bluuebunny/crossref_metadata_2025_split huggingface dataset:
Population Structures of Two Genotypes of
Vibrio vulnificus
in Oysters (
Crassostrea virginica
) and Seawater
Authors: Warner Elizabeth , Oliver James D. 
Published: November 2007

ABSTRACT

Vibrio vulnificus
biotype 1 strains can be classified into two genotypes based on the PCR analysis of variations in the virulence-correlated gene (
vcg
). Genotype has been correlated with human infection for 90% of isolates from human cases having the
vcgC
sequence type and 87% of environmental strains having the
vcgE
variant. In this study we examined the dynamics of
V. vulnificus
populations and the distribution of the two genotypes recovered from oysters and surrounding estuarine wasters. Analysis of 880 isolates recovered from oysters showed a disparity in the ratio of the two genotypes, with those of the
vcgE
(E) genotype accounting for 84.4% of the population. In contrast, 292 isolates recovered from the waters surrounding the oyster sites revealed an almost equal distribution of the two genotypes. The levels of
vcgC
(C genotype) strains from both sources increased as a percentage of the population as water temperatures increased, while no culturable
V. vulnificus
cells were recovered from December through February. Our results suggest that there is a selective advantage for strains of the E genotype within oysters while survival of the C genotype strains may be favored by increased water column temperatures. These data suggest that the low incidence of infections may be due to the comparatively rare consumption of an oyster that contains a greater number of
V. vulnificus vcgC
genotype strains than of
vcgE
genotype strains. Levels of the two genotypes as well as seasonal dynamics within both oyster tissue and the surrounding waters may aid in identifying risk factors associated with human infection.
Prevalence of Lysogeny among Soil Bacteria and Presence of 16S rRNA and
trzN
Genes in Viral-Community DNA
Authors: Ghosh Dhritiman , Roy Krishnakali , Williamson Kurt E. , White David C. , Wommack K. Eric , Sublette Kerry L. , Radosevich Mark 
Published: November 2007

ABSTRACT

Bacteriophages are very abundant in the biosphere, and viral infection is believed to affect the activity and genetic diversity of bacterial communities in aquatic environments. Lysogenic conversion, for example, can improve host fitness and lead to phage-mediated horizontal gene transfer. However, little is known about lysogeny and transduction in the soil environment. In this study we employed atrazine-impregnated Bio-Sep beads (a cell immobilization matrix) to sample active microbiota from soils with prior pesticide exposure history. Once recovered from soil, the bead communities were induced with mitomycin C (MC), and viral and bacterial abundances were determined to evaluate the incidence of inducible prophage in soil bacteria. The inducible fraction calculated within bead communities was high (ca. 85%) relative to other studies in aquatic and sedimentary environments. Moreover, the bacterial genes encoding 16S rRNA and
trzN
, a chlorohydrolase gene responsible for dehalogenation of atrazine, were detected by PCR in the viral DNA fraction purified from MC-induced bead communities. A diverse collection of actinobacterial 16S rRNA gene sequences occurred within the viral DNA fraction of induced, water-equilibrated beads. Similar results were observed in induced atrazine-equilibrated beads, where 77% of the cloned sequences were derived from actinobacterial lineages. Heterogeneous 16S rRNA gene sequences consisting of fragments from two different taxa were detected in the clone libraries. The results suggest that lysogeny is a prevalent reproductive strategy among soil bacteriophages and that the potential for horizontal gene transfer via transduction is significant in soil microbial communities.
Eucaryotic Diversity in a Hypersaline Microbial Mat
Authors: Feazel Leah M. , Spear John R. , Berger Alicia B. , Harris J. Kirk , Frank Daniel N. , Ley Ruth E. , Pace Norman R. 
Published: November 2007

ABSTRACT
To determine the eucaryotic diversity of the hypersaline Guerrero Negro microbial mat, we amplified 18S rRNA genes from DNA extracted from this mat and constructed and analyzed clone libraries. The extent of eucaryotic diversity detected was remarkably low, only 15 species among 890 clones analyzed. Six eucaryotic kingdoms were represented, as well as a novel cluster of sequences. Nematode sequences dominated the clone libraries.
Prokaryotic Suppression Subtractive Hybridization PCR cDNA Subtraction, a Targeted Method To Identify Differentially Expressed Genes
Authors: De Long Susan K. , Kinney Kerry A. , Kirisits Mary Jo 
Published: November 2007

ABSTRACT

Molecular biology tools can be used to monitor and optimize biological treatment systems, but the application of nucleic acid-based tools has been hindered by the lack of available sequences for environmentally relevant biodegradation genes. The objective of our work was to extend an existing molecular method for eukaryotes to prokaryotes, allowing us to rapidly identify differentially expressed genes for subsequent sequencing. Suppression subtractive hybridization (SSH) PCR cDNA subtraction is a technique that can be used to identify genes that are expressed under specific conditions (e.g., growth on a given pollutant). While excellent methods for eukaryotic SSH PCR cDNA subtraction are available, to our knowledge, no methods previously existed for prokaryotes. This work describes our methodology for prokaryotic SSH PCR cDNA subtraction, which we validated using a model system:
Pseudomonas putida
mt-2 degrading toluene. cDNA from
P. putida
mt-2 grown on toluene (model pollutant) or acetate (control substrate) was subjected to our prokaryotic SSH PCR cDNA subtraction protocol to generate subtraction clone libraries. Over 90% of the sequenced clones contained gene fragments encoding toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were sequenced. Based on these results, prokaryotic SSH PCR cDNA subtraction shows promise as a targeted method for gene identification.
LactobacillusStrain Diversity Based on Partialhsp60Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation
Authors: Blaiotta Giuseppe , Fusco Vincenzina , Ercolini Danilo , Aponte Maria , Pepe Olimpia , Villani Francesco 
Published: November 2007

ABSTRACTA phylogenetic tree showing diversities among 116 partial (499-bp)Lactobacillus hsp60(groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA andtufgene sequences. The topology of the tree produced in this study showed aLactobacillusspecies distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of thehsp60nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely relatedLactobacillusspecies. Therefore, our results suggest thathsp60can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genusLactobacillusand that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of thehsp60fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bphsp60partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43Lactobacillusspecies considered, with the exception of the pairL. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. Thehsp60PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wildLactobacillusstrains (including closely related species, such asL. caseiandL. rhamnosusorL. plantarumandL. pentosus) isolated from cheese and dry-fermented sausages.
Cloning of a Novel Gene Encoding β-1,3-Xylosidase from a Marine Bacterium,
Vibrio
sp. Strain XY-214, and Characterization of the Gene Product
Authors: Umemoto Yoshiaki , Onishi Ryosuke , Araki Toshiyoshi 
Published: November 2007

ABSTRACT

The β-1,3-xylosidase gene (
xloA
) of
Vibrio
sp. strain XY-214 was cloned and expressed in
Escherichia coli
. The
xloA
gene consisted of a 1,608-bp nucleotide sequence encoding a protein of 535 amino acids with a predicted molecular weight of 60,835. The recombinant β-1,3-xylosidase hydrolyzed β-1,3-xylooligosaccharides to
d
-xylose as a final product.
Cholest-4-En-3-One-Δ
1
-Dehydrogenase, a Flavoprotein Catalyzing the Second Step in Anoxic Cholesterol Metabolism
Authors: Chiang Yin-Ru , Ismail Wael , Gallien Sébastien , Heintz Dimitri , Van Dorsselaer Alain , Fuchs Georg 
Published: November 2007

ABSTRACT

The anoxic metabolism of cholesterol was studied in the denitrifying bacterium
Sterolibacterium denitrificans
, which was grown with cholesterol and nitrate. Cholest-4-en-3-one was identified before as the product of cholesterol dehydrogenase/isomerase, the first enzyme of the pathway. The postulated second enzyme, cholest-4-en-3-one-Δ
1
-dehydrogenase, was partially purified, and its N-terminal amino acid sequence and tryptic peptide sequences were determined. Based on this information, the corresponding gene was amplified and cloned and the His-tagged recombinant protein was overproduced, purified, and characterized. The recombinant enzyme catalyzes the expected Δ
1
-desaturation (cholest-4-en-3-one to cholesta-1,4-dien-3-one) under anoxic conditions. It contains approximately one molecule of FAD per 62-kDa subunit and forms high molecular aggregates in the absence of detergents. The enzyme accepts various artificial electron acceptors, including dichlorophenol indophenol and methylene blue. It oxidizes not only cholest-4-en-3-one, but also progesterone (with highest catalytic efficiency, androst-4-en-3,17-dione, testosterone, 19-nortestosterone, and cholest-5-en-3-one. Two steroids, corticosterone and estrone, act as competitive inhibitors. The dehydrogenase resembles 3-ketosteroid-Δ
1
-dehydrogenases from other organisms (highest amino acid sequence identity with that from
Pseudoalteromonas haloplanktis
), with some interesting differences. Due to its catalytic properties, the enzyme may be useful in steroid transformations.
A New Assay for Promoter Analysis in
Chlamydomonas
Reveals Roles for Heat Shock Elements and the TATA Box in
HSP70A
Promoter-Mediated Activation of Transgene Expression
Authors: Lodha Mukesh , Schulz-Raffelt Miriam , Schroda Michael 
Published: November 2007

ABSTRACT

The aim of this work was to identify
cis
-regulatory sequences within the
Chlamydomonas HSP70A
promoter that mediate its stimulatory effect on the expression of downstream promoters. For this, we deleted/mutated the
HSP70A
promoter and, using a new assay, quantified its stimulatory effect. Our results indicate that the effect is mediated largely by heat shock elements and the TATA box.
Sexual Development in
Cryptococcus neoformans
Requires
CLP1
, a Target of the Homeodomain Transcription Factors Sxi1α and Sxi2
a
Authors: Ekena Joanne L. , Stanton Brynne C. , Schiebe-Owens Jessica A. , Hull Christina M. 
Published: November 2007

ABSTRACT

Sexual development in the human fungal pathogen
Cryptococcus neoformans
is a multistep process that results in the formation of spores, the likely infectious particles. A critical step in this developmental process is the transition from bud-form growth to filamentous growth. This transition is controlled by the homeodomain transcription factors Sxi1α and Sxi2
a
, whose targets are largely unknown. Here we describe the discovery of a gene,
CLP1
, that is regulated by Sxi1α and Sxi2
a
and is essential for sexual development. In vitro binding studies also show that the
CLP1
promoter is bound directly by Sxi1α and Sxi2
a
. The deletion of
CLP1
leads to a block in sexual development after cell fusion but before filament formation, and cells without
CLP1
are unable to grow vegetatively after cell fusion. Our findings lead to a model in which
CLP1
is a downstream target of the Sxi proteins that functions to promote growth after mating and to establish the filamentous state, a critical step in the production of spores.
Campylobacter jejuniBiofilms Up-Regulated in the Absence of the Stringent Response Utilize a Calcofluor White-Reactive Polysaccharide
Authors: McLennan Meghan K. , Ringoir Danielle D. , Frirdich Emilisa , Svensson Sarah L. , Wells Derek H. , Jarrell Harold , Szymanski Christine M. , Gaynor Erin C. 
Published: November 2007

ABSTRACTThe enteric pathogenCampylobacter jejuniis a highly prevalent yet fastidious bacterium. Biofilms and surface polysaccharides participate in stress survival, transmission, and virulence inC. jejuni; thus, the identification and characterization of novel genes involved in each process have important implications for pathogenesis. We found thatC. jejunireacts with calcofluor white (CFW), indicating the presence of surface polysaccharides harboring β1-3 and/or β1-4 linkages. CFW reactivity increased with extended growth, under 42°C anaerobic conditions, and in a ΔspoTmutant defective for the stringent response (SR). Conversely, two newly isolateddimmutants exhibited diminished CFW reactivity as well as growth and serum sensitivity differences from the wild type. Genetic, biochemical, and nuclear magnetic resonance analyses suggested that differences in CFW reactivity between wild-type and ΔspoTanddimmutant strains were independent of well-characterized lipooligosaccharides, capsular polysaccharides, andN-linked polysaccharides. Targeted deletion ofcarBdownstream of thedim13mutation also resulted in CFW hyporeactivity, implicating a possible role for carbamoylphosphate synthase in the biosynthesis of this polysaccharide. Correlations between biofilm formation and production of the CFW-reactive polymer were demonstrated by crystal violet staining, scanning electron microscopy, and confocal microscopy, with theC. jejuniΔspoTmutant being the first SR mutant in any bacterial species identified as up-regulating biofilms. Together, these results provide new insight into genes and processes important for biofilm formation and polysaccharide production inC. jejuni.
Fur Regulates Expression of theSalmonellaPathogenicity Island 1 Type III Secretion System through HilD
Authors: Ellermeier Jeremy R. , Slauch James M. 
Published: November 2007

ABSTRACTThe invasion of intestinal epithelial cells bySalmonella entericaserovar Typhimurium is mediated by a type III secretion system (T3SS) encoded onSalmonellapathogenicity island 1 (SPI1). Expression of the SPI1 T3SS is tightly regulated by the combined action of HilC, HilD, and RtsA, three AraC family members that can independently activatehilA, which encodes the direct regulator of the SPI1 structural genes. Expression ofhilC,hilD, andrtsAis controlled by a number of regulators that respond to a variety of environmental signals. In this work, we show that one such signal is iron mediated by Fur (ferric uptake regulator). Fur activateshilAtranscription in a HilD-dependent manner. Fur regulation of HilD does not appear to be simply at the transcriptional or translational level but rather requires the presence of the HilD protein. Fur activation of SPI1 is not mediated through the Fur-regulated small RNAs RfrA and RfrB, which are theSalmonellaortholog and paralog of RyhB that control expression ofsodB. Fur regulation of HilD is also not mediated through the known SPI1 repressor HilE or the CsrABC system. Although understanding the direct mechanism of Fur action on HilD requires further analysis, this work is an important step toward elucidating how various global regulatory systems control SPI1.
Roles of SigB and SigF in theMycobacterium tuberculosisSigma Factor Network
Authors: Lee Jong-Hee , Karakousis Petros C. , Bishai William R. 
Published: November 2007

ABSTRACTTo characterize the roles of SigB and SigF in sigma factor regulation inMycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpresssigBandsigF.Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes aftersigBinduction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression ofsigBandsigF. Overexpression ofsigBresulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction ofsigBdid not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of theM. tuberculosissigma factor regulatory cascade. Analysis of the 5′-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N14-18-NNGNNG. This sequence appeared upstream of bothsigB(Rv2710) and the gene following it,ideR(Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression ofsigFrevealed that only thesigCgene was significantly upregulated 6 and 12 h aftersigFinduction. The previously identified SigF promoter consensus sequence AGTTTG-N15-GGGTTT was identified in the 5′ UTR of thesigCgene, and SigF-dependent in vitro transcription of the promoter upstream ofsigCwas confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression ofsigBandsigFresulted in reduced rates ofM. tuberculosisintracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.
A Multiprotein Bicarbonate Dehydration Complex Essential to Carboxysome Function in Cyanobacteria
Authors: Cot Swan S.-W. , So Anthony K.-C. , Espie George S. 
Published: November 2007

ABSTRACT

Carboxysomes are proteinaceous biochemical compartments that constitute the enzymatic “back end” of the cyanobacterial CO
2
-concentrating mechanism. These protein-bound organelles catalyze HCO
3
−
dehydration and photosynthetic CO
2
fixation. In
Synechocystis
sp. strain PCC6803 these reactions involve the β-class carbonic anhydrase (CA), CcaA, and Form 1B ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The surrounding shell is thought to be composed of proteins encoded by the
ccmKLMN
operon, although little is known about how structural and catalytic proteins integrate to form a functional carboxysome. Using biochemical activity assays and molecular approaches we have identified a catalytic, multiprotein HCO
3
−
dehydration complex (BDC) associated with the protein shell of
Synechocystis
carboxysomes. The complex was minimally composed of a CcmM73 trimer, CcaA dimer, and CcmN. Larger native complexes also contained RbcL, RbcS, and two or three immunologically identified smaller forms of CcmM (62, 52, and 36 kDa). Yeast two-hybrid analyses indicated that the BDC was associated with the carboxysome shell through CcmM73-specific protein interactions with CcmK and CcmL. Protein interactions between CcmM73 and CcaA, CcmM73 and CcmN, or CcmM73 and itself required the N-terminal γ-CA-like domain of CcmM73. The specificity of the CcmM73-CcaA interaction provided both a mechanism to integrate CcaA into the fabric of the carboxysome shell and a means to recruit this enzyme to the BDC during carboxysome biogenesis. Functionally, CcaA was the catalytic core of the BDC. CcmM73 bound H
14
CO
3
−
but was unable to catalyze HCO
3
−
dehydration, suggesting that it may potentially regulate BDC activity.
Transcriptome Profiling and Functional Analysis ofAgrobacterium tumefaciensReveals a General Conserved Response to Acidic Conditions (pH 5.5) and a Complex Acid-Mediated Signaling Involved inAgrobacterium-Plant Interactions
Authors: Yuan Ze-Chun , Liu Pu , Saenkham Panatda , Kerr Kathleen , Nester Eugene W. 
Published: November 2007

ABSTRACTAgrobacterium tumefacienstransferred DNA (T-DNA) transfer requires that the virulence genes (virregulon) on the tumor-inducing (Ti) plasmid be induced by plant phenolic signals in an acidic environment. Using transcriptome analysis, we found that these acidic conditions elicit two distinct responses: (i) a general and conserved response through whichAgrobacteriummodulates gene expression patterns to adapt to environmental acidification and (ii) a highly specialized acid-mediated signaling response involved inAgrobacterium-plant interactions. Overall, 78 genes were induced and 74 genes were repressed significantly under acidic conditions (pH 5.5) compared to neutral conditions (pH 7.0). Microarray analysis not only confirmed previously identified acid-inducible genes but also uncovered many new acid-induced genes which may be directly involved inAgrobacterium-plant interactions. These genes includevirE0,virE1,virH1, andvirH2. Further, thechvG-chvItwo-component system, previously shown to be critical for virulence, was also induced under acid conditions. Interestingly, acidic conditions induced a type VI secretion system and a putative nonheme catalase. We provide evidence suggesting that acid-induced gene expression was independent of the VirA-VirG two-component system. Our results, together with previous data, support the hypothesis that there is three-step sequential activation of thevirregulon. This process involves a cascade regulation and hierarchical signaling pathway featuring initial direct activation of the VirA-VirG system by the acid-activated ChvG-ChvI system. Our data strengthen the notion thatAgrobacteriumhas evolved a mechanism to perceive and subvert the acidic conditions of the rhizosphere to an important signal that initiates and directs the early virulence program, culminating in T-DNA transfer.
Multiple Phospholipid
N
-Methyltransferases with Distinct Substrate Specificities Are Encoded in
Bradyrhizobium japonicum
Authors: Hacker Stephanie , Sohlenkamp Christian , Aktas Meriyem , Geiger Otto , Narberhaus Franz 
Published: November 2007

ABSTRACT

Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a few prokaryotes including members of the family
Rhizobiaceae
. In these bacteria, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for
Agrobacterium tumefaciens
and
Bradyrhizobium japonicum
. At least two different phospholipid
N
-methyltransferases (PmtA and PmtX) have been postulated to convert phosphatidylethanolamine (PE) to PC in
B. japonicum
by three consecutive methylation reactions. However, apart from the known PmtA enzyme, we identified and characterized three additional
pmt
genes (
pmtX1
,
pmtX3
, and
pmtX4
), which can be functionally expressed in
Escherichia coli
, showing different substrate specificities.
B. japonicum
expressed only two of these
pmt
genes (
pmtA
and
pmtX1
) under all conditions tested. PmtA predominantly converts PE to monomethyl PE, whereas PmtX1 carries out both subsequent methylation steps.
B. japonicum
is the first bacterium known to use two functionally different Pmts. It also expresses a PC synthase, which produces PC via condensation of CDP-diacylglycerol and choline. Our study shows that PC biosynthesis in bacteria can be much more complex than previously anticipated.