bluuebunny/crossref_metadata_2025_split huggingface dataset:
An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca
Authors: Pugsley Anthony P. , Dupuy Bruno 
Published: October 2006

SummaryThe last gene (pulO) of the pulC–O pullulanase secretion gene operon of Klebsiella oxytoca codes for a protein that is 52% identical to the product of the pilD/xcpA gene required for extracellular protein secretion and type IV pilus biogenesis in Pseudomonas aeruginosa. The PilD/XcpA protein is known to remove the first six amino acids of the signal sequence of the type IV pilin precursor by cleaving after the glycine residue in the conserved sequence GF(M)XXXE (where X represents hydrophobic amino acids). This prepilin peptidase cleavage site is present in the products of four genes in the pulC–O operon (PulG, PulH, Pull and PulJ proteins). It is shown here that PulO processes the pulG gene product in vivo. Processing was maximal within 15 seconds, but experiments in which the expression of pulO was uncoupled from that of the other genes in the secretion operon suggest that processing can also occur post‐translationally. The products of two pulG derivatives with internal in‐frame deletions were also processed by PulO, but the three PulG–PhoA hybrids, two PulJ–PhoA hybrids and the single PulH–PhoA hybrid tested did not appear to be processed. Sucrose gradient fraction‐ation experiments showed that both precursor and mature forms of PulG appear to be associated with low‐density, outer membrane vesicles prepared by osmotic lysis of sphaeroplasts. Neither the xcpA gene nor the Bacillus subtilis gene comC, which is also homologous to pulO and codes for a protein with type IV prepilin peptidase activity, can correct the pullulanase secretion defect in an Escherichia coli strain carrying all of the genes required for secretion except pulO. Furthermore, neither XcpA nor ComC is able to process prePulG protein in vivo.
A leptomycin B resistance gene of Schizosaccharomyces pombe encodes a protein similar to the mammalian P‐glycoproteins
Authors: Nishi Kazunori , Yoshida Minoru , Nishimura Marie , Nishikawa Mitsuo , Nishiyama Makoto , Horinouchi Sueharu , Beppu Teruhiko 
Published: October 2006

SummaryScreening for leptomycin B (LMB)‐resistant transformants in a gene library constructed in Schizosaccharomyces pombe with the chromosomal DNA of an LMB‐resistant mutant of S. pombe and with multicopy plasmid pDB248′ as the vector led to the isolation of a gene, named pmd1+, encoding a 1362‐amino‐acid protein. This protein showed great similarity in amino acid sequence to the mammalian P‐glycoprotein encoded by the multidrug resistance gene, mdr, and the Saccharomyces cerevisiae a‐factor transporter encoded by STE6. In addition, computer analyses predicted that the protein encoded by pmd1+ formed an intramolecular duplicated structure and each of the halves contained six transmembrane regions as well as two ATP‐binding domains, as observed with the P‐glycoproteins and the STE6 product. Consistent with this was that S. pombe cells containing the pmd1+ gene on a multicopy plasmid showed resistance not only to LMB but also to several cytotoxic agents. The pmd1 null mutants derived by gene disruption were viable and hypersensitive to these agents. All these data suggest that the pmd1+ gene encodes a protein that is a structural and functional counterpart of mammalian mdr proteins.
Overproduction of a selenocysteine‐containing polypeptide in Escherichia coli: the fdhF gene product
Authors: Chen Giafen T. , Axley Milton J. , Hacia Joseph , Inouye Masayori 
Published: October 2006

SummaryThe fdhF gene of Escherichia coli codes for the selenocysteine‐including protein subunit of formate dehydrogenase H. The protein subunit consists of 715 amino acid residues containing a single selenocysteine residue at position 140 which is encoded by a UGA codon. The decoding of this opal termination codon occurs under anaerobic growth conditions by means of a specific tRNA, i.e. the selC gene product. The ability of E. coli cells to overproduce a seleno‐polypeptide was examined using the fdhF gene as a model system. Surprisingly, E. coli was able to synthesize the fdhF gene product at the level of approximately 12% of the total cellular protein. This was achieved by cloning fdhF in a multicopy plasmid together with a synthetic selC gene under the Ipp promoter. FdhF production was absolutely dependent upon the addition of selenium to the culture medium and was almost completely blocked in the presence of oxygen. The product was specifically labelled with 75Se, proving that it consisted of a selenoprotein. The product was purified to homogeneity and shown to exhibit the catalytic properties characteristic of formate dehydrogenase H.
A novel sensor‐regulator protein that belongs to the homologous family of signal‐transduction proteins involved in adaptive responses in Escherichia coli
Authors: Nagasawa S. , Tokishita S. , Aiba H. , Mizuno T. 
Published: October 2006

SummaryExpression of the Escherichia coli outer membrane porins, OmpC and OmpF, is regulated in response to changes in the medium osmolarity through the functions of the regulatory factors, EnvZ and OmpR. A 3.0 kilobase pair DNA fragment cloned from E. coli is able phenotypically to suppress the defect in ompC and ompF expression caused by an envZ deletion mutation, provided that a certain gene located in this fragment is expressed on a high copy‐number plasmid. Nucleotide sequencing revealed that the putative gene encodes a protein of 102452 Da. The deduced amino acid sequence of the protein shows a high degree of homology to those of both EnvZ and OmpR, i.e. it contains both a sensory kinase domain’ and a ‘response regulator domain’ in its primary amino acid sequence. The protein identified in this study is probably a novel member of the homologous family of proteins involved in bacterial adaptive responses. Hence, the gene encoding this novel sensor‐regulator protein was designated as barA (bacterial adaptive responses) and mapped at 60 min on the E. coli genetic map. The BarA protein in isolated membranes was demonstrated in vitro to undergo phosphorylation in the presence of ATP.
Rapid generation of directed and unmarked deletions in Xanthomonas
Authors: Kamoun Sophien , Tola Elisabetta , Kamdar Haresh , Kado Clarence I. 
Published: October 2006

SummaryWe have devised a rapid four‐step procedure for the generation of directed and unmarked chromosomal deletions in bacteria, based on the use of a novel cloning vector containing the Bacillus subtilis sacB gene that encodes levansucrase and confers sucrose sensitivity, which can be used for counter‐selection. Using this technique, we describe the construction of a 6.5 kb directed and unmarked deletion in a phytopathogenicity region of the chromosome in Xanthomonas campestris. This procedure allows rapid and easy transfer of a wide variety of mutant allelic DNA to the bacterial chromosome, and should be adaptable to various bacteria besides Xanthomonas spp.
Molecular basis of host epithelial cell recognition by Trichomonas vaginalis
Authors: Arroyo R. , Engbring J. , Alderete J. F. 
Published: October 2006

SummaryParasitism of host epithelial cells by Trichomonas vaginalis is a highly specific event. Four trichomonad surface proteins (adhesins) with molecular masses of 65000 daltons (65kDa; AP65), 51 kDa (AP51), 33kDa (AP33), and 23 kDa (AP23) mediate the interaction of T. vaginalis with epithelial cells. Fresh isolates, when compared with long‐term‐grown isolates, had greater amounts of adhesins, which corresponded with increased levels of cytoadherence. Anti‐adhesin antibodies reacted by immunobiol only with the respective protein and detected, by indirect immunofluore‐scence, each adhesin on the parasite surface. These antibodies inhibited the binding of live parasites to epithelial cells and protected epithelial cells from contact‐dependent cytotoxicity. The pretreatment of epithelial cells with a preparation of purified adhesins also blocked trichomonal cytoadherence. Moreover, HeLa cells possessed molecules which recognized and bound to adhesins on nitrocellulose blots.
Analysis of an Escherichia coli mutant strain resistant to the cell‐killing function encoded by the gef gene family
Authors: Poulsen Lars K. , Larsen Nethe W. , Molin Søren , Andersson Poul 
Published: October 2006

SummaryThe chromosomal genes gef and relF from Escherichia coli and the plasmid‐encoded genes hok, flmA, srnB, and pndA constitute the gef qene family, which encodes a cell‐killing function. In order to investigate the mechanism of cell killing we have isolated an E. coli mutant strain that is resistant to the overexpression of the toxic proteins encoded by the gef gene family. This phenotype requires at least two mutations, one of which has been mapped to 55.2 minutes. This mutation was sequenced and shown to represent a single base substitution in an open reading frame (ORF178) encoding a putative membrane protein having a molecular mass of 20.1 kDa. ORF178 and an upstream frame, ORF190, probably constitute an operon.
Isolation and characterization of conditional adherent and non‐type 1 fimbriated Salmonella typhimurium mutants
Authors: Lockman Hank A. , Curtiss Roy 
Published: October 2006

SummaryMutations in the genes encoding the type 1 fimbriae of Salmonella typhimurium were isolated by selecting for the deletion of Tn10 inserted adjacent to the chromosomal fim+ genes and screening for the loss of mannose‐sensitive haemagglutination (HA) activity. S. typhimurium strains with Tn10 insertions in ahp were hypersensitive to peroxides, and tetracycline‐sensitive derivatives of ahp::Tn10 mutants displayed two fim mutant phenotypes. The predominant class of fim mutants did not synthesize type 1 fimbriae. A second type of fim mutant synthesized type 1 fimbriae and exhibited a conditional lipoic acid requirement for HA. A fim–lip conditional mutant synthesized type 1 fimbriae when grown in Mueller‐Hinton broth but the haemagglutinating activity of the fimbriae was dependent upon the addition of lipoic acid to the growth medium. Independentiy isolated lip mutations did not demonstrate a similar pleiotropic effect on HA. Western blots of fimbriae extracted from a fim–lip conditional mutant that was grown under permissive and restrictive conditions indicated the presence of 33 and 36.6 kDa proteins in HA+ fimbriae that were absent in HA‐ fimbriae. The HA+ phenotype of both conditional and non‐fimbriafed mutants was restored by transformation with cloned genes encoding S. typhimurium type 1 fimbriae.
Organization and function of the mcrBC genes of Escherichia coli K‐12
Authors: Raleigh E. A. 
Published: October 2006

SummaryMany natural DNA sequences are restricted in Escherichia coli K‐12, not only by the classic Type I restriction system EcoK, but also by one of three modification‐specific restriction systems found in K‐12. The McrBC system is the best studied of these. We infer from the base composition of the mcrBC genes that they were imported from an evolutionarily distant source. The genes are located in a hypervariable cluster of restriction genes that may play a significant role in generation of species identity in enteric bacteria. Restriction activity requires the products of two genes for activity both in vivo and in vitro. The mcrB gene elaborates two protein products, only one of which is required for activity in vitro, but both of which contain a conserved amino acid sequence motif identified as a possible GTP‐binding site. The mcrC gene product contains a leucine heptad repeat that could play a role in protein‐protein interactions. McrBC activity in vivo and in vitro depends on the presence of modified cytosine in a specific sequence context; three different modifications are recognized. The in vitro activity of this novel multi‐subunit restriction enzyme displays an absolute requirement for GTP as a cofactor.
Characterization of cat messenger RNA decay suggests that turnover occurs by endonucleolytic cleavage in a 3′ to 5′ direction
Authors: Meyer Barbara J. , Schottel Janet L. 
Published: October 2006

SummaryTurnover of the chloramphenicol acetyltransferase (cat) messenger RNA in Escherichia coli was investigated by analysis of cellular mRNA decay intermediates and the transcript sequence. Analysis of the sequence has revealed the presence of a repetitive extragenic palindromic (REP) element at the 3′ end of the transcript as well as several 5′‐UUAU‐3′ sequences, two elements which have roles in modulating turnover of other E. coli mRNAs. For cat mRNA, however, removal of the REP sequence has no effect on the half‐life of the transcript, indicating that the REP sequence does not stabilize the upstream region of this message. Results from mapping of the mRNA decay products by several techniques suggest that the message is instead subject to endonucleolytic attack at five sites 5′ of the REP element. The sequence UUAU is present at three of these five sites. It also appears that the cat mRNA is sequentially cleaved in a 3′ to 5′ direction during turnover of this mRNA in vivo. A model for cat mRNA turnover is discussed.
Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL
Authors: Brown Kelly L. , Wood Steven , Buttner Mark J. 
Published: October 2006

SummaryThe promoter region of the agarase gene (dagA) of Streptomyces coelicolor A3(2) is complex; it consists of four distinct promoters with different ‐10 and ‐35 regions. We report the isolation of a form of RNA polymerase that mediates transcription in vitro from the dag Ap4 promoter. The core components of this RNA polymerase are associated with a polypeptide of c. 66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from the dag Ap4 and Bacillus subtilis veg promoters in vitro. Alignment of the DNA sequences of these two promoters shows that they have bases in common in the ‐10 and ‐35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria. N‐terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of the hrdB gene. Previous experiments showed that the predicted amino acid sequence of the hrdB gene product is very similar to the major sigma factors of other bacteria and suggested that disruption of the hrdB gene is lethal. These observations together lead to the conclusion that we have isolated the major RNA polymerase of Streptomyces coelicolor A3(2).We have developed an improved protocol for the renaturation of sigma factors that have been Isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS‐PAGE). This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL. We expect this protocol to find general application for renaturation of other polypeptides that have been subjected to SDS‐PAGE.
Biochemical activities of the ParA partition protein of the P1 plasmid
Authors: Davis M. A. , Martin K. A. , Austin S. J. 
Published: October 2006

SummaryThe unit‐copy P1 plasmid depends for stability on a plasmld‐encoded partition region called par, consisting of the parA and parB genes and the parS site. ParA is absolutely required for partition, but its partition‐critical role is not known. Purified ParA protein is shown to possess an ATPase activity in vitro which is specifically stimulated by purified ParB protein and by DNA. ParA is responsible for regulation of expression of parA and parB, and purified ParA has an ATP‐dependent, site‐specific DNA binding activity which recognizes a sequence that overlaps the parA promoter. The rote of the ATP‐dependence of the binding activity, as well as other possible functions of the ATPase activity in partition, is discussed.
High‐frequency rearrangements in the chromosome of Mycoplasma pulmonis correlate with phenotypic switching
Authors: Bhugra B. , Dybvig K. 
Published: October 2006

Summary Mycoplasma pulmonis is a murine pathogen that causes chronic respiratory disease in laboratory rats and mice. Several examples of high‐frequency phenotypic switching have been reported for M. pulmonis, the molecular basis of which is unknown. We report here that during growth the M. pulmonis chromosome undergoes DNA rearrangements at a high frequency. Some of the rearrangements we examined correlated with changes in the susceptibility of the cells to mycoplasma virus P1, an example of phenotypic switching involving changes in surface antigen structure. Other rearrangements, unrelated to phenotypic switching, involved a DNA element present in the chromosome in multiple copies. The high level of DNA recombination that occurred in M. pulmonis indicates that this may be one of the most variable genomes studied to date. High levels of DNA recombination may contribute to the unusually high rate of evolution that mycoplasmas are thought to be undergoing. Understanding the molecular basis for this phenomenon may provide an insight into the chronic nature of many mycoplasmal infections.
Mutants of Rhodobacter sphaeroides lacking one or more pigment‐protein complexes and complementation with reaction‐centre, LH1, and LH2 genes
Authors: Jones M. R. , Fowler G. J. S. , Gibson L. C. D. , Grief G. G. , Olsen J. D. , Crielaard W. , Hunter C. N. 
Published: October 2006

SummaryThe photosynthetic apparatus of Rhodobacter sphaeroides is comprised of three types of pigment‐protein complex: the photochemical reaction centre and its attendant LH1 and LH2 light‐harvesting complexes. To augment existing deletion/insertion mutants in the genes coding for these complexes we have constructed two further mutants, one of which is a novel double mutant which is devoid of all three types of complex. We have also constructed vectors for the expression of either LH1, LH2 or reaction‐centre genes. The resulting system allows each pigment‐protein complex to be studied either as part of an intact photosystem or as the sole complex in the cell. In this way we have demonstrated that reaction centres can assemble independently of either light‐harvesting complex in R sphaeroides. In addition, the isolation of derivatives of the deletion/insertion mutants exhibiting spontaneous mutations in carotenoid biosynthesis provides an avenue for examining the role of carotenoids in the assembly of the photo‐synthetic apparatus. We show that the LH1 complex is assembled regardless of the carotenoid background, and that the type of carotenoid present modifies the absorbance of the LH1 bacteriochlorophylls.
Identification and characterization of a novel Bacillus thuringiensis δ‐endotoxin entomocidal to coleopteran and lepidopteran larvae
Authors: Tailor Ravi , Tippett Jan , Gibb Graham , Pells Stephen , Jordan Linda , Ely Susan 
Published: October 2006

SummaryA new class of Bacillus thuringiensisδ‐endotoxins, or insecticidal control proteins (ICPs), is defined by an apparently cryptic protein with a unique primary structure and novel entomocidal specificity for certain coleopteran and lepidopteran species. The discovery of a new group of ICPs will extend the use of this natural insecticide in integrated pest‐management systems.
Morphological and cytoskeletal changes in epithelial cells occur immediately upon interaction with Salmonella typhimurium grown under low‐oxygen conditions
Authors: Francis C. L. , Starnbach M. N. , Falkow S. 
Published: October 2006

Summary Salmonella typhimurium grown under oxygen‐limiting conditions were found to enter into, elicit actin filament rearrangement in, and effect morphological changes upon HEp‐2 cells within 15 min after infection. Video microscopy revealed that host cell morphological changes associated with entry began within 1 min of productive adherence. Polarized Caco‐2 cell morphology was affected 40 s after infection with low‐oxygen‐grown S. typhimurium. Stationary‐phase S. typhimurium did not elicit these phenomena within this time‐period even when adherence was enhanced with the afimbrial adhesin, AFA‐I. Thus, environmental cues regulate S. typhimurium invasion factors, allowing for immediate entry into host cells. Additionally, actin filament rearrangement and morphological changes in the eukaryotic host cell are essential for entry and occur within minutes of infection.
Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes
Authors: Clepet C. , Borne F. , Krishnapillai V. , Baird C. , Patte J. C. , Cami B. 
Published: October 2006

SummaryThree genes from Pseudomonas aeruginosa involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrC genes lie at the thr locus of the P. aeruginosa chromosome map (31 min) and are likely to be organized in a bicistronic operon. The encoded proteins are quite similar to the Hom and TS proteins from other bacterial species. The thrB gene was located by pulsed‐field gel electrophoresis experiments at 10 min on the chromosome map. The product of this gene does not share any similarity with other known ThrB proteins. No phenotype could be detected when the chromosomal thrB gene was inactivated by an insertion. Therefore the existence of isozymes for this activity is postulated. HDH activity was feedback inhibited by threonine; the expression of all three genes was constitutive. The overall organization of these three genes appears to differ from that in other bacterial species.
DNA sequence determination and functional characterization of the OCT‐plasmid‐encoded alkJKL genes of Pseudomonas oleovorans
Authors: van Beilen Jan B. , Eggink Gerrit , Enequist Hans , Bos Rolf , Witholt Bernard 
Published: October 2006

SummaryThe alkBFGHJKL and alkST operons encode enzymes that allow Pseudomonas putida (oleovorans) to metabolize alkanes. In this paper we report the nucleotide sequence of a 4592 bp region of the alkBFGHJKL operon encoding the AlkJ, AlkK and AlkL polypeptides.The alkJ gene encodes a protein of 59 kilodaltons. The predicted amino acid sequence shows significant homology with four flavin proteins: choline dehydrogenase, a glucose dehydrogenase and two oxidases. AlkJ is membrane‐bound and converts aliphatic medium‐chain‐length alcohols into aldehydes. The properties of AlkJ suggest that it is linked to the electron transfer chain. AlkJ is necessary for growth on alkanes only in P. putida alcohol dehydrogenase (AlcA) mutants.AlkK is homologous to a range of proteins which act by an ATP‐dependent covalent binding of AMP to their substrate. This list includes the acetate, coumarate and long‐chain fatty acid CoA ligases. The alkK gene complements a fadD mutation in Escherichia coli, which shows that it indeed encodes an acyl‐CoA synthetase. AlkK is a 60 kilodalton protein located in the cytoplasm.AlkL is homologous to OmpW, a Vibrio cholerae outer membrane protein of unknown function, and a hypothetical polypeptide encoded by ytt4 in E. coli. AlkL, OmpW and Ytt4 all have a signal peptide and end with a sequence characteristic of outer membrane proteins. The alkL gene product was found in the outer membrane of E. coli W3110 containing the alk‐genes. The alkL gene can be deleted without a clear effect on growth rate. Its function remains unknown.The G+C content of the alkJKL genes is 45%, identical to that of the alkBFGH genes, and significantly lower than the G+C content of the OCT‐plasmid and the P. putida chromosome.