bluuebunny/crossref_metadata_2025_split huggingface dataset:
C‐terminal truncation of the transcriptional activator encoded by areA in Aspergillus nidulans results in both loss‐of‐function and gain‐of‐function phenotypes
Authors: Stankovich M. , Platt A. , Caddick M. X. , Langdon T. , Shaffer P. M. , Arst H. N. 
Published: October 2006

SummaryMutations truncating as many as 143 C‐terminal residues from the transcriptional activator encoded by the areA gene, mediating nitrogen metabolite repression in Aspergillus nidulans, do not significantly reduce the ability of the areA product to activate expression of most genes under areA control. Such mutations can even have a gain‐of‐function, derepressed phenotype, consistent with a critical role for this region in modulating the activity of the areA protein. However, expression of a few genes under areA control is substantially impaired by such C‐terminal truncations, indicating that regions of an activator protein can play differing roles in the control of different structural genes. This underlines the advantages of being able to monitor effects of areA mutations on expression of large numbers of structural genes. Additionally, it is shown that truncation of as many as 153 C‐terminal residues, virtually all amino acids C‐terminal to the DNA‐binding region, is compatible with retention of some areA function.
Cloning and molecular characterization of a gene involved in Salmonella adherence and invasion of cultured epithelial cells
Authors: Altmeyer Ralf M. , McNern John K. , Bossio J. C. , Rosenshine Ilan , Finlay B. Brett , Galán Jorge E. 
Published: October 2006

SummaryOur laboratories have independently identified a gene in Salmonella choleraesuis and Salmonella typhimurium that is necessary for efficient adherence and entry of these organisms into cultured epithelial cells. Introduction of a mutated gene into several Salmonella strains belonging to different serotypes rendered these organisms deficient for adherence and invasion of cultured cells. This effect was most pronounced in the host‐adapted serotypes Salmonella gallinarum, S. choleraesuis, and Salmonella typhi. The nucleotide sequence of this gene, which we have termed invH, encodes a predicted 147‐amino‐acid polypeptide containing a signal sequence. The InvH predicted polypeptide is highly conserved in S. typhimurium and S. choleraesuis, differing at only three residues. The invH gene was expressed in Escherichia coli using a T7 RNA polymerase expression system and a polypeptide of ∼16000 molecular weight was observed, in agreement with the predicted size of its gene product. Upon fractionation, the expressed polypeptide was localized in the bacterial membrane fraction. Southern and colony hybridization analyses indicated that the invH gene is present in all Salmonella strains tested (91 strains belonging to 37 serotypes) with the exception of strains of Salmonella arizonae. No homologous sequences were detected in Yersinia, Shigella, Proteus, and several strains of enteroinvasive and enteropathogenic E. coli. Downstream from the S. choleraesuis (but not S. typhimurium) invH gene, a region with extensive homology to the insertion sequence IS3 was detected.
A collagen receptor on Staphylococcus aureus strains isolated from patients with septic arthritis mediates adhesion to cartilage
Authors: Switalski Lech M. , Patti Joseph M. , Butcher Wade , Gristina Anthony G. , Speziale Pietro , Höök Magnus 
Published: October 2006

Summary Staphylococcus aureus strains isolated from patients with septic arthritis or osteomyelitis possess a collagen receptor present in two forms, which contains either two or three copies of a 187‐amino‐acid repeat motif. Collagen receptor‐positive strains adhered to both collagen substrata and cartilage in a time‐dependent process. Collagen receptor‐specific antibodies blocked bacterial adherence, as did preincubation of the substrate with a recombinant form of the receptor protein. Furthermore, polystyrene beads coated with the collagen receptor bound collagen and attached to cartilage. Taken together, these results suggest that the collagen receptor is both necessary and sufficient to mediate bacterial adherence to cartilage in a process that constitutes an important part of the pathogenic mechanism in septic arthritis.
Signal transduction between the two regulatory components involved in the regulation of the kdpABC operon in Escherichia coli: Phosphorylation‐dependent functioning of the positive regulator, KdpE
Authors: Nakashima K. , Sugiura A. , Kanamaru K. , Mizuno T. 
Published: October 2006

SummaryThe proteins KdpD and KdpE are crucial to the osmotic regulation of the kdpABC operon that is responsible for the high‐affinity K+ ion transport system in Escherichia coli. We demonstrated previously that the response regulator, KdpE, is capable of undergoing Phosphorylation mediated by the sensory protein kinase, KdpD. In this study, we obtained biochemical evidence supporting the view that when KdpE is phosphorylated, it takes on an active form that exhibits relatively high affinity for the kdpABC promoter, which in turn results in activation of the kdpABC operon. It was also suggested that the central hydrophobic domain of KdpD, which is conceivably responsible for membrane anchoring of this protein, plays a role in the signalling mechanism underlying KdpE Phosphorylation in response to hyperosmotic stress.
Identification and characterization of the exbB, exbD and tonB genes of Pseudomonas putida WCS358: their involvement in ferric‐pseudobactin transport
Authors: Bitter Wilbert , Tommassen Jan , Weisbeek Peter J. 
Published: October 2006

SummaryCatechol‐cephalosporins are siderophore‐like antibiotics which are taken up by cells of Pseudomonas putida WCS358 via the ferric‐siderophore transport pathway. Mutants of strain WCS358 were isolated that are resistant to high concentrations of these antibiotics. These mutants failed to grow under iron‐limiting conditions, and could not utilize different ferric‐siderophores. The mutants fall in three complementation groups. The nucleotide sequence determination identified three contiguous open reading frames, which were homologous to the exbB, exbD and tonB genes of Escherichia coli respectively. The deduced amino acid sequence of P. putida ExbB showed 58.6% homology with its E. coli homologue, but, unlike the E. coli protein, it has a N‐terminal extension of 91 amino acids. The ExbD proteins are 64.8% homologous, whereas the TonB proteins only show 27.7% homology. The P. putida exbB gene could complement an E. coli exbB mutation, but the TonB proteins were not interchangeable between the species. It is concluded that P. putida WCS358 contains an energy‐coupling system between the membranes for active transport across the outer membrane, which is comprised of a TonB‐like energy‐transducing protein and two accessory proteins. This system is similar to, but not completely compatible with, the E. coli system.
Topology of PhoE porin: the ‘eyelet’ region
Authors: Struyvé Marlies , Visser Jan , Adriaanse Henriëtte , Benz Roland , Tommassen Jan 
Published: October 2006

SummaryA model for the topology of the PhoE porin has been proposed according to which the polypeptide traverses the outer membrane sixteen times mostly as amphipathic β‐sheets, thereby exposing eight loops at the cell surface. Until now, no evidence has been obtained for the surface exposure of the third loop. Recently, the structure of porin of Rhodobacter capsulatus has been determined. The proposed model of PhoE is very similar to the structure of the R capsulatus porin, which has an ‘eyelet’ region, extending into the interior of the pore. The proposed third external loop of PhoE might form a similar ‘eyelet’ region. To determine the location of the predicted third external loop of PhoE, multiple copies of an oligonucleotide linker encoding an antigenic determinant of VP1 protein of foot‐and‐mouth disease virus (FMDV) were inserted. All hybrid proteins were properly inserted in the outer membrane. The monoclonal antibody MA11, directed against the linear FMDV epitope, was able to bind only to intact cells expressing a hybrid PhoE protein with at least three copies of the FMDV epitope present. Antibiotic sensitivity tests and single‐channel conductance measurements revealed that the insertions influenced the channel size. These results are consistent with a location of the third loop of PhoE within the pore channel.
Involvement of lipopolysaccharide in the secretion of Escherichia coli α haemolysin and Erwinia chrysanthemi proteases
Authors: Wandersman Cécile , Létoffé Sylvie 
Published: October 2006

SummaryThe presence of the α‐haemolysin secretion genes sensitizes Escherichia coli to vancomycin, a glycopeptide antibiotic that is normally excluded from the Gram‐negative envelope (owing to its large size) (Mr 1400). The selection of vancomycin mutants in strains carrying such genes was found to be a very powerful method for selecting non‐haemolytic mutants. In this way, mutations in the known secretion genes, hlyB, hlyD and tolC, were obtained. However additional mutations mapped in genes rfaH and galU which are required for lipopolysaccharide (LPS) biosynthesis. Mutations in rfaH and galU strongly reduced α‐haemolysin secretion as weli as the secretion of Erwinia chrysanthemi proteases in E. coli without affecting their synthesis. These mutations markedly lowered the content of TolC protein, required for haemolysin secretion and also of the PrtF protein necessary for protease secretion. These results raise the possibility that LPS is involved in the correct incorporation of the TolC and PrtF proteins into the cell envelope.
Definition of nitrite and nitrate response elements at the anaerobically inducible Escherichia coli nirB promoter: interactions between FNR and NarL
Authors: Tyson K. L. , Bell A. I. , Cole J. A. , Busby S. J. W. 
Published: October 2006

SummaryTranscription initiation at the Escherichia coli nirB promoter is induced by anaerobic growth and further increased by the presence of nitrite or nitrate in the growth medium. Expression from this promoter is totally dependent on the transcription factor, FNR, which binds between positions −52 and −30 upstream of the transcription startsite. The 20 base pairs from position −79 to −60 contain an inverted repeat of two 10‐base sequence elements that are related to sequences at the NarL‐binding site at the E. coli narG promoter. Comparison of these, and sequence elements at other promoters regulated by NarL, suggests a consensus NarL‐binding sequence. Mutations in the putative NarL‐binding site at the nirB promoter decrease FNR‐dependent anaerobic induction, suggesting that NarL acts as a helper to FNR during transcription activation. These mutations also suppress induction by nitrite: single mutations at symmetry‐related positions have similar effects, whilst double mutations have more severe effects, probably because two NarL subunits bind to the inverted repeat. Disruption of narL decreases nitrite induction of the nirB promoter whilst not suppressing induction by nitrate, suggesting that there may be a second nitrate‐responsive factor. Nitrate induction was, however, suppressed by double mutations at symmetry‐related positions in the NarL‐binding site, suggesting that this putative second factor may bind to sequences similar to those recognized by NarL.
Use of an ordered cosmid library to deduce the genomic organization of Mycobacterium leprae
Authors: Eiglmeier K. , Honoré N. , Woods S. A. , Caudron B. , Cole S. T. 
Published: October 2006

SummaryIn an attempt to unify the genetic and biological research on Mycobacterium leprae, the aetiological agent of leprosy, a cosmid library was constructed and then ordered by a combination of fingerprinting and hybridization techniques. The genome of M. leprae is represented by four contigs of overlapping clones which, together, account for nearly 2.B Mb of DNA. Several arguments suggest that the gaps between the contigs are small in size and that virtually complete coverage of the chromosome has been obtained. All of the cloned M. leprae genes have been positioned on the contig maps together with the 29 copies of the dispersed repetitive element, RLEP. These have been classified into four groups on the basis of differences in their organization. Several key housekeeping genes were identified and mapped by hybridization with heterologous probes, and the current genome map of this uncultivable pathogen comprises 72 loci.
Nucleotide sequence of the first cosmid from the Mycobacterium leprae genome project: structure and function of the Rif‐Str regions
Authors: Honoré N. , Bergh S. , Chanteau S. , Doucet‐Populaire F. , Eiglmeir K. , Garnier T. , Georges C. , Launois P. , Limpaiboon T. , Newton S. , Niang K. , del Portillo P. , Ramesh G. R. , Reddi P. , Ridel P. R. , Sittisombut N. , Wu‐Hunte S. , Cole S. T. 
Published: October 2006

SummaryThe nucleotide sequence of cosmid B1790, carrying the Rif‐Str regions of the Mycobacterium leprae chromosome, has been determined. Twelve open reading frames were identified in the 36716bp sequence, representing 40% of the coding capacity. Five ribosomal proteins, two elongation factors and the β and β'subunits of RNA polymerase have been characterized and two novel genes were found. One of these encodes a member of the so‐called ABC family of ATP‐binding proteins while the other appears to encode an enzyme involved in repairing genomic lesions caused by free radicals. This finding may well be significant as M. leprae, an intracellular pathogen, lives within macrophages.
The general amino acid control regulates MET4, which encodes a methionine‐pathway‐specific transcriptional activator of Saccharomyces cerevisiae
Authors: Mountain Harry A. , Byström Anders S. , Korch Christopher 
Published: October 2006

SummaryA met4 mutant of Saccharomyces cerevisiae was unable to transcribe a number of genes encoding enzymes of the methionine biosynthetic pathway. The sequence of the cloned MET4 gene allowed the previously sequenced flanking LEU4 and POL1 genes to be linked to MET4 into a 10 327 bp contiguous region of chromosome XIV. From the sequence and mapping of the transcriptional start points, MET4 is predicted to encode a protein of 634 amino acids (as opposed to 666 amino acids published by others) with a leucine zipper domain at the C‐terminus, preceded by both acidic and basic regions. Thus, MET4 belongs to the family of basic leucine zipper trans‐activator proteins. Disruption of MET4 resulted in methionine auxotrophy with no other phenotype. Transcriptional studies showed that MET4 was regulated by the general amino acid control and hence by another bZIP protein encoded by GCN4. GCN4 binding sequences are present between the divergently transcribed MET4 and LEU4 genes. Over‐expression of MET4 resulted in leaky expression from the otherwise tightly regulated MET3 promoter under its control. The presence of consensus sequences for other potential regulatory elements in the MET4 promoter suggests a complex regulation of this gene.
Basement membrane carbohydrate as a target for bacterial adhesion: binding of type I fimbriae of Salmonella enterica and Escherichia coli to laminin
Authors: Kukkonen Maini , Raunio Tiina , Virkola Ritva , Lähteenmäki Kaarina , Mäkelä P. Helena , Klemm Per , Clegg Steven , Korhonen Timo K. 
Published: October 2006

SummaryAdherence of type‐1‐fimbriate Salmonella enterica and Escherichia coli to immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type‐1‐fimbriate strain SH401 of S. enterica serovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non‐fimbriate strain of S. enterica serovar Typhimurium. A specific and mannoside‐inhibitable adhesion to laminin was exhibited by the recombinant E. coli strain HB101(plSF101) possessing fim genes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae‐coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type‐1 ‐fimbriate E. coli strain 2131 and the recombinant strain E. coli HB101(pPKL4) expressing the cloned type‐1‐fimbriae genes of E. coli. Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with the fimH mutant E. coli HB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type‐1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(plSF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type‐1 fimbriae of S. enterica and E. coli bind to oMgomannoside chains of the lamjnjn network in basement membranes.
Molecular cloning and characterization of the genetic determinants that express the complete Shigella serotype D (Shigella sonnei) lipopolysaccharide in heterologous live attenuated vaccine strains
Authors: Viret Jean‐François , Cryz Stanley J. , Lang Alois B. , Favre Didier 
Published: October 2006

SummaryThe genetic determinants for the complete Shigella sonnei lipopolysaccharide (LPS) have been cloned, characterized by restriction mapping, and expressed in heterologous genetic backgrounds, including Salmonella typhi and Vibrio cholerae live attenuated vaccine strains. The rfb/rfc locus encoding the polymerized serotype‐specific O polysaccharide was mapped within 23 kb of DNA isolated from S. sonnei virulence plasmid pWR105. A highly similar chromosomal DNA sequence was identified by Southern hybridization analysis in Plesiomonas shigelloides known to have the same O serotype specificity as S. sonnei. Expression studies of the rfb/rfc locus have shown that S. sonnei. O polysaccharide is covalently bound to LPS cores of both the K‐12 and RI types, but neither to Salmonella (Ra‐type) nor to V. cholerae O1 cores. In order to express a compatible core structure in the latter organisms, chromosomal rfa loci encoding R1‐type LPS were isolated from both an Escherichia coli R1 strain (rfaR1) and from S. sonnei (rfdsonnei). Restriction mapping and functional analysis of cloned DNA allowed us to localize the rfaR1 locus and to orient it with respect to the neighbouring cysE chromosomal marker. A high degree of sequence similarity was found at the DNA level between rfa loci of enterobacterial species characterized by Ri‐type LPS. Co‐expression studies involving S. sonnei rfb/rfc and rfa loci propagated on compatible plasmids have shown that, at most, 13 to 14 kb of r/api DNA are required for the expression of complete phase‐l‐like S. sonnei LPS in E. coli K‐12 and S. typhi, whereas an adjacent region of about 3.5 kb is needed in the more stringent host, V. cholerae, S. sonnei O antigen expressed in a V. eholerae recombinant vaccine strain is present on the cell surface in a form suitable for the induction of a specific antibody response in vaccinated rabbits.
Effects of tRNALeu1 overproduction in Escherichia coli
Authors: Wahab Samir Z. , Rowley Karla O. , Holmes W. Michael 
Published: October 2006

SummaryStrains of Escherichia coli have been produced which express very high levels of the tRNAleu1 isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNALeu1 gene. Most transformants grew very slowly and exhibited a 15‐fold increase in cellular concentrations of tRNALeu1 As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNALeu1. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two‐ and threefold. Analyses of leucyl tRNA isolated from these slow‐growing strains showed that at least 90% of the detectable tRNALeu1 was hypomodified as judged by altered mobility on RPC‐5 reverse‐phase columns, and by specific modification assays using tRNA(m1G)‐methyltransferase and pseudo‐uridylate synthetase. Analysis of fast‐growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild‐type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNALeu1 operon under the control of the T7 promoter was prepared which, when induced, produced six‐ to sevenfold increases in tRNALeu1 levels. This level of tRNALeu1 titrated the modification system as judged by RPC‐5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein‐synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.
Mutagenesis and functional analysis of the Escherichia coli tRNALeu1 promoter
Authors: Bauer Brian F. , Elford Roberta M. , Holmes W. Michael 
Published: October 2006

SummaryThe leuV promoter which produces tRNALeu1 in Escherichia coli has been extensively mutagenized in order to determine the effects of altered sequences on promoter efficiency (strength) and on growth‐rate‐dependent regulation (GDR). Each mutant promoter was ligated with a β‐galactosidase reporter gene into the chromosome of a host cell by phage lambda lysogenization. Reporter gene activities were measured for cells growing in selected media at various growth rates. Sequences which flank the −10 consensus region, when altered, caused remarkable up‐promoter effects, increasing efficiency in some cases almost 10‐fold. One up mutation which had five successive T residues in the‘discriminator’region completely abolished GDR, whereas several mutations with single base changes in the discriminator had little or no effect on GDR. Another mutation which changed one base in the −35 region to bring it to consensus increased promoter strength 18‐fold and sharply reduced GDR. Chimaeric promoters in which segments of leuV were replaced by segments of the his operon showed that only when the discriminator of leuV is replaced by the his discriminator was GDR‐disturbed. AM upstream sequences which were replaced by his sequences had little effect on GDR. Overall, there appeared to be little correlation between promoter efficiency and GDR.