bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of (R)-2-Hydroxyisocaproate Dehydrogenase and a Family III Coenzyme A Transferase Involved in Reduction ofl-Leucine to Isocaproate byClostridium difficile
Authors: Kim Jihoe , Darley Daniel , Selmer Thorsten , Buckel Wolfgang 
Published: September 2006

ABSTRACTThe strictly anaerobic pathogenic bacteriumClostridium difficileoccurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO2and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxyisocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates. The dehydratase requires activation by an ATP-dependent one-electron transfer (J. Kim, D. Darley, and W. Buckel, FEBS J. 272:550-561, 2005). Prior to the dehydration, a dehydrogenase and a CoA transferase are supposed to be involved in the formation of (R)-2-hydroxyisocaproyl-CoA. Deduced amino acid sequences ofldhAandhadAfrom the genome ofC. difficileshowed high identities tod-lactate dehydrogenase and family III CoA transferase, respectively. Both putative genes encoding the dehydrogenase and CoA transferase were cloned and overexpressed inEscherichia coli; the recombinant Strep tag II fusion proteins were purified to homogeneity and characterized. The substrate specificity of the monomeric LdhA (36.5 kDa) indicated that 2-oxoisocaproate (Km= 68 μM,kcat= 31 s−1) and NADH were the native substrates. For the reverse reaction, the enzyme accepted (R)- but not (S)-2-hydroxyisocaproate and therefore was named (R)-2-hydroxyisocaproate dehydrogenase. HadA showed CoA transferase activity with (R)-2-hydroxyisocaproyl-CoA as a donor and isocaproate or (E)-2-isocaprenoate as an acceptor. By site-directed mutagenesis, the conserved D171 was identified as an essential catalytic residue probably involved in the formation of a mixed anhydride with the acyl group of the thioester substrate. However, neither hydroxylamine nor sodium borohydride, both of which are inactivators of the CoA transferase, modified this residue. The dehydrogenase and the CoA transferase fit well into the proposed pathway of leucine reduction to isocaproate.
Survival of
Salmonella enterica
Serovar Newport in Manure and Manure-Amended Soils
Authors: You Youwen , Rankin Shelley C. , Aceto Helen W. , Benson Charles E. , Toth John D. , Dou Zhengxia 
Published: September 2006

ABSTRACT

Salmonella enterica
serovar Newport has undergone a rapid epidemic spread in dairy cattle. This provides an efficient mechanism for pathogen amplification and dissemination into the environment through manure spreading on agricultural land. The objective of this study was to determine the survival characteristics of
Salmonella
serovar Newport in manure and manure-amended soils where the pathogen may be amplified. A multidrug-resistant (MDR)
Salmonella
serovar Newport strain and a drug-susceptible (DS) strain, both bovine isolates, were inoculated into dairy manure that was incubated under constant temperature and moisture conditions alone or after being mixed with sterilized or nonsterilized soil.
Salmonella
serovar Newport concentrations increased by up to 400% in the first 1 to 3 days following inoculation, and a trend of steady decline followed. With manure treatment, a sharp decline in cell concentration occurred after day 35, possibly due to microbial antagonism. For all treatments, decreases in
Salmonella
serovar Newport concentrations over time fit a first-order kinetic model. Log reduction time was 14 to 32 days for 1 log
10
, 28 to 64 days for 2 log
10
, and 42 to 96 days for 3 log
10
declines in the organisms' populations from initially inoculated concentrations. Most-probable-number monitoring data indicated that the organisms persisted for 184, 332, and 405 days in manure, manure-amended nonsterilized soil, and manure-amended sterilized soil, respectively. The MDR strain and the DS strain had similar survival patterns.
Characterization of Phototrophic Purple Nonsulfur Bacteria Forming Colored Microbial Mats in a Swine Wastewater Ditch
Authors: Okubo Yoko , Futamata Hiroyuki , Hiraishi Akira 
Published: September 2006

ABSTRACTThe community structure of pink-colored microbial mats naturally occurring in a swine wastewater ditch was studied by culture-independent biomarker and molecular methods as well as by conventional cultivation methods. The wastewater in the ditch contained acetate and propionate as the major carbon nutrients. Thin-section electron microscopy revealed that the microbial mats were dominated by rod-shaped cells containing intracytoplasmic membranes of the lamellar type. Smaller numbers of oval cells with vesicular internal membranes were also found. Spectroscopic analyses of the cell extract from the biomats showed the presence of bacteriochlorophyllaand carotenoids of the spirilloxanthin series. Ubiquinone-10 was detected as the major quinone. A clone library of the photosynthetic gene,pufM, constructed from the bulk DNA of the biomats showed that all of the clones were derived from members of the generaRhodobacterandRhodopseudomonas. The dominant phototrophic bacteria from the microbial mats were isolated by cultivation methods and identified as being of the generaRhodobacterandRhodopseudomonasby studying 16S rRNA andpufMgene sequence information. Experiments of oxygen uptake with lower fatty acids revealed that the freshly collected microbial mats and theRhodopseudomonasisolates had a wider spectrum of carbon utilization and a higher affinity for acetate than did theRhodobacterisolates. These results demonstrate that the microbial mats were dominated by the purple nonsulfur bacteria of the generaRhodobacterandRhodopseudomonas, and the bioavailability of lower fatty acids in wastewater is a key factor allowing the formation of visible microbial mats with these phototrophs.
Occurrence of 1-Glyceryl-1-myo-Inosityl Phosphate in Hyperthermophiles
Authors: Lamosa Pedro , Gonçalves Luís G. , Rodrigues Marta V. , Martins Lígia O. , Raven Neil D. H. , Santos Helena 
Published: September 2006

ABSTRACTThe accumulation of compatible solutes was studied in the hyperthermophilic bacteriumAquifex pyrophilusas a function of the temperature and the NaCl concentration of the growth medium. Nuclear magnetic resonance analysis of cell extracts revealed the presence of α- and β-glutamate, di-mannosyl-di-myo-inositol phosphate, di-myo-inositol phosphate, and an additional compound here identified as 1-glyceryl-1-myo-inosityl phosphate. All solutes accumulated byA. pyrophilusare negatively charged at physiological pH. The intracellular levels of di-myo-inositol phosphate increased in response to supraoptimal growth temperature, while α- and β-glutamate accumulated in response to osmotic stress, especially at growth temperatures below the optimum. The newly discovered compound, 1-glyceryl-1-myo-inosityl phosphate, appears to play a double role in osmo- and thermoprotection, since its intracellular pool increased primarily in response to a combination of osmotic and heat stresses. This work also uncovered the nature of the unknown compound, previously detected inArchaeoglobus fulgidus(L. O. Martins et al., Appl. Environ. Microbiol. 63:896-902, 1997). The curious structural relationship between diglycerol phosphate (found only inArchaeoglobusspecies), di-myo-inositol phosphate (a canonical solute of hyperthermophiles), and the newly identified solute is highlighted. This is the first report on the occurrence of 1-glyceryl-1-myo-inosityl phosphate in living systems.
Transcriptomic Assessment of Isozymes in the Biphenyl Pathway of
Rhodococcus
sp. Strain RHA1
Authors: Gonçalves Edmilson R. , Hara Hirofumi , Miyazawa Daisuke , Davies Julian E. , Eltis Lindsay D. , Mohn William W. 
Published: September 2006

ABSTRACT

Rhodococcus
sp. RHA1 grows on a broad range of aromatic compounds and vigorously degrades polychlorinated biphenyls (PCBs). Previous work identified RHA1 genes encoding multiple isozymes for most of the seven steps of the biphenyl (BPH) pathway, provided evidence for coexpression of some of these isozymes, and indicated the involvement of some of these enzymes in the degradation of BPH, ethylbenzene (ETB), and PCBs. To investigate the expression of these isozymes and better understand how they contribute to the robust degradative capacity of RHA1, we comprehensively analyzed the 9.7-Mb genome of RHA1 for BPH pathway genes and characterized the transcriptome of RHA1 growing on benzoate (BEN), BPH, and ETB. Sequence analyses revealed 54 potential BPH pathway genes, including 28 not previously reported. Transcriptomic analysis with a DNA microarray containing 70-mer probes for 8,213 RHA1 genes revealed a suite of 320 genes of diverse functions that were upregulated during growth both on BPH and on ETB, relative to growth on the control substrate, pyruvate. By contrast, only 65 genes were upregulated during growth on BEN. Quantitative PCR assays confirmed microarray results for selected genes and indicated that some of the catabolic genes were upregulated over 10,000-fold. Our analysis suggests that up to 22 enzymes, including 8 newly identified ones, may function in the BPH pathway of RHA1. The relative expression levels of catabolic genes did not differ for BPH and ETB, suggesting a common regulatory mechanism. This study delineated a suite of catabolic enzymes for biphenyl and alkyl-benzenes in RHA1, which is larger than previously recognized and which may serve as a model for catabolism in other environmentally important bacteria having large genomes.
Detection of Microcystin-Producing Cyanobacteria in FinnishLakes with Genus-Specific Microcystin Synthetase Gene E (mcyE) PCR and Associations withEnvironmental Factors
Authors: Rantala Anne , Rajaniemi-Wacklin Pirjo , Lyra Christina , Lepistö Liisa , Rintala Jukka , Mankiewicz-Boczek Joanna , Sivonen Kaarina 
Published: September 2006

ABSTRACTWe studied the frequency and composition of potential microcystin (MC) producers in 70 Finnish lakes with general and genus-specific microcystin synthetase gene E (mcyE) PCR. Potential MC-producingMicrocystis,PlanktothrixandAnabaenaspp. existed in 70%, 63%, and 37% of the lake samples, respectively. Approximately two-thirds of the lake samples contained one or two potential MC producers, while all three genera existed in 24% of the samples. In oligotrophic lakes, the occurrence of only one MC producer was most common. The combination ofMicrocystisandPlanktothrixwas slightly more prevalent than others in mesotrophic lakes, and the cooccurrence of all three MC producers was most widespread in both eutrophic and hypertrophic lakes. The proportion of the three-producer lakes increased with the trophic status of the lakes. In correlation analysis, the presence of multiple MC-producing genera was associated with higher cyanobacterial and phytoplankton biomass, pH, chlorophylla, total nitrogen, and MC concentrations. Total nitrogen, pH, and the surface area of the lake predicted the occurrence probability ofmcyEgenes, whereas total phosphorus alone accounted for MC concentrations in the samples by logistic and linear regression analyses. In conclusion, the results suggested that eutrophication increased the cooccurrence of potentially MC-producing cyanobacterial genera, raising the risk of toxic-bloom formation.
Reductive Dehalogenase Gene Expression as a Biomarker for Physiological Activity of
Dehalococcoides
spp
Authors: Lee Patrick K. H. , Johnson David R. , Holmes Victor F. , He Jianzhong , Alvarez-Cohen Lisa 
Published: September 2006

ABSTRACT

This study characterizes the transcriptional expression of the reductive dehalogenase (RDase)-encoding
tceA
and
vcrA
genes and evaluates their applicability as potential biological markers of
Dehalococcoides
activity. When
Dehalococcoides ethenogenes
195 was provided with trichloroethene (TCE) as the electron acceptor, the expression of the
tceA
gene increased by 90-fold relative to that in cells starved of chlorinated ethenes, demonstrating that
tceA
gene expression is indicative of the active physiological state of this strain. In a
Dehalococcoides
-containing enrichment culture that contains both the
tceA
and
vcrA
genes, the
tceA
gene was up-regulated in response to TCE and
cis
-1,2-dichloroethene (cDCE) exposure, while the
vcrA
gene was up-regulated in response to TCE, cDCE, and vinyl chloride (VC). When chlorinated ethenes were depleted, the RDase-encoding gene transcripts decayed exponentially, with a half-life between 4.8 and 6.1 h, until they reached a stable background level after 2 days. We found that while gene expression correlated generally to the presence of chlorinated ethenes, there was no apparent direct relationship between RDase-encoding transcript numbers and respective rates of TCE, cDCE, and VC dechlorination activities. However, elevated
tceA
and
vcrA
expression did correlate with chlorinated-ethene reduction beyond cDCE, suggesting that elevated RDase-encoding transcript numbers could serve as a biomarker for the physiological ability of
Dehalococcoides
spp. to dechlorinate beyond cDCE.
Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of
Vibrio cholerae
Authors: Gubala Aneta J. , Proll David F. 
Published: September 2006

ABSTRACT

A multiplex real-time PCR assay was developed using molecular beacons for the detection of
Vibrio cholerae
by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.
Stability and Change in Estuarine Biofilm Bacterial Community Diversity
Authors: Moss Joseph A. , Nocker Andreas , Lepo Joe E. , Snyder Richard A. 
Published: September 2006

ABSTRACT

Biofouling communities contribute significantly to aquatic ecosystem productivity and biogeochemical cycling. Our knowledge of the distribution, composition, and activities of these microbially dominated communities is limited compared to other components of estuarine ecosystems. This study investigated the temporal stability and change of the dominant phylogenetic groups of the domain
Bacteria
in estuarine biofilm communities. Glass slides were deployed monthly over 1 year for 7-day incubations during peak tidal periods in East Sabine Bay, Fla. Community profiling was achieved by using 16S rRNA genes and terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes in combination with ribotyping, cloning, and sequencing to evaluate diversity and to identify dominant microorganisms. Bacterial community profiles from biofilms grown near the benthos showed distinct periods of constancy within winter and summer sampling periods. Similar periods of stability were also seen in T-RFLP patterns from floating biofilms. Alternating dominance of phylogenetic groups between seasons appeared to be associated with seasonal changes in temperature, nutrient availability, and light. The community structure appeared to be stable during these periods despite changes in salinity and in dissolved oxygen.
Rapid Quantitative Detection of
Lactobacillus sakei
in Meat and Fermented Sausages by Real-Time PCR
Authors: Martín Belén , Jofré Anna , Garriga Margarita , Pla Maria , Aymerich Teresa 
Published: September 2006

ABSTRACT

A quick and simple method for quantitative detection of
Lactobacillus sakei
in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a
Taq
Man fluorescence probe. Primers and probes were designed in the
L. sakei
16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using
L. sakei
genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate
L. sakei
in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of
L. sakei
colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (
P
> 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of
L. sakei
and evaluation of the implantation of starter strains of
L. sakei
in fermented sausages.
Biological Filtration Limits Carbon Availability and Affects Downstream Biofilm Formation and Community Structure
Authors: Pang Chee Meng , Liu Wen-Tso 
Published: September 2006

ABSTRACT

Carbon removal strategies have gained popularity in the mitigation of biofouling in water reuse processes, but current biofilm-monitoring practices based on organic-carbon concentrations may not provide an accurate representation of the in situ biofilm problem. This study evaluated a submerged microtiter plate assay for direct and rapid monitoring of biofilm formation by subjecting the plates to a continuous flow of either secondary effluent (SE) or biofilter-treated secondary effluent (BF). This method was very robust, based on a high correlation (
R
2
= 0.92) between the biomass (given by the
A
600
in the microtiter plate assay) and the biovolume (determined from independent biofilms developed on glass slides under identical conditions) measurements, and revealed that the biomasses in BF biofilms were consistently lower than those in SE biofilms. The influence of the organic-carbon content on the biofilm community composition and succession was further evaluated using molecular tools. Terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed a group of pioneer colonizers, possibly represented by
Sphingomonadaceae
and
Caulobacter
organisms, to be common in both SE and BF biofilms. However, differences in organic-carbon availabilities in the two water samples eventually led to the selection of distinct biofilm communities. Alphaproteobacterial populations were confirmed by fluorescence in situ hybridization to be enriched in SE biofilms, while
Betaproteobacteria
were dominant in BF biofilms. Cloning analyses further demonstrated that microorganisms adapted for survival under low-substrate conditions (e.g.,
Aquabacterium
,
Caulobacter
, and
Legionella
) were preferentially selected in the BF biofilm, suggesting that carbon limitation strategies may not achieve adequate biofouling control in the long run.
Multicopy Integration of Heterologous Genes, Using the Lactococcal Group II Intron Targeted to Bacterial Insertion Sequences
Authors: Rawsthorne Helen , Turner Kevin N. , Mills David A. 
Published: September 2006

ABSTRACTGroup II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome ofLactococcus lactisIL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, thetragene found in IS904(tra904),tra981, andtra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion oftra904,tra981, andtra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respectivetraalleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into thetra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade theL. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in differenttra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets oftra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.
Transformation of the Antibacterial Agent Norfloxacin by Environmental Mycobacteria
Authors: Adjei Michael D. , Heinze Thomas M. , Deck Joanna , Freeman James P. , Williams Anna J. , Sutherland John B. 
Published: September 2006

ABSTRACT

Because fluoroquinolone antimicrobial agents may be released into the environment, the potential for environmental bacteria to biotransform these drugs was investigated. Eight
Mycobacterium
sp. cultures in a sorbitol-yeast extract medium were dosed with 100 μg ml
−1
of norfloxacin and incubated for 7 days. The MICs of norfloxacin for these strains, tested by an agar dilution method, were 1.6 to 25 μg ml
−1
. Cultures were extracted with ethyl acetate, and potential metabolites in the extracts were purified by high-performance liquid chromatography. The metabolites were identified using mass spectrometry and nuclear magnetic resonance spectroscopy.
N
-Acetylnorfloxacin (5 to 50% of the total absorbance at 280 nm) was produced by the eight
Mycobacterium
strains.
N
-Nitrosonorfloxacin (5 to 30% of the total absorbance) was also produced by
Mycobacterium
sp. strain PYR100 and
Mycobacterium gilvum
PYR-GCK. The MICs of
N
-nitrosonorfloxacin and
N
-acetylnorfloxacin were 2- to 38- and 4- to 1,000-fold higher, respectively, than those of norfloxacin for several different bacteria, including the two strains that produced both metabolites. Although
N
-nitrosonorfloxacin had less antibacterial activity, nitrosamines are potentially carcinogenic. The biotransformation of fluoroquinolones by mycobacteria may serve as a resistance mechanism.
Detection of
Enterobacter sakazakii
in Dried Infant Milk Formula by Cationic-Magnetic-Bead Capture
Authors: Mullane N. R. , Murray J. , Drudy D. , Prentice N. , Whyte P. , Wall P. G. , Parton A. , Fanning S. 
Published: September 2006

ABSTRACT

Enterobacter sakazakii
has been associated with life-threatening infections in premature low-birth-weight infants. Contaminated infant milk formula (IMF) has been implicated in cases of
E. sakazakii
meningitis. Quick and sensitive methods to detect low-level contamination sporadically present in IMF preparations would positively contribute towards risk reduction across the infant formula food chain. Here we report on the development of a simple method, combining charged separation and growth on selective agar, to detect
E. sakazakii
in IMF. This protocol can reliably detect 1 to 5 CFU of
E. sakazakii
in 500 g of IMF in less than 24 h.