bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic identification of exported proteins in Streptococcus pneumoniae
Authors: Pearce B. J. , Yin Y. B. , Masure H. R. 
Published: October 2006

SummaryA 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

SummaryThe 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

SummaryA 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

SummaryThe 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

SummaryData 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

SummaryThe 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

Summary 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

SummaryThe 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

SummaryThe 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

SummaryProline 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

SummaryThe 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

SummaryMultidrug‐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

SummaryCytotoxic 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

SummaryEvidence 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

Summary 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

SummaryThe 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.
Surface‐associated, PrfA‐regulated proteins of Listeria monocytogenes synthesized under stress conditions
Authors: Sokolovic Zeljka , Riedel Jutta , Wuenscher Michael , Goebel Werner 
Published: October 2006

SummaryThe 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

SummaryThe 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.
Complex transcription of an operon encoding the Sail restriction‐modification system of Streptomyces albus G
Authors: Alvarez Miguel A. , Chater Keith F. , Rodicio M. Rosario 
Published: October 2006

SummaryHigh‐resolution SI nuclease mapping of mRNA synthesised in vivo, in vitro run‐off transcription with RNA polymerase from Streptomyces lividans and gene fusions were used to analyse the transcriptional organization of the Sall restriction‐modification system of Streptomyces albus G. The sallR and sallM genes that encode the restriction endonuclease and its cognate methyltransferase constitute an operon which is mainly transcribed from sal‐pR1, a promoter located immediately upstream of saIIR, with two possible minor promoters further upstream. Another promoter, sal‐pM, is within the 3′ end of the saIIR coding region, and allows expression of the modification gene in the absence of sal‐pR1. The sal‐pM promoter might be involved in the establishment of modification prior to restriction endonuclease activity. Sequences upstream of the apparent transcriptional start sites for sat‐pR1 and sal‐pM show similarity with the −10 region of typical vegetatively expressed eubacterial promoters, but appropriately centered −35 regions are absent.