bluuebunny/crossref_metadata_2025_split huggingface dataset:
Estimation of Bacterial Cell Numbers in Humic Acid-Rich Salt Marsh Sediments with Probes Directed to 16S Ribosomal DNA
Authors: Edgcomb Virginia P. , McDonald John H. , Devereux Richard , Smith David W. 
Published: December 2019

ABSTRACT

The feasibility of using probes directed towards ribosomal DNAs (rDNAs) as a quantitative approach to estimating cell numbers was examined and applied to study the structure of a bacterial community in humic acid-rich salt marsh sediments. Hybridizations were performed with membrane-bound nucleic acids by using seven group-specific DNA oligonucleotide probes complementary to 16S rRNA coding regions. These included a general eubacterial probe and probes encompassing most members of the gram-negative, mesophilic sulfate-reducing bacteria (SRB). DNA was extracted from sediment samples, and contaminating materials were removed by a series of steps. Efficiency of DNA extraction was 48% based on the recovery of tritiated plasmid DNA added to samples prior to extraction. Reproducibility of the extraction procedure was demonstrated by hybridizations to replicate samples. Numbers of target cells in samples were estimated by comparing the amount of hybridization to extracted DNA obtained with each probe to that obtained with a standard curve of genomic DNA for reference strains included on the same membrane. In June, numbers of SRB detected with an SRB-specific probe ranged from 6.0 × 10
7
to 2.5 × 10
9
(average, 1.1 × 10
9
± 5.2 × 10
8
) cells g of sediment
−1
. In September, numbers of SRB detected ranged from 5.4 × 10
8
to 7.3 × 10
9
(average, 2.5 × 10
9
± 1.5 × 10
9
) cells g of sediment
−1
. The capability of using rDNA probes to estimate cell numbers by hybridization to DNA extracted from complex matrices permits initiation of detailed studies on community composition and changes in communities based on cell numbers in formerly intractable environments.
β-Tubulin mRNA as a Marker of
Cryptosporidium parvum
Oocyst Viability
Authors: Widmer Giovanni , Orbacz Elizabeth A. , Tzipori Saul 
Published: December 2019

ABSTRACT

Determining the viability of waterborne
Cryptosporidium parvum
oocysts remains a technical challenge. rRNA and mRNA were evaluated in a reverse transcription (RT)-PCR assay as potential markers of oocyst viability. The rationale for this approach is the rapid turnover and postmortem decay of cellular RNA. The β-tubulin mRNA and an anonymous mRNA transcript were chosen as potential markers because they are the only mRNA species in
C. parvum
known to possess introns. This feature facilitated the distinction between genuine RT-PCR products and PCR products originating from copurifying DNA. Prolonged incubation at room temperature of initially viable oocysts resulted in a gradual decrease in mRNA levels, which correlated with the loss of oocyst infectivity to neonatal mice. In contrast, oocysts stored at 4°C for over 39 weeks maintained their infectivity and displayed no decrease in the level of β-tubulin RT-PCR product. The postmortem decay of two mRNA species demonstrates that RT-PCR analysis can provide information on the viability of
C. parvum
oocysts. The methodological similarity between PCR detection and RT-PCR viability analysis could facilitate the development of a combined detection and viability assay.
Cloning and Partial Characterization of Endopolygalacturonase Genes fromBotrytis cinerea
Authors: Wubben J. P. , Mulder W. , ten Have A. , van Kan J. A. L. , Visser J. 
Published: December 2019

ABSTRACTBotrytis cinereais a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene fromB. cinerea(Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes fromB. cinereaSAS56. The identity at the amino acid level between the six endoPGs ofB. cinereavaried from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins fromB. cinereaSAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains ofB. cinerea, as well as in otherBotrytisspecies. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.
Counting and Size Classification of Active Soil Bacteria by Fluorescence In Situ Hybridization with an rRNA Oligonucleotide Probe
Authors: Christensen Henrik , Hansen Michael , Sørensen Jan 
Published: December 2019

ABSTRACT

A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled oligonucleotide probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA probe, EUB338, for the domain
Bacteria
was performed with a soil slurry, and this was followed by collection of the bacteria by membrane filtration (pore size, 0.2 μm). A nonsense probe, NONEUB338 (which has a nucleotide sequence complementary to the nucleotide sequence of probe EUB338), was used as a control for nonspecific staining. Counting and size classification into groups of small, medium, and large bacteria were performed by fluorescence microscopy. To compensate for a difference in the relative staining intensities of the probes and for binding by the rhodamine part of the probe, control experiments in which excess unlabelled probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that nonspecific staining was due to binding of rhodamine to the bacteria. A value of 4.8 × 10
8
active bacteria per g of dry soil was obtained for bulk soil incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 × 10
8
active bacteria per g of dry soil was obtained for soil which had been air dried and subsequently rewetted. In both soils, the majority (68 to 77%) of actively growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 μm), but the active (and growing) bacteria still represented only approximately 5% of the total bacterial population determined by DAPI (4′,6-diamidino-2-phenylindole) staining. The FISH technique in which slurry hybridization is used holds great promise for use with phylogenetic probes and for automatic counting of soil bacteria.
Semiautomated Metabolic Staining Assay for
Bacillus cereus
Emetic Toxin
Authors: Finlay W. J. J. , Logan N. A. , Sutherland A. D. 
Published: December 2019

ABSTRACT

This paper describes a specific, sensitive, semiautomated, and quantitative Hep-2 cell culture-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for
Bacillus cereus
emetic toxin. Of nine
Bacillus
,
Brevibacillus
, and
Paenibacillus
species assessed for emetic toxin production, only
B. cereus
was cytotoxic.
Expression of Two Major Chitinase Genes of
Trichoderma atroviride
(
T. harzianum
P1) Is Triggered by Different Regulatory Signals
Authors: Mach Robert L. , Peterbauer Clemens K. , Payer Kathrin , Jaksits Sylvia , Woo Sheridan L. , Zeilinger Susanne , Kullnig Cornelia M. , Lorito Matteo , Kubicek Christian P. 
Published: December 2019

ABSTRACT

Regulation of the expression of the two major chitinase genes,
ech42
(encoding the CHIT42 endochitinase) and
nag1
(encoding the CHIT73
N
-acetyl-β-
d
-glucosaminidase), of the chitinolytic system of the mycoparasitic biocontrol fungus
Trichoderma atroviride
(=
Trichoderma harzianum
P1) was investigated by using a reporter system based on the
Aspergillus niger
glucose oxidase. Strains harboring fusions of the
ech42
or
nag1
5′ upstream noncoding sequences with the
A. niger goxA
gene displayed a glucose oxidase activity pattern that was consistent under various conditions with expression of the native
ech42
and
nag1
genes, as assayed by Northern analysis. The expression product of
goxA
in the mutants was completely secreted into the medium, detectable on Western blots, and quantifiable by enzyme-linked immunosorbent assay.
nag1
gene expression was triggered during growth on fungal (
Botrytis cinerea
) cell walls and on the chitin degradation product
N
-acetylglucosamine.
N
-Acetylglucosamine, di-
N
-acetylchitobiose, or tri-
N
-acetylchitotriose also induced
nag1
gene expression when added to mycelia pregrown on different carbon sources.
ech42
expression was also observed during growth on fungal cell walls but, in contrast, was not triggered by addition of chitooligomers to pregrown mycelia. Significant
ech42
expression was observed after prolonged carbon starvation, independent of the use of glucose or glycerol as a carbon source, suggesting that relief of carbon catabolite repression was not involved in induction during starvation. In addition,
ech42
gene transcription was triggered by physiological stress, such as low temperature, high osmotic pressure, or the addition of ethanol. Four copies of a putative stress response element (CCCCT) were found in the
ech42
promoter.
d
-Pantothenate Synthesis in
Corynebacterium glutamicum
and Use of
panBC
and Genes Encoding
l
-Valine Synthesis for
d
-Pantothenate Overproduction
Authors: Sahm Hermann , Eggeling Lothar 
Published: December 2019

ABSTRACT

d
-Pantothenate is synthesized via four enzymes from ketoisovalerate, which is an intermediate of branched-chain amino acid synthesis. We quantified three of these enzyme activities in
Corynebacterium glutamicum
and determined specific activities ranging from 0.00014 to 0.001 μmol/min mg (protein)
−1
. The genes encoding the ketopantoatehydroxymethyl transferase and the pantothenate synthetase were cloned, sequenced, and functionally characterized. These studies suggest that
panBC
constitutes an operon. By using
panC
, an assay system was developed to quantify
d
-pantothenate. The wild type of
C. glutamicum
was found to accumulate 9 μg of this vitamin per liter. A strain was constructed (i) to abolish
l
-isoleucine synthesis, (ii) to result in increased ketoisovalerate formation, and (iii) to enable its further conversion to
d
-pantothenate. The best resulting strain has
ilvA
deleted from its chromosome and has two plasmids to overexpress genes of ketoisovalerate (
ilvBNCD
) and
d
-pantothenate (
panBC
) synthesis. With this strain a
d
-pantothenate accumulation of up to 1 g/liter is achieved, which is a 10
5
-fold increase in concentration compared to that of the original wild-type strain. From the series of strains analyzed it follows that an increased ketoisovalerate availability is mandatory to direct the metabolite flux into the
d
-pantothenate-specific part of the pathway and that the availability of β-alanine is essential for
d
-pantothenate formation.
Study of the Response of a Biofilm Bacterial Community to UV Radiation
Authors: Elasri Mohamed O. , Miller Robert V. 
Published: December 2019

ABSTRACT

We have developed a bioluminescent whole-cell biosensor that can be incorporated into biofilm ecosystems. RM4440 is a
Pseudomonas aeruginosa
FRD1 derivative that carries a plasmid-based
recA-luxCDABE
fusion. We immobilized RM4440 in an alginate matrix to simulate a biofilm, and we studied its response to UV radiation damage. The biofilm showed a protective property by physical shielding against UV C, UV B, and UV A. Absorption of UV light by the alginate matrix translated into a higher survival rate than observed with planktonic cells at similar input fluences. UV A was shown to be effectively blocked by the biofilm matrix and to have no detectable effects on cells contained in the biofilm. However, in the presence of photosensitizers (i.e., psoralen), UV A was effective in inducing light production and cell death. RM4440 has proved to be a useful tool to study microbial communities in a noninvasive manner.
A New Sensitive Bioassay for Determination of Microbially Available Phosphorus in Water
Authors: Lehtola Markku J. , Miettinen Ilkka T. , Vartiainen Terttu , Martikainen Pertti J. 
Published: December 2019

ABSTRACT

The content of assimilable organic carbon has been proposed to control the growth of microbes in drinking water. However, recent results have shown that there are regions where it is predominantly phosphorus which determines the extent of microbial growth in drinking waters. Even a very low concentration of phosphorus (below 1 μg of P liter
−1
) can promote extensive microbial growth. We present here a new sensitive method to determine microbially available phosphorus concentrations in water down to 0.08 μg of P liter
−1
. The method is a bioassay in which the analysis of phosphorus in a water sample is based on maximum growth of
Pseudomonas fluorescens
P17 when the energy supply and inorganic nutrients, with the exception of phosphorus, do not limit bacterial growth. Maximum growth (CFU) in the water sample is related to the concentration of phosphorus with the factor 373,200 ± 9,400 CFU/μg of PO
4
-P. A linear relationship was found between cell growth and phosphorus concentration between 0.05 to 10 μg of PO
4
-P liter
−1
. The content of microbially available phosphorus in Finnish drinking waters varied from 0.1 to 10.2 μg of P liter
−1
(median, 0.60 μg of P liter
−1
).
Pullulanase Type I from
Fervidobacterium pennavorans
Ven5: Cloning, Sequencing, and Expression of the Gene and Biochemical Characterization of the Recombinant Enzyme
Authors: Bertoldo Costanzo , Duffner Fiona , Jorgensen Per L. , Antranikian Garabed 
Published: December 2019

ABSTRACT

The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium
Fervidobacterium pennavorans
Ven5 was cloned and sequenced in
Escherichia coli
. The
pulA
gene from
F. pennavorans
Ven5 had 50.1% pairwise amino acid identity with
pulA
from the anaerobic hyperthermophile
Thermotoga maritima
and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (
pulA
) encodes a protein of 849 amino acids with a 28-residue signal peptide. The
pulA
gene was subcloned without its signal sequence and overexpressed in
E. coli
under the control of the
trc
promoter. This clone,
E. coli
FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80°C and had a half-life of 2 h at 80°C. The rPulAs hydrolyzed α-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, α-β-limited dextrin. Interestingly, amylose, which contains only α-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.
Mutational Analysis of the Role of HPr in
Listeria monocytogenes
Authors: Christensen Douglas P. , Benson Andrew K. , Hutkins Robert W. 
Published: December 2019

ABSTRACT

The regulatory role of HPr, a protein of the phosphotransferase system (PTS), was investigated in
Listeria monocytogenes
. By constructing mutations in the conserved histidine 15 and serine 46 residues of HPr, we were able to examine how HPr regulates PTS activity. The results indicated that histidine 15 was phosphorylated in a phosphoenolpyruvate (PEP)-dependent manner and was essential for PTS activity. Serine 46 was phosphorylated in an ATP-dependent manner by a membrane-associated kinase. ATP-dependent phosphorylation of serine 46 was significantly enhanced in the presence of fructose 1,6-diphosphate and resulted in a reduction of PTS activity. The presence of a charge at position 15 did not inhibit ATP-dependent phosphorylation of serine 46, a finding unique to gram-positive PEP-dependent PTSs studied to this point. Finally, HPr phosphorylated at serine 46 does not appear to possess self-phosphatase activity, suggesting a specific phosphatase protein may be essential for the recycling of HPr to its active form.
Role of Methanogens and Other Bacteria in Degradation of Dimethyl Sulfide and Methanethiol in Anoxic Freshwater Sediments
Authors: Lomans Bart P. , Op den Camp Huub J. M. , Pol Arjan , van der Drift Chris , Vogels Godfried D. 
Published: December 2019

ABSTRACTThe roles of several trophic groups of organisms (methanogens and sulfate- and nitrate-reducing bacteria) in the microbial degradation of methanethiol (MT) and dimethyl sulfide (DMS) were studied in freshwater sediments. The incubation of DMS- and MT-amended slurries revealed that methanogens are the dominant DMS and MT utilizers in sulfate-poor freshwater systems. In sediment slurries, which were depleted of sulfate, 75 μmol of DMS was stoichiometrically converted into 112 μmol of methane. The addition of methanol or MT to DMS-degrading slurries at concentrations similar to that of DMS reduced DMS degradation rates. This indicates that the methanogens in freshwater sediments, which degrade DMS, are also consumers of methanol and MT. To verify whether a competition between sulfate-reducing and methanogenic bacteria for DMS or MT takes place in sulfate-rich freshwater systems, the effects of sulfate and inhibitors, like bromoethanesulfonic acid, molybdate, and tungstate, on the degradation of MT and DMS were studied. The results for these sulfate-rich and sulfate-amended slurry incubations clearly demonstrated that besides methanogens, sulfate-reducing bacteria take part in MT and DMS degradation in freshwater sediments, provided that sulfate is available. The possible involvement of an interspecies hydrogen transfer in these processes is discussed. In general, our study provides evidence for methanogenesis as a major sink for MT and DMS in freshwater sediments.
Construction and Characterization of Two Recombinant Bacteria That Grow on
ortho
- and
para
-Substituted Chlorobiphenyls
Authors: Hrywna Yarek , Tsoi Tamara V. , Maltseva Olga V. , Quensen John F. , Tiedje James M. 
Published: December 2019

ABSTRACT

Cloning and expression of the aromatic ring dehalogenation genes in biphenyl-growing, polychlorinated biphenyl (PCB)-cometabolizing
Comamonas testosteroni
VP44 resulted in recombinant pathways allowing growth on
ortho
- and
para
-chlorobiphenyls (CBs) as a sole carbon source. The recombinant variants were constructed by transformation of strain VP44 with plasmids carrying specific genes for dehalogenation of chlorobenzoates (CBAs). Plasmid pE43 carries the
Pseudomonas aeruginosa
142
ohb
genes coding for the terminal oxygenase (ISP
OHB
) of the
ortho
-halobenzoate 1,2-dioxygenase, whereas plasmid pPC3 contains the
Arthrobacter globiformis
KZT1
fcb
genes, which catalyze the hydrolytic
para
-dechlorination of 4-CBA. The parental strain, VP44, grew only on low concentrations of 2- and 4-CB by using the products from the fission of the nonchlorinated ring of the CBs (pentadiene) and accumulated stoichiometric amounts of the corresponding CBAs. The recombinant strains VP44(pPC3) and VP44(pE43) grew on, and completely dechlorinated high concentrations (up to 10 mM), of 4-CBA and 4-CB and 2-CBA and 2-CB, respectively. Cell protein yield corresponded to complete oxidation of both biphenyl rings, thus confirming mineralization of the CBs. Hence, the use of CBA dehalogenase genes appears to be an effective strategy for construction of organisms that will grow on at least some congeners important for remediation of PCBs.
Genetic Diversity of African and Worldwide Strains of
Ralstonia solanacearum
as Determined by PCR-Restriction Fragment Length Polymorphism Analysis of the
hrp
Gene Region
Authors: Poussier Stephane , Vandewalle Peggy , Luisetti Jacques 
Published: December 2019

ABSTRACT

The genetic diversity among a worldwide collection of 120 strains of
Ralstonia solanacearum
was assessed by restriction fragment length polymorphism (RFLP) analysis of amplified fragments from the
hrp
gene region. Five amplified fragments appeared to be specific to
R. solanacearum
. Fifteen different profiles were identified among the 120 bacterial strains, and a hierarchical cluster analysis distributed them into eight clusters. Each cluster included strains belonging to a single biovar, except for strains of biovars 3 and 4, which could not be separated. However, the biovar 1 strains showed rather extensive diversity since they were distributed into five clusters whereas the biovar 2 and the biovar 3 and 4 strains were gathered into one and two clusters, respectively. PCR-RFLP analysis of the
hrp
gene region confirmed the results of previous studies which split the species into an “Americanum” division including biovar 1 and 2 strains and an “Asiaticum” division including biovar 3 and 4 strains. However, the present study showed that most of the biovar 1 strains, originating from African countries (Reunion Island, Madagascar, Zimbabwe, and Angola) and being included in a separate cluster, belong to the “Asiaticum” rather than to the “Americanum” division. These African strains could thus have evolved separately from other biovar 1 strains originating from the Americas.
Differentiation of
Methanosaeta concilii
and
Methanosarcina barkeri
in Anaerobic Mesophilic Granular Sludge by Fluorescent In Situ Hybridization and Confocal Scanning Laser Microscopy
Authors: Rocheleau Sylvie , Greer Charles W. , Lawrence John R. , Cantin Christiane , Laramée Louise , Guiot Serge R. 
Published: December 2019

ABSTRACT

Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate
Methanosaeta concilii, Methanosarcina barkeri
, and mesophilic methanogens. All
M. concilii
oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific
M. concilii
oligonucleotide probe.
Methanosarcina barkeri
oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different
Methanosarcina
species. The MB4 probe specifically detected
Methanosarcina barkeri
, and the MB3 probe detected the presence of all mesophilic
Methanosarcina
species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of
M. concilii
and
Methanosarcina barkeri
, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of
M. concilii
. In contrast, a layered structure developed in methanol-enriched granules, where
M. barkeri
growth was induced in an outer layer. This outer layer was followed by a layer composed of
M. concilii
, with an inner core of
M. concilii
and other bacteria.
Detection of Bacteriocins by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Authors: Rose Natisha L. , Sporns Peter , McMullen Lynn M. 
Published: December 2019

ABSTRACT
The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the detection of bacteriocins was investigated. A 30-s water wash of the sample on the MALDI-TOF MS probe was effective in removing contaminants of the analyte. This method was used for rapid detection of nisin, pediocin, brochocin A and B, and enterocin A and B from culture supernatants and for detection of enterocin B throughout its purification.