bluuebunny/crossref_metadata_2025 huggingface dataset:
Topology of diphtheria toxin B fragment inserted in lipid vesicies
Authors: Cabiaux Veronique , Quertenmont Pierre , Conrath Karel , Brasseur Robert , Capiau Carina , Ruysschaert Jean‐Marie
Published: October 2006

Diphtheria toxin (DT) is a bacterial protein that crosses the membrane of endosomes of target cells In response to the low endosomal pH. In this paper, we have inserted diphtheria toxin in asolectin vesicles at pH 5.0 and treated the reconstituted system with pronase. The peptides that were protected from digestion were separated by gel electrophoresls, transferred to a membrane and their N‐terminal sequences were determined. All peptides belong to the B fragment of DT and cover residues 194–223, 266–375 and 429–528. The secondary structures of the peptides inserted in the membrane, determined by Fourier‐transformed infrared spectroscopy, were shown to be mostly α‐helices and β‐sheets (44% and 53%, respectively). On the basis of these data and the recently published X‐ray structure of DT, we are proposing a topology for the DTB fragment in the membrane.
A new class of proteins regulating gene expression in enterobacteria
Authors: Mikulskis Alvldas V. , Cornetis Guy R.
Published: October 2006

YmoA and Hha are highly similar bacterial proteins downregulating gene expression In Yersinia entero‐colitlca and Escherichia coli. respectively. The pheno‐type of ymoA mutants evokes that of mutants affected in some histone‐like proteins. This paper describes complementation of a ymoA mutation in Y. enterocolitica by the hha gene from E. coli. We show that YmoA and Hha are not only very similar proteins but that they are functionally Interchangeable. Genetic experiments indicate that Hha can also stimulate transposition events in vivo. By Southern Biol analysis we detected hha‐homologous genes at least in Citrobacter diversus, Shigella flexneri, Shigella dysenteriae, Klebsiella pneumoniae and Salmonella typhimurium. We suggest that both YmoA and Hha belong to a new family of proteins down regulating gene expression in different enterobacteria.
The citrulline biosynthetic operon, argC‐F, and a ribose transport operon, rbs, from Bacillus subtilis are negatively regulated by Spo0A
Authors: O'Reilly Mary , Woodson Karen , Dowds Barbara C. A. , Devine Kevin M.
Published: October 2006

A method Is described here that can be used to identify operons whose expression Is controlled by any particular regulator protein. This method was used to Identify operons whose expression is negatively regulated by Spo0A in Bacillus subtilis. Twenty‐eight strains were identified, each of which contains an operon‐lacZ transcriptional fusion, negatively regulated, either directly or Indirectly, by Spo0A. In one of these strains (CSA8), the lacZ gene is fused to the argC‐F operon positioned at 100° on the B. subtilis chromosome. The regulated expression of this operon by Spo0A ∼ P is mediated indirectly through the transition state regulator AbrB and is manifest only during growth on solid medium. In a second strain (CSA15), the lacZ gene is fused to an operon encoding a transport system which displays features characteristic of the ABC group of transporters, and which has a very high level of identity to the ribose transport system from Escherichia coli Expression of the ribose transport operon is directed by a single SigA‐type promoter. Transcription from this promoter is repressed by the phosphorylated form of Spo0A during the late‐exponential/transition phase of the growth cycle and this control is not mediated through the transition‐state regulator, AbrB.
Growth phase and low pH affect the thermal regulation of the Yersinia enterocolitica inv gene
Authors: Pepe Jeffrey C. , Badger Juile L. , Miller Virginia L.
Published: October 2006

The inv gene encodes the protein invasin, which is the primary invasion factor for Yersinia enterocolitica in vitro and in vivo. Previous studies of Yersinia species have shown that inv expression and entry into mammalian cells are temperature regulated. Invasin production is reduced at the host temperature of 37°C as compared to production at ambient temperature; consequently, this study was initiated to determine whether other host environmental signals might induce inv expression at 37°C. An inv::phoA translational fusion was recombined on to the Y. enterocolitica chromosome by allelic exchange to monitor inv expression. Molecular characterization of expression of the wild‐type inv gene and the inv phoA fusion showed that invasin is not produced until early stationary phase in bacteria grown at 23°C. Y. enterocolitica grown at 37°C and pH 5.5 showed levels of inv expression comparable to those observed in bacteria grown at 23 C. An increase in Na+ ions caused a slight increase in expression at 37 C. However, expression at 37°C was unaffected by anaerobiosis, growth’medium, calcium levels, or iron levels. Additionally, Y. enterocolitica expressed invasin in Peyer's patches two days after being introduced intragastrically into BALB/c mice. These results suggest that invasin expression in K enterocolitica may remain elevated eariy during interaction with the intestinal epithelium, a site at which invasin was shown to be necessary.
Structure and heterologous expression of the Ustilago maydis viral toxin KP4
Authors: Park Chung‐Mo , Bruenn Jeremy A. , Ganesa Chandrashekar , Flurkey William F. , Bozarth Robert F. , Koltin Yigal
Published: October 2006

Killer toxins are polypeptides secreted by some fungal species that kill sensitive cells of the same or related species, in the best‐characterized cases, they function by creating new pores in the ceil membrane and disrupting ion fluxes. Immunity or resistance to the toxins is conferred by the preprotoxins (or products thereof) or by nuclear resistance genes. In several cases, the toxins are encoded by one or more genomic segments of resident double‐stranded RNA viruses. The known toxins are composed of one to three polypeptides, usually present as multimers. We have further characterized the KP4 killer toxin from the maize smut fungus Ustilago maydis. This toxin is also encoded by a single viral double‐stranded RNA but differs from other known killer toxins in several respects: it has no N‐linked glycosylation either in the precursor or in the mature polypeptide, it is the first killer toxin demonstrated to be a single polypeptide, and h Is not processed by any of the known secretory protelnases (other than the signal peptidase). It is efficiently expressed in a heterologous fungal system.
Genetic characterization of a Rhizobium meliloti lactose utilization locus
Authors: Jelesko John G. , Leigh John A.
Published: October 2006

We identified several linked genes of a lactose regulon in Rhizobium meliloti. These were lacZ, the structural gene for β‐galactosidase; lacR, the lactose repressor gene; and two genes encoding proteins of unknown function. lacW and lacX. Insertion mutants in lacW and lacZ belonged to a single genetic compiementation group, and lacW appeared to lie upstream of lacZ in an operon. Expression of lacZ, lacW and lacX was repressed by lacR, and expression of lacZ and lacW was derepressed by lactose. lacZ was not required for Induction of lacW by lactose, suggesting that lactose itself, rather than a processed form of lactose, may be the actual Inducer molecule. Expression of all three genes was repressed by succinate, and the lacR independence of this repression showed that inducer exciusion could not be the sole mechanism. This pattern of lac gene organization and regulation differs in several ways from that observed in enteric bacteria.
The mutL repair gene of Escherichia coli K‐12 forms a superoperon with a gene encoding a new cell‐wall amidase
Authors: Tsui Ho‐Ching Tiffany , Zhao Genshi , Feng Gang , Leung Hon‐Chlu Eastwood , Winkler Malcolm E.
Published: October 2006

We report a molecular genetic analysis of the region Immediately upstream from the Escherichia coli mutL DNA repair gene at 94.8 min. An open reading frame ending 9bp upstream from the start of mutL corresponds to a 48kDa polypeptide detected previously in minicells. The predicted amino acid sequence of this 48kDa polypeptide shows homology to the major N‐acetylmuramoyl‐L‐alanine amidase autolysin of Bacillus subtilis, a known amidase of Bacillus licheniformis, and the product of a Salmonella typhimurium gene that maps near SO min. Insertions in this upstream gene, which we named AmiB, or in mutL did not affect cell shape or viability; however, overexpression of the AmiB potypeptide caused ceil lysis, hypersensitivity to osmotic shock and treatment with water, and temporary autolysis by low levels of antibiotics, which are all consistent with AmiB acting as a cell‐wall hydrolase. Analysis of chromosomal transcription demonstrated that amiB forms a complex operon with mutL and two additional upstream genes. mutL transcripts also originated from an internal promoter, designated PmutL, located in amiB 312bp upstream from the translational start of mutL. Together, these results suggest that E. coli contains a second amidase possibly involved in cell‐wall hydrolysis, septation, or recycling, and that transcription of this amidase is directly linked to a gene central for DNA repair.
Protein prenylation in eukaryotic microorganisms: genetics, biology and biochemistry
Authors: Omer C. A. , Gibbs J. B.
Published: October 2006

Modrfication of proteins at C‐terminal cysteine residue(s) by the isoprenoids farnesyl (C15) and geranylgeranyl (C20) is essential for the biological function of a number of eukaryotic proteins including fungal mating factors and the small, GTP‐binding proteins of the Ras superfamily. Three distinct enzymes, conserved between yeast and mammals, have been identified that prenylate proteins: farnesyl protein transferase, geranylgeranyl protein transferase type I and geranylgeranyl protein transferase type II. Each prenyl protein transferase has its own protein substrate specificity. Much has been learned about the biology, genetics and biochemistry of protein prenylation and prenyl protein transferases through studies of eukaryotic microorganisms, particularly Saccharo‐myces cerevisiae. The functional Importance of protein prenylation was first demonstrated with fungal mating factors. The initial genetic analysis of prenyl protein transferases was in S. cerewisiae with the isolation and subsequent characterization of mutations in the RAM1, RAM2, CDC43 and BET2 genes, each of which encodes a prenyl protein transferase subunit. We review here these and other studies on protein prenylation in eukaryotic microbes and how they relate to and have contributed to our knowledge about protein prenylation in all eukaryotic cells.
Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus
Authors: McDevitt D. , Francois P. , Vaudaux P. , Foster T. J.
Published: October 2006

Four mutants of Staphylococcus aureus strain Newman that were defective in the fibrinogen receptor (clumping factor) were isolated by transposon Tn917 mutagenesis. Southern hybridization analysis of the mutants identified transposon‐host DNA junction fragments, one of which was cloned and used to generate a probe to identify and clone the wild‐type clumping factor locus (clfA). The mutants failed to form clumps in soluble fibrinogen and adhered poorly to polymethylmethacrylate (PMMA) coverslips coated with fibrinogen. A single copy of the clfA gene, when introduced into the chromosome of the mutant strains, fuily compiemented the ciumping deficiency of these strains and restored the ability of these mutants to adhere to fibrinogen‐coated PMMA. in addition, the cloned clfA gene on a shuttle plasmid aiiowed the weakiy ciumping strain 8325‐4 to form clumps with the same avidity as the wild‐type strain Newman and also significantly enhanced the adherence of 8325‐4 strains. Thus the formation of clumps in soluble fibrinogen correlated with adherence of bacteria to solid‐phase fibrinogen. The clfA gene encodes a fibrinogen‐binding protein with an apparent molecular mass of c. 130 kDa. The amino acid sequence of the protein was deduced from the DNA sequence; it was predicted that a 896 residue protein (molecular mass 92 kDa) would be expressed. The putative ClfA protein has features that suggest that it is associated with the ceil surface. Furthermore it contains a novel 308 residue region comprising dipeptide repeats predominantly of Asp and Ser ending 28 residues upstream from the LPXTG motif common to wall‐associated proteins. Significant homology was found between the ClfA protein and the fibronectin‐binding proteins of S. S. aureus, particularly in the N‐and C‐termini.
The homologous operons for P1 and P7 plasmid partition are autoregulated from dissimilar operator sites
Authors: Hayes Finbarr , Radnedge Lyndsay , Davis Michael A. , Austin Stuart J.
Published: October 2006

The plasmid‐partition regions of the P1 and P7 plasmid prophages in Escherichia coli are homologues which each encode two partition proteins, ParA and ParB. The equivalent PI and P7 proteins are closely related. In each case, the proteins are encoded by an operon that is autoregulated by the ParA and ParB proteins in concert. This regulation is species‐specific, as the P1 proteins are unable to repress the P7 par operon and vice versa. The homologous ParA proteins are primarily responsible for repression and bind to regions that overlap the operon promoter in both cases. The DNA‐binding domain of the P7 auto‐repressor lies in the amino‐terminal end of the P7 ParA protein. This region includes a helix‐turn‐helix motif that has a clear counterpart in the P1 ParA sequence. However, despite the common regulatory mechanism and the similarity of the proteins involved in repression, the promoter‐operator sequences of these two operons are very different in sequence and organization. The operator is located downstream of the promoter in P1 and upstream of it in P7, and the two regions show little, if any, homology. How these differences may have arisen from a common ancestral form is discussed.
Trypanosoma cruzi glycoprotein of Mr 56000: characterization and assessment of its potential to protect against fatal parasite infections
Authors: Harth Günter , Mills Alea A. , Lin Teri , Araujo Fausto G.
Published: October 2006

A ∼ 56 000 Da membrane glycoprotein purified from epimastigotes of Trypanosoma cruzi was characterized biochemically and tested for its efficacy to induce protection in mice from a lethal challenge with this protozoan parasite. Immunofluorescence assays with live and formalin‐fixed epimastigotes and trypomastigotes localized the glycoprotein to the flagellum, the body of the parasite, and the cell membrane. Immunoblotting demonstrated the glyco‐protein's presence in nearly equal amounts in all developmental stages of several T. cruzi isolates. Mice immunized with the purified glycoprotein and challenged with 10000 infectious trypomastigote forms of isolate Y survived the controls by up to four days. This significant protection makes this antigen a potential candidate for a multi‐subunit vaccine against 7. cruzi.
Characterization of a 3‐dehydroquinase gene from Actinobacillus pleuropneumoniae with homology to the eukaryotic genes qa‐2 and QUTE
Authors: Lalonde G. , O'Hanley P. D. , Stacker B. A. D. , Denich K. T.
Published: October 2006

A gene was cloned from Actinobacillus pleuropneumoniae strain 4074 by complementation of an aroD strain of Escherichia coli. The E. coli gene aroD codes for a 3‐dehydroquinase enzyme of type I, active in the aromatic biosynthesis pathway. The A. pleuropneumoniae gene, termed aroQ, displays no base or ami no acid sequence homoiogy to aroD of E. coli. It is instead homologous to the QUTE and qa‐2 genes, respectively of Aspergillus niduians and Neurospora crassa. These genes code for 3‐dehydroquinase enzymes of type ii, involved in the catabolism of quinic acid. The 1.6 kb fragment, which includes aroQ, carries four overlapping or adjacent open reading frames: a dapD gene; aroQ; one without homology to sequences in GenBank; and one with homology to the C‐terminal 40% of chlN of E. coli.
Characterization of the dTDP‐rhamnose biosynthetic genes encoded in the rfb iocus of Shigella flexneri
Authors: Macpherson Debbie F. , Manning Paul A. , Morona Renato
Published: October 2006

The nucleotide sequence of the proximal half of the rfb region of Shigella flexneri has been determined, and the genes encoding enzymes involved in the biosynthesis of dTDP‐rhamnose have been identified. These genes show strong homology to the rfb genes encoding dTDP‐rhamnose biosynthesis in Salmonella enterica serovar typhimurium (strain LT2) and S. enterica serovar anatum (strain M32) (Jiang et al., 1991; Wang et al., 1992). An open reading frame upstream of rfbB was also identified which encoded a protein having strong similarity with GalU, and has been designated galF. GalF has 92% amino acid sequence identity with an S. enterica LT2 gene, orf2X8, which is similarly situated upstream of rfbB (Jiang et al., 1991). The T7 expression system was utilized to identify proteins corresponding to those predicted from DNA sequence analysis. The similarity of the predicted proteins with proteins that are functionally identical or related, and with others of unknown function from the Yersinia enterocolitica O3 rfb region, and in the Escherichia coli K‐12 rff region are also described. We have re‐addressed the assignment of each gene of the dTDP‐rhamnose pathway with the known enzymes of the pathway, in particular rfbC and rfbD. A reporter plasmid to detect genes encoding enzymes of the dTDP‐rhamnose pathway is described. An analysis of the intergenic region between galF and rfbB has been made, and comparison with the same region from S. enterica LT2 discussed.
In vivo study of engineered G‐domain mutants of Escherichia coli translation initiation factor IF2
Authors: Laalami Soumaya , Timofeev Andrei V. , Putzer Harald , Leautey Josette , Grunberg‐Manago Marianne
Published: October 2006

During the IF2‐catalysed formation of the 30S initiation complex, the GTP requirement and Its subsequent hydrolysis during 70S complex formation are considered to be essential for translation initiation in Escherichia coli. In order to clarify the role of certain amino acid residues believed to be crucial for the GTP hydrolytic activity of E. coli IF2, we have introduced seven single amino acid substitutions into its GTP‐binding site (Gly for Val‐400; Thr for Pro‐446; Gly, Glu, Gin for His‐448; and Asn, Glu for Asp‐501). These mutated IF2 proteins were expressed in vivo in physiological quantities and tested for their ability to maintain the growth of an E. coli strain from which the functional chromosomal copy of the infB gene has been deleted. Only one of the mutated proteins (Asp‐501 to Giu) was able to sustain cell viability and several displayed a dominant negative effect. These results emphasize that the amino acid residues we substituted are essential for the iF2 functions and demonstrate the importance of GTP hydrolysis in translation initiation. These findings are discussed in relation to a previously proposed theoretical model for the IF2 G‐domain.
The function of a ribosomal frameshifting signal from human immunodeficiency virus‐1 in Escherichia coli
Authors: Yelverton Elizabeth , Lindsley Dale , Yamauchi Phil , Gallant Jonathan A.
Published: October 2006

A 15‐17 nucleotide sequence from the gag‐pol ribosome frameshift site of HIV‐1 directs analogous ribosomal frameshifting in Escherichia coli. Limitation for leucine, which is encoded precisely at the frameshift site, dramatically increased the frequency of leftward frameshifting. Limitation for phenylaianine or arginine, which are encoded just before and just after the frameshift, did not significantly affect frameshifting. Protein sequence analysis demonstrated the occurrence of two closeiy related frameshift mechanisms. In the first, ribosomes appear to bind leucyl‐tRNA at the frameshift site and then slip leftward. This is the 'simultaneous slippage’mechanism. In the second, ribosomes appear to slip before binding amlnoacyl‐tRNA, and then bind phenylaianyl‐tRNA, which is encoded in the left‐shifted reading frame. This mechanism is identicai to the‘overlapping reading’we have demonstrated at other bacterial frameshift sites. The HIV‐1 sequence is prone to frame‐shifting by both mechanisms in E. coli.
Nodulating ability of Rhizobium tropici is conditioned by a plasmid‐encoded citrate synthase
Authors: Pardo Marco A. , Lagunez Jaime , Miranda Juan , Martinez Esperanza
Published: October 2006

Rhizobium species elicit the formation of nitrogen‐fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen‐fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen‐fixing nodules on the roots of beans (PhasBoius vuigaris) and trees (Leucaena spp.). Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl‐CoA and oxalo‐acetic acid to form citrate. R. tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5‐mob insertion causes the bacteria to form fewer nodules (30–50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented ail the phenotypic alterations of the pcsA mutant, and conferred Rhizobium iegumino‐sarum bv. phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.
Characterization of the mutX gene of Streptococcus pneumoniae as a homologue of Escherichia coli mutT, and tentative definition of a catalytic domain of the dGTP pyrophosphohydroiases
Authors: Mejean Vincent , Salles Catherine , Bullions Linda C. , Bessman Maurice J. , Claverys Jean‐Pierre
Published: October 2006

We show that deletion of a gene of Streptococcus pneumoniae, which we call mutX, confers a mutator phenotype to resistance to streptomycin. Analysis of the DNA sequence changes that occurred in several streptomycin‐resistant mutants showed that mutations are unidirectional AT to CG transversions. The mutX gene is located immediately downstream of the previously identified ung gene and genetic evidence suggests that the two genes are coordinately regulated. Nucleotide sequence determination reveals that the mutX gene encodes a 17870 Da protein (154 residues) which exhibits significant homology with the MutT protein of Escherichia coli, a nucleoside tri‐phosphatase (dGTP pyrophosphohydrolase). The mutX gene complements the E coli mutT mutator phenotype when introduced on a plasmid. Site‐directed mutagenesis and analysis of nitrosoguanidine‐induced mutT mutants suggest that a small region of high homology between the two proteins (61% identity over 23 residues) is part of the catalytic site of the nucleoside triphosphatase. Computer searching for sequence homology to MutX uncovered a second E. coli protein, the product of orf17, a gene of unknown function located near the ruvC gene. The region of high homology between MutX and MutT is also conserved in this protein, which raises the interesting possibility that the orf17 gene plays some role in determining mutation rates in E. coli. Finally, a small set of proteins, including a family of virus‐encoded proteins and two evolutionarily conserved proteins encoded by an antisense transcript from the Xenopus laevis and human bFGF genes, were also found to harbour significant homology to this highly conserved region.
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

The 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

Cellulases 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

The 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

Several 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

A 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

Previous 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

DNA 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

F 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

The 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

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

Nucleotide 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

The 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

Secretion 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

The 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.
An inner‐membrane‐associated virulence protein essential for T‐DNA transfer from Agrobacterium tumefaciens to plants exhibits ATPase activity and similarities to conjugative transfer genes
Authors: Shirasu Ken , Koukolíková‐Nicola Zdena , Hohn Barbara , Kado Clarence I.
Published: October 2006

The 9.5kb virB operon is the largest of the six major operons in the Ti plasmid vir region. This operon contains eleven genes, the largest of which is virB4. This gene encodes an 84kDa protein whose function has not been identified. Its roles in conferring virulence on Agrobacterium tumefaciens and in the T‐DNA transfer process were determined by generating non‐polar mutants by using the Tn5pvirB transposon in which the virB promoter is transcribed downstream of its position of insertion. Several independent mutants were isolated and each insertion site in virB4 was confirmed by nucleotide sequence analysis. These mutants were tested for T‐DNA transfer ability by agroinfection and for tumorigenicity by inoculation in Brassica and Datura. All mutants were agroinfection‐ and tumorigenicity‐negative. These data strongly suggest that virB4 is essential for both the interkingdom transfer of the T‐DNA and virulence. Furthermore, by using anti‐VirB4 serum, the protein product of virB4 was localized to the inner‐membrane fraction of A. tumefaciens. Purified VirB4 protein hydrolyses ATP and this activity was quenched by the anti‐VirB4 serum. The energy generated by VirB4 ATPase therefore may be used to transfer T‐DNA or to assemble the T‐DNA transfer apparatus on the bacterial membrane. Protein sequence analyses revealed striking similarities between VirB4 protein and the proteins required for conjugative transfer, which include TraC, TrwK, and TrbE of plasmids F, R388, and RP4, repectively. These findings suggest that VirB proteins play a direct role in the assembly of a conjugative transfer apparatus required for the transfer of the T‐DNA from A. tumefaciens to plant cells.