bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hydrophobic domains affect the collagen‐binding specificity and surface polymerization as well as the virulence potential of the YadA protein of Yersinia enterocolitica
Authors: Tamm Anu , Tarkkanen Ann‐Mari , Korhonen Timo K. , Kuusela Pentti , Toivanen Paavo , Skurnik Mikael 
Published: October 2006

SummaryThe YadA surface protein of enteropathogenic Yersinia species contains two highly hydrophobic regions: one close to the amino terminal, and the other at the carboxy‐terminal end of the YadA polypeptide. To study the role of these hydrophobic regions, we constructed 66 bp deletion mutants of the yadA genes of Yersinia enterocolitica serotype O:3 strain 6471/76 (YeO3) and of O:8 strain 8081 (YeO8). The mutant proteins, YadAYeO3‐Δ83–104 and YadAYeO8‐Δ80–101, lacked 22 amino acids from the amino‐terminal hydrophobic region, formed fibrillae and were expressed on the cell surface. Bacteria expressing the mutated protein lost their auto‐agglutination potential as well as their collagen‐binding property. Binding to fibronectin and laminin was affected differently in the YeO3 and the YeO8 constructs. The deletion did not influence YadA‐mediated complement inhibition. Loss of the collagen‐binding property was associated with loss of virulence in mice. We also constructed a number of YadAYeO3 deletion mutants lacking the hydrophobic carboxy‐terminal end of the protein. Deletions ranging from 19 to 79 amino acids from the carboxy terminus affected polymerization of the YadA subunits, and also resulted in the loss of the YadA expression on the cell surface. This suggests that the carboxy terminus of YadA is involved in transport of the protein to the bacterial outer surface.
Pilus‐facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin
Authors: Virji Mumtaz , Saunders Jon R. , Sims Gail , Makepeace Katherine , Maskell Duncan , Ferguson David J. P. 
Published: October 2006

SummaryAdherence of capsulate Neisseria meningitidis to endothelial and epithelial cells is facilitated in variants that express pili. Whereas piliated variants of N. meningitidis strain C311 adhered to endothelial cells in large numbers (<150 bacteria/cell), derivatives containing specific mutations that disrupt pilE encoding the pilin subunit were both non‐piliated and failed to adhere to endothelial cells (<1 bacterium/ cell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Variants of strain C311 were obtained that expressed pilins of reduced apparent Mr and exhibited a marked increase in adherence to epithelial cells. Structural analysis of pilins from two hyper‐adherent variants and the parent strain were carried out by DNA sequencing of their pilE genes. Deduced molecular weights of pilins were considerably tower compared with their apparent Mr values on SDS‐PAGE. Hyper‐adherent pilins shared unique changes in sequence including substitution of Asn‐113 for Asp‐113 and changes from Asn‐Asp‐Thr‐Asp to Thr‐Asp‐Ala‐Lys at residues 127‐130 in mature pilin. Asn residues 113 and 127 of‘parental’pilin both form part of the typical eukaryotic N‐glycosylation motif Asn‐X‐Ser/Thr and could potentially be glycosylated post‐translationally. The presence of carbohydrate on pilin was demonstrated and when pilins were deglycosylated, their migration on SDS‐PAGE increased, supporting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pilins produced by two fully piliated variants of a second strain (MC58) differed only in that the putative glycosylation motif Asn‐60‐Asn‐61‐Thr‐62 in an adherent variant was replaced with Asp‐60‐Asn‐61‐Ser‐62 in a non‐adherent variant. Fully adherent backswitchers obtained from the non‐adherent variant always regained Asn‐60 but retained Ser‐62. We propose, therefore, that functional variations in N. meningitidis pili may be modulated in large part by primary amino acid sequence changes that ablate or create N‐linked glycosylation sites on the pilin subunit.
Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompF and ompC in Escherichia coli
Authors: Delgado Jorge , Forst Steven , Harlocker Susan , Inouye Masayori 
Published: October 2006

SummaryIn Escherichia coli the OmpR and EnvZ proteins regulate the expression of the outer membrane porin proteins OmpC and OmpF. EnvZ and OmpR belong to a family of sensor/effector protein pairs that control adaptation to a variety of environmental conditions. EnvZ acts as the sensor protein that phosphorylates OmpR, which in turn regulates porin gene expression. The level of phosphorylated OmpR appears to be a determining factor for ompC and ompF regulation. Phosphorylation of OmpR is considered to occur at one or more aspartic acid residues (Asp‐11, Asp‐12 and/or Asp‐55) that are highly conserved among the effector proteins. In this report we biochemically characterized the aspartic acid residue(s) in OmpR that were phosphorylated by EnvZ. Reduction of aspartyl phosphate residues in the amino‐terminal domain of OmpR with [3H]‐NaBH4 indicated that Asp‐55 was a primary site of modification. We further studied the role of the highly conserved aspartate residues by creating OmpR mutants having aspartate to alanine substitutions at positions 11 (D11A), 12 (D12A) and 55 (D55A). Studies of ompF and ompC expression as well as in vivo and in vitro phosphorylation experiments also demonstrated that while Asp‐55 is the primary phosphate acceptor site in OmpR, Asp‐11 may also serve as a phosphorylation site, particularly in the absence of Asp‐55.
Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method
Authors: Groenen Peter M. A. , Bunschoten Annelies E. , Soolingen Dick van , Errtbden Jan D. A. van 
Published: October 2006

Summary Mycobacterium tuberculosis complex strains contain a unique chromosomal region, which consists of multiple 36bp direct repeats (DRs), which are interspersed by unique spacers 35 to 41 bp in length. In this study we investigated the nature of the DNA polymorphism of this DR cluster by sequencing part of this region in a large number of M. tuberculosis complex strains. Two types of genetic rearrangements were observed. One type consists of the variation in one or a few discrete, contiguous DRs plus spacer sequences. This variation is probably driven by homologous recombination between adjacent or distant DRs. The other type of polymorphism is probably driven by transpositional events of the insertion sequence, IS6110, which is almost invariably present in the DR cluster of M. tuberculosis complex strains. Based on the nature of the DNA polymorphism in the DR cluster, we developed a novel method of strain differentiation, direct variable repeat polymer chain reaction (DVR‐PCR), which enables typing of individual M. tuberculosis strains in a single PCR. The method allows an excellent differentiation of epidemiologically unrelated isolates and, in principle, the DVR‐PCR allows the detection of M. tuberculosis and strain differentiation at the same time.
Viability and preliminary in vivo characterization of site directed mutants of Escherichia coli single‐stranded DNA‐binding protein
Authors: Carlini Leslie E. , Porter Ronald D. , Curth Ute , Urbanke Claus 
Published: October 2006

SummarySite‐directed mutations involving selected amino acids of Escherichia coli single‐stranded DNA‐binding protein (SSB) were tested for their in vivo functionality when introduced into a chromosomal ssb deletion strain on a plasmid. All mutants complemented the ssb deletion for viability when present on a pSC101 derivative. The generation time with ssbW54S doubled in comparison to the ssb* control, and both the ssbW54S‐ and ssbH55K‐containing strains exhibited temperature sensitivity. ssbH55K, ssbW54S, ssbW88T, and ssbH55Y (ssb‐1) strains displayed reduced survival to ultraviolet irradiation, while ssbW40T and ssbF60L strains were comparable to the ssb+ control strain. This study represents the first investigation of the in vivo properties of ssb mutations constructed for in vitro analysis of DNA binding by SSB.
Sensory rhodopsin‐controlled release of the switch factor fumarate in Halobacterium salinarium
Authors: Montrone Marco , Marwan Wolfgang , Grünberg Heidi , Mußeleck Sabine , Starostzik Christine , Oesterhelt Dieter 
Published: October 2006

Summary Halobacterium salinarium responds to blue light by reversing its swimming direction. Fumarate has been proposed as one of the molecular components of this sensory system and is involved in the switching process of the flagellar motor. In order to obtain chemical proof for this role of fumarate, cells were stimulated with a pulse of blue light and lysed by rapid mixing with distilled water. The lysate contained fumarate in free and bound form, which were separated by ultrafiltration. The fumarate concentration in the low‐molecular‐mass fraction (<5 kDa) of the lysate was assayed enzymatically and a light‐induced increase was observed. Additionally, the total cellular fumarate content decreased in response to light, indicating that fumarate was released from a cellular pool rather than being formed by de novo synthesis. The light‐induced release was not detected in a mutant defective in sensory rhodopsin‐l and ‐II. Therefore it is concluded that photoreceptor activation rather than a direct effect of light on the activity of metabolic enzymes causes fumarate release. For each photoactivated sensory rhodopsin‐II molecule at least 350 molecules of fumarate were liberated demonstrating efficient amplification. The rate of light‐induced fumarate release is at least 10‐times faster than the fumarate turnover number of the citric acid cycle which was estimated as approximately 4300 per cell and second. Therefore this metabolic process is not expected to be part of the signal transduction chain in the halobacterial cell.
A gene encoding sn‐glycerol 3‐phosphate dehydrogenase (NAD+) complements an osmosensitive mutant of Saccharomyces cerevisiae
Authors: Larsson Katrin , Ansell Ricky , Eriksson Peter , Adler Lennart 
Published: October 2006

SummaryOsmoregulatory mutants of Saccharomyces cerevisiae with a defect in their capacity to readjust the cell volume/buoyant density after osmotically induced dehydration were enriched by density gradient centrifugation. Colonies derived from cells that remained dense after dehydration were screened for sensitivity to high concentrations of NaCl and defects in their osmotically induced production and intracellular accumulation of glycerol. The isolated osg (osmosensitive gtycerol defective) mutants were recessive in heterozygous diploids and fell into four complementation groups (osg1‐osg4). The osg1‐1 mutant, described in this work, is unable to grow at low water potential and shows a decreased capacity for glycerol production and a strongly reduced activity of NAD+‐dependent sn‐glycerol 3‐phosphate dehydrogenase (GPD), an enzyme in the glycerol‐producing pathway. Complementation of the osg1‐1 salt sensitivity defect with a low copy yeast genomic library led to the cloning of GPD1, encoding an S. cerevisiae GPD consisting of 391 amino acids and sharing 47‐50% identity with GPD from other sources. Micro‐sequencing of the N‐terminus of purified S. cerevisiae GPD revealed a 20‐amino‐acid sequence that was identical to a nucleotide‐deduced amino acid sequence in GPD1, but indicated that the enzyme is produced with an N‐terminal extension that is removed from the functional enzyme. Subcellular fractionation does not indicate, however, that the putative pre‐sequence targets GPD to any organelle; the enzyme appears to be located in the cytoplasm. Chromoblot and tetrad analysis were used to position the GPD1 gene to chromosome IV, with a distance of about 18 cM from trp1.
Molecular analysis of the Lactococcus lactis subspecies lactis CNRZ270 bidirectional theta replicating lactose plasmid pUCL22
Authors: Frère Jacques , Novel Madeleine , Novel Georges 
Published: October 2006

SummarypUCL22 is the lactose protease plasmid of Lactococcus lactis ssp. lactis CNRZ270. The nucleotide sequence of its replication region Rep22 contains a non‐transcribed region, the replication origin, followed by a gene encoding a putative 388‐amino‐acid protein named Rep22A. The promoter regions of the rep22A and pC194 cat genes share strong similarities and the pUCL22 replicon exerted trans or cis negative control on the pC194 cat gene expression in L. lactis. We suggest that Rep22A binds to its own promoter as well as to the pC194 cat promoter and thus is autoregulated. We show that pUCL22 replicates mainly by a bidirectional theta mechanism in L. lactis, and is representative of a widely distributed replicon family, members of which could be co‐resident. We propose that compatibility between these closely related replicons results from minor replication protein modifications coupled with base changes in their respective binding sites, supporting the co‐existence of numerous related replicons in lactococcal strains.
Hyper‐invasive mutants define a novel Pho‐regulated invasion pathway in Escherichia coli
Authors: Sinai A. P. , Bavoil P. M. 
Published: October 2006

SummaryWe have isolated two transposon insertion mutations of the pst–phoU operon which result in the constitutive expression of the phoA gene product, alkaline phosphatase. The two mutations also render Escherichia coli invasive towards cultured HEp‐2 cells and define a novel Pho‐regulated invasion pathway. The presence of the large‘invasion’plasmid derived from an entero‐invasive E. coli (EIEC) clinical isolate in these mutants leads to enhanced invasiveness toward cultured HEp‐2 cells, a phenomenon referred to as the‘hyper‐invasive’phenotype. Transduction of a pst–phoU insertion mutation into clinical isolates of EIEC and Shigella flexneri results in constitutive PhoA expression and coupled hyper‐invasiveness in the former but not the latter. We speculate that the Pho‐regulated invasion pathway described here, while silent in bacteria grown in standard laboratory rich media, may become functional in the host when invasive bacteria encounter nutrient starvation and/or other related stress conditions.
The Cro‐like Apl repressor of coliphage 186 is required for prophage excision and binds near the phage attachment site
Authors: Dodd Ian B. , Reed Michael R. , Egan J. Barry 
Published: October 2006

SummaryThe Apl protein of the temperate coliphage 186 represses transcription of the immunity repressor gene and down‐regulates lytic transcription. It is shown here that an apl‐ mutant is competent for lytic development and establishes lysogeny normally but is defective in excision of the prophage. The Apl protein binds between the lytic and lysogenic promoters and also near the phage attachment site, suggesting that its role in excision is direct. Apl thus appears to act as an excisionase as well as a repressor. The pattern of Apl‐induced DNase I enhancements indicates that the DNA is bent by Apl. Potential Apl recognition sequences are identified; these sequences are directly repeated several times across each binding region and are spaced 10 or 11 bases apart, suggesting that Apl binds to one face of the DNA helix.
Copper uptake and resistance in bacteria
Authors: Cooksey Donald A. 
Published: October 2006

SummaryCopper ions are essential for bacteria but can cause a number of toxic cellular effects if levels of free ions are not controlled. Investigations of copper‐resistant bacteria have revealed several mechanisms, mostly plasmid‐determined, that prevent cellular uptake of high levels of free copper ions. However, these studies have also revealed that bacteria apparently have efficient chromosomally encoded systems for uptake and management of trace levels of copper. This review will explore the relationship of copper uptake systems to resistance mechanisms and the possibility that copper resistance has evolved directly through modification of chromosomal copper uptake genes.
Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila
Authors: Berger Karen H. , Isberg Ralph R. 
Published: October 2006

Summary Legionella pneumophila mutants specifically defective for intracellular replication were isolated using an intracellular thymineless death enrichment strategy. Mutants belonging to two distinct phenotypic classes were unable to grow in macrophage‐like cultured cells. One class of mutants was defective for both inhibition of phagosome–lysosome fusion and association of host cell organelles with bacteria‐containing phagosomes (‘recruitment’). Another class of mutants was defective only for organelle recruitment, suggesting that recruitment may be necessary for intracellular growth. Recombinant clones were identified that complemented the intracellular growth defects of these mutants. A single genetic locus, designated dot (for defect in organelle trafficking), restored wild‐type phenotypes for intracellular growth, organelle recruitment, and inhibition of phagosome–lysosome fusion to mutants belonging to both phenotypic classes.
Membrane topology of MaIG, an inner membrane protein from the maltose transport system of Escherichia coli
Authors: Dassa Elie , Muir Susie 
Published: October 2006

SummaryIn Escherichia coli, the binding protein‐dependent transport system for maltose and maltodextrins is composed of five proteins — LamB, MaIE, MaIF, MaIG and MaIK — located in the three layers of the bacterial envelope. Proteins MaIF and MaIG are hydrophobic inner membrane components mediating the energy‐dependent translocation of substrates into the cytoplasm. In this paper, we analyse the topology of the MaIG protein by using methods based on the properties of fusions between maIG and‘phoA, a truncated gene encoding alkaline phosphatase lacking its translation initiation and exportation signals. Fusions were obtained by using either phage λTnphoA or by constructing in vitro fusions located randomly within the maIG gene. The deduced topological model suggests that MaIG spans the membrane six times and has its amino‐ and carboxy‐termini in the cytoplasm. These results will be helpful for the interpretation of the phenotypes of mutants in maIG.
Sequence–function relationships in MalG, an inner membrane protein from the maltose transport system in Escherichia coli
Authors: Dassa Elie 
Published: October 2006

SummaryThe maIG gene encodes a hydrophobic cytoplasmic membrane protein which is required for the energy‐dependent transport of maltose and maltodextrins in Escherichia coli. The MalG protein, together with MalF and MalK proteins, forms a multimeric complex in the membrane consisting of two MalK subunits for each MalF and MalG subunit. Fifteen mutations have been isolated in malG by random linker insertion mutagenesis. Two regions essential for maltose transport have been identified. In particular, a hydro philic region containing the peptidic motif EAA—G———I‐LP, highly conserved among inner membrane proteins from binding protein‐dependent transport systems, is essential for maltose transport.The results also show that several regions of MalG are not essential for function. A region (residues 30–50) encompassing the first predicted transmembrane segment and the first periplasmic loop in MalG may be modified extensively with little effect on maltose transport and no effect on the stability and the localization of the protein. A region located at the middle of the protein (residues 153–157) is not essential for the function of the protein. A region, essential for maltodextrin utilization but not for maltose transport, has been identified near the C‐terminus of the protein.
The strongly conserved carboxyl‐terminus glycine‐methionine motif of the Escherichia coli GroEL chaperonin is dispensable
Authors: McLennan N. F. , Girshovich A. S. , Lissin N. M. , Charters Y. , Masters M. 
Published: October 2006

SummaryThe universally distributed heat‐shock proteins (HSPs) are divided into classes based on molecular weight and sequence conservation. The members of at least two of these classes, the HSP60s and the HSP70S, have chaperone activity. Most HSP60s and many HSP70s feature a striking motif at or near the carboxyl terminus which consists of a string of repeated glycine and methionine residues. We have altered the groEL gene (encoding the essential Escherichia coli HSP60 chaperonin) so that the protein produced lacks its 16 final (including nine gly, and five met) residues. This truncated product behaves like the intact protein in several in vitro tests, the only discernible difference between the two proteins being in the rate at which ATP is hydrolysed. GroELtr can substitute for GroEL in vivo although cells dependent for survival on the truncated protein survive slightly less well during the stationary phase of growth. Elevated levels of the wild‐type protein can suppress a number of temperature‐sensitive mutations; the truncated protein lacks this ability.
MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri Ipa invasins
Authors: Allaoui Abdelmounaaïm , Sansonetti Philippe J. , Parsot Claude 
Published: October 2006

SummaryThe invasive phenotype of Shigella flexneri is conferred by a 220 kb virulence plasmid, pWR100, that encodes both the Ipa proteins, which are involved in the entry process, and factors which are required for the export and correct localization of the Ipa proteins. We have characterized the mxiD gene, whose expression, like that of the ipa operon, is regulated by temperature. After inactivation of mxiD, the mutant strain was unable to invade HeLa cells and to provoke keratoconjunctivitis in guinea‐pigs. Analysis of culture supernatants indicated that wild‐type S. flexneri secretes about nine polypeptides and that secretion of several of these, including IpaA, IpaB, and IpaC, is abolished in the mxiD mutant. Examination of the membrane proteins of the wild‐type and mxiD strains suggested that MxiD is an outer membrane protein. Amino acid sequence comparison revealed that MxiD is homologous to the YscC protein of Yersinia enterocolitica and to the C‐terminal region of the PulD protein of Klebsiella pneumoniae. Both YscC and PulD are involved in extracellular protein secretion. These results indicate that MxiD is an essential component of the Ipa secretion apparatus.
The essential Escherichia coli msbA gene, a multicopy suppressor of null mutations in the htrB gene, is related to the universally conserved family of ATP‐dependent translocators
Authors: Karow Margaret , Georgopoulos Costa 
Published: October 2006

SummaryWe report the characterization of the msbA gene, isolated as a multicopy suppressor of the HtrB temperature‐sensitive phenotype. The msbA gene maps to 20.5 min on the Escherichia coli genetic map and encodes a protein with an estimated molecular mass of 64460 Da, with the properties of an integral membrane protein. The amino acid sequence of MsbA is very similar to those of the family of ATP‐dependent translocators, which includes the haemolysin B protein of E. coli and the mammalian multidrug resistance (MDR) proteins. Mutational analysis of msbA indicates that it may form an operon with a downstream gene, orfE, and that both of these genes are essential for bacterial viability under all growth conditions tested.