bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake of inorganic carbon in the cyanobacterium Synechocystis PCC6803: physiological and genetic evidence for a high‐affinity uptake system
Authors: Bédu S. , Pozuelos P. , Cami B. , Joset F.
Published: March 2004

Synechocystis PCC6803 displays two inorganic carbon‐uptake processes, a low‐affinity one (apparent Km: 300–400 µM) functional in cells grown under standard or limiting inorganic carbon concentrations, and one with a higher affinity (60±12 µM), detected only in cells adapted to limiting inorganic carbon conditions. A mutational and screening procedure allowed the isolation of a mutant deficient in the high‐affinity system, but only slightly impaired in its growth capacities. The mutated genomic region revealed two open reading frames (ORFs), possibly belonging to an operonic structure. A clone in which the downstream ORF, hatR (high‐affinity transport), had been inactivated showed a phenotype close to that of the original mutant. Inactivation of the other ORF, hatA, yielded a clone unable to grow in limiting inorganic carbon conditions. The deduced HatA protein showed no homology with any registered protein. It possessed three hydrophobic domains, including a putative signal peptide. Several hypotheses are considered as to its role. The deduced HatR protein, which possessed the features characteristic of the response regulators of the two‐component regulatory systems ubiquitous in bacteria, might be a regulator controlling the activity of the high‐affinity transport process. It would belong to the subclass of these molecules lacking the DNA‐binding domain.
Identification of a plasminogen‐binding motif in PAM, a bacterial surface protein
Authors: Wistedt Annika Carlsson , Ringdahl Ulrika , Müller‐Esterl Werner , Sjöbring Ulf
Published: March 2004

Surface‐associated plasmin(ogen) may contribute to the invasive properties of various cells. Analysis of plasmin(ogen)‐binding surface proteins is therefore of interest. The N‐terminal variable regions of M‐like (ML) proteins from five different group A streptococcal serotypes (33,41,52,53 and 56) exhibiting the plasminogen‐binding phenotype were cloned and expressed in Escherichia coli. The recombinant proteins all bound plasminogen with high affinity. The binding involved the kringle domains of plasminogen and was blocked by a lysine analogue, 6‐aminohexanoic acid, indicating that lysine residues in the M‐like proteins participate in the interaction. Sequence analysis revealed that the proteins contain common 13–16‐amino‐acid tandem repeats, each with a single central lysine residue. Experiments with fusion proteins and a 30‐amino‐acid synthetic peptide demonstrated that these repeats harbour the major plasminogen‐binding site in the ML53 protein, as well as a binding site for the tissue‐type plasminogen activator. Replacement of the lysine in the first repeat with alanine reduced the plasminogen‐binding capacity of the ML53 protein by 80%. The results precisely localize the binding domain in a plasminogen surface receptor, thereby providing a unique ligand for the analysis of interactions between kringles and proteins with internal kringle‐binding determinants.
nasST, two genes involved in the induction of the assimilatory nitrite—nitrate reductase operon (nasAB) of Azotobacter vinelandii
Authors: Gutierrez Juan‐Carlos , Ramos Francisco , Ortner Ludwig , Tortolero María
Published: March 2004

An operon including two new genes (nasS and nasT) has been defined, cloned and sequenced. The deduced NASS protein is homologous to NRTA from Synechococcus sp. and to NASF from Klebsiella pneumoniae, two proteins involved in nitrate uptake. The predicted NAST polypeptide is homologous to the regulator proteins of the two‐component regulatory systems. NASS plays a negative regulatory role in the synthesis of the nitrate and nitrite reductase. NAST is required for the expression of the nitrite—nitrate reductase operon (nasAB). Expression of the nasST operon is not under the control of the NTR system and is not regulated by the nitrogen source. A Φ(nasA—lacZ) fusion has been used to analyse expression of the nasAB operon in three different genetic backgrounds with altered nitrate reductase activity. Beta‐galactosidase activity in two of them was independent of nitrate but in a mutant unable to reduce nitrate, nas‐4, it was normally induced by nitrate.
Photonic detection of bacterial pathogens in living hosts
Authors: Contag Chritopher H. , Contag Pamela R. , Mullins James I. , Spilman Stanley D. , Stevenson David K. , Benaron David A.
Published: March 2004

The study of pathogenic is often limited to ex vivo assays and cell‐culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurium that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bio‐luminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that the real time non‐invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo.
Organization of the Saccharomyces cerevisiae actin gene UAS: functional significance of reiterated REB1 binding sites and AT‐rich elements
Authors: McLean Michael , Hubberstey Andrew V. , Bouman Derek J. , Pece Nadia , Mastrangelo Peter , Wildeman Alan G.
Published: March 2004

The upstream activation sequence (UAS) in the Saccharomyces cerevisiae actin gene promoter contains three different motifs, specifically two AT‐rich tracts, two binding sites for the yeast protein REB1, and an Mlul site. Synthetic UAS elements containing individual motifs, or combinations of them, were inserted in place of the natural UAS, and assayed using a lacZ reporter gene. The REB1 binding sites were found to be essential for, and sufficient to restore partial, UAS activity. AT‐rich tracts alone were inactive. Multimerization of a REB1 binding site created a UAS that in galactose is more active, but in glucose less active, than a UAS having a single REB1 site with one AT‐rich tract. In general, transcription during growth in galactose or glycerol/lactate responds more to multimerization of motifs. The results suggest that the natural actin promoter UAS retains activity on these alternative carbon sources because of reiteration of sequence elements within it; the additional elements appear to be redundant when cells are grown on glucose. The mlul site, which is present upstream of a number of yeast genes involved in DNA synthesis and confers cell cycle periodicity to those genes, contributes to the activity of the synthetic UAS elements, but not in a cell‐cycle‐dependent manner.
Benefit of transcription‐coupled nucleotide excision repair for gene expression in u.v.‐damaged Escherichia coli
Authors: Li B.‐H. , Bockrath R.
Published: March 2004

Expression of the lactose operon upon induction by IPTG was studied with Escherichia coli B/r and K‐12 strains as a function of exposure to ultraviolet light. Patterns of expression inactivation were compared in cells with wild‐type UvrABC nucleotide excision repair, with transcription‐coupled excision repair (TCR) specifically defective because of a defect at mfd, or with excision repair (ER) and TCR eliminated by defects at uvrA or uvrC. Sets of inactivation patterns were also determined for cells expressing the lactose operon via the ‘UV5’ promoter, an alternative to the wild‐type promoter that eliminates dependence of expression on negative DNA supercoiling. The results demonstrated a major contribution by TCR to successful gene expression. Gene expression was more sensitive to u.v. inactivation when TCR was defective and similarly more sensitive when both ER and TCR were defective. Thus, TCR may be the only means of repairing transcription‐blocking damage at active genes. Contrasting results with wild‐type and UV5 promoters suggested that relaxed supercoiling might accompany repair and reduce expression even though a template lesion is removed. A test of mismatch repair defects on ultraviolet inactivation of gene expression found only limited interference with TCR as it benefits gene expression.
The effect of receptor rapid‐internalization signals on diphtheria toxin endocytosis and cell sensitivity
Authors: Almond Brian D. , Eidels Leon
Published: March 2004

Diphtheria toxin enters toxin‐sensitive mammalian cells by receptor‐mediated endocytosis employing the heparin‐binding EGF‐like growth factor precursor as its receptor. We reported previously (Almond and Eidels, 1994) that cytoplasmic domain mutants of the toxin receptor and cells expressing wild‐type receptor internalize toxin slowly, the rate being approximately that of normal turnover of the plasma membrane. To determine whether it was possible to increase toxin sensitivity by increasing the rate of toxin internalization, we constructed diphtheria toxin cytoplasmic domain mutant cell lines containing rapid‐internalization signals from either the low density lipoprotein receptor or from the lysosomal acid phosphatase precursor. Although cells transfected with mutant receptor genes internalized toxin at a faster rate than those expressing the wild‐type receptor, they showed a decrease in toxin sensitivity. This decreased sensitivity may be accounted for by an observed decrease in the number of toxin‐binding sites and by an increased rate of toxin internalization and degradation. These results suggest that the rate of toxin internalization may not be the rate‐limiting step in the cytotoxic process.
A bacteriocin‐like peptide induces bacteriocin synthesis in Lactobacillus plantarum C11
Authors: Diep Dzung Bao , H»varstein Leiv Sigve , Nes Ingolf F.
Published: March 2004

In this study, we show that bacteriocin production in Lactobacillus plantarum C11 is an inducible process triggered by a secreted protein factor produced by the bacteriocin producer itself. The induction factor was identified to be plantaricin A, a bacteriocin‐like peptide whose gene (plnA) is located in the same operon as a two‐component regulatory system (plnBCD). When L. plantarum C11 cultures were depleted for plantaricin A, either by growing individual colonies on agar plates or by starting a new culture with a highly diluted inoculum, no bacteriocin was produced during the following growth. When chemically synthesized plantaricin A or purified bacterially produced plantaricin A was added to non‐producing cultures, bacteriocin production was induced. Only 1 ng ml−1 plantaricin A is sufficient to induce the bacteriocin production in non‐producing L. plantarum C11, and bacteriocin activity appears in the growth medium approximately 150 min after induction. Northern analyses, using a plnA‐specific probe, demonstrated that plantaricin A is able to induce its own synthesis by transcription of the plnABCD operon, and this is observed approximately 15 min after adding plantaricin A. Furthermore, heterologous expression of the plnABCD operon in a Lactobacillus sake strain showed that the conditioned growth medium contained the active induction factor. Neither synthetic nor expressed plantaricin A from the heterologous system possesses any bacteriocin activity, suggesting that plantaricin A is primarily an induction factor and not a bacteriocin as claimed earlier.
Expression of the gltP gene of Escherichia coli in a glutamate transport‐deficient mutant of Rhodobacter sphaeroides restores chemotaxis to glutamate
Authors: Jacobs Mariken H.J. , Van Der Heide Tiemen , Tolner Berend , Driessen Arnold J.M. , Konings Wil N.
Published: March 2004

Rhodobacter sphaeroides is chemotactic to glutamate and most other amino acids. In Escherichia coli, chemotaxis involves a membrane‐bound sensor that either binds the amino acid directly or interacts with the binding protein loaded with the amino acid. In R. sphaeroides, chemotaxis is thought to require both the uptake and the metabolism of the amino acid. Glutamate is accumulated by the cells via a binding protein‐dependent system. To determine the role of the binding protein and transport in glutamate taxis, mutants were created by Tn5 insertion mutagenesis and selected for growth in the presence of the toxic glutamine analogue γ‐glutamyl‐hydrazide. One of the mutants, R. sphaeroides MJ7, was defective in glutamate uptake but showed wild‐type levels of binding protein. The mutant showed no chemotactic response to glutamate. Both glutamate uptake and chemotaxis were recovered when the gltP gene, coding for the H+‐linked glutamate carrier of E. coli, was expressed in R. sphaeroides MJ7. It is concluded that the chemotactic response to glutamate strictly requires uptake of glutamate, supporting the view that intracellular metabolism is needed for chemotaxis in R. sphaeroides.
Mycoplasma hyorhinis vlp gene transcription: critical role in phase variation and expression of surface lipoproteins
Authors: Citti Christine , Wise Kim S.
Published: March 2004

Mycoplasma hyorhinis contains clustered vlp genes encoding variable lipoproteins (Vlps), the major coat proteins and surface antigens of this wall‐less prokaryotic pathogen. vlp genes are subject to discrete, high frequency mutations independently affecting the size or the expression of variant Vlp products. Change in Vlp size occurs by mutations altering the number of tandem intragenic repeats at the 3′ end of each single‐copy vlp gene. In this report, phase‐variant Vlp expression is shown to result from altered vlp gene transcription. vlpA, vlpB and vlpC transcripts were monitored in a clonal lineage selected to display various Vlp phenotypes. Each vlp gene was expressed as a distinct transcript, which was subject to drastic ON/OFF switches associated with random insertion/ deletion mutations in a homopolymeric tract of adenine residues in the promoter region of all vlp genes. Unexpectedly, the level of vlp transcripts appeared to depend on the length of the corresponding genes in the ON configuration. Higher proportional levels of shorter vlp transcripts were shown to reflect a greater abundance of short Vlp lipoproteins present in l‐[35S]‐cysteine‐labelled membrane protein preparations. The vlp cluster provides a heritable, and highly mutable, locus for the generation of surface diversity through random promoter mutations affecting the expression of genes, whose products also vary in length and abundance, by virtue of separate mutations in structural regions of the genes.
Expression of two csg operons is required for production of fibronectin‐ and Congo red‐binding curli polymers in Escherichia coli K‐12
Authors: Hammar M»rten , Arnqvist Anna , Bian Zhao , Olsén Arne , Normark Staffan
Published: March 2004

Two divergently transcribed operons in Escherichia coli required for the expression of fibronectin‐ and Congo red‐binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein. The csgBA operon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA. A non‐polar csgB mutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers. A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre. The promoter for the oppositely transcribed csgDEFG operon was identified by primer extension and shown, like the csgBA promoter, to be dependent upon the alternate stationary phase‐specific sigma factor σs in wild‐type cells, but not in mutants lacking the nucleoid associated protein H‐NS. Insertions in csgD abolish completely trancription from the csgBA promoter. Therefore, any regulatory effect on the csgBA promoter might be secondary to events controlling the csgDEFG promoter and/or activation of CsgD. Insertions in csgE, csgF and csgG abolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein. No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a novel pathway.
Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection
Authors: Camilli Andrew , Mekalanos John J.
Published: March 2004

A complete understanding of host‐parasite interactions must necessarily include the identification and characterization of gene products expressed by both parties during the infectious process. We have developed a new screen to identify bacterial genes that are transcriptionally induced during infection of a host animal. The method is based on pre‐selection of strains carrying tnpR operon fusions (encoding resolvase, a site‐specific DNA recombinase) which are not expressed in vitro, followed by screening for a subset of these strains that subsequently express resolvase within the host environment. The latter subset was recognized as recombinants that had deleted a resolvase‐specific reporter construct. Thirteen transcription units of Vibrio cholerae were identified that were induced during infection in an infant mouse model of cholera. Five of these were predicted to encode polypeptides with diverse functions in metabolism, biosynthesis and motility; one encoded a secreted lipase; two appear to be antisense to genes involved in motility; and five are predicted to encode polypeptides of unknown function. Three of the transcripts were shown to be required for full virulence in infant mice, as assessed by competition experiments.
Characterization of Vibrio cholerae bacteriophage K139 and use of a novel mini‐transposon to identify a phage‐encoded virulence factor
Authors: Reidl Joachim , Mekalanos John J.
Published: March 2004

Temperate bacteriophage K139 was isolated from a Vibrio cholerae O139 isolate and characterized in this study. The phage genome consists of a 35 kbp, double‐stranded, linear DNA molecule that circularizes and integrates into the chromosome in a site‐specific manner. DNA sequences that cross‐hybridize with K139 phage DNA are present in all strains of V. cholerae serogroup O1 of the classical biotype examined and in some strains of the El Tor biotype. Phage K139 produces plaques on El Tor O1 strains that do not carry the K139‐related sequences but does not plaque on O139 strains that lack detectable phage DNA. This results suggests that O139 strains arose in part by horizontal gene transfer of the O139 antigen genes into an El Tor O1 strain that harboured a K139 prophage. Consistent with this interpretation, the morphology of K139 phage particles is identical to that displayed by the widely distributed family of O1 phages referred to as ‘kappa’. In order to test whether K139 phage is involved in lysogenic conversion of V. cholerae, we constructed a novel mini‐transposon, Tn10d‐bla, which was designed to produce β‐lactamase fusions to phage‐encoded, exported proteins. All Tn 10d‐bla insertions obtained were closely linked to one location on the K139 phage genome. DNA sequence determination of the fusion joints revealed an open reading frame (ORF1), encoding a gene product of 137 amino acids with a typical N‐terminal hydrophobic signal sequence. ORF1 was designated the glo gene (G protein‐like ORF) because its amino acid sequence shows similarity to eukaryotic Gs(α) protein (34.5% identity over an 81‐amino‐acid overlap) and its C‐terminus displays the consensus motif (CAAX) which is found in many small eukaryotic GTP‐binding proteins. LD50 assays with isogenic Glo+ and Glo− K139 lysogens suggest that glo encodes a secreted virulence determinant of V. cholerae
Mechanism of antigenic variation in Mycoplasma pulmonis: interwoven, site‐specific DNA inversions
Authors: Bhugra Bindu , Voelker LeRoy L. , Zou Nianxiang , Yu Huilan , Dybvig Kevin
Published: March 2004

The chromosome of the murine pathogen Mycoplasma pulmonis undergoes rearrangements at a high frequency. We show that some of these rearrangements regulate the phase‐variable expression of a cluster of genes (the vsa locus) that encode the variable V‐1 surface antigens. Only one vsa gene is associated with an expression site; the other vsa genes are transcriptionally silent. The silent genes lack the 5′ end region (promoter and ribosome‐binding site) that is present in the expressed gene, and DNA rearrangements regulate gene expression by reassorting the 5′ end region from an expressed gene with the 3′ end region from a previously silent gene. All vsa rearrangements identified so far are site‐specific DNA inversions that occur between copies of a specific 34 bp sequence that is conserved in each vsa gene. Interestingly, DNA inversions within the vsa locus apparently occur in concert with inversion of the hsd1 element, which regulates restriction and modification activity in M. pulmonis.
hilA is a novel ompR/toxR family member that activates the expression of Salmonella typhimurium invasion genes
Authors: Bajaj Vivek , Hwang Clara , Lee Catherine A.
Published: March 2004

During infection of its hosts, Salmonella enters intestinal epithelial cells. Many Salmonella typhimurium genes required for bacterial entry into host cells are encoded on a 40 kb ‘pathogenicity island’, We report here the identification of hilA, a gene within the ‘island’ that appears to encode an activator of invasion gene expression. By using a set of lacZY transcriptional fusions to S. typhimurium invasion genes, we found that hilA activates the expression of invasion genes located on the ‘pathogenicity island’. hilA is required for efficient entry into HEp‐2 cells in vitro. The predicted amino acid sequence of hilA shares significant homology with the DNA‐binding domains of the OmpR‐ToxR family of transcriptional activators. However, unlike OmpR and ToxR, HilA contains neither a phosphoryl acceptor nor a membrane‐spanning domain, and, therefore, its activity may be modulated by a novel mechanism. Many environmental conditions modulate the ability of Salmonella to enter non‐phagocytic mammalian cells. It has been proposed that induction of Salmonella invasion proteins in response to a combination of environmental cues ensures that bacterial entry is limited to specific sites and times during infection. Our results are consistent with the hypothesis that hilA plays a key role in the regulation of Salmonella invasion during infection.
Molecular analysis of a locus for the biosynthesis and phase‐variable expression of the lacto‐N‐neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis
Authors: Jennings Michael P. , Hood Derek W. , Peak Ian R.A. , Virji Mumtaz , Moxon E. Richard
Published: March 2004

Lipopolysaccharide (LPS) is a major determinant of Neisseria meningitidis virulence. A key feature of meningococcal LPS is the phase‐variable expression of terminal structures which are proposed to have disparate roles in pathogenesis. In order to identify the biosynthetic genes for terminal LPS structures and the control mechanisms for their phase‐variable expression, the lic2A gene, which is involved in LPS biosynthesis in Haemophilus influenzae, was used as a hybridization probe to identify a homologous gene in N. meningitidis strain MC58. The homologous region of DNA was cloned and nucleotide sequence analysis revealed three open reading frames (ORFs), two of which were homologous to the H. influenzae lic2A gene. All three ORFs were mutagenized by the insertion of antibiotic‐resistance cassettes and the LPS from these mutant strains was analysed to determine if the genes had a role in LPS biosynthesis. Immunological and tricine—SDS—PAGE analysis of LPS from the mutant strains indicated that all three genes were probably transferases in the biosynthesis of the terminal lacto‐N‐neotetraose structure of meningococcal LPS. The first ORF of the locus contains a homopolymeric tract of 14 guanosine residues within the 5′‐end of the coding sequence. As the lacto‐N‐neotetraose structure in meningococcal LPS is subject to phase‐variable expression, colonies that no longer expressed the terminal structure, as determined by monoclonal antibody binding, were isolated. Analysis of an ‘off’ phase variant revealed a change in the number of guanosine residues resulting in a frameshift mutation, indicating that a slipped‐strand mispairing mechanism, operating in the first ORF, controls the phase‐variable expression of lacto‐N‐neotetraose.
Opc‐ and pilus‐dependent interactions of meningococci with human endothelial cells: molecular mechanisms and modulation by surface polysaccharides
Authors: Virji Mumtaz , Makepeace Katherine , Peak Ian R.A. , Ferguson David J.P. , Jennings Michael P. , Moxon E. Richard
Published: March 2004

The interplay between four surface‐expressed virulence factors of Neisseria meningitidis (pili, Opc, capsule and lipopolysaccharide (LPS)) in host cell adhesion and invasion was examined using derivatives of a serogroup B strain, MC58, created by mutation (capsule, Opc) and selection of variants. To examine the role of Opc and of additional expression of pili, bacteria lacking the expression of Opa proteins were used. The effects of different LPS structures were examined in variants expressing either sialylated (L3 immunotype) or truncated non‐sialylated (L8 immuno‐type) LPS. Studies showed that (i) pili were essential for meningococcal interactions with host cells in both capsulate and acapsulate bacteria with the sialylated L3 LPS immunotype, (ii) the Opc‐mediated invasion of host cells by piliated and non‐piliated bacteria was observed only in acapsulate organisms with L8 LPS immunotype, and (iii) expression of pili in Opc‐expressing bacteria resulted in increased invasion. Investigations on the mechanisms of cellular invasion indicated that the Opc‐mediated invasion was dependent on the presence of serum in the incubation medium and was mediated by serum proteins with arginine‐glycine‐aspartic acid (RGD) sequence. Cellular invasion in piliated Opc+ phenotype also required bridging molecules containing the RGD recognition sequence and appeared to involve the integrin αvβ3 as a target receptor on endothelial cells. These studies extend the previous observations on variants of a serogroup A strain (C751) and show that Opc mediates cellular invasion in distinct meningococcal strains and provide confirmation of its mechanism of action. This is the first investigation that evaluates, using derivatives of a single strain, the interplay between four meningococcal surface virulence factors in host cell invasion.
Inactivation of mecA prevents recovery from the competent state and interferes with cell division and the partitioning of nucleoids in Bacillus subtilis
Authors: Hahn J. , Bylund J. , Haines M. , Higgins M. , Dubnau D.
Published: March 2004

The development of genetic competence in Bacillus subtilis requires the synthesis of ComK, a transcription factor, which is normally produced as a culture enters the stationary phase. This synthesis is known to be regulated in part by the protein MecA. Loss‐of‐function mutations in mecA result in overexpression of ComK and its appearance early during exponential growth. We show here that mecA inactivation also causes a loss of colony‐forming ability, especially during stationary phase. This loss is accompanied by the appearance of cells in which normal nucleoid separation has failed to occur. Renografin gradient fractionation of mecA cultures grown to competence reveals that nearly 100% of the cells band at the low buoyant density characteristic of competent cells, and that this low density is competence‐related. The loss of viability, the low buoyant density and the nucleoid separation defect, are all comK‐dependent. The loss of viability can be reversed by even the transient introduction of mecA+. It is proposed that these effects of ComK overexpression are related to the DNA replication arrest normally exhibited by the competent cell fraction and that MecA is needed to reverse this arrest and to permit escape from the competent state. The shift of nearly 100% of the cells to light buoyant density in a mecA mutant culture strongly suggests that the MecA protein is a regulator of the cell‐type‐specific expression of competence.
The type IV pre‐pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues
Authors: Hu Nien‐Tai , Lee Pei‐Fang , Chen Chanhen
Published: March 2004

Type IV pre‐pilin leader peptidase was demonstrated to be required for protein secretion, in addition to its involvement in biogenesis of type IV pili. The type IV pre‐pilin leader peptidase gene of Xanthomonas campestris pv. campestris was located on a 3 kb Accl fragment on account of its hybridization with the DNA fragment containing the type IV pre‐pilin leader‐peptidase gene pilD/xcpA of Pseudomonas aeruginosa. Sequencing of the cloned fragment revealed an open reading frame (ORF) (designated xpsO) of 287 amino acid residues. A protein with an apparent molecular mass of approximately 32.5 kDa was synthesized in vitro from a DNA fragment containing the xpsO gene. The amino acid sequence shares 50% identity with that of PilD throughout the entire sequence. Among other type IV pre‐pilin leader peptidases, XpsO is unique in not having the two conserved ‐CXXC‐ motifs in a cytoplasmic domain. Instead, new motifs were noted when the protein was compared with XpsE, which is another member of the extracellular protein‐secretion machinery. When the xpsO gene was introduced into the pilD mutant of P. aeruginosa, both the sensitivity against infection with the pilus‐specific phage PO4 and the ability to secrete extracellular protein were recovered. Furthermore, immunoblot analysis indicated that the P. aeruginosa pilin was apparently processed in vivo by the xpsO gene product.
Functional and regulatory analysis of the OmpF‐like porin, OpnP, of the symbiotic bacterium Xenorhabdus nematophilus
Authors: Forst Steven , Waukau Jill , Leisman Gary , Exner Maurice , Hancock Robert
Published: March 2004

The function and novel regulation of OpnP of the symbiotic/pathogenic bacterium, Xenorhabdus nematophilus was studied. In vitro pore‐function analysis of purified OpnP indicated that the single‐channel‐conductance values were similar to that measured for the porin protein, OmpF, of Esherichia coli. Nucleotide sequence analysis revealed that the mature OpnP protein contained 348 amino acid residues and shared 55% amino acid sequence identity with OmpF. Similar to ompF, opnP mapped between asnS and aspC. The 16 transmembrane β‐sheet structures and the internal loop 3 were highly conserved, while the remaining external loop domains were more divergent. Primer extension analysis identified the start site of transcription of opnP. A σ70‐type promoter, a perfect 20 bp OmpR‐binding site, and a binding site for the antisense molecule, micF RNA, were found in the upstream region of opnP. While the overall sequence identity of the asn—opnP—aspC region was high, the intergenic region between asnS and opnP had diverged markedly. The asnS—opnP region was 313 bp shorter than the intergenic region between asnS and ompF and lacked the OmpR‐binding site that is required for ompF repression by high osmolarity in E. coli. Results from osmolarity‐shift experiments indicated that OpnP was not repressed by high osmolarity. It was also found that OpnP was thermally regulated.
Unusual case of multicentric mixed‐type Castleman's disease presenting with exfoliative dermatitis
Authors: Yalcin Arzu D. , Keskin Ali , Erdogan Berna S. , Hekimgil Mine
Published: March 2004

Castleman's disease is a rare disorder that results in the underregulated growth of lymphoid tissue. It may present as benign involvement of one lymph node group or as multicentric disease with systemic symptoms. We here present a patient with Castleman's disease of mixed‐type and multicentric involvement.
HLA‐DQA1 and DQB1 alleles are associated with genetic susceptibility to psoriasis vulgaris in Chinese Han
Authors: Zhang Xuejun , Wei Shengcai , Yang Sen , Wang Zaixing , Zhang Anping , He Pingping , Wang Hongyan
Published: March 2004

Background  Psoriasis vulgaris is a chronic skin disorder characterized by infiltration of inflammatory elements, keratinocyte hyperproliferation and altered differentiation. Although the pathogenesis of psoriasis is not fully understood, there is solid evidence of a susceptibility locus in the human leukocyte antigen (HLA) region.

Objectives  To investigate whether HLA‐DQA1 and DQB1 alleles are associated with genetic susceptibility to psoriasis vulgaris in Chinese Han.

Patients and methods  The polymerase chain reaction–sequence‐specific primer (PCR‐SSP) method was used to analyse the distribution of HLA‐DQA1 and DQB1 alleles in 189 patients with psoriasis and 273 healthy controls.

Results  The HLA‐DQA1*0104 (OR = 2.33, P = 0.0001154, Pc = 2.0 × 10−3), DQA1*0201 (OR = 3.36, P < 1.0 × 10−7, Pc < 1.0 × 10−6), DQB1*0201 (OR = 1.64, P = 0.0192, Pc > 0.05) and DQB1*0303 (OR = 1.55, P = 0.0377, Pc > 0.05) alleles were more prevalent in patients with psoriasis vulgaris than in controls, and HLA‐DQA1*0501 (OR = 0.30, P = 0.0000039, Pc < 4.0 × 10−5) alleles were less prevalent. The HLA‐DQA1*0104 (OR = 2.42, P = 0.0001159, Pc < 2.0 × 10−3), DQA1*0201 (OR = 3.74, P < 1.0 × 10−7, Pc < 1.0 × 10−6) and DQA1*0501 (OR = 0.30, P = 0.0000374, Pc < 4.0 × 10−4) alleles were only associated with type I psoriasis. HLA‐DQA1*0104 and DQA1*0201 were more prevalent in patients with or without a family history of psoriasis. However, the DQA1*0501 allele was only more prevalent in patients without a family history of psoriasis.

Conclusion  HLA‐DQA1*0104 and DQA1*0201 alleles may be psoriasis susceptibility genes or may be in close linkage with the susceptibility genes. The HLA‐DQA1*0501 allele seems to have a protective effect against the development of psoriasis vulgaris in Chinese Han. There may be a difference in genetic background between psoriasis patients with and without a family history of psoriasis.
Functional analyses of placental protein 13/galectin‐13
Authors: Than Nandor G. , Pick Elah , Bellyei Szabolcs , Szigeti Andras , Burger Ora , Berente Zoltan , Janaky Tamas , Boronkai Arpad , Kliman Harvey , Meiri Hamutal , Bohn Hans , Than Gabor N. , Sumegi Balazs
Published: March 2004

Placental protein 13 (PP13) was cloned from human term placenta. As sequence analyses, alignments and computational modelling showed its conserved structural and functional homology to members of the galectin family, the protein was designated galectin‐13. Similar to human eosinophil Charcot–Leyden crystal protein/galectin‐10 but not other galectins, its weak lysophospholipase activity was confirmed by 31P‐NMR. In this study, recombinant PP13/galectin‐13 was expressed and specific monoclonal antibody to PP13 was developed. Endogenous lysophospholipase activity of both the purified and also the recombinant protein was verified. Sugar binding assays revealed that N‐acetyl‐lactosamine, mannose and N‐acetyl‐glucosamine residues widely expressed in human placenta had the strongest binding affinity to both the purified and recombinant PP13/galectin‐13, which also effectively agglutinated erythrocytes. The protein was found to be a homodimer of 16 kDa subunits linked together by disulphide bonds, a phenomenon differing from the noncovalent dimerization of previously known prototype galectins. Furthermore, reducing agents were shown to decrease its sugar binding activity and abolish its haemagglutination. Phosphorylation sites were computed on PP13/galectin‐13, and phosphorylation of the purified protein was confirmed. Using affinity chromatography, PAGE, MALDI‐TOF MS and post source decay, annexin II and beta/gamma actin were identified as proteins specifically bound to PP13/galectin‐13 in placenta and fetal hepatic cells. Perinuclear staining of the syncytiotrophoblasts showed its expression in these cells, while strong labelling of the syncytiotrophoblasts' brush border membrane confirmed its galectin‐like externalization to the cell surface. Knowing its colocalization and specific binding to annexin II, PP13/galectin‐13 was assumed to be secreted to the outer cell surface by ectocytosis, in microvesicles containing actin and annexin II. With regard to our functional and immunomorphological results, PP13/galectin‐13 may have special haemostatic and immunobiological functions at the lining of the common feto‐maternal blood‐spaces or developmental role in the placenta.
Type 2 isopentenyl diphosphate isomerase from a thermoacidophilic archaeon Sulfolobus shibatae
Authors: Yamashita Satoshi , Hemmi Hisashi , Ikeda Yosuke , Nakayama Toru , Nishino Tokuzo
Published: March 2004

Although isopentenyl diphosphate–dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. A novel type of the enzyme, which has no sequence similarity to the known, well‐studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg2+ for activity, similar to its bacterial homologues. Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH.
Two distinct heterodisulfide reductase‐like enzymes in the sulfate‐reducing archaeon Archaeoglobus profundus
Authors: Mander Gerd J. , Pierik Antonio J. , Huber Harald , Hedderich Reiner
Published: March 2004

Heterodisulfide reductase (Hdr) is a unique disulfide reductase that plays a key role in the energy metabolism of methanogenic archaea. Two types of Hdr have been identified and characterized from distantly related methanogens. Here we show that the sulfate‐reducing archaeon Archaeoglobus profundus cultivated on H2/sulfate forms enzymes related to both types of Hdr. From the membrane fraction of A. profundus, a two‐subunit enzyme (HmeCD) composed of a b‐type cytochrome and a hydrophilic iron–sulfur protein was isolated. The amino‐terminal sequences of these subunits revealed high sequence identities to subunits HmeC and HmeD of the Hme complex from A. fulgidus. HmeC and HmeD in turn are closely related to subunits HdrE and HdrD of Hdr from Methanosarcina spp. From the soluble fraction of A. profundus a six‐subunit enzyme complex (Mvh:Hdl) containing Ni, iron–sulfur clusters and FAD was isolated. Via amino‐terminal sequencing, the encoding genes were identified in the genome of the closely related species A. fulgidus in which these genes are clustered. They encode a three‐subunit [NiFe] hydrogenase with high sequence identity to the F420‐nonreducing hydrogenase from Methanothermobacter spp. while the remaining three polypeptides are related to the three‐subunit heterodisulfide reductase from Methanothermobacter spp. The oxidized enzyme exhibited an unusual EPR spectrum with gxyz = 2.014, 1.939 and 1.895 similar to that observed for oxidized Hme and Hdr. Upon reduction with H2 this signal was no longer detectable.
Effects of Escherichia coli ribosomal protein S12 mutations on cell‐free protein synthesis
Authors: Chumpolkulwong Namthip , Hori‐Takemoto Chie , Hosaka Takeshi , Inaoka Takashi , Kigawa Takanori , Shirouzu Mikako , Ochi Kozo , Yokoyama Shigeyuki
Published: March 2004

We examined the effects of Escherichia coli ribosomal protein S12 mutations on the efficiency of cell‐free protein synthesis. By screening 150 spontaneous streptomycin‐resistant isolates from E. coli BL21, we successfully obtained seven mutants of the S12 protein, including two streptomycin‐dependent mutants. The mutations occurred at Lys42, Lys87, Pro90 and Gly91 of the 30S ribosomal protein S12. We prepared S30 extracts from mutant cells harvested in the mid‐log phase. Their protein synthesis activities were compared by measuring the yields of the active chloramphenicol acetyltransferase. Higher protein production (1.3‐fold) than the wild‐type was observed with the mutant that replaced Lys42 with Thr (K42T). The K42R, K42N, and K42I strains showed lower activities, while the other mutant strains with Lys87, Pro90 and Pro91 did not show any significant difference from the wild‐type. We also assessed the frequency of Leu misincorporation in poly(U)‐dependent poly(Phe) synthesis. In this assay system, almost all mutants showed higher accuracy and lower activity than the wild‐type. However, K42T offered higher activity, in addition to high accuracy. Furthermore, when 14 mouse cDNA sequences were used as test templates, the protein yields of nine templates in the K42T system were 1.2–2 times higher than that of the wild‐type.
Isolation and characterization of the Xenopus HIVEP gene family
Authors: Dürr Ulrike , Henningfeld Kristine A. , Hollemann Thomas , Knöchel Walter , Pieler Tomas
Published: March 2004

The HIVEP gene family encodes for very large sequence‐specific DNA binding proteins containing multiple zinc fingers. Three mammalian paralogous genes have been identified, HIVEP1, ‐2 and ‐3, as well as the closely related Drosophila gene, Schnurri. These genes have been found to directly participate in the transcriptional regulation of a variety of genes. Mammalian HIVEP members have been implicated in signaling by TNF‐α and in the positive selection of thymocytes, while Schnurri has been shown to be an essential component of the TGF‐β signaling pathway. In this study, we describe the isolation of Xenopus HIVEP1, as well as partial cDNAs of HIVEP2 and ‐3. Analysis of the temporal and spatial expression of the XHIVEP transcripts during early embryogenesis revealed ubiquitous expression of the transcripts. Assays using Xenopus oocytes mapped XHIVEP1 domains that are responsible for nuclear export and import activity. The DNA binding specificity of XHIVEP was characterized using a PCR‐mediated selection and gel mobility shift assays.
Determination by electrospray mass spectrometry and 1H‐NMR spectroscopy of primary structures of variously fucosylated neutral oligosaccharides based on the iso‐lacto‐N‐octaose core
Authors: Kogelberg Heide , Piskarev Vladimir E. , Zhang Yibing , Lawson Alexander M. , Chai Wengang
Published: March 2004

We have isolated a nonfucosylated and three variously fucosylated neutral oligosaccharides from human milk that are based on the iso‐lacto‐N‐octaose core. Their structures were characterized by the combined use of electrospray mass spectrometry (ES‐MS) and NMR spectroscopy. The branching pattern and blood group‐related Lewis determinants, together with partial sequences and linkages of these oligosaccharides, were initially elucidated by high‐sensitivity ES‐MS/MS analysis, and then their full structure assignment was completed by methylation analysis and 1H‐NMR. Three new structures were identified. The nonfucosylated iso‐lacto‐N‐octaose, Galβ1–3GlcNAcβ1–3Galβ1–4GlcNAcβ1–6[Galβ1–3GlcNAcβ1–3]Galβ1–4Glc, has not previously been reported as an individual oligosaccharide. The monofucosylated and trifucosylated iso‐lacto‐N‐octaose, Galβ1–3GlcNAcβ1–3Galβ1–4(Fucα1–3) GlcNAcβ1–6[Galβ1–3GlcNAcβ1–3]Galβ1–4Glc and Galβ1–3(Fucα1–4)GlcNAcβ1–3Galβ1–4(Fucα1–3)GlcNAcβ1–6[Galβ1–3(Fucα1–4)GlcNAcβ1–3]Galβ1–4Glc, both containing an internal Lex epitope, are also novel structures.
The PAS fold
Authors: Hefti Marco H. , Françoijs Kees‐Jan , de Vries Sacco C. , Dixon Ray , Vervoort Jacques
Published: March 2004

In the postgenomic era it is essential that protein sequences are annotated correctly in order to help in the assignment of their putative functions. Over 1300 proteins in current protein sequence databases are predicted to contain a PAS domain based upon amino acid sequence alignments. One of the problems with the current annotation of the PAS domain is that this domain exhibits limited similarity at the amino acid sequence level. It is therefore essential, when using proteins with low‐sequence similarities, to apply profile hidden Markov model searches for the PAS domain‐containing proteins, as for the PFAM database. From recent 3D X‐ray and NMR structures, however, PAS domains appear to have a conserved 3D fold as shown here by structural alignment of the six representative 3D‐structures from the PDB database. Large‐scale modelling of the PAS sequences from the PFAM database against the 3D‐structures of these six structural prototypes was performed. All 3D models generated (> 5700) were evaluated using prosaii. We conclude from our large‐scale modelling studies that the PAS and PAC motifs (which are separately defined in the PFAM database) are directly linked and that these two motifs form the PAS fold. The existing subdivision in PAS and PAC motifs, as used by the PFAM and SMART databases, appears to be caused by major differences in sequences in the region connecting these two motifs. This region, as has been shown by Gardner and coworkers for human PAS kinase (Amezcua, C.A., Harper, S.M., Rutter, J. & Gardner, K.H. (2002) Structure10, 1349–1361, [1]), is very flexible and adopts different conformations depending on the bound ligand. Some PAS sequences present in the PFAM database did not produce a good structural model, even after realignment using a structure‐based alignment method, suggesting that these representatives are unlikely to have a fold resembling any of the structural prototypes of the PAS domain superfamily.
Further insights into the assembly of the yeast cytochrome bc1 complex based on analysis of single and double deletion mutants lacking supernumerary subunits and cytochrome b
Authors: Zara Vincenzo , Palmisano Ilaria , Conte Laura , Trumpower Bernard L.
Published: March 2004

The cytochrome bc1 complex of the yeast Saccharomyces cerevisiae is composed of 10 different subunits that are assembled as a symmetrical dimer in the inner mitochondrial membrane. Three of the subunits contain redox centers and participate in catalysis, whereas little is known about the function of the seven supernumerary subunits. To gain further insight into the function of the supernumerary subunits in the assembly process, we have examined the subunit composition of mitochondrial membranes isolated from yeast mutants in which the genes for supernumerary subunits and cytochrome b were deleted and from yeast mutants containing double deletions of supernumerary subunits. Deletion of any one of the genes encoding cytochrome b, subunit 7 or subunit 8 caused the loss of the other two subunits. This is consistent with the crystal structure of the cytochrome bc1 complex that shows that these three subunits comprise its core, around which the remaining subunits are assembled. Absence of the cytochrome b/subunit 7/subunit 8 core led to the loss of subunit 6, whereas cytochrome c1, iron–sulfur protein, core protein 1, core protein 2 and subunit 9 were still assembled in the membrane, although in reduced amounts. Parallel changes in the amounts of core protein 1 and core protein 2 in the mitochondrial membranes of all of the deletion mutants suggest that these can be assembled as a subcomplex in the mitochondrial membrane, independent of the presence of any other subunits. Likewise, evidence of interactions between subunit 6, subunit 9 and cytochrome c1 suggests that a subcomplex between these two supernumerary subunits and the cytochrome might exist.