bluuebunny/crossref_metadata_2025 huggingface dataset:
Colicin import and pore formation: a system for studying protein transport across membranes?
Authors: Lazdunski Claude J.
Published: October 2006

Pore‐forming colicins are a family of protein toxins (Mr40–70kDa) produced by Escherichia coli and related bacteria. They are bactericidal by virtue of their ability to form ion channels in the inner membrane of target cells. They provide a useful means of studying questions such as toxin action, polypeptide translocation across and into membranes, voltage‐gated channels and receptor function. These colicins bind to a receptor in the outer membrane before being translocated across the cell envelope with the aid of helper proteins that belong to nutrient‐uptake systems and the so‐called‘Tol’proteins, the function of which has not yet been properly defined. A distinct domain appears to be associated with each of three steps (receptor binding, translocation and formation of voltage‐gated channels). The Tol‐dependent uptake pathway is described here. The structures and interactions of TolA, B, Q and R have by now been quite clearly defined. Transmembrane α‐helix interactions are required for the functional assembly of the E. coli Tol complex, which is preferentially located at contact sites between the inner and outer membranes. The number of colicin translocation sites is about 1000 per cell. The role and the involvement of the OmpF porin (with colicins A and N) have been described in a recent study on the structural and functional interactions of a colicin‐resistant mutant of OmpF. The X‐ray crystal structure of the channel‐forming fragment of colicin A and that of the entire colicin la have provided the basis for biophysical and site‐directed muta‐genesis studies. Thanks to this powerful combination, it has been established that the interaction with the receptor in the outer membrane leads to a very substantial conformational change, as a result of which the N‐terminal domains of colicins interact with the lumen of the OmpF pore and then with the C‐terminal domain of TolA. A molten globular conformation of colicins probably constitutes the intermediate translocation/insertion competent state. Once the pore has formed, the polypeptide chain spans the whole cell envelope. Three distinct steps occur in the last stage of the process: (i) fast binding of the C‐terminal domain to the outer face of the cytoplasmic membrane; (ii) a slow insertion of the polypeptide chain into the outer face of the inner membrane in the absence of Δψ and (iii) a profound reorganization of the helix association, triggered by the transmembrane potential and resulting in the formation of the colicin channel.
Virus DNA packaging: the strategy used by phage λ
Authors: Catalano Carlos E. , Cue David , Feiss Michael
Published: October 2006

Phage λ, like a number of other large DNA bacterio‐phages and the herpesviruses, produces concatemeric DNA during DNA replication. The concatemeric DNA is processed to produce unit‐length, virion DNA by cutting at specific sites along the concatemer. DNA cutting is coordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the λ DNA packaging process is the phage‐encoded enzyme terminase, which is involved in (i) recognition of the concatemeric λ DNA; (ii) initiation of packaging, which includes the introduction of staggered nicks at cosN to generate the cohesive ends of virion DNA and the binding of the prohead; (iii) DNA packaging, possibly including the ATP‐driven DNA translocation; and (iv) following translocation, the cutting of the terminal cosN lo complete DNA packaging. To one side of cosN is the site cosB, which plays a role in the initiation of packaging; along with ATP, cosB stimulates the efficiency and adds fidelity to the endo‐nuclease activity of terminase in cutting cosN. cosB is essential for the formation of a post‐cleavage complex with terminase, complex I, that binds the prohead, forming a ternary assembly, complex II. Terminase interacts with cosN through its large subunit, gpA, and the small terminase subunit, gpNul, interacts with cosB. Packaging follows complex II formation. cosN is flanked on the other side by the site cosQ, which is needed for termination, but not initiation, of DNA packaging. cosQ is required for cutting of the second cosN, i.e. the cosN at which termination occurs. DNA packaging in λ has aspects that differ from other λ DNA transactions. Unlike the site‐specific recombination system of λ, for DNA packaging the initial site‐specific protein assemblage gives way to a mobile, translocating complete, and unlike the DNA replication system of λ, the same protein machinery is used for both initiation and translocation during λ DNA packaging.
Functional implications of the expression of PilC proteins in meningococci
Authors: Virji Mumtaz , Makepeace Katherine , Peak Ian , Payne Gail , Saunders Jon R. , Ferguson David J. P. , Moxon H. Richard
Published: October 2006

Multiple forms of PilC were found in Neisseria meningitidis (Nm) strains isolated from the oropharynx, blood or cerebrospinal fluid expressing either Class I or Class II pili. PilC expression was observed less frequently in case as opposed to carrier isolates. Moreover, PilC and pili were not always co‐expressed. Several heavily piliated strains had no detectable PilC protein as determined by Western blotting using an antiserum previously used to detect such proteins in adhesive variants (Nassif et al., 1994). Serogroup B strain MC58 produced large numbers of pili, but expressed barely detectable amounts of PilC. A clonal variant of this strain with increased expression of PilC concurrently exhibited increased adherence to Chang conjunctival epithelial cells and human umbilical vein endothelial cells (Huvecs), but with more rapid binding to the former. No alteration in pilin sequence occurred in this variant, suggesting the involvement of PilC in increased adhesion. A Pil‐ backswitcher isolated from the hyper‐adherent variant was PilC+ but was non‐adherent, indicating that any PilC adherence function requires pilus expression. Parental variant (low PilC) produced pili in bundles that were easily detached from the bacterial surface and were frequently associated with Huvec surfaces after bacteria had been sheared off, but pili infrequently replaced bacteria during infection with the PilC‐expressing variant. The hyper‐adherent variant, which appeared to produce morphologically distinct pilus bundles, was able to withstand considerable shearing force and remained firmly attached to Huvecs. This raises the possibility that the observed hyper‐adherence may arise from better anchorage of pili to the bacterial surface in addition to increased adhesion to some host cell surfaces.
The Cryptococcus neoformans GAL7 gene and its use as an inducible promoter
Authors: Wickes Brian L. , Edman Jeffrey C.
Published: October 2006

A Cryptococcus neoformans galactose auxotroph was created by ultraviolet light mutagenesis and complemented with a C. neoformans genomic library. The translated sequence of the complementing DNA revealed a high degree of simlarity to a number of UDP glucose‐D‐gatactose‐1‐phosphate uridylyitransferases. Expression of C. neoformans GAL7 mRNA followed a pattern similar to Saccharomyces cerevisiae expression; it was first observed within 2.5 min of induction and fully induced by 30 min. The gene was completely repressed in the presence of glucose. The GAL7 promoter was isolated and used to construct a promoter cassette. Two genes were tested in this cassette for galactose regulation by creating GAL7 promoter fusions with their coding regions. MFα, which encodes a pheromone, was found to produce filaments only in transformants that were induced by galactose. A second gene, β‐glucuronidase (gusA), which is a commonly used reporter gene, was tested and also found to be expressed. When the GAL7 p::GUS fusion was used to quantify inducibitity of the GAL7 promoter, the level of enzyme activity was at least 500‐fold greater for cells grown in galactose than for cells grown in glucose. The GAL7 promoter is the first inducible promoter characterized in C neoformans and the GUS gene is the first heterologous gene shown to be expressed in this yeast pathogen.
Regulation of ptsH and ptsl gene expression in Streptococcus salivarius ATCC 25975
Authors: Gagnon Guy , Vadeboncoeur Christian , Gauthier Lucie , Frenette Michel
Published: October 2006

The transcriptional regulation of the Streptococcus salivarius ptsH and ptsl genes coding for the general energy‐coupling proteins HPr and enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system were investigated. These genes form an operon with the gene order ptsH–ptsl. Three distinct mRNA species were detected: a 0.5 kb transcript specific for ptsH, and two long transcripts (2.2 arid 2.4 kb) covering the whole pts operon. Transcription of all these mRNAs initiated at the same nucleotide located 9 bp downstream from a promoter located immediately upstream from the ptsH gene. The presence of a high‐energy stem–loop structure (T0) located at the begining of ptsl was responsible for the premature transcrrption termination generating the 0.5 kb ptsH‐specific transcript. The long transcripts ended in the poly(U) region of two rho‐independent‐like terminators (T1 and T2) at the 3′ end of ptsl. Studies with a 2‐deoxyglucose‐resistant spontaneous mutant of S. salivarius (L26) that produces an HPr–El fusion protein suggest that the regulation of HPr and El expression involves transcriptional as well as translational mechanisms.
A central domain of Rhizobium NodE protein mediates host specificity by determining the hydrophobicity of fatty acyl moieties of nodulation factors
Authors: Bloemberg Guido V. , Kamst Eric , Harteveld Marga , van der Drift Koen M. G. M. , Haverkamp Johan , Thomas‐Oates Jane E. , Lugtenberg Ben J. J. , Spaink Herman P.
Published: October 2006

Previously, we have shown that the nodE gene is a major determinant of the difference in host range between Rhizobium leguminosarum biovars viciae and trifolii. A new genetic test system for stringent functional analysis of nodE genes was constructed. By testing chimeric nodE genes constructed by the exchange of poiymerase chain reaction (PCR)‐generated restriction cassettes, we show that a central domain, containing only 44 non‐conserved amino acid residues, determines the host specificity of the NodE protein (401 amino acid residues). Mass spectrometric analysis of the lipo‐chitin oligosaccharides (LCOs) produced by the new test strain containing the biovar viciae nodE gene shows that molecules containing a polyunsaturated C18:4 (trans‐2. trans‐4. trans‐6. cis‐11‐octadecatetraenoic) fatty acyl moiety are produced, as is the case for wild‐type R. leguminosarum bv. viciae. The LCOs determined by the biovar trifolii nodE gene, which was overproduced in our test strain, carry C1 8:2 and C18:3 fatty acyl chains containing two or three conjugated trans double bonds, respectively. Therefore, the main difference between the nodE‐determined LCOs of biovar viviae and trifolii in this system is the presence or absence of one cis double bond, resulting in the very different hydrophobicity of the LCOs. Using a newly developed spot application assay, we show that the 18:2‐ and C18:3‐containing LCOs are able to induce the formation of nodule primordia on roots of Trifolium pratense. On the basis of these and other recent results, we propose that the host range of nodulation of the R. leguminosarum biovars viciae and trifolii is determined by the degree of hydrophobicity of the poly‐unsaturated fatty acyl moieties of their LCOs, which is mediated by the host‐specific central domain of the NodE protein.
Molecular characterization of the lincomycin‐production gene cluster of Streptomyces lincolnensis 78‐11
Authors: Peschke Ursula , Schmidt Heike , Zhang Hui‐Zhan , Piepersberg Wolfgang
Published: October 2006

The lincomycin (LM)‐production gene cluster of the overproducing strain Streptomyces iincolnensis 78‐11 was cloned, analysed by hybridization, as well as by DNA sequencing, and compared with the respective genome segments of other lincomycin producers. The lmb/lmr gene cluster is composed of 27 open reading frames with putative biosynthetic or regulatory functions (lmb genes) and three resistance (lmr) genes, two of which, lmrA and lmrC, flank the cluster. A very similar overall organization of the lmb/lmr cluster seems to be conserved in four other LM producers, although the clusters are embedded in non‐homologous genomic surroundings, in the wild‐type strain (S. lincolnensis NRRL2936), the lmb/lmr‐cluster apparently is present only in single copy. However, in the industrial strain S. lincolnensis 78‐11 the non‐adjacent gene clusters for the production of LM and melanin (melC) both are duplicated on a large (0.45‐0.5 Mb) fragment, accompanied by deletion events. This indicates that enhanced gene dosage is one of the factors for the overproduction of LM and demonstrates that large‐scale genome rearrangements can be a result of classical strain improvement by mutagenesis. Only a minority of the putative Lmb proteins belong to known protein families. These include members of the γ‐glutamyl transferases (LmbA), amino acid acylases (LmbC), aromatic amino acid aminotransferases (LmbF), imidazoleglycerolphosphate dehydratases (LmbK), dTDP‐glucose synthases (LmbO), dTDP‐glucose 4,6‐dehydratases (LmbM) and (NDP‐) ketohexose (or ketocyclitol) aminotransferases (LmbS). In contrast to earlier proposals on the biosynthetic pathway of the C‐8 sugar moiety (methylthiolincosaminide), this branch of the LM pathway actually seems to be based on nucleotide‐activated sugars as precursors.
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

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

The 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

Stage 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

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

The 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

An 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

It 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

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

The 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

The 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

The 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

Sumnnary

Nonsense 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

The 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

Site‐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.
A novel chromatin‐forming histone H1 homologue is encoded by a dispensable and growth‐regulated gene in Bordetella pertussis
Authors: Scarlato Vincenzo , Aricó Beatrice , Goyard Sophie , Ricci Stefano , Manetti Riccardo , Prugnola Anna , Manetti Roberto , Polverino‐De‐Laureto Patrizia , Ullmann Agnes , Rappuoli Rino
Published: October 2006

We report the Identification of a protein homologous to a histone H1 in Bordetella pertussis. The B. pertussis histone homologue, BpH1, varies in size in different strains from 182 to 206 amino acids. The variability of the size of the protein is due to gene variability by insertion or deletion of DNA modules. Insertion of a kanamycin cassette into the bpH1 gene generates a BpH1 null mutant with phenotypic properties and growth rate similar to those of the wild‐type strain, showing that this gene is dispensable. In vitro, the BpH1 protein prevents chromosomal DNA degradation from DNase I and constrains supercoiled DNA. Transcription of the bpH1 gene is activated during exponential growth of the bacteria, whereas It is repressed during the stationary phase of growth, It is proposed that BpH1 plays a role in chromatin formation and condensation during DNA replication and that repression of transcription depends upon a reduced rate of DNA replication.
Action of receiver and activator modules of UhpA in transcriptional control of the Escherichia coli sugar phosphate transport system
Authors: Webber Carol A. , Kadner Robert J.
Published: October 2006

Induction of the sugar‐phosphate transport system in Escherichia coli by external glucose‐6‐phosphate is regulated by the UhpABC regulatory proteins. UhpA protein is required for uhpT transcription and is related to response regulators of two‐component regulatory systems. UhpA and its homologues appear to be composed of two modules: the receiver module which contains the putative site of phosphorylation, and the activation module whose predicted helix‐turn‐helix motif is related to that present in many transcription activators. The roles of the two modules were examined by analysis of the regulatory consequences of uhpA deletion mutations generated by in vitro manipulations and missense mutations selected for independence from the requirement for UhpB kinase activity. Deletion of even seven amino acids from the C‐terminus resulted in complete loss of transcription activation at the uhpT promoter. Overexpression of all C‐terminal truncations that left intact the receiver module (residues 1–120) exhibited strong dominant‐negative interference with a chromosomal uhpA+ allele. The genetic requirements for interference indicated that the overexpressed receiver module competed with intact UhpA for phosphate residues carried on UhpB. The site of phosphorylation of UhpA is not necessary for uhp activation by over‐expressed UhpA but is necessary for UhpA action at normal levels of UhpA or for interference by the truncated species.
Molecular cloning, expression in Streptomyces livdans, and analysis of a gene cluster from Arthrobacter simplex encoding 3‐ketosteroid‐Δ1‐dehydrogenase, 3‐ketosteroid‐Δ5‐isomerase and a hypothetical regulatory protein
Authors: Molnár István , Choi Kwang‐Pil , Yamashita Mitsuo , Murooka Yoshikatsu
Published: October 2006

The Arthrobacter simplex gene coding for 3‐ketosteroid‐Δ1‐dehydrogenase, a key enzyme in the degradation of the steroid nucleus, was cloned in Streptomyces lividans, Nucleotide sequence analysis revealed that the gene for 3‐ketosteroid‐Δ1‐dehydrogenase (ksdD) is clustered with at least two more genes possibly involved in steroid metabolism. Upstream of ksdD, we found a gene, ksdR, encoding a hypothetical regulatory protein that shows homologies to KdgR, the negative regulator of pectin biodegradation in Erwinia, and GylR, the activator for glycerol metabolism in Steptomyces. A helix‐turn‐helix DNA‐binding domain can be predicted at similar positions near the N‐terminal of KsdR, KdgR and GylR. ksdl adjoining downstream to ksdD codes for a protein that has strong similarities to 3‐ketosteroid‐Δ5‐isomerases. The highly conserved Tyr and Asp residues are present in the active‐centre motif of the enzyme. The translated ksdD gene product was found to be similar to the 3‐ketosteroid‐Δ1‐dehydrogenase of Pseudomonas testosteroni and to the fumarate reductase of Shewanella putrefaciens. A region highly conserved between the two steroid dehydrogenases can be aligned to the active‐centre motif of the fumarate reductase. S. lividans strains carrying the ksdD gene overexpressed 3‐ketosteroid‐Δ1‐dehydrogenase. The expression of 3‐ketosteroid‐Δ5‐isomerase, however, was barely detectable in recombinant S. lividans strains carrying the ksdl gene, or in the parental Arthrobacter strain.
Small clusters of divergent amino acids surrounding the effector domain mediate the varied phenotypes of EF‐G and LepA expression
Authors: Yaskowiak Edward S. , March Paul E.
Published: October 2006

Elongation factors G, Tu, and related proteins (including LepA) form a distinct subgroup within the GTPase superfamily. This observation is based primarily upon amino acid comparisons of the effector region (G2) of the GTP‐binding domain. To examine the functional importance of the highly conserved elongation factor G2 domain a series of chimeric proteins were constructed between Escherichia coli EF‐G and Micrococcus luteus EF‐G, and between E. coli EF‐G and LepA (a protein of unknown function). The M. luteus EF‐G/E. coli EF‐G hybrid, M. luteus EF‐G, and E. coli EF‐G efficiently complemented EF‐G function in an E. coli strain (PEM101) harbouring a temperature‐sensitive mutation in fusA (the gene encoding EF‐G). A comparison of the amino acid sequences of the M. luteus EF‐G and E. coli EF‐G indicated that groups of divergent amino acid residues (amino acids 1–9 and 72–80) were not important for function. LepA and LepA/EF‐G chimeric proteins were tested for the ability to complement EF‐G function in vivo, for cross‐linking to 8‐azido‐[γ‐32]‐GTP in vitro and for fusidic acid‐dependent co‐sedimentation with 70S ribosomes. With one exception, all chimeras could be readily cross‐linked to azido‐GTP in an EF‐G‐like manner, indicating that hybrid protein construction did not generally result in improperly folded GTP‐binding domains. However, the inability of such chimeras to complement EF‐G function in vivo indicates that the effector domains are not functionally interchangeable. All LepA/EF‐G chimeric proteins were severely defective in fusidic acid‐dependent complex formation with 70S ribosomes. A comparison of the amino acid sequences of all three proteins suggests that residues 30–33, 43–48, and 63–66 of E. coli EF‐G are important for EF‐G specific ribosome‐associated function.
Developmentally regulated expression of a Giardia lamblia cyst wall protein gene
Authors: Mowatt Michael R. , Luján Hugo D. , Cotten David B. , Bowers Blair , Yee Janet , Nash Theodore E. , Stibbs Henry H.
Published: October 2006

The protozoan Giardia lamblia is an obligate parasite of the mammalian small intestine. We studied the expression of a gene that encodes a protein component of the cyst wall, a complex structure assembled during the differentiation of trophozoites to cysts and which is critical to survival of the parasite outside its mammalian host. Transcripts from the cyst wall protein gene increase more than 100‐fold during encystation, reaching a maximum between 5 and 24 hours after induction. Cyst wall protein expression also increases dramatically during encystation, and, prior to its incorporation into the nascent cyst wall, the protein is contained within the encystation‐specific vesicles of encysting trophozoites. The sequence of the cloned gene predicts an acidic, leucine‐rich polypeptide of Mr, 26000 that contains 5.3 tandemly arranged copies of a degenerate 24‐amino‐acid repeat. A hydrophobic amino‐terminal peptide probably serves as the initial signal that targets this protein to a secretory pathway involving vesicular localization during encystation and, ultimately, secretion to form the cyst wall.
The positively acting amdA gene of Aspergillus nidulans encodes a protein with two C2H2 zinc‐finger motifs
Authors: Lints Robyn , Davis Meryl A. , Hynes Michael J.
Published: October 2006

Semi‐dominant mutations in the amdA gene iead to elevated expression of the gene encoding acetamidase, amdS. These mutations also cause constitutive expression of the acetate‐inducible gene, aciA. In the amdS 5′ regulatory region, two cis‐acting mutations, amd166 anti amd1666, have been isolated which specifically affect amdA activation of amdS. These mutations are a duplication and a triplication of an 18bp GA‐rich sequence, thought to define the amdA site of action within the amdS promoter region. Similar GA‐rich sequences have also been found in the 5′ region of aciA. This paper describes the cloning and initial functional characterization of the amdA gene and two of its mutant alleles. The wild‐type amdA gene has been cloned by a chromosome walk from genes gatA and alcC on linkage group VII and localized by complementation of an amdA loss‐of‐function mutation. Transcriptional analysis reveals that the gene is expressed constitutively at low levels under growth conditions which affect expression of amdS and aciA. The gene is predicted to encode an 880‐amino‐acid protein which contains two C2H2 zinc fingers, a nuclear localization sequence and two transcriptional activation domains. The amdA7 semi‐dominant gain‐of‐function mutation results in a glycine to aspartate substitution which would increase the acidity of one of these regions. Analysis of in vitro generated mutations in the 5′ region of amdS using an amdS::lacZ reporter has been used to localize the site of action of AmdA. The C2H2 zinc‐finger motifs identified in the protein are similar to those found in the carbon catabolite repressor protein, CreA, which also regulates amdS and recognizes sequences which overlap with the proposed site of action for AmdA.
The Escherichia coli DnaK chaperone machine and bacteriophage Mu iate transcription
Authors: Sand Olivier , Desmet Lucie , Toussaint Ariane , Pato Martin
Published: October 2006

Bacteriophage Mu does not grow on temperature‐sensitive E. coli dnaK mutants at elevated temperatures because of a defect in late transcription. As the Mu‐encoded C protein is required for activation of transcription from the phage late promoters, we attempted to determine if DnaK and its accessory proteins DnaJ and GrpE are required for synthesis of C protein or at a later step. We found that the chaperones act in Mu late transcription beyond C‐protein synthesis, and that C‐protein stability is decreased in the mutant hosts. This suggests that the DnaK chaperone machine may be required for the proper folding and/or multi‐merization of C protein.