bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Specific Chemoattractants and Genetic Complementation of a
Borrelia burgdorferi
Chemotaxis Mutant: Flow Cytometry-Based Capillary Tube Chemotaxis Assay
Authors: Bakker Richard G. , Li Chunhao , Miller Michael R. , Cunningham Cynthia , Charon Nyles W. 
Published: December 2006

ABSTRACT

Measuring the chemotactic response of
Borrelia burgdorferi
, the bacterial species that causes Lyme disease, is relatively more difficult than measuring that of other bacteria. Because these spirochetes have long generation times, enumerating cells that swim up a capillary tube containing an attractant by using colony counts is impractical. Furthermore, direct counts with a Petroff-Hausser chamber is problematic, as this method has a low throughput and necessitates a high cell density; the latter can lead to misinterpretation of results when assaying for specific attractants. Only rabbit serum and tick saliva have been reported to be chemoattractants for
B. burgdorferi
. These complex biological mixtures are limited in their utility for studying chemotaxis on a molecular level. Here we present a modified capillary tube chemotaxis assay for
B. burgdorferi
that enumerates cells by flow cytometry. Initial studies identified
N
-acetylglucosamine as a chemoattractant. The assay was then optimized with respect to cell concentration, incubation time, motility buffer composition, and growth phase. Besides
N
-acetylglucosamine, glucosamine, glucosamine dimers (chitosan), glutamate, and glucose also elicited significant chemoattractant responses, although the response obtained with glucose was weak and variable. Serine and glycine were nonchemotactic. To further validate and to exploit the use of this assay, a previously described nonchemotactic
cheA2
mutant was shown to be nonchemotactic by this assay; it also regained the wild-type phenotype when complemented in
trans
. This is the first report that identifies specific chemical attractants for
B. burgdorferi
and the use of flow cytometry for spirochete enumeration. The method should also be useful for assaying chemotaxis for other slow-growing prokaryotic species and in specific environments in nature.
Analysis ofThalassiosira pseudonanaSilicon Transporters Indicates Distinct Regulatory Levels and Transport Activity through the Cell Cycle
Authors: Thamatrakoln Kimberlee , Hildebrand Mark 
Published: December 2006

ABSTRACTAn analysis of the expression and activity of silicon transporters (SITs) was done on synchronously growing cultures of the diatomThalassiosira pseudonanato provide insight into the role these proteins play in cellular silicon metabolism during the cell cycle. The first SIT-specific polyclonal peptide antibody was generated and used in the immunoblot analysis of whole-cell protein lysates to monitor SIT protein levels during synchronized progression through the cell cycle. Peaks in SIT protein levels correlated with active periods of silica incorporation into cell wall substructures. Quantitative real-time PCR on each of the three distinctSITgenes (TpSIT1, TpSIT2, and TpSIT3) showed that mRNA levels for the most highly expressedSITgenes peaked during the S phase of the cell cycle, a period prior to maximal silicon uptake and during which cell wall silicification does not occur. Variations in protein and mRNA levels did not correlate, suggesting that a significant regulatory step of SITs is at the translational or posttranslational level. Surge uptake rates also did not correlate with SIT protein levels, suggesting that SIT activity is internally controlled by the rate of silica incorporation. This is the first study to characterize SIT mRNA and protein expression and cellular uptake kinetics during the course of the cell cycle and cell wall synthesis, and it provides novel insight into SIT regulation.
Pirin Regulates Pyruvate Catabolism by Interacting with the Pyruvate Dehydrogenase E1 Subunit and Modulating Pyruvate Dehydrogenase Activity
Authors: Soo Po-Chi , Horng Yu-Tze , Lai Meng-Jiun , Wei Jun-Rong , Hsieh Shang-Chen , Chang Yung-Lin , Tsai Yu-Huan , Lai Hsin-Chih 
Published: December 2006

ABSTRACT

The protein pirin, which is involved in a variety of biological processes, is conserved from prokaryotic microorganisms, fungi, and plants to mammals. It acts as a transcriptional cofactor or an apoptosis-related protein in mammals and is involved in seed germination and seedling development in plants. In prokaryotes, while pirin is stress induced in cyanobacteria and may act as a quercetinase in
Escherichia coli
, the functions of pirin orthologs remain mostly uncharacterized. We show that the
Serratia marcescens
pirin (pirin

Sm

) gene encodes an ortholog of pirin protein. Protein pull-down and bacterial two-hybrid assays followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization-tandem mass spectrometry analyses showed the pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with the pirin

Sm

gene. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity are inhibited by the pirin

Sm

gene in
S. marcescens
CH-1. The
S. marcescens
CH-1 pirin

Sm

gene was subsequently mutated by insertion-deletion homologous recombination. Accordingly, the PDH E1 and PDH enzyme complex activities and cellular ATP concentration increased up to 250%, 140%, and 220%, respectively, in the
S. marcescens
CH-1 pirin

Sm

mutant. Concomitantly, the cellular NADH/NAD
+
ratio increased in the pirin

Sm

mutant, indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that the pirin

Sm

gene plays a regulatory role in the process of pyruvate catabolism to acetyl coenzyme A through interaction with the PDH E1 subunit and inhibiting PDH enzyme complex activity in
S. marcescens
CH-1, and they suggest that pirin

Sm

is an important protein involved in determining the direction of pyruvate metabolism towards either the TCA cycle or the fermentation pathways.
Differentiation and Distribution of Colistin- and Sodium Dodecyl Sulfate-Tolerant Cells inPseudomonas aeruginosaBiofilms
Authors: Haagensen Janus A. J. , Klausen Mikkel , Ernst Robert K. , Miller Samuel I. , Folkesson Anders , Tolker-Nielsen Tim , Molin Søren 
Published: December 2006

ABSTRACTDuringPseudomonas aeruginosaflow cell biofilm development, the cell population differentiates into a nonmotile subpopulation which forms microcolonies and a migrating subpopulation which eventually colonizes the top of the microcolonies, resulting in the development of mushroom-shaped multicellular structures. The cap-forming subpopulation was found to develop tolerance to membrane-targeting antimicrobial agents, such as the cyclic cationic peptide colistin and the detergent sodium dodecyl sulfate. The stalk-forming subpopulation, on the other hand, was sensitive to the membrane-targeting antibacterial agents. All biofilm-associated cells were sensitive to the antibacterial agents when tested in standard plate assays. A mutation eliminating the production of type IV pili, and hence surface-associated motility, prevented the formation of regular mushroom-shaped structures in the flow cell biofilms, and the development of tolerance to the antimicrobial agents was found to be affected as well. Mutations in genes interfering with lipopolysaccharide modification (pmr) eliminated the biofilm-associated colistin tolerance phenotype. Experiments with a PAO1 strain harboring apmr-gfpfusion showed that only the cap-forming subpopulation in biofilms treated with colistin expresses thepmroperon. These results suggest that increased antibiotic tolerance in biofilms may be a consequence of differentiation into distinct subpopulations with different phenotypic properties.
β-
d
-Allose Inhibits Fruiting Body Formation and Sporulation in
Myxococcus xanthus
Authors: Chavira Marielena , Cao Nga , Le Karen , Riar Tanveer , Moradshahi Navid , McBride Melinda , Lux Renate , Shi Wenyuan 
Published: December 2006

ABSTRACT

Myxococcus xanthus
, a gram-negative soil bacterium, responds to amino acid starvation by entering a process of multicellular development which culminates in the assembly of spore-filled fruiting bodies. Previous studies utilizing developmental inhibitors (such as methionine, lysine, or threonine) have revealed important clues about the mechanisms involved in fruiting body formation. We used Biolog phenotype microarrays to screen 384 chemicals for complete inhibition of fruiting body development in
M. xanthus
. Here, we report the identification of a novel inhibitor of fruiting body formation and sporulation, β-
d
-allose. β-
d
-Allose, a rare sugar, is a member of the aldohexose family and a C3 epimer of glucose. Our studies show that β-
d
-allose does not affect cell growth, viability, agglutination, or motility. However, β-galactosidase reporters demonstrate that genes activated between 4 and 14 h of development show significantly lower expression levels in the presence of β-
d
-allose. Furthermore, inhibition of fruiting body formation occurs only when β-
d
-allose is added to submerged cultures before 12 h of development. In competition studies, high concentrations of galactose and xylose antagonize the nonfruiting response to β-
d
-allose, while glucose is capable of partial antagonism. Finally, a
magellan-4
transposon mutagenesis screen identified
glcK
, a putative glucokinase gene, required for β-
d
-allose-mediated inhibition of fruiting body formation. Subsequent glucokinase activity assays of the
glcK
mutant further supported the role of this protein in glucose phosphorylation.
Chlamydial Type III Secretion System Is Encoded on Ten Operons Preceded by Sigma 70-Like Promoter Elements
Authors: Hefty P. Scott , Stephens Richard S. 
Published: December 2006

ABSTRACTMany gram-negative bacterial pathogens employ type III secretion systems for infectious processes. Chlamydiae are obligate intracellular bacteria that encode a conserved type III secretion system that is likely requisite for growth. Typically, genes encoding type III secretion systems are located in a single locus; however, for chlamydiae these genes are scattered throughout the genome. Little is known regarding the gene regulatory mechanisms for this essential virulence determinant. To facilitate identification ofcis-acting transcriptional regulatory elements, the operon structure was determined. This analysis revealed 10 operons that contained 37 genes associated with the type III secretion system. Linkage within these operons suggests a role in type III secretion for each of these genes, including 13 genes encoding proteins with unknown function. The transcriptional start site for each operon was determined. In conjunction with promoter activity assays, this analysis revealed that the type III secretion system operons encode σ70-like promoter elements. Transcriptional initiation by a sigma factor responsible for constitutive gene expression indicates that undefined activators or repressors regulate developmental stage-specific expression of chlamydial type III secretion system genes.
Transcriptional Regulation of the
yghJ-pppA-yghG-
gspCDEFGHIJKLM
Cluster, Encoding the Type II Secretion Pathway in Enterotoxigenic
Escherichia coli
Authors: Yang Ji , Baldi Deborah L. , Tauschek Marija , Strugnell Richard A. , Robins-Browne Roy M. 
Published: December 2006

ABSTRACT

The gene cluster
gspCDEFGHIJKLM
codes for various structural components of the type II secretion pathway which is responsible for the secretion of heat-labile enterotoxin by enterotoxigenic
Escherichia coli
(ETEC). In this work, we used a variety of molecular approaches to elucidate the transcriptional organization of the ETEC type II secretion system and to unravel the mechanisms by which the expression of these genes is controlled. We showed that the
gspCDEFGHIJKLM
cluster and three other upstream genes,
yghJ
,
pppA
, and
yghG
, are cotranscribed and that a promoter located in the upstream region of
yghJ
plays a major role in the expression of this 14-gene transcriptional unit. Transcription of the
yghJ
promoter was repressed 168-fold upon a temperature downshift from 37°C to 22°C. This temperature-induced repression was mediated by the global regulatory proteins H-NS and StpA. Deletion mutagenesis showed that the promoter region encompassing positions −321 to +301 relative to the start site of transcription of
yghJ
was required for full repression. The
yghJ
promoter region is predicted to be highly curved and bound H-NS or StpA directly. The binding of H-NS or StpA blocked transcription initiation by inhibiting promoter open complex formation. Unraveling the mechanisms of regulation of type II secretion by ETEC enhances our understanding of the pathogenesis of ETEC and other pathogenic varieties of
E. coli
.
Molecular Evolution of the H-NS Protein: Interaction with Hha-Like Proteins Is Restricted toEnterobacteriaceae
Authors: Madrid Cristina , García Jesús , Pons Miquel , Juárez Antonio 
Published: December 2006

ABSTRACTWe show here that chromosomalhha-like genes are restricted to theEnterobacteriaceae. The H-NS N-terminal domain of members of this family includes an unaltered seven-amino-acid sequence located between helixes 1 and 2, termed the Hha signature, that contains key residues for H-NS-Hha interaction.
Characterization of
lptA
and
lptB
, Two Essential Genes Implicated in Lipopolysaccharide Transport to the Outer Membrane of
Escherichia coli
Authors: Sperandeo Paola , Cescutti Rachele , Villa Riccardo , Di Benedetto Cristiano , Candia Daniela , Dehò Gianni , Polissi Alessandra 
Published: December 2006

ABSTRACT

The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two
Escherichia coli
essential genes,
yhbN
and
yhbG
, now renamed
lptA
and
lptB
, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the
lptA-lptB
operon is controlled by the extracytoplasmic σ factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of
E. coli
.
FoxB of
Pseudomonas aeruginosa
Functions in the Utilization of the Xenosiderophores Ferrichrome, Ferrioxamine B, and Schizokinen: Evidence for Transport Redundancy at the Inner Membrane
Authors: Ó Cuív Páraic , Keogh Damien , Clarke Paul , O'Connell Michael 
Published: December 2006

ABSTRACT

Expression of the inner membrane protein FoxB (PA2465) of
Pseudomonas aeruginosa
in mutants of
Sinorhizobium meliloti
that are defective in the utilization of ferrichrome, ferrioxamine B, and schizokinen resulted in the restoration of siderophore utilization. Mutagenesis of
foxB
in
P. aeruginosa
did not abolish siderophore utilization, suggesting that the function is redundant.
Response of
Staphylococcus aureus
to Salicylate Challenge
Authors: Riordan James T. , Muthaiyan Arunachalam , Van Voorhies Wayne , Price Christopher T. , Graham James E. , Wilkinson Brian J. , Gustafson John E. 
Published: December 2006

ABSTRACT

Growth of
Staphylococcus aureus
with the nonsteroidal anti-inflammatory salicylate reduces susceptibility of the organism to multiple antimicrobials. Transcriptome analysis revealed that growth of
S. aureus
with salicylate leads to the induction of genes involved with gluconate and formate metabolism and represses genes required for gluconeogenesis and glycolysis. In addition, salicylate induction upregulates two antibiotic target genes and downregulates a multidrug efflux pump gene repressor (
mgrA
) and
sarR
, which represses a gene (
sarA
) important for intrinsic antimicrobial resistance. We hypothesize that these salicylate-induced alterations jointly represent a unique mechanism that allows
S. aureus
to resist antimicrobial stress and toxicity.
Analysis of the Roles of FlgP and FlgQ in Flagellar Motility of
Campylobacter jejuni
Authors: Sommerlad Shawn M. , Hendrixson David R. 
Published: December 2006

ABSTRACT

Flagellar motility is an important determinant of
Campylobacter jejuni
that is required for promoting interactions with various hosts to promote gastroenteritis in humans or commensal colonization of many animals. In a previous study, we identified a nonmotile mutant of
C. jejuni
81-176 with a transposon insertion in Cj1026c, but verification of the role of the encoded protein in motility was not determined. In this study, we have determined that Cj1026c and the gene immediately downstream, Cj1025c (here annotated as
flgP
and
flgQ
, respectively), are both required for motility of
C. jejuni
but not for flagellar biosynthesis. FlgP and FlgQ are not components of the transcriptional regulatory cascades to activate σ
28
- or σ
54
-dependent expression of flagellar genes. In addition, expression of
flgP
and
flgQ
is not largely dependent on σ
28
or σ
54
. Immunblot analyses revealed that the majority of FlgP in
C. jejuni
is associated with the outer membrane. However, in the absence of FlgQ, the amounts of FlgP in the outer membrane of
C. jejuni
are greatly reduced, suggesting that FlgQ may be required for localization or stability of FlgP at this location. This study provides insight into features of FlgP and FlgQ, two proteins with previously undefined functions that are required for the larger, multicomponent flagellar system of
C. jejuni
that is necessary for motility.
Glycosylation of
Pseudomonas aeruginosa
Strain Pa5196 Type IV Pilins with Mycobacterium-Like α-1,5-Linked
d
-Ara
f
Oligosaccharides
Authors: Voisin Sébastien , Kus Julianne V. , Houliston Scott , St-Michael Frank , Watson Dave , Cvitkovitch Dennis G. , Kelly John , Brisson Jean-Robert , Burrows Lori L. 
Published: December 2006

ABSTRACT

Pseudomonas aeruginosa
is a gram-negative bacterium that uses polar type IV pili for adherence to various materials and for rapid colonization of surfaces via twitching motility. Within the
P. aeruginosa
species, five distinct alleles encoding variants of the structural subunit PilA varying in amino acid sequence, length, and presence of posttranslational modifications have been identified. In this work, a combination of mass spectrometry and nuclear magnetic resonance spectroscopy was used to identify a novel glycan modification on the pilins of the group IV strain Pa5196. Group IV pilins continued to be modified in a lipopolysaccharide (
wbpM
) mutant of Pa5196, showing that, unlike group I strains, the pilins of group IV are not modified with the O-antigen unit of the background strain. Instead, the pilin glycan was determined to be an unusual homo-oligomer of α-1,5-linked
d
-arabinofuranose (
d
-Ara
f
). This sugar is uncommon in prokaryotes, occurring mainly in the cell wall arabinogalactan and lipoarabinomannan (LAM) polymers of mycobacteria, including
Mycobacterium tuberculosis
and
Mycobacterium leprae
. Antibodies raised against
M. tuberculosis
LAM specifically identified the glycosylated pilins from Pa5196, confirming that the glycan is antigenically, as well as chemically, identical to those of
Mycobacterium. P. aeruginosa
Pa5196, a rapidly growing strain of low virulence that expresses large amounts of glycosylated type IV pilins on its surface, represents a genetically tractable model system for elucidation of alternate pathways for biosynthesis of
d
-Ara
f
and its polymerization into mycobacterium-like α-1,5-linked oligosaccharides.
Export of the Pseudopilin XcpT of the
Pseudomonas aeruginosa
Type II Secretion System via the Signal Recognition Particle-Sec Pathway
Authors: Arts Jorik , van Boxtel Ria , Filloux Alain , Tommassen Jan , Koster Margot 
Published: December 2006

ABSTRACT

Type IV pilins and pseudopilins are found in various prokaryotic envelope protein complexes, including type IV pili and type II secretion machineries of gram-negative bacteria, competence systems of gram-positive bacteria, and flagella and sugar-binding structures in members of the archaeal kingdom. The precursors of these proteins have highly conserved N termini, consisting of a short, positively charged leader peptide, which is cleaved off by a dedicated peptidase during maturation, and a hydrophobic stretch of approximately 20 amino acid residues. Which pathway is involved in the inner membrane translocation of these proteins is unknown. We used XcpT, the major pseudopilin from the type II secretion machinery of
Pseudomonas aeruginosa
, as a model to study this process. Transport of an XcpT-PhoA hybrid was shown to occur in the absence of other Xcp components in
P. aeruginosa
and in
Escherichia coli
. Experiments with conditional
sec
mutants and reporter-protein fusions showed that this transport process involves the cotranslational signal recognition particle targeting route and is dependent on a functional Sec translocon.
Functional Analysis of the Carboxy-Terminal Region of
Bacillus subtilis
TnrA, a MerR Family Protein
Authors: Wray Lewis V. , Fisher Susan H. 
Published: December 2006

ABSTRACT

The
Bacillus subtilis
TnrA transcription factor belongs to the MerR family of proteins and regulates gene expression during nitrogen-limited growth. When
B. subtilis
cells are grown with excess nitrogen, feedback-inhibited glutamine synthetase forms a protein-protein complex with TnrA that prevents TnrA from binding to DNA. The C-terminal region of TnrA is required for the interaction with glutamine synthetase. Alanine scanning mutagenesis of the C-terminal region of TnrA identified three classes of mutants that altered the regulation by glutamine synthetase. While expression of the TnrA-regulated
amtB
gene was expressed constitutively in the class I (M96A, Q100A, and A103G) and class II (L97A, L101A, and F105A) mutants, the class II mutants were unable to grow on minimal medium unless a complex mixture of amino acids was present. The class III
tnrA
mutants (R93A, G99A, N102A, H104A, and Y107A mutants) were partially defective in the regulation of TnrA activity. In vitro experiments showed that feedback-inhibited glutamine synthetase had a significantly reduced ability to inhibit the DNA-binding activity of several class I and class II mutant TnrA proteins. A coiled-coil homology model of the C-terminal region of TnrA is used to explain the properties of the class I and II mutant proteins. The C-terminal region of TnrA corresponds to a dimerization domain in other MerR family proteins. Surprisingly, gel filtration and cross-linking analysis showed that a truncated TnrA protein which contained only the N-terminal DNA binding domain was dimeric. The implications of these results for the structure of TnrA are discussed.