bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cross‐regulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae
Authors: Martin Bernard , Prudhomme Marc , Alloing Geneviève , Granadel Chantal , Claverys Jean‐Pierre 
Published: March 2003

Two operons, comAB and comCDE, play a key role in the co‐ordination of spontaneous competence development in cultures of Streptococcus pneumoniae. ComAB is required for export of the comC‐encoded competence‐stimulating peptide (CSP). Upon CSP binding, the histidine kinase ComD activates ComE, its cognate response regulator, required for autoinduction of comCDE and for induction of the late competence genes. To understand better the early control of competence development, mutants upregulating comCDE (ComCDEUP) were isolated using a comC–lacZ transcriptional fusion. Mutants were generated by polymerase chain reaction mutagenesis of the comCDE region and by in vitro transposon mutagenesis of the chromosome. Both types of ComCDEUP mutants exhibited similar phenotypes. They differed from wild type in displaying trypsin‐resistant transformation, competence under acid growth conditions and expression of comCDE under microaerobiosis; increased production of CSP in the mutants could account for the various phenotypes. The ComCDEUP transposon mutations included four independent insertions in the ciaR gene, which encodes the response regulator of a two‐component system previously found to affect competence, and two immediately upstream of the comAB operon. The latter two resulted in comAB overexpression, indicating that CSP export is rate limiting. Among comDE point mutations, a single amino acid change in ComD (T233I) conferred constitutive, CSP‐independent competence and resulted in comAB overexpression, providing support for the hypothesis that ComE regulates comAB; a ComE mutant (R120S) exhibited altered kinetics of competence shut‐off. Collectively, these data indicate that pheromone autoinduction, cross‐regulation of the comAB and comCDE operons and, possibly, competence shut‐off contribute to the early control of competence development in S. pneumoniae. They argue for a metabolic control of competence, mediated directly or indirectly by CiaR, and they suggest that both comAB and comCDE are potential targets for regulation.
Cold‐temperature induction of Escherichia coli polynucleotide phosphorylase occurs by reversal of its autoregulation	†‡
Authors: Beran Rudolf K. , Simons Robert W. 
Published: March 2003

When Escherichia coli cells are shifted to low temperatures (e.g. 15°C), growth halts while the ‘cold shock response’ (CSR) genes are induced, after which growth resumes. One CSR gene, pnp, encodes polynucleotide phosphorylase (PNPase), a 3′‐exoribonuclease and component of the RNA degradosome. At 37°C, ribonuclease III (RNase III, encoded by rnc) cleaves the pnp untranslated leader, whereupon PNPase represses its own translation by an unknown mechanism. Here, we show that PNPase cold‐temperature induction involves several post‐transcriptional events, all of which require the intact pnp mRNA leader. The bulk of induction results from reversal of autoregulation at a step subsequent to RNase III cleavage of the pnp leader. We also found that pnp translation occurs throughout cold‐temperature adaptation, whereas lacZ+ translation was delayed. This difference is striking, as both mRNAs are greatly stabilized upon the shift to 15°C. However, unlike the lacZ+ mRNA, which remains stable during adaptation, pnp mRNA decay accelerates. Together with other evidence, these results suggest that mRNA is generally stabilized upon a shift to cold temperatures, but that a CSR mRNA‐specific decay process is initiated during adaptation.
Molecular and physiological characterization of the NAD‐dependent glycerol 3‐phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans
Authors: Fillinger Sabine , Ruijter George , Tamás Markus J. , Visser Jaap , Thevelein Johan M. , D'enfert Christophe 
Published: March 2003

In filamentous fungi, glycerol biosynthesis has been proposed to play an important role during conidiospore germination and in response to a hyperosmotic shock, but little is known about the genes involved. Here, we report on the characterization of the major Aspergillus nidulans glycerol 3‐phosphate dehydrogenase (G3PDH)‐encoding gene, gfdA. G3PDH is responsible for the conversion of dihydroxyacetone phosphate (DHAP) into glycerol 3‐phosphate (G3P), which is subsequently converted into glycerol by an as yet uncharacterized phosphatase. Inactivation of gfdA does not abolish glycerol biosynthesis, showing that the other pathway from DHAP, via dihydroxyacetone (DHA), to glycerol is also functional in A. nidulans. The gfdA null mutant displays reduced G3P levels and an osmoremediable growth defect on various carbon sources except glycerol. This growth defect is associated with an abnormal hyphal morphology that is reminiscent of a cell wall defect. Furthermore, the growth defect at low osmolarity is enhanced in the presence of the chitin‐interacting agent calcofluor and the membrane‐destabilizing agent sodium dodecyl sulphate (SDS). As inactivation of gfdA has no impact on phospholipid biosynthesis or glycolytic intermediates levels, as might be expected from reduced G3P levels, a previously unsuspected link between G3P and cell wall integrity is proposed to occur in filamentous fungi.
The variable P5 proteins of typeable and non‐typeable Haemophilus influenzae target human CEACAM1
Authors: Hill Darryl J. , Toleman Mark A. , Evans Debbie J. , Villullas Silvia , Van Alphen Loek , Virji Mumtaz 
Published: March 2003

Haemophilus influenzae, a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We have recently shown that numerous strains of capsulate (typeable) and acapsulate (non‐typeable) H. influenzae target the carcinoembryonic antigen (CEA) family of cell adhesion molecules (CEACAMs). Moreover, the ligands appeared to be antigenically variable and, when using viable typeable bacteria, their adhesive functions were inhibited by the presence of capsule. In this report, we show that the antigenically variable outer membrane protein, P5, expressed by typeable and non‐typeable H. influenzae targets human CEACAM1. Variants and mutants lacking the expression of P5 of all strains tested were unable to target purified soluble receptors. A non‐typeable strain that did not interact with CEACAM1 was made adherent to both the soluble receptors and CEACAM1‐transfected Chinese hamster ovary cells by transformation with the P5 gene derived from the adherent typeable strain Rd. However, several H. influenzae mutants lacking P5 expression continued to bind the cell‐bound CEACAM1 receptors. These observations suggest that (i) CEACAM1 alone can support P5 interactions and (ii) some strains contain additional ligands with the property to target CEACAM1 but require the receptor in the cellular context. The identification of a common ligand in diverse strains of H. influenzae and the presence of multiple ligands for the same receptor suggests that targeting of members of the CEACAM family of receptors may be of primary significance in colonization and pathogenesis of H. influenzae strains.
Characterization of the bacteriophage φ29‐encoded protein p16.7: a membrane protein involved in phage DNA replication
Authors: Meijer Wilfried J. J. , Serna‐Rico Alejandro , Salas Margarita 
Published: March 2003

An early expressed operon, located at the right end of the linear bacteriophage φ29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in φ29‐related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N‐terminally located transmembrane‐spanning domain is required for its membrane localization. The variant p16.7A, in which the N‐terminal membrane anchor was replaced by a histidine‐tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a φ29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivoφ29 DNA replication is discussed.
Dispensable nature of phosphatidylglycerol in Escherichia coli: dual roles of anionic phospholipids
Authors: Matsumoto Kouji 
Published: March 2003

The major anionic phospholipids of Escherichia coli, phosphatidylglycerol (PG) and cardiolipin (CL), have been considered to be indispensable for essential cellular functions, such as the initiation of DNA replication and translocation of proteins across the cytoplasmic membrane. However, we successfully constructed a null pgsA mutant of E. coli that had undetectable levels of PG and CL if the major outer membrane lipoprotein was deficient, clearly indicating that these anionic phospholipids are not indispensable. In the null mutant, we observed the accumulation of phosphatidic acid, an acidic biosynthetic precursor. This suggests a functionally substitutable nature of these anionic phospholipids and allows us to formulate a dual role model for the physiological roles of the anionic phospholipids in E. coli. The anionic phospholipids may play dual roles in E. coli as (i) substrates for head group‐specific enzyme reactions, albeit the viability of null PG mutants indicates that the products of head group‐specific reactions are not essential; and (ii) those that are replaceable, partly or entirely, by other phospholipids bearing net negative charges, because of their rather loose head group specificity. These two aspects of the physiological roles of anionic phospholipids are discussed with special reference to the phospholipids of other bacteria and eukaryotic organelles.
The Saccharomyces cerevisiae Sko1p transcription factor mediates HOG pathway‐dependent osmotic regulation of a set of genes encoding enzymes implicated in protection from oxidative damage
Authors: Rep Martijn , Proft Markus , Remize Fabienne , Tamás Markus , Serrano Ramón , Thevelein Johan M. , Hohmann Stefan 
Published: March 2003

A major part of the transcriptional response of yeast cells to osmotic shock is controlled by the HOG pathway and several downstream transcription factors. Sko1p is a repressor that mediates HOG pathway‐dependent regulation by binding to CRE sites in target promoters. Here, we report five target genes of Hog1p–Sko1p: GRE2, AHP1, SFA1, GLR1 and YML131w. The two CREs in the GRE2 promoter function as activating sequences and, hence, bind (an) activator protein(s). However, the two other yeast CRE‐binding proteins, Aca1p and Aca2p, are not involved in regulation of the GRE2 promoter under osmotic stress. In the absence of the co‐repressor complex Tup1p–Ssn6p/Cyc8p, which is recruited by Sko1p, stimulation by osmotic stress is still observed. These data indicate that Sko1p is not only required for repression, but also involved in induction upon osmotic shock. All five Sko1p targets encode oxidoreductases with demonstrated or predicted roles in repair of oxidative damage. Altered basal expression levels of these genes in hog1Δ and sko1Δ mutants may explain the oxidative stress phenotypes of these mutants. All five Sko1p target genes are induced by oxidative stress, and induction involves Yap1p. Although Sko1p and Yap1p appear to mediate osmotic and oxidative stress responses independently, Sko1p may affect Yap1p promoter access or activity. The five Sko1p target genes described here are suitable models for studying the interplay between osmotic and oxidative responses at the molecular and physiological levels.
Acetylation of the response regulator, CheY, is involved in bacterial chemotaxis
Authors: Barak Rina , Eisenbach Michael 
Published: March 2003

It is well established that the response regulator of the chemotaxis system of Escherichia coli, CheY, can undergo acetylation at lysine residues 92 and 109 via a reaction mediated by acetyl‐CoA synthetase (Acs). The outcome is activation of CheY, which results in increased clockwise rotation. Nevertheless, it has not been known whether CheY acetylation is involved in chemotaxis. To address this question, we examined the chemotactic behaviour of two mutants, one lacking the acetylating enzyme Acs, and the other having an arginine‐for‐lysine substitution at residue 92 of CheY – one of the acetylation sites. The Δacs mutant exhibited much reduced sensitivity to chemotactic stimuli (both attractants and repellents) in tethering assays and greatly reduced responses in ring‐forming, plug and capillary assays. Likewise, the cheY(92KR) mutant had reduced sensitivity to repellents in tethering assays and a reduced response in capillary assays. However, its response to the addition or removal of attractants was normal. These observations suggest that Acs‐mediated acetylation of CheY is involved in chemotaxis and that the acetylation site Lys‐92 is only involved in the response to repellents. The observation that, in the cheY(92KR) mutant, the addition of a repellent was not chemotactically equivalent to the removal of an attractant also suggests that there are different signalling pathways for attractants and repellents in E. coli.
Nuclease activity is essential for RecBCD recombination in Escherichia coli
Authors: Jockovich M. E. , Myers R. S. 
Published: March 2003

RecBCD has two conflicting roles in Escherichia coli. (i) As ExoV, it is a potent double‐stranded (ds)DNA exonuclease that destroys linear DNA produced by restriction of foreign DNA. (ii) As a recombinase, it promotes repair of dsDNA breaks and genetic recombination in the vicinity of χ recombination hot‐spots. These paradoxical roles are accommodated by χ‐dependent attenuation of RecBCD exonuclease activity and concomitant conversion of the enzyme to a recombinase. To challenge the proposal that χ converts RecBCD from a destructive exonuclease to a recombinogenic helicase, we mutated the nuclease catalytic centre of RecB and tested the resulting mutants for genetic recombination and DNA repair in vivo. We predicted that, if nuclease activity inhibits recombination and helicase activity is sufficient for recombination, the mutants would be constitutive recombinases, as has been seen in recD null mutants. Conversely, if nuclease activity is required, the mutants would be recombination deficient. Our results indicate that 5′→ 3′ exonuclease activity is essential for recombination by RecBCD at χ recombination hot‐spots and at dsDNA ends in recD mutants. In the absence of RecB‐dependent nuclease function, recombination becomes entirely dependent on the 5′ → 3′ single‐stranded (ss)DNA exonuclease activity of RecJ and the helicase activity of RecBC(D).
Leaderless mRNAs in bacteria: surprises in ribosomal recruitment and translational control
Authors: Moll Isabella , Grill Sonja , Gualerzi Claudio O. , Bläsi Udo 
Published: March 2003

SummaryIt is commonly believed that the translational efficiency of prokaryotic mRNAs is intrinsically determined by both primary and secondary structures of their translational initiation regions. However, for leaderless mRNAs starting with the AUG initiating codon occurring in bacteria, archaea and eukaryotes, there is no evidence for ribosomal recruitment signals downstream of the 5′‐terminal AUG that seems to
be the only necessary and constant element. Studies in Escherichia coli have brought to light that the ratio of initiation factors IF2 and IF3 plays a decisive role in translation initiation of leaderless mRNA, indicating that the translational efficiency of this mRNA class can be modulated depending on the availability of components of the translational machinery. Recent data suggested that the start codon of bacterial leaderless mRNAs is recognized by a ribosome‐IF2‐fMet‐tRNA complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes, which points to a conceptual similarity in all initiation pathways. In fact, the faithful translation of lead‐erless mRNAs in heterologous systems shows that the ability to translate leaderless mRNAs is an evo‐lutionarily conserved function of the translational apparatus.
Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli
Authors: Guzmán Elena C. , Caballero José Luis , Jiménez‐Sánchez Alfonso 
Published: March 2003

SummaryAlthough the nrdA101 allele codes for a ribonucleoside diphosphate (rNDP) reductase that is essentially destroyed in less than 2 min at 42°C, and chemical inhibition of the enzyme by hydroxyurea stops DNA synthesis at once, we found that incubation at 42°C of an Escherichia coli strain containing this allele allows DNA replication for about 40 min. This suggests that mutant rNDP reductase is protected from thermal inactivation by some hyperstructure. If, to‐gether with the temperature upshift, RNA or protein synthesis is inhibited, the thermostability time of the mutant rNDP reductase becomes at least as long as the replication time and residual DNA synthesis becomes a run‐out replication producing fully replicated chromosomes. This suggests that cessation of replication in the nrdA101 mutant strain is not the result of inactivation of its gene product but of the activity of a protein reflecting the presence of a partially altered enzyme. The absence of Tus protein, which specifically stops the replication complex by inhibiting replicative helicase activity, allows forks to replicate for a longer time at the restrictive temperature in the nrdA101 mutant strain. We therefore propose that rNDP reductase is a component of the replication complex, and that this association with other proteins protects the protein coded by allele nrdA101 from thermal inactivation.
Identification of novel factors involved in colonization and acid tolerance of Vibrio cholerae
Authors: Merrell D. Scott , Hava David L. , Camilli Andrew 
Published: March 2003

SummaryDespite over 100 years of study, the intestinal pathogen Vibrio cholerae
still causes epidemic disease in areas of the world where there is poor sanitation.
While cholera toxin and the toxin‐coregulated pilus (TCP) are known to be essential
for full virulence, the role that other factors play has remained ill‐defined. Herein,
we describe a large‐scale signature‐tagged mutagenesis (STM) screen utilizing 100
pools of 96 mutants each to identify factors involved in colonization of the infant
mouse small intestine. A total of 164 mutants representing transposition events into
95 different open reading frames were shown to be recovered at greatly reduced numbers
from the infant mouse model. Analysis of the sites of insertion revealed multiple
independent mutations within the rfb gene cluster, needed for synthesis of
lipopolysaccharide (LPS), and the tcp gene cluster, needed for synthesis of
the TCP. More importantly, in addition to these previously known colonization factors,
we identified many genes whose activity in colonization was not previously appreciated.
These can be divided into a number of functional groups, which include production
of factors involved in metabolic activities, regulation of cellular processes, transport,
adaptation to stress and unknown functions. In addition, we describe the reiterative
use of STM, whereby colonization‐defective mutants were assembled into virulence‐attenuated
pools (VAPs), which were used to begin to reveal roles that the identified virulence
factors play in the infection process. Nine new factors were shown to be crucial
for the V. cholerae acid tolerance response, which has previously been hypothesized
to be important for epidemic spread of cholera. Competition assays of these nine
acid tolerance response (ATR)‐defective mutants revealed that mutations in gshB,
hepA and recO result in a 1000‐fold reduction in colonization.
Identification of Pseudomonas syringae pv. tomato genes induced during infection of Arabidopsis thaliana
Authors: Boch Jens , Joardar Vinita , Gao Lisa , Robertson Tara L. , Lim Melisa , Kunkel Barbara N. 
Published: March 2003

SummaryPhytopathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. Many of these genes should be induced when the bacteria come in contact with plant tissue. We used a modified in vivo expression technology
(IVET) approach to identify genes from the plant pathogen Pseudomonas syringae
pv. tomato that are induced upon infection of Arabidopsis thaliana
and isolated over 500 in planta‐expressed (ipx) promoter fusions. Sequence
analysis of 79 fusions revealed several known and potential virulence genes, including
hrp/hrc, avr and coronatine biosynthetic genes. In addition, we identified
metabolic genes presumably important for adaptation to growth in plant tissue, as
well as several genes with unknown function that may encode novel virulence factors.
Many ipx fusions, including several corresponding to novel genes, are dependent
on HrpL, an alternative RNA polymerase sigma factor that regulates the expression
of virulence genes. Expression analysis indicated that several ipx fusions
are strongly induced upon inoculation into plant tissue. Disruption of one ipx
gene, conserved effector locus (CEL) orf1, encoding a putative lytic murein
transglycosylase, resulted in decreased virulence of P. syringae. Our results
demonstrate that this screen can be used successfully to isolate genes that are induced
in planta, including many novel genes potentially involved in pathogenesis.
Primosome assembly requirement for replication restart in the Escherichia coli holDG10 replication mutant
Authors: Flores Maria Jose , Ehrlich S. Dusko , Michel Bénédicte 
Published: March 2003

SummaryIn this report, we study the role of pre‐primosome proteins in a strain in which the frequency of replication arrest is increased because of a mutation in a replication protein. The holDG10 mutant was used, in which replication restart
involves replication fork reversal. As expected, PriA primosome assembly function
is essential for growth of the holDG10 mutant. The priA300
mutation, which inactivates only the helicase function of PriA in vitro, and
priB inactivation strongly impair viability. In contrast, priC inactivation
has no effect. Therefore, PriB is more important than PriC for PriA‐dependent replication
fork restart in vivo. The gain of function mutation dnaC809 restores
the viability of holDG10 priA and holDG10 priB
mutants only to some extent. The dnaC809 820 double mutation restores full
viability to the holDG10 mutant lacking either PriA or PriB. Similarly
to the holDG10 single mutant, the holDG10 priA dnaC809
820 strain is depend‐ent on RecBC for viability, indicating that facilitating
primosome assembly using the dnaC809 820 mutation does not allow bypass of replication fork reversal.