bluuebunny/crossref_metadata_2025 huggingface dataset:
Complex transcription of an operon encoding the Sail restriction‐modification system of Streptomyces albus G
Authors: Alvarez Miguel A. , Chater Keith F. , Rodicio M. Rosario
Published: October 2006

High‐resolution SI nuclease mapping of mRNA synthesised in vivo, in vitro run‐off transcription with RNA polymerase from Streptomyces lividans and gene fusions were used to analyse the transcriptional organization of the Sall restriction‐modification system of Streptomyces albus G. The sallR and sallM genes that encode the restriction endonuclease and its cognate methyltransferase constitute an operon which is mainly transcribed from sal‐pR1, a promoter located immediately upstream of saIIR, with two possible minor promoters further upstream. Another promoter, sal‐pM, is within the 3′ end of the saIIR coding region, and allows expression of the modification gene in the absence of sal‐pR1. The sal‐pM promoter might be involved in the establishment of modification prior to restriction endonuclease activity. Sequences upstream of the apparent transcriptional start sites for sat‐pR1 and sal‐pM show similarity with the −10 region of typical vegetatively expressed eubacterial promoters, but appropriately centered −35 regions are absent.
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

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

The 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

We 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

The 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

Antisera 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

Ability 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

It 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

Expression 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

The 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

Three 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

The 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

TonB 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

The 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

Iron‐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

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

Pertussis 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.
Intermolecular complementation of the kinase activity of CheA
Authors: Swanson Ronald V. , Bourret Robert B. , Simon Melvjn I.
Published: October 2006

CheA is a dimeric autophosphorylating protein kinase that plays a critical role in the signal transduction network controlling chemotaxis In Escherichia coli. The autophosphorylation reaction was analysed using mutant proteins defective in kinase and regulatory functions. Proteins in which the site of autophosphorylation was mutated (CheA48HQ) or missing (CheAs) were found to phosphorylate the kinase‐defective mutant, CheA470GK. The kinetics of this reaction support the hypothesis that autophosphorylation is the result of trans‐phosphorylation within a dimer. The carboxy‐terminal portion of CheA was previously shown to be dispensable for autophosphorylation, but required for regulation in response to environmental signals transmitted through a transducer and CheW. Mixing of CheA48HQ or CheA470GK with a truncated protein lacking this regulatory domain demonstrated that regulated autophosphoryltion requires the presence of both carboxy‐terminal portions in a CheA dimer. These results indicate that the dimeric form of CheA plays an integral role in signal transduction in bacterial chemotaxis.
Molecular cloning and characterization of 13 out genes from Erwinia carotovora subspecies carotovora: genes encoding members of a general secretion pathway (GSP) widespread in Gram‐negative bacteria
Authors: Reeves P.J. , Whitcombe D. , Wharam S. , Gibson M. , Allison G. , Bunce N. , Barallon R. , Douglas P. , Mulholland V. , Stevens S. , Walker D. , Salmond G. P. C.
Published: October 2006

The chemical mutagen ethylmethanesulphonate (EMS) has been used to generate mutants of Erwinia carotovora subspecies carotovora which are defective in the secretion of pectinases (Pel) and cellulases (Cel) but unaltered for protease (Prt) secretion. Such mutants, called Out− still synthesize Pel and Cel but these enzymes accumulate within the periplasm. Cosmid clones carrying wild‐type E. carotovra ssp. carotovora DNA, identified by their ability to restore the Out+ phenotype when transferred to some Out− mutants, were classified into six complementation groups using cosmids and cosmid derivatives. Analysis of the nucleotide sequence of a 12.7 kb DNA fragment, encompassing complementing cosmid inserts, revealed a coding capacity for 13 potential open reading frames (ORFs), and these were designated outC‐outO. Some of the out gene products were visualized using a T7 gene 10 expression system. The predicted Out proteins are highly similar to components of extracellular enzyme secretion systems from a diverse range of eubacteria including Erwinia chrysanthemi, Klebsiella oxytoca, Aeromonas hydrophila, Pseudomonas aeruginosa and Xanthomonas campestris. Lower levels of similarity exist between Ecc Out proteins and components of macromolecular trafficking systems from Bacillus subtilis, Haemophilus influenzae, Agrobacterium tumefaciens, Yersinia pestis and a protein involved in the morphogenesis of filamentous bacteriophages such as M13.
Identification of a two‐component regulatory system controlling methanol dehydrogenase synthesis in Paracoccus denitrificans
Authors: Harms Nellie , Reijnders Willem N.M. , Anazawa Hideharu , van der Palen Carol J. N. M. , van Spanning Rob J. M. , Oltmann L. Fred , Stouthamer Adriaan H.
Published: October 2006

Upstream of the moxFJGIR genes of Paracoccus denitrificans a regulatory region involved in methanol oxidation was identified. The nucleotide sequence of this region was determined and revealed three genes, moxZ, moxY and moxX, which are transcribed opposite to moxF and which encode proteins of 16.4, 48.2 and 24.5kDa, respectively. Computer alignment analysis revealed that the gene products of moxyand moxX have homology with the protein histidine kinases and the response regulators, respectively, forming the two‐component regulatory systems. No significant homology of the moxZ gene product with any known protein, sequenced thus far, was found. The MoxZ, MoxY and MoxX proteins were identified in Escherichia coli in a heterologous expression system. Mutants with an insertion of a kanamycin‐resistance marker in moxZ, moxY and moxX were isolated. These mutant strains were unable to grow on methanol while growth on methylamine was not affected. In the moxZ mutant both subunits of methanol dehydrogenase and cytochrome c5511 were not synthesized, methanol dehydrogenase activity was absent, and hardly any expression of a moxZ‐lacZ transcriptional fusion was found. Complementation of the mutation was observed after addition of the three genes moxZ, Y and X, in trans. This indicates that the two‐component regulatory system is involved in activation of the moxF promoter. A mutant with an unmarked deletion in moxZ was isolated. This mutant showed reduced growth on methanol relative to the wild type. Expression of the moxF‐lacZ transcriptional fusion gene and methanol dehydrogenase activity in this strain were also lower than those found in the wild type. Therefore, besides the two proteins of the two‐component regulatory pair, a third protein, MoxZ, appears to be involved in regulation of methanol dehydrogenase synthesis.
Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae
Authors: Rey Luis , Murillo Jesus , Hernando Yolanda , Hidalgo Elena , Cabrera Ezequiel , Imperial Juan , Ruiz‐Argüeso Tomás
Published: October 2006

The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX2C motifs characteristic of metal‐binding proteins. In addition, HypB bore a long histidine‐rich stretch of amino acids near the N‐terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX2CX81CX2C) with a capacity to form zinc finger‐like structures in the N‐terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.
Expression and analysis of two gyrB genes from the novobiocin producer, Streptomyces sphaeroides
Authors: Thiara Amrik S. , Cundliffe Eric
Published: October 2006

A novobiocin producer, Streptomyces sphaeroides, contains two genes, designated gyrBs and gyrBR, that encode novobiocin‐sensitive and ‐resistant DNA gyrase B proteins, respectively. The cloning of gyrBR was reported earlier; here, we describe the cloning of gyrBS. Both genes have been sequenced (the deduced products of gyrBs and gyrBR have Mr values of 87.6K and 86.5 K, respectively) and their transcripts have been mapped. Downstream of gyrBS, and co‐transcribed with it, is the sole gyrA gene (encoding DNA gyrase A protein). By constructing hybrid gyrBS genes, using fragments of gyrBS and gyrBR, a specific portion of the N‐terminal domain of the gyrase B protein (corresponding to amino acid residues 134–256 of Escherichia coli gyrase B) has been implicated in the binding of novobiocin.
Mechanism of initiation of transcription by Escherichia coli RNA polymerase on supercoiled template
Authors: Mishra Rakesh K. , Chatterji Dipankar
Published: October 2006

DNA supercoiling is known to influence the pattern of gene expression in prokaryotes. Thus the mechanism of transcription initiation and the topological state of the template are intimately related. Using in vitro reconstituted transcription assays, composed of purified RNA polymerase and promoters in their natural topological state, we have conducted a detailed study of transcription initiation from T7 early promoters including the following steps: the formation of ternary complexes, acquisition of rifampicin resistance, release of sigma factor and the capacity for RNA chain elongation in complexes. We determined the order of these events and the length of the transcripts when each step occurred during initiation of transcription on supercoiled templates. The length of the transcripts varied in a promoter‐specific manner. Analysis of abortive products formed during the initiation showed that stronger promoters go to the elongation mode at transcript lengths shorter than that required for weaker promoters.
Trichomonas vaginalis and early evolving DNA and protein sequences of the CDC2/28 protein kinase family
Authors: Riley Donald E. , Campbell Lee Ann , Puolakkainen Mirja , Krieger John N.
Published: October 2006

The human sexually transmittted parasite Trichomonas vaginalis is a representative of one of the three earliest evolving eukaryotic lineages. We investigated whether T. vaginalis has DNA sequences and peptides related to cell division control molecules universal among yeasts and higher eukaryotes. A T. vaginalis ceil division control (CDC2/28) homologue was amplified by the polymerase chain reaction and sequenced. The absolute similarity with other CDC2/28 genes was 47%, with conservative replacement similarity of 67%. Western blots demonstrated a single T. vaginalis peptide reactive with antiserum to the PSTAIRE peptide, an expressed component of CDC2/28 genes in higher eukaryotes. Although eukaryotic, T. vaginalis has properties similar to those of bacteria and is the earlist evolving eukaryote reported to possess CDC2/28 DNA and peptide homologues. These observations suggest that the molecular origins of cell division control in eukaroytes preceded mitochondria, 28S ribosomes and regulated glycolysis.
Transformation with katG restores isoniazid‐sensitivity in Mycobacterium tuberculosis isolates resistant to a range of drug concentrations
Authors: Zhang Ying , Garbe Thomas , Young Douglas
Published: October 2006

Isoniazid‐resistant isolates of Mycobacterium tuberculosis were transformed with a plasmid vector carrying the functional catalase‐peroxidase (katG) gene. Expression of katG restored full drug susceptibility in isolates initially resistant to concentrations ranging from 3.2 to >50μgml−1. Transformation with the corresponding katG gene from Escherichia coli resulted in low‐level expression of catalase and peroxidase activities and conferred partial isoniazid sensitivity.
Charged amino‐terminal amino acids affect the lethal capacity of Lambda lysis proteins S107 and S105
Authors: Steiner Martin , Bläsi Udo
Published: October 2006

The lysis inhibitor protein S107 and the lysis effector protein S105 start at Met codons 1 and 3 of the Lambda S gene, respectively. The antagonistic action of both proteins precisely schedules lysis by formation of a non‐specific lesion in the inner membrane through which the Lambda‐encoded murein transglycosylase can pass. Here, we show that the main difference between lysis—effector and lysis—inhibitor is the degree by which an energized membrane inhibits either protein from hole formation. To dissect the structural parameters responsible for intrinsic inhibition of both proteins, charged amino acids were replaced proximal to the first putative membrane‐spanning region in both S proteins. Our results show that the distribution of amino‐terminal charged amino acids as well as the total amino‐terminal net charge of S107 and S105 influence their lethal potential. The data are interpreted in terms of a model in which the electrostatic status of the amino‐terminus of both S107 and S105 is an important feature affecting their conf or mat ional change required for formation of the S‐dependent hole.
Regulation of the synthesis of unsaturated fatty acids by growth temperature in Bacillus subtilis
Authors: Grau Roberto , de Mendoza Diego
Published: October 2006

Bacillus subtilis synthesizes, almost exclusively, saturated fatty acids, when grown at 37° C. When cultures were transferred from 37° C to 20° C, a chloramphenicol‐ and rifampicin‐sensitive synthesis of a C‐16 unsaturated fatty acid was observed. Synthesis of this compound reached a plateau after 5 h at 20° C, reaching levels of 20% of the total fatty acid content. [14C]‐labelled fatty acids attached as thioesters to acyl‐carriers compounds, such as coenzyme A (CoA) or acyl‐carrier protein (ACP) synthesized de novo by glycerol‐requiring auxotrophs deprived of glycerol to arrest phospholipid synthesis, could not be desaturated at 20° C. Desaturation of these fatty acids was readily observed when glycerol was restored to the cultures allowing resumption of transfer of acyl‐moieties from acyl‐thioesters to phospholipid. It was also observed that depletion of the pools of CoA and ACP by starvation of pantothenate auxotrophs had no effect on the observed synthesis of unsaturated fatty acid at 20° C. The overall results indicate that synthesis of unsaturated fatty acids in B. subtilis is a cold‐inducible process and that phospholipids are obligate intermediates in this fatty acid desaturation pathway.
Nucleotide sequence of a 13.9 kb segment of the 90 kb virulence plasmid of Salmonella typhimurium: the presence of fimbriai biosynthetic genes
Authors: Friedrich Mary Jane , Kinsey Nicole E. , Vila Jordi , Kadner Robert J.
Published: October 2006

The 90 kb plasmid resident in Salmonella typhimurium confers increased virulence in mice by promoting the spread of infection after invasion of the intestinal epithelium. The nucleotide sequence of a 13.9 kb segment of this plasmid known to encode an outer membrane protein related in sequence to components of fimbrial biosynthesis in enteric bacteria was determined. This cloned segment between the repB and repC replicon regions programmed expression of abundant surface fimbriae in Escherichia coli and S. typhimurium cells. A 7 kb region contained seven open reading frames, the protein products of five of which were related in sequence to regulatory, structural, and assembly proteins of adherence fimbriae/pili, such as the P and K88 piM. These five genes and two adjacent ones which were not markedly related to proteins in the data bases comprise the pef (plasmid‐encoded fimbriae) locus. Transposon TnphoA insertions In four genes in the pef locus (pefA, pefC, orf5 and orf6) resulted in active PhoA fusions and blocked or reduced the surface presentation of fimbriae, indicating that the proteins encoded by these four genes are translocated at least across the cytoptasmic membrane and contribute to formation of the fimbrial structure. The differences in genetic organization and protein sequence relatedness from other fimbrial gene clusters suggest that the pef locus might encode a novel type of fimbria. Between the pef and the repB loci, there were five open reading frames, one of which (orfS) gave rise to active PhoA fusions but was not necessary for fimbriai expression. Two of the other proteins were homologous to transcription regulatory proteins and a third was the rck gene, which encodes an outer membrane protein that confers complement resistance to serum‐sensitive hosts.
Molecular genetics of phosphofructokinase in the yeast Kluyveromyces lactis
Authors: Heinisch Jürgeni , Kirchrath Lutz , Liesen Thomas , Vogelsang Kathrin , Hollenberg Cornelis P.
Published: October 2006

We have undertaken a study of phosphofructokinase (PFK; E.C. 2.7.1.11) in the yeast Kluyveromyces lactis. Like other eukaryotic PFKs, the K. lactis enzyme is activated by the allosteric effectors AMP and fructose‐2,6‐bisphosphate. PFK activity is induced in cells grown on glucose as compared to ethanol‐grown cells, in contrast to the constitutive expression of PFK in Saccharomyces cerevisiae. We show here that phosphofructokinase of the yeast K. lactis is composed of two non‐identical types of sub‐units, encoded by the genes KIPFK1 and KIPFK2. We have cloned and sequenced both genes. KIPFK1 and KIPFK2 encode the α‐ and the β‐PFK subunits with deduced molecular weights of 109.336 Da and 104.074Da, respectively. Sequence analysis indicates that the genes evolved from a double duplication event. Null mutants in either of the genes lack detectable PFK activity in vitro and the respective subunits cannot be detected on Western blots. In contrast to the situation in S. cerevisiae, Klpfk1 Klpfk2 double mutants retain the ability to grow on glucose. However. Klpfk2 mutants and the double mutants do not grow on glucose, when respiration is blocked. These data suggest that the pentose phosphate pathway and respiration play a substantial role in glucose utilization by K. lactis. The K. lactis PFK genes can be expressed independently in S. cerevisiae and each of them complements the glucose‐negative phenotype of pfk1 pfk2 double deletion mutants in this yeast. Expression of both K. lactis PFK genes simultaneously in S. cerevisiae pfk double deletion mutants complements for PFK activity. However, expression of a combination of PFK genes from K. lactis and S. cerevisiae does not lead to the production of a functional enzyme.
Streptomyces antibioticus contains at least three oleandomycin‐resistance determinants, one of which shows similarity with proteins of the ABC‐transporter superfamily
Authors: Ma Rodriguez Ana , Olano Carlos , Vilches Carmen , Méndez Carmen , Salas José A.
Published: October 2006

Three different DNA fragments of an oleandomycin producer, Streptomyces antibioticus, conferring oleandomycin resistance were cloned in plasmid pIJ702 and expressed in Streptomyces lividans and in Streptomyces albus. These oleandomycin resistance determinants were designated as oleA (pOR400), oleB (pOR501) and oleC (pOR800). oleA and oleC are closely linked in the chromosome as they were both obtained together in two cosmid clones that were isolated from a genomic library. Sequencing of the oleC resistance determinant revealed four complete open reading frames (ORFs) and the C‐terminal end of a fifth. The functions of orf1 and orf2 are unknown since they did not show significant similarity with other sequences in the data bases. The orf3 gene product has similarity with some proteins involved in iron and vitamin B12 uptake in bacteria. The orf4 gene product had a hydrophilic profile and showed important similarity with proteins containing typical ATP‐binding domains characteristic of the ABC‐transporter superfamily and involved in membrane transport and, particularly, with several genes conferring resistance to various macrolide antibiotics and anticancer drugs. The last gene, orf5, is translationally coupled to orf4 and codes for a hydrophobic polypeptide containing several trans‐membrane domains characteristic of integral membrane proteins. Subcloning and deletion experiments limited the resistance determinant to a 0.9kb Pst1‐Sph1 fragment and only orf4 is included in this fragment. These results suggest that resistance to oleandomycin conferred by oleC (orf4) is probably due to an efflux transport system of the ABC‐transporter superfamily.
Genetic analysis of the alginate biosynthetic gene cluster of Pseudomonas aeruginosa shows evidence of an operonic structure
Authors: Chitnis Chetan E. , Ohman Dennis E.
Published: October 2006

Strains of Pseudomonas aeruginosa causing pulmonary infections in cystic fibrosis patients have an unusual mucoid phenotype because of production of the capsule‐like exopolysaccharide, alginate. Transcriptional activation of algD, the first gene of a large alginate biosynthetic gene cluster, is associated with conversion to the alginate‐producing (Alg+) phenotype. In this study, we examined the regulation of alginate genes immediately downstream of algD. Mutants of the Alg+ strain FRD1 were constructed by gene replacement with defined Tn501 (8.2 kb) insertions in the alginate biosynthetic gene cluster, resulting in an Alg− phenotype. The Alg+ phenotype of these mutants was restored by integration of narrow‐host‐range plasmids containing DNA fragments from P. aeruginosa that reconstructed a continuous alginate gene cluster. A broad‐host‐range plasmid containing the entire alginate gene cluster except for the terminal gene, algA was unable to complement an aIG::Tn501 mutant unless algA was transcribed from a second plasmid. This indicated that any Tn501 insertion in the cluster was polar on downstream alginate genes. Northern blot hybridization experiments also showed that a transposon insertion downstream of algD adversely affected algG and algA transcription. These results provided evidence that the alginate biosynthetic gene cluster has an operonic structure and is cotranscribed from the algD promoter.
Identification and characterization of the pupB gene encoding an inducible ferric‐pseudobactin receptor of Pseudomonas putida WCS358
Authors: Koster Margot , van de Vossenberg Jack , Leong John , Weisbeek Peter J.
Published: October 2006

Pseudomonas putida WCS358 can transport iron complexed to a wide variety of pseudobactins produced by other Pseudomonas strains. The pupB gene encoding an outer membrane ferric‐pseudobactin receptor was isolated from a genomic library of P. putida WCS358. The PupB receptor facilitated iron transport via two distinct heterologous siderophores, i.e. pseudobactin BN8 and pseudobactin BN7. The amino acid sequence deduced from the nucleotide sequence consisted of 804 amino acids (molecular weight 88369) of which the N‐terminal part was very similar to a prokaryotic leader peptide. The mature protein shared significant homology with the receptor for ferric‐pseudobactin 358 (PupA) and contained three regions common to TonB‐dependent receptor proteins of Eschenchia coli. Interestingly, PupB expression was only observed in cells cultured in iron‐deficient medium containing pseudobactin BN8 or pseudobactin BN7. This expression required a transcriptional unit, pupR, identified upstream of the structurai pupB gene. Transposon Tn5 insertion mutants defective in PupB production still exhibited uptake of iron via pseudobactin BN8, although with reduced efficiency. Apparently, an additional transport system for this ferric‐siderophore complex operates in this strain. In addition to pseudobactin BN8 also other heterologous siderophores were capable of inducing synthesis of specific high‐molecular‐weight outer membrane proteins in strain WCS358, which suggests the existence of multiple siderophore‐inducible iron transport systems in this strain.