bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Nucleoside Uptake Systems in
Lactococcus lactis
: Competition between Purine Nucleosides and Cytidine Allows for Modulation of Intracellular Nucleotide Pools
Authors: Martinussen Jan , Wadskov-Hansen Steen L. L. , Hammer Karin
Published: February 2003

A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of
33
P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H
3
BO
3
-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in
Lactococcus lactis
were investigated by measuring the uptake of radioactively labeled nucleosides. The

K
m

for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.
Dual Role of Cysteine 172 in Redox Regulation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activity and Degradation
Authors: Marcus Yehouda , Altman-Gueta Hagit , Finkler Aliza , Gurevitz Michael
Published: February 2003

Alkylation and oxidation of cysteine residues significantly decrease the catalytic activity and stimulate the degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We analyzed the role of vicinal cysteine residues in redox regulation of RuBisCO from
Synechocystis
sp. strain PCC 6803. Cys172 and Cys192, which are adjacent to the catalytic site, and Cys247, which cross-links two large subunits, were replaced by alanine. Whereas all mutant cells (C172A, C192A, C172A-C192A, and C247A) and the wild type grew photoautotrophically at similar rates, the maximal photosynthesis rates of C172A mutants decreased 10 to 20% as a result of 40 to 60% declines in RuBisCO turnover number. Replacement of Cys172, but not replacement of Cys192, prominently decreased the effect of cysteine alkylation or oxidation on RuBisCO. Oxidants that react with vicinal thiols had a less inhibitory effect on the activity of either the C172A or C192A enzyme variants, suggesting that a disulfide bond was formed upon oxidation. Thiol oxidation induced RuBisCO dissociation into subunits. This effect was either reduced in the C172A and C192A mutant enzymes or eliminated by carboxypentitol bisphosphate (CPBP) binding to the activated enzyme form. The CPBP effect presumably resulted from a conformational change in the carbamylated CPBP-bound enzyme, as implied from an alteration in the electrophoretic mobility. Stress conditions, provoked by nitrate deprivation, decreased the RuBisCO contents and activities in the wild type and in the C192A and C247A mutants but not in the C172A and C172A-C192A mutants. These results suggest that although Cys172 does not participate in catalysis, it plays a role in redox regulation of RuBisCO activity and degradation.
Synergistic Effects of Cellulosomal Xylanase and Cellulases from
Clostridium cellulovorans
on Plant Cell Wall Degradation
Authors: Murashima Koichiro , Kosugi Akihiko , Doi Roy H.
Published: February 2003

Plant cell walls are comprised of cellulose and hemicellulose and other polymers that are intertwined, and this complex structure presents a barrier to degradation by pure cellulases or hemicellulases. In this study, we determined the synergistic effects on corn cell wall degradation by the action of cellulosomal xylanase XynA and cellulosomal cellulases from
Clostridium cellulovorans
. XynA minicellulosomes and cellulase minicellulosomes were found to degrade corn cell walls synergistically but not purified substrates such as xylan and crystalline cellulose. The mixture of XynA and cellulases at a molar ratio of 1:2 showed the highest synergistic effect of 1.6 on corn cell wall degradation. The amounts both of xylooligosaccharides and cellooligosaccharides liberated from corn cell walls were increased by the synergistic action of XynA and cellulases. Although synergistic effects on corn cell wall degradation were found in simultaneous reactions with XynA and cellulases, no synergistic effects were observed in sequential reactions. The possible mechanism of synergism between XynA and cellulases is discussed.
Genetic and Biochemical Characterization of the F-ATPase Operon from S
treptococcus sanguis
10904
Authors: Kuhnert Wendi L. , Quivey, Robert G.
Published: February 2003

Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species
Streptococcus sanguis
and
Streptococcus mutans
. Differences in the pH optima of the respective ATPases appears to be the main reason that
S. mutans
is more tolerant of low pH values than
S. sanguis
and hence pathogenic. We have recently reported the genetic arrangement for the
S. mutans
operon. For purposes of comparative structural biology we have also investigated the F-ATPase from
S. sanguis
. Here, we report the genetic characterization and expression in
Escherichia coli
of the
S. sanguis
ATPase operon. Sequence analysis showed a gene order of
atpEBFHAGDC
and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both
S. sanguis
and
S. mutans
; however, it is not induced to the same extent in the nonpathogenic
S. sanguis
. Expression studies with an
atpD
deletion strain of
E. coli
showed that
S. sanguis
-
E. coli
hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.
Efficient Turnover of Chlorocatechols Is Essential for Growth of
Ralstonia eutropha
JMP134(pJP4) in 3-Chlorobenzoic Acid
Authors: Pérez-Pantoja D. , Ledger T. , Pieper D. H. , González B.
Published: February 2003

Ralstonia eutropha
JMP134(pJP4) degrades 3-chlorobenzoate (3-CB) by using two not completely isofunctional, pJP4-encoded chlorocatechol degradation gene clusters,

tfdC
I

D
I

E
I

F
I

and

tfdD
II

C
II

E
II

F
II

. Introduction of several copies of each gene cluster into
R. eutropha
JMP222, which lacks pJP4 and thus accumulates chlorocatechols from 3-CB, allows the derivatives to grow in this substrate. However, JMP222 derivatives containing one chromosomal copy of each cluster did not grow in 3-CB. The failure to grow in 3-CB was the result of accumulation of chlorocatechols due to the limiting activity of chlorocatechol 1,2-dioxygenase (TfdC), the first enzyme in the chlorocatechol degradation pathway. Micromolar concentrations of 3- and 4-chlorocatechol inhibited the growth of strains JMP134 and JMP222 in benzoate, and cells of strain JMP222 exposed to 3 mM 3-CB exhibited a 2-order-of-magnitude decrease in viability. This toxicity effect was not observed with strain JMP222 harboring multiple copies of the

tfdC
I

gene, and the derivative of strain JMP222 containing

tfdC
I

D
I

E
I

F
I

plus multiple copies of the

tfdC
I

gene could efficiently grow in 3-CB. In addition,

tfdC
I

and

tfdC
II

gene mutants of strain JMP134 exhibited no growth and impaired growth in 3-CB, respectively. The introduction into strain JMP134 of the
xylS
-
xylXYZL
genes, encoding a broad-substrate-range benzoate 1,2-dioxygenase system and thus increasing the transformation of 3-CB into chlorocatechols, resulted in derivatives that exhibited a sharp decrease in the ability to grow in 3-CB. These observations indicate that the dosage of chlorocatechol-transforming genes is critical for growth in 3-CB. This effect depends on a delicate balance between chlorocatechol-producing and chlorocatechol-consuming reactions.
Polymorphism in the Collagen-Like Region of the
Bacillus anthracis
BclA Protein Leads to Variation in Exosporium Filament Length
Authors: Sylvestre Patricia , Couture-Tosi Evelyne , Mock Michèle
Published: February 2003

We recently identified a
Bacillus anthracis
glycoprotein which is a structural constituent of the exosporium filaments (P. Sylvestre, E. Couture-Tosi, and M. Mock, Mol. Microbiol.
45:
169-178, 2002). This
Bacillus
collagen-like protein (BclA) contains an internal collagen-like region (CLR) of GXX repeats which includes a large proportion of GPT triplets. Here, we report that the polymorphic marker Ceb-Bams13, for which there are nine alleles (P. Le Flèche et al., BMC Microbiol.
1:
2, 2001), maps within the open reading frame encoding BclA. The
bclA
gene in 11
B. anthracis
strains representative of seven Ceb-Bams13 alleles was sequenced and compared to the Ames
bclA
gene sequence. The amino- and carboxy-terminal sequences surrounding the CLR are conserved. The CLR itself is highly polymorphic: it contains between 17 and 91 GXX repeats and one to eight copies of the 21-amino-acid sequence (GPT)
5
GDTGTT, named the BclA repeat. The length of the filament on the spore surface differed between the strains. We exchanged the
bclA
gene between strains with different CLRs and examined the spore surfaces by electron microscopy analysis. The length of the BclA CLR is responsible for the variation in filament length.
Exogenous Glutathione Completes the Defense against Oxidative Stress in
Haemophilus influenzae
Authors: Vergauwen Bjorn , Pauwels Frederik , Vaneechoutte Mario , Van Beeumen Jozef J.
Published: February 2003

Since they are equipped with several strategies by which they evade the antimicrobial defense of host macrophages, it is surprising that members of the genus
Haemophilus
appear to be deficient in common antioxidant systems that are well established to protect prokaryotes against oxidative stress. Among others, no genetic evidence for glutathione (γ-Glu-Cys-Gly) (GSH) biosynthesis or for alkyl hydroperoxide reduction (e.g., the Ahp system characteristic or enteric bacteria) is apparent from the
Haemophilus influenzae
Rd genome sequence, suggesting that the organism relies on alternative systems to maintain redox homeostasis or to reduce small alkyl hydroperoxides. In this report we address this apparent paradox for the nontypeable
H. influenzae
type strain NCTC 8143. Instead of biosynthesis, we could show that this strain acquires GSH by importing the thiol tripeptide from the growth medium. Although such GSH accumulation had no effect on growth rates, the presence of cellular GSH protected against methylglyoxal,
tert
-butyl hydroperoxide (
t
-BuOOH), and
S
-nitrosoglutathione toxicity and regulated the activity of certain antioxidant enzymes.
H. influenzae
NCTC 8143 extracts were shown to contain GSH-dependent peroxidase activity with
t
-BuOOH as the peroxide substrate. The GSH-mediated protection against
t
-BuOOH stress is most probably catalyzed by the product of open reading frame HI0572 (Prx/Grx), which we isolated from a genomic DNA fragment that confers wild-type resistance to
t
-BuOOH toxicity in the Ahp-negative
Escherichia coli
strain TA4315 and that introduces GSH-dependent alkyl hydroperoxide reductase activity into naturally GSH peroxidase-negative
E. coli
. Finally, we demonstrated that cysteine is an essential amino acid for growth and that cystine, GSH, glutathione amide, and cysteinylglycine can be catabolized in order to complement cysteine deficiency.
The
Escherichia coli lipB
Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase
Authors: Jordan Sean W. , Cronan, John E.
Published: February 2003

In an earlier study (S. W. Jordan and J. E. Cronan, Jr., J. Biol. Chem. 272:17903-17906, 1997) we reported a new enzyme, lipoyl-[acyl carrier protein]-protein
N
-lipoyltransferase, in
Escherichia coli
and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that
E. coli lipB
mutants lack this enzyme activity, a finding consistent with
lipB
being the gene that encoded the lipoyltransferase. However, it remained possible that
lipB
encoded a positive regulator required for lipoyltransferase expression or action. We now report genetic and biochemical evidence demonstrating that
lipB
encodes the lipoyltransferase. A
lipB
temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the
lipB
-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these experiments the ATG initiation codon commonly assigned to
lipB
genes in genomic databases was shown to produce a nonfunctional
E. coli
LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein. Prior genetic results (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Bacteriol. 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions.
NovelspoIIEMutation That Causes Uncompartmentalized σFActivation inBacillus subtilis
Authors: Hilbert David W. , Piggot Patrick J.
Published: February 2003

During sporulation,Bacillus subtilisundergoes an asymmetric division that results in two cells with different fates, the larger mother cell and the smaller forespore. The protein phosphatase SpoIIE, which is required for activation of the forespore-specific transcription factor σF, is also required for optimal efficiency and timing of asymmetric division. We performed a genetic screen forspoIIEmutants that were impaired in sporulation but not σFactivity and isolated a strain with the mutationspoIIEV697A. The mutant exhibited a 10- to 40-fold reduction in sporulation and a sixfold reduction in asymmetric division compared to the parent. Transcription of the σF-dependentspoIIQpromoter was increased more than 10-fold and was no longer confined to the forespore. The excessive σFactivity persisted even when asymmetric division was prevented. Disruption ofspoIIGBdid not restore asymmetric division to thespoIIEV697Amutant, indicating that the deficiency is not a consequence of predivisional activation of the mother cell-specific transcription factor σE. Deletion of the gene encoding σF(spoIIAC)restored asymmetric division; however, a mutation that dramatically reduced the number of promoters responsive to σF,spoIIAC561(spoIIACV233 M), failed to do so. This result suggests that the block is due to expression of one of the small subset of σF-dependent genes expressed in this background or to unregulated interaction of σF with some other factor. Our results indicate that regulation of SpoIIE plays a critical role in coupling asymmetric division to σFactivation in order to ensure proper spatial and temporal expression of forespore-specific genes.
The Cyclic AMP-Cyclic AMP Receptor Protein Complex Regulates Activity of the
traJ
Promoter of the
Escherichia coli
Conjugative Plasmid pRK100
Authors: Starčič Marjanca , Žgur-Bertok Darja , Jordi Bart J. A. M. , Wösten Marc M. S. M. , Gaastra Wim , van Putten Jos P. M.
Published: February 2003

The TraJ protein is a central activator of F-like plasmid conjugal transfer. In a search for regulators of
traJ
expression, we studied the possible regulatory role of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex in
traJ
transcription using a
traJ-lacZ
reporter system. A comparison of the enzyme activities in the wild-type
Escherichia coli
strain MC4100 with those in
cya
and
crp
mutants indicated that disruption of the formation of the cAMP-CRP complex negatively influenced the activity of the
traJ
promoter of the F-like plasmid pRK100. The defect in the
cya
mutant was partially restored by addition of exogenous cAMP. Competitive reverse transcription-PCR performed with RNA isolated from the wild-type and mutant strains showed that the cAMP-CRP complex exerted its effect at the level of transcription. Electrophoretic mobility shift assays with purified CRP demonstrated that there was direct binding of CRP to the
traJ
promoter region. DNase I footprint experiments mapped the CRP binding site around position −67.5 upstream of the putative
traJ
promoter. Targeted mutagenesis of the
traJ
promoter region confirmed the location of the CRP binding site. Consistent with the demonstrated regulation of TraJ by the cAMP-CRP complex, mutants with defects in
cya
or
crp
exhibited reduced conjugal transfer from pRK100.
Role of the Cytoplasmic C Terminus of the FliF Motor Protein in Flagellar Assembly and Rotation
Authors: Grünenfelder Björn , Gehrig Stefanie , Jenal Urs
Published: February 2003

Twenty-six FliF monomers assemble into the MS ring, a central motor component of the bacterial flagellum that anchors the structure in the inner membrane. Approximately 100 amino acids at the C terminus of FliF are exposed to the cytoplasm and, through the interaction with the FliG switch protein, a component of the flagellar C ring, are essential for the assembly of the motor. In this study, we have dissected the entire cytoplasmic C terminus of the
Caulobacter crescentus
FliF protein by high-resolution mutational analysis and studied the mutant forms with regard to the assembly, checkpoint control, and function of the flagellum. Only nine amino acids at the very C terminus of FliF are essential for flagellar assembly. Deletion or substitution of about 10 amino acids preceding the very C terminus of FliF resulted in assembly-competent but nonfunctional flagella, making these the first
fliF
mutations described so far with a Fla
+
but Mot
−
phenotype. Removal of about 20 amino acids further upstream resulted in functional flagella, but cells carrying these mutations were not able to spread efficiently on semisolid agar plates. At least 61 amino acids located between the functionally relevant C terminus and the second membrane-spanning domain of FliF were not required for flagellar assembly and performance. A strict correlation was found between the ability of FliF mutant versions to assemble into a flagellum, flagellar class III gene expression, and a block in cell division. Motile suppressors could be isolated for nonmotile mutants but not for mutants lacking a flagellum. Several of these suppressor mutations were localized to the 5′ region of the
fliG
gene. These results provide genetic support for a model in which only a short stretch of amino acids at the immediate C terminus of FliF is required for flagellar assembly through stable interaction with the FliG switch protein.
Synthesis of a
Klebsiella pneumoniae
O-Antigen Heteropolysaccharide (O12) Requires an ABC 2 Transporter
Authors: Izquierdo Luis , Merino Susana , Regué Miguel , Rodriguez Florencia , Tomás Juan M.
Published: February 2003

A recombinant clone encoding enzymes for
Klebsiella pneumoniae
O12-antigen lipopolysaccharide (LPS) was found when we screened for serum resistance of a cosmid-based genomic library of
K. pneumoniae
KT776 (O12:K80) introduced into
Escherichia coli
DH5α. A total of eight open reading frames (ORFs) (
wb
O12
gene cluster) were necessary to produce
K. pneumoniae
O12-antigen LPS in
E. coli
K-12. A complete analysis of the
K. pneumoniae wb
O12
cluster revealed an interesting coincidence with the
wb
O4
cluster of
Serratia marcescens
from ORF5 to ORF8 (or WbbL to WbbA). This prompted us to generate mutants of
K. pneumoniae
strain KT776 (O12) and to study complementation between the two enterobacterial
wb
clusters using mutants of
S. marcescens
N28b (O4) obtained previously. Both
wb
gene clusters are examples of ABC 2 transporter-dependent pathways for O-antigen heteropolysaccharides. The
wzm
-
wzt
genes and the
wbbA
or
wbbB
genes were not interchangeable between the two gene clusters despite their high level of similarity. However, introduction of three cognate genes (
wzm
-
wzt
-
wbbA
or
wzm
-
wzt
-
wbbB
) into mutants unable to produce O antigen allowed production of the specific O antigen. The
K. pneumoniae
O12 WbbL protein performs the same function as WbbL from
S. marcescens
O4 in either the
S. marcescens
O4 or
E. coli
K-12 genetic background.
Rhamnogalacturonate Lyase RhiE Is Secreted by the Out System in
Erwinia chrysanthemi
Authors: Laatu Minna , Condemine Guy
Published: February 2003

Supernatants of rhamnose-induced
Erwinia chrysanthemi
strain 3937 cultures contain a principal secreted protein named RhiE. A
rhiE
mutant has been found among a set of rhamnose-induced MudI1681
lacZ
fusions. RhiE is a 62-kDa protein that has rhamnogalacturonate lyase activity on rhamnogalacturonan I (RG-I). It does not require a divalent cation for its activity and has an optimal pH of 6.0.
rhiE
expression is strongly induced in the presence of rhamnose but is also regulated by PecT and Crp, two regulators of the transcription of pectinolytic enzyme genes. RhiE is secreted through the type II Out secretion pathway. RhiE has no disulfide bond. The absence of RhiE secretion in a
dsb
mutant indicated that disulfide bond formation is required for the biogenesis of the secretion apparatus. RhiE was searched for in several
E. chrysanthemi
strains by using antibodies, and it was found to be present in one-third of the strains tested. However, the reduced virulence of the
rhiE
mutant indicates that degradation of the RG-I region of pectin is important for full virulence of
E. chrysanthemi
.
Functional Characterization of Penicillin-Binding Protein 1b fromStreptococcus pneumoniae
Authors: Di Guilmi Anne Marie , Dessen Andréa , Dideberg Otto , Vernet Thierry
Published: February 2003

The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by β-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region ofStreptococcus pneumoniaePBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.
Overexpression of the
waaZ
Gene Leads to Modification of the Structure of the Inner Core Region of
Escherichia coli
Lipopolysaccharide, Truncation of the Outer Core, and Reduction of the Amount of O Polysaccharide on the Cell Surface
Authors: Frirdich Emilisa , Lindner Buko , Holst Otto , Whitfield Chris
Published: February 2003

The
waa
gene cluster is responsible for the biosynthesis of the lipopolysaccharide (LPS) core region in
Escherichia coli
and
Salmonella.
Homologs of the
waaZ
gene product are encoded by the
waa
gene clusters of
Salmonella enterica
and
E. coli
strains with the K-12 and R2 core types. Overexpression of WaaZ in
E. coli
and
S. enterica
led to a modified LPS structure showing core truncations and (where relevant) to a reduction in the amount of O-polysaccharide side chains. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the predominant LPS structures in an
E. coli
isolate with an R1 core (
waaZ
is lacking from the type R1
waa
gene cluster) with a copy of the
waaZ
gene added on a plasmid. Novel truncated LPS structures, lacking up to 3 hexoses from the outer core, resulted from WaaZ overexpression. The truncated molecules also contained a KdoIII residue not normally found in the R1 core.
Complex Regulation of theBacillus subtilisAconitase Gene
Authors: Kim Hyun-Jin , Kim Sam-In , Ratnayake-Lecamwasam Manoja , Tachikawa Kiyoshi , Sonenshein Abraham L. , Strauch Mark
Published: February 2003

The roles of the CcpC, CodY, and AbrB proteins in regulation of theBacillus subtilisaconitase (citB) gene were found to be distinct and to vary with the conditions and phase of growth. CcpC, a citrate-inhibited repressor that is the primary factor regulatingcitBexpression in minimal-glucose-glutamine medium, also contributed to repression ofcitBduring exponential-phase growth in broth medium. A null mutation incodYhad no effect oncitBexpression during growth in minimal medium even when combined withccpCandabrBmutations. However, acodYmutation slightly relieved repression during exponential growth in broth medium and completely derepressedcitBexpression when combined with accpCmutation. AnabrBmutation led to decreased expression ofcitBduring stationary phase in both broth and minimal medium. All three proteins bound in vitro to specific and partially overlapping sites within thecitBregulatory region. Interaction of CcpC and CodY with thecitBpromoter region was partially competitive.
Conjugative Transfer of p42a from
Rhizobium etli
CFN42, Which Is Required for Mobilization of the Symbiotic Plasmid, Is Regulated by Quorum Sensing
Authors: Tun-Garrido Cristina , Bustos Patricia , González Víctor , Brom Susana
Published: February 2003

Rhizobium etli
CFN42 contains six plasmids. Only one of them, p42a, is self-conjugative at high frequency. This plasmid is strictly required for mobilization of the symbiotic plasmid (pSym). To study the transfer mechanism of p42a, a self-transmissible cosmid clone containing its transfer region was isolated. Its sequence showed that most of the
tra
genes are highly similar to genes of
Agrobacterium tumefaciens
pTiC58 and other related plasmids. Four putative regulatory genes were identified; three of these (
traI
,
traR
, and
cinR
) belong to the LuxR-LuxI family. Mutagenesis of these genes confirmed their requirement for p42a transfer. We found that the conjugative transfer of p42a is dependent on quorum sensing, and consequently pSym transfer also was found to be similarly regulated, establishing a complex link between environmental conditions and pSym transfer. Although
R. etli
has been shown to produce different
N
-acyl-homoserine lactones, only one of them, a 3-oxo-C
8
-homoserine lactone encoded by the
traI
gene described here, was involved in transfer. Mutagenesis of the fourth regulatory gene,
traM
, had no effect on transfer. Analysis of transcriptional fusions of the regulatory genes to a reporter gene suggests a complex regulation scheme for p42a conjugative transfer. Conjugal transfer gene expression was found to be directly upregulated by TraR and the 3-oxo-C
8
-homoserine lactone synthesized by TraI. The
traI
gene was autoregulated by these elements and positively regulated by CinR, while
cinR
expression required
traI
. Finally, we did not detect expression of
traM
, indicating that in p42a TraM may be expressed so weakly that it cannot inhibit conjugal transfer, leading to the unrepressed transfer of p42a.
P15 and P3, the Tail Completion Proteins of Bacteriophage T4, Both Form Hexameric Rings
Authors: Zhao Li , Kanamaru Shuji , Chaidirek Chatree'chalerm , Arisaka Fumio
Published: February 2003

Two proteins, gp15 and gp3 (gp for gene product), are required to complete the assembly of the T4 tail. gp15 forms the connector which enables the tail to bind to the head, whereas gp3 is involved in terminating the elongation of the tail tube. In this work, genes 15 and 3 were cloned and overexpressed, and the purified gene products were studied by analytical ultracentrifugation, electron microscopy, and circular dichroism. Determination of oligomerization state by sedimentation equilibrium revealed that both gp15 and gp3 are hexamers of the respective polypeptide chains. Electron microscopy of the negatively stained P15 and P3 (P denotes the oligomeric state of the gene product) revealed that both proteins form hexameric rings, the diameter of which is close to that of the tail tube. The differential roles between gp15 and gp3 upon completion of the tail are discussed.
The
Escherichia coli
Methyl-Directed Mismatch Repair System Repairs Base Pairs Containing Oxidative Lesions
Authors: Wyrzykowski Jennifer , Volkert Michael R.
Published: February 2003

A major role of the methyl-directed mismatch repair (MMR) system of
Escherichia coli
is to repair postreplicative errors. In this report, we provide evidence that MMR also acts on oxidized DNA, preventing mutagenesis. When cells deficient in MMR are grown anaerobically, spontaneous mutation frequencies are reduced compared with those of the same cells grown aerobically. In addition, we show that a
dam
mutant has an increased sensitivity to hydrogen peroxide treatment that can be suppressed by mutations that inactivate MMR. In a
dam
mutant, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted to single- and double-strand DNA breaks. Thus, base pairs containing oxidized bases will be converted to strand breaks if they are repaired by MMR. This is demonstrated by the increased peroxide sensitivity of a
dam
mutant and the finding that the sensitivity can be suppressed by mutations inactivating MMR. We demonstrate further that this repair activity results from MMR recognition of base pairs containing 8-oxoguanine (8-oxoG) based on the finding that overexpression of the MutM oxidative repair protein, which repairs 8-oxoG, can suppress the
mutH
-dependent increase in transversion mutations. These findings demonstrate that MMR has the ability to prevent oxidative mutagenesis either by removing 8-oxoG directly or by removing adenine misincorporated opposite 8-oxoG or both.
Characterization of a Cytosolic NiFe-Hydrogenase from the Hyperthermophilic Archaeon
Thermococcus kodakaraensis
KOD1
Authors: Kanai Tamotsu , Ito Sota , Imanaka Tadayuki
Published: February 2003

We have identified an NiFe-hydrogenase exclusively localized in the cytoplasm of the hyperthermophilic archaeon
Thermococcus kodakaraensis
KOD1 (
T. kodakaraensis
hydrogenase). A gene cluster encoding
T. kodakaraensis
hydrogenase was composed of four open reading frames (

hyhBGSL
Tk

), where the

hyhS
Tk

and

hyhL
Tk

gene products corresponded to the small and the large subunits of NiFe-hydrogenase, respectively. A putative open reading frame for hydrogenase-specific maturation endopeptidase (

hybD
Tk

) was found downstream of the cluster. Polyclonal antibodies raised against recombinant HyhL

Tk

were used for immunoaffinity purification of
T. kodakaraensis
hydrogenase, leading to a 259-fold concentration of hydrogenase activity. The purified
T. kodakaraensis
hydrogenase was composed of four subunits (β, γ, δ, and α), corresponding to the products of

hyhBGSL
Tk

, respectively. Each αβγδ unit contained 0.8 mol of Ni, 22.3 mol of Fe, 21.1 mol of acid-labile sulfide, and 1.01 mol of flavin adenine dinucleotide. The optimal temperature for the
T. kodakaraensis
hydrogenase was 95°C for H
2
uptake and 90°C for H
2
production with methyl viologen as the electron carrier. We found that NADP
+
and NADPH promoted high levels of uptake and evolution of H
2
, respectively, suggesting that the molecule is the electron carrier for the
T. kodakaraensis
hydrogenase.
DNA Microarray Analysis of Redox-Responsive Genes in the Genome of the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Hihara Yukako , Sonoike Kintake , Kanehisa Minoru , Ikeuchi Masahiko
Published: February 2003

Whole-genome DNA microarrays were used to evaluate the effect of the redox state of the photosynthetic electron transport chain on gene expression in
Synechocystis
sp. strain PCC 6803. Two specific inhibitors of electron transport, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-
p
-benzoquinone (DBMIB), were added to the cultures, and changes in accumulation of transcripts were examined. About 140 genes were highlighted as reproducibly affected by the change in the redox state of the photosynthetic electron transport chain. It was shown that some stress-responsive genes but not photosynthetic genes were under the control of the redox state of the plastoquinone pool in
Synechocystis
sp. strain PCC 6803.
tRNA
2
Thr
Complements Temperature Sensitivity Caused by Null Mutations in the
htrB
Gene in
Escherichia coli
Authors: Mohri Yoshio , Goto Simon , Nakahigashi Kenji , Inokuchi Hachiro
Published: February 2003

According to the wobble rule, tRNA2Thr is nonessential for protein synthesis, because the codon (ACG) that is recognized by tRNA2Thr is also recognized by tRNA4Thr. In order to investigate the reason that this nonessential tRNA nevertheless exists in
Escherichia coli
, we attempted to isolate tRNA2Thr-requiring mutants. Using strain JM101F
−
, which lacks the gene for tRNA2Thr, we succeeded in isolating two temperature-sensitive mutants whose temperature sensitivity was complemented by introduction of the gene for tRNA2Thr. These mutants had a mutation in the
htrB
gene, whose product is an enzyme involved in lipid A biosynthesis. Although it is known that some null mutations in the
htrB
gene give a temperature-sensitive phenotype, our mutants exhibited tighter temperature sensitivity. We discuss a possible mechanism for the requirement for tRNA2Thr.
Mechanism of
cis-trans
Isomerization of Unsaturated Fatty Acids in
Pseudomonas putida
Authors: von Wallbrunn Angelika , Richnow Hans Hermann , Neumann Grit , Meinhardt Friedhelm , Heipieper Hermann J.
Published: February 2003

We studied the pattern of the
cis
-
trans
isomerization of unsaturated fatty acids in cells of
Pseudomonas putida
S12 grown in a medium supplemented with oleic acid which was deuterated at both of the C atoms of its double bond. Direct evidence that isomerization does not include a transient saturation of the double bond was obtained. In addition, analysis of the amino acid sequences of the seven known Cti proteins identified them as heme-containing proteins of the cytochrome
c
type.
A Suppressor of the Menadione-Hypersensitive Phenotype of a
Xanthomonas campestris
pv. phaseoli
oxyR
Mutant Reveals a Novel Mechanism of Toxicity and the Protective Role of Alkyl Hydroperoxide Reductase
Authors: Vattanaviboon Paiboon , Whangsuk Wirongrong , Mongkolsuk Skorn
Published: February 2003

We isolated menadione-resistant mutants of
Xanthomonas campestris
pv. phaseoli
oxyR
(

oxyR
Xp

). The
oxyR
R2

Xp

mutant was hyperresistant to the superoxide generators menadione and plumbagin and was moderately resistant to H
2
O
2
and
tert
-butyl hydroperoxide. Analysis of enzymes involved in oxidative-stress protection in the
oxyR
R2

Xp

mutant revealed a >10-fold increase in AhpC and AhpF levels, while the levels of superoxide dismutase (SOD), catalase, and the organic hydroperoxide resistance protein (Ohr) were not significantly altered. Inactivation of
ahpC
in the
oxyR
R2

Xp

mutant resulted in increased sensitivity to menadione killing. Moreover, high levels of expression of cloned
ahpC
and
ahpF
in the

oxyR
Xp

mutant complemented the menadione hypersensitivity phenotype. High levels of other oxidant-scavenging enzymes such as catalase and SOD did not protect the cells from menadione toxicity. These data strongly suggest that the toxicity of superoxide generators could be mediated via organic peroxide production and that alkyl hydroperoxide reductase has an important novel function in the protection against the toxicity of these compounds in
X. campestris.
Hemin Binding, Functional Expression, and Complementation Analysis of Pap 31 fromBartonella henselae
Authors: Zimmermann Rainer , Kempf Volkhard A. J. , Schiltz Emile , Oberle Karin , Sander Anna
Published: February 2003

Growth ofBartonella henselaeis strongly heme dependent, andB. henselaeis unable to synthesize heme itself. At least five outer membrane-associated proteins fromB. henselaebind hemin, including the 31-kDa protein designated Pap31. The gene of this protein was heterologously expressed inEscherichia coliM15(pREP4) and detected with monoclonal antibodies in the outer membrane fraction. Complementation of thehemA-deficient mutantE. coliK-12 EB53 (aroB tsx malT hemA) withpap31demonstrated that this protein is involved in heme acquisition and may be an important virulence factor in the pathogenesis ofB. henselae.
Transposition of Tn
5367
in
Mycobacterium marinum
, Using a Conditionally Recombinant Mycobacteriophage
Authors: Rybniker Jan , Wolke Martina , Haefs Christiane , Plum Georg
Published: February 2003

Mycobacterium marinum
is a close relative of the obligate human pathogen
Mycobacterium tuberculosis
. As with
M. tuberculosis
,
M. marinum
causes intracellular infection of poikilothermic vertebrates and skin infection in humans. It is considered a valid model organism for the study of intracellular pathogenesis of mycobacteria. Low transformation efficiencies for this species have precluded approaches using mutant libraries in pathogenesis studies. We have adapted the conditionally replicating mycobacteriophage phAE94, originally developed as a transposon mutagenesis tool for
M. tuberculosis
, to meet the specific requirements of
M. marinum
. Conditions permissive for phage replication in
M. tuberculosis
facilitated highly efficient transposon delivery in
M. marinum
. Using this technique we succeeded in generating a representative mutant library of this species, and we conclude that TM4-derived mycobacteriophages are temperature-independent suicide vectors for
M. marinum
.
Molecular Cloning of Endo-β-
d
-1,4-Glucanase Genes,
rce1
,
rce2
, and
rce3
, from
Rhizopus oryzae
Authors: Moriya Tatsuki , Murashima Koichiro , Nakane Akitaka , Yanai Koji , Sumida Naomi , Koga Jinichiro , Murakami Takeshi , Kono Toshiaki
Published: February 2003

Three endoglucanase genes, designated the
rce1
,
rce2
, and
rce3
genes, were isolated from
Rhizopus oryzae
as the first cellulase genes from the subdivision
Zygomycota
. All the amino acid sequences deduced from the
rce1
,
rce2
, and
rce3
genes consisted of three distinct domains: cellulose binding domains, linker domains, and catalytic domains belonging to glycosyl hydrolase family 45. The
rce3
gene had two tandem repeated sequences of cellulose binding domains, while
rce1
and
rce2
had only one.
rce1
,
rce2
, and
rce3
had various lengths of linker sequences.
In-vehicle communication systems: the safety aspect
Authors: Pauzié A
Published: February 2003

Communication and information technology are developing very rapidly at present. At the same time, the number of older drivers is increasing. When designing systems for elderly drivers, it has been shown that: (1) simplifying a task reduces performance differences between old and young; and (2) the optimization of onboard systems (better legibility and intelligibility of the information, simplified dialogue) in relation to the abilities of elderly drivers benefits the rest of the user population. Elderly people do not automatically reject new information and assistance technologies especially when the systems are user friendly. However, the ergonomics of these new technologies must be studied, with particular attention to the specific needs of the elderly, in order not to marginalize them
Strategies for recovery and regeneration after brain and spinal cord injury
Authors: Tator C H
Published: February 2003

Current knowledge of the epidemiology and pathophysiology of neurotrauma and the key clinical and experimental strategies for promoting recovery and regeneration after brain and spinal cord trauma are reviewed. Brief overviews of the epidemiology and pathophysiology of neurotrauma are presented, and the key experimental and clinical treatments for the promotion of recovery and regeneration after brain and spinal cord trauma are discussed.
The uninsured and Medicaid Oregon tobacco user experience in a real world, phone based cessation programme
Authors: El-Bastawissi Ay , McAfee T , Zbikowski S M , Hollis J , Stark M , Wassum K , Clark N , Barwinski R , Broughton E
Published: February 2003

Objective:To describe the experience of uninsured and Medicaid Oregon tobacco users who registered in Free & Clear (F&C), a telephone based cessation programme including five scheduled outbound calls.

Design and setting:Using a retrospective cohort design, 1334 (423 uninsured, 806 Medicaid, and 105 commercially insured) Oregon tobacco users who registered in F&C between 18 November 1998 and 28 February 2000 were identified and followed for 12 months post-registration; 648 (48.6%) were successfully contacted at 12 months. Information was collected from the F&C database. Unconditional logistic regression, adjusted for race and education, was used.

Results:The seven day quit rate at 12 months, assuming non-respondents were smokers, was 14.8% (95% confidence interval (CI) 13.0 to 16.9). This rate was significantly higher among commercially insured participants (vMedicaid but not uninsured) and among participants who completed ⩾ 5 calls (v< 5 calls). The quit rate for those contacted at 12 months was 30.6% (95% CI 27.0% to 34.3%) and varied, however not significantly, by insurance and number of calls. After adjustment, respondents who completed ⩾ 5 calls were 60% more likely to quit tobacco (odds ratio (OR) 1.6, 95% CI 0.9 to 3.1), and uninsured respondents who completed ⩾ 5 calls were 70% more likely to quit tobacco (OR 1.7, 95% CI 0.9 to 3.5), relative to those who completed < 5 calls, but the difference was not significant.

Conclusions:The quit rates are similar to those reported in efficacy trials. The observed variation in quitting tobacco for respondents by number of calls completed and by insurance merits further investigation concentrating on increasing compliance with the call schedule, particularly for the uninsured.
Can parents who smoke socialise their children against smoking? Results from the Smoke-free Kids intervention trial
Authors: Jackson C , Dickinson D
Published: February 2003

Objective:To evaluateSmoke-free Kids, a new home based programme to assist parents who smoke in socialising their children against smoking.

Design:Two year randomised controlled trial.

Participants:At baseline, 887 adult smokers who had an abstinent child in the third grade (ages 7–8 years); 671 adults and children were retained through the 24 month follow up.

Intervention:Programme modules, newsletters, incentives, support calls.

Outcomes:Anti-smoking socialisation; susceptibility to smoking.

Results:Of 327 parents randomised to treatment, 210 obtained adequate treatment by using at least three of five core modules. Programme efficacy analyses, which compared these parents with controls (n = 344), showed that exposure to adequate treatment predicted significantly higher levels in nearly all categories of anti-smoking socialisation three months post-intervention. Two years post-baseline, children of parents who reported adequate treatment scored significantly higher than controls on attributes that reduce susceptibility to smoking, and they scored significantly lower than controls on attributes that raise susceptibility to smoking. Programme effectiveness analyses compared all parents randomised to treatment (n = 327) with controls (n = 344). Treatment effects were evident for several socialisation outcomes; however, these effects were smaller and less consistent than those from the efficacy analyses. Similarly, although treated children scored higher than controls on attributes that reduce susceptibility and lower than controls on attributes that raise susceptibility, several of these between-group differences were not significant.

Conclusions:Given adequate exposure to theSmoke-free Kidsprogramme, significant beneficial effects were observed on anti-smoking socialisation in households where parents smoke cigarettes, and significant beneficial effects were observed on children’s susceptibility to smoking after two years. Improving programme acceptance and utilisation is necessary before programme effectiveness can be demonstrated.
Smoking mothers and snuffing fathers: behavioural influences on youth tobacco use in a Swedish cohort
Authors: Rosendahl K I , Galanti M R , Gilljam H , Ahlbom A
Published: February 2003

Objective:To analyse the influences of parental use of cigarettes and snus (the Swedish variety of smokeless tobacco) on offspring’s behaviour.

Design:Prospective cohort study.

Setting:The Stockholm County of Sweden.

Subjects:2232 adolescents recruited in the fifth grade (mean age 11.6 years) with follow up in the eighth grade.

Main outcome measures:Self reported tobacco use (ever and current use of cigarettes and/or snus) in the eighth grade.

Results:Parents’ tobacco use was associated with adolescents’ current use of cigarettes and snus (odds ratio (OR) 2.7, 95% confidence interval (CI) 1.8 to 3.9 if both parents used tobaccovneither parent). Mother’s cigarette smoking was associated with adolescents’ current exclusive smoking (OR 2.4, 95% CI 1.6 to 3.6). Father’s use of snus was associated with current exclusive use of snus among boys (OR 3.0, 95% CI 1.4 to 6.4), but not with current cigarette use. The overall prevalence of current smoking was lower among children whose fathers used snus than among those whose fathers smoked.

Conclusions:Parental smoking, especially maternal smoking, enhances the risk of tobacco experimentation in youths, as does paternal use of smokeless tobacco. However, the transition to regular cigarette smoking is not likely to be affected by paternal use of smokeless tobacco. Contextual factors, in particular declining smoking trends and negative social acceptance of smoking, can explain most of these findings.