bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutational analysis of the Bordetella pertussis fim/fha gene cluster: identification of a gene with sequence similarities to haemolysin accessory genes involved in export of FHA
Authors: Willems Rob J. L. , Geuijen Cecile , van der Heide Han G. J. , Renauld Genevieve , Berlin Philippe , van den Akker Willem M. R. , Locht Camille , Mooi Frits R. 
Published: October 2006

SummaryThe chromosome of Bordetella pertussis harbours a region of 27 contiguous kb, which contains the bvg, fha and flm genes, involved in the co‐ordinate regulation of virulence genes, FHA production and fimbriae production, respectively. The linkage of FHA and fimbrial genes has resulted in some confusion concerning the existence and location of genes required for the production of FHA and the function of the fimbrial genes fimB‐D, which were proposed to be involved in both FHA and fimbriae biosynthesis. Through the use of non‐polar mutations in each of these genes, we found that fimB‐D are required for the production of both serotype 2 and 3 fimbriae, but not for FHA biosynthesis. Furthermore, a large open reading frame, designated fhaC, was identified downstream of fimD. It was shown that fhaC is essential for FHA production but not for fimbriae biogenesis. We propose that insertion mutations in fimB‐D affect FHA production because of polar effects on fhaC expression. An insertion in the region downstream of fhaC had only a slight effect on FHA and fimbriae production. The fhaC gene product shows homology with ShIB and HpmB, two outer membrane proteins involved in export and activation of the haemolysins, ShIA and HpmA, of Serratia marcescens and Proteus mirabilis, respectively. Homology is also observed between the N‐termini of FHA, ShIA and HpmA. Export of the haemolysins requires the Af‐termini of these molecules, and when this region was removed from FHA by an in‐frame deletion, FHA biosynthesis was abolished. These results suggest that the N‐terminus of FHA interacts with FhaC, and that as a result FHA is transported across the outer membrane.
Evidence for a general role for high‐affinity non‐catalytic cellulose binding domains in microbial plant ceil wall hydroiases
Authors: Millward‐Sadler S. J. , Poole D. M. , Henrissat B. , Hazlewood G. P. , Clarke J. H. , Gilbert H. J. 
Published: October 2006

SummaryCellulases expressed by Cellulomonas fimi consist of a catalytic domain and a discrete non‐catalytic cellulose‐binding domain (CBD). To establish whether CBDs are common features of plant cell‐wall hydroiases from C. fimi, the molecular architecture of xylanase D (XYLD) from this bacterium was investigated. The gene encoding XYLD, designated xynD, consisted of an open reading frame of 1936 bp encoding a protein of Mr 68000. The deduced primary sequence of XYLD was confirmed by the size (64kDa) and N‐terminal sequence of the purified recombinant xylanase. Biochemical analysis of the purified enzyme revealed that XYLD is an endo‐acting xylanase which displays no detectable activity against polysaccharides other than xylan. The predicted primary structure of XYLD comprised an /V‐terminal signal peptide followed by a 190‐residue domain that exhibited significant homology to Family‐G xylanases. Truncated derivatives of xynD, encoding the W‐terminal 193 amino acids of mature XYLD directed the synthesis of a functional xylanase, confirming that the 190‐residue N‐terminal sequence constitutes the catalytic domain. The remainder of the enzyme consisted of two approximately 90‐residue domains, which exhibited extensive homology with each other, and limited sequence identity with CBDs from other polysaccharide hydrolases. Between the two putative CBDs is a 197‐amino‐acid sequence that exhibits substantial homology with Rhizobium NodB proteins. The four discrete domains in XYLD were separated by either threonine/prolineor novel glycine‐rich linker regions. Although full‐length XYLD adsorbed to cellulose, truncated derivatives of the enzyme lacking the C‐terminal CBD hydrolysed xylan but did not bind to cellulose. Fusion of the C‐terminal domain to glutathione‐Stransferase generated hybrid proteins that bound to crystalline cellulose, but not to amorphous cellulose or xylan. The location of CBDs in a C. fimi xylanase indicates that domains of this type are not restricted to cellulases, but are widely distributed between hemicellutases also, and therefore play a pivotal role in the activity of the whole repertoire of plant cell‐wall hydrolases. The role of the NodB homologue in XYLD is less certain.
Escherichia coli periplasmic chaperone FaeE is a homodimer and the chaperone‐K88 subunit complex is a heterotrimer
Authors: Mol Olaf , Visschers Ronald W. , de Graaf Frits K. , Oudega Bauke 
Published: October 2006

SummaryThe interaction of FaeE, a periplasmic chaperone involved in K8B biosynthesis, and the major fimbrial subunit FaeG was Investigated. The genes encoding the two proteins were subcloned together in the expression vector pINIIIA1, Cells expressing the sub‐cloned genes accumulated in their periplasm a complex of FaeE and FaeG. This complex was purified by isoelectric focusing and anion‐exchange fast‐protein liquid chromatography. SOS‐PAGE, native gel etectrophoresis, immunoblotting and determination of the N‐terminal amino acid sequences and the molar ratio of the W‐terminal amino acid residues revealed that the complex is a heterotrimer consisting of two molecules of FaeE and one molecule of FaeG. The periplasmic chaperone FaeE was purified from the periplasm of cells expressing only the subcloned faeE gene. Gel filtration, protein cross‐linking analysis and a biophysical approach in which the rotation diffusion coefficient of the purified FaeE was determined led to the conclusion that the native FaeE chaperone is a homodimer.
Halobacterial megaplasmids are negatively supercoiled
Authors: López‐García Purificación , Antón Josefa , Abad Jose Pascua , Amils Ricardo 
Published: October 2006

SummarySeveral covalently closed circular halobacterial megaplasmids (up to more than 500 kb) from different strains of Halolerax mediterranei, have been resolved by orthogonal‐field alternating gel electro‐phoresis (OFAGE). These molecules seem to be negatively supercoiled in vivo, as deduced from the effect of intercalating agents affecting their topology and, therefore, their electrophoretic mobility. It has also been demonstrated that the topolsomerase II Inhibitor novobiocin affects the native topological state of halobacterial megaplasmids impeding their migration in OFAGE under standard conditions for resolution of large supercoiled molecules.
The receptor for Bacillus thuringiensis CrylA(c) delta‐endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N
Authors: Knight Peter J. K. , Crickmore Neil , Ellar David J. 
Published: October 2006

SummaryA 120 kDa glycoprotein in the larval midgut membrane of the Iepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) δ‐endotoxin, has been purified by a combination of protoxin affinity Chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin In the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N‐terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3‐fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.
A plasmid‐encoded rfbO:54 gene cluster is required for biosynthesis of the O:54 antigen in Salmonella enterica serovar Borreze
Authors: Keenleyside Wendy J. , Perry Malcolm , Maclean Leann , Poppe Cornelius , Whitfield Chris 
Published: October 2006

SummaryPrevious studies demonstrated that the presence of a 7–8 kb plasmid is correlated with expression of the lipopolysaccharide (LPS) O:54 antigen in several Salmonella enterica serovars. In this study, a 6.7 kb plasmid from a field isolate of S. enterica serovar Borreze was shown to encode enzymes responsible for the synthesis of the O:54 polysaccharide. Curing the plasmid results in simultaneous loss of smooth O‐polysaccharide‐substituted LPS molecules and O:54 serotype. SDS‐PAGE analysis of other 0:54 isolates indicated that the O:54 O‐polysaccharide can be co‐expressed with an additional O‐polysaccharide, likely encoded by chromosomal genes. The structure of the O:54 polysaccharide was determined by a combination of chemical and nuclear magnetic resonance (NMR) methods and was found to be an unusual homopolymer of N‐acetylmannosamine (D‐ManNAc) residues. The polysaccharide contained a disaccharide repeating unit with the structure:→4)‐β‐d‐MANpNAc‐(1→3)‐β‐ d‐ManpNAc‐(1 →This structure does not resemble other O‐polysaccharides in S. enterica. To examine the role played by plasmid functions in synthesis of the O:54 polysaccharide, the 6.7 kb plasmid was cloned to produce a hybrid plasmid (pWQ800) in pGEM‐7Zf(+). In Escherichia coli K‐12 Δrfb, pWQ800 directed the synthesis of authentic O:54 polysaccharide. Polymerized O:54 polysaccharide was also produced in S. enterica serovar Typhimurium rfb and rfc mutants. From these data, we conclude that pWQB00 carries the rfbO:54 gene cluster and synthesis of the O:54 poiysaccharides does not require host chromosomal rfb functions. However, synthesis of the O:54 polysaccharide requires the function of the rfe and rffE genes which are part of the gene cluster encoding enzymes involved in biosynthesis of enterobacterial common antigen. The rffE gene product synthesizes the O:54 precursor, uridine diphospho‐N‐acetylmannosamine. This is the first description of a plasmid‐encoded rfb gene cluster in Salmonella.
Initiation and termination of DNA transfer at F plasmid oriT
Authors: Gao Qian , Luo Yanan , Deonier Richard C. 
Published: October 2006

SummaryDNA sequences within the F plasmid transfer origin (oriT) were tested for their ability to initiate or terminate conjugal transfer. Mutant and wild‐type oriT elements were cloned as direct repetitions flanking the rpsL gene on a pBR322‐based plasmid, and the frequency of deletion of this segment during matings sponsored by F’lac (F42) with streptomycin‐resistant recipients was measured. Shortened oriT elements that lacked adjacent TraM‐binding sites allowed efficient initiation and termination. Some truncated orir segments lacking the TraM‐binding sites and the TraY‐binding site, sbyA, initiated transfer inefficiently, but nevertheless promoted efficient termination. Removal of TraM‐, TraY‐, and IHF‐binding sites severely reduced both nicking and termination. Point mutations that previously had been reported to prevent nicking caused reduced levels of both initiation and termination. These results indicate that regions of oriT supporting initiation are more extensive than those needed for termination, although some regions are required for both. Moreover, termination can be effective for some mutant loci that do not support efficient nicking.
Mutational and physical analysis of F plasmid traY protein binding to oriT
Authors: Luo Yanan , Gao Qian , Deonier Richard C. 
Published: October 2006

SummaryF plasmid traY protein binding to wild‐type or deleted regions containing the TraY‐binding site, sbyA, was studied in vitro. The principal DNA‐protein complex was formed with DNA segments including the sbyA site defined by footprinting and (with lesser affinity) with truncated segments that retained the leftward two‐thirds of sbyA. This located the major sequence determinants for TraY binding between bp 204 and 227 on the oriT map. For all sequences tested, bound TraY induced bending of approximateiy 50 to 55°, and centred between bp 214 and 221. Thermodynamic and mobility analyses indicated that two TraY protomers bind to sbyA. At higher TraY concentrations, additional TraY bound to the left of the sbyA in a region previously shown to bind IHF (site IHF A). TraY binding to this additional site (sbyC) was inhibited by IHF. Sequence similarities shared by sbyA, sbyB, and SbyC may include the critical base pairs for TraY binding.
VsrA, a second two‐component sensor regulating virulence genes of Pseudomonas solanacearum
Authors: Schell Mark A. , Denny Timothy P. , Huang Jianzhong 
Published: October 2006

SummaryThe wilt‐inducing phytopathogen Pseudomonas solanacearum produces several extracellular virulence factors, both polysaccharides (EPS I) and proteins (EXPs), which are independently regulated by a LysR‐type transcriptional regulator, PhcA, and a histidine kinase sensor, VsrB. Here we characterize a third locus, vsrA, which is also required for normal production of EPS I, some EXPs and wilt disease. Analysis of eps::lacZ reporters in vsrA mutants showed that, like vsrB and phcA, vsrA is required for maximal expression (transcription) of eps, which contains some of the genes necessary for production of EPS I. Unlike vsrB and phcA mutants, however, eps transcription (and EPS I production) by vsrA mutants varies from 3 to 17% of wild‐type levels, depending on growth conditions. Inactivation of vsrA also causes a dramatic reduction in production of three species of EXPs (28kDa, 48kDa, and 66kDa), and an apparent increase in production of a few other EXPs. Unlike most other EPS‐deficient P. solanacearum strains, vsrA mutants caused almost no disease symptoms when 104 cells were stem‐inoculated into tomato plants. This correlated with a greater than 10‐fold reduction in their ability to grow in plants. vsrA was cloned from a P. solanacearum genomic library by complementation of the vsrA mutant and was further subcloned on a 2.3kb DNA fragment. PhoA fusion analysis and subcellular localization of the vsrA gene product in Escherichia coli maxicells suggest that it is a 53 kDa membrane‐associated protein. Analysis of the nucleotide sequence of vsrA revealed a 502 residue open reading frame with homology to the histidine kinase domain of sensors in the two‐component regulator family. This discovery shows that EPS I production by P. solanacearum is simultaneously controlled by dual two‐component sensors.
Ordered cosmid library and high‐resolution physical—genetic map of Helicobacter pylori strain NCTC11638
Authors: Bukanov Nick O. , Berg Douglas E. 
Published: October 2006

Summary Helicobacter pylori is a Gram‐negative bacterium that infects the human gastric mucosa, causes gastritis and contributes to the development of peptic ulcers and gastric cancer. To facilitate molecular genetic analysis of this pathogen, we constructed a ∼20‐fold redundant cosmid library and physical/genetic map of strain NCTC11638. Genomic DNA fragments were cloned into the cosmid vector Lorist6, and clones were ordered by hybridization with several types of probes: (i) ends of cloned DNAs; (ii) chromosomal Notl digest fragments; (iii) cosmids containing Notl sites; and (iv) specific genes. Seven hundred and fifty‐one cosmids were mapped to one of thrM contigs covering > 90% of the chromosome, and are represented by a 68‐cosmid miniset. The order of cosmids was confirmed and extents of overlap among them were estimated by restriction analysis.All currently known H. pylori genes were mapped, including those for a cytotoxin (vacA), cytotoxin‐associated protein (cagA), urease and regulatory functions (ureAB, ureD and ureH), catalase (katA), major and minor flagellins (flaA and flaB), heat‐shock (stress) and chaperone proteins (dnaK, htrA, hspB (groEL)), prokaryotic ferritin (pfr), an adhesin subunit (hpaA), a surface protein (26kDa), and 16S and 23S ribosomal RNAs (two genes each). The orientations of eight genes or clusters were determined, and two repetitive sequences were also found. The gene order and rRNA gene copy number determined here differed from that reported for an unrelated strain, which suggests considerable flexibility in H. pylori genome organization.
Leucine‐responsive regulatory protein, IS 1 insertions, and the negative regulator FaeA control the expression of the fae (K88) operon in Escherichia coli
Authors: Huisman Tako T. , Bakker Douwe , Klaasen Pia , de Graaf Frits K. 
Published: October 2006

SummaryNucleotide sequence analysis of the fae operon encoding the biosynthesis of K88 fimbriae revealed the presence of two divergently transcribed regulatory genes, faeA and faeB, separated by two inverted iS 1 insertions. The amino acid sequences of the regulatory proteins FaeA and FaeB show similarity to the primary structure of corresponding regulatory proteins involved in the biosynthesis of Pap and S fimbriae. Expression of faeA is positively controlled by the FaeA protein, whereas K88 fimbriae production is negatively controlled by the co‐operative activity of FaeA and the leucine‐responsive regulatory protein (Lrp). Exchange of FaeA for Papl, a positive regulator of Pap fimbriae expression, also represses K88 production indicating that the combination Papl/Lrp has opposite effects on fae and pap expression. Mutations in faeB had no effect on the biosynthesis of K88 fimbriae. The presence of the two iS 1 insertions is hypothesized to neutralize part of the repression of K88 biosynthesis by FaeA/Lrp. Like pap, the fae operon does not respond to exogenous leucine.
Expression and role of the universal stress protein, UspA, of Escherichia coli during growth arrest
Authors: Nyström Thomas , Neidhardt Frederick C. 
Published: October 2006

SummaryThe synthesis of the small, cytoplasmic protein UspA universal stress protein A) of Escherichia coli is induced as soon as the cell growth rate falls below the maximal growth rate supported by the medium, regardless of the condition inhibiting growth. The increase in UspA synthesis appears to be the result of Induction of the monocistronic uspA gene. Induction of this gene during a heat‐shock treatment is demonstrated to be the result of transcriptional activation of σ70‐dependent promoter which has previously been shown to be activated also during carbon starvation‐induced growth arrest. Mutant cells lacking UspA grow at rates indistinguisible from the isogenic parent at different temperatures and in the presence of different growth inhibitors but are impaired in their ability to survive prolonged periods of complete growth inhibition caused by a variety of diverse stresses, including CdCl2, H2O2, DNP, CCCP exposure, and osmotic shock. Moreover, the uspA mutation results in an increased sensitivity of cells to carbon‐source starvation (i.e. glucose, glycerol or succinate depletion). Also, the mutation causes a marked alteration in the timing of starvation protein expression but protein expression during steady‐state growth appears to be normal. The results presented have prompted us to postulate that UspA may have a general protective function related to the growth arrest state.
Periplasmic disulphide bond formation is essential for cellulase secretion by the plant pathogen Erwinia chrysanthemi
Authors: Bortoli‐German Isabelle , Brun Emmanuel , Py Béatrice , Chippaux Marc , Barras Frédéric 
Published: October 2006

SummarySecretion to the cell exterior of cellulase EGZ and of at least six pectinases enables the Gram‐negative Erwinia chrysanthemi to cause severe plant disease. The C‐terminal cellulose‐binding domain (CBD) of EGZ was found to contain a disulphide bond which forms, in the periplasm, between residues Cys‐325 and Cys‐382. Dithiothreitol (DTT)‐treatment of native EGZ showed that the disulphide bond was dispensable, both for catalysis and cellulose binding. Adding DTT to E. chrysanthemi cultures led to immediate arrest of secretion of EGZ which accumulated in the periplasm where the CBD was eventually proteolysed. Site‐directed mutagenesis that affected Cys residues involved in disulphide bond formation resulted in molecules that were catalytically active and able to bind to cellulose but were no longer secreted, Instead they accumulated in the periplasm. Interestingly, the region around EGZ Cys‐325 is conserved in two pectinases secreted by the same pathway as EGZ. We conclude that the conserved Cys, and possibly adjacent residues, bear essential information for EGZ to be secreted and that periplasmic disulphide bond formation is an obligatory step which provides a pre‐folded functional form of EGZ with secretion competence.
Adhesin regulatory genes within large, unstable DNA regions of pathogenic Escherichia coli: cross‐talk between different adhesin gene clusters
Authors: Morschhäuser Joachim , Vetter Viktoria , Emödy Levente , Hacker Jörg 
Published: October 2006

SummaryThe uropathogenic Escherichia coli strain 536 possesses two large, unstable DNA regions on its chromosome, which were termed pathogenicity islands (pais). Deletions of pais, which occur with relatively high frequency in vitro and in vivo, lead to avirulent mutants. The genetic determinants for production of haemolysin (Hly) and P‐related fimbriae (Prf) are located in one of these islands. Deletion of this pathogenicity isiand (paill) not only removes the hly‐ and prf‐specific genes, but also represses S fimbriae (Sfa), although the sfa genes of this virulence factor are not located on paill. We have identified two regulatory genes, prfB and prfl, of the prf gene cluster that are homologous to the sfa regulatory genes staB and SfaC, respectively. Mutations in sfaB and sfaC that inhibit transcription of the major fimbrial subunit gene sfaA were complemented by the homologous prf genes, suggesting communication between the two fimbrial gene clusters in the wild‐type strain. Chromosomal mutagenesis of the two prf regulators in strain 536 repressed transcription of sfaA, detected by Northern hybridization and a chromosomal sfaA‐lacZ fusion. In addition, haemagglutination assays measured a lower level of S fimbriae in these mutants. Expression of the cloned prf regulators in trans reversed the effect of the mutations; furthermore, constitutive expression of prfB or prfl could also overcome the repression of S fimbriae in a strain that had lost the pathogenicity islands. Virulence assays in mice established that the prf mutants were less virulent than the wild‐type strain. The results demonstrate that cross‐regulation of two unlinked virulence gene clusters together with the co‐ordinate loss of large DNA regions significantly influences the virulence of an extraintestinal E. coli wild‐type isolate.