bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a membrane‐linked Ser/Thr protein kinase in Bacillus subtilis, implicated in developmental processes
Authors: Madec Edwige , Laszkiewicz Agnieszka , Iwanicki Adam , Obuchowski Michal , Séror Simone 
Published: March 2003

SummaryPrkC was shown to be a eukaryotic‐like (Hanks‐type) protein kinase from Bacillus subtilis with a struc­‐tural organization similar to that of the eukaryotic sensor Ser/Thr or Tyr kinases (e.g. the TGF β or PDGF receptors). The molecule consists of a catalytic domain located in the cytoplasm, joined by a single transmembrane‐spanning region (TMD) to a large extracellular domain. Using a genetic reporter system, involving the cI repressor of lambda, evidence was obtained indicating that PrkC forms a dimer, involving both the TMD and the external domain in dimerization. The purified catalytic domain of PrkC was shown to autophosphorylate and to phosphorylate an external target, MBP, in both cases on threonine. These two functions require the completely conserved K40 residue in subdomain II, which is essential for enzymatic activity. Importantly, both the mutant deleted for prkC and a K40R mutant exhibit decreased efficiency of sporulation and a significant reduction in biofilm formation, demonstrating that the catalytic activity of PrkC is necessary for these two developmental processes. In addition, we showed that the product of prpC, a PPM phosphatase encoded by the adjacent gene, co‐transcribed with prkC, is also required for normal biofilm and spore formation.
Regulation and mode of action of the second small RNA activator of RpoS translation, RprA
Authors: Majdalani Nadim , Hernandez David , Gottesman Susan 
Published: March 2003

SummaryTranslation of the stationary phase sigma factor RpoS is stimulated by at least two small RNAs, DsrA and RprA. DsrA disrupts an inhibitory secondary structure in the rpoS leader mRNA by pairing with the upstream RNA. Mutations in rprA and compensating mutations in the rpoS leader demonstrate that RprA interacts with the same region of the RpoS leader as DsrA. This is the first example of two different small RNAs regulating a common target. Regulation of these RNAs differs. DsrA synthesis is increased at low temperature. We find that RprA synthesis is regulated by the RcsC/RcsB phosphorelay system, previously found to regulate capsule synthesis and promoters of ftsZ and osmC. An rcsB null mutation abolishes the basal level, whereas mutations in rcsC that activate capsule synthesis also activate expression of the rprA promoter. An essential site with similarity to other RcsB‐regulated promoters was defined in the rprA promoter. Activation of the RcsC/RcsB system leads to increased RpoS synthesis, in an RprA‐dependent fashion. This work suggests a new signal for RpoS translation and extends the global regulation effected by the RcsC/RcsB system to coregulation of RpoS with capsule and FtsZ.
Elongation factor Tu and E1 β subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae
Authors: Dallo Shatha F. , Kannan T. R. , Blaylock Mark W. , Baseman Joel B. 
Published: March 2003

SummaryThe interactions between pathogenic bacteria and extracellular matrix (ECM) components markedly influence the initiation and establishment of infection. We have identified two surface proteins of virulent Mycoplasma pneumoniae with molecular masses of 45 and 30 kDa that bind to the ECM constituent, fibronectin (Fn). These Fn‐binding proteins (FnBPs) were purified to near homogeneity using Fn‐coupled Sepharose 4B‐affinity column chromatography, and amino acid sequence analysis of the 45 and the 30 kDa proteins identified them as elongation factor Tu (EF‐Tu) and pyruvate dehydrogenase E1 β subunit (PDH‐B) respectively. The genes for EF‐Tu and PDH‐B were cloned, and the entire EF‐Tu gene and NH2‐terminus of PDH‐B (NPDH (pyruvate dehydrogenase E1 β subunit from amino acid 1–244)‐B) gene were overexpressed in Escherichia coli. The recombinant proteins, rEF‐Tu and rNPDH‐B, were purified to homogeneity by His‐tag affinity column chromatography and used to immunize rabbits. Purified rEF‐Tu and rNPDH‐B bound to Fn using a ligand immunoblot assay and ELISA. Immunogold electron microscopy with polyclonal antibodies reactive against rEF‐Tu (antirEF‐Tu) and rNPDH‐B (antirNPDH‐B) and whole cell radioimmunoprecipitation (WCRIP) revealed the surface location of these proteins. Adherence of viable M. pneumoniae to immobilized Fn was inhibited by antirEF‐Tu and antirNPDH‐B antisera in a dose‐dependent and cumulative manner. These results demonstrate that M. pneumoniae EF‐Tu and PDH‐B, in addition to their major cytoplasmic biosynthetic and metabolic roles, can be surface translocated, which confers additional important biological functions.
Rst1 and Rst2 are required for the a/α diploid cell type in yeast
Authors: Gelli Angie 
Published: March 2003

SummaryIn the budding yeast Saccharomyces cerevisiae, the preservation of the mating competent haploid (a or α) and the mating incompetent diploid (a/α) is necessary to prevent aneuploidy. Once haploid cells respond to pheromone, the mating‐specific signal transduction pathway is activated, and the MAP kinase Fus3 phosphorylates two specific repressor proteins Rst1 and Rst2 (also known as Dig1 and Dig2) to promote Ste12‐dependent transcription of mating‐specific genes. In contrast, diploid cells cannot mate because genes that encode components of the mating pathway are repressed through the combined action of the Mata1–Matα2 and Matα2–Mcm1 repressors. Surprisingly, repression of Ste12 by Rst1 and Rst2 is essential for diploid sterility. Homozygous deletion of both RST1 and RST2 (rst–) causes a/α diploid cells constitutively to express a‐specific genes and mate preferentially as a‐cells. This phenotype is sensitive to Ste12 dosage, as removal of one copy of STE12 completely reduces the ectopic activation of a‐specific genes. The Matα2–Mcm1 complex, which normally represses a‐specific genes, is defective in rst– diploids because Matα2 is destabilized in rst– diploids, possibly as a consequence of its relocalization from the nucleus to the cytoplasm. This study finds that Rst1 and Rst2 are necessary for the a/α diploid cell type. Rst1 and Rst2 are required in order to prevent the amplification of a robust Ste12 transcriptional programme that appears to over‐ride Matα2‐dependent repression of haploid and a‐specific genes.
Fibronectin mediates Opc‐dependent internalization of Neisseria meningitidis in human brain microvascular endothelial cells
Authors: Unkmeir Alexandra , Latsch Kirsten , Dietrich Guido , Wintermeyer Eva , Schinke Birgitta , Schwender Stefan , Kim Kwang Sik , Eigenthaler Martin , Frosch Matthias 
Published: March 2003

SummaryA central step in the pathogenesis of bacterial meningitis caused by Neisseria meningitidis (the meningococcus) is the interaction of the bacteria with cells of the blood–brain barrier. In the present study, we analysed the invasive potential of two strains representing hypervirulent meningococcal lineages of the ET‐5 and ET‐37 complex in human brain‐derived endothelial cells (HBEMCs). In contrast to previous observations made with epithelial cells and human umbilical vein‐derived endothelial cells (HUVECs), significant internalization of encapsulated meningococci by HBMECs was observed. However, this uptake was found only for the ET‐5 complex isolate MC58, and not for an ET‐37 complex strain. Furthermore, the uptake of meningococci by HBMECs depended on the presence of human serum, whereas serum of bovine origin did not promote the internalization of meningococci in HBMECs. By mutagenesis experiments, we demonstrate that internalization depended on the expression of the opc gene, which is present in meningococci of the ET‐5 complex, but absent in ET‐37 complex meningococci. Chromatographic separation of human serum proteins revealed fibronectin as the uptake‐promoting serum factor, which binds to HBMECs via α5β1 integrin receptors. These data provide evidence for unique molecular mechanisms of the interaction of meningococci with endothelial cells of the blood–brain barrier and contribute to our understanding of the pathogenesis of meningitis caused by meningococci of different clonal lineages.
Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti
Authors: Pym Alexander S. , Brodin Priscille , Brosch Roland , Huerre Michel , Cole Stewart T. 
Published: March 2003

SummaryAlthough large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock‐in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1‐encoded proteins were localized in the cell wall, and two of them, the immunodominant T‐cell antigens ESAT‐6 and CFP‐10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock‐ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock‐in, whereas BCG controls were cleared. Knocking‐in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development.
Integron‐encoded IntI integrases preferentially recognize the adjacent cognate attI site in recombination with a 59‐be site
Authors: Collis Christina M. , Kim Mi‐Jurng , Stokes H. W. , Hall Ruth M. 
Published: March 2003

SummaryIntegrons have the capacity to capture small mobile elements known as gene cassettes, and this reaction is catalysed by integron‐encoded IntI integrases. IntI integrases form a distinct family within the tyrosine recombinase superfamily and include a characteristic additional domain that is well conserved. Two different IntI enzymes were used to examine their ability to recognize heterologous attI sites in both integration and excision assays. IntI1 and IntI3 are 59% identical and catalyse both integrative and excisive recombination between a cassette‐associated 59‐be site and the cognate attI1 or attI3 site. Integrative recombination events involving a 59‐be and a non‐cognate attI site, attI2 and attI3 for IntI1 or attI1 and attI2 for IntI3, were detected extremely rarely. In cassette excision assays, the non‐cognate attI3 site was recognized by IntI1, but attI1 was not well recognized by IntI3. The purified IntI1 and IntI3 proteins bound strongly only to their cognate attI site.
Intestinal short‐chain fatty acids alter Salmonella typhimurium invasion gene expression and virulence through BarA/SirA
Authors: Lawhon Sara D. , Maurer Russell , Suyemoto Mitsu , Altier Craig 
Published: March 2003

Summary Salmonella typhimurium causes enteric and systemic disease by invading the intestinal epithelium of the distal ileum, a process requiring the invasion genes of Salmonella pathogenicity island 1 (SPI‐1). BarA, a sensor kinase postulated to interact with the response regulator SirA, is required for the expression of SPI‐1 invasion genes. We found, however, that a barA null mutation had little effect on virulence using the mouse model for septicaemia. This confounding result led us to seek environmental signals present in the distal ileum that might supplant the need for BarA. We found that acetate restored the expression of invasion genes in the barA mutant, but had no effect on a sirA mutant. Acetate had its effect only at a pH that allowed its accumulation within the bacterial cytoplasm and not with the deletion of ackA and pta, the two genes required to produce acetyl‐phosphate. These results suggest that the rising concentration of acetate in the distal ileum provides a signal for invasion gene expression by the production of acetyl‐phosphate in the bacterial cytoplasm, a pathway that bypasses barA. We also found that a Δ(ackA–pta) mutation alone had no effect on virulence but, in combination with Δ(barA), it increased the oral LD50 24‐fold. Thus, the combined loss of the BarA‐ and acetate‐dependent pathways is required to reduce virulence. Two other short‐chain fatty acids (SCFA), propionate and butyrate, present in high concentrations in the caecum and colon, had effects opposite to those of acetate: neither restored invasion gene expression in the barA mutant, and both, in fact, reduced expression in the wild‐type strain. Further, a combination of SCFAs found in the distal ileum restored invasion gene expression in the barA mutant, whereas colonic conditions failed to do so and also reduced expression in the wild‐type strain. These results suggest that the concentration and composition of SCFAs in the distal ileum provide a signal for productive infection by Salmonella, whereas those of the large intestine inhibit invasion.
Ectopic RNase E sites promote bypass of 5′‐end‐dependent mRNA decay in Escherichia coli
Authors: Baker Kristian E. , Mackie George A. 
Published: March 2003

SummaryIn Escherichia coli, 5′‐terminal stem–loops form major impediments to mRNA decay, yet conditions that determine their effectiveness or the use of alternative decay pathway(s) are unclear. A synthetic 5′‐terminal hairpin stabilizes the rpsT mRNA sixfold. This stabilization is dependent on efficient translational initiation and ribosome transit through at least two‐thirds of the coding sequence past a major RNase E cleavage site in the rpsT mRNA. Insertion of a 12–15 residue ‘ectopic’ RNase E cleavage site from either the rne leader or 9S pre‐rRNA into the 5′‐non‐coding region of the rpsT mRNA significantly reduces the stabilizing effect of the terminal stem–loop, dependent on RNase E. A similar insertion into the rpsT coding sequence is partially destabilizing. These findings demonstrate that RNase E can bypass an interaction with the 5′‐terminus, and exploit an alternative ‘internal entry’ pathway. We propose a model for degradation of the rpsT mRNA, which explains the hierarchy of protection afforded by different 5′‐termini, the use of internal entry for bypass of barriers to decay, ‘ectopic sites’ and the role of translating ribosomes.
Copper‐mediated reversal of defective laccase in a Δvph1 avirulent mutant of Cryptococcus neoformans
Authors: Zhu Xudong , Gibbons Jack , Zhang Shirong , Williamson Peter R. 
Published: March 2003

SummaryPrevious studies have shown that a Δvph1 Cryptococcus neoformans mutant defective in vesicular acidification lacked several important virulence factors including a copper‐containing laccase and was avirulent in a mouse model. In the present studies, we characterized laccase transcription and protein production to obtain insights into the mechanism of the vph1 mutation in this pathogen. Although transcription and protein expression were somewhat reduced, laccase protein was found to be successfully translated and correctly targeted to the cell wall in the Δvph1 mutant as shown by Western blot and immuno‐electron microscopy, despite a complete lack of laccase activity. Laccase activity was substantially restored in metabolically active Δvph1 cells at 30°C by addition of 100 µM copper sulphate. This restoration by copper was found to occur through both transcriptional and post‐translational mechanisms. Laccase transcriptional induction by copper was found to be dependent on enhancer region II within the 5′‐untranslated region of CNLAC1. Copper was also found to restore partial activity to Δvph1 cells at 0°C, suggesting that cell wall laccase was expressed in the mutant as an apo‐enzyme. Apo‐laccase restoration by copper was found to be facilitated by an acidic environment, consistent with a role for the vacuolar (H+)‐ATPase proton pump in copper assembly of laccase in C. neoformans.
Transcriptional organization and regulation of the Escherichia coli K30 group 1 capsule biosynthesis (cps) gene cluster
Authors: Rahn Andrea , Whitfield Chris 
Published: March 2003

Summary Escherichia coli
group 1 capsules are important virulence determinants, yet little is known about the transcriptional organization or regulation of their biosynthetic (
cps
) operons. Transcription of the prototype serotype K30 cluster is modulated by the JUMPStart–RfaH antitermination mechanism, with the
cps
promoter being localized to a region immediately upstream of the JUMPStart sequence. A putative stem–loop structure located within the K30
cps
cluster separates conserved genes with products that are required for surface expression of capsule from serotype‐specific genes encoding enzymes for polymer repeat‐unit synthesis and polymerization. This putative stem–loop structure significantly reduces transcription in a termination‐probe vector and may contribute to differential expression of the
cps
genes. Previous work indicated that increased amounts of group 1 capsular polysaccharide synthesis resulted from the overexpression of the Rcs (
r
egulator of
c
apsule
s
ynthesis) proteins. However, neither overexpression of the transcriptional activator RcsB nor an
rcsB
::
aadA
chromosomal insertion altered the level of transcription measured by
cps::lacZ
fusions. In the group 1 strains examined, an RcsAB box was found immediately upstream of
galF
, a gene involved in the production of sugar nucleotide precursors. Overexpression of RcsB was found to result in a threefold increase in transcription of a
galF::lacZ
chromosomal fusion. Moreover, overexpression of GalF gave rise to a two‐ to threefold increase in cell‐free as well as cell‐associated capsule, without affecting
cps::lacZ
activity. These results indicate that transcription of the
E. coli
group 1 capsule cluster itself is not regulated by the Rcs system and may, in fact, be constitutive. However, the Rcs system can potentially influence levels of capsular polysaccharide production by increasing
galF transcription and influencing the available pool of biosynthetic precursors.
Trigger Factor and DnaK possess overlapping substrate pools and binding specificities
Authors: Deuerling Elke , Patzelt Holger , Vorderwülbecke Sonja , Rauch Thomas , Kramer Günter , Schaffitzel Elke , Mogk Axel , Schulze‐Specking Agnes , Langen Hanno , Bukau Bernd 
Published: March 2003

SummaryRibosome‐associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF‐deficient cells, Δtig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37°C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety‐four aggregated proteins isolated from DnaK‐ and DnaJ‐depleted Δtig cells were identified by mass spectrometry and found to include essential cytosolic proteins.Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co‐operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.
Cytochrome c oxidase genes required for nitrogenase activity and diazotrophic growth in Anabaena sp. PCC 7120
Authors: Valladares Ana , Herrero Antonia , Pils Dietmar , Schmetterer Georg , Flores Enrique 
Published: March 2003

SummaryN2 fixation is an O2‐sensitive process and some filamentous diazotrophic cyanobacteria that grow performing oxygenic photosynthesis confine their N2 fixation machinery to heterocysts, specialized cells that maintain a reducing environment adequate for N2 fixation. Respiration is thought to contribute to the diazotrophic metabolism of heterocysts and the genome of the heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120 bears three gene clusters putatively encoding cytochrome c oxidases. Transcript analysis of these cox gene clusters through RNA/DNA hybridization identified two cox operons, cox2 and cox3, that are induced after nitrogen step‐down in an NtcA‐ and HetR‐dependent manner and appear to be expressed specifically in heterocysts. In contrast, cox1 was expressed only in vegetative cells. Expression of cox2 and cox3 occurred at an intermediate stage (about 9 h) during the process of heterocyst development following nitrogen step‐down. Inactivation of genes in the two inducible cox operons, but not separately in either of them, strongly reduced nitrogenase activity and prevented diazotrophic growth in aerobic conditions. These results show that the nitrogen‐regulated cytochrome c oxidase‐type respiratory terminal oxidases Cox2 and Cox3 are essential for heterocyst function in Anabaena sp. PCC 7120.
An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi
Authors: Cohen Georges N. , Barbe Valérie , Flament Didier , Galperin Michael , Heilig Roland , Lecompte Odile , Poch Olivier , Prieur Daniel , Quérellou Joël , Ripp Raymond , Thierry Jean‐Claude , Van der Oost John , Weissenbach Jean , Zivanovic Yvan , Forterre Patrick 
Published: March 2003

SummaryThe hyperthermophilic euryarchaeon Pyrococcus abyssi and the related species Pyrococcus furiosus and Pyrococcus horikoshii, whose genomes have been completely sequenced, are presently used as model organisms in different laboratories to study archaeal DNA replication and gene expression and to develop genetic tools for hyperthermophiles. We have performed an extensive re‐annotation of the genome of P. abyssi to obtain an integrated view of its phylogeny, molecular biology and physiology. Many new functions are predicted for both informational and operational proteins. Moreover, several candidate genes have been identified that might encode missing links in key metabolic pathways, some of which have unique biochemical features. The great majority of Pyrococcus proteins are typical archaeal proteins and their phylogenetic pattern agrees with its position near the root of the archaeal tree. However, proteins probably from bacterial origin, including some from mesophilic bacteria, are also present in the P. abyssi genome.