bluuebunny/crossref_metadata_2025_split huggingface dataset:
Amino acid replacements in the Serratia marcescens haemolysin ShlA define sites involved in activation and secretion
Authors: Schönherr Roland , Tsolis Renée , Focareta Tony , Braun Volkmar 
Published: October 2006

SummaryThe haemolysin of Serratia marcescens (ShlA) is translocated through the cytoplasmic membrane by the signal peptide‐dependent export apparatus. Translocation across the outer membrane (secretion) is mediated by the ShIB protein. Only the secreted form of ShlA is haemolytic. ShIB also converts in vitro inactive ShlA (ShlA*), synthesized in the absence of ShIB, into the haemolytic form (a process termed activation). To define regions in ShlA involved in both processes, ShlA derivatives were isolated and tested for secretion and activation. Analysis of C‐terminally truncated proteins (ShlA) assigned the secretion signal to the amino‐terminal 238 residues of ShlA. Trypsin cleavage of a secreted ShlA' derivative yielded a 15kDa N‐terminal fragment, by which a haemolytically inactive ShlA* protein could be activated in vitro. It is suggested that the haemolysin activation site is located in this N‐terminal fragment. Replacement of asparagine‐69 and asparagine‐109 by isoleucine yielded inactive haemolysin derivatives. Both asparagine residues are part of two short sequence motifs, reading Ala‐Asn‐Pro‐Asn, which are critical to both activation and secretion. These point mutants as well as N‐terminal deletion derivatives which were not activated by ShIB were activated by adding a non‐haemolytic N‐terminal fragment synthesized in an ShIB+ strain (complementation). Apparently the activated N‐terminal fragment substituted for the missing activation of the ShlA derivatives and directed them into the erythrocyte membrane, where they formed pores. It is concluded that activation is only required for initiation of pore formation, and that in vivo activation and secretion are tightly coupled processes. Complementation may also indicate that haemolysin oligomers form the pores.
Regulation and sequence of the structural gene for cytochrome C552 from Escherichia coli: not a hexahaem but a 50kDa tetrahaem nitrite reductase
Authors: Darwin Andrew , Hussain Haitham , Griffiths Lesley , Grove Jane , Sambongi Yoshihiro , Busby Stephen , Cole Jeff 
Published: October 2006

SummaryThe structural gene, nrfA, for cytochrome C552, which is the terminal reductase of the formate‐dependent pathway for nitrite reduction to ammonia, has been located at co‐ordinate 4366 on the physical map of the Escherichia coli chromosome. The DNA sequence of nrfA encodes a tetrahaem c‐type cytochrome with a predicted Mr for the unprocessed product of 53788. Cleavage of the putative signal peptide at Ala‐26 would result in a mature, periplasmic cytochrome of Mr 50580 rather than a larger hexahaem cytochrome, as has been widely reported previously. A cytochrome of this size was detected by staining SDS‐polyacryla‐mide gels for covalently bound haem. This cytochrome was partially purified by anion exchange chromatography and confirmed to be cytochrome C552 by difference spectroscopy. Similar cytochromes were detected in five other E. coli strains including strain ST 249, which was used previously to purify and characterize the protein. A plasmid with an in‐phase deletion within nrfA directed the synthesis of a truncated haemoprotein of the predicted mass. In‐phase translational fusions to lacZ were used to locate the nrfA translation start, and the transcription start site was found by S1 mapping.Expression from the FNR‐dependent nrfA promoter was almost totally repressed during aerobic growth, partially induced during anaerobic growth in the absence of nitrite or in the presence of nitrate, but fully induced only during anaerobic growth in the presence of nitrite. No nitrate repression was detected in a narL mutant, but nitrite induction was unaffected, indicating that the nitrite‐sensing mechanism is independent of the NarL protein. Expression from the nrfA promoter was subject to glucose repression but regulation was independent of the CRP‐cAMP complex.
Activation of potassium channels during metabolite detoxification in Escherichia coli
Authors: Ferguson Gail P. , Munro Andrew W. , Douglas Roseileen M. , McLaggan Debra , Booth Ian R. 
Published: October 2006

SummaryIn bacteria the detoxification of compounds as diverse as methylglyoxal and chlorodinitrobenzene proceeds through the formation of a glutathione adduct. In the Gram‐negative bacteria, e.g. Escherichia coli, such glutathione adducts activate one, or both, of a pair of potassium efflux systems KefB and KefC. These systems share many of the properties of cation‐translocating channels in eukaryotes. The activity of these systems has been found to be present in a range of Gram‐negative bacteria, but not in the glutathione‐deficient species of Gram‐positive organisms. The conservation of the activity of these systems in a diverse range of organisms suggested a physiological role for these systems. Here we demonstrate that in E. coli cells activation of the KefB efflux system is essential for the survival of exposure to methylglyoxal. Methylglyoxal can be added to the growth medium or its synthesis can be stimulated in the cytoplasm. Under both sets of conditions survival is aided by the activity of KefB. Inhibition of KefB activity by the addition of 10 mM potassium to the growth medium stimulates methylglyoxal‐induced cell death. This establishes an essential physiological function for the KefB system.
Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression
Authors: Strom A. R. , Kaasen I. 
Published: October 2006

SummaryEndogenously synthesized trehalose is a stress protectant in Escherichia coli. Externally supplied trehalose does not serve as a stress protectant, but it can be utilized as the sole source of carbon and energy. Mutants defective in trehalose synthesis display an impaired osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary‐phaseinduced heat tolerance. Mechanisms for stress protection by trehalose are discussed. The genes for trehalose‐6‐phosphate synthase (otsA) and anabolic trehalose‐6‐phosphate phosphatase (otsB) constitute an operon. Their expression is induced both by osmotic stress and by growth into the stationary phase and depend on the sigma factor encoded by rpoS (katF). rpoS is amber‐mutated in E. coli K‐12 and its DNA sequence varies among K‐12 strains. For trehalose catabolism under osmotic stress E. coli depends on the osmoticcally inducible periplasmic trehalase (TreA). In the absence of osmotic stress, trehalose induces the formation of an enzyme IITre (TreB) of the group translocation system, a catabolic trehalose‐6‐phosphate phosphatase (TreE), and an amylotrehalase (TreC) which converts trehalose to free glucose and a glucose polymer.
Spatial and temporal controls of the Aspergillus brlA developmental regulatory gene
Authors: Aguirre Jesus 
Published: October 2006

SummaryThe Aspergillus nidulans brlA gene encodes a transcriptional regulator of central importance in controlling conidiophore development. I have determined the effects of mutations in other developmental regulatory genes on expression of a brlA‐lacZ fusion gene. Deletion of brlA reduced ß;‐galactosidase levels by half and led to delocalization of enzyme accumulation. The medA 26 and abaA2 developmental mutations led to overexpression of the fusion gene without altering spatial specificity. In contrast, the stuA1 mutation did not affect the timing or levels of brlA expression during induction, but Instead resulted in spatial derangement of expression. These results and the phenotypes of the mutants suggest a model in which subsets of morphogenetic loci are controlled by differing levels and combinations of regulatory gene products, which are themselves determined by interactions among the regulatory genes.
The Escherichia coli cAMP receptor protein (CRP) represses the Rhizobium meliloti dctA promoter in a cAMP‐dependent fashion
Authors: Wang Yi‐Ping , Giblin Linda , Boesten Bert , O'Gara Fergal 
Published: October 2006

SummaryThe expression of the Rhizobium meliloti C4‐dicarboxytic acid permease gene (dctA) is controlled by the sensor DctB and the transcriptional regulator, DctD. The R. meliloti Dct system has been reconstituted in Escherichia coli. Expression of the dctA promoter is DctBD dependent and is induced in the presence of C4‐dicarboxyrtc acids (dCA). Other carbon sources also influence dctA expression. We demonstrate that the cAMP receptor protein (CRP) has a repressive effect on the dctA promoter. A mutated CRP molecule (CRP‐H159L), unable to activate catabolic promoters (but still proficient in ONA binding), gives similar results. This suggests that the CRP‐cAMP complex represses the dctA promoter activity by direct interaction with the DNA. Direct binding of the CRP‐cAMP complex to the dctA promoter was confirmed in vitro by gel mobility‐shift assays. Sequence analysis of the dctA promoter indicates that the most likely binding sites for CRP are the two confirmed DctD‐binding sites. It is proposed that the CRP‐cAMP complex competes with DctD for occupancy of these sites. Since in the presence of CRP‐cAMP complex the uninduced levels of dctA expression are reduced, whereas induced levels are largely unaffected, such competition appears to be an essential regulatory feature of dctA expression.
Phospholipid substitution of capsular polysaccharides and mechanisms of capsule formation in Neisseria meningitidis
Authors: Frosch Matthias , Müller Astrid 
Published: October 2006

SummaryWithin the capsule gene complex (cps) of Neisseria meningitidis two functional regions B and C are involved in surface translocation of the cytoplasmically synthesized capsular polysaccharide, which is a homopolymer of α‐2,8 polyneuraminic acid. The region‐C gene products share characteristics with transporter proteins of the ABC (ATP‐binding cassette) superfamily of active transporters. For analysis of the role of region B in surface translocation of the capsular polysaccharide we purified the polysaccharides of region B‐ and region C‐defective Escherichia coli clones by affinity chromatography. The molecular weights of the polysaccharides were determined by gel filtration and the polysaccharides were analysed for phospholipid substitution by polyacrylamide gel electrophoresis and immunoblotting. The results indicate that the full‐size capsular polysaccharide with a phospholipid anchor is synthesized intracellularly and that lipid modification is a strong requirement for translocation of the poly saccharide to the cell surface. Proteins encoded by region B are involved in phospholipid substitution of the capsular polysaccharide. Nucleotide sequence analysis of region B revealed two open reading frames, which encode proteins with molecular masses of 45.1 and 48.7 kDa.
Orphan enzyme or patriarch of a new tribe: the arsenic resistance ATPase of bacterial plasmids
Authors: Silver Simon , Ji Guangyong , Bröer Stefan , Dey Saibal , Dou Dexian , Rosen Barry P. 
Published: October 2006

SummaryThe plasmid‐determined arsenite and antimonite efflux ATPase of bacteria differs from other membrane transport ATPases, which are classified into several families (such as the F0F1‐type H+‐translocating ATP synthases, the related vacuolar H+‐translocating ATPases, the P‐type cation‐translocating ATPases, and the superfamily which includes the periplasmic binding‐protein‐dependent systems in Gram‐negative bacteria, the human multidrug resistance P‐glycoprotein, and the cystic fibrosis transport regulator). The amino acid sequences of the components of the arsenic resistance system are not similar to known ATPase proteins. New findings with the arsenic resistance operons of bacterial plasmids suggest that instead of being an orphan the Ars system will now be the first recognized member of a new class of ATPases. Furthermore, fundamental questions of energy‐coupling (ATP‐driven or chemiosmotic) have recently been raised and the finding that the arsC gene product is a soluble enzyme that reduces arsenate to arsenite changes the previous picture of the functioning of this widespread bacterial system.
Expression of listeriolysin and phosphatidylinositol‐specific phospholipase C is repressed by the plant‐derived molecule cellobiose in Listeria monocytogenes
Authors: Park Simon F. , Kroll Rohan G. 
Published: October 2006

SummaryThe primary habitat of the intracellular pathogen Listeria monocytogenes is considered to be soil and decaying vegetation. As an opportunistic pathogen it must be able to recognize its entry into host tissue and, in response, co‐ordinately induce the expression of virulence factors. No signature molecule, which facilitates this regulation, has been identified for any human pathogen. Our studies have demonstrated for the first time that the expression of major virulence determinants in L. monocytogenes can be repressed by an environmentally ubiquitous molecule. Transcriptional hlyA and plcA fusions to luxAB were used to monitor virulent gene expression in the presence of various disaccharides. These studies revealed that the expression of listeriolysin O and phosphatidylinositol‐specific phospholipase C is repressed specifically by the plant‐derived disaccharide, cellobiose.
Phosphate starvation and low temperature as well as ultraviolet irradiation transcriptionally induce the Escherichia coli LexA‐controlled gene sfiA
Authors: Dri Anna‐Maria , Moreau Patrice L. 
Published: October 2006

SummaryThe LexA repressor controls the expression of several SOS genes, such as lexA, recA and sfiA, which are induced by DNA damage. Induction results from the activation of the RecA protein that favours the cleavage and thus the inactivation of LexA. It has been shown that the activation of RecA results from its binding to damaged DNA. It is therefore believed that in growing bacteria, in the absence of any DNA‐damaging treatment, the intracellular level of LexA remains stable at a high basal; level and, hence, SOS genes are expressed at relatively low basal levels. In contrast, we show here that the intracellular level of LexA and the rate of transcription of the sfiA gene may vary markedly throughout the growth cycle of wild‐type Escherichia coli. We provide evidence that such changes result from two superimposed processes: proteolytic cleavage of LexA upon dilution of stationary phase bacteria, and increase in strength of the promoters of the lexA and sfiA genes when bacteria approach the stationary phase. We show that a signal which strongly increases the strength of the SfiA gene promoter is starvation for phosphate. Such induction was not significantly affected by mutations either in phoB (encoding the transcriptional regulator for the phosphate regulon) or rpoS (encoding a putative stationary phase‐specific sigma factor). However, sfiA induction by phosphate starvation appeared to be markedly inhibited by the presence of the osmZ205 mutation which alters the histone‐like protein H‐NS, suggesting that changes in the DNA structure may play a role in signal transduction during phosphate starvation. As previously shown for several processes which are controlled by H‐NS, induction of sfiA was modulated by growth temperature.
Structural heterogeneity of the emm gene cluster in group A streptococci
Authors: Hollingshead Susan K. , Readdy Terri L. , Li Yung Der , Bessen Debra E. 
Published: October 2006

SummaryOne or more distinct copies of emm genes lie within a gene cluster that is located downstream from a transcriptional regulatory gene (mry). Mry is a positive regulator for the genes in this cluster and for the downstream gene, scpA. The objective of this study is to examine the structure of this cluster and the distribution of specific alleles within the cluster among group A streptococcal isolates of 32 different serotypes. The peptidoglycan (PG)‐spanning domain, which exists in four divergent forms, was used to identify specific alleles of the genes within the emm cluster. Gene content of the cluster was determined by Southern hybridization with allele‐specific oligonucleotides. Five different chromosomal patterns for this cluster were observed. Sequence heterogeneity in the adjacent mry locus was demonstrated by the ability of some of the isolates to hybridize with a whole mry gene probe, but not with mry‐based oligonucleotide probes. A PCR‐based chromosomal mapping technique was used to examine further the gene order within the emm gene clusters. Structural heterogeneity of the emm gene cluster was found within class I isolates in this study, while class II isolates were relatively homogeneous at this chromosomal locus and distinct from class I.
Growth and translation elongation rate are sensitive to the concentration of EF‐Tu
Authors: Tubulekas Ioannis , Hughes Diarmaid 
Published: October 2006

SummaryWe have used quantitative immunoblotting to estimate the amount of EF‐Tu in a variety of S. typhimurium strains with wild‐type, mutant, insertionally inactivated or plasmid‐borne tuf genes. In the same strains we have measured translation elongation rate, exponential growth rate and the level of nonsense codon readthrough. In the wild‐type strain, at moderate to fast growth rates, our data show that EF‐Tu makes up 8–9% of total cell protein. Strains with either of the tuf genes insertionally inactivated have 65% of the wild‐type EF‐Tu level, irrespective of which tuf gene remains active, or whether that gene is wild‐type or a kirromycin‐resistant mutant. Strains with only one active tuf gene have reduced growth and translation elongation rates. From the magnitude of the reduction in elongation rate relative to the level of EF‐Tu we calculate that in glucose minimal medium the in vivo saturation level of wild‐type ribosomes by ternary complexes is only 63%. Strains with a ribosome mutation causing a poor interaction with ternary complex are non‐viable on minimal medium when the level of EF‐Tu is reduced.
Characterization of the srfA locus of Bacillus subtilis: only the valine‐activating domain of srfA is involved in the establishment of genetic competence
Authors: van Sinderen Douwe , Galli Giuliano , Cosmina Paola , de Ferra Francesca , Withoff Sebo , Venema Gerard , Grandi Guido 
Published: October 2006

Summary srfA is a locus required for the production of the lipopeptide antibiotic surfactin. This locus is also necessary for efficient sporulation and competence development. Mutations in the 5′ portion of the srfA operon affect all three of these processes, whereas mutations in the 3′ portion of srfA only affect sporulation and surfactin production. Analysis of the proteins encoded by the srfA locus revealed seven large domains which are likely to be responsible for the activation and binding of the seven amino acids of surfactin. Identification of the amino acid that is activated by the srfA domains was determined by amino acid‐dependent pyrophosphate exchange reactions on partially purified cell extracts of strains carrying different srfA mutations. These results indicate colin‐earity between the order of the domains in the srfA locus and the amino acid sequence of surfactin. The minimal genetic element of srfA required for the establishment of competence was shown to be the 5′ region of the second open reading of srfA, which encodes the valine activation domain. This portion of srfA, when cloned on a plasmid, complemented the competence deficiency of a srfA deletion mutant in trans.
Ligand‐based histochemical localization and capture of cells expressing heat‐stable enterotoxin receptors
Authors: Almenoff June S. , Williams Sabrina I. , Scheving Lawrence A. , Judd Amrit K. , Schoolnik Gary K. 
Published: October 2006

SummaryThe heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors. Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology. To accomplish this, we quantitatively coupled biotin to the N‐terminus of ST1b using biotin‐X‐X‐N‐hydroxysuccinimide ester. The derivatized toxin (BST) has an apparent Kd of 11.7±10 nM for rat brush border receptors. We used BST in an affinity panning cell‐capture system, to validate its ability to discriminate between receptor‐positive and receptor‐negative cells. Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase‐C (GC‐C) ST receptor cDNA) were captured to streptavidin and anti‐biotin‐coated plates with high efficiency and specificity. This system provides a novel approach to screening cells for the presence of unique ST‐binding proteins. BST was then used with streptavidin‐gold to demonstrate the cellular topography of ST receptors at the light microscopic level. Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine. Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes. Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes. Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts. Thus, ST receptors, are concentrated in villus entercoytes, while guanylin appears to be produced at the base of the crypts. This topographical arrangement suggests that there are autocrine or paracrine pathways by which ST receptors interact with endogenous ligands.
Bacillus subtilis PrsA is required in vivo as an extracytoplasmic chaperone for secretion of active enzymes synthesized either with or without pro‐sequences
Authors: Jacobs M. , Andersen J. B. , Kontinen V. , Sarvas M. 
Published: October 2006

SummaryIn prsA (protein secretion) mutants of Bacillus subtilis, decreased levels of exoproteins, including α‐amylase and subtilisins, are found extracellularly. The effect of prsA on subtilisin secretion is elaborated here. Extracytoplasmic folding and secretion of active subtilisin is assisted by the N‐terminal pro‐sequence of its precursor. In this paper we present evidence that the product of the prsA gene is additionally required for these processes in vivo. We examined inducible expression of different subtilisin‐alkaline phosphatase fusion genes in the prsA3 mutant. We found massive degradation of the fusion proteins, and a lack of enzymatic activity in the protein secreted. We suggest that PrsA is a novel chaperone with a predicted extracytoplasmic location, and is important in vivo for the proper conformation of various exoproteins, including those with pro‐sequence (like subtilisin) and those without (like α‐amylase).
Modification of tRNA as a regulatory device
Authors: Persson Britt C. 
Published: October 2006

SummaryOur knowledge of the different biological roles of tRNA modification has increased considerably in recent years. Not only have we learned about how modified nucleosides affect the performance of tRNA in translation, but also how they influence regulation of intermediary metabolism, antibiotics production, gene expression in eukaryotic viruses, cell division, cell‐cycle control, u.v. sensitivity, and mutation frequency. This review summarizes our current understanding of the role of tRNA modification.