bluuebunny/crossref_metadata_2025_split huggingface dataset:
Presence of Pathogens and Indicator Microbes at a Non-Point Source Subtropical Recreational Marine Beach
Authors: Abdelzaher Amir M. , Wright Mary E. , Ortega Cristina , Solo-Gabriele Helena M. , Miller Gary , Elmir Samir , Newman Xihui , Shih Peter , Bonilla J. Alfredo , Bonilla Tonya D. , Palmer Carol J. , Scott Troy , Lukasik Jerzy , Harwood Valerie J. , McQuaig Shannon , Sinigalliano Chris , Gidley Maribeth , Plano Lisa R. W. , Zhu Xiaofang , Wang John D. , Fleming Lora E. 
Published: December 2009

ABSTRACT

Swimming in ocean water, including ocean water at beaches not impacted by known point sources of pollution, is an increasing health concern. This study was an initial evaluation of the presence of indicator microbes and pathogens and the association among the indicator microbes, pathogens, and environmental conditions at a subtropical, recreational marine beach in south Florida impacted by non-point sources of pollution. Twelve water and eight sand samples were collected during four sampling events at high or low tide under elevated or reduced solar insolation conditions. The analyses performed included analyses of fecal indicator bacteria (FIB) (fecal coliforms,
Escherichia coli
, enterococci, and
Clostridium perfringens
), human-associated microbial source tracking (MST) markers (human polyomaviruses [HPyVs] and
Enterococcus faecium esp
gene), and pathogens (
Vibrio vulnificus
,
Staphylococcus aureus
, enterovirus, norovirus, hepatitis A virus,
Cryptosporidium
spp., and
Giardia
spp.). The enterococcus concentrations in water and sand determined by quantitative PCR were greater than the concentrations determined by membrane filtration measurement. The FIB concentrations in water were below the recreational water quality standards for three of the four sampling events, when pathogens and MST markers were also generally undetectable. The FIB levels exceeded regulatory guidelines during one event, and this was accompanied by detection of HPyVs and pathogens, including detection of the autochthonous bacterium
V. vulnificus
in sand and water, detection of the allochthonous protozoans
Giardia
spp. in water, and detection of
Cryptosporidium
spp. in sand samples. The elevated microbial levels were detected at high tide and under low-solar-insolation conditions. Additional sampling should be conducted to further explore the relationships between tidal and solar insolation conditions and between indicator microbes and pathogens in subtropical recreational marine waters impacted by non-point source pollution.
Use of
rpsL
as a Counterselectable Marker in
Borrelia burgdorferi
Authors: Drecktrah Dan , Douglas J. Miles , Samuels D. Scott 
Published: December 2009

ABSTRACT

We have demonstrated that
rpsL
, encoding the S12 protein of the small ribosomal subunit, can be used as a counterselectable marker in
Borrelia burgdorferi
, the causative agent of Lyme disease. Mutations in
rpsL
confer streptomycin resistance. Streptomycin susceptibility is dominant in an
rpsL
merodiploid, and streptomycin selects for the loss of wild-type
rpsL
carried in
trans
. This is the first description of a counterselectable marker in
B. burgdorferi
.
Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of the
tdh
and
trh
Genes of
Vibrio parahaemolyticus
and Related
Vibrio
Species
Authors: Yamazaki Wataru , Kumeda Yuko , Misawa Naoaki , Nakaguchi Yoshitsugu , Nishibuchi Mitsuaki 
Published: December 2009

ABSTRACT

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of
Vibrio parahaemolyticus
. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the
tdh
,
trh
1, and
trh
2 genes of
V. parahaemolyticus
. The LAMP assay was designed for both combined and individual detection of the
tdh
,
trh
1, and
trh
2 genes and combined detection of the
trh
1 and
trh
2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of
V. parahaemolyticus
, 3 strains of
Grimontia hollisae
, and 2 strains of
Vibrio mimicus
carrying the
tdh
,
trh
1, and
trh
2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the
tdh
,
trh
1, and
trh
2 genes. The sensitivities of the LAMP assay for detection of
tdh
-,
trh
1-, and
trh
2-carrying
V. parahaemolyticus
strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the
tdh
,
trh
1, and
trh
2 genes of
V. parahaemolyticus
and related
Vibrio
species.
New Architectures for Tet-On and Tet-Off Regulation in
Staphylococcus aureus
Authors: Stary Elena , Gaupp Rosmarie , Lechner Sabrina , Leibig Martina , Tichy Evelyn , Kolb Martina , Bertram Ralph 
Published: December 2009

ABSTRACT

Inducible expression is a valuable approach for the elucidation of gene functions. Here, we present new configurations of the tetracycline-dependent gene regulation (
tet
) system for
Staphylococcus aureus
. To provide improved and expanded modes of control, strains and plasmids were constructed for the constitutive expression of
tetR
or a variant allele, rev
-tetR
r2
. The encoded regulators respond differently to the effector anhydrotetracycline (ATc), which causes target gene expression to be induced with TetR or repressed with rev
-
TetR. To quantify and compare regulation mediated by episomal or chromosomal (rev-)
tetR
constructs, expression from a chromosomal P
xyl/tet
-
gfpmut2
fusion was measured. Chromosomally encoded TetR showed tight repression and allowed high levels of dose-dependent gene expression in response to ATc. Regulatory abilities were further verified using a strain in which a native
S. aureus
gene (
zwf
) was put under
tet
control in its native chromosomal location. Tight repression was reflected by transcript amounts, which were barely detectable under repressed conditions and high in ATc-treated cells. In reporter gene assays, this type of control, termed Tet-on, was more efficient than Tet-off regulation, in which addition of ATc causes downregulation of a target gene. The latter was achieved and quantified by direct rev
-
TetR control of P

xyl/tet

-
gfpmut2
. Additionally, TetR was used in
trans
to control the expression of antisense RNA for posttranscriptional gene silencing. Induction of antisense RNA expression of the
fabI
gene caused pronounced growth retardation lasting several hours. These results demonstrate the efficiency of the new
tet
systems and their flexible use for different purposes.
Double Labeling of Oligonucleotide Probes for Fluorescence
In Situ
Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility
Authors: Stoecker Kilian , Dorninger Christiane , Daims Holger , Wagner Michael 
Published: December 2009

ABSTRACT

Fluorescence
in situ
hybridization (FISH) with singly labeled rRNA-targeted oligonucleotide probes is widely applied for direct identification of microbes in the environment or in clinical specimens. Here we show that a replacement of singly labeled oligonucleotide probes with 5′-, 3′-doubly labeled probes at least doubles FISH signal intensity without causing specificity problems. Furthermore, Cy3-doubly labeled probes strongly increase
in situ
accessibility of rRNA target sites and thus provide more flexibility for probe design.
Multiple Genetic Mechanisms Lead to Loss of Functional TbAT1 Expression in Drug-Resistant Trypanosomes
Authors: Stewart Mhairi L. , Burchmore Richard J. S. , Clucas Caroline , Hertz-Fowler Christiane , Brooks Karen , Tait A. , MacLeod A. , Turner C. Michael R. , De Koning Harry P. , Wong Pui Ee , Barrett Michael P. 
Published: December 2009

ABSTRACT

The P2 aminopurine transporter, encoded by Tb
AT1
in African trypanosomes in the
Trypanosoma brucei
group, carries melaminophenyl arsenical and diamidine drugs into these parasites. Loss of this transporter contributes to drug resistance. We identified the genomic location of Tb
AT1
to be in the subtelomeric region of chromosome 5 and determined the status of the Tb
AT1
gene in two trypanosome lines selected for resistance to the melaminophenyl arsenical, melarsamine hydrochloride (Cymelarsan), and in a
Trypanosoma equiperdum
clone selected for resistance to the diamidine, diminazene aceturate. In the
Trypanosoma brucei
gambiense
STIB 386 melarsamine hydrochloride-resistant line, Tb
AT1
is deleted, while in the
Trypanosoma brucei brucei
STIB 247 melarsamine hydrochloride-resistant and
T. equiperdum
diminazene-resistant lines, Tb
AT1
is present, but expression at the RNA level is no longer detectable. Further characterization of Tb
AT1
in
T. equiperdum
revealed that a loss of heterozygosity at the Tb
AT1
locus accompanied loss of expression and that P2-mediated uptake of [
3
H]diminazene is lost in drug-resistant
T. equiperdum
. Adenine-inhibitable adenosine uptake is still detectable in a ΔTb
at1
T. b. brucei
mutant, although at a greatly reduced capacity compared to that of the wild type, indicating that an additional adenine-inhibitable adenosine permease, distinct from P2, is present in these cells.
The NDR Kinase DBF-2 Is Involved in Regulation of Mitosis, Conidial Development, and Glycogen Metabolism in Neurospora crassa
Authors: Dvash Efrat , Kra-Oz Galia , Ziv Carmit , Carmeli Shmuel , Yarden Oded 
Published: December 2009

ABSTRACT

Neurospora crassa
dbf-2
encodes an NDR (nuclear Dbf2-related) protein kinase, homologous to LATS1, a core component of the Hippo pathway. This pathway plays important roles in restraining cell proliferation and promoting apoptosis in differentiating cells. Here, we demonstrate that DBF-2 is involved in three fundamental processes in a filamentous fungus: cell cycle regulation, glycogen biosynthesis, and conidiation. DBF-2 is predominantly localized to the nucleus, and most (approximately 60%)
dbf-2
null mutant nuclei are delayed in mitosis, indicating that DBF-2 activity is required for properly completing the cell cycle. The
dbf-2
mutant exhibits reduced basal hyphal extension rates accompanied by a carbon/nitrogen ratio-dependent bursting of hyphal tips, vast glycogen leakage, defects in aerial hypha formation, and impairment of all three asexual conidiation pathways in
N. crassa
. Our findings also indicate that DBF-2 is essential for sexual reproduction in a filamentous fungus. Defects in other Hippo and glycogen metabolism pathway components (
mob-1
,
ccr-4
,
mst-1
, and
gsk-3
) share similar phenotypes such as mitotic delay and decreased CDC-2 (cell division cycle 2) protein levels, massive hyphal swellings, hyphal tip bursting, glycogen leakage, and impaired conidiation. We propose that DBF-2 functions as a link between Hippo and glycogen metabolism pathways.
The S Helix Mediates Signal Transmission as a HAMP Domain Coiled-Coil Extension in the NarX Nitrate Sensor fromEscherichia coliK-12
Authors: Stewart Valley , Chen Li-Ling 
Published: December 2009

ABSTRACTIn the nitrate-responsive, homodimeric NarX sensor, two cytoplasmic membrane α-helices delimit the periplasmic ligand-binding domain. The HAMP domain, a four-helix parallel coiled-coil built from two α-helices (HD1 and HD2), immediately follows the second transmembrane helix. Previous computational studies identified a likely coiled-coil-forming α-helix, the signaling helix (S helix), in a range of signaling proteins, including eucaryal receptor guanylyl cyclases, but its function remains obscure. In NarX, the HAMP HD2 and S-helix regions overlap and apparently form a continuous coiled-coil marked by a heptad repeat stutter discontinuity at the distal boundary of HD2. Similar composite HD2-S-helix elements are present in other sensors, such as Sln1p fromSaccharomyces cerevisiae. We constructed deletions and missense substitutions in the NarX S helix. Most caused constitutive signaling phenotypes. However, strongly impaired induction phenotypes were conferred by heptad deletions within the S-helix conserved core and also by deletions that remove the heptad stutter. The latter observation illuminates a key element of the dynamic bundle hypothesis for signaling across the heptad stutter adjacent to the HAMP domain in methyl-accepting chemotaxis proteins (Q. Zhou, P. Ames, and J. S. Parkinson, Mol. Microbiol. 73:801-814, 2009). Sequence comparisons identified other examples of heptad stutters between a HAMP domain and a contiguous coiled-coil-like heptad repeat sequence in conventional sensors, such as CpxA, EnvZ, PhoQ, and QseC; other S-helix-containing sensors, such as BarA and TorS; and theNeurospora crassaNik-1 (Os-1) sensor that contains a tandem array of alternating HAMP and HAMP-like elements. Therefore, stutter elements may be broadly important for HAMP function.
Analysis of the Mobilization Functions of the Vancomycin Resistance Transposon Tn
1549
, a Member of a New Family of Conjugative Elements
Authors: Tsvetkova Krassimira , Marvaud Jean-Christophe , Lambert Thierry 
Published: December 2009

ABSTRACT

Conjugative transfer from
Clostridium symbiosum
to enterococci of Tn
1549
, which confers VanB-type vancomycin resistance, has been reported. This indicates the presence of a transfer origin (
oriT
) in the element. Transcription analysis of Tn
1549
indicated that
orf29
,
orf28
,
orfz
, and
orf27
were cotranscribed. A pACYC184 derivative containing 250 bp intergenic to
orf29-orf30
of Tn
1549
was mobilized in
Escherichia coli recA
::RP4::Δ
nic
provided that
orf28
and
orf29
were delivered simultaneously. These open reading frame (ORF) genes were able to promote mobilization in
trans
, but a
cis
-acting preference was observed. On the basis of a mobilization assay, a minimal 28-bp
oriT
was delimited, although the frequency of transfer was significantly reduced compared to that of a 130-bp
oriT
fragment. The minimal
oriT
contained an inverted repeat and a core, which was homologous to the cleavage sequence found in certain Gram-positive rolling-circle replicating (RCR) plasmids. While Orf29 was a mobilization accessory component similar to MobC proteins, Orf28 was identified as a relaxase belonging to a new phyletic cluster of the MOB
p
superfamily. The nick site was identified within
oriT
by an oligonucleotide cleavage assay. Closely related
oriT
s linked to mobilization genes were detected in data banks; they were found in various integrative and conjugative elements (ICEs) originating mainly from anaerobes. These results support the notion that Tn
1549
is a member of a MOB
p
clade. Interestingly, the Tn
1549
-derived constructs were mobilized by RP4 in
E. coli
, suggesting that a relaxosome resulting from DNA cleavage by Orf28 interacted with the coupling protein TraG. This demonstrates the capacity of Tn
1549
to be mobilized by a heterologous transfer system.
Glycerol Is Metabolized in a Complex and Strain-Dependent Manner in
Enterococcus faecalis
Authors: Bizzini Alain , Zhao Chen , Budin-Verneuil Aurélie , Sauvageot Nicolas , Giard Jean-Christophe , Auffray Yanick , Hartke Axel 
Published: December 2009

ABSTRACT

Enterococcus faecalis
is equipped with two pathways of glycerol dissimilation. Glycerol can either first be phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase (the
glpK
pathway) or first be oxidized by glycerol dehydrogenase and then phosphorylated by dihydroxyacetone kinase (the
dhaK
pathway). Both pathways lead to the formation of dihydroxyacetone phosphate, an intermediate of glycolysis. It was assumed that the
glpK
pathway operates during aerobiosis and that the
dhaK
pathway operates under anaerobic conditions. Because this had not been analyzed by a genetic study, we constructed mutants of strain JH2-2 affected in both pathways. The growth of these mutants on glycerol under aerobic and anaerobic conditions was monitored. In contrast to the former model, results strongly suggest that glycerol is catabolized simultaneously by both pathways in the
E. faecalis
JH2-2 strain in the presence of oxygen. In accordance with the former model, glycerol is metabolized by the
dhaK
pathway under anaerobic conditions. Comparison of different
E. faecalis
isolates revealed an impressive diversity of growth behaviors on glycerol. Analysis by BLAST searching and real-time reverse transcriptase PCR revealed that this diversity is based not on different gene contents but rather on differences in gene expression. Some strains used preferentially the
glpK
pathway whereas others probably exclusively the
dhaK
pathway under aerobic conditions. Our results demonstrate that the species
E. faecalis
cannot be represented by only one model of aerobic glycerol catabolism.
Identification, Functional Studies, and Genomic Comparisons of New Members of the NnrR Regulon in
Rhodobacter sphaeroides
Authors: Hartsock Angela , Shapleigh James P. 
Published: December 2009

ABSTRACT

Analysis of the
Rhodobacter sphaeroides
2.4.3 genome revealed four previously unidentified sequences similar to the binding site of the transcriptional regulator NnrR. Expression studies demonstrated that three of these sequences are within the promoters of genes, designated
paz
,
norEF
, and
cdgA
, in the NnrR regulon, while the status of the fourth sequence, within the
tat
operon promoter, remains uncertain.
nnrV
, under control of a previously identified NnrR site, was also identified.
paz
encodes a pseudoazurin that is a donor of electrons to nitrite reductase.
paz
inactivation did not decrease nitrite reductase activity, but loss of pseudoazurin and cytochrome

c
2

together reduced nitrite reduction. Inactivation of
norEF
reduced nitrite and nitric oxide reductase activity and increased the sensitivity to nitrite in a taxis assay. This suggests that loss of
norEF
increases NO production as a result of decreased nitric oxide reductase activity. 2.4.3 is the only strain of
R. sphaeroides
with
norEF
, even though all four of the strains whose genomes have been sequenced have the
norCBQD
operon and
nnrR
.
norEF
was shown to provide resistance to nitrite when it was mobilized into
R. sphaeroides
strain 2.4.1 containing
nirK
. Inactivation of the other identified genes did not reveal any detectable denitrification-related phenotype. The distribution of members of the NnrR regulon in
R. sphaeroides
revealed patterns of coselection of structural genes with the ancillary genes identified here. The strong coselection of these genes indicates their functional importance under real-world conditions, even though inactivation of the majority of them does not impact denitrification under laboratory conditions.
fpr
, a Deficient Xer Recombination Site from a
Salmonella
Plasmid, Fails To Confer Stability by Dimer Resolution: Comparative Studies with the pJHCMW1
mwr
Site
Authors: Tran Tung , Sherratt David J. , Tolmasky Marcelo E. 
Published: December 2009

ABSTRACT

Salmonella
plasmid pFPTB1 includes a Tn
3
-like transposon and a Xer recombination site,
fpr
, which mediates site-specific recombination at efficiencies lower than those required for stabilizing a plasmid by dimer resolution. Mutagenesis and comparative studies with
mwr
, a site closely related to
fpr
, indicate that there is an interdependence of the sequences in the XerC binding region and the central region in Xer site-specific recombination sites.
Reconstitution Experiments and Gene Deletions Reveal the Existence of Two-Component Major Cell Wall Channels in the GenusCorynebacterium
Authors: Barth Enrico , Barceló Miriam Agulló , Kläckta Christian , Benz Roland 
Published: December 2009

ABSTRACTTwo small polypeptides, PorA and PorH, are known to form cell wall channels inCorynebacteriumglutamicumand inCorynebacteriumefficiens. The genes coding for both polypeptides are localized in close proximity to one another between the genes coding for GroEl2 and a polyphosphate kinase (PKK2). In this study, we investigated the relationship of PorA and PorH to one another. The results suggested that the major cell wall channels ofCorynebacteriumglutamicum,Corynebacteriumefficiens, andCorynebacteriumdiphtheriaeneed the obligatory presence of two distinct polypeptides, one of class PorA and one of class PorH, to form an active cell wall channel. Identification of genes coding for homologous proteins in the chromosome ofCorynebacteriumcallunaesuggested a similar result for this strain. Contrary to our previous reports on channel-forming proteins in these strains, a heterooligomeric structure composed of PorA and PorH is needed in all of them to form the major cell wall channel. This was concluded from complementation experiments using aporH- andporA-deficientC.glutamicumstrain. The stringent necessity of proteins of either class to recover the wild-type channels was demonstrated by black lipid bilayer experiments using detergent or organic solvent extracts of the complementedporH- andporA-deficientC.glutamicumstrain. The channel-forming capability of recombinant expressed, affinity-purified PorA and PorH proteins ofC.glutamicumrevealed that the channels consisted solely of these two components. This agreed with results obtained from a transcript coding for both channel-forming components identified inC.glutamicumby Northern blot analysis and reverse transcription-PCR analysis. The transcription start point of the genes was determined by the rapid amplification of cDNA ends approach, allowing the prediction of the −35 and −10 regions of the promoter. The results demonstrate that the cell wall channels within the genusCorynebacteriummay be formed by two-component oligomers.
Noncoding RNA of Glutamine Synthetase I Modulates Antibiotic Production inStreptomyces coelicolorA3(2)
Authors: D'Alia Davide , Nieselt Kay , Steigele Stephan , Müller Jonas , Verburg Ilse , Takano Eriko 
Published: December 2009

ABSTRACTOverexpression of antisense chromosomalcis-encoded noncoding RNAss (ncRNAs) in glutamine synthetase I resulted in a decrease in growth, protein synthesis, and antibiotic production inStreptomyces coelicolor. In addition, we predicted 3,597cis-encoded ncRNAs and validated 13 of them experimentally, including several ncRNAs that are differentially expressed in bacterial hormone-defective mutants.