bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane
Authors: van der Wolk J. , Klose M. , Breukink E. , Demel R. A. , de Kruijff B. , Freudl R. , Driessen A. J. M.
Published: October 2006

SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton‐motive‐force‐driven translocation across the cytoplasmic membrane. A putative ATP‐binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A‐type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine‐rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site‐directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild‐type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (kcat/km) of the K106N SecA for lipid‐stimulated ATP hydrolysis was only 1% of that of the wild‐type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild‐type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid mono‐layer, in contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton‐motive‐force‐dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino‐terminal domain of SecA is part of the catalytic ATP‐binding site. This site may be involved in the ATP‐driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.
Cell differentiation of Proteus mirabilis is initiated by glutamine, a specific chemoattractant for swarming cells
Authors: Allison Clive , Lai Hsin‐Chih , Gygi Daniel , Hughes Colin
Published: October 2006

Swarming by Proteus mirabilis involves differentiation of typical short vegetative rods into filamentous hyper‐flagellated swarm cells which undergo cycles of rapid and co‐ordinated population migration across surfaces and exhibit high levels of virulence gene expression. By supplementing a minimal growth medium (MGM) unable to support swarming migration we identified a single amino acid, glutamine, as sufficient to signal initiation of cell differentiation and migration. Bacteria isolated from the migrating edge of colonies grown for 8h with glutamine as the only amino acid were filamentous and synthesized the characteristic high levels of flagellin and haemolysin. In contrast, addition of the other 19 common amino acids (excluding glutamine) individually or in combination did not initiate differentiation even after 24 h, cells remaining typical vegetative rods with basal levels of haemolysin and flagellin. The glutamine analogue γ‐glutamyl hydroxamate (GH) inhibited swarming but not growth of P. mirabilis on glutamine MGM and transposon mutants defective in glutamine uptake retained their response to glutamine signalling and its inhibition by GH, suggesting that differentiation signalling by glutamine may be transduced independently of the cellular glutamine transport system. Levels of mRNA transcribed from the haemolysin (hpmA) and flagellin (fliC) genes were low in vegetative cells grown on MGM without glutamine or with glutamine and GH, but were specifically increased c. 40‐fold during glutamine‐dependent differentiation. In liquid glutamine—MGM cultures, differentiation to filamentous hyper‐flagellated hyper‐haemorytic swarm cells occurred early in the exponential phase of growth, and increased concomitantly with the concentration of glutamine from a 0.1 mM threshold up to 10 mM. Differentiation in liquid culture was completely inhibited by GH but was further stimulated c. 30% in the absence of GH by the viscosity agent polyvinylpy‐rollidone (PVP). Chemotaxis assays of bacterial cells in which the viscosity of liquid media was varied by PVP to allow either swimming or swarming motility demonstrated that glutamine was chemoattractive specifically to differentiated swarming cells.
Sensor and regulator proteins from the cyanobacterium Synechococcus species PCC7942 that belong to the bacterial signal‐transduction protein families: implication in the adaptive response to phosphate limitation
Authors: Aiba Hirofumi , Nagaya Masahiro , Mizuno Takeshi
Published: October 2006

A 1.2kb DNA fragment was cloned from Synechococcus sp. PCC7942, which is able phenotypicalty to complement a phoRcreC Escherichia coli mutant for the expression of alkaline phosphatase. A 2.5kb DNA fragment encompassing the putative gene was then cloned and its complete nucleotide sequence determined. Nucleotide sequencing revealed that the intact gene encodes a protein of 46389 Da, and that the deduced amino acid sequence shows a high degree of homology to those of the bacterial sensory kinase family. In the determined nucleotide sequence, another gene was adjacently located, which encodes a protein of 29012Da. This protein shows a high degree of homology to those of the response regulator family. Thus, we succeeded in the cloning of a pair of genes encoding the sensory kinase and response regulator, respectively, in a cyanobacterium. Mutant strains that lack these genes were constructed, and demonstrated to be defective in their ability to produce alkaline phosphatase and some inducible proteins in response to phosphate‐limitation in the medium. These results imply that the gene products identified in this study are probably involved, either directly or indirectly, in the signal‐transduction mechanism underlying regulation of the phosphate regulon in Synechococcus sp. PCC7942. Hence, the genes encoding the sensory kinase and response regulator were designated as sphS and sphR, respectively (Synechococcusphosphate regulon). The SphS protein was demonstrated in vitro to undergo phosphorylation in the presence of ATP.
High‐resolution mapping of genetic loci of Anabaena PCC 7120 required for photosynthesis and nitrogen fixation
Authors: Kuritz Tanya , Ernst Anneliese , Black Todd A. , Wolk C. Peter
Published: October 2006

A physical map of the Anabaena genome permitted the localization of its genes to chromosomal fragments generated by rarely cutting restriction endonucleases and separated by pulsed‐field gel electrophoresis. We introduce a novel means of mapping more precisely to c. 20 kb by use of rare restriction sites within vectors bearing cloned sequences that undergo homologous recombination with the genome. We thereby localize and orient genes encoding principal photosynthetic pigments. The relative spacing of loci within a single restriction fragment was determined with even higher resolution, as illustrated for genes required for heterocyst development and nitrogen fixation that were marked with transposons. Small, newly visualized restriction fragments of the chromosome were also mapped.
Iron‐hydroxamate uptake systems in Bacillus subtilis: identification of a lipoprotein as part of a binding protein‐dependent transport system
Authors: Schneider Richard , Hantke Klaus
Published: October 2006

Bacillus subtilis was shown to utilize three types of hydroxamate siderophores, ferrichromes, ferrioxamines and shizokinen, each of which is taken up by different transport systems. Mutants deficient in the uptake of ferrichrome and/or ferrioxamine B were isolated. The gene fhuD, which was able to complement a mutant defective in ferrichrome uptake, was cloned. The deduced sequence of FhuD showed low but significant homology to the binding proteins FepB, FecB and FhuD of Escherichia coli, which are all components of binding protein‐dependent, ferric siderophore transport systems. The first 23 amino acids of FhuD of B. subtilis possessed all characteristics of a lipoprotein signal sequence. The processing of FhuD in E. coli was inhibited by globomycin. Inhibition by globomycin indicated a lipid modification at the N‐terminal cysteine in E. coli. It is highly likely that this step may also take place in B. subtilis. As in other binding protein‐dependent transport systems of Gram‐positive organisms it is proposed that the lack of a periplasm is compensated for by the lipid through which the binding protein is anchored to the cytoplasmic membrane.
Rhizobium phaseoli cytochrome c‐deficient mutant induces empty nodules on Phaseolus vulgaris L.
Authors: Soberón Mario , Aguilar Germán R. , Sánchez Federico
Published: October 2006

A Rhizobium phaseoli cytochrome mutant, unable to oxidize N,N,N′,N′ ‐tetramethyl‐p‐phenylend(amine (TMPD), was isolated after Mu‐dl (Kan lac) mutagenesis of the wild‐type strain CE‐3. Mutant strain CFN4202 had sixfold less haem‐c but similar levels of b type, o and aa3 cytochromes than the wild‐type strain. CFN402 strain also showed reduced NADH‐ and TMPD‐oxidase activity than the wild‐type strain. Succinate‐oxidase activities were very similar. Western blot experiments, using antiserum against bovine c1 and c cytochromes, revealed that both proteins were present in CFN4202 membranes, suggesting a defect of haem binding to cytochrome c. Nodules formed by this strain in Phaseolus vulgaris did not contain bacteroids. These data suggest that the cytochrome c‐aa3 chain or some other respiratory chain, containing c‐type cytochromes in R. phaseoli, is essential for bacterial division during the early steps of the symbiotic interaction with the legume‐host.
AUA1, a gene involved in ammonia regulation of amino acid transport in Saccharomyces cerevisiae
Authors: Sophianopoulou Vicky , Diallinas George
Published: October 2006

In Saccharomyces cerevisiae the general amino acid (GAP1) permease catalyses active transport of apparently all amino acids across the plasma membrane. GAP1 activity is regulated by control of synthesis and control of activity in response to the nitrogen source supplied; ammonia and giutamine Inactivate GAP1 function while proline and urea allow its maximum expression. We have isolated and characterized a gene. AUA1, involved in ammonia regulation of GAP1 activity. AUA1 is not essential for growth but over‐expression of the AUA1 transcript in a high‐copy vector or due to a regulatory mutation. aua1‐1, present ∼10 bp upstream from the start of AUA1 transcription, releases GAP1 activity from ammonia‐inactivation without affecting GAP1 transcription. The aua1‐1 mutation has no phenotype when ammonia is replaced by proline or glutamate as the nitrogen source or when it is present in a gap1 background. AUA1 expression is itself ammonia repressible in a wild‐type strain but not in the aua1‐1 mutant. The AUA1 gene sequence contains a unique short open reading frame of 94 codons corresponding to a polypeptide of 11 714 Da. This polypeptide is highly hydrophilic and extremely basic. The AUA1 product shows no significant similarity with any previously known protein sequence. Interestingly, a 10‐amino acid segment of AUA1 is directly repeated in the most basic segment of the protein. Possible roles of AUA1 are discussed.
Sequence requirements for target activity in site‐specific recombination mediated by the Int protein of transposon Tn 1545
Authors: Trieu‐Cuot P. , Poyart‐Salmeron C. , Carlier C. , Courvalin P.
Published: October 2006

Excision and integration of Tn 1545 occur by reciprocal site‐specific recombination between 6 (or 7)bp variable sequences present in the recombining attachment (atf) sites and designated overlap regions. We devised an assay for Tn1545 transposition in which derivatives containing the cis‐acting transposition sequences (atfTn 1545) integrate into a target replicon when complemented in trans by the transposon‐encoded integrase Int‐Tn. This assay was used to determine the characteristics of the DNA sequence that influence target site selection. Characterization of several integration sites indicated that a 20 bp segment, designated attB, contains the sequences required for target activity. It also appeared that (i) the target activity depends upon the extent of homology between the 7bp segments flanking the overlap regions in attB and attTn1545, and (ii) the degree of homology between the two recombining overlap regions does not affect the level of target activity and has no influence on integration orientation.
Identification of a nitrogen‐regulated promoter controlling expression of Klebsiella pneumoniae urease genes
Authors: Collins Carleen M. , Gutman Delia M. , Laman Heike
Published: October 2006

Synthesis of urease by Klebsiella species is known to be induced when the nitrogen source of the growth medium is limiting, suggesting that urease gene expression is controlled by the nitrogen regulatory (ntr) system. This study showed that K. pneumoniae with mutations in either ntrA or ntrC, two integral components of the ntr system, were phenotypically urease‐negative. These mutants could be complemented back to a urease positive phenotype with recombinant plasmids encoding the corresponding ntr gene. A series of ure–lacZYA transcriptional fusions, in conjunction with primer extension analysis, identified a DNA region that encoded a nitrogen‐regulated promoter. This promoter region controlled transcription of ureD, the first gene in the Klebsiella pneumoniae urease gene cluster, and ureA, a gene that resides immediately downstream of ureD. A high level of transcription from the ureD promoter required NAC, a recently characterized member of the nitrogen regulatory cascade. NAC is a Lys R‐like transcriptional regulator that can act at σ;70 promoters; expression from nac itself is dependent upon NTRA. Therefore, expression of K. pneumoniae urease was dependent upon the nitrogen regulatory cascade, and transcription of at least two urease genes was from a promoter that was positively regulated by NAC.
The sialidase superfamily and its spread by horizontal gene transfer
Authors: Roggentin Peter , Schauer Roland , Hoyer Lois L. , Vimr Eric R.
Published: October 2006

Sialidases (neuraminidases, EC 3.2.1.18) belong to a class of glycosyl hydrolases that release terminal N‐acylneuraminate (slalic acid) residues from glycoproteins, glycolipids, and polysaccharides. These enzymes are common in animals of the deuterostomate lineage (Echinodermata through Mammalia) and also in diverse microorganisms that mostly exist as animal commensals or pathogens. Sialidases, and their sialyl substrates, appear to be absent from plants and most other metazoans. Even among bacteria, sialidase is found irregularly so that related species or even strains of one species differ in this property. This unusual phylogenetic distribution makes sialidases interesting for evolutionary studies. The biochemical diversity among bacterial sialidases does not indicate close relationships. However, at the molecular level, homologies are detectable, supporting the hypothesis of a common sialidase origin and thus of a sialidase super family. Some findings indicate that sialidase genes were recently transferred via phages among bacteria. The proposal of a sialidase origin in higher animals is suggested by the presence of apparently homologous enzymes in this kingdom, supporting the idea that some microbes may have acquired the genetic information during association with their animal hosts.
Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways
Authors: Marqués Silvia , Ramos Juan L.
Published: October 2006

TOL plasmid pWWO of Pseudomonas putida contains two operons that specify a pathway for the degradation of aromatic hydrocarbons. The upper pathway operon encodes the enzymes for the oxidation of toluene/xylenes to benzoate/toluates, and the meta‐cleavage pathway operon encodes the enzymes for the further oxidation of these compounds to Krebs cycle intermediates. Their expression is controlled by the gene products of two divergently transcribed regulatory genes, xylR and xylS. The XylR protein, which belongs to the NtrC family of regulators, is expressed from two tandem promoters and autoregulates its synthesis. XylR stimulates transcription from the xylS gene promoter (Ps) and the upper pathway operon promoter (Pu) in the presence of pathway substrates. Both promoters are a54 dependent, and Pu also requires the presence of integration host factor (IHF) for activation of transcription. Binding sites for XylR and IHF in the Pu promoter and for XylR in the Ps promoters have been defined. The XylS protein, which belongs to the AraC family of regulators, stimulates transcription from the mefa‐cleavage pathway operon promoter (Pm) in the presence of benzoates. The effector binding pocket and ONA‐binding region of XylS have been defined through the isolation of mutants that exhibit altered effector specificity and modified transcriptional patterns, respectively. Expression of the mefa‐cleavage pathway operon is also induced by xylene‐activated XylR protein via a cascade regulatory system in which this protein, in combination with σ;54, stimulates the expression from the xylS promoter. The increased concentration of XylS in turn leads to high level expression of the
Internalin‐mediated invasion of epithelial cells by Listeria monocytogenes is regulated by the bacterial growth state, temperature and the pleiotropic activator prfA
Authors: Dramsi S. , Kocks C. , Forestier C. , Cossart P.
Published: October 2006

Entry of Listeria monocytogenes into epithelial cells requires expression of inlA, the first gene of an operon comprising two genes: inlA, which encodes internalin, a 800‐amino‐acid protein, and inlB, which encodes a 630‐amino‐acid protein. We report here that the inl locus is transcribed on two transcripts in constant relative ratio: a 5 kb transcript spanning inlA and inlB, and a 2.9 kb transcript that covers only inlA. The promoter is located 397 bp from the GTG initiator of inlA and displays in its ‐35 region a palindrome similar to that found in promoters controlled by the pleiotropic activator prfA. Transcription of the inl locus is, as are several other L. monocytogenes virulence genes, activated by prfA and regulated by temperature—with higher expression at 37°C versus 25°C — and bacterial growth state. It is maximal during exponential growth and correlates with maximal invasivity of the bacteria in the human epithelial cell line Caco‐2. It also correlates with maximum amounts of internalin present on the bacterial surface. Internalin is also detected in substantial amounts in culture supernatants. Taken together, these data suggest that surface‐bound internalin plays an important role in bacterial entry but do not exclude a role for the released form.
A C‐terminal truncation results in high‐level expression of the functional photoreceptor sensory rhodopsin I in the archaeon Halobacterium salinarium
Authors: Ferrando‐May Elisa , Brustmann Bettina , Oesterhelt Dieter
Published: October 2006

Expression of the gene encoding the halobacterial photoreceptor sensory rhodopsin I (SRI), sopl, was studied by means of homologous gene targeting. A sopr− Halobacterium salinarium mutant strain was constructed by homologous replacement of sopl with a novobiocin‐resistant gyrB from Haloferax Aa 2.2. Cells bearing gyrB were resistant to novobiocin, indicating that the Haloferax gene is functional in H. salinarium. Complementation of this deletion strain with sopl fused to the bacterio‐opsin promoter resulted in the recovery of all phenotypical attributes of SRI. This establishes the first direct correlation between sopl and the function of its gene product, in the complemented deletion strain, functional expression of sopl occurred from the bop locus, where sopl had Integrated by homologous recombination. This shows that cotranscription of sopl and the gene encoding the SRI signal transducer, htrl, which is found in the wild type, is not a prerequisite for photosensory activity. Deletion of the last 43 bp at the 3′ end of sopl resulted in a 10‐fold increase in the amount of SRI, without affecting the activity of the pigment. The mRNA level of the truncated gene was not affected as compared to that of the wild type. We propose that regulation occurs at the protein level, probably through a negative determinant of protein stability located in the C‐terminus of SRI. Replacement of the last 26 amino acids of bacteriorhodopsin by the last 29 amino acids of SRI results in a decrease of the bacteriorhodopsin, supporting our observations. The C‐terminus of SRI is the first domain with a downregulating influence on protein levels thus far identified in H. salinarium. The system for SRI overexpression we present here greatly facilitates biochemical and biophysical studies on the photoreceptor and allows
Roles in inhibitor recognition and quinol oxidation of the amino acid side chains at positions of cytb providing resistance to Qo‐inhibitors of the bc1 complex from Rhodobacter capsulatus
Authors: Tokito Mariko K. , Daldal Fevzi
Published: October 2006

The substitutions M140I, F144S and L, G152S, T163A and V333A in cytochrome b of the ubiquinol‐cytochrome c oxidoreductase (bc1 complex) from Rhodobacter capsulatus provide resistance to the quinol oxidation (Qo inhibitors myxothiazol, mucidin and stigmatellin. Site‐directed mutagenesis with degenerate primers was used to define the role of these positions in inhibitor recognition and quinol oxidation, and a collection of various substitutions at each of these positions was obtained. The effects of these mutations on quinol oxidation, nature and level of inhibitor resistance, prosthetic group incorporation and assembly of the complex were analysed. Most of these mutations, unlike those at position 158 reported earlier, yielded functional bc1, complexes able to support the photosynthetic growth of R. capsulatus. However, they perturbed steady‐state quinol oxidation and inhibitor recognition indicating that they are important for the function of the Qo site. In particular, the presence of a methyl group on the β‐carbon (He and Val residues) at position 140, the absence of an aromatic ring (Phe, Tyr and Trp residues) at position 144 and the loss of residues with small side chains (Gly and Ala) at position 152 correlated with resistance to myxothiazol. On the other hand, no myxothiazol resistance was observed with the substitutions at positions 163 and 333 suggesting that they affected solely the recognition of stigmatellin. Five substitutions, M140R, F144H and R, G152P and T163R, yielded photosynthesis‐deficient mutants with assembled but impaired bc1 complexes. Unexpectedly, two substitutions at position 163 (T to F or P) yielded mutants lacking the three subunits of
The catR gene encoding a catalase from Aspergillus niger primary structure and elevated expression through increased gene copy number and use of a strong promoter
Authors: Fowler Timothy , Rey Michael W. , Vähä‐Vahe Peka , Power Scott D. , Berka Randy M.
Published: October 2006

Synthetic oligonucleotide probes based on amino acid sequence data were used to identify and clone cDNA sequences encoding a catalase (catalase‐R) of Aspergillus niger. One cDNA clone was subsequently used to isolate the corresponding genomic DNA sequences (designated catR). Nucleotide sequence analysis of both genomic and cDNA clones suggested that the catR coding region consists of five exons interrupted by four small introns. The deduced amino acid sequence of catalase‐R spans 730 residues which show significant homology to both prokaryotic and eukaryotic catalases, particularly in regions involved in catalytic activity and binding of the haem prosthetic group. Increased expression of the catR gene was obtained by transformation of an A. niger host strain with an integrative vector carrying the cloned genomic DNA segment. Several of these transformants produced three‐ to fivefold higher levels of catalase than the untransformed parent strain. Hybridization analyses indicated that these strains contained multiple copies of catR integrated into the genome. A second expression vector was constructed in which the catR coding region was functionally joined to the promoter and terminator elements of the A. niger glucoamylase (glaA) gene. A. niger transformants containing this vector produced from three‐ to 10‐fold higher levels of catalase‐R than the untransformed parent strain.
Nucleotide sequence and expression of the Pseudomonas aeruginosa algF gene controlling acetylation of alginate
Authors: Shinabarger Dean , May Thomas B. , Boyd Aoife , Ghosh Mita , Chakrabarty A. M.
Published: October 2006

Colonization of the cystic fibrosis lung by Pseudomonas aeruginosa is greatly facilitated by the production of an exopolysaccharide called alginate. In this study we determined the nucleotide sequence of an alginate modification gene, algF, which controls the addition of acetyl groups to alginate. Expression of algF using a T7 promoter‐expression system showed that algF codes for a 24.5 kDa polypeptide (predicted size 22 832 Da) that is processed to 19.5 kDa. The N‐terminus of the processed polypeptide matched the predicted amino acid sequence of AlgF starting at Asp‐29. An algF mutant failed to produce alginate owing to a polar effect on the downstream algA gene. Although the algA gene, provided in trans, restored synthesis of alginate, the alginate was non‐acetylated. We show that a plasmid containing both the algF and algA gene complements the alginate acetylation defect of the algF mutant strain.
Genetic identification of exported proteins in Streptococcus pneumoniae
Authors: Pearce B. J. , Yin Y. B. , Masure H. R.
Published: October 2006

A strategy was developed to mutate and genetically identify exported proteins in Streptococcus pneumoniae. Vectors were created and used to screen pneumococcal DNA in Escherichia coli and S. pneumoniae for translational gene fusions to alkaline phosphatase (PhoA), Twenty five PhoA+ pneumococcal mutants were isolated and the loci from eight of these mutants showed similarity to known exported or membrane‐associated proteins. Homologues were found to: (i) protein‐dependent peptide permeases, (ii) penicillin‐binding proteins, (iii) Cip proteases, (iv) two‐component sensor regulators, (v) the phospho‐enolpyruvate:carbohydrate phosphotransferase permeases, (vi) membrane‐associated dehydrogenases, (vii) P‐type (E1E2‐type) cation transport ATPases, (viii) ABC transporters responsible for the translocation of the RTX class of bacterial toxins. Unexpectedly one PhoA+ mutant contained a fusion to a member of the DEAD protein family of ATP‐dependent RNA helicases suggesting export of these proteins.
Regulation of mobilization of the broad‐host‐range plasmid pTF‐FC2
Authors: Rohrer Jurg , Rawlings Douglas E.
Published: October 2006

The mobilization region of the broad‐host‐range plasmid pTF‐FC2 consists of an oriT and five genes arranged in two operons that are divergently transcribed from the oriT. The transcriptional starts of both operons were identified and the quantity of transcript from the mobC‐mobE promoter (PI) was at least 10‐fold greater than that from the mobA‐mobB promoter (P2). A translational fusion between the first protein of each operon and a lacZ reporter gene was constructed and used to demonstrate that mob gene expression was autoregulated. Analysis of the oriT resulted in the detection of a putative integration host factor (IHF)‐binding site and an intrinsically bent region. In the absence of IHF, the mobilization frequency and expression from P1 were reduced. The presence of ssi sites on both strands within the oriT region was demonstrated by using an M13 phage mutant, defective in its mechanism for priming DNA replication. Initiation of DNA synthesis at the oriT did not require a plasmid‐encoded primase.
The contribution of the cell wall to a transmembrane calcium gradient could play a key role in Bacillus subtilis protein secretion
Authors: Petit‐Glatron Marie‐Françoise , Grajcar Lydie , Munz Anette , Chambert Régis
Published: October 2006

A weak Ca2+‐binding site (Ka= 0.8× 103 M−1, at pH7) was identified in the mature part of levansucrase. An amino acid substitution (Thr‐236 →lle) in this site alters simultaneously the affinity for calcium, the folding transition and the efficiency of the secretion process of levansucrase. Moreover, the ability of the Bacillus subtilis cell wall to concentrate calcium ions present in the culture medium was studied. We confirm the results of Beveridge and Murray who showed that the concentration factor is about 100 to 120 times. This property preserves a high concentration of Ca2+ (>2 mM) on the external side of the cytoplasmic membrane, even in the absence of further Ca2+ supplementation in the growth medium. Such local conditions allow the spontaneous unfolding folding transition of levansucrase en route for secretion. Since several exocellular proteins of B. subtilis are calcium‐binding proteins, we propose that the high concentration of calcium ion in the microenvironment of the cell wall may play a key role in the ultimate step of their secretion process.
The function of the upstream region of the σ;54‐dependent Klebsiella pneumoniae nifL promoter is sensitive to DNA supercoiling
Authors: Whitehall Simon , Austin Sara , Dixon Ray
Published: October 2006

The positive control protein NTRC activates transcription from the σ;54‐dependent nifL and glnAp2 promoters of Klebsiella pneumoniae by binding to upstream enhancer‐like sequences and contacting downstream bound σ;54‐RNA polymerase via looping of the intervening DNA. In contrast to the glnAp2 promoter, the activity of the nifL promoter is very sensitive to changes in DNA supercoiling both in vivo and in vitro. We have shown previously that the downstream elements of the nifL promoter are involved in the supercoiling response. In this study we find that the upstream region of nifL influences the supercoiling response of a hybrid nifL‐ginAp2 promoter both in vivo and in vitro, demonstrating that the nifL upstream region also confers supercoiling sensitivity. DNA supercoiling did not appear to influence binding of NTRC to its sites in the nifL upstream region, suggesting that another function of this region, most probably DNA loop formation, is sensitive to changes in DNA topology.
Processes at the nick region link conjugation, T‐DNA transfer and rolling circle replication
Authors: Waters Virginia L. , Guiney Donald G.
Published: October 2006

Data from prokaryotic replicative and conjugative systems, which interrelate DNA processing events initiated by a site‐specific nick, are reviewed. While the replicative systems have been established in accordance with the rolling circle replication model, the mechanism of conjugative replication has not been elucidated experimentally. We summarize data involving random point mutagenesis of the RK2 transfer origin (oriT), which yielded relaxation‐deficient and transfer‐deficient derivatives having mutations exclusively in a 10bp region defined as the nick region. Features of the RK2 (IncP) nick region, including the DNA sequence, nick site position, and 5′ covalent attachment of the nicking protein, have striking parallels in other systems involving nicking and mobilization of single‐stranded DNA from a supercoiled substrate. These other systems include T‐DNA transfer occurring in Agrobacterium tumefaciens Ti plasmid‐mediated tumorigenesis in plants, and the rolling circle replication of plasmids of Gram‐positive bacteria and of φX174‐like bacteriophage. The structural and functional similarities suggest that IncP conjugative replication, originating at the oriT, and T‐DNA transfer replication, originating at the T‐DNA border, produce continuous strands via a rolling circle‐type replication.
PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of CoIE1‐related plasmids
Authors: He Lin , Söderbom Fredrik , Wagner E. Gerhart H. , Binnie Uta , Binns Nigel , Masters Millicent
Published: October 2006

The replication of CoIE1‐related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for pcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5′‐end processing by RNAse E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST‐ PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.
A Rhizobium tropici DNA region carrying the amino‐terminal half of a nodD gene and a nod‐box‐like sequence confers host‐range extension
Authors: Sousa Carolina , Folch Jorge Luis , Boloix Purificación , Megías Manuel , Nava Noreide , Quinto Carmen
Published: October 2006

Rhizobium tropici CIAT899 is a broad‐host‐range strain that, in addition to Phaseolus, nodulates other plant legumes such as Leucaena and Macroptilium. The narrow‐host‐range of Rhizobium leguminosarum biovars phaseoli (strain CE3) and trifolii (strain RS1051) can be extended to Leucaena esculents and Phaseolus vulgaris plants, respectively, by the introduction of a DNA fragment 521 bp long, which carries 128 amino acids of the amino‐terminal region of a nodD gene from R. tropici, as well as a putative nod‐box‐like sequence, divergently oriented. The 521 bp fragment, in the presence of L. esculenta or P. vulgaris root exudates, induced a R. leguminosarum bv. viciae nodA‐lacZ fusion in either a CE3 or RS1051 background, respectively.
The nik operon of Escherichia coli encodes a periplasmic binding‐protein‐dependent transport system for nickel
Authors: Navarro Clarisse , Wu Long‐Fei , Mandrand‐Berthelot Marie‐Andrée
Published: October 2006

The complete nucleotide sequence of the Escherichia coli nik locus, which has been suggested to encode the specific transport system for nickel, has been determined. It was found to contain five overlapping open reading frames that form a single transcription unit. Deduced amino acid sequence of the nik operon shows that its five gene products, NikA to NikE, are highly homologous to components of oligopeptide‐and dipeptide‐binding protein‐dependent transport systems from several Gram‐negative and Gram‐positive species. NikA represents the periplasmic binding protein, NikB and NikC are similar to integral membrane components of periplasmic permeases, and NikD and NikE possess typical ATP‐binding domains that suggest their energy coupling role to the transport process. Insertion mutations in nik genes totally abolished the nickel‐containing hydrogenase activity under nickel limitation and markedly altered the rate of nickel transport. Taken together, these data support the notion that the nik operon encodes a typical periplasmic binding‐protein‐dependent transport system for nickel.
Rubisco but not Rubisco activase is clustered in the carboxysomes of the cyanobacterium Synechococcus sp. PCC 7942: Mud‐induced carboxysomeless mutants
Authors: Friedberg D. , Jager K. M. , Kessel M. , Silman N. J. , Bergman B.
Published: October 2006

The Mud technology of Groisman and Casadaban was adapted to the cyanobacterium Synechococcus sp. PCC 7942. A new high‐CO2‐requiring (hcr) mutant, hcr Mu28 was isolated following the integration of the Mud element 89 bp upstream of ORFI, at the 5′‐flanking region of the rbc operon, which encodes RuBP carboxylase/oxygenase (Rubisco). The integration involved a 7 bp duplication that formed a direct repeat at the integration site, as previously shown in Escherichia coli The mutant was devoid of apparent carboxysome bodies, which are considered to be important for the availability of CO2 for Rubisco. Immunolabelling studies demonstrated that Rubisco was distributed throughout hcr Mu28 cells, while in the wild type (WT) and in the carboxysome aberrant mutant hcr 0221, Rubisco was markedly associated with the carboxysomes. Rubisco activase, however, was evenly distributed throughout the cytosol of the hcr and WT cells, without any preferential association with the apparent carboxysomes.
Molecular cloning and characterization of a proline iminopeptidase gene from Neisseria gonorrhoeae
Authors: Albertson N. H. , Koomey M.
Published: October 2006

Proline iminopeptidase (Pip) is a hydrolase elaborated by virtually all strains of Neisseria gonorrhoeae that selectively removes N‐terminal proline residues from peptides. Escherichia coli clones expressing the gonococcal gene coding for Pip were identified in a genomic cosmid library using a synthetic colorimetric substrate. Nucleotide sequence determination and analyses of polypeptides detected by coupled in vitro transcription/translation reactions revealed that Pip is a 311‐amino‐acid polypeptide with a Mr of 35 kDa and a pi of 5.4. Southern hybridization showed that the pip gene is present in a single copy on the chromosome of N. gonorrhoeae strain MS11 which maps immediately upstream of the previously identified opaA locus. The transcriptional start site of pip in E. coli, determined by primer extension analysis, was characteristic of an NtrA or sigma‐54‐dependent pro‐motor. Complementation of an E. coli mutant deficient in both proline biosynthesis and dipeptide uptake confirmed that Pip is capable of releasing biologically active proline from peptides. Pip expression was found to be non‐essential for in vitro growth of N. gonorrhoeae, based on the viability of a Pip− gonococcal mutant.
The ORF1 of the gentamicin‐resistance operon (aac) of Pseudomonas aeruginosa encodes adenosine 5′‐phosphosulphate kinase
Authors: Satishchandran C. , Taylor John C. , Markham George D.
Published: October 2006

The gentamicin‐resistance operon of Pseudomonas aeruginosa (aac) contains two cistrons for which only the second gene product has an identified function. The 813bp second cistron (ORF2) encodes a protein that confers gentamicin resistance by catalysis of the transfer of an acetyl group from acetyl Coenzyme A to gentamicin. The first open reading frame (ORF1) encodes a 23.9 kDa protein that we have found, by enzyme activity and immunological reactivity, to be adenosine‐5′‐phosphosulphate (APS) kinase. APS kinase catalyses the transfer of the gamma phosphoryl group of ATP to the 3′‐hydroxyl group of APS. The 70% sequence similarity between the Pseudomonas and Escherichia coli APS kinases suggests that the Pseudomonas enzyme may catalyse phosphoryl transfer to the 3′‐hydroxyl group of other nucleotides such as dephosphocoenzyme A, as does the purified E. coli APS kinase. In extracts of pseudomonad cells we have also detected a higher molecular mass (70 kDa) protein that cross‐reacts with an anti‐E. coli APS kinase antibody. This cross‐reactive protein is also present in Pseudomonas strains lacking the gentamicin‐resistance plasmid, and apparently reflects an APS kinase analogous to the nodQ‐encoded high‐molecular‐weight APS kinase present in Rhizobium meliloti. Production of the Pseudomonas aac APS kinase was repressed by cysteine when expressed in E. coli, as is E. coli APS kinase. However, cysteine did not repress production of the Pseudomonas enzyme when the aac ORF1 ‐encoded enzyme was expressed in a Pseudomonas strain, indicating differential regulation of gene expression in the two organisms.
Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNA pseudoknot
Authors: Finken Marion , Kirschner Philip , Meier Albrecht , Wrede Annette , Böttger Erik C.
Published: October 2006

Multidrug‐resistant strains of Mycobacterium tuberculosis have resulted in several recent outbreaks. Recognition of drug resistance is important both for treatment and to prevent further transmission. Here we use molecular biology techniques to study the basis of streptomycin resistance in single and multi‐drug‐resistant M. tuberculosis. We demonstrate that streptomycin resistance is associated with mutations implicated in ribosomal resistance. The mutations found either lead to amino acid changes in ribosomal protein SI2 or alter the primary structure of the 16S rRNA. The 16S rRNA region mutated perturbs a pseudoknot structure in a region which has been linked to ribosomal S12 protein.
Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor type1
Authors: Falzano Loredana , Fiorentini Carla , Donelli Gianfranco , Michel Eric , Kocks Christine , Cossart Pascale , Cabanié Lucien , Oswald Eric , Boquet Patrice
Published: October 2006

Cytotoxic necrotizing factor type 1 (CNF1) from strains of pathogenic Escherichia coli induces in human epithelial HEp‐2 cells, a profound reorganization of the actin cytoskeleton into prominent stress fibres and membrane ruffles. We report here that this process is associated with induction of phagocytic‐like activity. CNF1‐treated cells acquired the ability to ingest latex beads as well as non‐invasive bacteria such as Listeria innocua, which were taken as a model system. Uptake of bacteria was similar to pathogen‐induced phagocytosis, since L. innocua transformed with DNA coding for the pore‐forming toxin listeriolysln O behaved, with respect to intracellular growth, like the invasive, pathogenic species L. monocytogenes. Our results raise the possibility that, in vivo, pathogenic CNF1 ‐producing E. coli may invade epithelia by this novel induced phagocytic‐like mechanism.
Multiple novel promoters from the early region in the Streptomyces temperate phage φC31 are activated during lytic development
Authors: Ingham C. J. , Crombie H. J. , Bruton C. J. , Chater K. F. , Hartley N. M. , Murphy G. J. P. , Smith M. C. M.
Published: October 2006

Evidence is presented that transcription of most of the early genes in the Streptomyces coelicolor A3(2) phage φC31 is from a series of unusual promoters that depend on a function expressed early in the φC31 lytic cycle. Primer extension analysis on the 5′ ends of three early mRNAs, from samples prepared 10min after induction of a thermosensitive φC31 lysogen, showed that the 5′ ends all mapped close to highly similar sequences, which are proposed to be an important part of phage‐specific promoters. In a shotgun cloning experiment, a fragment containing one of these sequences strongly activated transcription of the xylE reporter gene in plaques of a φC31‐derived promoter‐probe vector. Another of the sequences was inserted into a xylE‐containing promoter‐probe plasmsid vector, and promoted xylE expression only when the host was supporting the lytic cycle of φ C31. This suggested that a transcription factor needed for activity of the promoters was present only in φC31 ‐infected cells. Examination of published and unpublished φ C31 sequence data revealed several more sequences that closely resemble the conserved region of the characterized promoters. Most of these are found in positions close to apparent transcription start sites mapped previously by low‐resolution S1 mapping. An overall consensus sequence for the conserved region suggests a general organization (though not a primary sequence) resembling that of promoters recognized in other bacteria by the σ;54 form of RNA polymerase.
The role of a repetitive DNA motif (5′‐CAAT‐3′) in the variable expression of the Haemophilus influenzae lipopolysaccharide epitope αGal(1–4)βGal
Authors: High Nicola J. , Deadman Mary E. , Moxon E. Richard
Published: October 2006

Haemophilus influenzae lipopolysaccharide (LPS) contains structures, defined by monoclonal antibodies, which undergo phase variation. This investigation reports the nucleotide sequence of lic2A, which is required for the expression of at least three phase‐variable LPS epitopes, one of which has the structure αGal(1–4)βGal. lic2A contains multiple tandem repeats of the tetramer 5′‐CAAT‐3′ Previous studies have correlated changes in the number of 5′‐CAAT‐3′ repeats with the phase‐variable expression of the αGal(1–4)βGal epitope. To obtain direct evidence for this, the 5′‐CAAT‐3′ repeat region from lic2A was amplified directly from immunostained colonies and sequenced. This demonstrated that the variable expression of LPS epitopes, including αGal(1–4)βGal, is in part directly dependent upon the number of copies of 5′‐CAAT‐3′ within lic2A.
The IS4 family of insertion sequences: evidence for a conserved transposase motif
Authors: Rezsöhazy René , Hallet Bernard , Delcour Jean , Mahillon Jacques
Published: October 2006

The eight IS 231 variants characterized so far (IS 231 A‐F, V and W) display similar transposases with an overall 40% identity. Comparison with all the proka‐ryotic transposable elements sequenced so far revealed that the IS231 transposases share two conserved regions with those of 35 other insertion sequences of wide origins. These insertion sequences, defining the IS4 family, have a common bipartite organization of their ends and are divided into two similarity groups. Interestingly, the transposase domains conserved within this family display similarities with the well known integrase domain shared by transposases of the IS3 and IS15 families, and integrases of retroelements. This domain is also found in IS30‐ related elements and Tn7 TnsB protein. Amino acid residues conserved throughout all these prokaryotic and eukaryotic mobile genetic elements define a major transposase/integrase motif, likely to play an important role in the transposition process.
Micro Correspondence
Authors: Imperial J. , Rey L , Palacios J. M. , Ruiz‐Argueso T.
Published: October 2006

HupK, a hydrogenase‐ancillary protein from Rhizobium leguminosarum, shares structural motifs with the large subunit of NiFe hydrogenases and could be a scaffolding protein for hydrogenase metal cofactor assembly
Surface‐associated, PrfA‐regulated proteins of Listeria monocytogenes synthesized under stress conditions
Authors: Sokolovic Zeljka , Riedel Jutta , Wuenscher Michael , Goebel Werner
Published: October 2006

The expression of the five clustered genes of Listeria monocytogenes: plcA, hly, mpl, actA and plcB is under the control of the positive regulation factor PrfA. Listeriolysin, encoded by the hly gene, is the only prominent PrfA‐controlled gene product observed when L. monocytogenes strain NCTC 7973 is cultured in a rich medium at 37°C to the logarithmic growth phase. Stress conditions such as heat‐shock or stationary culture conditions lead to the induction of additional PrfA‐dependent proteins (PdPs): ActA (92 kDa), a 38kDa protein of unknown function and a 34kDa protein which probably represents PlcA. Under nutrient‐stress conditions PdPs are preferentially synthesized and in addition to the already known PdPs at least five new, not yet functionally identified PdPs are detected. All PdPs are either secreted or are localized at the cell surface. Differences in the amount as well as the sizes of the PdPs are observed in different L. monocytogenes strains.
Secretion of Serratia liquefaciens phospholipase from Escherichia coli
Authors: Givskov Michael , Molin Søren
Published: October 2006

The Serratia liquefaciens phospholipase (PhIA) is secreted to the medium from its natural host. Here we present results which indicate that, when cloned and expressed in Escherichia coli, secretion can be mediated by a putative host‐encoded pathway, expression of which is controlled by FlhD (formerly FlbB), the master regulator of the flagellar/ chemotaxis regulon. In the absence of this secretion pathway, the synthesized phospholipase accumulates inside the host cell where it forms a complex with the PhlB protein. PhlB, which is encoded from the promoter distal gene of the phospholipase operon, inhibits the phospholipase activity of PhlA. Formation of this enzymatically inactive PhlA/PhlB complex is required for maintenance of cell viability.