bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Epidemiology of
Campylobacter jejuni
in Broiler Flocks Using Randomly Amplified Polymorphic DNA-PCR and 23S rRNA-PCR and Role of Litter in Its Transmission
Authors: Payne Randy E. , Lee Margie D. , Dreesen David W. , Barnhart Harold M.
Published: December 2019

Poultry has long been cited as a reservoir for
Campylobacter
spp., and litter has been implicated as a vehicle in their transmission. Chicks were raised on litter removed from a broiler house positive for
Campylobacter jejuni
. Litter was removed from the house on days 0, 3, and 9 after birds were removed for slaughter. Chicks were raised on these three litters under controlled conditions in flocks of 25. None of these birds yielded
C. jejuni
in their cecal droppings through 7 weeks. Two successive flocks from the same
Campylobacter
-positive broiler house were monitored for
Campylobacter
colonization.
Campylobacter jejuni
prevalence rates were determined for each flock. Randomly amplified polymorphic DNA (RAPD)-PCR and 23S rRNA-PCR typing methods were used to group isolates. A high prevalence (60%) of
C. jejuni
in flock 1 coincided with the presence of an RAPD profile not appearing in flock 2, which had a lower rate of prevalence (28%). A 23S rRNA-PCR typing method was used to determine if strains with different RAPD profiles and different prevalence rates contained different 23S sequences. RAPD profiles detected with higher prevalence rates contained a spacer in the 23S rRNA region 100% of the time, while RAPD profiles found with lower prevalence rates contained an intervening sequence less than 2% of the time. Data suggest varying colonizing potentials of different RAPD profiles and a source other than previously used litter as a means of transmission of
C. jejuni
. These molecular typing methods demonstrate their usefulness, when used together, in this epidemiologic investigation.
New Mobilizable Vectors Suitable for Gene Replacement in Gram-Negative Bacteria and Their Use in Mapping of the 3′ End of the
Xanthomonas campestris
pv. campestris
gum
Operon
Authors: Katzen Federico , Becker Anke , Ielmini M. Verónica , Oddo Cristian G. , Ielpi Luis
Published: December 2019

We describe useful vectors to select double-crossover events directly in site-directed marker exchange mutagenesis in gram-negative bacteria. These vectors contain the
gusA
marker gene, providing colorimetric screens to identify bacteria harboring those sequences. The applicability of these vectors was shown by mapping the 3′ end of the
Xanthomonas campestris gum
operon, involved in biosynthesis of xanthan.
Rapid Detection of
Ophiostoma piceae
and
O. quercus
in Stained Wood by PCR
Authors: Kim Seong Hwan , Uzunovic Adnan , Breuil Colette
Published: December 2019

A rapid, sensitive, and simple method was developed to detect the sapstain fungi
Ophiostoma piceae
and
O. quercus
in stained wood. By using microwave heating for DNA extraction and PCR with internal transcribed spacer-derived-specific primers, detection was feasible within 4 h, even with DNA obtained from a single synnema. This method can easily be extended for the detection of other wood-inhabiting fungi.
Organic Solvent Tolerance of
Escherichia coli
Is Independent of OmpF Levels in the Membrane
Authors: Asako Hiroyuki , Kobayashi Kei , Aono Rikizo
Published: December 2019

The organic solvent tolerance of
Escherichia coli
was measured under conditions in which OmpF levels were controlled by various means as follows: alteration of NaCl concentration in the medium, transformation with a stress-responsive gene (
marA
,
robA
, or
soxS
), or disruption of the
ompF
gene. It was shown that solvent tolerance of
E. coli
did not depend upon OmpF levels in the membrane.
The α Subunit of Toluene Dioxygenase from
Pseudomonas putida
F1 Can Accept Electrons from Reduced Ferredoxin
TOL
but Is Catalytically Inactive in the Absence of the β Subunit
Authors: Jiang Haiyan , Parales Rebecca E. , Gibson David T.
Published: December 2019

The oxygenase component of toluene dioxygenase from
Pseudomonas putida
F1 is an iron-sulfur protein (ISP
TOL
) consisting of α (TodC1) and β (TodC2) subunits. Purified TodC1 gave absorbance and electron paramagnetic resonance spectra identical to those given by purified ISP
TOL
. TodC1 was reduced by NADH and catalytic amounts of Reductase
TOL
and Ferredoxin
TOL
. Reduced TodC1 did not oxidize toluene, and catalysis was strictly dependent on the presence of purified TodC2.
Bacterial Leaching of Metal Sulfides Proceeds by Two Indirect Mechanisms via Thiosulfate or via Polysulfides and Sulfur
Authors: Schippers Axel , Sand Wolfgang
Published: December 2019

The acid-insoluble metal sulfides FeS
2
, MoS
2
, and WS
2
are chemically attacked by iron(III) hexahydrate ions, generating thiosulfate, which is oxidized to sulfuric acid. Other metal sulfides are attacked by iron(III) ions and by protons, resulting in the formation of elemental sulfur via intermediary polysulfides. Sulfur is biooxidized to sulfuric acid. This explains leaching of metal sulfides by
Thiobacillus thiooxidans
.
Sources of
Vibrio mimicus
Contamination of Turtle Eggs
Authors: Acuña María T. , Díaz Gerardo , Bolaños Hilda , Barquero Candy , Sánchez Olga , Sánchez Luz M. , Mora Grettel , Chaves Anny , Campos Elena
Published: December 2019

Vibrio mimicus
contamination of sand increased significantly during the arrival of the olive ridley sea turtles (
Lepidochelys olivacea
) at Ostional anidation beach, Costa Rica. Statistical analysis supports that eggs are contaminated with
V. mimicus
by contact with the sand nest.
V. mimicus
was isolated from eggs of all nests tested, and
ctxA
+
strains were found in 31% of the nests, all of which were near the estuary.
Persistence of Colonization of Human Colonic Mucosa by a Probiotic Strain,
Lactobacillus rhamnosus
GG, after Oral Consumption
Authors: Alander Minna , Satokari Reetta , Korpela Riitta , Saxelin Maija , Vilpponen-Salmela Terttu , Mattila-Sandholm Tiina , von Wright Atte
Published: December 2019

Lactobacillus rhamnosus
GG is one of the most thoroughly studied probiotic strains. Its advantages in the treatment of gastrointestinal disorders are well documented. The aim of the present study was to demonstrate with colonic biopsies the attachment of strain GG to human intestinal mucosae and the persistence of the attachment after discontinuation of GG administration. A whey drink fermented with strain GG was fed to human volunteers for 12 days. Fecal samples were collected before, during, and after consumption.
L. rhamnosus
GG-like colonies were detected in both fecal and colonic biopsy samples. Strain GG was identified by its characteristic colony morphology, a lactose fermentation test, and PCR. This study showed that strain GG was able to attach in vivo to colonic mucosae and, although the attachment was temporary, to remain for more than a week after discontinuation of GG administration. The results demonstrate that the study of fecal samples alone is not sufficient in evaluating colonization by a probiotic strain.
Potential Role of
Thiobacillus caldus
in Arsenopyrite Bioleaching
Authors: Dopson Mark , Lindström E. Börje
Published: December 2019

We investigated the potential role of the three strains of
Thiobacillus caldus
(KU, BC13, and C-SH12) in arsenopyrite leaching in combination with a moderately thermophilic iron oxidizer,
Sulfobacillus thermosulfidooxidans
. Pure cultures of
T. caldus
and
S. thermosulfidooxidans
were used as well as defined mixed cultures. By measuring released iron, tetrathionate, and sulfur concentrations, we found that the presence of
T. caldus
KU and BC13 in the defined mixed culture lowered the concentration of sulfur, and levels of tetrathionate were comparable to or lower than those in the presence of
S. thermosulfidooxidans
. This suggests that
T. caldus
grows on the sulfur compounds that build up during leaching, increasing the arsenopyrite-leaching efficiency. This result was similar to leaching arsenopyrite with a pure culture of
S. thermosulfidooxidans
in the presence of yeast extract. Therefore, three possible roles of
T. caldus
in the leaching environment can be hypothesized: to remove the buildup of solid sulfur that can cause an inhibitory layer on the surface of the mineral, to aid heterotrophic and mixotrophic growth by the release of organic chemicals, and to solubilize solid sulfur by the production of surface-active agents. The results showed that
T. caldus
KU was the most efficient at leaching arsenopyrite under the conditions tested, followed by BC13, and finally C-SH12.
Incidence of
Fusarium
spp. and Levels of Fumonisin B
1
in Maize in Western Kenya
Authors: Kedera C. J. , Plattner R. D. , Desjardins A. E.
Published: December 2019

Maize kernel samples were collected in 1996 from smallholder farm storages in the districts of Bomet, Bungoma, Kakamega, Kericho, Kisii, Nandi, Siaya, Trans Nzoia, and Vihiga in the tropical highlands of western Kenya. Two-thirds of the samples were good-quality maize, and one-third were poor-quality maize with a high incidence of visibly diseased kernels. One hundred fifty-three maize samples were assessed for
Fusarium
infection by culturing kernels on a selective medium. The isolates obtained were identified to the species level based on morphology and on formation of the sexual stage in
Gibberella fujikuroi
mating population tests.
Fusarium moniliforme
(
G. fujikuroi
mating population A) was isolated most frequently, but
F. subglutinans
(
G. fujikuroi
mating population E),
F. graminearum
,
F. oxysporum
,
F. solani
, and other
Fusarium
species were also isolated. The high incidence of kernel infection with the fumonisin-producing species
F. moniliforme
indicated a potential for fumonisin contamination of Kenyan maize. However, analysis of 197 maize kernel samples by high-performance liquid chromatography found little fumonisin B
1
in most of the samples. Forty-seven percent of the samples contained fumonisin B
1
at levels above the detection limit (100 ng/g), but only 5% were above 1,000 ng/g, a proposed level of concern for human consumption. The four most-contaminated samples, with fumonisin B
1
levels ranging from 3,600 to 11,600 ng/g, were from poor-quality maize collected in the Kisii district. Many samples with a high incidence of visibly diseased kernels contained little or no fumonisin B
1
, despite the presence of
F. moniliforme
. This result may be attributable to the inability of
F. moniliforme
isolates present in Kenyan maize to produce fumonisins, to the presence of other ear rot fungi, and/or to environmental conditions unfavorable for fumonisin production.
Enumeration of Marine Viruses in Culture and Natural Samples by Flow Cytometry
Authors: Marie Dominique , Brussaard Corina P. D. , Thyrhaug Runar , Bratbak Gunnar , Vaulot Daniel
Published: December 2019

Flow cytometry (FCM) was successfully used to enumerate viruses in seawater after staining with the nucleic acid-specific dye SYBR Green-I. The technique was first optimized by using the
Phaeocystis
lytic virus PpV-01. Then it was used to analyze natural samples from different oceanic locations. Virus samples were fixed with 0.5% glutaraldehyde and deep frozen for delayed analysis. The samples were then diluted in Tris-EDTA buffer and analyzed in the presence of SYBR Green-I. A duplicate sample was heated at 80°C in the presence of detergent before analysis. Virus counts obtained by FCM were highly correlated to, although slightly higher than, those obtained by epifluorescence microscopy or by transmission electron microscopy (
r
= 0.937,
n
= 14, and
r
= 0.96,
n
= 8, respectively). Analysis of a depth profile from the Mediterranean Sea revealed that the abundance of viruses displayed the same vertical trend as that of planktonic cells. FCM permits us to distinguish between at least two and sometimes three virus populations in natural samples. Because of its speed and accuracy, FCM should prove very useful for studies of virus infection in cultures and should allow us to better understand the structure and dynamics of virus populations in natural waters.
Use of the
KlADH4
Promoter for Ethanol-Dependent Production of Recombinant Human Serum Albumin in
Kluyveromyces lactis
Authors: Saliola Michele , Mazzoni Cristina , Solimando Nicola , Crisà Alessandra , Falcone Claudio , Jung Gerard , Fleer Reinhard
Published: December 2019

KlADH4
is a gene of
Kluyveromyces lactis
encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in
K. lactis
, a very promising organism which can be used as an alternative host to
Saccharomyces cerevisiae
due to its good secretory properties. In this paper we report the ethanol-driven expression in
K. lactis
of the bacterial β-glucuronidase and of the human serum albumin (HSA) genes under the control of the
KlADH4
promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of
KlADH4 trans
-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UAS
E
) which is sufficient for the induction of
KlADH4
by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as
PGK
and
LAC4
. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source.
Effects of Carbon Substrates on Nitrite Accumulation in Freshwater Sediments
Authors: Kelso Beverley H. L. , Smith Roger V. , Laughlin Ronald J.
Published: December 2019

The contribution of the biochemical pathways nitrification, denitrification, and dissimilatory NO
3
−
reduction to NH
4
+
(DNRA) to the accumulation of NO
2
−
in freshwaters is governed by the species compositions of the bacterial populations resident in the sediments, available carbon (C) and nitrogen (N) substrates, and environmental conditions. Recent studies of major rivers in Northern Ireland have shown that high NO
2
−
concentrations found in summer, under warm, slow-flowing conditions, arise from anaerobic NO
3
−
reduction. Locally, agricultural pollutants entering rivers are important C and N sources, providing ideal substrates for the aquatic bacteria involved in cycling of N. In this study a range of organic C compounds commonly found in agricultural pollutants were provided as energy sources in 48-h incubation experiments to investigate if the chemical compositions of the pollutants affected which NO
3
−
reduction pathway was followed and influenced subsequent NO
2
−
accumulation. Carbon stored within the sediments was sufficient to support DNRA and denitrifier populations, and the resulting NO
2
−
peak (80 μg of N liter
−1
[approximate]) observed at 24 h was indicative of the simultaneous activities of both bacterial groups. The value of glycine as an energy source for denitrification or DNRA appeared to be limited, but glycine was an important source of additional N. Glucose was an efficient substrate for both the denitrification and DNRA pathways, with a NO
2
−
peak of 160 μg of N liter
−1
noted at 24 h. Addition of formate and acetate stimulated continuous NO
2
−
production throughout the 48-h period, caused by partial inhibition of the denitrification pathway. The formate treatment resulted in a high NO
2
−
accumulation (1,300 μg of N liter
−1
[approximate]), and acetate treatment resulted in a low NO
2
−
concentration (<100 μg of N liter
−1
).
A Method of Profiling Microbial Communities Based on a Most-Probable-Number Assay That Uses BIOLOG Plates and Multiple Sole Carbon Sources
Authors: Gamo Masashi , Shoji Tadashi
Published: December 2019

A new approach to the community-level BIOLOG assay was proposed. This assay, which we call the BIOLOG-MPN assay, is a most-probable-number (MPN) assay that uses BIOLOG plates and multiple sole carbon sources, and the profiles obtained by this assay consist of MPNs estimated for the substrates in the BIOLOG plates. In order to demonstrate the performance of the BIOLOG-MPN assay, it was applied to pure cultures, model bacterial communities that contain two strains in different ratios, and microbial community samples. MPN estimation using BIOLOG plates worked well for the substrates on which utilizers can grow at a sufficiently high rate for color development under the conditions of the assay procedure. Furthermore, the results obtained using model communities showed that the MPNs obtained reflected the mixing ratios of pure cultures in the model communities. The profiles obtained using model communities and community samples were differentiated properly by statistical analyses. The results suggest that the BIOLOG-MPN assay is a promising procedure for obtaining a quantitative picture of the community structure.
Identification of
Enterococcus
spp. with a Biochemical Key
Authors: Manero Albert , Blanch Anicet R.
Published: December 2019

A six-step biochemical key is presented for the identification of all recognized
Enterococcus
spp. The key consists of 12 tests, but no more than 6 are needed for the most complicated identification. The reliability of the key has been evaluated with collection type strains and clinical and environmental isolates. This key has fewer tests than those reported in previous studies. There is no commercial kit that includes the whole set of tests. However, some of the tests are included in enzyme activity-based kits that could be used with the proposed key. The key is designed for use in routine applications, especially in environmental and clinical studies with a high number of isolates.
Enhanced Production of Pediocin PA-1 and Coproduction of Nisin and Pediocin PA-1 by
Lactococcus lactis
Authors: Horn Nikki , Martínez María I. , Martínez José M. , Hernández Pablo E. , Gasson Michael J. , Rodríguez Juan M. , Dodd Helen M.
Published: December 2019

The production and secretion of class II bacteriocins share a number of features that allow the interchange of genetic determinants between certain members of this group of antimicrobial peptides.
Lactococcus lactis
IL1403 encodes translocatory functions able to recognize and mediate secretion of lactococcin A. The ability of this strain to also produce the pediococcal bacteriocin pediocin PA-1, has been demonstrated previously by the introduction of a chimeric gene, composed of sequences encoding the leader of lactococcin A and the mature part of pediocin PA-1 (N. Horn, M. I. Martı́nez, J. M. Martı́nez, P. E. Hernández, M. J. Gasson, J. M. Rodrı́guez, and H. M. Dodd, Appl. Environ. Microbiol. 64:818–823, 1998). This heterologous expression system has been developed further with the introduction of the lactococcin A-dedicated translocatory function genes,
lcnC
and
lcnD
, and their effect on bacteriocin yields in various lactococcal hosts was assessed. The copy number of
lcnC
and
lcnD
influenced production levels, as did the particular strain employed as host. Highest yields were achieved with
L. lactis
IL1403, which generated pediocin PA-1 at a level similar to that for the parental strain,
Pediococcus acidilactici
347, representing a significant improvement over previous systems. The genetic determinants required for production of pediocin PA-1 were introduced into the nisin-producing strain
L. lactis
FI5876, where both pediocin PA-1 and nisin A were simultaneously produced. The implications of coproduction of these two industrially relevant antimicrobial agents by a food-grade organism are discussed.
Enhanced Biotransformation of Carbon Tetrachloride by
Acetobacterium woodii
upon Addition of Hydroxocobalamin and Fructose
Authors: Hashsham Syed A. , Freedman David L.
Published: December 2019

The objective of this study was to evaluate the effect of hydroxocobalamin (OH-Cbl) on transformation of high concentrations of carbon tetrachloride (CT) by
Acetobacterium woodii
(ATCC 29683). Complete transformation of 470 μM (72 mg/liter [aqueous]) CT was achieved by
A. woodii
within 2.5 days, when 10 μM OH-Cbl was added along with 25.2 mM fructose. This was approximately 30 times faster than
A. woodii
cultures (live or autoclaved) and medium that did not receive OH-Cbl and 5 times faster than those controls that did receive OH-Cbl, but either live
A. woodii
or fructose was missing. CT transformation in treatments with only OH-Cbl was indicative of the important contribution of nonenzymatic reactions. Besides increasing the rate of CT transformation, addition of fructose and OH-Cbl to live cultures increased the percentage of [
14
C]CT transformed to
14
CO
2
(up to 31%) and
14
C-labeled soluble materials (principally
l
-lactate and acetate), while decreasing the percentage of CT reduced to chloroform and abiotically transformed to carbon disulfide.
14
CS
2
represented more than 35% of the [
14
C]CT in the presence of reduced medium and OH-Cbl. Conversion of CT to CO was a predominant pathway in formation of CO
2
in the presence of live cells and added fructose and OH-Cbl. These results indicate that the rate and distribution of products during cometabolic transformation of CT by
A. woodii
can be improved by the addition of fructose and OH-Cbl.
Effect of Milk Proteins on Adhesion of Bacteria to Stainless Steel Surfaces
Authors: Barnes L.-M. , Lo M. F. , Adams M. R. , Chamberlain A. H. L.
Published: December 2019

Stainless steel coupons were treated with skim milk and subsequently challenged with individual bacterial suspensions of
Staphylococcus aureus
,
Pseudomonas fragi
,
Escherichia coli
,
Listeria monocytogenes
, and
Serratia marcescens
. The numbers of attached bacteria were determined by direct epifluorescence microscopy and compared with the attachment levels on clean stainless steel with two different surface finishes. Skim milk was found to reduce adhesion of
S. aureus
,
L. monocytogenes
, and
S. marcescens
.
P. fragi
and
E. coli
attached in very small numbers to the clear surfaces, making the effect of any adsorbed protein layer difficult to assess. Individual milk proteins α-casein, β-casein, κ-casein, and α-lactalbumin were also found to reduce the adhesion of
S. aureus
and
L. monocytogenes
. The adhesion of bacteria to samples treated with milk dilutions up to 0.001% was investigated. X-ray photoelectron spectroscopy was used to determine the proportion of nitrogen in the adsorbed films. Attached bacterial numbers were inversely related to the relative atomic percentage of nitrogen on the surface. A comparison of two types of stainless steel surface, a 2B and a no. 8 mirror finish, indicated that the difference in these levels of surface roughness did not greatly affect bacterial attachment, and reduction in adhesion to a milk-treated surface was still observed. Cross-linking of adsorbed proteins partially reversed the inhibition of bacterial attachment, indicating that protein chain mobility and steric exclusion may be important in this phenomenon.
Automated Approach for Ribosomal Intergenic Spacer Analysis of Microbial Diversity and Its Application to Freshwater Bacterial Communities
Authors: Fisher Madeline M. , Triplett Eric W.
Published: December 2019

An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.
A Method To Detect Low Levels of Enteric Viruses in Contaminated Oysters
Authors: Shieh Y.-S. Carol , Calci Kevin R. , Baric Ralph S.
Published: December 2019

Direct isolation and identification of pathogenic viruses from oysters implicated in gastroenteritis outbreaks are hampered by inefficient methods for recovering viruses, naturally occurring PCR inhibitors, and low levels of virus contamination. In this study we focused on developing rapid and efficient oyster-processing procedures that can be used for sensitive PCR detection of viruses in raw oysters. Poliovirus type 3 (PV3) Sabin strain was used to evaluate the efficacy of virus recovery and the removal of PCR inhibitors during oyster-processing procedures. These procedures included elution, polyethylene glycol precipitation, solvent extraction, and RNA extraction. Acid adsorption-elution in which glycine buffer (pH 7.5) was used was found to retain fewer inhibitors than direct elution in which glycine buffer (pH 9.5) was used. RNA extraction in which a silica gel membrane was used was more effective than single-step RNA precipitation for removing additional nonspecific PCR inhibitors. The final 10-μl volume of RNA concentrates obtained from 2 g of oyster tissue (concentration factor, 200-fold) was satisfactory for efficient reverse transcription-PCR detection of virus. The overall detection sensitivity of our method was 1 PFU/g of oyster tissue initially seeded with PV3. The method was utilized to investigate a 1998 gastroenteritis outbreak in California in which contaminated oysters were the suspected disease transmission vehicle. A genogroup II Norwalk-like virus was found in two of three recalled oyster samples linked by tags to the harvest dates and areas associated with the majority of cases. The method described here improves the response to outbreaks and can be used for rapid and sensitive detection of viral agents in outbreak-implicated oysters.
Quantification of Syntrophic Fatty Acid-β-Oxidizing Bacteria in a Mesophilic Biogas Reactor by Oligonucleotide Probe Hybridization
Authors: Hansen Kaare H. , Ahring Birgitte K. , Raskin Lutgarde
Published: December 2019

Small-subunit rRNA sequences were obtained for two saturated fatty acid-β-oxidizing syntrophic bacteria,
Syntrophomonas sapovorans
and
Syntrophomonas wolfei
LYB, and sequence analysis confirmed their classification as members of the family
Syntrophomonadaceae
.
S. wolfei
LYB was closely related to
S. wolfei
subsp.
wolfei
, but
S. sapovorans
did not cluster with the other members of the genus
Syntrophomonas
. Five oligonucleotide probes targeting the small-subunit rRNA of different groups within the family
Syntrophomonadaceae
, which contains all currently known saturated fatty acid-β-oxidizing syntrophic bacteria, were developed and characterized. The probes were designed to be specific at the family, genus, and species levels and were characterized by temperature-of-dissociation and specificity studies. To demonstrate the usefulness of the probes for the detection and quantification of saturated fatty acid-β-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria and methanogens were compared to specific methanogenic activities and microbial numbers determined with most-probable-number estimates. Most of the methanogenic rRNA was comprised of
Methanomicrobiales
rRNA, suggesting that members of this order served as the main hydrogen-utilizing microorganisms. Between 0.2 and 1% of the rRNA was attributed to the
Syntrophomonadaceae
, of which the majority was accounted for by the genus
Syntrophomonas
.
Potential of Three-Way Randomly Amplified Polymorphic DNA Analysis as a Typing Method for Twelve
Salmonella
Serotypes
Authors: Soto S. M. , Guerra B. , González-Hevia M. A. , Mendoza M. C.
Published: December 2019

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing
Salmonella enterica
strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0.96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying
Salmonella
organisms and as a typing method that is complementary to serotyping and phage typing methods.
Culture-Independent Characterization of a Gene Responsible for Nitrogen Fixation in the Symbiotic Microbial Community in the Gut of the Termite
Neotermes koshunensis
Authors: Noda Satoko , Ohkuma Moriya , Usami Ron , Horikoshi Koki , Kudo Toshiaki
Published: December 2019

Expression of the nitrogen fixation gene,
nifH
, in the gut of the termite
Neotermes koshunensis
was characterized without cultivation.
nifH
cDNA was directly amplified from mRNA of the mixed microbial population in the gut by reverse transcription (RT)-PCR. Analyses of the RT-PCR products revealed that, among the diverse
nifH
sequences, only a few corresponding to an alternative nitrogenase (encoded by the
anf
gene) were preferentially transcribed in the termite gut. Expression of the
anf
gene was further investigated quantitatively under several termite feeding conditions by competitive PCR. The levels of expression of the
anf
gene were largely congruent with the nitrogen fixation activity displayed by the termite. The amounts of the genomic
anf
gene in the population showed no significant change, indicating that the level of expression was critical for nitrogen fixation activity. Interestingly, no significant decrease in the expression level was observed when the diet contained molybdenum (Mo), which represses ordinary
anf
genes. A 3.6-kb DNA region downstream of the
anf
gene was isolated and found to contain reading frames homologous to
anfH
,
anfD
, and
anfG
of the
Bacteria
domain which encode subunits of an alternative nitrogenase having no Mo as a cofactor. This DNA region also contained reading frames encoding
glnB
-like proteins, which is a common feature of the nitrogenase genes of the
Archaea
domain. These results indicate that the
anf
group of nitrogenase genes is the most important group of genes responsible for nitrogen fixation in
N. koshunensis
and that the
anf
gene possesses novel features with respect to the regulation of its expression and its gene organization.
Impacts of the Reduction of Nutrient Levels on Bacterial Water Quality in Distribution Systems
Authors: Volk Christian J. , LeChevallier Mark W.
Published: December 2019

This study evaluated the impacts of reducing nutrient levels on bacterial water quality in drinking water. Two American Water System facilities (sites NJ102a and IN610) with histories of coliform problems were involved, and each water utility received two pilot distribution systems (annular reactors). One reactor simulated the conventional treatment conditions (control), while the other reactor was used to assess the effect of biological filtration and subsequent reduced biodegradable organic matter levels on suspended (water column) and biofilm bacterial concentrations in the distribution systems. Biodegradable organic matter levels were reduced approximately by half after biological treatment. For site NJ102a, the geometric mean of the assimilable organic carbon concentrations was 217 μg/liter in the plant effluent and 91 μg/liter after biological filtration. For both sites, plant effluent biodegradable dissolved organic carbon levels averaged 0.45 mg/liter, versus 0.19 to 0.22 mg/liter following biological treatment. Biological treatment improved the stability of free chlorine residuals, while it had little effect on chloramine consumption patterns. High bacterial levels from the biological filters resulted in higher bacterial concentrations entering the test reactors than entering the control reactors. On average, biofilms in the model systems were reduced by 1 log unit (from 1.4 × 10
5
to 1.4 × 10
4
CFU/cm
2
) and 0.5-log unit (from 2.7 × 10
5
to 7.8 × 10
4
CFU/cm
2
) by biological treatment at sites NJ102a and IN610, respectively. Interestingly, it required several months of biological treatment before there was an observable impact on bacterial water quality in the system, suggesting that the effect of the treatment change was influenced by other factors (i.e., pipe conditions or disinfection, etc.).
Lactobacillus salivarius
CTC2197 Prevents
Salmonella enteritidis
Colonization in Chickens
Authors: Pascual Mònica , Hugas Marta , Badiola Jose Ignacio , Monfort Josep Maria , Garriga Margarita
Published: December 2019

A rifampin-resistant
Lactobacillus salivarius
strain, CTC2197, was assessed as a probiotic in poultry, by studying its ability to prevent
Salmonella enteritidis
C-114 colonization in chickens. When the probiotic strain was dosed by oral gavage together with
S. enteritidis
C-114 directly into the proventriculus in 1-day-old Leghorn chickens, the pathogen was completely removed from the birds after 21 days. The same results were obtained when the probiotic strain was also administered through the feed and the drinking water apart from direct inoculation into the proventriculus. The inclusion of
L. salivarius
CTC2197 in the first day chicken feed revealed that a concentration of 10
5
CFU g
−1
was enough to ensure the colonization of the gastrointestinal tract of the birds after 1 week. However, between 21 and 28 days,
L. salivarius
CTC2197 was undetectable in the gastrointestinal tract of some birds, showing that more than one dose would be necessary to ensure its presence till the end of the rearing time. Freeze-drying and freezing with glycerol or skim milk as cryoprotective agents, appeared to be suitable methods to preserve the probiotic strain. The inclusion of the
L. salivarius
CTC2197 in a commercial feed mixture seemed to be a good way to supply it on the farm, although the strain showed sensitivity to the temperatures used during the feed mixture storage and in the chicken incubator rooms. Moreover, survival had been improved after several reinoculations in chicken feed mixture.
Isolation, Oxygen Sensitivity, and Virulence of NADH Oxidase Mutants of the Anaerobic SpirocheteBrachyspira(Serpulina)hyodysenteriae, Etiologic Agent of Swine Dysentery
Authors: Stanton Thad B. , Rosey Everett L. , Kennedy Michael J. , Jensen Neil S. , Bosworth Brad T.
Published: December 2019

Brachyspira(Serpulina)hyodysenteriae, the etiologic agent of swine dysentery, uses the enzyme NADH oxidase to consume oxygen. To investigate possible roles for NADH oxidase in the growth and virulence of this anaerobic spirochete, mutant strains deficient in oxidase activity were isolated and characterized. The cloned NADH oxidase gene (nox; GenBank accession no.U19610) on plasmid pER218 was inactivated by replacing 321 bp of coding sequence with either a gene for chloramphenicol resistance (cat) or a gene for kanamycin resistance (kan). The resulting plasmids, respectively, pCmΔNOX and pKmΔNOX, were used to transform wild-typeB. hyodysenteriaeB204 cells and generate the antibiotic-resistant strains Nox-Cm and Nox-Km. PCR and Southern hybridization analyses indicated that the chromosomal wild-typenoxgenes in these strains had been replaced, through allelic exchange, by the inactivatednoxgene containingcatorkan. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis revealed that bothnoxmutant cell lysates were missing the 48-kDa Nox protein. Soluble NADH oxidase activity levels in cell lysates of Nox-Cm and Nox-Km were reduced 92 to 96% compared to the activity level in parent strain B204. In an aerotolerance test, cells of bothnoxmutants were at least 100-fold more sensitive to oxygen exposure than were cells of the wild-type parent strain B204. In swine experimental infections, bothnoxmutants were less virulent than strain B204 in that fewer animals were colonized by the mutant cells and infected animals displayed mild, transient signs of disease, with no deaths. These results provide evidence that NADH oxidase serves to protectB. hyodysenteriaecells against oxygen toxicity and that the enzyme, in that role, contributes to the pathogenic ability of the spirochete.
Metabolism of Acrylate to β-Hydroxypropionate and Its Role in Dimethylsulfoniopropionate Lyase Induction by a Salt Marsh Sediment Bacterium,
Alcaligenes faecalis
M3A
Authors: Ansede John H. , Pellechia Perry J. , Yoch Duane C.
Published: December 2019

Dimethylsulfoniopropionate (DMSP) is degraded to dimethylsulfide (DMS) and acrylate by the enzyme DMSP lyase. DMS or acrylate can serve as a carbon source for both free-living and endophytic bacteria in the marine environment. In this study, we report on the mechanism of DMSP-acrylate metabolism by
Alcaligenes faecalis
M3A. Suspensions of citrate-grown cells expressed a low level of DMSP lyase activity that could be induced to much higher levels in the presence of DMSP, acrylate, and its metabolic product, β-hydroxypropionate. DMSP was degraded outside the cell, resulting in an extracellular accumulation of acrylate, which in suspensions of citrate-grown cells was then metabolized at a low endogenous rate. The inducible nature of acrylate metabolism was evidenced by both an increase in the rate of its degradation over time and the ability of acrylate-grown cells to metabolize this molecule at about an eight times higher rate than citrate-grown cells. Therefore, acrylate induces both its production (from DMSP) and its degradation by an acrylase enzyme.
1
H and
13
C nuclear magnetic resonance analyses were used to identify the products resulting from [1-
13
C]acrylate metabolism. The results indicated that
A. faecalis
first metabolized acrylate to β-hydroxypropionate outside the cell, which was followed by its intracellular accumulation and subsequent induction of DMSP lyase activity. In summary, the mechanism of DMSP degradation to acrylate and the subsequent degradation of acrylate to β-hydroxypropionate in the aerobic β-Proteobacterium
A. faecalis
has been described.
Acetogenic and Sulfate-Reducing Bacteria Inhabiting the Rhizoplane and Deep Cortex Cells of the Sea Grass
Halodule wrightii
Authors: Küsel Kirsten , Pinkart Holly C. , Drake Harold L. , Devereux Richard
Published: December 2019

Recent declines in sea grass distribution underscore the importance of understanding microbial community structure-function relationships in sea grass rhizospheres that might affect the viability of these plants. Phospholipid fatty acid analyses showed that sulfate-reducing bacteria and clostridia were enriched in sediments colonized by the sea grasses
Halodule wrightii
and
Thalassia testudinum
compared to an adjacent unvegetated sediment. Most-probable-number analyses found that in contrast to butyrate-producing clostridia, acetogens and acetate-utilizing sulfate reducers were enriched by an order of magnitude in rhizosphere sediments. Although sea grass roots are oxygenated in the daytime, colorimetric root incubation studies demonstrated that acetogenic O-demethylation and sulfidogenic iron precipitation activities were tightly associated with washed, sediment-free
H. wrightii
roots. This suggests that the associated anaerobes are able to tolerate exposure to oxygen. To localize and quantify the anaerobic microbial colonization, root thin sections were hybridized with newly developed
33
P-labeled probes that targeted (i) low-G+C-content gram-positive bacteria, (ii) cluster I species of clostridia, (iii) species of
Acetobacterium
, and (iv) species of
Desulfovibrio
. Microautoradiography revealed intercellular colonization of the roots by
Acetobacterium
and
Desulfovibrio
species. Acetogenic bacteria occurred mostly in the rhizoplane and outermost cortex cell layers, and high numbers of sulfate reducers were detected on all epidermal cells and inward, colonizing some 60% of the deepest cortex cells. Approximately 30% of epidermal cells were colonized by bacteria that hybridized with an archaeal probe, strongly suggesting the presence of methanogens. Obligate anaerobes within the roots might contribute to the vitality of sea grasses and other aquatic plants and to the biogeochemistry of the surrounding sediment.
A New Selective Medium for
Bifidobacterium
spp
Authors: Nebra Y. , Blanch A. R.
Published: December 2019

A new selective antibiotic-free medium for
Bifidobacterium
spp. is defined. This medium has lactulose as the main carbon source and includes methylene blue, propionic acid, and lithium chloride as inhibitors of some related bacterial species. The low pH of the medium contributes to the inhibition of the growth of
Enterobacteriaceae
. This new selective medium has a simple composition, and the level of recovery it yields is similar to those yielded by nonselective media for
Bifidobacterium
strains. It could thus be used for routine analysis in environmental or food microbiology.
Soluble Methane Monooxygenase Gene Clusters from Trichloroethylene-Degrading
Methylomonas
sp. Strains and Detection of Methanotrophs during In Situ Bioremediation
Authors: Shigematsu Toru , Hanada Satoshi , Eguchi Masahiro , Kamagata Yoichi , Kanagawa Takahiro , Kurane Ryuichiro
Published: December 2019

The soluble MMO (sMMO) gene clusters from group I methanotrophs were characterized. An 8.1-kb
Kpn
I fragment from
Methylomonas
sp. strain KSWIII and a 7.5-kb
Sal
I fragment from
Methylomonas
sp. strain KSPIII which contained the sMMO gene clusters were cloned and sequenced. The sequences of these two fragments were almost identical. The sMMO gene clusters in the fragment consisted of six open reading frames which were 52 to 79% similar to the corresponding genes of previously described sMMO gene clusters of the group II and group X methanotrophs. The phylogenetic analysis of the predicted amino acid sequences of sMMO demonstrated that the sMMOs from these strains were closer to that from
M. capsulatus
Bath in the group X methanotrophs than to those from
Methylosinus trichosporium
OB3b and
Methylocystis
sp. strain M in the group II methanotrophs. Based on the sequence data of sMMO genes of our strains and other methanotrophs, we designed a new PCR primer to amplify sMMO gene fragments of all the known methanotrophs harboring the
mmoX
gene. The primer set was successfully used for detecting methanotrophs in the groundwater of trichloroethylene-contaminated sites during in situ-biostimulation treatments.
Swimming MarineSynechococcusStrains with Widely Different Photosynthetic Pigment Ratios Form a Monophyletic Group
Authors: Toledo Gerardo , Palenik B. , Brahamsha B.
Published: December 2019

Unicellular marine cyanobacteria are ubiquitous in both coastal and oligotrophic regimes. The contribution of these organisms to primary production and nutrient cycling is substantial on a global scale. Natural populations of marineSynechococcusstrains include multiple genetic lineages, but the link, if any, between unique phenotypic traits and specific genetic groups is still not understood. We studied the genetic diversity (as determined by the DNA-dependent RNA polymeraserpoC1gene sequence) of a set of marineSynechococcusisolates that are able to swim. Our results show that these isolates form a monophyletic group. This finding represents the first example of correspondence between a physiological trait and a phylogenetic group in marineSynechococcus. In contrast, the phycourobilin (PUB)/phycoerythrobilin (PEB) pigment ratios of members of the motile clade varied considerably. An isolate obtained from the California Current (strain CC9703) displayed a pigment signature identical to that of nonmotile strain WH7803, which is considered a model for low-PUB/PEB-ratio strains, whereas several motile strains had higher PUB/PEB ratios than strain WH8103, which is considered a model for high-PUB/PEB-ratio strains. These findings indicate that the PUB/PEB pigment ratio is not a useful characteristic for defining phylogenetic groups of marineSynechococcusstrains.
Attributes of Atmospheric Carbon Monoxide Oxidation by Maine Forest Soils
Authors: King G. M.
Published: December 2019

CO, one of the most important trace gases, regulates tropospheric methane, hydroxyl radical, and ozone contents. Ten to 25% of the estimated global CO flux may be consumed by soils annually. Depth profiles for
14
CO oxidation and CO concentration indicated that CO oxidation occurred primarily in surface soils and that photooxidation of soil organic matter did not necessarily contribute significantly to CO fluxes. Kinetic analyses revealed that the apparent

K
m

was about 18 nM (17 ppm) and the
V
max
was 6.9 μmol g (fresh weight)
−1
h
−1
; the apparent

K
m

was similar to the apparent

K
m

for atmospheric methane consumption, but the
V
max
was more than 100 times higher. Atmospheric CO oxidation responded sensitively to soil water regimes; decreases in water content in initially saturated soils resulted in increased uptake, and optimum uptake occurred at water contents of 30 to 60%. However, extended drying led to decreased uptake and net CO production. Rewetting could restore CO uptake, albeit with a pronounced hysteresis. The responses to changing temperatures indicated that the optimum temperature for net uptake was between 20 and 25°C and that there was a transition to net production at temperatures above 30°C. The responses to methyl fluoride and acetylene indicated that populations other than ammonia oxidizers and methanotrophs must be involved in forest soils. The response to acetylene was notable, since the strong initial inhibition was reversed after 12 h of incubation; in contrast, methyl fluoride did not have an inhibitory effect. Ammonium did not inhibit CO uptake; the level of nitrite inhibition was initially substantial, but nitrite inhibition was reversible over time. Nitrite inhibition appeared to occur through indirect effects based on abiological formation of NO.