bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thioredoxin 1 Participates in the Activity of theSalmonella entericaSerovar Typhimurium Pathogenicity Island 2 Type III Secretion System
Authors: Negrea Aurel , Bjur Eva , Puiac Speranta , Ygberg Sofia Eriksson , Åslund Fredrik , Rhen Mikael 
Published: September 2009

ABSTRACTThe facultative intracellular pathogenSalmonella entericaserovar Typhimurium relies on itsSalmonellapathogenicity island 2 (SPI2) type III secretion system (T3SS) for intracellular replication and virulence. We report that the oxidoreductase thioredoxin 1 (TrxA) and SPI2 are coinduced for expression under in vitro conditions that mimic an intravacuolar environment, that TrxA is needed for proper SPI2 activity under these conditions, and that TrxA is indispensable for SPI2 activity in both phagocytic and epithelial cells. Infection experiments in mice demonstrated that SPI2 strongly contributed to virulence in a TrxA-proficient background whereas SPI2 did not affect virulence in atrxAmutant. Complementation analyses using wild-typetrxAor a genetically engineeredtrxAcoding for noncatalytic TrxA showed that the catalytic activity of TrxA is essential for SPI2 activity in phagocytic cells whereas a noncatalytic variant of TrxA partially sustained SPI2 activity in epithelial cells and virulence in mice. These results show that TrxA is needed for the intracellular induction of SPI2 and provide new insights into the functional integration between catalytic and noncatalytic activities of TrxA and a bacterial T3SS in different settings of intracellular infections.
A Protein Important for Antimicrobial Peptide Resistance, YdeI/OmdA, Is in the Periplasm and Interacts with OmpD/NmpC
Authors: Pilonieta M. Carolina , Erickson Kimberly D. , Ernst Robert K. , Detweiler Corrella S. 
Published: September 2009

ABSTRACTAntimicrobial peptides (AMPs) kill or prevent the growth of microbes. AMPs are made by virtually all single and multicellular organisms and are encountered by bacteria in diverse environments, including within a host. Bacteria use sensor-kinase systems to respond to AMPs or damage caused by AMPs.Salmonella entericadeploys at least three different sensor-kinase systems to modify gene expression in the presence of AMPs: PhoP-PhoQ, PmrA-PmrB, and RcsB-RcsC-RcsD. TheydeIgene is regulated by the RcsB-RcsC-RcsD pathway and encodes a 14-kDa predicted oligosaccharide/oligonucleotide binding-fold (OB-fold) protein important for polymyxin B resistance in broth and also for virulence in mice. We report here thatydeIis additionally regulated by the PhoP-PhoQ and PmrA-PmrB sensor-kinase systems, which confer resistance to cationic AMPs by modifying lipopolysaccharide (LPS).ydeI, however, is not important for known LPS modifications. Two independent biochemical methods found that YdeI copurifies with OmpD/NmpC, a member of the trimeric β-barrel outer membrane general porin family. Genetic analysis indicates thatompDcontributes to polymyxin B resistance, and bothydeIandompDare important for resistance to cathelicidin antimicrobial peptide, a mouse AMP produced by multiple cell types and expressed in the gut. YdeI localizes to the periplasm, where it could interact with OmpD. A second predicted periplasmic OB-fold protein, YgiW, and OmpF, another general porin, also contribute to polymyxin B resistance. Collectively, the data suggest that periplasmic OB-fold proteins can interact with porins to increase bacterial resistance to AMPs.
A 5′ Leader Sequence Regulates Expression of Methanosarcinal CO Dehydrogenase/Acetyl Coenzyme A Synthase
Authors: Anderson Kimberly L. , Apolinario Ethel E. , MacAuley Sheridan R. , Sowers Kevin R. 
Published: September 2009

ABSTRACT

In vivo expression of CO dehydrogenase/acetyl coenzyme A synthase in
Methanosarcina
spp. is coordinately regulated in response to substrate by at least two mechanisms: differential transcription initiation and early elongation termination near the 3′ end of a 371-bp leader sequence. This is the first report of regulation of transcription elongation in the
Archaea
.
Functional Analysis of theHelicobacter pyloriFlagellar Switch Proteins
Authors: Lowenthal Andrew C. , Hill Marla , Sycuro Laura K. , Mehmood Khalid , Salama Nina R. , Ottemann Karen M. 
Published: September 2009

ABSTRACTHelicobacter pyloriuses flagellum-mediated chemotaxis to promote infection. Bacterial flagella change rotational direction by changing the state of the flagellar motor via a subcomplex referred to as the switch. Intriguingly, theH. pylorigenome encodes four switch complex proteins, FliM, FliN, FliY, and FliG, instead of the more typical three ofEscherichia coliorBacillus subtilis. Our goal was to examine whether and how all four switch proteins participate in flagellation. Previous work determined that FliG was required for flagellation, and we extend those findings to show that all four switch proteins are necessary for normal numbers of flagellated cells. Furthermore, whilefliYandfliNare partially redundant with each other, both are needed for wild-type levels of flagellation. We also report the isolation of anH. pyloristrain containing an R54C substitution infliM, resulting in bacteria that swim constantly and do not change direction. Along with data demonstrating that CheY-phosphate interacts with FliM, these findings suggest that FliM functions inH. pylorimuch as it does in other organisms.
Structural and Genetic Basis for the Serological Differentiation ofPasteurella multocidaHeddleston Serotypes 2 and 5
Authors: St. Michael Frank , Harper Marina , Parnas Henrietta , John Marietta , Stupak Jacek , Vinogradov Evgeny , Adler Ben , Boyce John D. , Cox Andrew D. 
Published: September 2009

ABSTRACTPasteurella multocidais classified into 16 serotypes according to the Heddleston typing scheme. As part of a comprehensive study to define the structural and genetic basis of this scheme, we have determined the structure of the lipopolysaccharide (LPS) produced byP. multocidastrains M1404 (B:2) and P1702 (E:5), the type strains for serotypes 2 and 5, respectively. The only difference between the LPS structures made by these two strains was the absence of a phosphoethanolamine (PEtn) moiety at the 3 position of the second heptose (Hep II) in M1404. Analysis of thelpt-3gene, required for the addition of this PEtn residue, revealed that the gene was intact in P1702 but contained a nonsense mutation in M1404. Expression of an intact copy oflpt-3in M1404 resulted in the attachment of a PEtn residue to the 3 position of the Hep II residue, generating an LPS structure identical to that produced by P1702. We identified and characterized each of the glycosyltransferase genes required for assembly of the serotype 2 and 5 LPS outer core. Monoclonal antibodies raised against serotype 2 LPS recognized the serotype 2/5-specific outer core LPS structure, but recognition of this structure was inhibited by the PEtn residue on Hep II. These data indicate that the serological classification of strains into Heddleston serotypes 2 and 5 is dependent on the presence or absence of PEtn on Hep II.
Functional Characterization of SsaE, a Novel Chaperone Protein of the Type III Secretion System Encoded by
Salmonella
Pathogenicity Island 2
Authors: Miki Tsuyoshi , Shibagaki Yoshio , Danbara Hirofumi , Okada Nobuhiko 
Published: September 2009

ABSTRACT

The type III secretion system (T3SS) encoded by
Salmonella
pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of
Salmonella enterica
serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An
S. enterica
serovar Typhimurium
ssaE
mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an
sseA
deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast,
ssaE
mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an
ssaE
(
I55G
) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for
Salmonella
virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery.
ThedltOperon ofBacillus cereusIs Required for Resistance to Cationic Antimicrobial Peptides and for Virulence in Insects
Authors: Abi Khattar Z. , Rejasse A. , Destoumieux-Garzón D. , Escoubas J. M. , Sanchis V. , Lereclus D. , Givaudan A. , Kallassy M. , Nielsen-Leroux C. , Gaudriault S. 
Published: September 2009

ABSTRACTThedltoperon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of thedltoperon in insect virulence by inactivating this operon inBacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The ΔdltBcmutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B fromSpodoptera frugiperda. ΔdltBcwas as pathogenic as the wild-type strain in oral infections ofGalleria mellonellabut much less virulent when injected into the hemocoels ofG. mellonellaandSpodoptera littoralis. We detected thedltoperon in three gram-negative genera:Erwinia(Erwinia carotovora),Bordetella(Bordetella pertussis,Bordetella parapertussis, andBordetella bronchiseptica), andPhotorhabdus(the entomopathogenic bacteriumPhotorhabdus luminescensTT01, thedltoperon of which did not restore cationic AMP resistance in ΔdltBc). We suggest that thedltoperon protectsB. cereusagainst insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.
Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface
Authors: Mihajlovic Sanja , Lang Silvia , Sut Marta V. , Strohmaier Heimo , Gruber Christian J. , Koraimann Günther , Cabezón Elena , Moncalián Gabriel , de la Cruz Fernando , Zechner Ellen L. 
Published: September 2009

ABSTRACT

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDΔN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of
oriT
plasmid DNA in vivo (Δ

L
k

= −4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDΔN130 on supercoiled DNA in vitro confirmed localized unwinding at
nic
but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.
Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1
Authors: Sut Marta V. , Mihajlovic Sanja , Lang Silvia , Gruber Christian J. , Zechner Ellen L. 
Published: September 2009

ABSTRACT

The mechanisms controlling progression of conjugative DNA processing from a preinitiation stage of specific plasmid strand cleavage at the transfer origin to a stage competent for unwinding the DNA strand destined for transfer remain obscure. Linear heteroduplex substrates containing double-stranded DNA binding sites for plasmid R1 relaxosome proteins and various regions of open duplex for TraI helicase loading were constructed to model putative intermediate structures in the initiation pathway. The activity of TraI was compared in steady-state multiple turnover experiments that measured the net production of unwound DNA as well as transesterase-catalyzed cleavage at
nic
. Helicase efficiency was enhanced by the relaxosome components TraM and integration host factor. The magnitude of stimulation depended on the proximity of the specific protein binding sites to the position of open DNA. The cytoplasmic domain of the R1 coupling protein, TraDΔN130, stimulated helicase efficiency on all substrates in a manner consistent with cooperative interaction and sequence-independent DNA binding. Variation in the position of duplex opening also revealed an unsuspected autoinhibition of the unwinding reaction catalyzed by full-length TraI. The activity reduction was sequence dependent and was not observed with a truncated helicase, TraIΔN308, lacking the site-specific DNA binding transesterase domain. Given that transesterase and helicase domains are physically tethered in the wild-type protein, this observation suggests that an intramolecular switch controls helicase activation. The data support a model where protein-protein and DNA ligand interactions at the coupling protein interface coordinate the transition initiating production and uptake of the nucleoprotein secretion substrate.
Physiology and Posttranscriptional Regulation of Methanol:Coenzyme M Methyltransferase Isozymes in
Methanosarcina acetivorans
C2A
Authors: Opulencia Rina B. , Bose Arpita , Metcalf William W. 
Published: September 2009

ABSTRACT

Methanosarcina
species possess three operons (
mtaCB1
,
mtaCB2
, and
mtaCB3
) encoding methanol-specific methyltransferase 1 (MT1) isozymes and two genes (
mtaA1
and
mtaA2
) with the potential to encode a methanol-specific methyltransferase 2 (MT2). Previous genetic studies showed that these genes are differentially regulated and encode enzymes with distinct levels of methyltransferase activity. Here, the effects of promoter strength on growth and on the rate of methane production were examined by constructing strains in which the
mtaCB
promoters were exchanged. When expressed from the strong P
mtaC1
or P
mtaC2
promoter, each of the MtaC and MtaB proteins supported growth and methane production at wild-type levels. In contrast, all
mtaCB
operons exhibited poorer growth and lower rates of methane production when P
mtaC3
controlled their expression. Thus, previously observed phenotypic differences can be attributed largely to differences in promoter activity. Strains carrying various combinations of
mtaC
,
mtaB
, and
mtaA
expressed from the strong, tetracycline-regulated P
mcrB
(
tetO1
) promoter exhibited similar growth characteristics on methanol, showing that all combinations of MtaC, MtaB, and MtaA can form functional MT1/MT2 complexes. However, an in vitro assay of coupled MT1/MT2 activity showed significant variation between the strains. Surprisingly, these variations in activity correlated with differences in protein abundance, despite the fact that all the encoding genes were expressed from the same promoter. Quantitative reverse transcriptase PCR and reporter gene fusion data suggest that the
mtaCBA
transcripts show different stabilities, which are strongly influenced by the growth substrate.
A Novel Regulatory Protein Involved in Motility of
Vibrio cholerae
Authors: Moisi Manuel , Jenul Christian , Butler Susan M. , New Aaron , Tutz Sarah , Reidl Joachim , Klose Karl E. , Camilli Andrew , Schild Stefan 
Published: September 2009

ABSTRACT

The facultative pathogen
Vibrio cholerae
is the causative agent of the human intestinal disease cholera. Both motility and chemotaxis of
V. cholerae
have been shown to contribute to the virulence and spread of cholera. The flagellar gene operons are organized into a hierarchy composed of four classes (I to IV) based on their temporal expression patterns. Some regulatory elements involved in flagellar gene expression have been elucidated, but regulation is complex and flagellar biogenesis in
V. cholerae
is not completely understood. In this study, we determined that the virulence defect of a
V. cholerae cheW1
deletion mutant was due to polar effects on the downstream open reading frame VC2058 (
flrD
). Expression of
flrD
in
trans
restored the virulence defect of the
cheW1
deletion mutant, and deletion of
flrD
resulted in a
V. cholerae
strain attenuated for virulence, as determined by using the infant mouse intestinal colonization model. The
flrD
mutant strain exhibited decreased transcription of class III and IV flagellar genes and reduced motility. Transcription of the
flrD
promoter, which lies within the coding sequence of
cheW1
, is independent of the flagellar transcriptional activators FlrA and RpoN, which activate class II genes, indicating that
flrD
does not fit into any of the four flagellar gene classes. Genetic epistasis studies revealed that the two-component system FlrBC, which is required for class III and IV flagellar gene transcription, acts downstream of
flrD
. We hypothesize that the inner membrane protein FlrD interacts with the cytoplasmic FlrBC complex to activate class III and IV gene transcription.
Role of the Transcriptional Regulator RamB (Rv0465c) in the Control of the Glyoxylate Cycle in
Mycobacterium tuberculosis
Authors: Micklinghoff Julia C. , Breitinger Katrin J. , Schmidt Mascha , Geffers Robert , Eikmanns Bernhard J. , Bange Franz-Christoph 
Published: September 2009

ABSTRACT

Mycobacterium tuberculosis
generally is assumed to depend on lipids as a major carbon and energy source when persisting within the host. The utilization of fatty acids requires a functional glyoxylate cycle with the key enzymes isocitrate lyase (Icl) and malate synthase. The open reading frame Rv0465c of
M. tuberculosis
H37Rv encodes a protein with significant sequence similarity to the transcriptional regulator RamB, which in
Corynebacterium glutamicum
controls the expression of several genes involved in acetate metabolism, i.e., those encoding enzymes of acetate activation and the glyoxylate cycle. We show here that the
M. tuberculosis
Rv0465c protein can functionally complement RamB in
C. glutamicum
and that it binds to the promoter regions of
M. tuberculosis icl1
and Rv0465c. Construction and subsequent transcriptional and enzymatic analysis of a defined Rv0465c deletion mutant in
M. tuberculosis
revealed that the Rv0465c protein, now designated RamB, represses
icl1
expression during growth with glucose and negatively autoregulates the expression of its own operon. Whole-genome microarray analysis of the
M. tuberculosis ramB
(

ramB
MT

) mutant and the wild type furthermore showed that apart from
icl1
and the

ramB
MT

operon, the expression of all other
M. tuberculosis
genes involved in acetate metabolism remain unchanged in the mutant. Thus, RamB
MT
has a more specific regulatory function as RamB from
C. glutamicum
and is confined to expression control of
icl1
and the

ramB
MT

operon.
Complete Genome Sequence of the Ethanol Producer
Zymomonas mobilis
NCIMB 11163
Authors: Kouvelis Vassili N. , Saunders Elizabeth , Brettin Thomas S. , Bruce David , Detter Chris , Han Cliff , Typas Milton A. , Pappas Katherine M. 
Published: September 2009

ABSTRACT

Zymomonas mobilis
is an ethanol-producing alphaproteobacterium currently considered a major candidate organism for bioethanol production. Here we report the finished and annotated genome sequence of
Z. mobilis
subsp.
mobilis
strain NCIMB 11163, a British ale-infecting isolate. This is the first
Z. mobilis
strain whose genome, chromosomal and plasmid, is presented in its entirety.
Occupational exposure to silica and lung cancer risk in the Netherlands
Authors: Preller Liesbeth , van den Bosch Linda M C , van den Brandt Piet A , Kauppinen T , Goldbohm Alexandra 
Published: September 2009

ObjectivesThe lung cancer carcinogenicity of crystalline silica dust remains the subject of discussion. Epidemiological evidence is based on occupational cohort studies and population-based case–control studies. The aim of this study was to assess associations between male lung cancer risk and silica exposure in a population-based cohort study.MethodsThe study was conducted among men aged 55–69 years (n=58 279) from the Netherlands Cohort Study, which included self-reported, life-time job histories. Job titles were linked to the occupational groups of the external Finnish Job Exposure Matrix (FINJEM), including probability and level of silica exposure, each for specific time periods. 1667 incident lung cancer cases with known silica exposure status (210 exposed) were available after 11.3 years of follow-up. Risks were estimated based on a case–cohort design, and using Cox proportional hazards models.ResultsAdjusted for smoking and other confounders, elevated risks were observed for exposure duration (RR 1.65, 95% CI 1.14 to 2.41 for 26–51 years vs no exposure) and cumulative exposure (RR 1.47, 95% CI 0.93 to 2.33 for ≥3 vs <3 mg/m3.year). Associations with average exposure levels were weaker. Associations were stronger for occupations with an exposure probability of ≥90%. Adjustment for asbestos exposure slightly increased the risk.ConclusionsResults from this prospective population-based cohort study corroborate the classification of crystalline silica as a lung carcinogen. Associations could not be explained by smoking or by asbestos exposure.