bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evolutionary relationship of uptake systems for biopolymers in Escherichia coli: cross‐complementation between the TonB‐ExbB‐ExbD and the TolA‐TolQ‐TolR proteins
Authors: Braun V. , Herrmann C. 
Published: October 2006

Summary Escherichia coli possesses two energy‐coupled import systems through which substances of low concentration and of a size too large to permit diffusion through the porins are translocated across the outer membrane. Group B colicins, ferric siderophores and vitamin B12 are taken up via the TonB‐ExbB‐ExbD, group A colicins via the TolA‐TolQ‐TolR system. Cross‐complementation between the two systems was demonstrated in that tolQ tolR mutants transformed with plasmids carrying exbB exbD became sensitive to group A colicins, and exbB exbD mutants transformed with plasmid‐encoded tolQ tolR became sensitive to group B colicins. TolQ‐TolR interacted through TonB, and ExbB‐ExbD interacted through TolA with the outer membrane receptors and colicins. Activity of ExbB ExbD via TolA was higher in cells laciting TonB, and activity of TolQ TolR via TonB was increased when TolA was missing. The very distinct TolA and TonB proteins mediate exclusive interaction with group A and group B receptors, respectively. ExbB‐TolR and ExbD‐TolQ mixtures showed little if any complementation of exbB exbD and tolQ tolR mutants indicating coevolution of ExbB with ExbD and TolQ with ToIR. Sequence homology and mutual functional substitution of ExbB‐ExbD and TolQ‐TolR suggest the evolution of the two import systems from a single import system.
Topology of the PhoR protein of Escherichia coli and functional analysis of internal deletion mutants
Authors: Scholten Monica , Tommassen Jan 
Published: October 2006

SummaryThe PhoR protein of Escherichia coli K‐12 belongs to a family of structurally related sensor‐kinases that regulate responses to environmental stimuli. These proteins are often located in the inner membrane with two membrane‐spanning segments that are separated by a periplasmic domain, which is supposed to sense the environmental stimuli. However, the hydrophobicity plot of PhoR suggests a somewhat different topology in which a large periplasmic domain is lacking and an extended cytoplasmic domain is present besides the kinase domain. In protease‐accessibility experiments and by using phoR‐phoA gene fusions, the topology of PhoR was investigated and the absence of a large periplasmic domain was confirmed. Furthermore, the function of the extended cytoptasmic domain was studied by creating internal deletions. The mutations in this domain resulted in a constitutive expression of the pho regulon, indicating that the mutant PhoR proteins are locked in their kinase function. We propose that this extended cytoplasmic domain functions by sensing an internal signal that represses the kinase function of the PhoR protein.
Mycobacterium smegmatis RNA polymerase: DNA supercoiling, action of rifampicin and mechanism of rifampicin resistance
Authors: Levin Margaret E. , Hatfull Graham F. 
Published: October 2006

SummaryWe have isolated RNA polymerase from Mycobacterium smegmatis and established conditions for specific transcription initiation in vitro. The M. smegmatis enzyme has a strong dependence on supercoiling of the DNA substrate for transcription from mycobacterial promoters. We also show that RNA polymerase is the target for rifampicin, and that this antibiotic specifically inhibits the transition from synthesis of short oligoribonucleotides to full‐length transcripts. RNA polymerase isolated from a rifampicin‐resistant mutant of M. smegmatis is less sensitive to rifampicin in vitro, confirming that one mechanism of rifampicin resistance in mycobacteria is through alteration of RNA polymerase. This in vitro transcription system provides a simple method for the characterization of gene expression in mycobacteria including the pathogens Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium leprae. It also provides a system for evaluating potential anti‐mycobacterial drugs.
Core RNA polymerase assists binding of the transcription factor σ;54 to promoter DNA
Authors: Cannon Wendy , Claverie‐Martin Felix , Austin Sara , Buck Martin 
Published: October 2006

SummaryThe Sigma subunit of bacterial RNA polymerase Is necessary for the specific binding of RNA polymerase holoenzyme to promoter DNA. Promoter complexes which form with holoenzyme containing σ;54 remain as closed complexes unless they are activated by one class of enhancer binding protein. The σ;54 transcription factor can bind specifically to certain promoter sites in the absence of the core RNA polymerase subunits. This property has allowed demonstration of a new role for core polymerase in transcription, namely that it assists the binding of σ;54 to promoter DNA, An altered form of σ;54 with a deletion within the amino‐terminal region showed increased affinity for specific DNA‐binding sites. Although able to complex with core RNA polymerase the mutant σ;54 failed to respond to core polymerase in the manner characteristic of the wild‐type σ;54 by altering its footprint. This result indicates that σ;54 has a latent DNA‐binding activity which is revealed by core RNA polymerase, and possibly involves a change in σ;54 conformation. Promoter complexes which formed with σ;54‐holoenzyme appeared to be qualitatively different, depending upon the target promoter sequence, suggesting that different activatable complexes form at different promoter sequences.
Molecular analysis of three major wall‐associated proteins of Bacillus subtilis 168: evidence for processing of the product of a gene encoding a 258 kDa precursor two‐domain ligand‐binding protein
Authors: Foster Simon J. 
Published: October 2006

SummaryAntisera raised to a 109 kDa wall‐associated protein (WAP) of Bacillus subtilis 168 cross‐reacts with two other WAPs of 220 and 58 kDa. The structural gene for the 109 kDa WAP (designated wapA) was cloned, sequenced, mapped at around 340° on the B. subtilis 168 chromosome and found to encode a precursor of all three wall‐bound forms (2334 amino acids and 258 329 Da). The protein has two ligand‐binding domains; the N‐terminal domain has three direct repeats of 102 residues with 40% identity, which are responsible for wall binding. The C‐terminal domain consists of two blocks of residues with a conserved motif repeated a total of 31 times. The motif consensus sequence GXXXX(Y,F)XYDXXG is almost identical to that of the Escherichia coli rearrangement hot spot family and shows similarity to a carbohydrate‐binding motif of a number of Gram‐positive secreted proteins. A mutant insertionally inactivated in the wapA gene had no distinguishable phenotype apart from lacking the three WAPs. The possible role of WAPA and its two‐domain relationship with other ligand‐binding proteins is discussed.
Yersinia pestis pH 6 antigen forms fimbriae and is induced by intracellular association with macrophages
Authors: Lindler Luther E. , Tall Ben D. 
Published: October 2006

SummaryAbility to express pH 6 antigen (Ag) is necessary for full virulence of Yersinia pestis; however, the function of the Ag in pathogenesis remains unclear. We determined the nucleotide sequence of a 4232 bp region of Y. pestis DNA which encoded the pH 6 Ag structural gene (psaA) and accessory loci necessary for Ag synthesis. Protein sequences encoded by the Y. pestis DNA were similar to accessory proteins which function in the biosynthesis of Escherichia coli fimbriae Pap, K88, K99 and CS3 as well as the molecular chaperone for the Y. pestis capsule protein. Electron microscopy and immunogold labelling studies revealed that pH 6 Ag expressing E coU or Yersinia produced flexible‘fibrillar’organelles composed of individual linear strands, multiple strand bundles or wiry aggregates of PsaA. Y. pestis associated with the murine macrophage‐like cell line, RAW264.7, expressed pH 6 Ag in an intracellular acidification‐dependent manner. Together with an earlier study showing that a Y. pestis psaA mutant was reduced in virulence, these results demonstrate that the expression of fimbriae which are induced in host macrophages is involved in plague pathogenesis.
Sequencing of a Clostridium thermocellum gene (cipA) encoding the cellulosomal SL‐protein reveals an unusual degree of internal homology
Authors: Gerngross U.T. , Romaniec M.P.M. , Kobayashi T. , Huskisson N. S. , Demain A. L. 
Published: October 2006

SummaryIt is known that two proteins of the cellulosomal complex of Clostridium thermocellum (SL and Ss) together degrade crystalline cellulose. SL is a glycoprotein of 210000Da which enhances the binding to cellulose and the activity of Ss, an endoglucanase of 83000 Da. We have previously reported the cloning of a DNA fragment encoding the N‐terminal end of the SL protein using antibodies raised against the native protein. A chromosomal walking approach using an EcoRI and a BamHI‐Sau3A gene library allowed us to isolate the C‐terminal end of the gene. Sequencing of both fragments revealed the existence of a leader peptide as has been found in cellulases of the same organism. This leader sequence is followed by a stretch of 14 amino acids that is identical to the N‐terminal amino acid sequence of the native secreted protein. The open reading frame (ORF) of this gene encodes a protein of 196800 Da and is followed by a hairpin loop that could be involved in transcription termination. Within the open reading frame (ORF), we found nine internal repeated elements (IREs) of about 500 nucleotides each. Seven of these sequences displayed 98–100% homology and were located adjacent to each other within the structural gene without intervening regions. The remaining two, located on the N‐terminal end of the gene, showed a significantly lower homology. Bearing in mind the inherent instability of reiterated regions, we confirmed the authenticity of our clones by Southern blot analysis using chromosomal C. thermocellum DNA and ruled out the possibility of rearrangements during the cloning and sequencing process. The sequenced gene is designated clpA and the encoded SL protein CipA.
Contributions of promoter context and structure to regulated expression of the F plasmid traY promoter in Escherichia coli K‐12
Authors: Gaudin Helen M. , Silverman Philip M. 
Published: October 2006

SummaryExpression of the F plasmid traY promoter in vivo requires both host (E. coli) and plasmid encoded proteins. As judged by transcript size and primer extension analyses, the F plasmid traY promoter was utilized in vitro by purified E. coliσ;70 RNA polymerase in the absence of other proteins. However, in vitro transcription required supercoiled templates. Endonuclease protection experiments showed that RNA polymerase is unable to form a stable complex at the traY promoter in linear or relaxed circular templates, in vitro transcription with linear templates could be elicited by altering the traY‐10 and −35 hexamers to the consensus sequences. Alterations that reduced the effect of template supercoiling on apparent promoter strength in vitro also reduced the effect of the F plasmid TraJ protein on traY expression in vivo. Apparent traY promoter strength in vitro, estimated in template competition experiments, was unaltered by deletion of tra DNA normally upstream of the promoter, a change in promoter context that elicited high levels of promoter activity in TraJ− cells. These data suggest a model for regulated traY promoter activity in which a nucleoprotein complex involving tra DNA immediately upstream locally relaxes traY promoter DNA. TraJ and perhaps other activators could disrupt the complex, allowing promoter DNA to equilibrate at the prevailing negative superhelical density and thereby eliciting transcription initiation.
Conservation and evolution of the rpsU‐dnaG‐rpoD macromolecular synthesis operon in bacteria
Authors: Versalovic James , Koeuth Thearith , Britton Robert , Geszvain Kati , Lupski James R. 
Published: October 2006

SummaryThe macromolecular synthesis (MMS) operon contains three essential genes (rpsU, dnaG, rpoD) whose products (S21, primase, sigma‐70) are necessary for the initiation of protein, DNA, and RNA synthesis respectively. PCR amplifications with primers complementary to conserved regions within these three genes, and subsequent DNA sequencing of rpsU—dnaG PCR products, demonstrate that the three genes appear to be contiguous in 11 different Gram‐negative species. Within the Gram‐negative enteric bacterial lineage, the S21 amino acid sequence is absolutely conserved in 10 species examined. The putative nuteq antiterminator sequence in rpsU consists of two motifs, boxA and boxB, conserved in primary sequence and secondary structure. The terminator sequence, Ti, located between rpsU and dnaG is conserved at 31 positions in nine enterobacterial species, suggesting the importance of primary sequence in addition to secondary structure for transcription termination. The intergenic region between rpsU and dnaG varies in size owing to the presence or absence of the Enterobacterial Repetitive Intergenic Consensus (ERIC) DNA element. The rpoD gene contains rearrangements involving a divergent sequence, although two carboxy‐terminal regions which encode functional domains are conserved in primary sequence and spacing. Our data suggest that primary sequence divergence and DNA rearrangements in both coding and non‐coding sequences account for the interspecies variation in operon structure. However, MMS operon gene organization and cis‐acting regulatory sequences appear to be conserved in diverse bacteria.
Conservation of genes encoding components of a type IV pilus assembly/two‐step protein export pathway in Neisseria gonorrhoeae
Authors: Lauer Peter , Albertson Nan H. , Koomey Michael 
Published: October 2006

SummaryThree gonococcal genes have been identified which encode proteins with substantial similarities to known components of the type IV pilus biogenesis pathway in Pseudomonas aeruginosa. Two of the genes were identified based on their hybridization with a DNA probe derived from the pilB gene of P. aeruginosa under conditions of reduced stringency. The product of the gonococcal pilF gene is most closely related to the pilus assembly protein PilB of P. aeruginosa while the product of the gonococcal pilT gene is most similar to the PilT protein of P. aeruginosa which is involved in pilus‐associated twitching motility and colony morphology. The products of both of these genes display canonical nucleoside triphosphate binding sites and are predicted to be to cytoplasmically localized based on their overall hydrophilicity. The gonococcal pilD gene, identified by virtue of its linkage to the pilF gene, is homologous to a family of prepilin leader peptidase genes. When expressed in Escherichia coli, the gonococcal PilD protein functions to process gonococcal prepilin in a manner consistent with its being gonococcal prepilin peptidase. These results suggest that Neisseria gonorrhoeae is capable of expressing many of the essential elements of a highly conserved protein translocation system and that these gene products are probably involved in pilus biogenesis.
The NodI and NodJ proteins from Rhizobium and Bradyrhizobium strains are similar to capsular polysaccharide secretion proteins from Gram‐negative bacteria
Authors: Vázquez Martha , Santana Olivia , Quinto Carmen 
Published: October 2006

SummaryThe NodI and NodJ nodulation proteins have been described in different Rhizobium and Bradyrhizobium species. The NodIJ genes belong to the nod regulon. Other genes from this regulon are involved in the biosynthesis and modification of lipo‐oligosaccharide molecule(s) which are morphogénic signals when acting on legume roots. It has been proposed that the NodI and NodJ proteins belong to a bacterial inner membrane transport system of small molecules. Nucleotide sequencing of MudII PR 13 insertions in the nodulation region of the symbiotic plasmid from a Rhizobium leguminosarum bv. phaseoli strain CE3 has revealed the presence of NodI and nodJ related sequences downstream of nodC. Computer nucleotide sequence analysis of the entire NodI and NodJ sequences from R. leguminosarum bv. viciae and Bradyrhizobium japonicum strains show that both proteins are similar to the KpsT and KpsM proteins from Escherichia coli Kl and K5 strains, to the BexB and BexA proteins from Haemophilis influenzae and to the CtrD and CtrC proteins from Neisseria meningitidis, respectively. Except for the NodI and NodJ proteins, all of them have been involved in the mechanism of secretion of polysaccharides in each of their harbouring species. On the basis of the similarity found, we propose that the NodI and the NodJ proteins could be involved in the export of a lipo‐oligosaccharide.
A sequence‐specific function for the N‐terminal signal‐like sequence of the TonB protein
Authors: Karlsson Margareta , Hannavy Kevin , Higgins Christopher F. 
Published: October 2006

SummaryTonB is a proline‐rich protein which provides a functional link between the inner and outer membranes of Gram‐negative bacteria. TonB is anchored to the inner membrane via an N‐terminal signal‐like sequence and spans the periplasm, interacting with transport receptors in the outer membrane. We have investigated the role of the N‐terminal signal‐like peptide in TonB function. Replacement of the N‐terminal sequence with heterologous sequences indicates that it has at least three distinct rotes in TonB function: (i) to facilitate translocation of TonB across the cytoplasmic membrane; (ii) to anchor TonB to the cytoplasmic membrane; (iii) a sequence‐specific functional interaction with the ExbBD proteins.
ExbB acts as a chaperone‐like protein to stabilize TonB in the cytoplasm
Authors: Karlsson Margareta , Hannavy Kevin , Higgins Christopher F. 
Published: October 2006

SummaryThe TonB protein is required to transduce energy from the cytoplasmic membrane to outer membrane transport proteins of Gram‐negative bacteria. Two accessory proteins, ExbB and ExbD, are required for TonB function and it has been suggested that TonB and ExbBD form a complex in the membrane. In this paper we demonstrate that there are two spatially distinct, functional interactions between ExbBD and TonB. First, there is an interaction between ExbBD and the N‐terminal signal‐like peptide of TonB, probabiy the formation of a stable complex in the membrane. Second, ExbB interacts with TonB in the cytoplasm. This interaction involves the domain of TonB that is normally periplasmic. Thus, this is a transient interaction which occurs during the synthesis and/or localization of TonB, implying a chaperone‐like role for ExbB. The transmembrane topology of ExbB was shown to be consistent with this role.
Virulence of Yersinia enterocolitica is closely associated with siderophore production, expression of an iron‐repressible outer membrane polypeptide of 65 000 Da and pesticin sensitivity
Authors: Heesemann Jürgen , Hantke Klaus , Vocke Tilman , Saken Elizabeth , Rakin Alexander , Stojiljkovic Igor , Berner Reinhard 
Published: October 2006

SummaryIron‐repressible outer membrane proteins (Irp) and siderophore production of Yersinia enterocolitica, serotype 08, were subjected to analysis. Here four Irps of apparent molecular weights of 62000, 65000, 74000 and 75000 could be identified which were expressed constitutively by a fur mutant. Production of a novel catechol‐containing siderophore (denoted yersiniabactin) was detected by siderophore‐indicator agar (chrome azurol S) and feeding experiments. Growth support by yersiniabactin under iron‐restricted conditions was TonB‐ and Irp65‐dependent and correlated with pesticin‐sensitivity of Yersinia enterocolitica and Escherichia coliø. From these results we conclude that Irp65 of Y. enterocolitica functions as yersiniabactin receptor (FyuA) and as pesticin receptor. By immunoblotting using rabbit antibodies against Irp65 and chrome azurol S‐agar, we were able to demonstrate that all tested mouse‐lethai Y. enterocoiitica and Yersinia pseudotuberculosis strains of different serotypes express siderophores and Irp65. Moreover, the anti‐lrp65 rabbit serum did not cross‐react with the known iron‐repressible high‐molecular‐weight proteins (HMWPs). Evidently, the mouse lethality trait in enteropathogenic Yersinia spp. is closely associated with a novel iron‐uptake system, comprising the production of a siderophore and a siderophore receptor of apparent molecular mass 65 000 Da.
Activity domains of the TonB protein
Authors: Traub I. , Gaisser S. , Braun V. 
Published: October 2006

Summary Escherichia coli and related Gram‐negative bacteria contain an energy‐coupied transport system through the outer membrane which consists of the proteins TonB, ExbB, ExbD anchored in the cytoplasmic membrane and receptors in the outer membrane. Differences in the activities of the Escherichia coli and the Serratia marcescens TonB proteins were used to identify TonB functional domains. In E. coli TonB segments were replaced by equivalent fragments of S. marcescens TonB and the activities of the resulting chimaeric proteins were determined. In addition, E. coli TonB was truncated at the C‐terminal end, and point mutants were generated using bisulphite. From the results obtained we draw the following conclusions: an important site of interaction between TonB and ExbB is located in the M‐terminal region of TonB within or close to the cytoplasmic membrane since an N‐terminal 44‐residue fragment of TonB was stabilized by ExbB and interfered with wild‐type TonB activity. In addition, the activity of a TonB derivative in which histidine residue 20 was replaced by arginine was strongly reduced, and a double mutant containing arginine‐7 to histidine and alanine‐22 to threonine substitutions displayed an impaired uptake of ferrichrome. Furthermore, the domain around residue 160 is involved in TonB activity. S. marcescens TonB segments of this region in E. coli TonB conferred S. marcescens TonB activities, and E. coli TonB pöint mutants displayed strongly impaired activities for the uptake of colicin B and M and ferric siderophores. Plasmid‐encoded tonB mutants of this region showed negative complementation of chromosomal wild‐type tonB, and certain tonB mutants suppressed colicin B TonB‐box mutants. Uptake of colicins required different domains in TonB, for colicin B and M around residue 160 and for colicin la, a domain closer to the C‐terminal end. Tandem duplication of the E. coli (EP)X(KP) region by insertion of the S. marcescens (EP)×(KP) region (38 residues) and replacement of lysine residue 91 by glutamate did not alter TonB activity so that no evidence was obtained for this region to be implicated in receptor binding. The aberrant electrophoretic mobility of TonB was caused by the praline‐rich sequence since its removal resulted in a normal mobility.
Pertussis toxin export requires accessory genes located downstream from the pertussis toxin operon
Authors: Covacci Antonello , Rappuoli Rino 
Published: October 2006

SummaryPertussis toxin, a major virulence factor of Bordetella pertussis, is an oligomeric protein composed of five different subunits that are exported individually to the periplasmic space by the signal peptide–dependent pathway. After assembly, the protein is exported from the periplasm to the extracellular compartment. We show that pertussis toxin secretion across the outer membrane requires the gene product of at least one gene (ptlC) that is located downstream from the pertussis toxin operon. The amino acid sequence of PtlC shows a high degree of homology to VirB4, a protein encoded by the virB operon, which contains 11 open reading frames that are involved in the transfer of T‐DNA from Agrobaderium tumefaciens to the plant cells. This is a novel mechanism of protein export in Gram‐negative bacteria.