyotype instability, which were dependent on both the medium and the strain, during vegetative growth. In all cases, the mtDNA restriction pattern was conserved in strains kept under the same conditions. Analysis of different repetitive sequences in their genomes suggested that ribosomal DNA repeats play an important role in the changes in size observed in chromosome XII, whereas SUC genes or Ty elements did not show amplification or transposition processes that could be related to rearrangements of the chromosomes showing these sequences. Karyotype changes also occurred in monosporidic diploid derivatives. We propose that these changes originated mainly from ectopic recombination between repeated sequences interspersed in the genome. None of the rearranged karyotypes provided a selective advantage strong enough to allow the strains to displace the parental strains. The nature and frequency of these changes suggest that they may play an important role in the establishment and maintenance of the genetic diversity observed inS. cerevisiaewild populations.
Isolation and Characterization of a Second Subunit of Molecular Chaperonin from
Pyrococcus kodakaraensis
KOD1: Analysis of an ATPase-Deficient Mutant Enzyme
Authors: Izumi Michi , Fujiwara Shinsuke , Takagi Masahiro , Kanaya Shigenori , Imanaka Tadayuki
Published: December 2019

The
cpkA
gene encoding a second (α) subunit of archaeal chaperonin from
Pyrococcus kodakaraensis
KOD1 was cloned, sequenced, and expressed in
Escherichia coli
. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (β subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from
P. kodakaraensis
) in
E. coli
. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp
95
to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in
E. coli
without requiring energy from ATP hydrolysis.