bluuebunny/crossref_metadata_2025_split huggingface dataset:
CooB is required for assembly but not transport of CS1 pilin
Authors: Scott J. R. , Wakefield J. C. , Russell P. W. , Orndorff P. E. , Froehlich B. J. 
Published: October 2006

SummaryCS1 pili are filamentous proteinaceous appendages found on many enterotoxigenic Escherichia coli (ETEC) strains isolated from human diarrhoeal disease. They are thought to effect colonization of the upper intestine by facilitating binding to human ileal epithelial cells. We have identified a gene, cooB, which lies directly upstream of cooA, the gene that encodes the major structural CS1 protein. When translated in vitro, the protein product of cooB migrates in sodium dodecyl sulphate/polyacrylamide gel with an apparent molecular mass of 26 kDa, which is consistent with that predicted from its DNA sequence. We constructed a mutant allele (cooB‐1) by insertion of the omega fragment, which inhibits transcription and translation, into the cooB gene in vitro. In a derivative of an ETEC strain with the cooB‐1 mutation (JEF100) and a plasmid that encodes Rns (pEU2030), the positive regulator required for CS1 expression, no cooB and a greatly reduced level of cooA product was detectable in total cell extracts. The reduction of cooA in this strain appears to result from polarity of the cooB mutation because introduction of the wild‐type cooA gene in trans causes production of CooA protein, which is found in cell pellet extracts, in extracts containing only surface proteins and in the culture supernatant. Therefore, in the absence of CooB, CooA is stable and it is transported through both inner and outer membranes. However, the cooB‐1 strain with cooA in trans does not cause haemagglutination of bovine erythrocytes (the model system used to assay adherence mediated by coli surface antigen 1 (CS1) pili). Electron microscopy reveals that there are no pili present on the cell surface or in the culture medium in which this strain was grown. Thus, although it is not required for stability or transport of the major CS1 pilin protein, CooB is needed for assembly of CooA into pili. The relationship of these gene products to those of other pili is discussed.
Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs
Authors: Gonzàlez A. , Membrillo‐Hernández J. , Olivera H. , Aranda C. , Macino G. , Ballario P. 
Published: October 2006

SummaryA Saccharomyces cerevisiae glutamate auxotroph, lacking NADP‐glutamate dehydrogenase (NADP‐GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP‐GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH‐less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.
Cloning of an autonomously replicating sequence (ars) from the Bacillus subtilis chromosome
Authors: Moriya S. , Atlung T. , Hansen F. G. , Yoshikawa H. , Ogasawara N. 
Published: October 2006

SummaryCloning of an autonomously replicating sequence (ars) from the origin region of Bacillus subtilis was previously unsuccessful because of the strong incompatibility exerted by sequences located within the oriC region. Using an ars searching vector which would be selective for drug resistance even at one copy per cell, and by cloning large fragments covering as much as possible of the oriC region, we have succeeded in isolating ars fragments from the origin region of the chromosome. The minimum essential fragment contains two DnaA‐box regions (non‐translatable regions containing multiple repeats of DnaA‐box) separated by the dnaA gene. Neither one of the DnaA‐box regions by itself showed ars activity. When constructed as oriC plasmids, the dnaA coding region could be removed without affecting ars activity. The minimum distance between the two DnaA‐box regions obtained so far is 274 bp. The copy number of the oriC plasmid is estimated as one per replicating chromosome. These plasmids are unstable and tend to be lost or integrated into chromosome.
Zinc and iron regulate translation of the gene encoding Pseudomonas aeruginosa elastase
Authors: Brumlik M. J. , Storey D. G. 
Published: October 2006

SummaryA lasB–lacZ translational fusion (pTS400) was used to examine expression of the elastase gene (lasB) in Pseudomonas aeruginosa strain PAO1. Expression from the lasB–lacZ fusion was enhanced when PAO1(pTS400) was grown in a defined medium containing elevated levels of zinc (6.0μg ml‐1). Transcript accumulation studies on PAO1(pTS400) and PAO1 showed that the addition of zinc had a slight negative effect on lasB transcription. These results indicated that zinc regulates the expression of elastase at the translational level. A comparison between zinc regulation and iron regulation was also made. Iron has a negative effect on lasB–lacZ expression. When PAO1(pTS400) was grown in a defined medium with a low iron content (0.1 μg ml‐1) the bacteria still responded to zinc. The independent effects of low iron and high zinc concentrations suggest separate control mechanisms for the two factors. Transcript accumulation studies on PA01 and PAO1(pTS400) indicated that early in the growth curve iron did not influence transcription of tasB or lasB–lacZ. Later in the growth curve a slight increase in lasB–lacZ transcription was observed only in PAO1(pTS400) grown in low iron. These results suggest that the iron regulation of lasB occurs predominantly at the transtational level. Finally, when PAO1(pTS400) was grown in a complex peptone‐based medium, a high level of transcript accumulation accounted for elastase expression. Alterations of iron and zinc concentrations of this medium did not affect the expression of elastase. These results suggest that there may be additional environmental cues regulating lasB transcription.
Retron‐Ec107 is inserted into the Escherichia coli genome by replacing a palindromic 34bp intergenic sequence
Authors: Herzer P. J. , Inouye S. , Inouye M. 
Published: October 2006

SummarySome natural isolates of Escherichia coli have been shown to produce a unique branched RNA‐linked single‐stranded DNA called msDNA. These bacteria contain a retro‐element called retron consisting of the msr–msd region and the gene for reverse transcriptase (RT). All three E. coli retrons characterized to date have been shown to be integrated into a prophage or to be associated with phage‐related genes. In this report, we identified a new msDNA from an E. coli wild strain. Using the msDNA as a probe, the retron for the msDNA was cloned and its DNA sequence was determined. The retron was found to consist of a 1.3 kb DNA fragment, making it the smallest retron isolated to date. The msDNA produced from the retron consists of a 107 base single‐stranded DNA, which is considered to be branched out from the 18th G residue of a 75‐base RNA molecule by a 2′,5′‐phosphodiester linkage. Thus, the msDNA and the retron were designated msDNA‐Ec107 and retron‐Ec107, respectively. Most significantly, retron‐Ec107 was inserted into the E. coli genome by replacing a 34bp intergenic sequence between the pyrE and ttk genes located at 82 min on the E. coli chromosome. Interestingly, the retron contains palindromic structures at both ends and the E. Coli 34bp intergenic sequence also contains a 10bp inverted repeat structure. These palindromic structures might have played a role in the integration of retron‐Ec107 into the E. coli genome.
Efficient repression by a heterodimeric repressor in Escherichia coli
Authors: Webster C. , Merryweather A. , Brammar W. 
Published: October 2006

SummaryPrevious studies with purified variants of the 434 repressor having different operator‐binding specificities have demonstrated the interactions of a heterodimeric repressor with a hybrid operator site. The present study investigates the interactions between the 434 repressor and its operator site. The optimum 434 operator half‐site is used with a P22 operator half‐site to create a hybrid 434/P22 operator. We show that this hybrid operator can be efficiently bound by a heterodimeric repressor, consisting of one wild‐type 434 repressor monomer and one 434 repressor monomer with the binding specificity of the P22 repressor, to bring about repression in Escherichia coli.
Role of lipid modification on a starch‐debranching enzyme, Klebsieila pullulanase: comparison of properties of lipid‐modified and unmodified pullulanases
Authors: Yamashita M. , Nakagawa A. , Katsuragi N. , Murooka Y. 
Published: October 2006

Summary Klebsiella pullulanase is a lipoprotein synthesized as a precursor with a signal peptide, which is processed by lipoprotein signal peptidase. To clarify the role of lipid modification of pullulanase, we purified lipid‐modified wild‐type and the unmodified (mutant) pullulanases and compared their properties. The Km and Vmax values of both pullulanases for pullulan were the same. The optimal pH and temperature, the stabilities over pH and temperature ranges, the specificity of substrates, and the patterns of inhibition of the lipid‐modified and unmodified pullulanases were also the same. However, we found that the wild‐type pullulanase formed trimers whereas the unmodified enzyme did not, and that the migrations of the two enzymes on sodium dodecyl sulphate/electrophoresis were different when the samples were applied on the gel without heating. The results presented in this paper and in previous work show that the correct processing and translocation of pullulanase in K. aerogenes require modification of lipid. However, the enzymatic properties and physical stabilities of pullulanase were not affected by the lipid modification.
Role of the transcriptional activator, VirF, and temperature in the expression of the pYV plasmid genes of Yersinia enterocolitica
Authors: de Rouvroit C. Lambert , Sluiters C. , Cornelis G. R. 
Published: October 2006

SummaryUpon incubation at 37°C in the absence of Ca2+ ions, pathogenic yersiniae release high amounts of pYV plasmid‐encoded proteins called Yops, involved in pathogenesis. Yersinia enterocolitica also express two outer membrane proteins, an adhesin called YadA and a lipoprotein called YIpA. The production of the Yops is co‐ordinately regulated by a 20kb region of the plasmid referred to as the ‘Ca2+ dependence region’ and containing at least four loci called virA, virB, virC, and virF. The 8.5kb virC region, involved in the specific transport of the Yops, is a single operon containing 13 open reading frames called yscA to yscM. Gene virF encodes a key transcriptional activator of the yop, yadA and ylpA genes. It is only transcribed at 37°C and its expression is modulated by a chromosome‐encoded histone‐like protein called YmoA. We show here that virF also controls the virC operon. By contrast, VirF is not essential for the induction of virA and virB. The VirF protein binds specifically to yop promoters. In particular, it protects the region spanning nucleotides ‐64 to ‐34 of yopH. In order to analyse the role of temperature in the induction of the yop regulon, we constructed Y. enterocolitica strains expressing virF from the tac promoter. In spite of the fact that virF was transcribed at 25°C, neither the Yops nor YadA were expressed at that temperature. This poor response to VirF at 25°C was at least partially due to a weak and slow transcription of the genes controlled by virF. Surprisingly, when cloned on pACYC184, gene yadA was expressed even in absence of VirF, but remained thermodependent. Hence temperature and virF are both required for the induction of the yop regulon. Among other possible roles, temperature could modify the structure of either the activator itself or the yop promoter. The fact that VirF binds in vitro to yop promoters at 25°C rules out the first hypothesis. In order to test the second hypothesis, we studied, in vivo, the activity of the yopH promoter in ymoA mutants. The yopH promoter became active in the absence of VirF, indicating that yop promoter activity depends upon chromatin structure. We conclude from these two observations that, in vivo, temperature is required to modify the DNA structure of the yop promoters in order to allow the action of the transcriptional activator.
Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7
Authors: Yu J. , Kaper J. B. 
Published: October 2006

SummaryThe eae (Escherichia collattaching and effacing) gene from enteropathogenic Escherichia coli (EPEC) was previously shown to be essential for production of the ‘attaching and effacing’ histopathology characteristic of EPEC infections (Jerse et al., 1990). We have now cloned the eae gene from enterohaemorrhagic E. coli (EHEC) which, in addition to producing Shiga‐like cytotoxins, also produces the attaching and effacing effect. The sequence homology between the EPEC and EHEC sequences was 86% and 83% at the nucleotide and amino acid levels, respectively. The predicted amino acid sequence of the EHEC eae gene shared 31% identity and 51 % similarity with invasin of Yersinia pseudotuberculosis. Alignment of the EPEC and EHEC Eae proteins and the Y. pseudotuberculosis and Y. enterocoltica invasins shows striking regions of identity with the greatest divergence at the C‐terminal end, the putative receptor‐binding portion of invasin.
DNA loops: structural and functional properties of scaffold‐attached regions
Authors: Roberge Michel , Gasser Susan M. 
Published: October 2006

SummaryThe long DNA molecules of eukaryotic genomes appear to be organized into large loops formed by the binding of dispersed DNA sequences to non‐histone proteins. This partitioning of DNA into topologically constrained units constitutes one of the highest orders of DNA packing in chromosomes. DNA loops are likely to define functional units as well as topological domains, contributing to the regulation of gene expression and DNA replication. This review presents recent work on the properties of the DNA sequences and proteins thought to be involved in loop formation, and on their possible significance for replication and transcription.
Control of bacterial DNA supercoiling
Authors: Drlica Karl 
Published: October 2006

SummaryTwo DNA topoisomerases control the level of negative supercoiling in bacterial cells. DNA gyrase introduces supercoils, and DNA topoisomerase I prevents super‐coiling from reaching unacceptably high levels. Perturbations of supercoiling are corrected by the substrate preferences of these topoisomerases with respect to DNA topology and by changes in expression of the genes encoding the enzymes. However, super‐coiling changes when the growth environment is altered in ways that also affect cellular energetics. The ratio of [ATP] to [ADP], to which gyrase is sensitive, may be involved in the response of supercoiling to growth conditions. Inside cells, supercoiling is partitioned into two components, superhelical tension and restrained supercoils. Shifts in superhelical tension elicited by nicking or by salt shock do not rapidly change the level of restrained supercoiling. However, a steady‐state change in supercoiling caused by mutation of topA does alter both tension and restrained supercoils. This communication between the two compartments may play a role in the control of supercoiling.
Co‐ordinate expression of virulence genes by ToxR in Vibrio cholerae
Authors: DiRita Victor J. 
Published: October 2006

SummaryEvolution of complex regulatory pathways that control virulence factor expression in pathogenic bacteria indicates the importance to these organisms of being able to distinguish time and place. In the human intestinal pathogen Vibrio cholerae, control over many virulence genes identified to date is the responsibility of the ToxR protein. ToxR, in conjunction with a second regulatory protein called ToxS, directly activates the genes encoding the cholera toxin; other ToxR regulated genes are not activated directly by ToxR. For some of these genes, ToxR manifests its control through another activator called ToxT. Expression of toxT, which encodes a member of the AraC family of bacterial transcriptional activators, is ToxR dependent and is modulated by in vitro growth conditions that modulate expression of the ToxR virulence regulon. Thus, as in other regulatory circuits, co‐ordinate expression of several genes in V. cholerae results from the activity of a cascading system of regulatory factors.
The filA (rpoF) gene of Pseudomonas aeruginosa encodes an alternative sigma factor required for flagellin synthesis
Authors: Starnbach M. N. , Lory S. 
Published: October 2006

SummaryIn order to better understand the regulation of Pseudomonas aeruginosa flagellin expression we cloned the sigma factor of RNA polymerase used to transcribe the flagellin gene. It is a member of the σ28 class of alternative sigma factors described in several bacterial genera. Using the published sequence of the filA gene encoding the σ28 from Salmonella typhimurium, we designed two oligonucleotides and, using the polymerase chain reaction, isolated the fliA gene from S. typhimurium chromosomal DNA. This heterologous probe was used in the DNA blot analysis of restriction digests of P. aeruginosa DNA. A 1.7 kb SalI‐EcoRI fragment reacted with the probe and this fragment was cloned into the pBluescript vectors. The P. aeruginosa fliA gene was able to complement the motility defect of an Escherichia coli fliA mutant, but only when transcription was driven from the vector promoter. Insertional inactivation of the fliA gene with a gentamicin gene cassette rendered P. aeruginosa non‐motile and unable to express the flagellin gene. The 1.7 kb cloned fragment was sequenced and shown to contain the entire fliA gene. P. aeruginosa FliA shares 67% amino acid similarity with the homologous S. typhimurium sequence. Transcriptional analysis of the fliA gene showed that its expression was not dependent on RpoN, a sigma factor shown also to be required for flagellin synthesis. A reading frame downstream of fliA was found to encode the P. aeruginosa homologue of the enterobacterial cheY gene.
The adsorption protein of phage IKe. Localization by deletion mutagenesis of domains involved in infectivity
Authors: Endemann H. , Bross P. , Rasched I. 
Published: October 2006

SummaryWe constructed a set of plasmid‐encoded internal deletion mutants within the gene for the adsorption protein (g3p) of phage IKe. All mutant proteins still contain the signal and membrane anchor sequence, as those are known to be indispensable for proper localization and hence assembly of the g3p into phage. These various deletions comprise ail internal parts of the protein and are properly incorporated into phage, which remarkably shows that signal and anchor sequence are sufficient for incorporation of g3p.The data furthermore reveal that two separate sections within the IKe g3p are essential for infection: one amino‐terminal, preceding the glycine‐rich stretch, and the other carboxy‐terminal. We conclude that this latter domain is involved in penetration because mutants tacking it are not infectious, but still bind to the receptor. The amino‐terminal region, essential for infection, bears the receptor‐recognizing domain and a sequence homologous to the penetration domain of the evolutionary related Ff phages, which is probably also involved in penetration of phage IKe.The prominent glycine‐rich stretch of the IKe g3p is not essential for infection but significantly promotes it.
Suppression of the ndv mutant phenotype of Rhizobium meliloti by cloned exo genes
Authors: Nagpal Punita , Khanuja Suman P. S. , Stanfield Sharon W. 
Published: October 2006

SummaryThe ndv A and ndvB genes of Rhizobium meliloti are involved in the export and synthesis, respectively, of the small cyclic polysaccharide β(1,2)glucan. We have previously shown that spontaneous symbiotic pseudorevertants of ndv mutants do not produce periplasmic β(1,2)glucan. Here we show that the pseudorevertants also do not produce extracellular β(1,2)glucan, but do show alterations in the amount of the major acidic exopolysaccharide produced. This exopolysaccharide is not detectably different from that produced by the wild type or by the ndv mutants. A cosmid which suppresses the symbiotic defect of both ndvA and ndvB mutants was isolated from a gene bank prepared from DNA of an ndvA pseudo‐revertant. This cosmid contains a number of exo genes, including exoH and exoF. Subcloning and Tn5 mutagenesis were used to show that the widely separated exoH and exoF genes are both involved in suppression of the ndv mutant phenotype and that the 3.5 kb DNA fragment which contains the exoH gene does not carry the mutation responsible for second site suppression.
Kex2‐dependent processing of yeast K1 killer preprotoxin includes cleavage at ProArg‐44
Authors: Zhu Yun‐Song , Zhang Xia‐Ying , Cartwright Charles P. , Tipper Donald J. 
Published: October 2006

SummaryThe K1 killer toxin of Saccharomyces cerevisiae consists of 103‐ and 83‐residue α and β components whose derivation, from a 316‐residue precursor preprotoxin, requires processing at the αN‐terminus (after ProArg‐44), the αC‐terminus (after ArgArg‐149) and at the βN‐terminus (after LysArg–233). These processing events occur after translocation to the Golgi and have been investigated using β‐lactamase fusions. Signal peptidase cleavage of the precursor, predicted to occur after Ala‐26, was confirmed by N‐terminal sequence analysis of Ala‐34 and IIe‐52 fusions. Cleavage at all of the other predicted processing sites, including ProArg‐44, is dependent on activity of the Kex2 protease. A fourth Kex2‐dependent cleavage occurs at LysArg‐188. Implications for the specificity of Kex2 cleavage and preprotoxin processing are discussed.
A gene near the plasmid pSa origin of replication encodes a nuclease
Authors: Closed S. M. , Kado C. I. 
Published: October 2006

SummaryWe have cloned and sequenced a gene (nuc) from the IncW plasmid pSa which shows amino acid sequence similarity to staphylococcal nuclease (EC 3.1.4.7) and to the parB locus of plasmid RP4. The 525bp open reading frame encodes a 174‐amino‐acid potential polypeptide of 19.7kDa. Expression of the gene was confirmed using an in vitro transcription‐translation assay which produced a protein of identical size. Nuclease activity was demonstrated using DNA as the substrate in toluidine blue‐DNA agar plates. The deduced amino acid sequence revealed a signal sequence, and TnphoA insertion within the open reading frame indicated that a portion of the protein is transported across the bacterial cell membrane.