bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System Mediates Resistance of
Vibrio cholerae
O1 Strains to Multiple Environmental Bacteriophages
Authors: Zahid M. Shamim Hasan , Waise T. M. Zaved , Kamruzzaman M. , Ghosh Amar N. , Nair G. Balakrish , Mekalanos John J. , Faruque Shah M. 
Published: May 2010

ABSTRACT

Toxigenic
Vibrio cholerae
, the causative agent of the epidemic diarrheal disease cholera, interacts with diverse environmental bacteriophages. These interactions promote genetic diversity or cause selective enrichment of phage-resistant bacterial clones. To identify bacterial genes involved in mediating the phage-resistant phenotype, we screened a transposon insertion library of
V. cholerae
O1 El Tor biotype strain C6706 to identify mutants showing altered susceptibility to a panel of phages isolated from surface waters in Bangladesh. Mutants with insertion in
cyaA
or
crp
genes encoding adenylate cyclase or cyclic AMP (cAMP) receptor protein (CRP), respectively, were susceptible to a phage designated JSF9 to which the parent strain was completely resistant. Application of the
cyaA
mutant as an indicator strain in environmental phage monitoring enhanced phage detection, and we identified 3 additional phages to which the parent strain was resistant. Incorporation of the
cyaA
or
crp
mutations into other
V. cholerae
O1 strains caused similar alterations in their phage susceptibility patterns, and the susceptibility correlated with the ability of the bacteria to adsorb these phages. Our results suggest that cAMP-CRP-mediated downregulation of phage adsorption may contribute to a mechanism for the
V. cholerae
O1 strains to survive predation by multiple environmental phages. Furthermore, the
cyaA
or
crp
mutant strains may be used as suitable indicators in monitoring cholera phages in the water.
Novel Carbohydrate-Binding Module Identified in a Ruminal Metagenomic Endoglucanase
Authors: Duan Cheng-Jie , Liu Jun-Liang , Wu Xi , Tang Ji-Liang , Feng Jia-Xun 
Published: May 2010

ABSTRACT
Endoglucanase C5614-1 comprises a catalytic module (CM) and an X module (XM). The XM showed no significant homology with known carbohydrate-binding modules (CBMs). Recombinant full-length endoglucanase could bind Avicel, whereas the CM could not. The XM could bind various polysaccharides. The results demonstrated that the XM was a new CBM.
Presence of
Coxiella burnetii
DNA in the Environment of the United States, 2006 to 2008
Authors: Kersh Gilbert J. , Wolfe Teresa M. , Fitzpatrick Kelly A. , Candee Amanda J. , Oliver Lindsay D. , Patterson Nicole E. , Self Joshua S. , Priestley Rachael A. , Loftis Amanda D. , Massung Robert F. 
Published: May 2010

ABSTRACT

Coxiella burnetii
is an obligate intracellular bacterium that causes the zoonotic disease Q fever. Because
C. burnetii
is highly infectious, can survive under a variety of environmental conditions, and has been weaponized in the past, it is classified as a select agent and is considered a potential bioweapon. The agent is known to be present in domestic livestock and in wild animal populations, but the background levels of
C. burnetii
in the environment have not been reported. To better understand the amount of
C. burnetii
present in the environment of the United States, more than 1,600 environmental samples were collected from six geographically diverse parts of the United States in the years 2006 to 2008. DNA was purified from these samples, and the presence of
C. burnetii
DNA was evaluated by quantitative PCR of the IS
1111
repetitive element. Overall, 23.8% of the samples were positive for C.
burnetii
DNA. The prevalence in the different states ranged from 6 to 44%.
C. burnetii
DNA was detected in locations with livestock and also in locations with primarily human activity (post offices, stores, schools, etc.). This study demonstrates that
C. burnetii
is fairly common in the environment in the United States, and any analysis of
C. burnetii
after a suspected intentional release should be interpreted in light of these background levels. It also suggests that human exposure to
C. burnetii
may be more common than what is suggested by the number of reported cases of Q fever.
Production of Indole froml-Tryptophan and Effects of These Compounds on Biofilm Formation byFusobacterium nucleatumATCC 25586
Authors: Sasaki-Imamura Takako , Yano Akira , Yoshida Yasuo 
Published: May 2010

ABSTRACTThel-tryptophan degradation product indole is a purported extracellular signaling molecule that influences biofilm formation in various bacteria. Here we analyzed the mechanisms of indole production inFusobacterium nucleatumand the effects of tryptophan and indole onF. nucleatumplanktonic and biofilm cells. The amino acid sequence deduced from thefn1943gene inF. nucleatumATCC 25586 was 28% identical to that deduced fromtnaAinEscherichia coli, which encodes tryptophanase catalyzing the β-elimination ofl-tryptophan to produce indole. Thefn1943gene was cotranscribed with the downstream genefn1944, which is a homolog oftnaBencoding low-affinity tryptophan permease. The transcript started at position −68 or −153 from the first nucleotide of thefn1943translation initiation codon. Real-time quantitative PCR showed that much moreF. nucleatum fn1943transcripts were obtained from log-phase cells than from stationary-phase cells. Indole production by the purified recombinant protein encoded byfn1943was examined using high-performance liquid chromatography. TheKmandkcatof the enzyme were 0.26 ± 0.03 mM and 0.74 ± 0.04 s−1, respectively.F. nucleatumbiofilm formation and the biofilm supernatant concentration of indole increased dose dependently with increasing tryptophan concentrations. Exogenous indole also increasedF. nucleatumbiofilm formation in a dose-dependent manner. Even at very high concentrations, tryptophan did not affectfn1943expression, whereas similar indole concentrations decreased expression. Thus, exogenous tryptophan and indole were suggested to increaseF. nucleatumbiofilms.
Animal-to-Animal Variation in Fecal Microbial Diversity among Beef Cattle
Authors: Durso Lisa M. , Harhay Gregory P. , Smith Timothy P. L. , Bono James L. , DeSantis Todd Z. , Harhay Dayna M. , Andersen Gary L. , Keen James E. , Laegreid William W. , Clawson Michael L. 
Published: May 2010

ABSTRACT
The intestinal microbiota of beef cattle are important for animal health, food safety, and methane emissions. This full-length sequencing survey of 11,171 16S rRNA genes reveals animal-to-animal variation in communities that cannot be attributed to breed, gender, diet, age, or weather. Beef communities differ from those of dairy. Core bovine taxa are identified.
Adaptation in a Mouse Colony Monoassociated with
Escherichia coli
K-12 for More than 1,000 Days
Authors: Lee Sean M. , Wyse Aaron , Lesher Aaron , Everett Mary Lou , Lou Linda , Holzknecht Zoie E. , Whitesides John F. , Spears Patricia A. , Bowles Dawn E. , Lin Shu S. , Tonkonogy Susan L. , Orndorff Paul E. , Bollinger R. Randal , Parker William 
Published: May 2010

ABSTRACT

Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with
Escherichia coli
K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of
E. coli
was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent
E. coli
. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of
E. coli
, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis
in vivo
, and an increased tendency for spontaneous lysis
in vitro
. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.
Natural Competence in
Thermoanaerobacter
and
Thermoanaerobacterium
Species
Authors: Shaw A. Joe , Hogsett David A. , Lynd Lee R. 
Published: May 2010

ABSTRACT

Low-G+C thermophilic obligate anaerobes in the class
Clostridia
are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13
Thermoanaerobacter
and
Thermoanaerobacterium
strains previously believed to be difficult to transform or genetically recalcitrant. In
Thermoanaerobacterium saccharolyticum
JW/SL-YS485, natural competence-mediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In
T. saccharolyticum
, disruptions of genes similar to
comEA
,
comEC
, and a type IV pilus (T4P) gene operon result in strains unable to incorporate further DNA, suggesting that natural competence occurs via a conserved Gram-positive mechanism. The relative ease of employing natural competence for gene transfer should foster genetic engineering in these industrially relevant organisms, and understanding the mechanisms underlying natural competence may be useful in increasing the applicability of genetic tools to difficult-to-transform organisms.
Influence of Low Oxygen Tensions and Sorption to Sediment Black Carbon on Biodegradation of Pyrene
Authors: Ortega-Calvo José-Julio , Gschwend Philip M. 
Published: May 2010

ABSTRACT

Sorption to sediment black carbon (BC) may limit the aerobic biodegradation of polycyclic aromatic hydrocarbons (PAHs) in resuspension events and intact sediment beds. We examined this hypothesis experimentally under conditions that were realistic in terms of oxygen concentrations and BC content. A new method, based on synchronous fluorescence observations of
14
C-pyrene, was developed for continuously measuring the uptake of dissolved pyrene by
Mycobacterium gilvum
VM552, a representative degrader of PAHs. The effect of oxygen and pyrene concentrations on pyrene uptake followed Michaelis-Menten kinetics, resulting in a dissolved oxygen half-saturation constant (
K
om
) of 14.1 μM and a dissolved pyrene half-saturation constant (
K
pm
) of 6 nM. The fluorescence of
14
C-pyrene in air-saturated suspensions of sediments and induced cells followed time courses that reflected simultaneous desorption and biodegradation of pyrene, ultimately causing a quasi-steady-state concentration of dissolved pyrene balancing desorptive inputs and biodegradation removals. The increasing concentrations of
14
CO
2
in these suspensions, as determined with liquid scintillation, evidenced the strong impact of sorption to BC-rich sediments on the biodegradation rate. Using the best-fit parameter values, we integrated oxygen and sorption effects and showed that oxygen tensions far below saturation levels in water are sufficient to enable significant decreases in the steady-state concentrations of aqueous-phase pyrene. These findings may be relevant for bioaccumulation scenarios that consider the effect of sediment resuspension events on exposure to water column and sediment pore water, as well as the direct uptake of PAHs from sediments.
Escherichia coli
Strains Engineered for Homofermentative Production of
d
-Lactic Acid from Glycerol
Authors: Mazumdar Suman , Clomburg James M. , Gonzalez Ramon 
Published: May 2010

ABSTRACT

Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in
Escherichia coli
under anaerobic and microaerobic conditions. In this work, we engineer
E. coli
for the efficient conversion of glycerol to
d
-lactic acid (
d
-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for
d
-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of
d
-lactate from pyruvate (
d
-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (Δ
pflB
) and fumarate reductase (Δ
frdA
) (strain LA01) and (ii) inactivation of fumarate reductase (Δ
frdA
), phosphate acetyltransferase (Δ
pta
), and alcohol/acetaldehyde dehydrogenase (Δ
adhE
) (strain LA02). A mutation that blocked the aerobic
d
-lactate dehydrogenase (Δ
dld
) also was introduced in both LA01 and LA02 to prevent the utilization of
d
-lactate. The most efficient strain (LA02Δ
dld
, with GlpK-GlpD overexpressed) produced 32 g/liter of
d
-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for
d
-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of
d
-lactate and is redox balanced, thus representing a viable metabolic pathway.
Genetic and Metabolic Divergence within aRhizobium leguminosarumbv.trifoliiPopulation Recovered from Clover Nodules
Authors: Wielbo Jerzy , Marek-Kozaczuk Monika , Mazur Andrzej , Kubik-Komar Agnieszka , Skorupska Anna 
Published: May 2010

ABSTRACTRhizobia are able to establish symbiosis with leguminous plants and usually occupy highly complex soil habitats. The large size and complexity of their genomes are considered advantageous, possibly enhancing their metabolic and adaptive potential and, in consequence, their competitiveness. A population ofRhizobium leguminosarumbv.trifoliiorganisms recovered from nodules of several clover plants growing in each other's vicinity in the soil was examined regarding possible relationships between their metabolic-physiological properties and their prevalence in such a local population. Genetic and metabolic variability within theR. leguminosarumbv.trifoliistrains occupying nodules of several plants was of special interest, and both types were found to be considerable. Moreover, a prevalence of metabolically versatile strains, i.e., those not specializing in utilization of any group of substrates, was observed by combining statistical analyses of Biolog test results with the frequency of occurrence of genetically distinct strains. Metabolic versatility with regard to nutritional requirements was not directly advantageous for effectiveness in the symbiotic interaction with clover: rhizobia with specialized metabolism were more effective in symbiosis but rarely occurred in the population. The significance of genetic and, especially, metabolic complexity of bacteria constituting a nodule population is discussed in the context of strategies employed by bacteria in competition.
Application of the
Saccharomyces cerevisiae
FLP/
FRT
Recombination System in Filamentous Fungi for Marker Recycling and Construction of Knockout Strains Devoid of Heterologous Genes
Authors: Kopke Katarina , Hoff Birgit , Kück Ulrich 
Published: May 2010

ABSTRACT

To overcome the limited availability of antibiotic resistance markers in filamentous fungi, we adapted the FLP/
FRT
recombination system from the yeast
Saccharomyces cerevisiae
for marker recycling. We tested this system in the penicillin producer
Penicillium chrysogenum
using different experimental approaches. In a two-step application, we first integrated ectopically a nourseothricin resistance cassette flanked by the
FRT
sequences in direct repeat orientation (
FRT
-
nat1
cassette) into a
P. chrysogenum
recipient. In the second step, the gene for the native yeast FLP recombinase, and in parallel, a codon-optimized
P. chrysogenum flp
(
Pcflp
) recombinase gene, were transferred into the
P. chrysogenum
strain carrying the
FRT
-
nat1
cassette. The corresponding transformants were analyzed by PCR, growth tests, and sequencing to verify successful recombination events. Our analysis of several single- and multicopy transformants showed that only when the codon-optimized recombinase was present could a fully functional recombination system be generated in
P. chrysogenum
. As a proof of application of this system, we constructed a ΔPcku70 knockout strain devoid of any heterologous genes. To further improve the FLP/
FRT
system, we produced a flipper cassette carrying the
FRT
sites as well as the
Pcflp
gene together with a resistance marker. This cassette allows the controlled expression of the recombinase gene for one-step marker excision. Moreover, the applicability of the optimized FLP/
FRT
recombination system in other fungi was further demonstrated by marker recycling in the ascomycete
Sordaria macrospora
. Here, we discuss the application of the optimized FLP/
FRT
recombination system as a molecular tool for the genetic manipulation of filamentous fungi.
γ-Glutamylmethylamide Is an Essential Intermediate in the Metabolism of Methylamine by
Methylocella silvestris
Authors: Chen Yin , Scanlan Julie , Song Lijiang , Crombie Andrew , Rahman M. Tanvir , Schäfer Hendrik , Murrell J. Colin 
Published: May 2010

ABSTRACT

Methylocella silvestris
BL2, a facultative methane utilizer, can grow on monomethylamine (MMA) as a sole carbon and nitrogen source. No activity of MMA dehydrogenase was detectable. Instead, this bacterium utilizes a methylated amino acid pathway (γ-glutamylmethylamide [GMA] and
N
-methylglutamate [NMG]) for MMA metabolism. The activities of the two key enzymes in this pathway, GMA synthetase and NMG dehydrogenase, were found when the bacterium was grown on MMA. GMA was detected by high-performance liquid chromatography-mass spectrometry only when the bacterium was grown on MMA but not when it was grown on methanol. Proteomic analysis of soluble and membrane fractions of the proteome from MMA- and methanol-grown cultures revealed that an eight-gene cluster (Msil2632 to Msil2639) was induced by MMA and cotranscribed as an operon, as shown by reverse transcription-PCR. GMA-dissimilating enzyme activity was also detected when it was grown on MMA. Formaldehyde and ammonium production from GMA was dependent on glutamate but not on α-ketoglutarate. Marker exchange mutagenesis of a putative GMAS gene homologue (
gmas
, Msil2635) within this eight-gene cluster, with a kanamycin gene cassette, abolished growth of
M. silvestris
on MMA as either a sole carbon or a sole nitrogen source. Overall, our results suggest that
gmas
is essential in MMA metabolism by
M. silvestris
.
Expression of
Xylella fastidiosa
Fimbrial and Afimbrial Proteins during Biofilm Formation
Authors: Caserta R. , Takita M. A. , Targon M. L. , Rosselli-Murai L. K. , de Souza A. P. , Peroni L. , Stach-Machado D. R. , Andrade A. , Labate C. A. , Kitajima E. W. , Machado M. A. , de Souza A. A. 
Published: May 2010

ABSTRACT

Complete sequencing of the
Xylella fastidiosa
genome revealed characteristics that have not been described previously for a phytopathogen. One characteristic of this genome was the abundance of genes encoding proteins with adhesion functions related to biofilm formation, an essential step for colonization of a plant host or an insect vector. We examined four of the proteins belonging to this class encoded by genes in the genome of
X. fastidiosa
: the PilA2 and PilC fimbrial proteins, which are components of the type IV pili, and XadA1 and XadA2, which are afimbrial adhesins. Polyclonal antibodies were raised against these four proteins, and their behavior during biofilm development was assessed by Western blotting and immunofluorescence assays. In addition, immunogold electron microscopy was used to detect these proteins in bacteria present in xylem vessels of three different hosts (citrus, periwinkle, and hibiscus). We verified that these proteins are present in
X. fastidiosa
biofilms but have differential regulation since the amounts varied temporally during biofilm formation, as well as spatially within the biofilms. The proteins were also detected in bacteria colonizing the xylem vessels of infected plants.
Combined Use of Fluorescent Dyes and Flow Cytometry To Quantify the Physiological State ofPichia pastorisduring the Production of Heterologous Proteins in High-Cell-Density Fed-Batch Cultures
Authors: Hyka Petr , Züllig Thomas , Ruth Claudia , Looser Verena , Meier Christian , Klein Joachim , Melzoch Karel , Meyer Hans-Peter , Glieder Anton , Kovar Karin 
Published: May 2010

ABSTRACTMatching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinantPichia pastorisMut+(methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.