bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of the C-Terminal Cytoplasmic Domain of FlhA in Bacterial Flagellar Type III Protein Export
Authors: Minamino Tohru , Shimada Masafumi , Okabe Mayuko , Saijo-Hamano Yumiko , Imada Katsumi , Kihara May , Namba Keiichi 
Published: January 2010

ABSTRACT

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA
C
) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive
Salmonella enterica
mutant, the
flhA
(
G368C
) mutant, which has a mutation in the sequence encoding FlhA
C
. The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA
C
consists of at least three subdomains, a flexible linker, FlhA
CN
, and FlhA
CC
, and that FlhA
CN
becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA
C
not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.
Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor
Authors: Bahl Christopher D. , Morisseau Christophe , Bomberger Jennifer M. , Stanton Bruce A. , Hammock Bruce D. , O'Toole George A. , Madden Dean R. 
Published: January 2010

ABSTRACT

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by
Pseudomonas aeruginosa
that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other α/β hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-Å resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and
cis
-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of α/β hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.
Characterization of the CI Repressor Protein Encoded by the Temperate Lactococcal Phage TP901-1
Authors: Pedersen Margit , Ligowska Małgorzata , Hammer Karin 
Published: January 2010

ABSTRACT

The gene regulatory mechanism determining the developmental pathway of the temperate bacteriophage TP901-1 is regulated by two phage-encoded proteins, CI and MOR. Functional domains of the CI repressor were investigated by introducing linkers of 15 bp at various positions in
cI
and by limited proteolysis of purified CI protein. We show that insertions of five amino acids at positions in the N-terminal half of CI resulted in mutant proteins that could no longer repress transcription from the lytic promoter, P
L
. We confirmed that the N-terminal domain of CI contains the DNA binding site, and we showed that this part of the protein is tightly folded, whereas the central part and the C-terminal part of CI seem to contain more flexible structures. Furthermore, insertions at several different positions in the central part of the CI protein reduced the cooperative binding of CI to the operator sites and possibly altered the interaction with MOR.
Role for CysJ Flavin Reductase in Molybdenum Cofactor-Dependent Resistance of
Escherichia coli
to 6-
N
-Hydroxylaminopurine
Authors: Kozmin Stanislav G. , Wang Jian , Schaaper Roel M. 
Published: January 2010

ABSTRACT

We have previously described a novel
Escherichia coli
detoxification system for the removal of toxic and mutagenic N-hydroxylated nucleobases and related compounds that requires the molybdenum cofactor. Two subpathways (
ycbX
and
yiiM
) were identified, each employing a novel molybdo activity capable of inactivating N-hydroxylated compounds by reduction to the corresponding amine. In the present study, we identify the
cysJ
gene product as one additional component of this system. While the CysJ protein has been identified as the NADPH:flavin oxidoreductase component of the CysJI sulfite reductase complex (CysJ
8
I
4
), we show that the role of CysJ in base analog detoxification is unique and independent of CysI and sulfite reductase. We further show that CysJ functions as a specific partner of the YcbX molybdoenzyme. We postulate that the function of CysJ in this pathway is to provide, via its NADPH:flavin reductase activity, the reducing equivalents needed for the detoxification reaction at the YcbX molybdocenter. In support of the proposed interaction of the CysJ and YcbX proteins, we show that an apparent CysJ-YcbX “hybrid” protein from two
Vibrio
species is capable of compensating for a double
cysJ ycbX
defect in
E. coli
.
Lipopolysaccharide as Shield and Receptor for R-Pyocin-Mediated Killing in
Pseudomonas aeruginosa
Authors: Köhler Thilo , Donner Viviane , van Delden Christian 
Published: January 2010

ABSTRACT

Pseudomonas aeruginosa
produces three different types of bacteriocins: the soluble S-pyocins and the bacteriophage-like F- and R-pyocins. R-pyocins kill susceptible bacteria of the same or closely related species with high efficiency. Five different types of R-pyocins (R1- to R5-pyocins) have been described based on their killing spectra and tail fiber protein sequences. We analyzed the distribution of R-pyocin genes in a collection of clinical
P. aeruginosa
isolates. We found similar percentages of isolates not containing R-pyocins (28%) and isolates containing genes encoding R1-pyocins (25%), R2-pyocins (17%), and R5-pyocins (29%). The R-pyocin-deficient isolates were susceptible to R1-, R2-, and R5-pyocins, while most R2- and R5- pyocin producers were resistant. Determination of the O serotypes revealed that the R-pyocin-susceptible isolates belonged to serotypes O1, O3, and O6, while the R-pyocin-resistant isolates were serotype O10, O11, and O12 isolates. We hypothesized that O-serotype-specific lipopolysaccharide (LPS) packaging densities may account for the distinct accessibilities of R-pyocins to their receptors at the cell surface. Using genetically defined LPS mutants, we showed that the
l
-Rha residue and two distinct
d
-Glc residues of the outer core are part of the receptor sites for R1-, R2-, and R5-pyocins, respectively. To illustrate R-pyocin-mediated intraspecies biological warfare, we monitored the population dynamics of two different R-pyocin-producing
P. aeruginosa
clones of sequential respiratory isolates obtained from a colonized patient. The results of this study highlight the potential role of R-pyocins in shaping bacterial populations during host colonization and support use of these molecules as specific and potent bactericidal agents.
Archaeal Eukaryote-Like Serine/Threonine Protein Kinase Interacts with and Phosphorylates a Forkhead-Associated-Domain-Containing Protein
Authors: Wang Bin , Yang Shifan , Zhang Lei , He Zheng-Guo 
Published: January 2010

ABSTRACT

Protein phosphorylation plays an important role in cell signaling. However, in the
Archaea
, little is known about which proteins are phosphorylated and which kinases are involved. In this study, we identified, for the first time, a typical eukaryote-like Ser/Thr protein kinase and its protein partner, a forkhead-associated (FHA)-domain-containing protein, from the archaeon
Sulfolobus tokodaii
strain 7. This protein kinase, ST1565, physically interacted with the FHA-domain-containing protein, ST0829, both
in vivo
and
in vitro
. ST1565 preferred Mn
2+
as a cofactor for autophosphorylation and for substrate phosphorylation; the optimal temperature for this was 45°C, and the optimal pH was 5.5 to 7.5. The critical amino acid residues of the conserved FHA and kinase domain sites were identified by performing a series of mutation assays. Thr329 was part of a major activation site in the kinase, while Thr326 was a negative regulation site. Several mutants with amino acid substitutions in the conserved FHA domain sites of ST0829 did not physically interact with ST1565. A structural model for the FHA domain demonstrated that the mutation sites were located at the edge of the protein and thus were in the domain that potentially interacts with ST1565. This report describes pioneering work on the third domain of life, the
Archaea
, showing that a protein kinase interacts with and phosphorylates an FHA-domain-containing protein. Our data provide critical information on the structural or functional characteristics of archaeal proteins and could help increase our understanding of fundamental signaling mechanisms in all three domains of life.
Histidine Kinase-Mediated Production and Autoassembly of
Porphyromonas gingivalis
Fimbriae
Authors: Nishikawa Kiyoshi , Duncan Margaret J. 
Published: January 2010

ABSTRACT

Porphyromonas gingivalis
, a Gram-negative oral anaerobe, is strongly associated with chronic adult periodontitis, and it utilizes FimA fimbriae to persistently colonize and evade host defenses in the periodontal crevice. The FimA-related gene cluster (the
fim
gene cluster) is positively regulated by the FimS-FimR two-component system. In this study, comparative analyses between fimbriate type strain ATCC 33277 and fimbria-deficient strain W83 revealed differences in their
fimS
loci, which encode FimS histidine kinase. Using a reciprocal gene exchange system, we established that FimS from W83 is malfunctional. Complementation analysis with chimeric
fimS
constructs revealed that W83 FimS has a defective kinase domain due to a truncated conserved G3 box motif that provides an ATP-binding pocket. The introduction of the functional
fimS
from 33277 restored the production, but not polymerization, of endogenous FimA subunits in W83. Further analyses with a
fimA
-exchanged W83 isogenic strain showed that even the fimbria-deficient W83 retains the ability to polymerize FimA from 33277, indicating the assembly of mature FimA by a primary structure-dependent mechanism. It also was shown that the substantial expression of 33277-type FimA fimbriae in the W83 derivative requires the introduction and expression of the functional 33277
fimS
. These findings indicate that FimSR is the unique and universal regulatory system that activates the
fim
gene cluster in a
fimA
genotype-independent manner.
The Pneumococcal Cell Envelope Stress-Sensing System LiaFSR Is Activated by Murein Hydrolases and Lipid II-Interacting Antibiotics
Authors: Eldholm Vegard , Gutt Beatrice , Johnsborg Ola , Brückner Reinhold , Maurer Patrick , Hakenbeck Regine , Mascher Thorsten , Håvarstein Leiv Sigve 
Published: January 2010

ABSTRACTIn theFirmicutes, two-component regulatory systems of the LiaSR type sense and orchestrate the response to various agents that perturb cell envelope functions, in particular lipid II cycle inhibitors. In the current study, we found that the corresponding system inStreptococcus pneumoniaedisplays similar properties but, in addition, responds to cell envelope stress elicited by murein hydrolases. During competence for genetic transformation, pneumococci attack and lyse noncompetent siblings present in the same environment. This phenomenon, termed fratricide, increases the efficiency of horizontal gene transferin vitroand is believed to stimulate gene exchange also under natural conditions. Lysis of noncompetent target cells is mediated by the putative murein hydrolase CbpD, the key effector of the fratricide mechanism, and the autolysins LytA and LytC. To avoid succumbing to their own lysins, competent attacker cells must possess a protective mechanism rendering them immune. The most important component of this mechanism is ComM, an integral membrane protein of unknown function that is expressed only in competent cells. Here, we show that a second layer of self-protection is provided by the pneumococcal LiaFSR system, which senses the damage inflicted to the cell wall by CbpD, LytA, and LytC. Two members of the LiaFSR regulon,spr0810 and PcpC (spr0351), were shown to contribute to the LiaFSR-coordinated protection against fratricide-induced self-lysis.
Responses of
Escherichia coli
Bacteria to Two Opposing Chemoattractant Gradients Depend on the Chemoreceptor Ratio
Authors: Kalinin Yevgeniy , Neumann Silke , Sourjik Victor , Wu Mingming 
Published: January 2010

ABSTRACT

Escherichia coli
chemotaxis has long served as a simple model of environmental signal processing, and bacterial responses to single chemical gradients are relatively well understood. Less is known about the chemotactic behavior of
E. coli
in multiple chemical gradients. In their native environment, cells are often exposed to multiple chemical stimuli. Using a recently developed microfluidic chemotaxis device, we exposed
E. coli
cells to two opposing but equally potent gradients of major attractants, methyl-aspartate and serine. The responses of
E. coli
cells demonstrated that chemotactic decisions depended on the ratio of the respective receptor number of Tar/Tsr. In addition, the ratio of Tar to Tsr was found to vary with cells’ growth conditions, whereby it depended on the culture density but not on the growth duration. These results provide biological insights into the decision-making processes of chemotactic bacteria that are subjected to multiple chemical stimuli and demonstrate the importance of the cellular microenvironment in determining phenotypic behavior.
Analysis of the
dbpBA
Upstream Regulatory Region Controlled by RpoS in
Borrelia burgdorferi
Authors: Ouyang Zhiming , Haq Shayma , Norgard Michael V. 
Published: January 2010

ABSTRACT

Decorin-binding proteins B and A (DbpB and DbpA) are thought to play important roles in
Borrelia burgdorferi
pathogenesis by serving as adhesins for the extracellular matrix. It has been established that the expression of DbpBA is governed by the Rrp2-RpoN-RpoS regulatory pathway. However, the precise mechanism underlying the control of DbpBA expression has been unclear. In particular, it has been unknown whether RpoS influences DbpBA expression directly or indirectly (through an additional regulatory molecule[s]). Here, employing a wild-type
B. burgdorferi
strain and a
dbpBA
-deficient mutant, we analyzed the 5′ genetic elements of the
dbpBA
operon using deletion analysis, coupled with luciferase reporter assays, quantitative reverse transcription PCR, and immunoblot analyses. A minimal promoter, encompassed within 70 bp upstream of the ATG start codon of
dbpBA
, was identified and found to be necessary and sufficient to initiate
dbpBA
transcription. The minimal
dbpBA
promoter was responsive to environmental stimuli such as temperature, pH, and whole blood. Two
in silico
-identified inverted repeat elements were not involved in the response of
dbpBA
expression to
in vitro
stimulation by environmental factors. The expression of
dbpBA
from the minimal promoter was abolished when
rpoS
was inactivated. In addition, the targeted mutagenesis of a C at position −14 within the extended −10 region of
dbpBA
, which has been postulated to be strategic for Eσ
S
binding in
Escherichia coli
, abolished
dbpBA
expression in
B. burgdorferi
. These combined data suggest that the Rrp2-RpoN-RpoS pathway controls
dbpBA
expression by the direct binding of RpoS to an RpoS-dependent promoter. However, given that there remains a distinct difference between the expression of DbpBA and other genes under the direct control of RpoS (e.g., OspC), our findings do not preclude the existence of another layer of gene regulation that may contribute to the modulation of DbpBA expression via an as-yet unknown mechanism.
Flagellated but Not Hyperfimbriated
Salmonella enterica
Serovar Typhimurium Attaches to and Forms Biofilms on Cholesterol-Coated Surfaces
Authors: Crawford Robert W. , Reeve Kristin E. , Gunn John S. 
Published: January 2010

ABSTRACT

The asymptomatic, chronic carrier state of
Salmonella enterica
serovar Typhi occurs in the bile-rich gallbladder and is frequently associated with the presence of cholesterol gallstones. We have previously demonstrated that salmonellae form biofilms on human gallstones and cholesterol-coated surfaces
in vitro
and that bile-induced biofilm formation on cholesterol gallstones promotes gallbladder colonization and maintenance of the carrier state. Random transposon mutants of
S. enterica
serovar Typhimurium were screened for impaired adherence to and biofilm formation on cholesterol-coated Eppendorf tubes but not on glass and plastic surfaces. We identified 49 mutants with this phenotype. The results indicate that genes involved in flagellum biosynthesis and structure primarily mediated attachment to cholesterol. Subsequent analysis suggested that the presence of the flagellar filament enhanced binding and biofilm formation in the presence of bile, while flagellar motility and expression of type 1 fimbriae were unimportant. Purified
Salmonella
flagellar proteins used in a modified enzyme-linked immunosorbent assay (ELISA) showed that FliC was the critical subunit mediating binding to cholesterol. These studies provide a better understanding of early events during biofilm development, specifically how salmonellae bind to cholesterol, and suggest a target for therapies that may alleviate biofilm formation on cholesterol gallstones and the chronic carrier state.
Establishing and Maintaining Sequestration of Dam Target Sites for Phase Variation ofagn43inEscherichia coli
Authors: Kaminska Renata , van der Woude Marjan W. 
Published: January 2010

ABSTRACTPhase variation of the outer membrane protein Ag43 encoded byagn43inEscherichia coliis controlled by an epigenetic mechanism. Sequestration of the regulatory region from Dam-dependent methylation has to be established and maintained throughout a generation to obtain and maintain the OFF phase. This work shows that hemimethylated DNA, which is formed by the passage of the DNA replication fork in an ON-phase cell, can be sequestered from methylation by OxyR binding, which is thus a key event for the switch from ON to OFF. No evidence was found that the protein SeqA, which also binds to the region, is involved in sequestration. To facilitate the dissection of this process further, a novel approach was introduced that does not alter the sequence of the regulatory region or the cellular concentration of Dam or OxyR, which consists of inserting auxiliary OxyR binding sites upstream of the regulatory region. Using this strategy, it was shown that the ON-to-OFF switch frequency can be modulated without changing the OFF-to-ON frequency. The data support a model in which in an ON-phase cell, the subcellular OxyR availability at the replication fork as it passes through theagn43regulatory region is key for initiating an ON-to-OFF switch. In contrast, this availability is not a determining factor for the switch from OFF to ON. This finding shows that different variables affect these two stochastic events. This provides new insight into the events determining the stochastic nature of epigenetic phase variation.
Functional Analysis of the RdxA and RdxB Nitroreductases of
Campylobacter jejuni
Reveals that Mutations in
rdxA
Confer Metronidazole Resistance
Authors: Ribardo Deborah A. , Bingham-Ramos Lacey K. , Hendrixson David R. 
Published: January 2010

ABSTRACT

Campylobacter jejuni
is a leading cause of gastroenteritis in humans and a commensal bacterium of the intestinal tracts of many wild and agriculturally significant animals. We identified and characterized a locus, which we annotated as
rdxAB
, encoding two nitroreductases. RdxA was found to be responsible for sensitivity to metronidazole (Mtz), a common therapeutic agent for another epsilonproteobacterium,
Helicobacter pylori
. Multiple, independently derived mutations in
rdxA
but not
rdxB
resulted in resistance to Mtz (Mtz
r
), suggesting that, unlike the case in
H. pylori
, Mtz
r
might not be a polygenic trait. Similarly, Mtz
r
C. jejuni
was isolated after both
in vitro
and
in vivo
growth in the absence of selection that contained frameshift, point, insertion, or deletion mutations within
rdxA
, possibly revealing genetic variability of this trait in
C. jejuni
due to spontaneous DNA replication errors occurring during normal growth of the bacterium. Similar to previous findings with
H. pylori
RdxA, biochemical analysis of
C. jejuni
RdxA showed strong oxidase activity, with reduction of Mtz occurring only under anaerobic conditions. RdxB showed similar characteristics but at levels lower than those for RdxA. Genetic analysis confirmed that
rdxA
and
rdxB
are cotranscribed and induced during
in vivo
growth in the chick intestinal tract, but an absence of these genes did not strongly impair
C. jejuni
for commensal colonization. Further studies indicate that
rdxA
is a convenient locus for complementation of mutants in
cis
. Our work contributes to the growing knowledge of determinants contributing to susceptibility to Mtz (Mtz
s
) and supports previous observations of the fundamental differences in the activities of nitroreductases from epsilonproteobacteria.
The
hdrRM
Operon of
Streptococcus mutans
Encodes a Novel Regulatory System for Coordinated Competence Development and Bacteriocin Production
Authors: Okinaga Toshinori , Niu Guoqing , Xie Zhoujie , Qi Fengxia , Merritt Justin 
Published: January 2010

ABSTRACT

The
Streptococcus mutans hdrRM
operon encodes a novel two-gene regulatory system induced by high cell density. Previous studies identified
hdrM
as the only known negative regulator of competence development in
S
.
mutans
. In the present study, we demonstrated that the HdrRM system bypasses the prototypical competence gene regulators ComC and ComDE in the transcriptional regulation of the competence-specific sigma factor
comX
and the late competence genes. Similarly, the HdrRM system can abrogate the requirement for ComE to produce the bacteriocin mutacin IV. To further probe the regulatory mechanism of
hdrRM
, we created an
hdrR
overexpression strain and showed that it could reproduce each of the
hdrM
competence and mutacin phenotypes, indicating that HdrM acts as a negative regulator of HdrR activity. Using a mutacin IV-luciferase reporter, we also demonstrated that the
hdrRM
system utilizes the same promoter elements recognized by ComE and thus appears to comprise a novel regulatory pathway parallel to ComCDE.