bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of
Methylophaga
spp. from Marine Dimethylsulfide-Degrading Enrichment Cultures and Identification of Polypeptides Induced during Growth on Dimethylsulfide
Authors: Schäfer Hendrik 
Published: February 2007

ABSTRACT

Dimethylsulfide (DMS)-degrading enrichment cultures were established from samples of coastal seawater, nonaxenic
Emiliania huxleyi
cultures, and mixed marine methyl halide-degrading enrichment cultures. Bacterial populations from a broad phylogenetic range were identified in the mixed DMS-degrading enrichment cultures by denaturing gradient gel electrophoresis (DGGE). Sequences of dominant DGGE bands were similar to those of members of the genera
Methylophaga
and
Alcanivorax
. Several closely related
Methylophaga
strains were obtained that were able to grow on DMS as the carbon and energy source.
Roseobacter
-related populations were detected in some of the enrichment cultures; however, none of the
Roseobacter
group isolates that were tested were able to grow on DMS. Oxidation of DMS by
Methylophaga
sp. strain DMS010 was not affected by addition of the inhibitor chloroform or methyl
tert
-butyl ether, suggesting that DMS metabolism may occur by a route different from those described for
Thiobacillus
species and other unidentified marine isolates. Addition of DMS and methanethiol to whole-cell suspensions of strain DMS010 induced oxygen uptake when strain DMS010 was grown on DMS but not in cells grown on methanol. The apparent

K
m

s of strain DMS010 for DMS and for methanethiol were 2.1 and 4.6 μM, respectively, when grown on DMS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the biomass of strain DMS010 and analysis of peptide bands by mass spectrometry techniques and N-terminal sequencing provided the first insight into the identity of polypeptides induced during growth on DMS. These included XoxF, a homolog of the large subunit of methanol dehydrogenase for which a biological role has not been identified previously.
Development of a Real-Time PCR Probe for Quantification of the Heterotrophic Dinoflagellate
Cryptoperidiniopsis brodyi
(Dinophyceae) in Environmental Samples
Authors: Park Tae-Gyu , de Salas Miguel F. , Bolch Christopher J. S. , Hallegraeff Gustaaf M. 
Published: February 2007

ABSTRACT

A TaqMan format real-time PCR probe was developed against the internal transcribed spacer 2 ribosomal DNA region for the specific detection and quantification of
Cryptoperidiniopsis brodyi
in environmental samples. The assay specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR amplicons from natural water samples. Phylogenetic analysis of the sequenced environmental samples also showed that this assay is specific to
C. brodyi
. The
C. brodyi
-specific assay was used in conjunction with
Pfiesteria piscicida-
and
Pfiesteria shumwayae
-specific real-time PCR assays to investigate the temporal variations of
C. brodyi
,
P. piscicida
, and
P. shumwayae
abundance in the Derwent estuary, Tasmania. The 18-month field survey from November 2004 to April 2006 revealed that
C. brodyi
occurred in all seasons at very low densities, mostly below 25 cells liter
−1
, with higher abundance (maximum, 112 cells liter
−1
) in April and May.
P. piscicida
was detected only once, in May 2005 at 60 cells liter
−1
.
P. shumwayae
was not detected during the survey.
Analysis of Homothallic
Saccharomyces cerevisiae
Strain Mating during Must Fermentation
Authors: Ambrona Jesús , Ramírez Manuel 
Published: February 2007

ABSTRACT

Genetic instability and genome renewal may cause loss of heterozygosity (LOH) in homothallic wine yeasts (
Saccharomyces cerevisiae
), leading to the elimination of the recessive lethal or deleterious alleles that decrease yeast fitness. LOH was not detected in genetically stable wine yeasts during must fermentation. However, after sporulation, the heterozygosity of the new yeast population decreased during must fermentation. The frequency of mating between just-germinated haploid cells from different tetrads was very low, and the mating of haploid cells from the same ascus was favored because of the physical proximity. Also, mating restriction between haploid cells from the same ascus was found, leading to a very low frequency of self spore clone mating. This mating restriction slowed down the LOH process of the yeast population, maintaining the heterozygote frequency higher than would be expected assuming a fully random mating of the haploid yeasts or according to the Mortimer genome renewal proposal. The observed LOH occurs because of the linkage of the locus
MAT
to the chromosome III centromere, without the necessity for self spore clone mating or the high frequency of gene conversion and rapid asymmetric LOH observed in genetically unstable yeasts. This phenomenon is enough in itself to explain the high level of homozygosis found in natural populations of wine yeasts. The LOH process for centromere-linked markers would be slower than that for the nonlinked markers, because the linkage decreases the frequency of newly originated heterozygous yeasts after each round of sporulation and mating. This phenomenon is interesting in yeast evolution and may cause important sudden phenotype changes in genetically stable wine yeasts.
Generation by a Widely Applicable Approach of a Hybrid Dioxygenase Showing Improved Oxidation of Polychlorobiphenyls
Authors: Cámara Beatriz , Seeger Michael , González Myriam , Standfuß-Gabisch Christine , Kahl Silke , Hofer Bernd 
Published: February 2007

ABSTRACT

Recently, a sequence-based approach has been developed for the fast isolation and characterization of class II aryl-hydroxylating dioxygenase activities (S. Kahl and B. Hofer, Microbiology
149:
1475-1481, 2003). It comprises the PCR amplification of segments of alpha subunit genes of unknown sequence that encode the catalytic center and their fusion with sequences of the
bphA
gene cluster of
Burkholderia xenovorans
LB400. One of the resulting chimeric enzymes, harboring the core segment of a dioxygenase from
Pseudomonas
sp. strain B4-Magdeburg, has now been characterized with respect to the oxidation of chlorobiphenyls (CBs). Its substrate and product specificities differed favorably from those of the parental dioxygenase of strain LB400. The hybrid possessed a higher regiospecificity and yielded less unproductive dioxygenations at
meta
and
para
carbons. It attacked
ortho
-,
meta
-, and
para
-chlorinated rings with comparable efficiencies. It gave significantly higher yields in
ortho
,
meta
-dioxygenation of recalcitrant congeners containing a doubly
ortho
-chlorinated ring. While the parental enzyme yielded mainly unproductive
meta
,
para
dioxygenation of 2,5,4′-CB, the hybrid predominantly converted this congener into an
ortho
,
meta
-dioxygenated product. The subsequent enzymes of the LB400 catabolic pathway were able to transform most of the metabolites formed by the novel dioxygenase, indicating that the substrate ranges of these biocatalysts are not adapted to that of their initial pathway enzyme. Some of the catabolites, however, were identified as problematic for further degradation. Our results demonstrate that the outlined approach can successfully be applied to obtain novel dioxygenase specificities that favorably complement or supplement known ones.
Genetic Characterization of 2,4,6-Trichlorophenol Degradation in
Cupriavidus necator
JMP134
Authors: Sánchez M. A. , González B. 
Published: February 2007

ABSTRACT

The degradation pathway of 2,4,6-trichlorophenol (2,4,6-TCP), a hazardous pollutant, in the aerobic bacterium
Cupriavidus necator
JMP134(pJP4) (formerly
Ralstonia eutropha
JMP134) is encoded by the
tcp
genes. These genes are located in a genetic context,
tcpRXABCYD
, which resembles a putative catabolic operon. In this work, these gene sequences were individually disrupted and mutant strains were evaluated for their ability to grow on or degrade 2,4,6-TCP. The
tcpX
and
tcpA
mutants completely failed to degrade this compound. Although the
tcpC
mutant was also unable to grow on 2,4,6-TCP, it still transformed this chlorophenol to 6-chlorohydroquinol. In contrast, the
tcpD
mutant grew on 2,4,6-TCP, suggesting the presence of additional maleylacetate reductase-encoding genes. Five other open reading frames encoding maleylacetate reductases, in addition to the
tcpD
gene, were found in the genome of
C. necator
, and two of them provide this function in the
tcpD
mutant. The
tcpR
gene, encoding a putative LysR-type transcriptional regulator, was disrupted, and this mutant strain completely failed to grow on 2,4,6-TCP. Transcriptional fusion studies demonstrated that TcpR activates the expression of the
tcp
genes, responding specifically to 2,4,6-TCP. The transcriptional start of the
tcp
operon was mapped, and a putative σ
70
-type promoter was identified.
Ferrous Iron- and Sulfur-Induced Genes in
Sulfolobus metallicus
Authors: Bathe Stephan , Norris Paul R. 
Published: February 2007

ABSTRACT

Genes of
Sulfolobus metallicus
that appeared to be upregulated in relation to growth on either ferrous iron or sulfur were identified using subtractive hybridization of cDNAs. The genes upregulated during growth on ferrous iron were found in a cluster, and most were predicted to encode membrane proteins. Quantitative reverse transcription-PCR of cDNA showed upregulation of most of these genes during growth on ferrous iron and pyrite compared to results during growth on sulfur. The highest expression levels observed included those for genes encoding proteins with similarities to cytochrome
c
oxidase subunits and a CbsA-like cytochrome. The genes identified here that may be involved in oxidation of ferrous iron by
S. metallicus
are termed
fox
genes. Of three available genomes of
Sulfolobus
species (
S. tokodaii
,
S. acidocaldarius
, and
S. solfataricus
), only that of
S. tokodaii
has a cluster of highly similar open reading frames, and only
S. tokodaii
of these three species was also able to oxidize ferrous iron. A gene encoding sulfur oxygenase-reductase was identified as the source of the dominant transcript in sulfur-grown cells of
S. metallicus
, with the predicted protein showing high identities to the previously described examples from
S. tokodaii
and species of
Acidianus
.
Gene Targeting in Gram-Negative Bacteria by Use of a Mobile Group II Intron (“Targetron”) Expressed from a Broad-Host-Range Vector
Authors: Yao Jun , Lambowitz Alan M. 
Published: February 2007

ABSTRACTMobile group II introns (“targetrons”) can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via anm-toluic acid-inducible promoter from a broad-host-range vector containing an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-negative bacteria, includingEscherichia coli,Pseudomonas aeruginosa, andAgrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in theE. coli lacZ,P. aeruginosa pqsAandpqsH, andA. tumefaciens aopBandchvIgenes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.
The
MAT1
Locus of
Histoplasma capsulatum
Is Responsive in a Mating Type-Specific Manner
Authors: Bubnick Meggan , Smulian A. George 
Published: February 2007

ABSTRACT

Recombination events associated with sexual replication in pathogens may generate new strains with altered virulence.
Histoplasma capsulatum
is a mating-competent, pathogenic fungus with two described phenotypic mating types, + and −. The mating (
MAT
) locus of
H. capsulatum
was identified to facilitate molecular studies of mating in this organism. Through syntenic analysis of the
H. capsulatum
genomic sequence databases, a
MAT1-1
idiomorph region was identified in
H. capsulatum
strains G217B and WU24, and a
MAT1-2
idiomorph region was identified in the strain G186AR. A mating type-specific PCR assay was developed, and two clinical isolates of opposite genotypic mating type, UH1 and VA1, were identified. A known − mating type strain, T-3-1 (ATCC 22635), was demonstrated to be of
MAT1-2
genotypic mating type. The clinical isolates UH1 and VA1 were found to be mating compatible and also displayed mating-type-dependent regulation of the
MAT
transcription factors in response to extracts predicted to contain mating pheromones. These studies support a role for the identified
MAT1
locus in determining mating type in
H. capsulatum
.
Basic Helix-Loop-Helix Transcription Factor Heterocomplex of Yas1p and Yas2p Regulates Cytochrome P450 Expression in Response to Alkanes in the Yeast
Yarrowia lipolytica
Authors: Endoh-Yamagami Setsu , Hirakawa Kiyoshi , Morioka Daisuke , Fukuda Ryouichi , Ohta Akinori 
Published: February 2007

ABSTRACT

The expression of the
ALK1
gene, which encodes cytochrome P450, catalyzing the first step of alkane oxidation in the alkane-assimilating yeast
Yarrowia lipolytica
, is highly regulated and can be induced by alkanes. Previously, we identified a
cis
-acting element (alkane-responsive element 1 [ARE1]) in the
ALK1
promoter. We showed that a basic helix-loop-helix (bHLH) protein, Yas1p, binds to ARE1 in vivo and mediates alkane-dependent transcription induction. Yas1p, however, does not bind to ARE1 by itself in vitro, suggesting that Yas1p requires another bHLH protein partner for its DNA binding, as many bHLH transcription factors function by forming heterodimers. To identify such a binding partner of Yas1p, here we screened open reading frames encoding proteins with the bHLH motif from the
Y. lipolytica
genome database and identified the
YAS2
gene. The deletion of the
YAS2
gene abolished the alkane-responsive induction of
ALK1
transcription and the growth of the yeast on alkanes. We revealed that Yas2p has transactivation activity. Furthermore, Yas1p and Yas2p formed a protein complex that was required for the binding of these proteins to ARE1. These findings allow us to postulate a model in which bHLH transcription factors Yas1p and Yas2p form a heterocomplex and mediate the transcription induction in response to alkanes.
Regulators of the
Bacillus subtilis cydABCD
Operon: Identification of a Negative Regulator, CcpA, and a Positive Regulator, ResD
Authors: Puri-Taneja Ankita , Schau Matthew , Chen Yinghua , Hulett F. Marion 
Published: February 2007

ABSTRACT

The
cydABCD
operon of
Bacillus subtilis
encodes products required for the production of cytochrome
bd
oxidase. Previous work has shown that one regulatory protein, YdiH (Rex), is involved in the repression of this operon. The work reported here confirms the role of Rex in the negative regulation of the
cydABCD
operon. Two additional regulatory proteins for the
cydABCD
operon were identified, namely, ResD, a response regulator involved in the regulation of respiration genes, and CcpA, the carbon catabolite regulator protein. ResD, but not ResE, was required for full expression of the
cydA
promoter in vivo. ResD binding to the
cydA
promoter between positions −58 and −107, a region which includes ResD consensus binding sequences, was not enhanced by phosphorylation. A
ccpA
mutant had increased expression from the full-length
cydA
promoter during stationary growth compared to the wild-type strain. Maximal expression in a
ccpA
mutant was observed from a 3′-deleted
cydA
promoter fusion that lacked the Rex binding region, suggesting that the effect of the two repressors, Rex and CcpA, was cumulative. CcpA binds directly to the
cydA
promoter, protecting the region from positions −4 to −33, which contains sequences similar to the CcpA consensus binding sequence, the
cre
box. CcpA binding was enhanced upon addition of glucose-6-phosphate, a putative cofactor for CcpA. Mutation of a conserved residue in the
cre
box reduced CcpA binding 10-fold in vitro and increased
cydA
expression in vivo. Thus, CcpA and ResD, along with the previously identified
cydA
regulator Rex (YdiH), affect the expression of the
cydABCD
operon. Low-level induction of the
cydA
promoter was observed in vivo in the absence of its regulatory proteins, Rex, CcpA, and ResD. This complex regulation suggests that the
cydA
promoter is tightly regulated to allow its expression only at the appropriate time and under the appropriate conditions.
Comparative Analysis of
hmuO
Function and Expression in
Corynebacterium
Species
Authors: Kunkle Carey A. , Schmitt Michael P. 
Published: February 2007

ABSTRACT

We have constructed defined deletions in the
hmuO
gene from
Corynebacterium diphtheriae
and
Corynebacterium ulcerans
and show that the
C. ulcerans hmuO
mutation results in a significant reduction in hemoglobin-iron utilization, whereas in
C. diphtheriae
strains, deletion of
hmuO
caused no or only partial reduction in the utilization of heme as an iron source. We also show that expression from the
C. ulcerans hmuO
promoter exhibits minimal regulation by iron and heme whereas transcription from the
C. diphtheriae hmuO
promoter shows both significant iron repression and heme-dependent activation. These findings indicate that variability in HmuO function and expression exists among
Corynebacterium
species.
Interaction of Related Tn
916
-Like Transposons: Analysis of Excision Events Promoted by Tn
916
and Tn
5386
Integrases
Authors: Rice Louis B. , Carias Lenore L. , Hutton-Thomas Rebecca , Rudin Susan 
Published: February 2007

ABSTRACT

In recent work, we described the excision of a large genomic region from
Enterococcus faecium
D344R in which the sequence from “joint” regions suggested that excision resulted from the interaction of conjugative transposon Tn
916
and the related mobile element Tn
5386
. In the present study, we examined the ability of integrases and integrase-excisase combinations from Tn
916
and Tn
5386
to promote the excision of constructs consisting of the termini of Tn
916
, Tn
5386
, and the VanB mobile element Tn
5382
. Integrases alone from either Tn
916
or Tn
5386
promoted the circularization of constructs from the three different transposons, even when the different termini used in the constructs were discordant in their transposon of origin. The termini of Tn
916
and Tn
5382
found in all joints were consistent with previously identified Tn
916
and Tn
5382
termini. Substantial variation was seen in the integrase terminus of Tn
5386
used to form joints, regardless of the integrase that was responsible for circularization. Variability was observed in joints formed from Tn
5386
constructs, in contrast to joints observed with the termini of Tn
916
or Tn
5382
. The coexpression of excisase yielded some variability in the joint regions observed. These data confirm that integrases from some Tn
916
-like elements can promote circularization with termini derived from heterologous transposons and, as such, could promote excision of large genomic regions flanked by such elements. These findings also raise interesting questions about the sequence specificities of the C terminals of Tn
916
-like integrases, which bind to the ends and facilitate strand exchange.
Defective Ribonucleoside Diphosphate Reductase Impairs Replication Fork Progression inEscherichia coli
Authors: Guarino Estrella , Jiménez-Sánchez Alfonso , Guzmán Elena C. 
Published: February 2007

ABSTRACTThe observed lengthening of theCperiod in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in thenrdA101mutant strain. We show here that thenrdA101mutation induces DNA double-strand breaks at the permissive temperature in arecB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in thenrdA101strain in a Rec+context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named “replication fork reversal.” Viability results supported the occurrence of this process, as specific lethality was observed in thenrdA101 recBdouble mutant and was suppressed by the additional inactivation ofruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in thenrdA101strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in thenrdA+strain growing under limited (2-μg/ml) or under optimal (5-μg/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of theCperiod in thenrdA101mutant at the permissive temperature.
Peptide Transport in
Helicobacter pylori
: Roles of Dpp and Opp Systems and Evidence for Additional Peptide Transporters
Authors: Weinberg Michael V. , Maier Robert J. 
Published: February 2007

ABSTRACT

Despite research into the nutritional requirements of
Helicobacter pylori
, little is known regarding its use of complex substrates, such as peptides. Analysis of genome sequences revealed putative ABC-type transporter genes for dipeptide (
dppABCDF
) and oligopeptide (
oppABCD
) transport. Genes from each system were PCR amplified, cloned, and disrupted by cassette insertion either individually (
dppA, dppB, dppC, oppA, oppB
, and
oppC
) or to create double mutants (
dppA oppA, dppB oppB, dppB dppC
, and
oppB oppC
). Peptide-utilizing abilities of the strains were assessed by monitoring growth in a chemically defined medium where the only source of the essential amino acid isoleucine was from peptides of various lengths (two to nine amino acids long). The dipeptide system mutants lacked the ability to use certain dipeptides, hexapeptides, and nonapeptides. However, these mutants retained some ability to grow with other dipeptides, tripeptides, and tetrapeptides. Of the oligopeptide mutants, only the
oppB
strain differed significantly from the wild type. This strain showed a wild-type phenotype for growth with longer peptides (hexa- and nonapeptides) while having a decreased ability to utilize di-, tri-, and tetrapeptides. The
dppA oppA
and
dppB oppB
mutants showed similar phenotypes to those of the
dppA
and
dppB
mutants, respectively. Peptide digestion by metalloproteases was ruled out as the cause for residual peptide transport by growing mutant strains in the presence of either EDTA or EGTA. Degradation products associated with a fluorescein isothiocyanate-labeled hexapeptide (plus cells) were minimal. An as yet unidentified peptide transport system(s) in
H. pylori
is proposed to be responsible for the residual transport.