bluuebunny/crossref_metadata_2025_split huggingface dataset:
Three Proliferating Cell Nuclear Antigen-Like Proteins Found in the Hyperthermophilic Archaeon
Aeropyrum pernix
: Interactions with the Two DNA Polymerases
Authors: Daimon Katsuya , Kawarabayasi Yutaka , Kikuchi Hisashi , Sako Yoshihiko , Ishino Yoshizumi 
Published: April 2009

ABSTRACT

Proliferating cell nuclear antigen (PCNA) is an essential component in the eukaryotic DNA replication machinery, in which it works for tethering DNA polymerases on the DNA template to accomplish processive DNA synthesis. The PCNA also interacts with many other proteins in important cellular processes, including cell cycle control, DNA repair, and an apoptotic pathway in the domain
Eucarya
. We identified three genes encoding PCNA-like sequences in the genome of
Aeropyrum pernix
, a crenarchaeal archaeon. We cloned and expressed these genes in
Escherichia coli
and analyzed the gene products. All three PCNA homologs stimulated the primer extension activities of the two DNA polymerases, polymerase I (Pol I) and Pol II, identified in
A. pernix
to various extents, among which
A. pernix
PCNA 3 (ApePCNA3) provided a most remarkable effect on both Pol I and Pol II. The three proteins were confirmed to exist in the
A. pernix
cells. These results suggest that the three PCNAs work as the processivity factor of DNA polymerases in
A. pernix
cells under different conditions. In
Eucarya
, three checkpoint proteins, Hus1, Rad1, and Rad9, have been proposed to form a PCNA-like ring structure and may work as a sliding clamp for the translesion DNA polymerases. Therefore, it is very interesting that three active PCNAs were found in one archaeal cell. Further analyses are necessary to determine whether each PCNA has specific roles, and moreover, how they reveal different functions in the cells.
Identification of the Periplasmic Cobalamin-Binding Protein BtuF ofEscherichia coli
Authors: Cadieux Nathalie , Bradbeer Clive , Reeger-Schneider Eva , Köster Wolfgang , Mohanty Arun K. , Wiener Michael C. , Kadner Robert J. 
Published: April 2009

ABSTRACTCells ofEscherichia colitake up vitamin B12(cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to thebtuCEDoperon. The open reading frame termedyadTin theE. coligenomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake inSalmonella. TheE. coli yadTproduct, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His6tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation inbtuF, but not in the flanking genespfsandyadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of thebtuFmutant was much stronger than the slight impairment previously described forbtuC,btuD, orbtuFmutants. Hence, null mutations inbtuCandbtuDwere constructed and revealed that thebtuCmutant had a strong impairment similar to that of thebtuFmutant, whereas thebtuDdefect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.
Isolation and Characterization of BTF-37: Chromosomal DNA Captured from
Bacteroides fragilis
That Confers Self-Transferability and Expresses a Pilus-Like Structure in
Bacteroides
spp. and
Escherichia coli
Authors: Vedantam Gayatri , Hecht David W. 
Published: April 2009

ABSTRACT

We report the isolation and preliminary characterization of BTF-37, a new 52-kb transfer factor isolated from
Bacteroides fragilis
clinical isolate LV23. BTF-37 was obtained by the capture of new DNA in the nonmobilizable
Bacteroides-Escherichia coli
shuttle vector pGAT400ΔBglII using a functional assay. BTF-37 is self-transferable within and from
Bacteroides
and also self-transfers in
E. coli
. Partial DNA sequencing, colony hybridization, and PCR revealed the presence of Tet element-specific sequences in BTF-37. In addition, Tn
5520
, a small mobilizable transposon that we described previously (G. Vedantam, T. J. Novicki, and D. W. Hecht, J. Bacteriol. 181:2564–2571, 1999), was also coisolated within BTF-37. Scanning and transmission electron microscopy of Tet element-containing
Bacteroides
spp. and BTF-37-harboring
Bacteroides
and
E. coli
strains revealed the presence of pilus-like cell surface structures. These structures were visualized in
Bacteroides
spp. only when BTF-37 and Tet element strains were induced with subinhibitory concentrations of tetracycline and resembled those encoded by
E. coli
broad-host-range plasmids. We conclude that we have captured a new, self-transferable transfer factor from
B. fragilis
LV23 and that this new factor encodes a tetracycline-inducible
Bacteroides
sp. conjugation apparatus.
Integration of Global Regulation of Two Aromatic-Responsive σ54-Dependent Systems: a Common Phenotype by Different Mechanisms
Authors: Sze Chun Chau , Bernardo Lisandro M. D. , Shingler Victoria 
Published: April 2009

ABSTRACTPseudomonas-derived regulators DmpR and XylR are structurally and mechanistically related σ54-dependent activators that control transcription of genes involved in catabolism of aromatic compounds. The binding of distinct sets of aromatic effectors to these regulatory proteins results in release of a repressive interdomain interaction and consequently allows the activators to promote transcription from their cognate target promoters. The DmpR-controlled Po promoter region and the XylR-controlled Pu promoter region are also similar, although homology is limited to three discrete DNA signatures for binding σ54RNA polymerase, the integration host factor, and the regulator. These common properties allow cross-regulation of Pu and Po by DmpR and XylR in response to appropriate aromatic effectors. In vivo, transcription of both the DmpR/Po and XylR/Pu regulatory circuits is subject to dominant global regulation, which results in repression of transcription during growth in rich media. Here, we comparatively assess the contribution of (p)ppGpp, the FtsH protease, and a component of an alternative phosphoenolpyruvate-sugar phosphotransferase system, which have been independently implicated in mediating this level of regulation. Further, by exploiting the cross-regulatory abilities of these two circuits, we identify the target component(s) that are intercepted in each case. The results show that (i) contrary to previous speculation, FtsH is not universally required for transcription of σ54-dependent systems; (ii) the two factors found to impact the XylR/Pu regulatory circuit do not intercept the DmpR/Po circuit; and (iii) (p)ppGpp impacts the DmpR/Po system to a greater extent than the XylR/Pu system in both the nativePseudomonas putidaand a heterologousEscherichia colihost. The data demonstrate that, despite the similarities of the specific regulatory circuits, the host global regulatory network latches onto and dominates over these specific circuits by exploiting their different properties. The mechanistic implications of how each of the host factors exerts its action are discussed.
rpoS
Mutations and Loss of General Stress Resistance in
Escherichia coli
Populations as a Consequence of Conflict between Competing Stress Responses
Authors: Notley-McRobb Lucinda , King Thea , Ferenci Thomas 
Published: April 2009

ABSTRACT

The general stress resistance of
Escherichia coli
is controlled by the RpoS sigma factor (φ
S
), but mutations in
rpoS
are surprisingly common in natural and laboratory populations. Evidence for the selective advantage of losing
rpoS
was obtained from experiments with nutrient-limited bacteria at different growth rates. Wild-type bacteria were rapidly displaced by
rpoS
mutants in both glucose- and nitrogen-limited chemostat populations. Nutrient limitation led to selection and sweeps of
rpoS
null mutations and loss of general stress resistance. The rate of takeover by
rpoS
mutants was most rapid (within 10 generations of culture) in slower-growing populations that initially express higher φ
S
levels. Competition for core RNA polymerase is the likeliest explanation for reduced expression from distinct promoters dependent on φ
70
and involved in the hunger response to nutrient limitation. Indeed, the mutation of
rpoS
led to significantly higher expression of genes contributing to the high-affinity glucose scavenging system required for the hunger response. Hence,
rpoS
polymorphism in
E. coli
populations may be viewed as the result of competition between the hunger response, which requires sigma factors other than φ
S
for expression, and the maintenance of the ability to withstand external stresses. The extent of external stress significantly influences the spread of
rpoS
mutations. When acid stress was simultaneously applied to glucose-limited cultures, both the phenotype and frequency of
rpoS
mutations were attenuated in line with the level of stress. The conflict between the hunger response and maintenance of stress resistance is a potential weakness in bacterial regulation.
Cytochrome
aa
3
in
Haloferax volcanii
Authors: Tanaka Mikiei , Ogawa Naohide , Ihara Kunio , Sugiyama Yasuo , Mukohata Yasuo 
Published: April 2009

ABSTRACT

A cytochrome in an extremely halophilic archaeon,
Haloferax volcanii
, was purified to homogeneity. This protein displayed a redox difference spectrum that is characteristic of
a
-type cytochromes and a CN
−
complex spectrum that indicates the presence of heme
a
and heme
a
3
. This cytochrome
aa
3
consisted of 44- and 35-kDa subunits. The amino acid sequence of the 44-kDa subunit was similar to that of the heme-copper oxidase subunit I, and critical amino acid residues for metal binding, such as histidines, were highly conserved. The reduced cytochrome
c
partially purified from the bacterial membrane fraction was oxidized by the cytochrome
aa
3
, providing physiological evidence for electron transfer from cytochrome
c
to cytochrome
aa
3
in archaea.
Molecular and Physical Characterization of
Burkholderia mallei
O Antigens
Authors: Burtnick Mary N. , Brett Paul J. , Woods Donald E. 
Published: April 2009

ABSTRACT

Burkholderia mallei
lipopolysaccharide (LPS) has been previously shown to cross-react with polyclonal antibodies raised against
B. pseudomallei
LPS; however, we observed that
B. mallei
LPS does not react with a monoclonal antibody (Pp-PS-W) specific for
B. pseudomallei
O polysaccharide (O-PS). In this study, we identified the O-PS biosynthetic gene cluster from
B. mallei
ATCC 23344 and subsequently characterized the molecular structure of the O-PS produced by this organism.
NADPH-Dependent
l
-Sorbose Reductase Is Responsible for
l
-Sorbose Assimilation in
Gluconobacter suboxydans
IFO 3291
Authors: Shinjoh Masako , Tazoe Masaaki , Hoshino Tatsuo 
Published: April 2009

ABSTRACT

The NADPH-dependent
l
-sorbose reductase (SR) of
l
-sorbose-producing
Gluconobacter suboxydans
IFO 3291 contributes to intracellular
l
-sorbose assimilation. The gene disruptant showed no SR activity and did not assimilate the once-produced
l
-sorbose, indicating that the SR functions mainly as an
l
-sorbose-reducing enzyme in vivo and not as a
d
-sorbitol-oxidizing enzyme.
ZupT Is a Zn(II) Uptake System in
Escherichia coli
Authors: Grass Gregor , Wong Marco D. , Rosen Barry P. , Smith Ron L. , Rensing Christopher 
Published: April 2009

ABSTRACT

Escherichia coli zupT
(
ygiE
), encoding a ZIP family member, mediated zinc uptake. Growth of cells disrupted in both
zupT
and the
znuABC
operon was inhibited by EDTA at a much lower concentration than a single mutant or the wild type. Cells expressing ZupT from a plasmid exhibited increased uptake of
65
Zn
2+
.
The Flavoprotein MrsD Catalyzes the Oxidative Decarboxylation Reaction Involved in Formation of the Peptidoglycan Biosynthesis Inhibitor Mersacidin
Authors: Majer Florian , Schmid Dietmar G. , Altena Karsten , Bierbaum Gabriele , Kupke Thomas 
Published: April 2009

ABSTRACT
The lantibiotic mersacidin inhibits peptidoglycan biosynthesis by binding to the peptidoglycan precursor lipid II. Mersacidin contains an unsaturated thioether bridge, which is proposed to be synthesized by posttranslational modifications of threonine residue +15 and the COOH-terminal cysteine residue of the mersacidin precursor peptide MrsA. We show that the flavoprotein MrsD catalyzes the oxidative decarboxylation of the COOH-terminal cysteine residue of MrsA to an aminoenethiol residue. MrsD belongs to the recently described family of homo-oligomeric flavin-containing Cys decarboxylases (i.e., the HFCD protein family). Members of this protein family include the bacterial Dfp proteins (which are involved in coenzyme A biosynthesis), eukaryotic salt tolerance proteins, and further oxidative decarboxylases such as EpiD. In contrast to EpiD and Dfp, MrsD is a FAD and not an FMN-dependent flavoprotein. HFCD enzymes are characterized by a conserved His residue which is part of the active site. Exchange of this His residue for Asn led to inactivation of MrsD. The lantibiotic-synthesizing enzymes EpiD and MrsD have different substrate specificities.
PduA Is a Shell Protein of Polyhedral Organelles Involved in Coenzyme B
12
-Dependent Degradation of 1,2-Propanediol in
Salmonella enterica
Serovar Typhimurium LT2
Authors: Havemann Gregory D. , Sampson Edith M. , Bobik Thomas A. 
Published: April 2009

ABSTRACT

Salmonella enterica
forms polyhedral organelles involved in coenzyme B
12
-dependent 1,2-propanediol degradation. These organelles are thought to consist of a proteinaceous shell that encases coenzyme B
12
-dependent diol dehydratase and perhaps other enzymes involved in 1,2-propanediol degradation. The function of these organelles is unknown, and no detailed studies of their structure have been reported. Genes needed for organelle formation and for 1,2-propanediol degradation are located at the 1,2-propanediol utilization (
pdu
) locus, but the specific genes involved in organelle formation have not been identified. Here, we show that the
pduA
gene encodes a shell protein required for the formation of polyhedral organelles involved in coenzyme B
12
-dependent 1,2-propanediol degradation. A His
6
-PduA fusion protein was purified from a recombinant
Escherichia coli
strain and used for the preparation of polyclonal antibodies. The anti-PduA antibodies obtained were partially purified by a subtraction procedure and used to demonstrate that the PduA protein localized to the shell of the polyhedral organelles. In addition, electron microscopy studies established that strains with nonpolar
pduA
mutations were unable to form organelles. These results show that the
pduA
gene is essential for organelle formation and indicate that the PduA protein is a structural component of the shell of these organelles. Physiological studies of nonpolar
pduA
mutants were also conducted. Such mutants grew similarly to the wild-type strain at low concentrations of 1,2-propanediol but exhibited a period of interrupted growth in the presence of higher concentrations of this growth substrate. Growth tests also showed that a nonpolar
pduA
deletion mutant grew faster than the wild-type strain at low vitamin B
12
concentrations. These results suggest that the polyhedral organelles formed by
S. enterica
during growth on 1,2-propanediol are not involved in the concentration of 1,2-propanediol or coenzyme B
12
, but are consistent with the hypothesis that these organelles moderate aldehyde production to minimize toxicity.
Reconstitution of the Trimethylamine Oxide Reductase Regulatory Elements of
Shewanella oneidensis
in
Escherichia coli
Authors: Gon Stéphanie , Patte Jean-Claude , Dos Santos Jean-Philippe , Méjean Vincent 
Published: April 2009

ABSTRACT

Several bacteria can grow by using small organic compounds such as trimethylamine oxide (TMAO) as electron acceptors. In
Shewanella
species, the TMAO reductase respiratory system is encoded by the
torECAD
operon. We showed that production of the TMAO reductase of
S. oneidensis
was induced by TMAO and repressed by oxygen, and we noticed that a three-gene cluster (
torSTR
) encoding a complex two-component regulatory system was present downstream of the
torECAD
operon. We introduced the
torSTR
gene cluster into
Escherichia coli
and showed that this regulatory gene cluster is involved in TMAO induction of the
torE
promoter but plays no role in the oxygen control. The TorR response regulator was purified, and gel shift and footprinting experiments revealed that TorR binds to a single region located about 70 bases upstream of the transcription start site of the
tor
structural operon. By deletion analysis, we confirmed that the TorR operator site is required for induction of the
tor
structural promoter. As the TMAO regulatory system of
S. oneidensis
is homologous to that of
E. coli
, we investigated a possible complementation between the TMAO regulatory components of the two bacteria. Interestingly, TorS
ec
, the TMAO sensor of
E. coli
, was able to transphosphorylate TorR
so
, the TMAO response regulator of
S. oneidensis
.
Salmonella enterica
Serovar Typhimurium Resistance to Bile: Identification and Characterization of the
tolQRA
Cluster
Authors: Prouty Angela M. , Van Velkinburgh Jennifer C. , Gunn John S. 
Published: April 2009

ABSTRACT

Salmonella enterica
serovar Typhimurium is resistant to the action of bile salts, and resistance to bile is enhanced in strains in which the PhoP-PhoQ (PhoPQ) two-component regulatory system has been activated. To identify genes necessary for bile resistance, MudJ transposon mutagenesis was performed on a strain containing a
phoP
mutation that results in constitutive expression of PhoP-activated genes. After screening >10,000 mutants for the loss of growth on Luria-Bertani broth-bile plates, 14 bile-sensitive mutants were identified. Of these 14 mutants, 3 were found to retain the bile sensitivity phenotype upon P22 transduction, to possess wild-type growth characteristics, and to contain a smooth lipopolysaccharide. Southern hybridization experiments showed that all three strains contained unique insertions. DNA sequencing of the transposon-chromosomal-DNA fusion junctions of these strains showed all to be linked to the putative
Salmonella orf1-tolQRA
operon, with insertions in
tolQ
,
orf1
, and a gene upstream of the
orf1-tolQRA
operon not previously associated with Tol function (
orfX
). Through the use of transcriptional fusions, none of the putative
tol
(or
tol
-associated) genes were shown to be regulated by PhoPQ, bile, or the RcsC-RcsB two-component system; however, all of the genes (
orfX
,
orf1
,
tolQRA
) are predicted to be cotranscribed. This is the first identification of
Salmonella
serovar Typhimurium Tol homologs and the first demonstration of their role in bile resistance in this organism. In addition, the observed regulation, operon arrangement, and phenotypes associated with these
tol
genes demonstrate significant differences from their
Escherichia coli
homologs.
Allelic Diversity and Population Structure in
Vibrio cholerae
O139 Bengal Based on Nucleotide Sequence Analysis
Authors: Farfán M. , Miñana-Galbis D. , Fusté M. C. , Lorén J. G. 
Published: April 2009

ABSTRACT

Comparative analysis of gene fragments of six housekeeping loci, distributed around the two chromosomes of
Vibrio cholerae
, has been carried out for a collection of 29
V. cholerae
O139 Bengal strains isolated from India during the first epidemic period (1992 to 1993). A toxigenic O1 ElTor strain from the seventh pandemic and an environmental non-O1/non-O139 strain were also included in this study. All loci studied were polymorphic, with a small number of polymorphic sites in the sequenced fragments. The genetic diversity determined for our O139 population is concordant with a previous multilocus enzyme electrophoresis study in which we analyzed the same
V. cholerae
O139 strains. In both studies we have found a higher genetic diversity than reported previously in other molecular studies. The results of the present work showed that O139 strains clustered in several lineages of the dendrogram generated from the matrix of allelic mismatches between the different genotypes, a finding which does not support the hypothesis previously reported that the O139 serogroup is a unique clone. The statistical analysis performed in the
V. cholerae
O139 isolates suggested a clonal population structure. Moreover, the application of the Sawyer's test and split decomposition to detect intragenic recombination in the sequenced gene fragments did not indicate the existence of recombination in our O139 population.
Trinucleotide GAA Repeats Dictate pMGA Gene Expression in
Mycoplasma gallisepticum
by Affecting Spacing between Flanking Regions
Authors: Liu Li , Panangala Victor S. , Dybvig Kevin 
Published: April 2009

ABSTRACT

The pMGA genes of the avian respiratory pathogen
Mycoplasma gallisepticum
encode a family of hemagglutinins that are subject to phase variation. A trinucleotide GAA repeat region is located upstream of the pMGA transcription start site. The length of the repeat region varies at a high frequency due to changes in the number of repeat units. Previous studies have shown that pMGA genes are transcribed when 12 GAA repeats are present but are not transcribed when the number of repeats is not 12. To further analyze the mechanism of gene regulation, the pMGA promoter region was modified either by deleting the nucleotides 5" of the GAA repeats or by inserting linkers of 10 or 12 bp at a position 3" of the repeats. The modified promoter region was fused to a promoterless
lacZ
gene and transformed into
M. gallisepticum
by using transposon Tn
4001
as a vector. Transformants and successive generations of progeny were analyzed with 5-bromo-4-chloro-3-indolyl-β-
d
-galactopyranoside (X-Gal) to monitor β-galactosidase activity. For the transformants of
M. gallisepticum
containing the reporter with deletion of nucleotides 5" of the GAA repeats, GAA-dependent pMGA gene regulation was abolished. For the transformants containing the reporter with an addition of 10- or 12-bp linkers,
lacZ
was expressed only when eight GAA repeats were present. These data indicate that the nucleotides 5" of the GAA repeats as well as the spacing between the GAA repeats and sequences downstream (3") of the repeats are important for pMGA gene expression.