bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937
Authors: Lojkowska Ewa , Masclaux Celine , Boccara Martine , Robert‐Baudouy Janine , Hugouvieux‐Cotte‐Pattat Nicole 
Published: October 2006

Summary Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pelA, pelB, pelC, pelD and pelE genes. Recently, a new set of pectate lyases was identified in E. chrysanthemi mutants deleted of those pel genes. We cloned the pelL gene, encoding one of these secondary pectate lyases of E. chrysanthemi 3937, from a genomic bank of a strain deleted of the five major pel genes. The nucleotide sequence of the region containing the pelL gene was determined. The pelL reading frame is 1275 bases long, corresponding to a protein of 425 amino acids including a typical amino‐terminal signal sequence of 25 amino acids. Comparison of the amino acid sequences of PelL and the exo‐pectate lyase PelX of E. chrysanthemi EC16 revealed a low homology, limited to 220 residues of the central part of the proteins. No homology was detected with other bacterial pectinolytic enzymes. Regulation of pelL transcription was analysed using gene fusion. As shown for the other pel genes, the transcription of pelL is dependent on various environmental conditions. It is induced by pectic catabolic products and affected by growth phase, temperature, iron starvation, osmolarity, anaerobiosis, nitrogen starvation and catabolite repression. Regulation of pelL expression appeared to be independent of the KdgR repressor, which controls all the steps of pectin catabolism. In contrast, the pecS gene, which is involved in regulation of the synthesis of the major pectate lyases and of cellulase, also appeared to be involved in pelL expression. The PelL protein is able to macerate plant tissue. This enzyme has a basic isoelectric point, presents an endo‐cleaving activity on polygalacturonate or partially methylated pectin, with a basic pH optimum and an absolute requirement for Ca2+. The pelL mutant displayed a reduced virulence on potato tubers and Saintpaulia ionantha plants, demonstrating the important role of this enzyme in soft‐rot disease.
Substitution of two histidine residues in YadA protein of Yersinia enterocolitica abrogates collagen binding, cell adherence and mouse virulence
Authors: Roggenkamp Andreas , Neuberger Hans‐Ruprecht , Flügel Alexander , Schmoll Thomas , Heesemann Jürgen 
Published: October 2006

SummaryThe plasmid‐encoded surface protein YadA of Yersinia enterocolitica mediates binding to diverse extracellular matrix (ECM) proteins, adherence to epithelial cell lines, resistance to complement lysis, autoagglutination, and is required for mouse virulence. Using site‐directed mutagenesis we attempted to analyse the relationship between structural domains and functions of YadA. In a first approach we could abrogate collagen binding by chemical modification of histidyl residues of YadA protein. This result prompted us to substitute histidyl residues (His) of conserved regions of YadA protein of Y. enterocolitica 08 by tyrosine residues using site‐directed mutagenesis. Substitution of His‐156 and His‐159 (YadA‐2 mutant) resulted in abrogation of binding to ECM proteins, of cell adherence, and in reduction of mouse virulence, whereas autoagglutination, serum complement resistance and oligomer formation remained unaffected. A striking result was obtained from the orogastric mouse‐infection model: the YadA‐2 mutant retained the ability to colonize the small intestine and to invade and multiply within the Peyer's patches but was impaired in colonizing mesenteric lymph nodes and spleen in comparison to the wild‐type strain.
Cloning and characterization of a bradyzoite‐specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat‐shock proteins of plants
Authors: Bohne Wolfgang , Gross Uwe , Ferguson David J. P. , Heesemann Jürgen 
Published: October 2006

SummaryStage conversion between the tachyzoite and bradyzoite forms of the protozoan parasite Toxoplasma gondii is an important aspect in the pathogenesis of toxoplasmosis. In an initial investigation of molecular regulation of stage conversion in T. gondii, we describe the cloning and characterization of a bradyzoite‐specifically expressed gene (hsp30/bag1). Bradyzoite formation was induced in cell culture by alkaline pH, and this was followed by purification of this parasitic stage using magnetic cell sorting. A bradyzoite cDNA library was constructed by random amplification using the polymerase chain reaction. Screening with a bradyzoite‐specific monoclonal antibody identified a reactive clone. The amino acid sequence derived from the 687 bp open reading frame showed similarity to the conserved C‐terminal region of small heat‐shock proteins from plants. Stage‐specific expression of the naturally occurring 30kDa antigen in bradyzoites was confirmed by polyclonal antisera generated against the recombinant antigen, Immuno‐electron microscopy indicated a cytosolic location of this antigen in bradyzoites. The expression of HSP30/BAG1 seems to be regulated at the mRNA level, since reverse polymerase chain reaction using bradyzoite‐specific primers amplified transcripts in bradyzoites only, not in tachyzoites.
Heterogeneous endolysins in Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes
Authors: Loessner Martin J. , Wendlinger Günther , Scherer Siegfried 
Published: October 2006

Summary Listeria monocytogenes bacteriophages A118, A500 and A511 are members of three distinct phage groups with characteristic host ranges. Their endolysin (ply) genes were colonies and expressed in Escherichia coli as demonstrated by the conferred lytic phenotype when colonies of recombinant cells were overlaid with a lawn of Listeria ceils. The nucleotide sequences of the cloned DNA fragments were determined and the individual enzymes (PLY118, 30.8 kDa; PLY500, 33.4 kDa; PLY511, 36.5 kDa) were shown to have varying degrees of homology within their N‐terminal or C‐terminal domains. Transcriptional analysis revealed them to be‘late’genes with transcription beginning 15–20 min post‐infection. The enzymes were over‐expressed and partially purified and their individual specificities examined. When applied exogenously, the lysins induced rapid lysis of Listeria strains from all species but generally did not affect other bacteria. Using hydrolysis of purified listerial cell walls, PLY511 was characterized as an N‐acetylmuramoyl‐L‐alanine amidase (EC 3.5.1.28) and shows homology in its N‐terminal domain to other enzymes of this type. In contrast, PLY118 and PLY500 were shown to represent a new class of cell wall lytic enzymes which cleave between the L‐alanine and D‐glutamate residues of listerial peptidoglycan; these were designated as L‐alanoyl‐D‐glutamate peptidases. These two enzymes share homology in the N‐terminal domain which we propose determines hydrolytic specificity. Highly conserved holin (hol) gene sequences are present upstream of ply118 and ply500. They encode proteins of structural similarity to the product of phage lambda gene S, and are predicted to be membrane proteins which form pores to allow access of the lysins to their peptidoglycan substrates. This arrangement of conserved holin genes with downstream lysin genes among the siphoviral lysis cassettes explains why the cytoplasmic endolysins alone are not lethal, since they require a specific transport function across the cell membrane.
Subcellular localization and topology of the K88 usher FaeD in Escherichia coli
Authors: Valent Quido A. , Zaal Jelle , de Graaf Frits K. , Oudega Bauke 
Published: October 2006

SummaryThe subcellular localization of the K88 usher FaeD was studied in Escherichia coli whole ceils by using iso‐pycnic sucrose density gradient centrifugation of isolated membranes, the detergents Triton X‐100 and sodium lauryl sarcosinate and immunoblotting with a specific FaeD antiserum. Cells containing the complete K88 operon, as well as cells containing the sub‐cloned faeD gene in various expression vectors, were used. Most of the FaeD was present in the outer membranes in a detergent‐resistant form. Agglutination experiments with E coli cells expressing FaeD confirmed an outer membrane localization and indicated the presence of FaeD at the cell surface. Automated Edman degradation indicated that the mature FaeD contained 777 amino acid residues and confirmed that FaeD is synthesized with a rather long signal sequence of 35 amino acid residues. Twelve different FaeD–PhoA fusion proteins were prepared and characterized by nucleotide sequencing and immuno‐blotting. Most of these fusion sites were located in the amino‐terminal and carboxyl‐terminal regions of FaeD. Six amino‐terminal fusion proteins were soluble proteins in the peripiasm, whereas the other fusion proteins were associated with the outer membrane. The protease accessibility of FaeD and of the six outer membrane‐bound FaeD–PhoA fusion proteins was studied using whole cells, cells with permeabilized outer membranes, and isolated membranes. Collagenase H, kallikrein, trypsin and proteinase K were used. Based on the results of these experiments and computer predictions, a model for the membrane topology of FaeD was developed in which FaeD contains a large central domain containing 24 membrane‐spanning segments and two relatively large periplasmic regions, at the amino‐terminal and carboxyl‐terminal end of the protein, respectively.
Identification of GroEL as a constituent of an mRNA‐protection complex in Escherichia coli
Authors: Georgellis Dimitris , Sohlberg Björn , Hartl F. Ulrich , von Gabain Alexander 
Published: October 2006

SummaryAn RNA‐binding activity has been identified in Escherichia coli that provides physical protection of RNA against ribonucleases in an ATP‐ and Mg2+‐dependent manner. This binding activity is stimulated under growth conditions known to cause a decrease in the rate of mRNA decay. RNA protection is mediated by a protein complex that contains a modified form of the chaperonin GroEL as an indispensable constituent. These results suggest a new role for GroEL as an RNA chaperone.
On microbial states of growth†
Authors: Fishov Itzhak , Zaritsky Arieh , Grover N. B. 
Published: October 2006

SummaryIt is crucial to the reproducibility of results and their proper interpretation that the conditions under which experiments are carried out be defined with rigour and consistency, in this review we attempt to clarify the differences and interrelationships among steady, balanced and exponential states of culture growth. Basic thermodynamic concepts are used to introduce the idea of steady‐state growth in open, biological systems. The classical, sometimes conflicting, definitions of steady‐state and balanced growth are presented, and a consistent terminology is proposed. The conditions under which a culture in balanced growth is also in exponential growth and in steady‐state growth are indicated. It is pointed out that steady‐state growth always implies both balanced and exponential growth, and examples in which the converse does not hold are described. More complex situations are then characterized and the terminology extended accordingly. This leads to the notion of normal growth and growth that can be synchronous or otherwise unbalanced but still reproducible, and to the condition of approximate steady state manifested by growth in batch culture and by asymmetrically dividing cells, which is analysed in some detail.
Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport
Authors: Parker Corrine , Barnell Wendy O. , Snoep Jacky L. , Ingram Lonnie O. , Conway Tyrrell 
Published: October 2006

Summary Zymomonas mobilis is known to transport glucose by a facilitated diffusion process. A putative glucose facilitator gene (glf), closely related to a large family of glucose transporters, is located in a cluster of genes that code for enzymes of glucose metabolism. The Z. mobilis glf gene is able to complement glucose transport in an Escherichia coli strain that is defective in native glucose transport and glucokinase. In this study, the recombinant E coli was shown to be capable of influx counterflow when preloaded with glucose and had an apparent Km for glucose of approximately 1.1 ‐ 2.9 mM, consistent with the function of Gif as a low‐affinity glucose facilitator. The ability of glucokinase mutants expressing glf to transport glucose made it clear that glucokinase activity was not required for Glf‐dependent glucose transport. The possibility that glucokinase can interact with Glf to improve the affinity for glucose was not supported since expression of the Z mobilis glucokinase gene, in addition to glf, did not affect the Km of Glf for glucose in recombinant E. coli The inability of various sugars to compete with glucose during glucose transport by recombinant E. coli expressing glf indicated that Glf is specific for glucose. While the results of fructose transport assays did not completely rule out the possibility of very low affinity for fructose, the apparent specificity of Gif for glucose makes it possible that Z. mobilis utilizes a different transporter(s) for fructose.
Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene
Authors: Kok Ruben G. , van Thor Jasper J. , Nugteren‐Roodzant Inge M. , Brouwer Marc B. W. , Egmond Maarten R. , Nudel Clara B. , Vosman Ben , Hellingwerf Klaas J. 
Published: October 2006

SummaryThe extracellular lipase from Acinetobacter calcoaceticus BD413 was purified to homogeneity, via hydrophobic‐interaction fast performance liquid chromatography (FPLC), from cultures grown in mineral medium with hexadecane as the sole carbon source. The enzyme has an apparent molecular mass of 32 kDa on SDS‐polyacrylamide gels and hydrolyses long acyl chain p‐nitrophenol (pNP) esters, like pNP palmitate (pNPP), with optimal activity between pH 7.8 and 8.8. Additionally, the enzyme shows activity towards triglycerides such as olive oil and tributyrin and towards egg‐yolk emulsions. The N‐terminal amino acid sequence of the mature protein was determined, and via reverse genetics the structural lipase gene was cloned from a gene library of A. calcoaceticus DNA in Escherichia coli phage M13. Sequence analysis of a 2.1 kb chromosomal DNA fragment revealed one complete open reading frame, lipA, encoding a mature protein with a predicted molecular mass of 32.1 kDa. This protein shows high similarity to known lipases, especially Pseudomonas lipases, that are exported in a two‐step secretion mechanism and require a lipase‐specific chaperone. The identification of an export signai sequence at the N‐terminus of the mature lipase suggests that the lipase of Acinetobacter is also exported via a two‐step translocation mechanism. However, no chaperone‐encoding gene was found downstream of lipA, unlike the situation in Pseudomonas. Analysis of an A. calcoaceticus mutant showing reduced lipase production revealed that a periplasmic disutphide oxidoreductase is involved in processing of the lipase. Via sequence alignments, based upon the crystal structure of the closely related Pseudomonas glumae lipase, a model has been made of the secondary‐structure elements in AcLipA. The active site serine of AcLipA was changed to an alanine, via site‐directed mutagenesis, resulting in production of an inactive extracellular lipase.
Differential DNA bending introduced by the Pseudomonas putida LysR‐type regulator, CatR, at the plasmid‐borne pheBA and chromosomal catBC promoters
Authors: Parsek M. R. , Kivisaar M. , Chakrabarty A. M. 
Published: October 2006

SummaryThe plasmid‐borne pheBA operon of Pseudomonas putida strain PaW85 allows growth of the host cells on phenol. The promoter of this operon is activated by the chromosomally encoded LysR‐type regulator CatR, in the presence of the inducer cis, cis‐muconate. cis, cis ‐muconate is an intermediate of catechol degradation by the chromosomally encoded ortho or β‐ketoadipate pathway. The catBC operon encodes two enzymes of the β‐ketoadipate pathway and also requires CatR and cis, cis‐muconate for its expression. The promoters of the pheBA and catBC operons are highly homologous, and since both respond to CatR, it is likely that the pheBA promoter was recruited from the ancestral catBC promoter. Gel shift assays and DNase I footprinting have shown that the pheBA promoter has a higher binding affinity for CatR than the catBC promoter. Like the catBC promoter, the pheBA promoter forms two complexes (C1 and C2) with CatR in the absence of cis, cis‐muconate, but only forms a single complex (C2) in the presence of cis, cis‐muconate. Like the catBC promoter CatR repression binding site (RBS) and activation binding site (ABS) arrangement, the pheBA promoter demonstrates the presence of a 26 bp segment highly homologous to the RBS that is protected by CatR from DNase I digestion in the absence of the inducer. An additional 16 bp sequence, similar to the catBC promoter ABS, is protected only when the inducer cis‐cis‐muconate is present. The binding of CatR in absence of cis, cis ‐muconate bends the catBC and pheBA promoter regions to significantly different degrees, but CatR binding in the presence of cis, cis‐muconate results in a similar degree of DNA bending. The evolutionary implications of the interactions of CatR with these two promoters are discussed.
Boundaries of the nicking region for the F plasmid transfer origin, oriT
Authors: Luo Yanan , Gao Qian , Deonier Richard C. 
Published: October 2006

SummaryThe extent of the F plasmid oriT nicking region was determined from the properties of successive substitution mutations in the region from base pair 121 to base pair 174 and from KMnO4 probing of DNA structural distortions induced in vivo by tra gene products. Nicking and transfer assays indicated that the left margin of oriT Wes predominantly at the nick site, and that the nicking domain primarily lies within 17bp to the right of the nick. Some mutants that were proficient for nicking showed reduced frequencies of termination, indicating that oriT nicking does not guarantee efficient termination. DNA in the vicinity of the nick (G137, T138, G140, and T141 on the nicked strand) showed elevated sensitivity to KMnO4 when tra gene products were present in the donor. Bases C145, C146, C147, C149, and G150 on the un‐nicked strand also became more sensitive to oxidation under tra+ conditions. The bases preferentially oxidized by KMnO4 lie within the nicking domain, as defined by the substitution mutants, and they include dinucleotides that can produce kinks in the DNA. Base pairs in the nicking region are calculated to be more thermodynamically stable than base pairs in the flanking regions.
Isolation of Lactococcus lactis nonsense suppressors and construction of a food‐grade cloning vector
Authors: Dickely Fraçoise , Nilsson Dan , Hansen Egon Bech , Johansen Eric 
Published: October 2006

SumnnaryNonsense suppressor strains of Lactococcus lactis were isolated using plasmids containing nonsense mutations or as revertants of a nonsense auxotrophic mutant. The nonsense suppressor gene was cloned from two suppressor strains and the DNA sequence determined. One suppressor is an ochre suppressor with an altered tRNAgin and the other an amber suppressor with an altered tRNAser. The nonsense suppressors allowed isolation of nonsense mutants of a lytic bacteriophage and suppressible auxotrophic mutants of L. lactis MG1363. A food‐grade cloning vector based totally on DNA from Lactococcus and a synthetic polylinker with 11 unique restriction sites was constructed using the ochre suppressor as a selectable marker. Selection, following etectroporation of a suppressible purine auxotroph, can be done on purine‐free medium. The pepN gene from L. lactis Wg2 was subcloned resulting in a food‐grade plasmid giving a four‐ to fivefold increase in lysine aminopeptidase activity.
ThefimAlocus ofStreptococcus parasanguisencodes an ATP‐binding membrane transport system
Authors: Fenno J. Christopher , Shaikh Aisha , Spatafora Grace , Fives‐Taylor Paula 
Published: October 2006

SummaryThe gene encodingfimA, a 36 kDa fimbrial adhesin ofStreptococcus parasanguisFW213, is highly conserved in all four genetic groups of sanguis streptococci. FimA‐like peptides were produced by all strains tested. The nucleotide sequence directly upstream offimAcontains two open reading frames, ORF5 and ORF1, whose deduced protein products are homologous to members of a superfamily of ATP‐binding cassette membrane transport proteins, including both prokaryotic and aukaryotic uptake and export systems. The amino acid sequence of FimA contains the consensus prolipoprotein cleavage site (LxxC) common to the‘periplasmic’ binding proteins of Gram‐positive transport systems. The deduced product of ORF5 is a 28.6 kDa membrane‐associated protein that has the consensus binding site for ATP (GxxGxGKS). It shares significant homology with AmiE ofStreptococcus pneumoniaeas well as withEscherichia coliproteins involved in iron(III) uptake. Allelic‐replacement mutagenesis of ORF5 resulted in greatly Increased resistance to aminopterin. These data demonstrate functionality with theamiElocus as well. The deduced product of ORF1 is an extremely hydrophobic integral membrane protein of 30.8 kDa with a pattern of six potential membrane‐spanning regions, typical of a component of these types of transport system. The nucleotide sequence downstream offimA.ORF3, encodes a 20 kDa protein having 78% identity with the 20 kDa protein encoded downstream ofssaB, afimAhomologue inS. sanguis12. It also exhibits significant homology with bacterioferritin co‐migratory protein (Bcp) ofE. coliK‐12. Allelic‐replacement mutagenesis in thefimAlocus of FW213 showed that (i) expression offimAwas initiated at a site far upstream of thefimAstart codon, and (ii) expression offimAwas not linked to expression of ORF3. Northern blots probed with internal fragments of ORF5, ORF1,fimAor ORF3 hybridized to the same transcript of 3.3 kb, which suggested that these loci were transcribed as a polycistronic message. The ORF3 probe also hybridized to a 540 bp transcript consistent with the size of ORF3 alone and supportive of the mutagenesis data of non‐linkage. Strains mutated infimAcontinued to produce fimbriae, indicating that FimA was not the fimbrial structural subunit. Immunoelectron microscopy revealed FimA was localized at the tips of the fimbriae of FW213. This is the first study that demonstrates that an adhesin which binds a bacterial cell to a substrate is associated with an ATP‐binding cassette.
The tyrosine‐6 hydroxyl of γδ resolvase is not required for the DNA cleavage and rejoining reactions
Authors: Leschziner Andres E. , Boocock Martin R. , Grindley Nigel D. F. 
Published: October 2006

SummarySite‐specific recombinases of the resolvase and DNA invertase family all contain a tyrosine residue close to the N‐terminus, and four residues away from a serine that has been implicated in catalysis of DNA strand breakage and reunion. To examine the role of this tyrosine in recombination, we have constructed a mutant of γδ resolvase in which the tyrosine (residue 6) is replaced by phenytalanine. Characterization of the Y6F mutant protein in vitro indicated that although it was highly defective in recombination, it could cleave ONA at the cross‐over site, form a covalent resolvase‐DNA complex and rejoin the cleaved cross‐over site (usually restoring the parental site). These data rule out a direct role of the Tyr‐6 hydroxyl as the nucleophile In the DNA cleavage reaction and strengthen the conclusion that this nucleophile is the nearby invariant serine residue, Ser‐10. We conclude that Tyr‐6 is essential for fully coordinated strand cleavage and exchange, but is dispensable for individual strand cleavage and religation reactions.