bluuebunny/crossref_metadata_2025 huggingface dataset:
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

The 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

Within 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

The 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

The 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

The 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

One 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

We 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

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

The 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

In 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

Our 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.
Tn5‐mediated bleomycin resistance in Escherichia coli requires the expression of host genes
Authors: Blot Michel , Heitman Joseph , Arber Werner
Published: October 2006

The transposon Tn5 expresses a gene, ble, whose product increases the viability of Escherichia coli and also confers resistance to the DNA‐cleaving antibiotic bleomycin and the DNA‐alkylating agent ethyl‐methanesulphonate. We find that the Ble protein induces expression of an alkylation inducible gene, aidC, and that both the AidC gene product and DNA polymerase I are required for Ble to confer bleomycin resistance. These findings support models in which Ble enhances DNA repair and suggest that Tn5 confers a fitness advantage to the host bacterium by increasing the repair of spontaneous DNA lesions. Such co‐operation between a transposon and its host suggests that Tn5 is a symbiotic rather than a selfish DNA element.
Physical organization of lipids in the cell wall of Mycobacterium chelonae
Authors: Nikaido Hiroshi , Kim Sung‐Hou , Rosenberg Emiko Y.
Published: October 2006

Mycobacterial cell wall functions as an effective permeability barrier, making these bacteria resistant to most antibacterial agents. It has been assumed that this low permeability was due to the presence of a large amount of unusual lipids in the cell wall, but it was not known how these lipids are able to produce such an exceptional barrier. We report here the first experimental evidence on the physical arrangement of these lipids based on X‐ray diffraction studies of purified Mycobacterium chelonae cell wall, a result suggesting that the hydrocarbon chains of the cell‐wall lipids are arranged predominantly in a direction perpendicular to the cell wall surface, probably producing an asymmetric bilayer structure.
Molecular genetic analysis of the moa operon of Escherichia coli K‐12 required for molybdenum cofactor biosynthesis
Authors: Rivers S. L. , McNairn E. , Blasco F. , Giordano G. , Boxer D. H.
Published: October 2006

A 3.2 kb chromosomal DNA fragment which complements the defects in a series of twelve moa::Mucts insertion mutants has been sequenced. Five open reading frames (ORFs) were identified and these are arranged in a manner consistent with their forming an operon. The encoded proteins (MoaA‐MoaE) have predicted molecular weights of 37346, 18665, 17234, 8843 and 16981 respectively. Examination of subclones of the whole locus in an expression system demonstrated the predicted products. N‐terminal amino acid sequences for the moa A, B, C and E products confirmed the translational starts. Genetic analysis distinguished four classes of moa mutants corresponding to genes moaA, C, D and E. Potential promoter sequences upstream of moaA and a possible transcription termination signal have been identified. Genetic analysis of the chlA1 and chlM mutants, which have been biochemically characterized as defective in molybdopterin biosynthesis, indicates that these carry lesions in moaA and moaD respectively. The moa locus is orientated clockwise at 17.7 minutes in the chromosome.
The obligate intracellular bacterium Chlamydia trachomatis is auxotrophic for three of the four ribonucleoside triphosphates
Authors: Tipples Graham , McClarty Grant
Published: October 2006

Using we11‐characterized mutant host cell lines, deficient in specific enzymes of energy and nucleotide metabolism, we addressed numerous questions regarding nucleotide metabolism in the obligate intracellular bacterium Chlamydia trachomatis. The results presented indicate that C. trachomatis: (i) does not absolutely depend on mitochondrial generated ATP for survival; (ii) does have a significant draw on host‐cell NTP pools but does not have a detrimental effect on the ability of the host cell to maintain its energy charge; (iii) lacks the ability to synthesize purine and pyrimidine nucleotides de novo; (iv) is not capable of interconverting purine nucleotides; and (v) possesses the pyrimidine metabolic‐pathway enzymes CTP synthetase and deoxycytidine nucleotide deaminase. In total our results indicate that C. trachomatis is auxotrophic for host‐cell ATP, GTP and UTP. In contrast, CTP can be obtained from the host cell or it can be synthesized from UTP by the parasite.
ATPase activity and ATP/ADP‐induced conformational change in the soluble domain of the bacterial protein translocator HlyB
Authors: Koronakis Vassilis , Hughes Colin , Koronakis Eva
Published: October 2006

The haemolysin exporter HlyB and its homologues are central to the unconventional signal‐peptide‐independent secretion of toxins, proteases and nodulation proteins by bacteria. HlyB is a member of the ATP‐binding cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug‐resistance P‐glycoprotein, combining both integral membrane and cytosolic domains. Overproduction of the HlyB cytopiasmic domain as a C ‐terminal peptide fused to glutathione S‐transferase allowed the direct affinity purification and concentration of 30‐50 mg ml−1 of soluble protein (GST‐Bctp) in an apparently dimeric form possessing both transferase and ATPase activity. GST‐Bctp bound to ADP‐agarose and was eluted specifically by ATP and ADP, affinity behaviour which was confirmed in both the full‐length HlyB and the unfused HlyB cytoplasmic domain synthesized in vitro. The stoichiometry of binding to MgATP and MgADP was close to equimolar and both ligands induced substantial conformational change in the protein. Mg2+ ‐dependent ATPase activity of GST‐Bctp Vmax 17mu;mol min−1 mg−1, Km 0.2 mM) was comparable with the activity of the bacterial importer MalK and human P‐glycoprotein reconstituted into proteoliposomes, and over an order of magnitude higher than in vitro measurements of disaggregated MalK purified from inclusion bodies. Activity was unaffected by inhibitors of F‐ and V‐type ATPases, non‐hydrolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P‐type) ATPases, and the acidic phospholipid phosphatidyl glycerol.
The role of mRNA degradation in the regulated expression of bacterial photosynthesis genes
Authors: Klug Gabriele
Published: October 2006

Regulation of gene expression in bacteria, as in eukaryotic cells, is often achieved by variation of mRNA levels. Since the steady state levels of mRNA depend on both the rate of synthesis and the rate of decay, both mechanisms are important for gene regulation. After considerable effort undertaken over many years to understand the regulation of transcription, mRNA degradation has recently gained Increasing attention as an important step in the regulation of some bacterial genes, and many investigations have addressed the mechanisms involved in mRNA decay. The puf mRNA of Rhodobacter capsulatus encoding pigment binding proteins has become a model system to study decay of a polycistronic mRNA and the role of mRNA degradation in gene expression. Individual segments of the polycistronic puf mRNA display extremely different half‐lives. These differences in stability of mRNA segments are involved in the differential expression of puf encoded genes and consequently contribute to the stoichiometry of light‐harvesting I and reaction centre complexes that results in optimal growth. In addition, control of mRNA stability is involved in the oxygen‐dependent regulation of photosynthesis genes. High oxygen tension results in decreased stability of the reaction‐centre specific puf mRNA segment, most likely by affecting the rate of endonucleolytic cleavage within the reaction centre coding region. The results obtained from studying puf mRNA degradation in R. capsulatus and Escherichia coli suggest that a specific distribution of decay promoting and decay impeding mRNA elements along the polycistronic mRNA is responsible for the different half‐lives of individual puf segments.
Adaptation of Escherichia coli to redox environments by gene expression
Authors: Iuchi S. , Lin E. C. C.
Published: October 2006

Escherichia coli is adroit in exploiting environmental energy sources to its greatest profit. A key strategy is to channel electron transport from donors to a terminal acceptor(s) so that the voltage drop is maximal. At the level of transcription, the goal is achieved by the interaction of three global regulatory systems, Fnr, NarL/NarX and ArcB/ArcA. In addition, the regulator FhlA is involved in a cascade‐controlled pathway for the formate branch of the pyruvate fermentation pathway.
Molecular cloning and characterization of a sym plasmid locus that regulates cultivar‐specific nodulation of soybean by Rhizobium fredii USDA257
Authors: Meinhardt L. W. , Krishnan H. B. , Balatti P. A. , Pueppke S. G.
Published: October 2006

Rhizobium fredii strain USDA257 produces nitrogen‐fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposonmutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain U5DA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolWand nolX are of the opposite polarity. The initiation codon for nolW lies 155bp upstream from that of nolB, and it is separated from nolXby 281 bp. The predicted NolT and NolW proteins have putative membrane‐spanning regions. The N‐terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site‐directed mutagenesis with mudll1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU anti nolX is induced as much as 30‐fold by flavonoid signal molecules, even though these genes lack nod‐box promoters. Histochemical staining of McCall roots inoculated with nolB–, nolU–, or nolX–lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU–ORF4–nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad‐host‐range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.
S‐layer protein from Thermus thermophilus HB8 assembles into porin‐like structures
Authors: Castón José R. , Berenguer José , de Pedro Miguel A. , Carrascosa José L.
Published: October 2006

The cells of the extreme thermophile Thermus thermophilus are surounded by a regular layer (S‐layer) built up by a protein with an apparent molecular mass of 100 kDa (P100). From purified membrane fractions, three different class of two‐dimensional crystals can be obtained by following alternative extractive procedures. One of these crystals, with p6 symmetry, clearly represents the native S‐layer detected by freeze etching on whole cells, while the other two, showing p2 and p3 symmetries respectively, closely resemble aggregates of bacterial porins. We demonstrate here by limited protreolysis and Western blotting the surprising fact that the protein component of the three crystals is the P100 protein. Our biochemical data also show how this protein forms Ca2+‐stabilized trimers in each crystal, which support the structural analysis that points to p3 units as the common structural block in all of them, and again resembles the situation found in bacterial porins.
Cloning and nucleotide sequence of frpC, a second gene from Neisseria meningitidis encoding a protein similar to RTX cytotoxins
Authors: Thompson Stuart A. , Wang Lisa L. , Sparling P. Frederick
Published: October 2006

Neisseria meningitidis FAM20 has recently been shown to produce two Fe‐regulated proteins (FrpA and FrpC) related to the RTX family of cytotoxins. Here we report the cloning and DNA sequence of the locus containing the gene encoding the larger meningococcal RTX protein FrpC. FrpC was highly similar to FrpA throughout much of the predicted protein, with two main differences. Whereas the FrpA protein had 13 copies of the nine‐amino‐acid repeat units typical of RTX proteins, FrpC had 43 copies. The additional copies in FrpC apparently arose from a threefold tandem amplification of a 600bp DNA fragment encoding the repeats. In addition, the frpC gene lacked good promoter consensus sequences. An open reading frame (0RF1) of unknown function was found immediately upstream of frpC, suggesting the possibility that frpC was cotranscribed with ORF1. A probable promoter was found 300 bp upstream of ORF1, and it contained a Fur protein‐binding sequence found in the promoters of Fe‐regulated Escherichia coli genes. DNA upstream of the ORF 1/frpC promoter was homologous to IStO76‐like elements surrounding capsulation loci of strains of Haemophilus influenzae. A FrpC‐like protein (reactive in immunoblots with monoclonal antibody 9D4; multiple reactive bands of about 200 to 120kDa) was found in five out of eight meningococcal strains but only in one out of 14 other Neisseria, suggesting that FrpC may participate in the pathogenesis of meningococcal disease.
Characterization of the cspB gene encoding PS2, an ordered surface‐layer protein in Corynebacterium glutamicum
Authors: Peyret J. L. , Bayan N. , Joliff G. , Gulik‐Krzywicki T. , Mathieu L. , Shechter E. , Leblon G.
Published: October 2006

PS2 is one of two major proteins detected in the culture media of various Corynebacterium glutamicum strains. The coding and promoter regions of the cspB gene encoding PS2 were cloned in lambda gt11 using polyclonal antibodies raised against PS2 for screening. Expression of the cspB gene in Escherichia coli led to the production of a major anti‐PS2 labelled peptide of 63 000 Da, corresponding presumably to the mature form of PS2. It was detected in the cytoplasm, periplasm and surrounding medium of E. coli. Three other slower migrating bands of 65000, 68 000 and 72 000 Da were detected. The largest one probably corresponds to the precursor form of PS2 in E. coli. Analysis of the nucleotide sequence revealed an open reading frame (ORF) of 1533 nucleotides. The deduced 510‐amino‐acid polypeptide had a calculated molecular mass of 55 426 Da. According to the predicted amino acid sequence, PS2 is synthesized with a W‐terminal segment of 30‐amino‐acid residues reminiscent of eukaryotic and prokaryotic signal pep‐tides, and a hydrophobic domain of 21 residues near the C‐terminus. Although no significant homologies were found with other proteins, it appears that some characteristics and the amino acid composition of PS2 share several common features with surface‐layer proteins. The cspB gene was then disrupted in C. glutamicum by gene replacement. Freeze‐etching electron microscopy performed on the wild‐type strain indicated that the cell wall of C. glutamicum is covered with an ordered surface of proteins (surface layer, S‐layer) which is in very close contact with other cell‐wall components. These structures are absent from the cspB‐disrupted strain but are present after reintroduction of the cspB gene on a plasmid into this mutant. Thus we demonstrate that the Slayer protein is the product of the cspB gene.
Complementation of an Enterococcus hirae (Streptococcus faecalis) mutant in the alpha subunit of the H+‐ATPase by cloned genes from the same and different species
Authors: Suzuki Tomoko , Shibata Chiyoko , Yamaguchi Akie , Igarashi Kazuei , Kobayashi Hiroshi
Published: October 2006

We isolated an Enterococcus hirae (formerly Streptococcus faecalis) mutant, designated MS117, in which ‘G’ at position 301 of the alpha‐subunit gene of the F1F0 type of H+‐ATPase was deleted. MS117 had low H+‐ATPase activity, was deficient in the regulatory system of cytoplasmic pH, and was unable to grow at pH6.0. When the alpha‐subunit gene of E. hirae H+‐ATPase was ligated with the shuttle vector pHY300PLK at the downstream region of the tet gene of the vector, it was expressed without its own promoter in MS117, and the mutation of MS117 was complemented; the mutant harbouring the plasmid had the ability to maintain a neutral cytoplasm and grew at pH6.0. We next transformed MS117 with pHY300PLK containing the alpha‐subunit gene of Bacillus megaterium F1F0‐ATPase constructed in the same way. The transformant grew at pH 6.0, and the ATP hydrolysis activity was recovered. These results suggested that an active hybrid H+‐ATPase containing the B. megaterium alpha subunit was produced, and that the hybrid enzyme regulated the enterococcal cytoplasmic pH, although the function of the B. megaterium enzyme did not include pH regulation. Thus, our present results support the previous proposal that the enterococcal cytoplasmic pH is regulated by the F1F0 type of H+‐ATPase.
Conservation of motifs within the unusually variable polypeptide sequences of type I restriction and modification enzymes
Authors: Murray Noreen E. , Daniel Anne S. , Cowan Gill M. , Sharp Paul M.
Published: October 2006

Type I restriction enzymes comprise three subunits encoded by genes designated hsdR, hsdM, and hsdS; S confers sequence specificity. Three families of enzymes are known and within families, but not between, hsdM and hsdR are conserved. Consequently, interfamily comparisons of M and R sequences focus on regions of putative functional significance, while both inter‐ and intrafamily comparisons address the origin, nature and role of diversity of type I restriction systems. We have determined the sequence of the hsdR gene for EcoA, thus making available sequences of all three hsd genes of one representative from each family. The predicted R polypeptide sequences share conserved regions with one superfamily of putative helicases, so‐called ‘DEAD box’ proteins; these conserved sequences may be associated with the ATP‐dependent translocation of DNA that precedes restriction. We also present hsdM and hsdR sequences for EcoE, a member of the same family as EcoA. The sequences of the M and R genes of EcoA and EcoE are at least as divergent as typical genes from Escherichia coli and Salmonella, perhaps as the result of selection favouring diversity of restriction specificities combined with lateral transfer among different species.
Regulation of the ansB gene of Salmonella enterica
Authors: Jennings Michael P. , Scott Shaun P. , Beacham Ifor R.
Published: October 2006

The expression of l‐asparaginase II (encoded by ansB) in Salmonella enterica was found to be positively regulated by the cAMP receptor protein (CRP) and anaerobiosis. The anaerobic regulation of the S. enterica ansB gene is not mediated by the anaerobic transcriptional activator FNR. This is unlike the situation of the ansB gene of Escherichia coli, which is dependent on both CRP and FNR. To investigate this fundamental difference in the regulation of l‐asparaginase II expression in S. enterica, the ansB gene was cloned and the nucleotide sequence of the promoter region determined. Sequence analysis and transcript mapping of the 5′ promoter region revealed a single transcriptional start point (tsp) and two regulatory sites with substantial homology with those found in E. coli. One site, centred –90.5 bp from the tsp, is homologous to a hybrid CRP/FNR (‘CF’) site which is the site of CRP regulation in the E. coli promoter. The other site, centred 40.5 bp upstream of the tsp, is homologous to the FNR binding site of the E. coli promoter. Significantly, however, a single base‐pair difference exists in this site, at a position of the related CRP and FNR DNA‐binding site consensus sequences known to be involved in CRP versus FNR specificity. Site‐directed mutagenesis indicates that this single difference, relative to the homologous E. coli site, results in a CRP binding site and the observed FNR‐independent ansB expression in S. enterica. Thus, not only may CRP and FNR sites be interconverted experimentally, with few nucleotide changes, but this has apparently contributed to the evolutionary divergence of ansB regulation in E. coli and S. enterica.
Analysis of diphtheria toxin repressor‐operator interactions and characterization of a mutant repressor with decreased binding activity for divalent metals
Authors: Schmitt Michael P. , Holmes Randall K.
Published: October 2006

The diphtheria toxin repressor (DtxR) is an Fe2+‐activated protein with sequence‐specific DNA‐binding activity for the diphtheria toxin (tox) operator. Under high‐iron conditions in Corynebacterium diphtheriae, DtxR represses toxin and siderophore biosynthesis as well as iron uptake. DtxR and a mutant repressor with His–47 substituted for Arg–47, designated DtxR‐R47H, were purified and compared. Six different divalent cations (Cd2+, Co2+, Fe2+, Mn2+, Ni2+, and Zn2+) activated the sequence‐specific DNA‐binding activity of DtxR and enabled it to protect the fox operator from DNase I digestion, but Cu2+ failed to activate DtxR. Hydroxyl radical footprinting experiments indicated that DtxR binds symmetrically about the dyad axis of the tox operator. Methylation protection experiments demonstrated that DtxR binding alters the susceptibility to methylation of three G residues within the AT‐rich tox operator. These findings suggest that two or more monomers of DtxR are involved in binding to the tox operator, with symmetrical DNA‐protein interactions occurring at each end of the palindromic operator. In this regard, DtxR resembles several other well‐characterized prokaryotic repressor proteins but differs dramatically from the Fe2+‐activated ferric uptake repressor protein (Fur) of Escherichia coli. The concentration of Co2+ required to activate DtxR‐R47H was at least 10‐foid greater than that needed to activate DtxR, but the sequence‐specific DNA binding of activated DtxR‐R47H was indistinguishable from that of wild‐type DtxR. The markedly deficient repressor activity of DtxR‐R47H is consistent with a significant decrease in its binding activity for divalent cations.
Structure–function correlation for the EcoRV restriction enzyme: from non‐specific binding to specific DNA cleavage
Authors: Vipond I. Barry , Halford Stephen E.
Published: October 2006

The EcoRV restriction endonuclease cleaves DNA at its recognition sequence at least a million times faster than at any other DNA sequence. The only cofactor it requires for activity is Mg2+: but in binding to DNA in the absence of Mg2+, the EcoRV enzyme shows no specificity for its recognition site. Instead, the reason why EcoRV cuts one DNA sequence faster than any other is that the rate of cleavage is controlled by the binding of Mg2+ to EcoRV‐DNA complexes: the complex at the recognition site has a high affinity for Mg2+, while the complexes at other DNA sequences have low affinities for Mg2+. The structures of the EcoRV endonuclease, and of its complexes with either 8pecific or non‐specific DNA, have been solved by X‐ray crystallography. In the specific complex, the protein interacts with the bases in the recognition sequence and the DNA takes up a highly distorted structure. In the non‐specific complex with an unrelated DNA sequence, there are virtually no interactions with the bases and the DNA retains a B‐like structure. Since the free energy changes for the formation of specific and non‐specific complexes are the same, the energy from the specific interactions balances that required for the distortion of the DNA. The distortion inserts the phosphate at the scissile bond into the active site of the enzyme, where it forms part of the binding site for Mg2+. Without this distortion, the EcoRV–DNA complex would be unable to bind Mg2+ and thus unable to cleave DNA. The specificity of the EcoRV restriction enzyme is therefore governed, not by DNA binding as such, but by its ability to organize the structure of the DNA to which it is bound.
Double, independent mutational events in the rpsL gene of Escherichia coli: an example of hypermutability?
Authors: Timms Andrew R. , Bridges Bryn A.
Published: October 2006

A proportion (up to 20%) of newly arising streptomycin‐dependent (SmD) colonies of Escherichia coli WP2 contain bacteria where, in addition to a known SmD‐determining (primary) mutation in the rpsL gene, there is a further ancillary mutation in the same gene. These ancillary mutations occur at 10 sites between 11 and 201 bp away from the primary mutation. Ancillary mutations have been found in mutant colonies arising both spontaneously and after treatment with ultraviolet light and some have been found repeatedly. Ancillary mutations were frequently found to occur in mixed clones with an excess of bacteria carrying only the primary SmD mutation. No ancillary mutations were found in an adjacent non‐coding region and there were no coding sequence changes that did not alter the amino acid specified. Although a selective advantage for bacteria containing ancillary mutations could not be demonstrated directly in every case, some small advantage must be presumed to have occurred to explain the absence of mutations at the other sites and particularly at third (wobble) codon positions. Ancillary mutations appear to occur fairly early in the life of a newly arisen SmD mutant clone in some sort of hypermutable process. Whether they are noticed appears to depend on their conferment of some selective advantage on the bacteria carrying them. While the ancillary mutations within rpsL lie close to, and may be consequent upon the formation of a primary SmD mutation, their mechanism of formation appears to be at least to some extent independent and does not involve the recA or umuC genes. Ancillary mutations were also detected in bacteria lacking reverse transcriptase or transcription repair coupling factor.
Retarded RNA turnover in Escherichia coli: a means of maintaining gene expression during anaerobiosis
Authors: Georgellis Dimitris , Barlow Tamara , Arvidson Staffan , von Gabain Alexander
Published: October 2006

In this study, we extend earlier observations on the influence of growth rate on mRNA stability and rRNA processing in Escherichia coli during continuous culture, to the effect of anaerobiosis. During slow anaerobic growth (generation time 700 min) both ompA and bla mRNA had a prolonged half‐life compared to that during slow aerobic growth and the processing of 9S RNA was even more profoundly retarded, which indicated a general slowing of mRNA turnover. The latter was confirmed by a nearly fourfold increase in the functional half‐life of bulk mRNA. In spite of this difference in stability, steady state levels of RNA, as judged by those of the ompA and 9S transcripts, were the same in aerobic and anaerobic cells at a given growth rate. Furthermore, we found that RNA synthesis during anaerobiosis was a fraction of that observed during slow aerobic growth and it is proposed that this offsets the general increase in mRNA stability. Our data therefore suggest that a constant level of RNA is maintained by matching the rate of decay to the level of RNA synthesis.
FtsZ ring in bacterial cytokinesis
Authors: Lutkenhaus Joe
Published: October 2006

FtsZ is localized to a cytokinetic ring at the cell division site in bacteria. In this review a model is discussed that suggests that FtsZ self assembles into a ring at a nucleation site formed on the cytoplasmic membrane under cell‐cycle control. This model suggests that formation of the cytokinetic FtsZ ring initiates and coordinates the circumferential invagination of the cytoplasmic membrane and cell wall, leading to formation of the septum. It is also suggested that this process may be conserved among the peptidoglycan‐containing eubacteria. In addition, similarities between FtsZ and tubulin are discussed.
Translation elongation factor 3: a fungus‐specific translation factor?
Authors: Belfield Graham P. , Tuite Mick F.
Published: October 2006

Fungi appear to be unique in their requirement for a third soluble translation elongation factor. This factor, designated elongation factor 3 (EF‐3), was first described in the yeast Saccharomycescerevisiae and has subsequently been identified in a wide range of fungal species Including Candida albicans and Schizo‐saccharomyces pombe. EF‐3 exhibits ribosome‐dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome, but which are essential for translation elongation. Recent studies on the structure of EF‐3 from several fungal species have shown that it consists of a repeated domain, with each domain containing the expected putative ATP‐ and GTP‐binding motifs. Overall, EF‐3 shows striking amino acid similarity to members of the ATP‐binding Cassette (ABC) family of membrane‐associated transport proteins although EF‐3 is not itself directly membrane‐associated. Regions of the EF‐3 polypeptide also show structural homology with other translation‐associated factors including aminoacyl‐tRNA synthetases and the Escherichia coli ribosomal protein S5. While the precise role of EF‐3 in the translation elongation cycle remains to be defined, recent evidence suggests that it may be involved in optimizing accuracy during mRNA decoding at the ribosomal A site. Furthermore, the essential nature of EF‐3 with respect to the fungal cell indicates that it may be an effective antifungal target. Its apparently ubiquitous occurrence throughout the fungal kingdom also suggests that it may be a useful fungal taxonomic marker.
Assembly of the Escherichia coli F1F0 ATPase, a large multimeric membrane‐bound enzyme
Authors: Brusilow William S. A.
Published: October 2006

The F1F0 proton translocating ATPase of Escherichia coli is a large membrane‐bound enzyme complex consisting of more than 20 polypeptides that are encoded by the unc operon. Besides being a system for analysing the enzymology of ATP synthesis and energy coupling, the ATPase is a model system for determining how large oligomeric membrane‐bound proteins are synthesized and assembled. The assembly of the ATPase involves differential gene expression and assembly of the subunits within the membrane and with each other. This review discusses the influence of F1 subunits on the assembly and proton permeability of the F0 proton channel, and the possible advantages to assembly of the particular arrangement of genes in the unc operon.
Orientation of porin channels in the outer membrane of Bordetella pertussis
Authors: Kocsis E. , Trus B. L. , Steven A. C. , Smith P. R. , Hannah J. H. , Brennan M. J. , Kessel M.
Published: October 2006

We have examined the surface topography and channel connectivity of a naturally crystalline porin that is known to be functional, and whose structure has not been perturbed by detergent extraction, A three‐dimensional density map, calculated from two independent tilt series of negatively stained cell envelopes, reveals three separate channels per trimer on one side (the ‘smooth’ side), and a single common opening at the other (‘rough’) side. This arrangement is consistent with the molecular structures recently determined at high resolution by X‐ray crystallography for three other porins after detergent solubilization, and implies that the Bordetella pertussis porin may have the same kind of folding. Surface relief maps calculated from electron micrographs of cell envelopes contrasted by unidirectional shadowing clearly show that the side with single opening (i.e. the rough side) represents the external surface.
Erwinia stewartii WtsA, a positive regulator of pathogenicity gene expression, is similar to Pseudomonas syringae pv. phaseolicola HrpS
Authors: Frederick R. D. , Majerczak D. R. , Coplin D. L.
Published: October 2006

Erwinia Stewartii contains a large cluster of wts genes that are required by this bacterium for pathogenicity on com plants. Three complementation groups within the right half of this cluster, wtsA, wtsC, and ivtsB, were previously identified. In this study, WtsA was found to be a positive activator of ivfsB::lacZ expression. The WtsA locus was sequenced and a single open reading frame is present within the wtsA locus, which has the capacity to encode a 323 amino acid polypeptide. A corresponding 38kDa protein was observed in Escherichia coli minicells containing the cloned wtsA gene. The predicted WtsA polypeptide has significant similarity to HrpS from Pseudomonas syringae pv. phaseolicola, as well as other members of the NtrC class of prokaryotic regulatory proteins. Similar to other genes activated by NtrC regulators, wtsB::lacZ expression in E. coli was dependent upon rpoN.