bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Mutation in
Flavobacterium psychrophilum tlpB
Inhibits Gliding Motility and Induces Biofilm Formation
Authors: Álvarez B. , Secades P. , Prieto M. , McBride M. J. , Guijarro J. A. 
Published: June 2006

ABSTRACT

Flavobacterium psychrophilum
is a psychrotrophic, fish-pathogenic bacterium belonging to the
Cytophaga-Flavobacterium-Bacteroides
group. Tn
4351
-induced mutants deficient in gliding motility, growth on iron-depleted media, and extracellular proteolytic activity were isolated. Some of these mutants were affected in only one of these characteristics, whereas others had defects in two or more. FP523, a mutant deficient in all of these properties, was studied further. FP523 had a Tn
4351
insertion in
tlpB
(thiol oxidoreductase-like protein gene), which encodes a 41.4-kDa protein whose sequence does not exhibit high levels of similar to the sequences of proteins having known functions. TlpB has two domains; the N-terminal domains has five transmembrane regions, whereas the C-terminal domains has the Cys-X-X-Cys motif and other conserved motifs characteristic of thiol:disulfide oxidoreductases. Quantitative analysis of the thiol groups of periplasmic proteins revealed that TlpB is required for reduction of these groups. The
tlpB
gene is part of the
fpt
(
F. psychrophilum
thiol oxidoreductase) operon that contains two other genes,
tlpA
and
tpiA
, which encode a thiol:disulfide oxidoreductase and a triosephosphate isomerase, respectively. FP523 exhibited enhanced biofilm formation and decreased virulence and cytotoxicity. Complementation with the
tlpB
loci restored the wild-type phenotype. Gliding motility and biofilm formation appear to be antagonistic properties, which are both affected by TlpB.
Fluorescence In Situ Hybridization-Flow Cytometry-Cell Sorting-Based Method for Separation and Enrichment of Type I and Type II Methanotroph Populations
Authors: Kalyuzhnaya Marina G. , Zabinsky Rebecca , Bowerman Sarah , Baker David R. , Lidstrom Mary E. , Chistoserdova Ludmila 
Published: June 2006

ABSTRACT

A fluorescence in situ hybridization-flow cytometry (FISH/FC)-based method was optimized using artificial mixtures of pure cultures of methanotrophic bacteria. Traditional oligonucleotide probes targeting 16S rRNAs of type I (MG84/705 probe) and type II (MA450 probe) methanotrophs were labeled with fluorescein or Alexa fluor and used for FISH, followed by fluorescence-activated FC analysis and cell sorting (FACS). The method resulted in efficient separation of target cells (type I or type II methanotrophs) from the artificial mixtures. The method was then applied for detection and enrichment of type I and type II methanotroph populations from a natural sample, Lake Washington sediment. Cells were extracted from the sediment, fixed, and subjected to FISH/FC/FACS. The resulting subpopulations were analyzed by reverse transcriptase PCR surveys of 16S rRNA,
pmoA
(encoding a subunit of particulate methane monooxygenase), and
fae
(encoding formaldehyde-activating enzyme) genes. The functional gene analysis indicated specific separation of the type I and type II methanotroph populations. 16S rRNA gene analysis revealed that type I methanotrophs comprised 59% of the subpopulation separated using the type I-specific probe and that type II methanotrophs comprised 47.5% of the subpopulation separated using the type II-specific probe. Our data indicate that the FISH/FC/FACS protocol described can provide significant enrichment of microbial populations of interest from complex natural communities and that these can be used for genetic tests. We further tested the possibility of direct whole-genome amplification (WGA) from limited numbers of sorted cells, using artificial mixtures of microbes whose genome sequences are known. We demonstrated that efficient WGA can be achieved using 10
4
or more cells separated by 16S rRNA-specific FISH/FC/FACS, while fewer cells resulted in less specific WGA.
Quantification of In Vitro and In Vivo
Cryptosporidium parvum
Infection by Using Real-Time PCR
Authors: Godiwala Nihal T. , Vandewalle Alain , Ward Honorine D. , Leav Brett A. 
Published: June 2006

ABSTRACT

Established methods for quantifying experimental
Cryptosporidium
infection are highly variable and subjective. We describe a new technique using quantitative real-time PCR (qPCR) that can be used to measure in vitro and in vivo laboratory infections with
Cryptosporidium
. We show for the first time that qPCR permits absolute quantification of the parasite while simultaneously controlling for the amount of host tissue and correlates significantly with established methods of quantification in in vitro and in vivo laboratory models of infection.
Thermophilic Temperature Optimum for Crenarchaeol Synthesis and Its Implication for Archaeal Evolution
Authors: Zhang Chuanlun L. , Pearson Ann , Li Yi-Liang , Mills Gary , Wiegel Juergen 
Published: June 2006

ABSTRACT

The isoprenoid lipid crenarchaeol is widespread in hot springs of California and Nevada. Terrestrial and marine data together suggest a maximum relative abundance of crenarchaeol at ∼40°C. This warm temperature optimum may have facilitated colonization of the ocean by (hyper)thermophilic
Archaea
and the major marine radiation of
Crenarchaeota
.
Nitrification in a Biofilm at Low pH Values: Role of In Situ Microenvironments and Acid Tolerance
Authors: Gieseke Armin , Tarre Sheldon , Green Michal , de Beer Dirk 
Published: June 2006

ABSTRACT

The sensitivity of nitrifying bacteria to acidic conditions is a well-known phenomenon and generally attributed to the lack and/or toxicity of substrates (NH
3
and HNO
2
) with decreasing pHs. In contrast, we observed strong nitrification at a pH around 4 in biofilms grown on chalk particles and investigated the following hypotheses: the presence of less acidic microenvironments and/or the existence of acid-tolerant nitrifiers. Microelectrode measurements (in situ and under various experimental conditions) showed no evidence of a neutral microenvironment, either within the highly active biofilm colonizing the chalk surface or within a control biofilm grown on a nonbuffering (i.e., sintered glass) surface under acidic pH. A 16S rRNA approach (clone libraries and fluorescence in situ hybridizations) did not reveal uncommon nitrifying (potentially acid-tolerant) strains. Instead, we found a strongly acidic microenvironment, evidence for a clear adaptation to the low pH in situ, and the presence of nitrifying populations related to subgroups with low

K
m

s for ammonia (
Nitrosopira
spp.,
Nitrosomonas oligotropha
, and
Nitrospira
spp.). Acid-consuming (chalk dissolution) and acid-producing (ammonia oxidation) processes are equilibrated on a low-pH steady state that is controlled by mass transfer limitation through the biofilm. Strong affinity to ammonia and possibly the expression of additional functions, e.g., ammonium transporters, are adaptations that allow nitrifiers to cope with acidic conditions in biofilms and other habitats.
Three Enzymes for Trehalose Synthesis in
Bradyrhizobium
Cultured Bacteria and in Bacteroids from Soybean Nodules
Authors: Streeter J. G. , Gomez M. L. 
Published: June 2006

ABSTRACT

α,α-Trehalose is a disaccharide accumulated by many microorganisms, including rhizobia, and a common role for trehalose is protection of membrane and protein structure during periods of stress, such as desiccation. Cultured
Bradyrhizobium japonicum
and
B. elkanii
were found to have three enzymes for trehalose synthesis: trehalose synthase (TS), maltooligosyltrehalose synthase (MOTS), and trehalose-6-phosphate synthetase. The activity level of the latter enzyme was much higher than those of the other two in cultured bacteria, but the reverse was true in bacteroids from nodules. Although TS was the dominant enzyme in bacteroids, the source of maltose, the substrate for TS, is not clear; i.e., the maltose concentration in nodules was very low and no maltose was formed by bacteroid protein preparations from maltooligosaccharides. Because bacteroid protein preparations contained high trehalase activity, it was imperative to inhibit this enzyme in studies of TS and MOTS in bacteroids. Validamycin A, a commonly used trehalase inhibitor, was found to also inhibit TS and MOTS, and other trehalase inhibitors, such as trehazolin, must be used in studies of these enzymes in nodules. The results of a survey of five other species of rhizobia indicated that most species sampled had only one major mechanism for trehalose synthesis. The presence of three totally independent mechanisms for the synthesis of trehalose by
Bradyrhizobium
species suggests that this disaccharide is important in the function of this organism both in the free-living state and in symbiosis.
Identification of the Leucine-to-2-Methylbutyric Acid Catabolic Pathway of
Lactococcus lactis
Authors: Ganesan Balasubramanian , Dobrowolski Piotr , Weimer Bart C. 
Published: June 2006

ABSTRACT
Nutrient starvation and nonculturability in bacteria lead to changes in metabolism not found during the logarithmic phase. Substrates alternate to those used during growth are metabolized in these physiological states, yielding secondary metabolites. In firmicutes and actinobacteria, amino acid catabolic pathways are induced during starvation and nonculturability. Examination of lactococci showed that the population entered a nonculturable state after carbohydrate depletion and was incapable of growth on solid media; however, the cells gained the ability to produce branched-chain fatty acids from amino acids. Gene expression profiling and in silico pathway analysis coupled with nuclear magnetic resonance spectroscopy were used to delineate the leucine catabolic pathway. Lactococci produced acetic and propionic acid during logarithmic growth and starvation. At the onset of nonculturability, 2-methylbutyric acid was produced via hydroxymethyl-glutaryl-coenzyme A (CoA) and acetyl-CoA, along with ATP and oxidation/reduction precursors. Gene expression profiling and genome sequence analysis showed that lactococci contained redundant genes for branched-chain fatty acid production that were regulated by an unknown mechanism linked to carbon metabolism. This work demonstrated the ability of a firmicute to induce new metabolic capabilities in the nonculturable state for producing energy and intermediates needed for transcription and translation. Phylogenetic analyses showed that homologues of these enzymes and their functional motifs were widespread across the domains of life.
The
PKS4
Gene of
Fusarium graminearum
Is Essential for Zearalenone Production
Authors: Lysøe Erik , Klemsdal Sonja S. , Bone Karen R. , Frandsen Rasmus J. N. , Johansen Thomas , Thrane Ulf , Giese Henriette 
Published: June 2006

ABSTRACT

Zearalenones are produced by several
Fusarium
species and can cause reproductive problems in animals. Some aurofusarin mutants of
Fusarium pseudograminearum
produce elevated levels of zearalenone (ZON), one of the estrogenic mycotoxins comprising the zearalenones. An analysis of transcripts from polyketide synthase genes identified in the
Fusarium graminearum
database was carried out for these mutants.
PKS4
was the only gene with an enoyl reductase domain that had a higher level of transcription in the aurofusarin mutants than in the wild type. An
Agrobacterium tumefaciens-
mediated transformation protocol was used to replace the central part of the
PKS4
gene with a
hygB
resistance gene through double homologous recombination in an
F. graminearum
strain producing a high level of ZON. PCR and Southern analysis of transformants were used to identify isolates with single insertional replacements of
PKS4
. High-performance liquid chromatography analysis showed that the
PKS4
replacement mutant did not produce ZON. Thus,
PKS4
encodes an enzyme required for the production of ZON in
F. graminearum
. Barley root infection studies revealed no alteration in the pathogenicity of the
PKS4
mutant compared to the pathogenicity of the wild type. The expression of
PKS13
, which is located in the same cluster as
PKS4
, decreased dramatically in the mutant, while transcription of
PKS4
was unchanged. This differential expression may indicate that ZON or its derivatives do not regulate expression of
PKS4
and that the
PKS4
-encoded protein or its product stimulates expression of
PKS13
. Furthermore, both the lack of aurofusarin and ZON influenced the expression of other polyketide synthases, demonstrating that one polyketide can influence the expression of others.
Inactivation ofMycobacterium aviumsubsp.paratuberculosisin Cow's Milk by Means of High Hydrostatic Pressure at Mild Temperatures
Authors: López-Pedemonte Tomás , Sevilla Iker , Garrido Joseba M. , Aduriz Gorka , Guamis Buenaventura , Juste Ramón A. , Roig-Sagués Artur X. 
Published: June 2006

ABSTRACTTwo strains ofMycobacterium aviumsubsp.paratuberculosis(3644/02 and ATCC 19698) were inoculated (approximately 6 log CFU/ml) into sterilized milk to evaluate inactivation by high hydrostatic pressure. Reductions ofM. aviumsubsp.paratuberculosisincreased with pressure level. Significant differences were also found betweenM. aviumsubsp.paratuberculosisstrains and between the media used. Average reductions of 4 log CFU/ml after treatment with 500 MPa are comparable to those caused by thermal treatments.
Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR
Authors: Wolffs Petra F. G , Glencross Kari , Thibaudeau Romain , Griffiths Mansel W. 
Published: June 2006

ABSTRACT

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the
invA
gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 μm to remove large particles and of 0.22 μm to recover the
Salmonella
cells. After this, the
Salmonella
cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 × 10
2
CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.
Analysis of Amino Acid Residues Involved in Catalysis of Polyethylene Glycol Dehydrogenase from
Sphingopyxis terrae
, Using Three-Dimensional Molecular Modeling-Based Kinetic Characterization of Mutants
Authors: Ohta Takeshi , Kawabata Takeshi , Nishikawa Ken , Tani Akio , Kimbara Kazuhide , Kawai Fusako 
Published: June 2006

ABSTRACT

Polyethylene glycol dehydrogenase (PEGDH) from
Sphingopyxis terrae
(formerly
Sphingomonas terrae
) is composed of 535 amino acid residues and one flavin adenine dinucleotide per monomer protein in a homodimeric structure. Its amino acid sequence shows 28.5 to 30.5% identity with glucose oxidases from
Aspergillus niger
and
Penicillium amagasakiense
. The ADP-binding site and the signature 1 and 2 consensus sequences of glucose-methanol-choline oxidoreductases are present in PEGDH. Based on three-dimensional molecular modeling and kinetic characterization of wild-type PEGDH and mutant PEGDHs constructed by site-directed mutagenesis, residues potentially involved in catalysis and substrate binding were found in the vicinity of the flavin ring. The catalytically important active sites were assigned to His-467 and Asn-511. One disulfide bridge between Cys-379 and Cys-382 existed in PEGDH and seemed to play roles in both substrate binding and electron mediation. The Cys-297 mutant showed decreased activity, suggesting the residue's importance in both substrate binding and electron mediation, as well as Cys-379 and Cys-382. PEGDH also contains a motif of a ubiquinone-binding site, and coenzyme Q
10
was utilized as an electron acceptor. Thus, we propose several important amino acid residues involved in the electron transfer pathway from the substrate to ubiquinone.
Comparison of Methods of Extracting
Salmonella enterica
Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control
Authors: Klerks M. M. , van Bruggen A. H. C. , Zijlstra C. , Donnikov M. 
Published: June 2006

ABSTRACT

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of
Salmonella enterica
serovar Enteritidis from soil, manure, and compost and uses an
Escherichia coli
strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of
S. enterica
serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 × 10
3
to 1.8 × 10
3
CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent
S. enterica
-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.
Quantifying the Significance of Phage Attack on Starter Cultures: a Mechanistic Model for Population Dynamics of Phage and Their Hosts Isolated from Fermenting Sauerkraut
Authors: Mudgal P. , Breidt F. , Lubkin S. R. , Sandeep K. P. 
Published: June 2006

ABSTRACT

We investigated the possibility of using starter cultures in sauerkraut fermentation and thereby reducing the quantity of salt used in the process. This, in turn, would reduce the amount of waste salt that would enter in our water resources. Phage, naturally present in sauerkraut fermentation, could potentially affect the starter cultures introduced. Thus, a mechanistic mathematical model was developed to quantify the growth kinetics of the phage and starter cultures. The model was validated by independent experiments with two
Leuconostoc mesenteroides
strains isolated from sauerkraut and their corresponding phage. Model simulations and experimental evidence showed the presence of phage-resistant cell populations in starter cultures which replaced phage-sensitive cells, even when the initial phage density (
P
0
) and multiplicity of infection (MOI) were low (
P
0
< 1 × 10
3
PFU/ml; MOI < 10
−4
) in the MRS media. Based on the results of model simulation and parameter optimization, it was suggested that the kinetic parameters of phage-host interaction, especially the adsorption rate, vary with the initial phage and host densities and with time. The model was validated in MRS broth. Therefore, the effects of heterogeneity and other environmental factors, such as temperature and pH, should be considered to make the model applicable to commercial fermentations.
Mycobacterium aviumsubsp.paratuberculosisin Lake Catchments, in River Water Abstracted for Domestic Use, and in Effluent from Domestic Sewage Treatment Works: Diverse Opportunities for Environmental Cycling and Human Exposure
Authors: Pickup R. W. , Rhodes G. , Bull T. J. , Arnott S. , Sidi-Boumedine K. , Hurley M. , Hermon-Taylor J. 
Published: June 2006

ABSTRACTMycobacterium aviumsubsp.paratuberculosisfrom infected animals enters surface waters and rivers in runoff from contaminated pastures. We studied the River Tywi in South Wales, United Kingdom, whose catchment comprises 1,100 km2containing more than a million dairy and beef cattle and more than 1.3 million sheep. The River Tywi is abstracted for the domestic water supply. Between August 2002 and April 2003, 48 of 70 (68.8%) twice-weekly river water samples tested positive by IS900PCR. In river water, the organisms were associated with a suspended solid which was depleted by the water treatment process. Disposal of contaminated slurry back onto the land established a cycle of environmental persistence. A concentrate from 100 liters of finished water tested negative, but 1 of 54 domestic cold water tanks tested positive, indicating the potential for these pathogens to access domestic outlets. In the separate English Lake District region, with hills up to 980 m, tests forM. aviumsubsp.paratuberculosisin the high hill lakes and sediments were usually negative, but streams and sediments became positive lower down the catchment. Sediments from 9 of 10 major lakes receiving inflow from these catchments were positive, with sediment cores indicating deposition over at least 40 to 50 years. Two of 12 monthly 1-liter samples of effluent and a single 100-liter sample from the Ambleside sewage treatment works were positive forM. aviumsubsp.paratuberculosis. Since Lake Ambleside discharges into Lake Windermere, which is available for domestic supply, there is a potential for these organisms to cycle within human populations.
In Vitro Fermentation of Breast Milk Oligosaccharides by
Bifidobacterium infantis
and
Lactobacillus gasseri
Authors: Ward Robert E. , Niñonuevo Milady , Mills David A. , Lebrilla Carlito B. , German J. Bruce 
Published: June 2006

ABSTRACT

It has been proposed that human milk oligosaccharides (HMO) function as a prebiotic for bifidobacteria, yet this activity has not been adequately investigated. In this study,
Bifidobacterium infantis
was shown to ferment purified HMO as a sole carbon source, while another gut commensal,
Lactobacillus gasseri
, did not ferment HMO. Our results support the hypothesis that HMO selectively amplify bacterial populations in the infant intestine.
Reduction of Acid Tolerance by Tetracycline in
Escherichia coli
Expressing
tetA
(C) Is Reversed by Cations
Authors: Hung Kai F. , Byrd Jeffrey J. , Bose Jeffrey L. , Kaspar Charles W. 
Published: June 2006

ABSTRACT

When tetracycline was present,
tetA
(C) reduced acid tolerance, suppressed
rpoS
expression, and increased the concentration of total soluble proteins in stationary-phase
Escherichia coli
. The suppression of acid tolerance was reversed by 85 mM sodium, potassium, magnesium, and calcium ions but not by 85 mM sucrose. Implications for using TetA(C) are discussed.