bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a Multigene-Encoded Sodium/Hydrogen Antiporter (Sha) from
Pseudomonas aeruginosa
: Its Involvement in Pathogenesis
Authors: Kosono Saori , Haga Koki , Tomizawa Rui , Kajiyama Yusuke , Hatano Kazuo , Takeda Shinobu , Wakai Yoshimi , Hino Motohiro , Kudo Toshiaki 
Published: July 2005

ABSTRACT

Sha (also known as Mrp/Mnh/Pha) is a Na
+
/H
+
antiporter encoded by a cluster of six or seven genes that probably form a multisubunit transport complex. The Sha system is important for the homeostasis of H
+
, Na
+
, and other monovalent cations and plays a critical role in various functions, including alkaliphily, sporulation, and symbiosis. Here, we characterized the
sha
homologue genes from the opportunistic pathogen
Pseudomonas aeruginosa
, which exist as a cluster of six genes (PA1054 to PA1059). The gene cluster PA1054 to PA1059, but not the cluster with a deletion of PA1054, complemented a growth defect in the presence of 0.2 M NaCl and a defect in Na
+
/H
+
antiport activity of the
Escherichia coli
TO114 mutant lacking the three major Na
+
/H
+
antiporters, indicating that genes PA1054 to PA1059 are responsible for Na
+
/H
+
antiport activity. We disrupted PA1054 (a
shaA
homologue gene) and determined its effect on Na
+
tolerance during growth, Na
+
efflux, and pathogenicity in mice. Disruption of PA1054 resulted in severe Na
+
sensitivity during growth and decreased Na
+
efflux activity. In mice, the deletion mutant of PA1054 also exhibited an attenuated virulence in systemic, pulmonary, and urinary tract infections and also a decrease in colonization of the infected organs. From these results, we conclude that the genes PA1054 to PA1059 encode a Na
+
/H
+
antiporter that is largely responsible for Na
+
extrusion in
P. aeruginosa
and has a role in the infection of the pathogen. We propose to designate PA1054 to PA1059 as the
sha
(sodium hydrogen antiporter) genes,
shaABCDEFG
.
Identification of Genes Required for Recycling Reducing Power during Photosynthetic Growth
Authors: Tavano Christine L. , Podevels Angela M. , Donohue Timothy J. 
Published: July 2005

ABSTRACT

Photosynthetic organisms have the unique ability to transform light energy into reducing power. We study the requirements for photosynthesis in the α-proteobacterium
Rhodobacter sphaeroides
. Global gene expression analysis found that ∼50 uncharacterized genes were regulated by changes in light intensity and O
2
tension, similar to the expression of genes known to be required for photosynthetic growth of this bacterium. These uncharacterized genes included RSP4157 to -4159, which appeared to be cotranscribed and map to plasmid P004. A mutant containing a polar insertion in RSP4157, CT01, was able to grow via photosynthesis under autotrophic conditions using H
2
as an electron donor and CO
2
as a carbon source. However, CT01 was unable to grow photoheterotrophically in a succinate-based medium unless compounds that could be used to recycle reducing power (the external electron acceptor dimethyl sulfoxide (DMSO) or CO
2
) were provided. This suggests that the insertion in RSP4157 caused a defect in recycling reducing power during photosynthetic growth when a fixed carbon source was present. CT01 had decreased levels of RNA for genes encoding putative glycolate degradation functions. We found that exogenous glycolate also rescued photoheterotrophic growth of CT01, leading us to propose that CO
2
produced from glycolate metabolism can be used by the Calvin cycle to recycle reducing power generated in the photosynthetic apparatus. The ability of glycolate, CO
2
, or DMSO to support photoheterotrophic growth of CT01 suggests that one or more products of RSP4157 to -4159 serve a previously unknown role in recycling reducing power under photosynthetic conditions.
Functional Analysis of the
glycero-manno
-Heptose 7-Phosphate Kinase Domain from the Bifunctional HldE Protein, Which Is Involved in ADP-
l
-
glycero
-
d
-
manno
-Heptose Biosynthesis
Authors: McArthur Fiona , Andersson C. Evalena , Loutet Slade , Mowbray Sherry L. , Valvano Miguel A. 
Published: July 2005

ABSTRACT

The core oligosaccharide component of the lipopolysaccharide can be subdivided into inner and outer core regions. In
Escherichia coli
, the inner core consists of two 3-
deoxy
-
d
-
manno-
octulosonic acid and three
glycero
-
manno
-heptose residues. The HldE protein participates in the biosynthesis of ADP-
glycero
-
manno
-heptose precursors used in the assembly of the inner core. HldE comprises two functional domains: an N-terminal region with homology to the ribokinase superfamily (HldE1 domain) and a C-terminal region with homology to the cytidylyltransferase superfamily (HldE2 domain). We have employed the structure of the
E. coli
ribokinase as a template to model the HldE1 domain and predict critical amino acids required for enzyme activity. Mutation of these residues renders the protein inactive as determined in vivo by functional complementation analysis. However, these mutations did not affect the secondary or tertiary structure of purified HldE1, as judged by fluorescence spectroscopy and circular dichroism. Furthermore, in vivo coexpression of wild-type, chromosomally encoded HldE and mutant HldE1 proteins with amino acid substitutions in the predicted ATP binding site caused a dominant negative phenotype as revealed by increased bacterial sensitivity to novobiocin. Copurification experiments demonstrated that HldE and HldE1 form a complex in vivo. Gel filtration chromatography resulted in the detection of a dimer as the predominant form of the native HldE1 protein. Altogether, our data support the notions that the HldE functional unit is a dimer and that structural components present in each HldE1 monomer are required for enzymatic activity.
Rot Repression of Enterotoxin B Expression in
Staphylococcus aureus
Authors: Tseng Ching Wen , Stewart George C. 
Published: July 2005

ABSTRACT

The accessory gene regulator (Agr) system is a quorum-sensing system of
Staphylococcus aureus
responsible for upregulation of certain exoprotein genes and downregulation of certain cell-wall associated proteins during the post-exponential phase of growth. The enterotoxin B (
seb
) determinant is upregulated by the Agr system. Agr-regulated
ci
s elements within the
seb
promoter region were examined by deletion analyses of the
seb
promoter by a hybrid promoter approach utilizing the staphylococcal
lac
operon promoter. To identify the regulatory pathway for enterotoxin B expression, the
seb
promoter fused to the chloramphenicol acetyltransferase reporter gene was introduced into mutants of
S. aureus
lacking
agr
or different members of the Sar family of transcriptional regulators. Agr control of
seb
promoter activity was found to be dependent upon the presence of a functional Rot protein, and Rot was shown to be able to bind to the
seb
promoter. Therefore, the Agr-mediated post-exponential-phase increase in
seb
transcription results from the Agr system's inactivation of Rot repressor activity.
Swarming Differentiation and Swimming Motility inBacillus subtilisAre Controlled byswrA, a Newly Identified Dicistronic Operon
Authors: Calvio Cinzia , Celandroni Francesco , Ghelardi Emilia , Amati Giuseppe , Salvetti Sara , Ceciliani Fabrizio , Galizzi Alessandro , Senesi Sonia 
Published: July 2005

ABSTRACTThe number and disposition of flagella harbored by eubacteria are regulated by a specific trait successfully maintained over generations. The genes governing the number of flagella inBacillus subtilishave never been identified, although theifmlocus has long been recognized to influence the motility phenotype of this microorganism. The characterization of a spontaneousifmmutant ofB. subtilis, displaying diverse degrees of cell flagellation in both liquid and solid media, raised the question of how theifmlocus governs the number and assembly of functional flagella. The major finding of this investigation is the characterization of a newly identified dicistronic operon, namedswrA, that controls both swimming motility and swarming differentiation inB. subtilis. Functional analysis of theswrAoperon allowedswrAA(previously namedswrA[D. B. Kearns, F. Chu, R. Rudner, and R. Losick, Mol. Microbiol.52:357-369, 2004]) to be the first gene identified inB. subtilisthat controls the number of flagella in liquid environments and the assembly of flagella in response to cell contact with solid surfaces. Evidence is given that the second gene of the operon,swrAB, is essential for enabling the surface-adhering cells to undergo swarming differentiation. Preliminary data point to a molecular interaction between the two gene products.
Expression of Canonical SOS Genes Is Not under LexA Repression inBdellovibrio bacteriovorus
Authors: Campoy Susana , Salvador Noelia , Cortés Pilar , Erill Ivan , Barbé Jordi 
Published: July 2005

ABSTRACTThe here-reported identification of the LexA-binding sequence ofBdellovibrio bacteriovorus, a bacterial predator belonging to the δ-Proteobacteria, has made possible a detailed study of its LexA regulatory network. Surprisingly, only thelexAgene and a multiple gene cassette includingdinPanddnaEhomologues are regulated by the LexA protein in this bacterium. In vivo expression analyses have confirmed that this gene cassette indeed forms a polycistronic unit that, like thelexAgene, is DNA damage inducible inB. bacteriovorus. Conversely, genes such asrecA,uvrA,ruvCAB, andssb, which constitute the canonical core of theProteobacteriaSOS system, are not repressed by the LexA protein in this organism, hinting at a persistent selective pressure to maintain both thelexAgene and its regulation on the reported multiple gene cassette. In turn, in vitro experiments show that theB. bacteriovorusLexA-binding sequence is not recognized by other δ-ProteobacteriaLexA proteins but binds to the cyanobacterial LexA repressor. This placesB. bacteriovorusLexA at the base of the δ-ProteobacteriaLexA family, revealing a high degree of conservation in the LexA regulatory sequence prior to the diversification and specialization seen in deeper groups of theProteobacteriaphylum.
NanA, a Neuraminidase fromStreptococcus pneumoniae, Shows High Levels of Sequence Diversity, at Least in Part through Recombination withStreptococcus oralis
Authors: King Samantha J. , Whatmore Adrian M. , Dowson Christopher G. 
Published: July 2005

ABSTRACTStreptococcus pneumoniae, an important human pathogen, contains at least two genes,nanAandnanB, that express sialidase activity. NanA is a virulence determinant of pneumococci which is important in animal models of colonization and middle ear infections. The gene encoding NanA was detected in all 106 pneumococcal strains screened that represented 59 restriction profiles. Sequencing confirmed a high level of diversity, up to 17.2% at the nucleotide level and 14.8% at the amino acid level. NanA diversity is due to a number of mechanisms including insertions, point mutations, and recombination generating mosaic genes. The level of nucleotide divergence for each recombinant block is greater than 30% and much higher than the 20% identified within mosaicpbpgenes, suggesting that a high selective pressure exists for these alterations. These data indicate that at least one of the four recombinant blocks identified originated from aStreptococcus oralisisolate, demonstrating for the first time that protein virulence determinants of pneumococci have, as identified previously for genes encoding penicillin binding proteins, evolved by recombination with oral streptococci. No amino acid alterations were identified within the aspartic boxes or predicted active site, suggesting that sequence variation may be important in evading the adaptive immune response. Furthermore, this suggests thatnanAis an important target of the immune system in the interaction between the pneumococcus and host.
The Holin Protein of Bacteriophage PRD1 Forms a Pore for Small-Molecule and Endolysin Translocation
Authors: Žiedaitė Gabija , Daugelavičius Rimantas , Bamford Jaana K. H. , Bamford Dennis H. 
Published: July 2005

ABSTRACT

PRD1 is a bacteriophage with an icosahedral outer protein layer surrounding the viral membrane, which encloses the linear double-stranded DNA genome. PRD1 infects gram-negative cells harboring a conjugative IncP plasmid. Here we studied the lytic functions of PRD1. Using infected cells and plasmid-borne lysis genes, we demonstrated that a two-component lysis system (holin-endolysin) operates to release progeny phage particles from the host cell. Monitoring of ion fluxes and the ATP content of the infected cells allowed us to build a model of the sequence of lysis-related physiological changes. A decrease in the intracellular level of ATP is the earliest indicator of cell lysis, followed by the leakage of K
+
from the cytosol approximately 20 min prior to the decrease in culture turbidity. However, the K
+
efflux does not immediately lead to the depolarization of the cytoplasmic membrane or leakage of the intracellular ATP. These effects are observed only ∼5 to 10 min prior to cell lysis. Similar results were obtained using cells expressing the holin and endolysin genes from plasmids.
YycH Regulates the Activity of the Essential YycFG Two-Component System in
Bacillus subtilis
Authors: Szurmant Hendrik , Nelson Kristine , Kim Eun-Ja , Perego Marta , Hoch James A. 
Published: July 2005

ABSTRACT

Of the numerous two-component signal transduction systems found in bacteria, only a very few have proven to be essential for cell viability. Among these is the YycF (response regulator)-YycG (histidine kinase) system, which is highly conserved in and specific to the low-G+C content gram-positive bacteria. Given the pathogenic nature of several members of this class of bacteria, the YycF-YycG system has been suggested as a prime antimicrobial target. In an attempt to identify genes involved in regulation of this two-component system, a transposon mutagenesis study was designed to identify suppressors of a temperature-sensitive YycF mutant in
Bacillus subtilis
. Suppressors could be identified, and the prime target was the
yycH
gene located adjacent to
yycG
and within the same operon. A
lacZ
reporter assay revealed that YycF-regulated gene expression was elevated in a
yycH
strain, whereas disruption of any of the three downstream genes within the operon,
yycI
,
yycJ
, and
yycK
, showed no such effect. The concentrations of both YycG and YycF, assayed immunologically, remained unchanged between the wild-type and the
yycH
strain as determined by immunoassay. Alkaline phosphatase fusion studies showed that YycH is located external to the cell membrane, suggesting that it acts in the regulation of the sensor domain of the YycG sensor histidine kinase. The
yycH
strain showed a characteristic cell wall defect consistent with the previously suggested notion that the YycF-YycG system is involved in regulating cell wall homeostasis and indicating that either up- or down-regulation of YycF activity affects this homeostatic mechanism.
Amino Acids in Positions 48, 52, and 73 Differentiate the Substrate Specificities of the Highly Homologous Chlorocatechol 1,2-Dioxygenases CbnA and TcbC
Authors: Liu Shenghao , Ogawa Naoto , Senda Toshiya , Hasebe Akira , Miyashita Kiyotaka 
Published: July 2005

ABSTRACT

Chlorocatechol 1,2-dioxygenase (CCD) is the first-step enzyme of the chlorocatechol
ortho
-cleavage pathway, which plays a central role in the degradation of various chloroaromatic compounds. Two CCDs, CbnA from the 3-chlorobenzoate-degrader
Ralstonia eutropha
NH9 and TcbC from the 1,2,4-trichlorobenzene-degrader
Pseudomonas
sp. strain P51, are highly homologous, having only 12 different amino acid residues out of identical lengths of 251 amino acids. But CbnA and TcbC are different in substrate specificities against dichlorocatechols, favoring 3,5-dichlorocatechol (3,5-DC) and 3,4-dichlorocatechol (3,4-DC), respectively. A study of chimeric mutants constructed from the two CCDs indicated that the N-terminal parts of the enzymes were responsible for the difference in the substrate specificities. Site-directed mutagenesis studies further identified the amino acid in position 48 (Leu in CbnA and Val in TcbC) as critical in differentiating the substrate specificities of the enzymes, which agreed well with molecular modeling of the two enzymes. Mutagenesis studies also demonstrated that Ile-73 of CbnA and Ala-52 of TcbC were important for their high levels of activity towards 3,5-DC and 3,4-DC, respectively. The importance of Ile-73 for 3,5-DC specificity determination was also shown with other CCDs such as TfdC from
Burkholderia
sp. NK8 and TfdC from
Alcaligenes
sp. CSV90 (identical to TfdC from
R. eutropha
JMP134), which convert 3,5-DC preferentially. Together with amino acid sequence comparisons indicating high conservation of Leu-48 and Ile-73 among CCDs, these results suggested that TcbC of strain P51 had diverged from other CCDs to be adapted to conversion of 3,4-DC.
Functional Replacement of the Oligomerization Domain of H-NS by the Hha Protein of
Escherichia coli
Authors: Rodríguez Sonia , Nieto José María , Madrid Cristina , Juárez Antonio 
Published: July 2005

ABSTRACT

Members of the H-NS family of proteins play a relevant role as modulators of gene expression in gram-negative bacteria. Interaction of these proteins with members of the Hha/YmoA family of proteins has been previously reported. It has been hypothesized that the latter proteins are functionally equivalent to the N-terminal domain of H-NS-like proteins. In this report we test this assumption by replacing the N-terminal domain of
Escherichia coli
H-NS by Hha. It has been possible to obtain a functional protein that can compensate for some of the
hns-
induced phenotypes. These results highlight the relevance of H-NS-Hha interactions to generate heterooligomeric complexes that modulate gene expression in gram-negative bacteria.
Effective Symbiosis between
Rhizobium etli
and
Phaseolus vulgaris
Requires the Alarmone ppGpp
Authors: Moris Martine , Braeken Kristien , Schoeters Eric , Verreth Christel , Beullens Serge , Vanderleyden Jos , Michiels Jan 
Published: July 2005

ABSTRACT

The symbiotic interaction between
Rhizobium etli
and
Phaseolus vulgaris
, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The
R. etli relA
gene was identified through a genome-wide screening for
R. etli
symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an
R. etli relA
mutant strain under conditions of amino acid starvation. Plants nodulated by an
R. etli relA
mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of
relA
mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the σ
N
-dependent nitrogen fixation genes
rpoN2
and
iscN
was considerably reduced in the
relA
mutant. In addition, the expression of the
relA
gene was negatively regulated by RpoN2, the symbiosis-specific σ
N
copy of
R. etli
. Therefore, an autoregulatory loop controlling the expression of
relA
and
rpoN2
seems operative in bacteroids. The production of long- and short-chain acyl-homoserine-lactones by the
cinIR
and
raiIR
systems was decreased in an
R. etli relA
mutant. Our results suggest that
relA
may play an important role in the regulation of gene expression in
R. etli
bacteroids and in the adaptation of bacteroid physiology.
Functional Analysis of Conserved Gene Products Involved in Assembly ofEscherichia coliCapsules and Exopolysaccharides: Evidence for Molecular Recognition between Wza and Wzc for Colanic Acid Biosynthesis
Authors: Reid Anne N. , Whitfield Chris 
Published: July 2005

ABSTRACTGroup 1 capsular polysaccharides (CPSs) ofEscherichia coliand some loosely cell-associated exopolysaccharides (EPSs), such as colanic acid, are assembled by a Wzy-dependent polymerization system. In this biosynthesis pathway, Wza, Wzb, and Wzc homologues are required for surface expression of wild-type CPS or EPS. Multimeric complexes of Wza in the outer membrane are believed to provide a channel for polymer export; Wzc is an inner membrane tyrosine autokinase and Wzb is its cognate phosphatase. This study was performed to determine whether the Wza, Wzb, and Wzc proteins for colanic acid expression inE. coliK-12 could function in theE. coliK30 prototype group 1 capsule system. When expressed together, colanic acid Wza, Wzb, and Wzc could complement awza-wzb-wzcdefect inE. coliK30, suggesting conservation in their collective function in Wzy-dependent CPS and EPS systems. Expressed individually, colanic acid Wza and Wzb could also function in K30 CPS expression. In contrast, the structural requirements for Wzc function were more stringent because colanic acid Wzc could restore translocation of K30 CPS to the cell surface only when expressed with its cognate Wza protein. Chimeric colanic acid-K30 Wzc proteins were constructed to further study this interaction. These proteins could restore K30 biosynthesis but were unable to couple synthesis to export. The chimeric protein comprising the periplasmic domain of colanic acid Wzc was functional for effective K30 CPS surface expression only when coexpressed with colanic acid Wza. These data highlight the importance of Wza-Wzc interactions in group 1 CPS assembly.
Histones in
Crenarchaea
Authors: Čuboňová L'ubomíra , Sandman Kathleen , Hallam Steven J. , DeLong Edward F. , Reeve John N. 
Published: July 2005

ABSTRACT

Archaeal histone-encoding genes have been identified in marine
Crenarchaea
. The protein encoded by a representative of these genes, synthesized in vitro and expressed in
Escherichia coli
, binds DNA and forms complexes with properties typical of an archaeal histone. The discovery of histones in
Crenarchaea
supports the argument that histones evolved before the divergence of
Archaea
and
Eukarya
.
Global Regulation of Gene Expression by ArlRS, a Two-Component Signal Transduction Regulatory System of
Staphylococcus aureus
Authors: Liang Xudong , Zheng Li , Landwehr Christina , Lunsford Dwayne , Holmes David , Ji Yinduo 
Published: July 2005

ABSTRACT

Staphylococcus aureus
expresses various cell wall-associated and extracellular virulence factors, coordinately controlled by different two-component signal transduction systems and transcriptional regulators. In this study, we used microarray technology to identify the genes regulated by ArlR. The microarray data indicate that ArlR functions as a positive regulator and also as a negative repressor to directly and/or indirectly mediate the expression of at least 114 genes involved in different functions, including autolysis, cell division, growth, and pathogenesis.
Phenotypic Alteration and Target Gene Identification Using Combinatorial Libraries of Zinc Finger Proteins in Prokaryotic Cells
Authors: Park Kyung-Soon , Jang Young-Soon , Lee Horim , Kim Jin-Soo 
Published: July 2005

ABSTRACT

We have developed a method with prokaryotic organisms that uses randomized libraries of zinc finger-containing artificial transcription factors to induce phenotypic variations and to identify genes involved in the generation of a specific phenotype of interest. Combining chromatin immunoprecipitation experiments and in silico prediction of target DNA binding sequences for the artificial transcription factors, we identified
ubiX
, whose down-regulation correlates with the thermotolerance phenotype in
Escherichia coli
. Our results show that randomized libraries of artificial transcription factors are powerful tools for functional genomic studies.