bluuebunny/crossref_metadata_2025 huggingface dataset:
Gram-Positive and Gram-Negative Bacteria Do Not Trigger Monocytic Cytokine Production through Similar Intracellular Pathways
Authors: Rabehi Lila , Irinopoulou Théano , Cholley Béatrice , Haeffner-Cavaillon Nicole , Carreno Marie-Paule
Published: July 2002

Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) andStaphylococcus aureusCowan (SAC), suggesting that gram-positive and gram-negative bacteria may trigger similar intracellular events. Treatment with specific kinase inhibitors prior to cell stimulation dramatically decreased LPS-induced cytokine production. Blocking of the p38 pathway prior to LPS stimulation decreased interleukin-1α (IL-1α), IL-1ra, and tumor necrosis factor alpha (TNF-α) production, whereas blocking of the ERK1/2 pathways inhibited IL-1α, IL-1β, and IL-1ra but not TNF-α production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1α production was decreased in the presence of ERK kinase inhibitor. We also demonstrated that although LPS and SAC have been shown to bind to CD14 before transmitting signals to TLR4 and TLR2, respectively, internalization of CD14 occurred only in monocytes triggered by LPS. Pretreatment of the cells with SB203580, U0126, or a mixture of both inhibitors did not affect internalization of CD14. Altogether, these results suggest that TLR2 signaling does not involve p38 mitogen-activated protein kinase signaling pathways, indicating that divergent pathways are triggered by gram-positive and gram-negative bacteria, thereby inducing cytokine production.
Rgg Influences the Expression of Multiple Regulatory Loci To Coregulate Virulence Factor Expression in
Streptococcus pyogenes
Authors: Chaussee Michael S. , Sylva Gail L. , Sturdevant Daniel E. , Smoot Laura M. , Graham Morag R. , Watson Robert O. , Musser James M.
Published: July 2002

The human pathogen
Streptococcus pyogenes
secretes many proteins to the cell wall and extracellular environment that contribute to virulence. Rgg regulates the expression of several exoproteins including a cysteine protease (SPE B), a nuclease (MF-1), a putative nuclease (MF-3), and autolysin. The functional heterogeneity of Rgg-regulated exoproteins and the lack of a conserved regulatory motif in the promoter regions of the genes suggested that Rgg interacts with additional regulatory networks to influence gene expression. DNA microarrays were used to test this hypothesis by comparing genomewide transcript profiles of
S. pyogenes
NZ131 and isogenic derivative NZ131
rgg
during the exponential phase of growth. Transcripts of known and putative virulence-associated genes were more abundant in the
rgg
mutant, including
emm
,
scpA
,
orfX
,
scl1
,
hasAB
,
slo
,
sagA
,
ska
,
speH
,
grab
,
mac
,
mf-1
, and
mf-3
. Increased transcription of
emm
,
scpA
, and
orfX
in the
rgg
mutant was associated with increased production of the corresponding proteins. Differences in the expression of virulence-associated genes were associated with changes in the expression of several regulatory genes, including
mga
,
sagA
,
csrRS
, and
fasBCA
. The results show that Rgg influences the expression of multiple regulatory networks to coregulate virulence factor expression in
S. pyogenes
.
Binding Component of
Clostridium perfringens
Iota-Toxin Induces Endocytosis in Vero Cells
Authors: Nagahama Masahiro , Nagayasu Koichi , Kobayashi Keiko , Sakurai Jun
Published: July 2002

Clostridium perfringens
iota-toxin is a binary toxin consisting of two individual proteins, the binding component (Ib) and the enzyme component (Ia). Wild-type Ib bound to Vero cells at 4 and 37°C and formed oligomers at 37°C but not at 4°C. The Ib-induced K
+
release from the cells was dependent on the oligomer formation of Ib in the cells, but the oligomer formation did not induce rounding activity or cytotoxicity. After incubation of the cells with recombinant Ib (rIb) at 37°C, the Ib oligomer in the cell became resistant to pronase treatment with time, but the Ib monomer was sensitive to the treatment. Furthermore, treatment of Vero cells with rIb in the presence of bafilomycin, methylamine, or ethylamine resulted in accumulation of the oligomer in the cells but had no effect on K
+
release. Moreover, incubation with Ib plus Ia in the presence of these agents caused no rounding in the cells. These observations suggest that Ib binds to Vero cells, itself oligomerizing to form ion-permeable channels and that the formation of oligomer then induces endocytosis.
Intimin Facilitates Colonization byEscherichia coliO157:H7 in Adult Ruminants
Authors: Cornick Nancy A. , Booher Sheridan L. , Moon Harley W.
Published: July 2002

We compared the magnitude and duration of fecal shedding of wild-typeEscherichia coliO157:H7 to that of an isogenic intimin mutant in young adult cattle and sheep. In both ruminant species, wild-typeE. coliO157:H7 was shed in greater numbers and for a longer duration than was the intimin mutant.
Essential Role for Cyclic AMP and Its Receptor Protein in
Yersinia enterocolitica
Virulence
Authors: Petersen Shane , Young Glenn M.
Published: July 2002

Insertion mutations were isolated in
cya
and
crp
of
Yersinia enterocolitica
, which encode adenylate cyclase and the cyclic AMP (cAMP) receptor protein (CRP). The
cya
and
crp
mutants were affected for the production of proteins exported by the Ysc, Ysa, and flagellar type III secretion systems (TTSS). Protein production by each TTSS was restored when the respective mutation was complemented by a plasmid-encoded copy of the wild-type gene. Both
cya
and
crp
mutants exhibited reduced virulence for orally infected BALB/c mice in a 50% lethal dose analysis. Examination of bacterial survival in host tissues showed that
cya
and
crp
mutants colonized Peyer's patches and, to a lesser extent, mesenteric lymph nodes. However, the mutants did not appear to disseminate to the liver and spleen of infected mice. An initial examination of the effectiveness of
Y. enterocolitica cya
and
crp
mutants to stimulate protective immunity against subsequent challenge with virulent bacteria in mice was promising. The results indicate that the cAMP-CRP regulatory system is required for
Y. enterocolitica
virulence.
Molecular Cloning of a Vaccine Antigen against Infection with the Larval Stage of
Echinococcus multilocularis
Authors: Gauci Charles , Merli Michael , Muller Volker , Chow Conan , Yagi Kinpei , Mackenstedt Ute , Lightowlers Marshall W.
Published: July 2002

Alveolar and cystic hydatidosis are caused by infection with the larval stages of
Echinococcus multilocularis
and
Echinococcus granulosus
, respectively. A host-protective antigen has been identified in
E. granulosus
. Here we identify the presence of a closely related protein
in E. multilocularis
, characterize and express a cDNA encoding the antigen (designated EM95), determine the structure of the
em
95 gene, and demonstrate that the EM95 recombinant protein can be used to induce significant levels of protection against challenge infection with
E. multilocularis
eggs in mice.
Regulation of Acetyl Coenzyme A Synthetase in
Escherichia coli
Authors: Kumari Suman , Beatty Christine M. , Browning Douglas F. , Busby Stephen J. W. , Simel Erica J. , Hovel-Miner Galadriel , Wolfe Alan J.
Published: July 2002

Cells of
Escherichia coli
growing on sugars that result in catabolite repression or amino acids that feed into glycolysis undergo a metabolic switch associated with the production and utilization of acetate. As they divide exponentially, these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. Here, we present evidence that this switch occurs primarily through the induction of
acs
and that the timing and magnitude of this induction depend, in part, on the direct action of the carbon regulator cyclic AMP receptor protein (CRP) and the oxygen regulator FNR. It also depends, probably indirectly, upon the glyoxylate shunt repressor IclR, its activator FadR, and many enzymes involved in acetate metabolism. On the basis of these results, we propose that cells induce
acs
, and thus their ability to assimilate acetate, in response to rising cyclic AMP levels, falling oxygen partial pressure, and the flux of carbon through acetate-associated pathways.
Role of FliJ in Flagellar Protein Export in
Salmonella
Authors: Minamino Tohru , Chu Ryan , Yamaguchi Shigeru , Macnab Robert M.
Published: July 2002

We isolated and characterized spontaneous mutants with defects in the 147-amino-acid
Salmonella
protein FliJ, which is a cytoplasmic component of the type III flagellar export apparatus. These mutants, including ones with null mutations, have the ability to form swarms on motility agar plates after prolonged incubation at 30°C; i.e., they display a leaky motile phenotype. One mutant, SJW277, which formed significantly bigger swarms than the others, encoded only the N-terminal 73 amino acids of FliJ, one-half of the protein. At 30°C, overproduction of this mutant protein improved, to wild-type levels, both motility and the ability to export both rod/hook-type (FlgD; hook capping protein) and filament-type (FliC; flagellin) substrates. At 42°C, however, export was inhibited, indicating that the mutant FliJ protein was temperature sensitive. Taking advantage of this, we performed temperature upshift experiments, which demonstrated that FliJ is directly required for the export of FliC. Co-overproduction of FliJ and either of two export substrates, FliE or FlgG, hindered their aggregation in the cytoplasm. We conclude that FliJ is a general component of the flagellar export apparatus and has a chaperone-like activity for both rod/hook-type and filament-type substrates.
Developmental Control of Stress Stimulons in
Streptomyces coelicolor
Revealed by Statistical Analyses of Global Gene Expression Patterns
Authors: Vohradsky J. , Li X.-M. , Dale G. , Folcher M. , Nguyen L. , Viollier P. H. , Thompson C. J.
Published: July 2002

Stress-induced regulatory networks coordinated with a procaryotic developmental program were revealed by two-dimensional gel analyses of global gene expression. Four developmental stages were identified by their distinctive protein synthesis patterns using principal component analysis. Statistical analyses focused on five stress stimulons (induced by heat, cold, salt, ethanol, or antibiotic shock) and their synthesis during development. Unlike other bacteria, for which various stresses induce expression of similar sets of protein spots, in
Streptomyces coelicolor
heat, salt, and ethanol stimulons were composed of independent sets of proteins. This suggested independent control by different physiological stress signals and their corresponding regulatory systems. These stress proteins were also under developmental control. Cluster analysis of stress protein synthesis profiles identified 10 different developmental patterns or “synexpression groups.” Proteins induced by cold, heat, or salt shock were enriched in three developmental synexpression groups. In addition, certain proteins belonging to the heat and salt shock stimulons were coregulated during development. Thus, stress regulatory systems controlling these stimulons were implicated as integral parts of the developmental program. This correlation suggested that thermal shock and salt shock stress response regulatory systems either allow the cell to adapt to stresses associated with development or directly control the developmental program.
Multiprobe RNase Protection Assay Analysis of mRNA Levels for the
Escherichia coli
Oxidative DNA Glycosylase Genes under Conditions of Oxidative Stress
Authors: Gifford Christine M. , Blaisdell Jeffrey O. , Wallace Susan S.
Published: July 2002

Escherichia coli
formamidopyrimidine DNA glycosylase (Fpg), MutY DNA glycosylase, endonuclease VIII, and endonuclease III are oxidative base excision repair DNA glycosylases that remove oxidized bases from DNA, or an incorrect base paired with an oxidized base in the case of MutY. Since genes encoding other base excision repair proteins have been shown to be part of adaptive responses in
E. coli
, we wanted to determine whether the oxidative DNA glycosylase genes are induced in response to conditions that cause the type of damage their encoded proteins remove. The genes
fpg
,
mutY
,
nei
, and
nth
encode Fpg, MutY, endonuclease VIII, and endonuclease III, respectively. Multiprobe RNase protection assays were used to examine the transcript levels of these genes under conditions that induce the SoxRS, OxyR, and SOS regulons after a shift from anaerobic to aerobic growth and at different stages along the growth curve. Transcript levels for all four genes decreased as cells progressed from log-phase growth to stationary phase and increased after cells were shifted from anaerobic to aerobic growth. None of the genes were induced by hydrogen peroxide, paraquat, X rays, or conditions that induce the SOS response.
Mutational Analysis of a Role for Salicylic Acid in Iron Metabolism of
Mycobacterium smegmatis
Authors: Adilakshmi Tadepalli , Ayling Peter D. , Ratledge Colin
Published: July 2002

The role of salicylic acid in iron metabolism was examined in two wild-type strains (mc
2
155 and NCIMB 8548) and three mutant strains (mc
2
1292 [lacking exochelin], SM3 [lacking iron-dependent repressor protein IdeR] and S99 [a salicylate-requiring auxotroph derived in this study]) of
Mycobacterium smegmatis
. Synthesis of salicylate in SM3 was derepressed even in the presence of iron, as was synthesis of the siderophores exochelin, mycobactin, and carboxymycobactin. S99 was dependent on salicylate for growth and failed to grow with the three ferrisiderophores, suggesting that salicylate fulfills an additional function(s) other than being a precursor of mycobactin and carboxymycobactin. Salicylic acid at 100 μg/ml repressed the formation of a 29-kDa cell envelope protein (putative exochelin receptor protein) in S99 grown both iron deficiently and iron sufficiently. In contrast, synthesis of this protein was affected only under iron-limited conditions in the parent strain, mc
2
155, and remained unaltered in SM3, suggesting an interaction between the IdeR protein and salicylate. Thus, salicylate may also function as a signal molecule for recognition of cellular iron status. Growth of all strains and mutants with
p
-aminosalicylate (PAS) at 100 μg/ml increased salicylate accumulation between three- and eightfold under both iron-limited and iron-sufficient growth conditions and decreased mycobactin accumulation by 40 to 80% but increased carboxymycobactin accumulation by 50 to 55%. Thus, although PAS inhibited salicylate conversion to mycobactin, presumptively by blocking salicylate AMP kinase, PAS also interferes with the additional functions of salicylate, as its effect was heightened in S99 when the salicylate concentration was minimal.
ssgA
Is Essential for Sporulation of
Streptomyces coelicolor
A3(2) and Affects Hyphal Development by Stimulating Septum Formation
Authors: van Wezel Gilles P. , van der Meulen Jannes , Kawamoto Shinichi , Luiten Ruud G. M. , Koerten Henk K. , Kraal Barend
Published: July 2002

The role of
ssgA
in cell division and development of streptomycetes was analyzed. An
ssgA
null mutant of
Streptomyces coelicolor
produced aerial hyphae but failed to sporulate, and
ssgA
can therefore be regarded as a novel
whi
gene. In addition to the morphological changes, antibiotic production was also disturbed, with strongly reduced actinorhodin production. These defects could be complemented by plasmid-borne
ssgA
. In the wild-type strain, transcription of
ssgA
was induced by nutritional shift-down and was shown to be linked to that of the upstream-located gene
ssgR
, which belongs to the family of
iclR
-type transcriptional regulator genes. Analysis of mycelium harvested from liquid-grown cultures by transmission electron microscopy showed that septum formation had strongly increased in
ssgA
-overexpressing strains in comparison to wild-type
S. coelicolor
and that spore-like compartments were produced at high frequency. Furthermore, the hyphae were significantly wider and contained irregular and often extremely thick septa. These data underline the important role for
ssgA
in
Streptomyces
cell division.
Null Mutation of thedamorseqAGene Suppresses Temperature-Sensitive Lethality but Not Hypersensitivity to Novobiocin ofmukNull Mutants
Authors: Onogi Toshinari , Yamazoe Mitsuyoshi , Ichinose Chiyome , Niki Hironori , Hiraga Sota
Published: July 2002

Escherichia coli mukF, mukE, andmukBnull mutants have common phenotypes such as temperature-dependent colony formation, anucleate cell production, chromosome cutting by septum closure, and abnormal localization of SeqA-DNA clusters. We show here that the associatedmuknull mutations cause hypersensitivity to novobiocin. Null mutation of eitherdamorseqAsuppressed partially the temperature-sensitive lethality but failed to suppress the anucleate cell production and the hypersensitivity to novobiocin caused bymuknull mutations.
Identification by Genetic Suppression of
Escherichia coli
TolB Residues Important for TolB-Pal Interaction
Authors: Ray Marie-Céline , Germon Pierre , Vianney Anne , Portalier Raymond , Lazzaroni Jean Claude
Published: July 2002

The Tol-Pal system of
Escherichia coli
is involved in maintaining outer membrane stability. Mutations in
tolQ
,
tolR
,
tolA
,
tolB
, or
pal
genes result in sensitivity to bile salts and the leakage of periplasmic proteins. Moreover, some of the
tol
genes are necessary for the entry of group A colicins and the DNA of filamentous bacteriophages. TolQ, TolR, and TolA are located in the cytoplasmic membrane where they interact with each other via their transmembrane domains. TolB and Pal form a periplasmic complex near the outer membrane. We used suppressor genetics to identify the regions important for the interaction between TolB and Pal. Intragenic suppressor mutations were characterized in a domain of Pal that was shown to be involved in interactions with TolB and peptidoglycan. Extragenic suppressor mutations were located in
tolB
gene. The C-terminal region of TolB predicted to adopt a β-propeller structure was shown to be responsible for the interaction of the protein with Pal. Unexpectedly, none of the suppressor mutations was able to restore a correct association between Pal and peptidoglycan, suggesting that interactions between Pal and other components such as TolB may also be important for outer membrane stability.
Fragmentation of 23S rRNA in Strains of
Proteus
and
Providencia
Results from Intervening Sequences in the
rrn
(rRNA) Genes
Authors: Miller Wayne L. , Pabbaraju Kanti , Sanderson Kenneth E.
Published: July 2002

Intervening sequences (IVSs) were originally identified in the
rrl
genes for 23S rRNA (
rrl
genes, for large ribosomal subunit, part of
rrn
operon encoding rRNA) of
Salmonella enterica
serovars Typhimurium LT2 and Arizonae. These sequences are transcribed but later removed during RNase III processing of the rRNA, resulting in fragmentation of the 23S species; IVSs are uncommon, but have been reported in at least 10 bacterial genera. Through PCR amplification of IVS-containing regions of the
rrl
genes we showed that most
Proteus
and
Providencia
strains contain IVSs similar to those of serovar Typhimurium in distribution and location in
rrl
genes. By extraction and Northern blotting of rRNA, we also found that these IVSs result in rRNA fragmentation. We report the first finding of two very different sizes of IVS (113 bp and 183 to 187 bp) in different
rrl
genes in the same strain, in helix 25 of
Proteus
and
Providencia
spp.; IVSs from helix 45 are 113 to 123 bp in size. Analysis of IVS sequence and postulated secondary structure reveals striking similarities of
Proteus
and
Providencia
IVSs to those of serovar Typhimurium, with the stems of the smaller IVSs from helix 25 being similar to those of
Salmonella
helix 25 IVSs and with both the stem and the central loop domain of helix 45 IVSs being similar. Thus, IVSs of related sequences are widely distributed throughout the
Enterobacteriaceae
, in
Salmonella
,
Yersinia
,
Proteus
, and
Providencia
spp., but we did not find them in
Escherichia coli
,
Citrobacter
,
Enterobacter
,
Klebsiella
, or
Morganella
spp.; the sporadic distribution of IVSs of related sequence indicates that lateral genetic transfer has occurred.
Respiration of 2,4,6-Trinitrotoluene by
Pseudomonas
sp. Strain JLR11
Authors: Esteve-Nuñez Abraham , Lucchesi Gloria , Philipp Bodo , Schink Bernhard , Ramos Juan L.
Published: July 2002

Under anoxic conditions
Pseudomonas
sp. strain JLR11 can use 2,4,6-trinitrotoluene (TNT) as the sole N source, releasing nitrite from the aromatic ring and subsequently reducing it to ammonium and incorporating it into C skeletons. This study shows that TNT can also be used as a terminal electron acceptor in respiratory chains under anoxic conditions by
Pseudomonas
sp. strain JLR11. TNT-dependent proton translocation coupled to the reduction of TNT to aminonitrotoluenes has been observed in TNT-grown cells. This extrusion did not occur in nitrate-grown cells or in anaerobic TNT-grown cells treated with cyanide, a respiratory chain inhibitor. We have shown that in a membrane fraction prepared from
Pseudomonas
sp. strain JLR11 grown on TNT under anaerobic conditions, the synthesis of ATP was coupled to the oxidation of molecular hydrogen and to the reduction of TNT. This phosphorylation was uncoupled by gramicidin. Respiration by
Pseudomonas
sp. strain JLR11 is potentially useful for the biotreatment of TNT in polluted waters and soils, particularly in phytorhizoremediation, in which bacterial cells are transported to the deepest root zones, which are poor in oxygen.
A Novel Phenanthrene Dioxygenase from
Nocardioides
sp. Strain KP7: Expression in
Escherichia coli
Authors: Saito Atsushi , Iwabuchi Tokuro , Harayama Shigeaki
Published: July 2002

Nocardioides
sp. strain KP7 grows on phenanthrene but not on naphthalene. This organism degrades phenanthrene via 1-hydroxy-2-naphthoate,
o
-phthalate, and protocatechuate. The genes responsible for the degradation of phenanthrene to
o
-phthalate (
phd
) were found by Southern hybridization to reside on the chromosome. A 10.6-kb DNA fragment containing eight
phd
genes was cloned and sequenced. The
phdA
,
phdB
,
phdC
, and
phdD
genes, which encode the α and β subunits of the oxygenase component, a ferredoxin, and a ferredoxin reductase, respectively, of phenanthrene dioxygenase were identified. The gene cluster,
phdAB
, was located 8.3 kb downstream of the previously characterized
phdK
gene, which encodes 2-carboxybenzaldehyde dehydrogenase. The
phdCD
gene cluster was located 2.9 kb downstream of the
phdB
gene. PhdA and PhdB exhibited moderate (less than 60%) sequence identity to the α and β subunits of other ring-hydroxylating dioxygenases. The PhdC sequence showed features of a [3Fe-4S] or [4Fe-4S] type of ferredoxin, not of the [2Fe-2S] type of ferredoxin that has been found in most of the reported ring-hydroxylating dioxygenases. PhdD also showed moderate (less than 40%) sequence identity to known reductases. The
phdABCD
genes were expressed poorly in
Escherichia coli
, even when placed under the control of strong promoters. The introduction of a Shine-Dalgarno sequence upstream of each initiation codon of the
phdABCD
genes improved their expression in
E. coli. E. coli
cells carrying
phdBCD
or
phdACD
exhibited no phenanthrene-degrading activity, and those carrying
phdABD
or
phdABC
exhibited phenanthrene-degrading activity which was significantly less than that in cells carrying the
phdABCD
genes. It was thus concluded that all of the
phdABCD
genes are necessary for the efficient expression of phenanthrene-degrading activity. The genetic organization of the
phd
genes, the phylogenetically diverged positions of these genes, and an unusual type of ferredoxin component suggest phenanthrene dioxygenase in
Nocardioides
sp. strain KP7 to be a new class of aromatic ring-hydroxylating dioxygenases.
Multiple Hexose Transporters ofSchizosaccharomyces pombe
Authors: Heiland Sylvia , Radovanovic Nada , Höfer Milan , Winderickx Joris , Lichtenberg Hella
Published: July 2002

We have identified a family of six hexose transporter genes (Ght1toGht6) in the fission yeastSchizosaccharomyces pombe. Sequence homology toSaccharomyces cerevisiaeand mammalian hexose transporters (Hxtp and GLUTp, respectively) and secondary-structure predictions of 12 transmembrane domains for each of the Ght proteins place them into the sugar porter subfamily within the major facilitator superfamily. Interestingly, among this sugar porter family, the emergingS. pombehexose transporter family clusters are separate from monosaccharide transporters of other yeasts (S. cerevisiae,Kluyveromyces lactis, andCandida albicans) and of humans, suggesting that these proteins form a distinct structural family of hexose transporters. Expression of theGht1,Ght2,Ght5, andGht6genes in theS. cerevisiaemutant RE700A may functionally complement itsd-glucose uptake-deficient phenotype. Northern blot analysis and reverse transcription-PCR showed that among allGht's ofS. pombe,Ght5is the most prominently expressed hexose transporter. Ght1p, Ght2p, and Ght5p displayed significantly higher specificities ford-glucose than ford-fructose. Analysis of the previously describedS. pombed-glucose transport-deficient mutant YGS-5 revealed that this strain is defective in theGht1,Ght5, andGht6genes. Based on an analysis of threeS. pombestrains bearing single or double mutations inGht3andGht4, we conclude that the Ght3p function is required ford-gluconate transport inS. pombe. The function of Ght4p remains to be clarified. Ght6p exhibited a slightly higher affinity tod-fructose than tod-glucose, and among the Ght's it is the transporter with the highest specificity ford-fructose.
Recombinational Repair Is Critical for Survival of
Escherichia coli
Exposed to Nitric Oxide
Authors: Spek Erik J. , Wright Teresa L. , Stitt Molly S. , Taghizadeh Nazbeh R. , Tannenbaum Steven R. , Marinus Martin G. , Engelward Bevin P.
Published: July 2002

Nitric oxide (NO
⋅
) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO
⋅
-induced DNA damage on
Escherichia coli
. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO
⋅
-induced toxicity was determined.
E. coli
strains lacking either NER or DNA glycosylases (including those that repair alkylation damage [
alkA tag
strain], oxidative damage [
fpg nei nth
strain], and deaminated cytosine [
ung
strain]) showed essentially wild-type levels of NO
⋅
resistance. However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (
xth nfo
strain) were very sensitive to killing by NO
⋅
, which indicates that normal processing of abasic sites is critical for defense against NO
⋅
. In addition,
recA
mutant cells were exquisitely sensitive to NO
⋅
-induced killing. Both SOS-deficient (
lexA3
) and Holliday junction resolvase-deficient (
ruvC
) cells were very sensitive to NO
⋅
, indicating that both SOS and recombinational repair play important roles in defense against NO
⋅
. Furthermore, strains specifically lacking double-strand end repair (
recBCD
strains) were very sensitive to NO
⋅
, which suggests that NO
⋅
exposure leads to the formation of double-strand ends. One consequence of these double-strand ends is that NO
⋅
induces homologous recombination at a genetically engineered substrate. Taken together, it is now clear that, in addition to the known point mutagenic effects of NO
⋅
, it is also important to consider recombination events among the spectrum of genetic changes that NO
⋅
can induce. Furthermore, the importance of recombinational repair for cellular survival of NO
⋅
exposure reveals a potential susceptibility factor for invading microbes.
Glucose-Induced Monoubiquitination of the
Saccharomyces cerevisiae
Galactose Transporter Is Sufficient To Signal Its Internalization
Authors: Horak Jaroslav , Wolf Dieter H.
Published: July 2002

In
Saccharomyces cerevisiae
, the addition of glucose to cells growing on galactose induces internalization of the galactose transporter Gal2p and its subsequent proteolysis in the vacuole. Here we report that the essential step in Gal2p down-regulation is its ubiquitination through the Ubc1p-Ubc4p-Ubc5p triad of ubiquitin-conjugating enzymes and Npi1/Rsp5p ubiquitin-protein ligase. Moreover, Gal2p appears to be stabilized in mutant cells defective in the ubiquitin-hydrolase Npi2p/Doa4p, and the mutant phenotype can be reversed by overexpression of ubiquitin. An analysis of the fate of Gal2p in cells overexpressing wild-type ubiquitin as well as its variants incompetent to form polyubiquitin chains showed that monoubiquitination of Gal2p is sufficient to signal internalization of the protein into the endocytic pathway.
Tagging Morphogenetic Genes by Insertional Mutagenesis in the Yeast
Yarrowia lipolytica
Authors: Richard Mathias , Quijano Raymundo Rosas , Bezzate Samira , Bordon-Pallier Florence , Gaillardin Claude
Published: July 2002

The yeast
Yarrowia lipolytica
is distantly related to
Saccharomyces cerevisiae
, can be genetically modified, and can grow in both haploid and diploid states in either yeast, pseudomycelial, or mycelial forms, depending on environmental conditions. Previous results have indicated that the
STE
and
RIM
pathways, which mediate cellular switching in other dimorphic yeasts, are not required for
Y. lipolytica
morphogenesis. To identify the pathways involved in morphogenesis, we mutagenized a wild-type strain of
Y. lipolytica
with a Tn
3
derivative. We isolated eight tagged mutants, entirely defective in hyphal formation, from a total of 40,000 mutants and identified seven genes homologous to
S. cerevisiae CDC25, RAS2, BUD6, KEX2, GPI7, SNF5
, and
PPH21
. We analyzed their abilities to invade agar and to form pseudomycelium or hyphae under inducing conditions and their sensitivity to temperature and to Calcofluor white. Chitin staining was used to detect defects in their cell walls. Our results indicate that a functional Ras-cyclic AMP pathway is required for the formation of hyphae in
Y. lipolytica
and that perturbations in the processing of extracellular, possibly parietal, proteins result in morphogenetic defects.
Transcriptional Pattern of Genes Coding for the Proteolytic System of
Lactococcus lactis
and Evidence for Coordinated Regulation of Key Enzymes by Peptide Supply
Authors: Guédon Eric , Renault Pierre , Ehrlich S. Dusko , Delorme Christine
Published: July 2002

The transcription of 16 genes encoding 12 peptidases (
pepC, pepN, pepX, pepP, pepA, pepF2, pepDA1, pepDA2, pepQ, pepT, pepM
, and
pepO1
), P
I
and P
III
proteinases (
prtP1
and
prtP3
), and three transport systems (
dtpT, dtpP,
and
opp-pepO1
) of
Lactococcus lactis
MG1363 was analyzed in response to different environmental factors. Promoter fusions with luciferase reporter genes and/or mRNA analysis were used to study the effects of sugar sources, growth at 37°C, and peptide supply on the transcription of these genes. Only transcription of the
pepP
gene is modulated by the source of sugar. The presence of potential catabolite-responsive element (CRE) boxes in its promoter region suggests that expression of this gene is directly controlled by catabolic repression. Elevated temperature had no significant effect on the level of transcription of these genes.
prtP1, prtP3, pepC, pepN, pepX,
and the
opp-pepO1
operon are the most highly expressed genes in chemically defined medium, and their expression is repressed 5- to 150-fold by addition of peptide sources such as Casitone in the medium. Moreover, the transcription of
prtP1, prtP3, pepC, pepN
, and the
opp-pepO1
operon is repressed two- to eight-fold by the dipeptides leucylproline and prolylleucine. The transcription of
pepDA2
might also be repressed by the peptide sources, but this effect is not observed on the regulation of
dtpT, pepP, pepA, pepF2, pepDA1, pepQ, pepT, pepM
, and the
dtpP
operon. The significance of these results with respect to the functions of different components of the proteolytic system in
L. lactis
are discussed.
Plasmid-Encoded Phthalate Catabolic Pathway in
Arthrobacter keyseri
12B
Authors: Eaton Richard W.
Published: July 2002

Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown
Arthrobacter keyseri
(formerly
Micrococcus
sp.) 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates). Because these products lack a carboxyl group at the 2 position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated. When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates. This chromogenic response was subsequently used to identify recombinant
Escherichia coli
strains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130-kbp plasmid pRE1 of strain 12B. Beginning with the initially cloned 8.14-kbp
Pst
I fragment of pRE824 as a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases. From these plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units:
tnpR, pcm
operon,
ptr
genes,
pehA, norA
fragment, and
pht
operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively. Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant
E. coli
strains.
Role of a Ferredoxin Gene Cotranscribed with the
nifHDK
Operon in N
2
Fixation and Nitrogenase “Switch-Off” of
Azoarcus
sp. Strain BH72
Authors: Egener Tanja , Martin Dietmar E. , Sarkar Abhijit , Reinhold-Hurek Barbara
Published: July 2002

The endophytic diazotroph
Azoarcus
sp. strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (
nif
) genes there. In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (
nifHDK
) were cloned and sequenced. The sequence analysis revealed an unusual gene organization: downstream of
nifHDK
, a ferredoxin gene (
fdxN
; 59% amino acid sequence identity to
R. capsulatus
FdxN) and open reading frames showing 52 and 36% amino acid sequence identity to
nifY
of
Pseudomonas stutzeri
A15 and ORF1 of
Azotobacter vinelandii
were located. Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a ς
54
-type promoter. Shorter transcripts sequentially missing genes of the 3′ part of the full-length mRNA were more abundantly detected. Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase. An in-frame deletion of
fdxN
resulted in reduced growth rates (59% ± 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type. Nitrogenase activity was fully complemented in an
fdxN
mutant which carried a
nifH
promoter-driven
fdxN
gene in
trans
. Also, in coculture with the ascomycete
Acremonium alternatum
, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the
fdxN
deletion mutant was decreased to 56% of the wild-type level. Surprisingly, the
fdxN
deletion also had an effect on the rapid “switch-off” of nitrogenase activity in response to ammonium. Wild-type strain BH72 and the deletion mutant complemented with
fdxN
in
trans
showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene. In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be involved in the mechanism of physiological nitrogenase switch-off, our results suggest that the ferredoxin may be a component involved in this process.
Glycerol 3-Phosphate Inhibits Swarming and Aggregation of
Myxococcus xanthus
Authors: Moraleda-Muñoz Aurelio , Carrero-Lérida Juana , Extremera Antonio L. , Arias José M. , Muñoz-Dorado José
Published: July 2002

We have cloned a gene of
Myxococcus xanthus
with similarities to the permease for glycerol 3-phosphate (G3P) of other bacteria. Expression of the gene increased significantly during the first hours of starvation. Swarming of the wild-type strain was inhibited and aggregation was delayed by G3P. Conversely, a Δ
glpT
strain aggregated even on rich medium. These results indicate that G3P may function to regulate the timing of aggregation in
M. xanthus
.
Complementation of the Essential Peptidoglycan Transpeptidase Function of Penicillin-Binding Protein 2 (PBP2) by the Drug Resistance Protein PBP2A in
Staphylococcus aureus
Authors: Pinho Mariana G. , Filipe Sérgio R. , de Lencastre Hermı́nia , Tomasz Alexander
Published: July 2002

The essential function of penicillin-binding protein 2 (PBP2) in methicillin-susceptible
Staphylococcus aureus
RN4220 was clearly established by placing the
pbp2
gene under control of the inducible P
spac
promoter; the resulting bacteria were unable to grow in the absence of inducer. In contrast, the deficit in PBP2 caused by inhibition of transcription of the
pbp2
gene did not block growth of a methicillin-resistant
S. aureus
strain expressing the extra penicillin-binding protein PBP2A, a protein of extraspecies origin that is central to the mechanism of methicillin resistance. Several lines of evidence indicate that the essential function of PBP2 that can be compensated for by PBP2A is the transpeptidase activity. This provides direct genetic evidence that PBP2A has transpeptidase activity.
Identification of a Genomic Island Present in the Majority of Pathogenic Isolates of
Pseudomonas aeruginosa
Authors: Liang Xiaoyou , Pham Xuan-Quynh T. , Olson Maynard V. , Lory Stephen
Published: July 2002

Pseudomonas aeruginosa
, a ubiquitous gram-negative bacterium, is capable of colonizing a wide range of environmental niches and can also cause serious infections in humans. In order to understand the genetic makeup of pathogenic
P. aeruginosa
strains, a method of differential hybridization of arrayed libraries of cloned DNA fragments was developed. An M13 library of DNA from strain X24509, isolated from a patient with a urinary tract infection, was screened using a DNA probe from
P. aeruginosa
strain PAO1. The genome of PAO1 has been recently sequenced and can be used as a reference for comparisons of genetic organization in different strains. M13 clones that did not react with a DNA probe from PAO1 carried X24509-specific inserts. When a similar array hybridization analysis with DNA probes from different strains was used, a set of M13 clones which carried sequences present in the majority of human
P. aeruginosa
isolates from a wide range of clinical sources was identified. The inserts of these clones were used to identify cosmids encompassing a contiguous 48.9-kb region of the X24509 chromosome called PAGI-1 (for “
P. aeruginosa
genomic island 1”). PAGI-1 is incorporated in the X24509 chromosome at a locus that shows a deletion of a 6,729-bp region present in strain PAO1. Survey of the incidence of PAGI-1 revealed that this island is present in 85% of the strains from clinical sources. Approximately half of the PAGI-1-carrying strains show the same deletion as X24509, while the remaining strains contain both the PAGI-1 sequences and the 6,729-bp PAO1 segment. Sequence analysis of PAGI-1 revealed that it contains 51 predicted open reading frames. Several of these genes encoded products with predictable function based on their sequence similarities to known genes, including insertion sequences, determinants of regulatory proteins, a number of dehydrogenase gene homologs, and two for proteins of implicated in detoxification of reactive oxygen species. It is very likely that PAGI-1 was acquired by a large number of
P. aeruginosa
isolates through horizontal gene transfer. The selection for its maintenance may be the consequence of expression of any one of the genes of unknown function or the genes which allow
P. aeruginosa
to survive under the conditions that generate reactive oxygen species. Alternatively, one or both of the transcriptional regulators encoded in PAGI-1 may control the expression of genes in the
P. aeruginosa
chromosome, which provides a selective advantage for strains that have acquired this genomic island.
Purification of the RelB and RelE Proteins of
Escherichia coli
: RelE Binds to RelB and to Ribosomes
Authors: Galvani Cheryl , Terry Jefferson , Ishiguro Edward E.
Published: July 2002

The direct interaction of the
Escherichia coli
cytotoxin RelE with its specific antidote, RelB, was demonstrated in two ways: (i) copurification of the two proteins and (ii) a positive yeast two-hybrid assay involving the
relB
and
relE
genes. In addition, the purified RelE protein exhibited ribosome-binding activity in an in vitro assay, supporting previous observations suggesting that it is an inhibitor of translation.
Streptococcus salivarius
Fimbriae Are Composed of a Glycoprotein Containing a Repeated Motif Assembled into a Filamentous Nondissociable Structure
Authors: Lévesque Céline , Vadeboncoeur Christian , Chandad Fatiha , Frenette Michel
Published: July 2002

Streptococcus salivarius
, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of
S. salivarius
fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of
S. salivarius
ATCC 25975) with glass beads. Preliminary experiments showed that
S. salivarius
fimbriae did not dissociate when they were incubated at 100°C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 × 10
6
to 40 × 10
6
Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 μm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that
S. salivarius
fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/φ, where X represents a modified amino acid residue and φ represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species,
Streptococcus mitis
and
Streptococcus constellatus
.
The Active Component of the Bioemulsifier Alasan from
Acinetobacter radioresistens
KA53 Is an OmpA-Like Protein
Authors: Toren Amir , Orr Elisha , Paitan Yossi , Ron Eliora Z. , Rosenberg Eugene
Published: July 2002

The bioemulsifier of
Acinetobacter radioresistens
KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. Recently, one of the alasan proteins, with an apparent molecular mass of 45 kDa, was purified and shown to constitute most of the emulsifying activity. The N-terminal sequence of the 45-kDa protein showed high homology to an OmpA-like protein from
Acinetobacter
spp. In the research described here the gene coding for the 45-kDa protein was cloned, sequenced, and expressed in
Escherichia coli
. Recombinant protein AlnA (35.77 kDa without the leader sequence) had an amino acid sequence homologous to that of
E. coli
OmpA and contained 70% of the specific (hydrocarbon-in-water) emulsifying activity of the native 45-kDa protein and 2.4 times that of the alasan complex. In addition to their emulsifying activity, both the native 45-kDa protein and the recombinant AlnA were highly effective in solubilizing phenanthrene, ca. 80 μg per mg of protein, corresponding to 15 to 19 molecules of phenanthrene per molecule of protein.
E. coli
OmpA had no significant emulsifying or phenanthrene-solubilizing activity. The production of a recombinant surface-active protein (emulsification and solubilization of hydrocarbons in water) from a defined gene makes possible for the first time structure-function studies of a bioemulsan.
Characterization of aLegionella pneumophila relAInsertion Mutant and Roles of RelA and RpoS in Virulence Gene Expression
Authors: Zusman Tal , Gal-Mor Ohad , Segal Gil
Published: July 2002

To investigate the involvement of RelA in the regulation ofLegionella pneumophilavirulence, a deletion substitution was constructed in therelAgene. TherelAknockout resulted in an undetectable level of ppGpp in the cells during the stationary phase, but the original level was restored when therelAgene product was supplied on a plasmid. The effect of therelAmutation was examined with two systems that are known to be expressed during the stationary phase inL. pneumophila. Pigment production was found to be dependent on therelAgene product, and only one-half as much pigment was produced by therelAmutant as by the wild-type strain. Flagellum gene expression was also found to be dependent on therelAgene product, as determined with aflaA::lacZfusion. However, therelAgene product was found to be dispensable for intracellular growth both in HL-60-derived human macrophages and in the protozoan hostAcanthamoeba castellanii. To determine the involvement of therelAgene product in expression ofL. pneumophilagenes required for intracellular growth (icm/dotgenes), nineicm::lacZfusions were constructed, and expression of these fusions in the wild-type strain was compared with their expression inrelAmutant strains. Expression of only one of theicm::lacZfusions was moderately reduced in therelAmutant strain. Expression of the nineicm::lacZfusions was also examined in a strain containing an insertion in the gene that codes for the stationary-phase sigma factor RpoS, and similar results were obtained. We concluded that RelA is dispensable for intracellular growth ofL. pneumophilain the two hosts examined and that both RelA and RpoS play minor roles inL. pneumophila icm/dotgene expression.