bluuebunny/crossref_metadata_2025_split huggingface dataset:
The phoP locus influences processing and presentation of Salmonella typhimurium antigens by activated macrophages
Authors: Jo Wick Mary , Harding Clifford V. , Twesten Nicholas J. , Normark Staffan J. , Pfeifer John D. 
Published: October 2006

SummaryThe destruction and processing of bacteria by activated macrophages facilitates the presentation of antigens to T cells and thereby promotes the induction of specific immunity. The PhoP‐PhoQ regulatory system that controls the synthesis of many Salmonella proteins required for virulence and survival within macrophages is one mechanism that this particular intracellular pathogen has evolved to resist destruction. To address whether the phoP locus also influences antigen processing during the interaction of Salmonella typhimurium with macrophages, we tested the effect of phoP mutations on the processing and presentation of model antigens expressed by the bacteria. Activated macrophages processed phoP− bacteria with greater efficiency than wild‐type bacteria, as measured by the response of antigen‐specific T‐hybridoma cells; Salmonella constitutively expressing PhoP were processed even less efficiently than wild‐type Salmonella. After heat‐inactivation, however, both wild‐type and phoP− bacteria were efficiently processed. The altered processing and presentation efficiency was not due to differences in the level of antigen expressed by the bacteria or differences in the level of bacterial uptake by the macrophages. In addition, phoP‐regulated gene expression was shown to influence processing of antigen phagocytosed independently of the bacteria. Thus, phoP‐regulated gene products decrease the processing and presentation of S. typhimurium antigens, demonstrating a rote 1or this virulence locus in the inhibition of the induction of specific immunity.
Identification of a gene, pilV, required for type 4 fimbrial biogenesis in Pseudomonas aeruginosa, whose product possesses a pre‐pilin‐like leader sequence
Authors: Alm Richard A. , Mattick John S. 
Published: October 2006

SummaryType 4 fimbriae are important colonization factors in Pseudomonas aeruginosa and other pathogens that mediate attachment to epithelial cells of the host. They are also responsible for a form of translocation termed ‘twitching motility’ and are implicated in the susceptibility to fimbrial‐specific bacteriophage. Analysis of a transposon mutant which lacks functional fimbriae has identified a new gene which is required for fimbrial biogenesis. This gene, termed pilV, is located on chromosomal Spel fragment E, 2 kb downstream of the previously characterized pilSR genes involved in transcriptional activation of the fimbrial subunit gene. The pilV gene encodes a 20kDa membrane‐located protein with considerable amino‐terminal homology to the type 4 consensus pre‐pilin leader sequence, suggesting that it is processed by a leader peptidase. Site‐directed mutagenesis has shown that PilV requires such cleavage to be functional. PilV also exhibits close similarity to a group of proteins involved in extracellular protein secretion from a number of Gram‐negative bacteria, suggesting that the biogenesis of type 4 fimbriae may have a similar basis.
Characterization of a five‐cluster required for the biogenesis of type 4 fimbriae in Pseudomonas aeruginosa
Authors: Martin Paul R. , Watson Alison A. , McCaul Thomas F. , Mattick John S. 
Published: October 2006

SummaryThe opportunistic pathogen Pseudomonas aeruginosa produces type 4 fimbriae which promote adhesion to epithelial cells and are associated with a form of surface translocation called twitching motility. Transposon mutagenesis was used to identify loci required for fimbrial assembly or function by screening for mutants that lack the spreading colony morphology characteristic of twitching motility. Six mutants were isolated that contain transposon insertions upstream of the previously characterized gene pilQ. This region contains four genes: pilM‐P, which encode proteins with predicted sizes of 37.9, 22.2, 22.8 and 19.0 kDa, respectively, pilM‐P appear to form an operon and to be expressed from a promoter in the intergenic region between pilM and the divergently transcribed upstream gene ponA. PilM‐P were found to be required for fimbrial biogenesis by complementation studies using twitching motility and sensitivity to fimbrial‐specific phage as indicators of the presence of functional fimbriae. This was confirmed by electron microscopy. PilO and PilP did not have homologues in the sequence databases, but the predicted PilN amino acid sequence displayed similarity to XpsL from Xanthamonas campestris, a protein required for protein secretion. PilP contained a hydro‐phobic leader sequence characteristic of lipoproteins, while PilN and PilO have long internal hydrophobic domains which may serve to localize them to the cytoplasmic membrane. PilM has shared sequence motifs with the cell division protein FtsA from Bacillus subtilis and Escherichia coli, as well as the rod‐shapedetermining protein MreB from E. coli. These motifs are also conserved in eukaryotic actin, in which they are involved in forming an ATPase domain. Deletion mutants of pilM and pilQ displayed a dominant negative phenotype when transformed into wild‐type cells, suggesting that these genes encode proteins involved in multimeric structures.
Molecular population genetic analysis of the streptokinase gene of Streptococcus pyogenes: mosaic alleles generated by recombination
Authors: Kapur Vivek , Kanjilal Sagarika , Hamrick Marcie R. , Li Ling‐Ling , Whittam Thomas S. , Sawyer Stanley A. , Musser James M. 
Published: October 2006

SummaryTo understand the mechanisms governing molecular evolution of the streptokinase gene (skn), a 384 bp DNA fragment encoding two variable regions of the molecule was characterized in 47 isolates of Streptococcus pyogenes. The results reveal that alleles of the streptokinase gene have a mosaic structure, and provide strong evidence for intragenic recombination. Moreover, organisms that are well differentiated in overall chromosomal character have identical skn alleles, which suggests that horizontal gene transfer and recombination have participated in the evolution of this locus. No simple relationship between skn allele and serum opacity factor production or specific disease was identified. The predicted amino acid sequences of highly divergent skn alleles are strikingly similar in hydrophobicity and hydrophobicity profiles, distribution of amphipathic and flexible regions, surface probability plots, and antigenic indices, indicating that despite extensive nucleotide polymorphism in the two skn variable regions, selective pressure has constrained overall structural divergence. These results add to an important emerging theme that intragenic recombination plays a critical role in diversifying genes coding for streptococcal virulence factors.
Transcriptional regulation of the proton translocating NADH dehydrogenase (nuoA‐N) of Escherichia coli by electron acceptors, electron donors and gene regulators
Authors: Bongaerts Johannes , Zoske Sascha , Weidner Uwe , Linden Gottfried 
Published: October 2006

SummaryThe promoter region and transcriptional regulation of the nuoA‐N gene locus encoding the proton‐translocating NADH:quinone oxidoreductase was analysed. A 560 bp intergenic region upstream of the nuo locus was followed by a gene (designated irhA for LysRhomo‐logue A) coding for a gene regulator similar to those of the LysR family. Disruption of irhA did not affect growth (respiratory or non‐respiratory) or expression of nuo significantly. Transcriptional regulation of nuo by electron acceptors, electron donors and the transcriptional regulators ArcA, FNR, NarL and NarP, and by IHF (integration host factor) was studied with protein and operon fusions containing the promoter region up to base pair ‐277 (‘nuo277’) or up to base pair ‐899 (‘nuo899’). The expression of the nuo277‐lacZ fusions was subject to ArcA‐mediated anaerobic repression and NarL(+nitrate)‐mediated anaerobic activation. FNR and IHF acted as weak repressors under anaerobic conditions. Expression of nuo899‐lacZ was stimulated during anaerobic fumarate respiration and aerobically by C4 dicarboxylates. Therefore, expression of nuo is regulated by O2 and nitrate via ArcA, NarL, FNR and IHF at sites within the ‐277 region, and by other factors including C4 dicarboxylates at a site between ‐277 and ‐899. A physiological role for the transcriptional stimulation by O2 and nitrate is suggested.
Molecular genetics of the chloramphenicol‐resistance transposon Tn4451 from Clostridium perfringens: the TnpX site‐specific recombinase excises a circular transposon molecule
Authors: Bannam Trudi L , Crellin Paul K. , Rood Julian I. 
Published: October 2006

SummaryThe chloramphenicol‐resistance transposon Tn4451 undergoes precise conjugative deletion from its parent plasmid piP401 in Clostridium perfringens and precise spontaneous excision from multicopy plasmids in Escherichia coli. The complete nucleotide sequence of the 6338 bp transposon was determined and it was found to encode six genes. Genetic analysis demonstrated that the largest Tn4451‐encoded gene, tnpX, was required for the spontaneous excision of the transposon in both E. coli and C. perfringens, since a Tn4451 derivative that lacked a functional tnpX gene was completely stable in both organisms. Because the ability of this derivative to excise was restored by providing the tnpX gene on a compatible plasmid, it was concluded that this gene encoded a trans‐acting site‐specific recombinase. Allelic exchange was used to introduce the tnpXΔ allele onto plP401 and it was shown that TnpX was also required for the conjugative excision of Tn4451 in C. perfringens. It was also shown by hybridization and polymerase chain reaction (PCR) studies that TnpX‐mediated transposon excision resulted in the formation of a circular form of the transposon. The TnpX recombinase was unique because it potentially contained the motifs of two independent site‐specific recombinase families, namely the resolvase/invertase and integrase families. Sequence analysis indicated that the resolvase/invertase domain of TnpX was likely to be involved in the excision process by catalysing the formation of a 2bp staggered nick on either side of the GA dinucleotide located at the ends of the transposon and at the junction of the circular form. The other Tn4451‐encoded genes include tnpZ, which appears to encode a second potential site‐specific recombinase. This protein has similarity to plasmid‐encoded Mob/Pre proteins, which are involved in plasmid mobilization and multimer formation. Located upstream of the tnpZ gene was a region with similarity to the site of interaction of these mobilization proteins.
Characterization of the osmotically inducible gene osmE of Escherichia coli K‐12
Authors: Gutierrez Claude , Gordia Sylvie , Bonnassie Sylvie 
Published: October 2006

Summary osmE, an osmotically inducible gene of Escherichia coli, was physically mapped on the bacterial chromosome, cloned and sequenced. osmE appeared to encode a 12 021 Da protein of unknown function, with a lipoprotein‐type signal sequence at the amino‐terminus. The osmE reading frame was confirmed by sequencing the junction of an osmE‐phoA gene fusion. osmE was demonstrated to be transcribed as a single cistron. A φ[osmEp–lac] operon fusion was constructed, and analysis of its expression demonstrated that osmE osmotic regulation probably occurs at the transcriptional level. The osmE promoter was identified by both S1 nuclease and primer extension mapping of the 5′ end of the osmE mRNA, by deletion analysis and by identification of a point mutation reducing its activity. Sequence information sufficient for expression and osmotic regulation is present on a DNA fragment extending from positions ‐37 to + 52 with respect to the osmE transcription start. Unin‐duced expression of the osmE‐lac fusion was increased in the presence of mutations in the hns and himA genes. The osmE promoter overlaps a promoter for a gene transcribed in the opposite direction, efg. Transcription from the efg promoter is only weakly affected by osmotic pressure and is independent of the presence of an intact OsmE protein.
Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype O5)
Authors: de Kievit Teresa R. , Dasgupta Tapashi , Schweizer Herbert , Lam Joseph S. 
Published: October 2006

SummaryPrevious work from our laboratory has shown that cosmid clone pFVl00, containing a 26 kb insert, is able to restore O‐antigen synthesis in serotype O5 rough mutants of Pseudomonas aeruginosa. Mobilization of pFV100 into two P. aeruginosa semi‐rough (SR) mutants, AK14O1 and rd7513, resulted in O‐antigen expression, indicating that pFV100 may contain an O‐polymerase (rfc) gene. pFV.TK6, a subclone of pFVl00 that contains a 5.6 kb chromosomal insert, was able to complement O‐antigen expression in these SR mutants. Mutagenesis of pFV.TK6 using Tn1000 exposed a 1.5 kb region that was essential for complementing O‐antigen expression in AK14O1. A 2.0 kb Xhol‐HindIII fragment, containing this region, was cloned into vector pUCP26 and the resulting plasmid called pFV.TK8. In Southern analysis of the 20 P aeruginosa serotypes using a probe generated from the 1.5 kb Xhol fragment of pFV.TK8, the rfc probe hybridized to a common fragment of the cross‐reactive O2‐O5‐O16‐O18‐O20 serogroup, suggesting that these serotypes may share a common O‐polymerase gene. In functional studies of the rfc gene, the PAOl (serotype O5) chromosomal rfc was mutated using a gene‐replacement strategy. These knockout mutants expressed the SR lipopolysaccharide (LPS) phenotype, which indicated that they were no longer producing a functional O‐polymerase enzyme. Nucleotide sequence analysis of the insert DNA of pFV.TK8 revealed one open reading frame (ORF), designated ORF48.9, which could code for a 48.9 kDa protein. In comparisons of the P. aeruginosa rfc nucleotide and amino acid sequences with DNA and protein databases, no significant homology was found. However, the deduced structure of the P. aeruginosa Rfc protein indicated that it is very hydrophobic and contains 11 putative membrane‐spanning domains. Therefore, the predicted structure is similar to that of other reported Rfc proteins. Furthermore, comparison of the amino acid composition and codon usage of the P. aeruginosa Rfc with other Rfc proteins revealed significant similarity between them.
Purification of ArcA and analysis of is specific interaction with the pfl promoter‐regulatory region
Authors: Drapal Nikola , Sawers Gary 
Published: October 2006

SummaryArcA is one of several transcription factors required for optimal anaerobic induction of the pyruvate formatelyase (pfl) operon. To aid the study at the molecular level of the interaction of ArcA with the pfl promoter‐regulatory region we developed a procedure for the isolation of ArcA. The purification of ArcA involved chromatography in heparin agarose, hydroxylapatite and Mono‐Q matrices and delivered a protein that was >95% pure. Gel retardation assays demonstrated that ArcA bound specifically to the pfl regulatory region. Three distinct ArcA–DNA complexes could be resolved depending on the ArcA concentration used. This finding suggested that either multiple ArcA‐binding sites are present in the regulatory region or that ArcA can oligomerize at one or more sites. The DNA‐binding activity of ArcA could be increased an estimated 10‐fold by prior incubation of the protein with carbamoyl phosphate, suggesting that phosphorylation activates DNA binding or oligomerisation. DNase I footprint analyses identified four sites that were protected by ArcA from cleavage. Two of these sites spanned the transcription start site and —10 regions of promoters 6 and 7, while a third site partially overlapped the characterized binding site of integration host factor (IHF). ArcA exhibited the highest affinity for a stretch of DNA located between the IHF site and the transcription start site of promoter 7. These results are congruent with the hypothesis that a higher‐order nucleoprotein complex comprising several proteins, including ArcA, is required to activate transcription from the multiple promoters of the pfl operon.
Pro‐sequence‐assisted protein folding
Authors: Eder Jörg , Fersht Alan R. 
Published: October 2006

SummaryMany proteins, including proteases and growth factors, are synthesized as precursors in the form of prepro‐proteins. Whereas the pre‐sequences usually act as signal peptides for transport, the pro‐sequences of an increasing number of these proteins have been found to be essential for the correct folding of their associated proteins. In contrast to the action of molecular chaperones, pro‐sequences appear to catalyse the protein‐folding reaction directly. The similarity between the pro‐sequence‐assisted folding mechanisms of different proteases supports the hypothesis that a common folding mechanism has developed through convergent evolution. Further, the frequent requirement of the pro‐sequences for both folding and intracellular transport or secretion suggests that these two functionalities are intimately related.
The bacterial ‘enigma’: cracking the code of cell–cell communication
Authors: Salmond G. P. C. , Bycroft B. W. , Stewart G. S. A. B. , Williams P. 
Published: October 2006

SummaryIn recent years it has become clear that the production of N‐acyl homoserine lactones (N‐AHLs) is widespread in Gram‐negative bacteria. These molecules act as diffusible chemical communication signals (bacterial pheromones) which regulate diverse physiological processes including bioluminescence, antibiotic production, piasmid conjugal transfer and synthesis of exoenzyme virulence factors in plant and animal pathogens. The paradigm for N‐AHL production is in the bioluminescence (lux) phenotype of Photobacterium fischeri (formerly classified as Vibrio fischeri) where the signalling molecule N‐(3‐oxohexanoyl)‐L‐homoserine lactone (OHHL) is synthesized by the action of the Luxl protein. OHHL is thought to bind to the LuxR protein, allowing it to act as a positive transcriptional activator in an autoinduction process that physiologically couples cell density (and growth phase) to the expression of the bioluminescence genes. Based on the growing information on Luxl and LuxR homologues in other N‐AHL‐producing bacterial species such as Erwinia carotovora, Pseudomonas aeruginosa, Yersinia enterocolitica, Agrobacterium tumefaciens and Rhizobium legumino‐sarum, it seems that analogues of the P. fischeri lux autoinducer sensing system are widely distributed in bacteria. The general physiological function of these simple chemical signalling systems appears to be the modulation of discrete and diverse metabolic processes in concert with cell density. In an evolutionary sense, the elaboration and action of these bacterial pheromones can be viewed as an example of multi‐cellularity in prokaryotic populations.
Tracheal colonization factor: a Bordetella pertussis secreted virulence determinant
Authors: Finn Theresa M. , Stevens Lisa A. 
Published: October 2006

SummaryWe report here the identification of a virulence‐associated factor, Tcf, (tracheal colonization factor), produced by strains of Bordetella pertussis but not Bordetella parapertussis or Bordetella bronchiseptica. This protein is encoded by the tcfA gene. When a strain of B. pertussis 18323 lacking this protein is used to infect mice with an aerosol challenge, the number of bacteria isolated from the tracheas is decreased 10‐foid when compared with the parent 18323. The derived amino acid sequence of tcfA predicts a 68 kDa RGD‐containing, proline‐rich protein, which after cleavage of a typical prokaryotic signal sequence would be 64 kDa. Amino acid sequence analysis demonstrates that the C‐terminal 30 kDa of this protein shows 50% identity to the 30 kDa C‐terminus of another Bordetella protein, the pertactin precursor. The N‐terminal 34 kDa region contains the three amino‐acid motif RGD and is 16.5% proline. Coupled in vitro transcription and translation analysis indicates that the tcfA gene product migrates as two bands of approximately 90 kDa. A fusion protein of the N‐terminal, 34 kDa portion of Tcf to maltose‐binding protein migrates, on SDS–PAGE, 30 kDa higher than expected from the combined molecular weights. Polyclonal antisera raised against the unique N‐terminal portion of Tcf recognizes 90 kDa and 60 kDa bands in immunoblots of whole‐cell lysates of strains of B. pertussis; it does not recognize any protein in whole‐cell lysates of B. bronchiseptica or B. parapertussis. Supernatants of cultures of B. pertussis 18323 contain the 60 kDa form of the protein. Southern blot analysis of chromosomal DNA from strains of B. bronchiseptica and B. parapertussis, using a probe derived from tcfA, shows strong hybridization only to B. pertussis DNA. Thus, Tcf appears to be a unique virulence factor of B. pertussis.
Organization of host‐inducible transcripts on the symbiotic plasmid of Rhizobium sp. NGR234
Authors: Fellay R. , Perret X. , Viprey V. , Broughton W. J. , Brenner S. 
Published: October 2006

SummaryIn a systematic approach to identify genes involved in the early steps of the legume—Rhizobium symbiosis, we studied transcription patterns of symbiotic plasmid‐borne loci. A competitive hybridization procedure was used to identify DNA restriction fragments carrying genes whose expression is enhanced by plant root exudates or by purified flavonoids. Fragments containing induced genes were then located on the physical map of the 500 kb pNGR234a. New inducible loci as well as previously described genes were identified and their time course of induction determined. After initial induction, transcription of loci such as nodABC and the host‐specificity genes nodSU decreased to undetectable levels 24 h after incubation with purified flavonoids. In contrast, expression of other loci is detectable only after several hours of induction. Surprisingly, many genes remained transcribed in the nodD1 mutant suggesting the presence of other flavonoid‐dependent activators in NGR234. The hsnl region, which is involved in host specificity, was shown to carry several inducible but independently regulated transcripts. Sequencing analysis revealed several open reading frames whose products, based on sequence similarities, may be involved in L‐fucose metabolism and its adjunction to the Nod factors.