bluuebunny/crossref_metadata_2025_split huggingface dataset:
Drug-Lipid A Interactions on the
Escherichia coli
ABC Transporter MsbA
Authors: Woebking Barbara , Reuter Galya , Shilling Richard A. , Velamakanni Saroj , Shahi Sanjay , Venter Henrietta , Balakrishnan Lekshmy , van Veen Hendrik W. 
Published: September 2005

ABSTRACT

MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gram-negative
Escherichia coli
and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive
Lactococcus lactis
, our recent work in
E. coli
suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in
L. lactis
. MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with

K
i

values of 16 and 11 μM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a

K
i

of 57 μM, in a similar range as the

K
i

for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbA-mediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary
E. coli
proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from
Salmonella enterica
serovar Typhimurium (C. L. Reyes and G. Chang, Science 308:1028-1031, 2005).
Bacterial Cyanide Oxygenase Is a Suite of Enzymes Catalyzing the Scavenging and Adventitious Utilization of Cyanide as a Nitrogenous Growth Substrate
Authors: Fernandez Ruby F. , Kunz Daniel A. 
Published: September 2005

ABSTRACT

Cyanide oxygenase (CNO) from
Pseudomonas fluorescens
NCIMB 11764 catalyzes the pterin-dependent oxygenolytic cleavage of cyanide (CN) to formic acid and ammonia. CNO was resolved into four protein components (P1 to P4), each of which along with a source of pterin cofactor was obligately required for CNO activity. Component P1 was characterized as a multimeric 230-kDa flavoprotein exhibiting the properties of a peroxide-forming NADH oxidase (oxidoreductase) (Nox). P2 consisted of a 49.7-kDa homodimer that showed 100% amino acid identity at its N terminus to NADH peroxidase (Npx) from
Enterococcus faecali
s. Enzyme assays further confirmed the identities of both Nox and Npx enzymes (specific activity, 1 U/mg). P3 was characterized as a large oligomeric protein (∼300 kDa) that exhibited cyanide dihydratase (CynD) activity (specific activity, 100 U/mg). Two polypeptides of 38 kDa and 43 kDa were each detected in the isolated enzyme, the former believed to confer catalytic activity based on its similar size to other CynD enzymes. The amino acid sequence of an internal peptide of the 43-kDa protein was 100% identical to bacterial elongation factor Tu, suggesting a role as a possible chaperone in the assembly of CynD or a multienzyme CNO complex. The remaining P4 component consisted of a 28.9-kDa homodimer and was identified as carbonic anhydrase (specific activity, 2,000 U/mg). While the function of participating pterin and the roles of Nox, Npx, CynD, and CA in the CNO-catalyzed scavenging of CN remain to be determined, this is the first report describing the collective involvement of these four enzymes in the metabolic detoxification and utilization of CN as a bacterial nitrogenous growth substrate.
Nitrate-Dependent Activation of the Dif Signaling Pathway ofMyxococcus xanthusMediated by a NarX-DifA Interspecies Chimera
Authors: Xu Qian , Black Wesley P. , Ward Scott M. , Yang Zhaomin 
Published: September 2005

ABSTRACTMyxococcus xanthusfibril exopolysaccharide (EPS), essential for the social gliding motility and development of this bacterium, is regulated by the Dif chemotaxis-like pathway. DifA, an MCP homolog, is proposed to mediate signal input to the Dif pathway. However, DifA lacks a prominent periplasmic domain, which in classical chemoreceptors is responsible for signal perception and for initiating transmembrane signaling. To investigate the signaling properties of DifA, we constructed a NarX-DifA (NafA) chimera from the sensory module ofEscherichia coliNarX and the signaling module ofM. xanthusDifA. We report here the first functional chimeric signal transducer constructed using genes from organisms in two different phylogenetic subdivisions. When expressed inM. xanthus, NafA restored fruiting body formation, EPS production, and S-motility todifAmutants in the presence of nitrate. Studies with various double mutants indicate that NafA requires the downstream Dif proteins to function. We propose that signal inputs to the Dif pathway and transmembrane signaling by DifA are essential for the regulation of EPS production inM. xanthus. Despite the apparent structural differences, DifA appears to share similar transmembrane signaling mechanisms with enteric sensor kinases and chemoreceptors.
Regulation of Tryptophan Operon Expression in the Archaeon
Methanothermobacter thermautotrophicus
Authors: Xie Yunwei , Reeve John N. 
Published: September 2005

ABSTRACT

Conserved
trp
genes encode enzymes that catalyze tryptophan biosynthesis in all three biological domains, and studies of their expression in
Bacteria
and eukaryotes have revealed a variety of different regulatory mechanisms. The results reported here provide the first detailed description of an archaeal
trp
gene regulatory system. We have established that the
trpEGCFBAD
operon in
Methanothermobacter thermautotrophicus
is transcribed divergently from a gene (designated
trpY
) that encodes a tryptophan-sensitive transcription regulator. TrpY binds to TRP box sequences (consensus, TGTACA) located in the overlapping promoter regions between
trpY
and
trpE
, inhibiting
trpY
transcription in the absence of tryptophan and both
trpY
and
trpEGCFBAD
transcription in the presence of tryptophan. TrpY apparently inhibits
trpY
transcription by blocking RNA polymerase access to the site of
trpY
transcription initiation and represses
trpEGCFBAD
transcription by preventing TATA box binding protein (TBP) binding to the TATA box sequence. Given that residue 2 (W2) is the only tryptophan in TrpY and in TrpY homologues in other
Euryarchaea
and that there is only one tryptophan codon in the entire
trpEGCFBAD
operon (
trpB
encodes W175), expression of the
trp
operon may also be regulated in vivo by the supply of charged tRNA
Trp
available to translate the second codon of the
trpY
mRNA.
Positive Autoregulation of
c
I Is a Dispensable Feature of the Phage λ Gene Regulatory Circuitry
Authors: Michalowski Christine B. , Little John W. 
Published: September 2005

ABSTRACT

Complex gene regulatory circuits contain many features that are likely to contribute to their operation. It is unclear, however, whether all these features are necessary for proper circuit behavior or whether certain ones are refinements that make the circuit work better but are dispensable for qualitatively normal behavior. We have addressed this question using the phage λ regulatory circuit, which can persist in two stable states, the lytic state and the lysogenic state. In the lysogenic state, the CI repressor positively regulates its own expression by stimulating transcription from the
P
RM
promoter. We tested whether this feature is an essential part of the regulatory circuitry. Several phages with a
c
I mutation preventing positive autoregulation and an up mutation in the
P
RM
promoter showed near-normal behavior. We conclude that positive autoregulation is not necessary for proper operation of the λ circuitry and speculate that it serves a partially redundant function of stabilizing a bistable circuit, a form of redundancy we term “circuit-level redundancy.” We discuss our findings in the context of a two-stage model for evolution and elaboration of regulatory circuits from simpler to more complex forms.
Analysis of PutativeChlamydia trachomatisChaperones Scc2 and Scc3 and Their Use in the Identification of Type III Secretion Substrates
Authors: Fields Kenneth A. , Fischer Elizabeth R. , Mead David J. , Hackstadt Ted 
Published: September 2005

ABSTRACTThe obligate intracellular pathogenChlamydia trachomatisexpresses a type III secretion system (T3SS) which has the potential to contribute significantly to pathogenesis. Based on a demonstrated role of type III secretion (T3S)-specific chaperones in the secretion of antihost proteins by gram-negative pathogens, we initiated a study of selected putativeChlamydiaT3S chaperones in an effort to gain mechanistic insight into theChlamydiaT3SS and to potentially identifyChlamydia-specific secreted products.C. trachomatisScc2 and Scc3 are homologous to SycD ofYersiniaspp. Functional studies of the heterologousYersiniaT3SS indicated that although neither Scc2 nor Scc3 was able to fully complement asycDnull mutant, both have SycD-like characteristics. Both were able to associate with the translocator protein YopD, and Scc3 expression restored limited secretion of YopD in in vitro studies of T3S. CopB (CT578) and CopB2 (CT861) are encoded adjacent toscc2andscc3, respectively, and have structural similarities with the YopB family of T3S translocators. Either Scc2 or Scc3 coprecipitates with CopB fromC. trachomatisextracts. Expression of CopB or CopB2 inYersiniaresulted in their type III-dependent secretion, and localization studies withC. trachomatis-infected cells indicated that both were secreted byChlamydia.
Structural Characterization of a Flavonoid-Inducible
Pseudomonas aeruginosa
A-Band-Like O Antigen of
Rhizobium
sp. Strain NGR234, Required for the Formation of Nitrogen-Fixing Nodules
Authors: Reuhs Bradley L. , Relić Biserka , Forsberg L. Scott , Marie Corinne , Ojanen-Reuhs Tuula , Stephens Samuel B. , Wong Chee-Hoong , Jabbouri Saïd , Broughton William J. 
Published: September 2005

ABSTRACT

Rhizobium
(
Sinorhizobium
) sp. strain NGR234 contains three replicons, the smallest of which (pNGR234
a
) carries most symbiotic genes, including those required for nodulation and lipo-chito-oligosaccharide (Nod factor) biosynthesis. Activation of
nod
gene expression depends on plant-derived flavonoids, NodD transcriptional activators, and
nod
box promoter elements. Nod boxes NB6 and NB7 delimit six different types of genes, one of which (
fixF
) is essential for the formation of effective nodules on
Vigna unguiculata
. In vegetative culture, wild-type NGR234 produces a distinct, flavonoid-inducible lipopolysaccharide (LPS) that is not produced by the mutant (NGRΩ
fixF
); this LPS is also found in nitrogen-fixing bacteroids isolated from
V. unguiculata
infected with NGR234. Electron microscopy showed that peribacteroid membrane formation is perturbed in nodule cells infected by the
fixF
mutant. LPSs were purified from free-living NGR234 cultured in the presence of apigenin. Structural analyses showed that the polysaccharide portions of these LPSs are specialized, rhamnose-containing O antigens attached to a modified core-lipid A carrier. The primary sequence of the O antigen is [-3)-α-
l
-Rha
p
-(1,3)-α-
l
-Rha
p
-(1,2)-α-
l
-Rha
p
-(1-]
n
, and the LPS core region lacks the acidic sugars commonly associated with the antigenic outer core of LPS from noninduced cells. This rhamnan O antigen, which is absent from noninduced cells, has the same primary sequence as the A-band O antigen of
Pseudomonas aeruginosa
, except that it is composed of
l
-rhamnose rather than the
d
-rhamnose characteristic of the latter. It is noteworthy that A-band LPS is selectively maintained on the
P. aeruginosa
cell surface during chronic cystic fibrosis lung infection, where it is associated with an increased duration of infection.
Whole-Genome Sequence Analysis of
Pseudomonas syringae
pv. phaseolicola 1448A Reveals Divergence among Pathovars in Genes Involved in Virulence and Transposition
Authors: Joardar Vinita , Lindeberg Magdalen , Jackson Robert W. , Selengut Jeremy , Dodson Robert , Brinkac Lauren M. , Daugherty Sean C. , DeBoy Robert , Durkin A. Scott , Giglio Michelle Gwinn , Madupu Ramana , Nelson William C. , Rosovitz M. J. , Sullivan Steven , Crabtree Jonathan , Creasy Todd , Davidsen Tanja , Haft Dan H. , Zafar Nikhat , Zhou Liwei , Halpin Rebecca , Holley Tara , Khouri Hoda , Feldblyum Tamara , White Owen , Fraser Claire M. , Chatterjee Arun K. , Cartinhour Sam , Schneider David J. , Mansfield John , Collmer Alan , Buell C. Robin 
Published: September 2005

ABSTRACT

Pseudomonas syringae
pv. phaseolicola, a gram-negative bacterial plant pathogen, is the causal agent of halo blight of bean. In this study, we report on the genome sequence of
P. syringae
pv. phaseolicola isolate 1448A, which encodes 5,353 open reading frames (ORFs) on one circular chromosome (5,928,787 bp) and two plasmids (131,950 bp and 51,711 bp). Comparative analyses with a phylogenetically divergent pathovar,
P. syringae
pv. tomato DC3000, revealed a strong degree of conservation at the gene and genome levels. In total, 4,133 ORFs were identified as putative orthologs in these two pathovars using a reciprocal best-hit method, with 3,941 ORFs present in conserved, syntenic blocks. Although these two pathovars are highly similar at the physiological level, they have distinct host ranges; 1448A causes disease in beans, and DC3000 is pathogenic on tomato and
Arabidopsis
. Examination of the complement of ORFs encoding virulence, fitness, and survival factors revealed a substantial, but not complete, overlap between these two pathovars. Another distinguishing feature between the two pathovars is their distinctive sets of transposable elements. With access to a fifth complete pseudomonad genome sequence, we were able to identify 3,567 ORFs that likely comprise the core
Pseudomonas
genome and 365 ORFs that are
P. syringae
specific.
TP0453, a Concealed Outer Membrane Protein of
Treponema pallidum
, Enhances Membrane Permeability
Authors: Hazlett Karsten R. O. , Cox David L. , Decaffmeyer Marc , Bennett Michael P. , Desrosiers Daniel C. , La Vake Carson J. , La Vake Morgan E. , Bourell Kenneth W. , Robinson Esther J. , Brasseur Robert , Radolf Justin D. 
Published: September 2005

ABSTRACT

The outer membrane of
Treponema pallidum
, the noncultivable agent of venereal syphilis, contains a paucity of protein(s) which has yet to be definitively identified. In contrast, the outer membranes of gram-negative bacteria contain abundant immunogenic membrane-spanning β-barrel proteins mainly involved in nutrient transport. The absence of orthologs of gram-negative porins and outer membrane nutrient-specific transporters in the
T. pallidum
genome predicts that nutrient transport across the outer membrane must differ fundamentally in
T. pallidum
and gram-negative bacteria. Here we describe a
T. pallidum
outer membrane protein (TP0453) that, in contrast to all integral outer membrane proteins of known structure, lacks extensive β-sheet structure and does not traverse the outer membrane to become surface exposed. TP0453 is a lipoprotein with an amphiphilic polypeptide containing multiple membrane-inserting, amphipathic α-helices. Insertion of the recombinant, nonlipidated protein into artificial membranes results in bilayer destabilization and enhanced permeability. Our findings lead us to hypothesize that TP0453 is a novel type of bacterial outer membrane protein which may render the
T. pallidum
outer membrane permeable to nutrients while remaining inaccessible to antibody.
Characterization of a Novel Porin Protein from
Moraxella catarrhalis
and Identification of an Immunodominant Surface Loop
Authors: Easton Donna M. , Smith Adam , Gallego Sara Gomez , Foxwell A. Ruth , Cripps Allan W. , Kyd Jennelle M. 
Published: September 2005

ABSTRACT

Moraxella catarrhalis
is a gram-negative bacterium that is mainly responsible for respiratory tract infections. In this study we report a novel outer membrane protein (OMP), designated M35, with a molecular mass of 36.1 kDa. This protein was structurally homologous to classic gram-negative porins, such as OMP C from
Escherichia coli
and OMP K36 from
Klebsiella pneumoniae
, with a predicted structure of 8 surface loops and 16 antiparallel β-sheets. The DNA sequences of the genes from 18 diverse clinical isolates showed that the gene was highly conserved (99.6 to 100% of nucleotides), with only one isolate (ID78LN266) having base variations that resulted in amino acid substitutions. Electrophoresis and analysis of recognition of the protein using mouse anti-M35 sera showed that M35 was expressed on the bacterial surface and constitutively expressed across
M. catarrhalis
isolates, with only ID78LN266 showing poor antibody recognition. Our results showed that the single amino acid mutation in loop 3 significantly affected antibody recognition, indicating that loop 3 appeared to contain an immunodominant B-cell epitope. The antibody specificity to loop 3 may be a potential mechanism for evasion of host immune responses targeted to M35, since loop 3 should theoretically orientate into the porin channel. Thus, M35 is a highly conserved, surface-expressed protein that is of significance for its potential functional role as an
M. catarrhalis
porin and is of interest as a vaccine candidate.
Cell Division inBacillus subtilis: FtsZ and FtsA Association Is Z-Ring Independent, and FtsA Is Required for Efficient Midcell Z-Ring Assembly
Authors: Jensen S. O. , Thompson L. S. , Harry E. J. 
Published: September 2005

ABSTRACTThe earliest stage in cell division in bacteria is the assembly of a Z ring at the division site at midcell. Other division proteins are also recruited to this site to orchestrate the septation process. FtsA is a cytosolic division protein that interacts directly with FtsZ. Its function remains unknown. It is generally believed that FtsA localization to the division site occurs immediately after Z-ring formation or concomitantly with it and that FtsA is responsible for recruiting the later-assembling membrane-bound division proteins to the division site. Here, we report the development of an in vivo chemical cross-linking assay to examine the association between FtsZ and FtsA inBacillus subtiliscells. We subsequently use this assay in a synchronous cell cycle to show that these two proteins can interact prior to Z-ring formation. We further show that in aB. subtilisstrain containing anftsAdeletion, FtsZ localized at regular intervals along the filament but the majority of Z rings were abnormal. FtsA in this organism is therefore critical for the efficient formation of functional Z rings. This is the first report of abnormal Z-ring formation resulting from the loss of a single septation protein. These results suggest that in this organism, and perhaps others, FtsA ensures recruitment of the membrane-bound division proteins by ensuring correct formation of the Z ring.
Differences in Gene Content betweenSalmonella entericaSerovar Enteritidis Isolates and Comparison to Closely Related Serovars Gallinarum and Dublin
Authors: Porwollik S. , Santiviago C. A. , Cheng P. , Florea L. , McClelland M. 
Published: September 2005

ABSTRACTSalmonella entericaserovar Enteritidis is often transmitted into the human food supply through eggs of hens that appear healthy. This pathogen became far more prevalent in poultry following eradication of the fowl pathogenS. entericaserovar Gallinarum in the mid-20th century. To investigate whether changes in serovar Enteritidis gene content contributed to this increased prevalence, and to evaluate genetic heterogeneity within the serovar, comparative genomic hybridization was performed on eight 60-year-old and nineteen 10- to 20-year-old serovar Enteritidis strains from various hosts, using aSalmonella-specific microarray. Overall, almost all the serovar Enteritidis genomes were very similar to each other. Excluding two rare strains classified as serovar Enteritidis in theSalmonellareference collection B, only eleven regions of the serovar Enteritidis phage type 4 (PT4) chromosome (sequenced at the Sanger Center) were absent or divergent in any of the other serovar Enteritidis strains tested. The more recent isolates did not have consistent differences from 60-year-old field isolates, suggesting that no large genomic additions on a whole-gene scale were needed for serovar Enteritidis to become more prevalent in domestic fowl. Cross-hybridization of phage genes on the array with related genes in the examined genomes grouped the serovar Enteritidis isolates into two major lineages. Microarray comparisons of the sequenced serovar Enteritidis PT4 to isolates of the closely related serovars Dublin and Gallinarum (biovars Gallinarum and Pullorum) revealed several genomic areas that distinguished them from serovar Enteritidis and from each other. These differences in gene content could be useful in DNA-based typing and in understanding the different phenotypes of these related serovars.
Evolutionary and Diagnostic Implications of Intragenomic Heterogeneity in the 16S rRNA Gene inAeromonasStrains
Authors: Morandi Alessia , Zhaxybayeva Olga , Gogarten J. Peter , Graf Joerg 
Published: September 2005

ABSTRACTSequencing 16S rRNA genes (SSU) cloned fromAeromonasstrains revealed that strains contained up to six copies differing by ≤1.5%. The SSU copies fromAeromonas veroniiLMG13695 clustered with sequences from fourAeromonasspecies. These results demonstrate intragenomic heterogeneity of SSU and suggest caution when using SSU to identify aeromonads.
Transcriptome Analysis ofPseudomonas aeruginosaGrowth: Comparison of Gene Expression in Planktonic Cultures and Developing and Mature Biofilms
Authors: Waite Richard D. , Papakonstantinopoulou Anastasia , Littler Eddie , Curtis Michael A. 
Published: September 2005

ABSTRACTThe transcriptomes of logarithmic- and stationary-phasePseudomonas aeruginosaplanktonic cultures and static biofilms of different stages of development were compared. Developing and confluent biofilm transcriptomes were found to be related to those of logarithmic- and stationary-phase planktonic cultures, respectively. In addition, a number of novel genes were up-regulated in developing and confluent biofilms, including genes encoding putative solute transport proteins and transcriptional regulators, respectively.
Extended −10 Motif Is Critical for Activity of thecspAPromoter but Does Not Contribute to Low-Temperature Transcription
Authors: Phadtare Sangita , Severinov Konstantin 
Published: September 2005

ABSTRACTBacterial promoters belonging to the extended −10 class contain a conserved TGn motif upstream of the −10 promoter consensus element. Open promoter complexes can be formed on some extended −10Escherichia colipromoters at temperatures as low as 6°C, when complexes on most promoters are closed. The promoter ofcspA, a gene that codes for the major cold shock protein CspA ofE. coli, contains an extended −10 motif. CspA is dramatically induced upon temperature downshift from 37 to 15°C, and its cold shock induction has been attributed to transcription, translation, and mRNA stabilization effects. Here, we show that though the extended −10 motif is critical for high-level expression ofcspA, it does not contribute to low-temperature expression. In fact, transcription from the wild-typecspApromoter is cold sensitive in vitro and in vivo. Thus, transcription appears to play little or no role in low-temperature induction ofcspAexpression.