bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transfer of the Symbiotic Plasmid of
Rhizobium etli
CFN42 Requires Cointegration with p42a, Which May Be Mediated by Site-Specific Recombination
Authors: Brom Susana , Girard Lourdes , Tun-Garrido Cristina , García-de los Santos Alejandro , Bustos Patricia , González Víctor , Romero David 
Published: October 2004

ABSTRACT

Plasmid p42a from
Rhizobium etli
CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym). Most pSym transconjugants also inherit p42a. pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms*. RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions. These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, and self-transmissible. The symbiotic features of derivatives simultaneously containing a wild-type pSym and an RpSym* were analyzed. Structural analysis of 10 RpSyms* showed that 7 shared one of the two pSym-p42a junctions. Sequencing of this common junction revealed a 53-bp region that was 90% identical in pSym and p42a, including a 5-bp central region flanked by 9- to 11-bp inverted repeats reminiscent of bacterial and phage attachment sites. A gene encoding an integrase-like protein (
intA
) was localized downstream of the attachment site on p42a. Mutation or the absence of
intA
abolished pSym transfer from a
recA
mutant donor. Complementation with the wild-type
intA
gene restored transfer of pSym. We propose that pSym-p42a cointegration is required for pSym transfer; cointegration may be achieved either through homologous recombination among large reiterated sequences or through IntA-mediated site-specific recombination between the attachment sites. Cointegrates formed through the site-specific system but resolved through RecA-dependent recombination or vice versa generate RpSyms*. A site-specific recombination system for plasmid cointegration is a novel feature of these large plasmids and implies that there is unique regulation which affects the distribution of pSym in nature due to the role of the cointegrate in conjugative transfer.
Decaprenyl Diphosphate Synthesis in
Mycobacterium tuberculosis
Authors: Kaur Devinder , Brennan Patrick J. , Crick Dean C. 
Published: October 2004

ABSTRACT

Z
-prenyl diphosphate synthases catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphates to synthesize polyprenyl diphosphates. In mycobacteria, these are precursors of decaprenyl phosphate, a molecule which plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan. Recently, it was demonstrated that open reading frame
Rv2361c
of the
Mycobacterium tuberculosis
H
37
Rv genome encodes a unique prenyl diphosphate synthase (M. C. Schulbach, P. J. Brennan, and D. C. Crick, J. Biol. Chem.
275:
22876-22881, 2000). We have now purified the enzyme to near homogeneity by using an
Escherichia coli
expression system and have shown that the product of this enzyme is decaprenyl diphosphate. Rv2361c has an absolute requirement for divalent cations and an optimal pH range of 7.5 to 8.5, and the activity is stimulated by both detergent and dithiothreitol. The enzyme catalyzes the addition of isopentenyl diphosphate to geranyl diphosphate, neryl diphosphate, ω,
E
,
E
-farnesyl diphosphate, ω,
E
,
Z
-farnesyl diphosphate, or ω,
E
,
E
,
E
-geranylgeranyl diphosphate, with

K
m

values for the allylic substrates of 490, 29, 84, 290, and 40 μM, respectively. The

K
m

value for isopentenyl diphosphate is 89 μM. The catalytic efficiency is greatest when ω,
E
,
Z
-farnesyl diphosphate is used as the allylic acceptor, suggesting that this is the natural substrate in vivo, a conclusion that is supported by previous structural studies of decaprenyl phosphoryl mannose isolated from
M. tuberculosis
. This is the first report of a bacterial
Z
-prenyl diphosphate synthase that preferentially utilizes an allylic diphosphate primer having the α-isoprene unit in the
Z
configuration, indicating that Rv1086 (ω,
E
,
Z
-farnesyl diphosphate synthase) and Rv2361c act sequentially in the biosynthetic pathway that leads to the formation of decaprenyl phosphate in
M. tuberculosis
.
Analysis of the Cytoplasmic Domains ofSalmonellaFlhA and Interactions with Components of the Flagellar Export Machinery
Authors: McMurry Jonathan L. , Van Arnam John S. , Kihara May , Macnab Robert M. 
Published: October 2004

ABSTRACTMost flagellar proteins are exported via a type III export apparatus which, in part, consists of the membrane proteins FlhA, FlhB, FliO, FliP, FliQ, and FliR and is housed within the membrane-supramembrane ring formed by FliF subunits.SalmonellaFlhA is a 692-residue integral membrane protein with eight predicted transmembrane spans. Its function is not understood, but it is necessary for flagellar export. We have created mutants in which potentially important sequences were deleted. FlhA lacking the amino-terminal sequence prior to the first transmembrane span failed to complement and was dominant negative, suggesting that the sequence is required for function. Similar effects were seen in a variant lacking a highly conserved domain (FHIPEP) within a putative cytoplasmic loop. Scanning deletion analysis of the cytoplasmic domain (FlhAc) demonstrated that substantially all of FlhAc is required for efficient function. Affinity blotting showed that FlhA interacts with several other export apparatus membrane proteins. The implications of these findings are discussed, and a model of FlhA within the export apparatus is presented.
Continuous Control in Bacterial Regulatory Circuits
Authors: Batchelor Eric , Silhavy Thomas J. , Goulian Mark 
Published: October 2004

ABSTRACT

We show that for two well-characterized regulatory circuits in
Escherichia coli
, Tn
10
tetracycline resistance and porin osmoregulation, the transcriptional outputs in individual cells are graded functions of the applied stimuli. These systems are therefore examples of naturally occurring regulatory circuits that exhibit continuous control of transcription. Surprisingly, however, we find that porin osmoregulation is open loop; i.e., the porin expression level does not feed back into the regulatory circuit. This mode of control is particularly interesting for an organism such as
E. coli
, which proliferates in diverse environments, and raises important questions regarding the biologically relevant inputs and outputs for this system.
Lack of YggX Results in Chronic Oxidative Stress and Uncovers Subtle Defects in Fe-S Cluster Metabolism in
Salmonella enterica
Authors: Skovran Elizabeth , Lauhon C. T. , Downs D. M. 
Published: October 2004

ABSTRACT

As components involved in Fe-S cluster metabolism are described, the challenge becomes defining the integrated process that occurs in vivo based on the individual functions characterized in vitro. Strains lacking
yggX
have been used here to mimic chronic oxidative stress and uncover subtle defects in Fe-S cluster metabolism. We describe the in vivo similarities and differences between
isc
mutants, which have a known function in cluster assembly, and mutants disrupted in four additional loci,
gshA, apbC, apbE
, and
rseC
. The latter mutants share similarities with
isc
mutants: (i) a sensitivity to oxidative stress, (ii) a thiamine auxotrophy in the absence of the YggX protein, and (iii) decreased activities of Fe-S proteins, including aconitase, succinate dehydrogenase, and MiaB. However, they differ from
isc
mutants by displaying a phenotypic dependence on metals and a distinct defect in the SoxRS response to superoxides. Results presented herein support the proposed role of YggX in iron trafficking and protection against oxidative stress, describe additional phenotypes of
isc
mutants, and suggest a working model in which the ApbC, ApbE, and RseC proteins and glutathione participate in Fe-S cluster repair.
Purification and Characterization of the Repressor of the Shiga Toxin-Encoding Bacteriophage 933W: DNA Binding, Gene Regulation, and Autocleavage
Authors: Koudelka Astrid P. , Hufnagel Lisa A. , Koudelka Gerald B. 
Published: October 2004

ABSTRACT

The genes encoding Shiga toxin (stx), the major virulence factor of Shiga toxin-encoding
Escherichia coli
(STEC) strains, are carried on lambdoid prophages resident in all known STEC strains. The
stx
genes are expressed only during lytic growth of these temperate bacteriophages. We cloned the gene encoding the repressor of the Shiga toxin-encoding bacteriophage 933W and examined the DNA binding and transcriptional regulatory activities of the overexpressed, purified protein. Typical of nearly all lambdoid phage repressors, 933W repressor binds to three sites in 933W right operator (O
R
). Also typical, when bound at O
R
, 933W repressor functions as an activator at the P
RM
promoter and a repressor at the P
R
promoter. In contrast to other lambdoid bacteriophages, 933W left operator (O
L
) contains only two repressor binding sites, but the O
L
-bound repressor still efficiently represses P
L
transcription. Lambdoid prophage induction requires inactivation of the repressor's DNA binding activity. In all phages examined thus far, this inactivation requires a RecA-stimulated repressor autoproteolysis event, with cleavage occurring precisely in an Ala-Gly dipeptide sequence that is found within a “linker ” region that joins the two domains of these proteins. However, 933W repressor protein contains neither an Ala-Gly nor an alternative Cys-Gly dipeptide cleavage site anywhere in its linker sequence. We show here that the autocleavage occurs at a Leu-Gly dipeptide. Thus, the specificity of the repressor autocleavage site is more variable than thought previously.
8-Oxo-7,8-Dihydroguanine Is Removed by a Nucleotide Excision Repair-Like Mechanism in
Porphyromonas gingivalis
W83
Authors: Johnson N. A. , McKenzie R. , McLean L. , Sowers L. C. , Fletcher H. M. 
Published: October 2004

ABSTRACT

A consequence of oxidative stress is DNA damage. The survival of
Porphyromonas gingivalis
in the inflammatory microenvironment of the periodontal pocket requires an ability to overcome oxidative stress caused by reactive oxygen species (ROS). 8-Oxo-7,8-dihydroguanine (8-oxoG) is typical of oxidative damage induced by ROS. There is no information on the presence of 8-oxoG in
P. gingivalis
under oxidative stress conditions or on a putative mechanism for its repair. High-pressure liquid chromatography with electrochemical detection analysis of chromosomal DNA revealed higher levels of 8-oxoG in
P. gingivalis
FLL92, a nonpigmented isogenic mutant, than in the wild-type strain. 8-OxoG repair activity was also increased in cell extracts from
P. gingivalis
FLL92 compared to those from the parent strain. Enzymatic removal of 8-oxoG was catalyzed by a nucleotide excision repair (NER)-like mechanism rather than the base excision repair (BER) observed in
Escherichia coli
. In addition, in comparison with other anaerobic periodontal pathogens, the removal of 8-oxoG was unique to
P. gingivalis
. Taken together, the increased 8-oxoG levels in
P. gingivalis
FLL92 could further support a role for the hemin layer as a unique mechanism in oxidative stress resistance in this organism. In addition, this is the first observation of an NER-like mechanism as the major mechanism for removal of 8-oxoG in
P. gingivalis
.
Transcription of the Rod-Shaped Viruses SIRV1 and SIRV2 of the Hyperthermophilic ArchaeonSulfolobus
Authors: Kessler Alexandra , Brinkman Arie B. , van der Oost John , Prangishvili David 
Published: October 2004

ABSTRACTThe double-stranded DNA genomes of the crenarchaeal rudiviruses SIRV1 (32 kb) and SIRV2 (35 kb) were previously sequenced. Here we present results of the analysis of gene expression of these viruses at different time points after infection of the host cell,Sulfolobus islandicus, and of the mapping of transcriptional start sites. Transcription of both genomes starts simultaneously at multiple sites spread over the total length of the genome and from both strands. The earliest time point when viral transcripts could be detected in cells was 30 min after infection. At this time point all the viral genes, except one, were transcribed. Many genes were clustered and appeared to be transcribed as polycistronic messengers. Although the coat protein-encoding gene was initially also transcribed as a polycistronic messenger, an abundant monocistronic transcript of this gene was detected 2 to 3 h after infection, just before assembly of viral particles. The expression of a single gene, adjacent to the coat protein gene, was upregulated at the late phase of infection, suggesting that it might be involved in specific processing and activation of the coat protein messenger. Start sites of 13 transcripts from the SIRV1 genome have been mapped by primer extension, and promoter sequences have been identified. Similar to host promoters, these viral promoters all contain potential binding sites for the archaeal transcription factors TATA binding protein and transcription factor B. In addition, most of them contain a virus-specific consensus element, suggesting the involvement of alternative transcription factors.
Identification and Functional Verification of Archaeal-Type Phosphoenolpyruvate Carboxylase, a Missing Link in Archaeal Central Carbohydrate Metabolism
Authors: Ettema Thijs J. G. , Makarova Kira S. , Jellema Gera L. , Gierman Hinco J. , Koonin Eugene V. , Huynen Martijn A. , de Vos Willem M. , van der Oost John 
Published: October 2004

ABSTRACT

Despite the fact that phosphoenolpyruvate carboxylase (PEPC) activity has been measured and in some cases even purified from some
Archaea
, the gene responsible for this activity has not been elucidated. Using sensitive sequence comparison methods, we detected a highly conserved, uncharacterized archaeal gene family that is distantly related to the catalytic core of the canonical PEPC. To verify the predicted function of this archaeal gene family, we cloned a representative from the hyperthermophilic acidophile
Sulfolobus solfataricus
and functionally produced the corresponding enzyme as a fusion with the
Escherichia coli
maltose-binding protein. The purified fusion protein indeed displayed highly thermostable PEPC activity. The structural and biochemical properties of the characterized archaeal-type PEPC (atPEPC) from
S. solfataricus
are in good agreement with previously reported biochemical analyses of other archaeal PEPC enzymes. The newly identified atPEPC, with its distinct properties, constitutes yet another example of the versatility of the enzymes of the central carbon metabolic pathways in the archaeal domain.
Single-Molecule Approach to Bacterial Genomic Comparisons via Optical Mapping
Authors: Zhou Shiguo , Kile Andrew , Bechner Michael , Place Michael , Kvikstad Erika , Deng Wen , Wei Jun , Severin Jessica , Runnheim Rodney , Churas Christopher , Forrest Daniel , Dimalanta Eileen T. , Lamers Casey , Burland Valerie , Blattner Frederick R. , Schwartz David C. 
Published: October 2004

ABSTRACTModern comparative genomics has been established, in part, by the sequencing and annotation of a broad range of microbial species. To gain further insights, new sequencing efforts are now dealing with the variety of strains or isolates that gives a species definition and range; however, this number vastly outstrips our ability to sequence them. Given the availability of a large number of microbial species, new whole genome approaches must be developed to fully leverage this information at the level of strain diversity that maximize discovery. Here, we describe how optical mapping, a single-molecule system, was used to identify and annotate chromosomal alterations between bacterial strains represented by several species. Since whole-genome optical maps are ordered restriction maps, sequenced strains ofShigella flexneriserotype 2a (2457T and 301),Yersinia pestis(CO 92 and KIM), andEscherichia coliwere aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations. Importantly, an unsequencedShigella flexneristrain (serotype Y strain AMC[328Y]) was optically mapped and aligned with two sequenced ones to reveal one novel locus implicated in serotype conversion and several other loci containing insertion sequence elements or phage-related gene insertions. Our results suggest that genomic rearrangements and chromosomal breakpoints are readily identified and annotated against a prototypic sequenced strain by using the tools of optical mapping.
Functional Characterization of a Catabolic Plasmid from Polychlorinated- Biphenyl-Degrading
Rhodococcus
sp. Strain RHA1
Authors: Warren René , Hsiao William W. L. , Kudo Hisashi , Myhre Matt , Dosanjh Manisha , Petrescu Anca , Kobayashi Hiroyuki , Shimizu Satoru , Miyauchi Keisuke , Masai Eiji , Yang George , Stott Jeff M. , Schein Jacquie E. , Shin Heesun , Khattra Jaswinder , Smailus Duane , Butterfield Yaron S. , Siddiqui Asim , Holt Robert , Marra Marco A. , Jones Steven J. M. , Mohn William W. , Brinkman Fiona S. L. , Fukuda Masao , Davies Julian , Eltis Lindsay D. 
Published: October 2004

ABSTRACT

Rhodococcus
sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial
rep
gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative “genomic islands” (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.
Cuprous Oxidase Activity of CueO from
Escherichia coli
Authors: Singh Satish K. , Grass Gregor , Rensing Christopher , Montfort William R. 
Published: October 2004

ABSTRACT

We have found CueO from
Escherichia coli
to have a robust cuprous oxidase activity, severalfold higher than any homologue. These data suggest that a functional role for CueO in protecting against copper toxicity in vivo includes the removal of Cu(I).
Assessment of18F-fluorodeoxyglucose dual-head gamma camera in asbestos lung diseases
Authors: Melloni B. , Monteil J. , Vincent F. , Bertin F. , Gaillard S. , Ducloux T. , Verbeke S. , Maubon A. , Vandroux J-C. , Bonnaud F. 
Published: October 2004

The purpose of this study was to evaluate the performance of18F-fluorodeoxyglucose (18FDG) imagingviacoincidence detection emission tomography (CDET) in identifying malignant lesions in subjects exposed to asbestos.A total of 30 patients exposed to asbestos underwent18FDG-CDET between January 2000 and June 2003. A CDET scan of the thorax and abdomen was performed 60 min after injection of18FDG in fasting patients, and results were obtained in slices in three axes. The CDET results were compared to those from computed tomography (CT), and pleural or surgical biopsy in patients with positive18FDG-CDET results.All primary malignant mesotheliomas accumulated18FDG (n=6), and, in two patients, CDET findings were superior to those of CT, allowing early detection. In two cases, lung carcinomas with malignant pleural effusion were also detected. There were five false positive CDET results: three unilateral pleural thickening, one rounded atelectasis, and one benign lung nodule. All patients with pleural plaques showed no significant18FDG uptake. Malignant diseases were detected by18FDG-CDET imaging with a sensitivity of 89% and specificity of 71%.Coincidence detection emission tomography can identify malignant mesothelioma in selected subjects exposed to asbestos. Coincidence detection emission tomography appears to be a useful noninvasive method for the follow-up of subjects with exposure risk of asbestosis.
Oesophageal pH monitoring is of limited value in the diagnosis of “reflux-cough”
Authors: Patterson R.N. , Johnston B.T. , MacMahon J. , Heaney L.G. , McGarvey L.P.A. 
Published: October 2004

Reflux-cough is a diagnosis based on demonstrating both gastro-oesophageal reflux and a positive response to anti-reflux therapy. The authors sought to determine early and long-term response to therapy in patients with a “positive” 24 h oesophageal pH study, and identify any features which might predict response.Patients with chronic cough were recruited from July 1998 to July 2002. Those with a positive pH study were given dietary advice and an 8-week trial of omeprazole (20 mgb.i.d.). Response was judged after 8 weeks (clinical follow-up), and at long-term follow-up (telephone questionnaire).A total of 146 patients underwent pH monitoring with 82 (56.2%) “positive” studies. Follow-up data was available in 60 patients, with 49 receiving anti-reflux therapy, of which 20 (40.8%) reported a positive treatment response. At long-term follow-up (median 30 months), there was a significantly lower response (14 out of 49, 28.5%), with no significant difference in either acid exposure times (distal/proximal) or symptom correlation between responders and nonresponders at early or long-term follow-up.In conclusion, despite “positive” pH studies, over half of the patients (55.1%) failed torespond to therapy. No features on pH monitoring accurately predicted response. Short-term response did not predict long-term response. The precise role for pH monitoring in the assessment of chronic cough remains to be defined.
Morning levels of fibrinogen in children with sleep-disordered breathing
Authors: Kaditis A.G. , Alexopoulos E.I. , Kalampouka E. , Kostadima E. , Angelopoulos N. , Germenis A. , Zintzaras E. , Gourgoulianis K. 
Published: October 2004

Elevated fibrinogen level is a predictor of cardiovascular disease in adults, and it is associated with sleep-disordered breathing. Levels of fibrinogen in adults are affected by other co-existing cardiovascular risk factors, which are not usually present in children.To investigate the association between fibrinogen and sleep-disordered breathing, a case-control study was carried out in children with and without habitual snoring. All snoring children underwent polysomnography.Morning fibrinogen values in 30 children with snoring and an apnoea-hypopnoea index (AHI) ≥5 episodes·h−1(median (interquartile range) 318 mg·dL−1(290–374)) were similar to values in 61 children with snoring and an AHI <5 episodes·h−1(307 (269–346)). Both groups had higher fibrinogen values than those in 23 controls without snoring (271 mg·dL−1(244–294)). There was no correlation between fibrinogen values and AHI, respiratory movement/arousal index or haemoglobin desaturation index.In conclusion, fibrinogen values are higher in children with snoring than in controls, but there is no association between these values and polysomnography indices.