bluuebunny/crossref_metadata_2025_split huggingface dataset:
Galactose‐binding site in Escherichia coli heat‐labile enterotoxin (LT) and cholera toxin (CT)
Authors: Merritt Ethan A. , Sixma Titia K. , Kalk Kor H. , van Zanten Ben A. M. , Hol Wim G. J. 
Published: October 2006

SummaryThe galactose‐binding site in cholera toxin and the closely related heat‐labile enterotoxin (LT) from Escherichia coli is an attractive target for the rational design of potential anti‐cholera drugs, in this paper we analyse the molecular structure of this binding site as seen in several crystal structures, including that of an LT: galactose complex which we report here at 2.2 Å resolution. The binding surface on the free toxin contains several tightly associated water molecules and a relatively flexible loop consisting of residues 51–60 of the B subunit. During receptor binding this loop becomes tightly ordered by forming hydrogen bonds jointly to the GM1 pentasaccharide and to a set of water molecules which stabilize the toxin: receptor complex.
The bacterial phosphotransferase system: new frontiers 30 years later
Authors: Saler Milton H. , Reizer Jonathan 
Published: October 2006

SummaryIn 1964, Kundig, Ghosh and Roseman reported the discovery of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Thirty years later, we find that the PTS functions not only as a sugar‐phosphorylating system, but also as a complex protein kinase system that regulates a wide variety of metabolic processes and controls the expression of numerous genes. As a result of recent operon‐ and genome‐sequencing projects, novel PTS protein‐encoding genes have been discovered, most of which have yet to be functionally defined. Some of them appear to be involved in cellular processes distinct from those recognized previously. Fundamental aspects of past and current PTS research are briefly reviewed, and recent advances are integrated into conceptual pictures that provide guides for future research.
Signal peptides: exquisitely designed transport promoters
Authors: Izard Jennifer W. , Kendall Debra A. 
Published: October 2006

SummaryProkaryotic proteins destined for transport out of the cytoplasm typically contain an N‐terminal extension sequence, called the signal peptide, which is required for export, it is evident that many secretory proteins utilize a common export system, yet the signal sequences themselves display very little primary sequence homology. in attempting to understand how different signal peptides are able to promote protein secretion through the same pathway, the physical features of natural signal sequences have been extensively examined for similarities that might play a part in function. Experimental data have confirmed statistical analyses which highlighted dominant features of natural signal sequences in Escherichia coli: a net positive charge in the N‐terminus increases efficiency of transport; the core region must maintain a threshold level of hydrophoblcity within a range of length limitations; the central portion adopts an α‐hellcal conformation in hydrophobic environments; and the signal cleavage region is ideally six residues long, with small side‐chain amino acids in the −1 and −3 positions. This review focuses on the parallels between signal peptide physical features and their functions, which emerge when the results of a variety of experimental approaches are combined. The requirement for each property may be ascribed to a potential interaction that is critical for efficient protein export. The summation of the key physical features produces signal peptides with the flexibility to function in multiple roles in order to expedite secretion. In this way, nature has indeed evolved exquisitely tuned signal sequences.
Molecular characterization of the cai operon necessary for carnitine metabolism in Escherichia coii
Authors: Eichler Knut , Bourgis Fabienne , Buchet Anne , Kleber Hans‐Peter , Mandrand‐Berthelot Marie‐Andrée 
Published: October 2006

SummaryThe sequence encompassing the cai genes of Escherichia coli, which encode the carnitine pathway, has been determined. Apart from the already identified caiB gene coding for the carnitine dehydratase, five additional open reading frames were identified. They belong to the caiTABCDE operon, which was shown to be located at the first minute on the chromosome and transcribed during anaerobic growth in the presence of carnitine. The activity of carnitine dehydratase was dependent on the CRP regulatory protein and strongly enhanced in the absence of a functional H‐NS protein, in relation to the consensus sequences detected in the promoter region of the cai operon. In vivo expression studies led to the synthesis of five polypeptides in addition to CaiB, with predicted molecular masses of 56 613 Da (CaiT), 42 564 Da (CaiA), 59311 Da (CaiC), 32 329 Da (CaiD) and 21 930 Da (CaiE). Amino acid sequence similarity or enzymatic analysis supported the function assigned to each protein. CaiT was suggested to be the transport system for carnitine or betaines, CaiA an oxidoreduction enzyme, and CaiC a crotonobetaine/carnitine CoA ligase. CaiD bears strong homology with enoyl hydratases/isomerases. Overproduction of CaiE was shown to stimulate the carnitine racemase activity of the CaiD protein and to markedly increase the basal level of carnitine dehydratase activity. It is inferred that CaiE is an enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine racemase activities. Taken together, these data suggest that the carnitine pathway in E. coli resembles that found in a strain situated between Agrobacterium and Rhizobium.
Universal barrier to lateral spread of specific genes among microorganisms
Authors: Diaz Eduardo , Munthali Mathildah , de Lorenzo Víctor , Timmis Kenneth N. 
Published: October 2006

SummaryA genetic circuit to suppress the lateral spread of cloned genes from recombinant to indigenous microorganisms in the environment has been developed. It is based on the endonucleolytic activity of the bacterial toxin colicin E3, which has a distinct target at the 3 end of the 16S ribosomal RNA; this sequence is conserved in virtually all prokaryotic and many eukaryotic genera. Cleavage at this sequence separates the mRNA binding sites from the remainder of the 16S rRNA, thereby inhibiting protein synthesis. While host bacteria carrying the genes for both colicin production and colicin immunity are perfectly viable, lateral transfer of the E3 gene to non‐immune reciptents results in killing of such recipients. This genetic circuit decreases operational transfer frequencies of cloned genes linked to the E3 gene among a variety of bacterial genera by four to five orders of magnitude, in combination with transposon cloning vectors, the circuit is predicted to reduce the rate of lateral spread of specific genes to ecologically insignificant levels. This system therefore represents a useful tool both to explore the evolutionary and ecological consequences of experimentally reducing lateral gene spread among microorganisms, and to increase the ecological predictability of novel recombinant microorganisms.
A phosphorylated DNA‐binding protein is specific for the red‐light signal during complementary chromatic adaptation in cyanobacteria
Authors: Sobczyk André , Bely Alexandra , de Marsac Nicole Tandeau , Houmard Jean 
Published: October 2006

SummaryComplementary chromatic adaptation is a mechanism by which some cyanobacteria that are able to synthesize phycoerythrin can adapt their pigment (phycobili‐protein) content to the incident wavelengths of the light. In Calothrix sp. PCC 7601 it concerns phycoerythrin (cpe operon), synthesized under green light, and phycocyanin‐2 (cpc2 operon), expressed under red light, and involves transcriptional controls. With cell‐free extracts from Calothrix sp. PCC 7601 grown under various light regimes, a protein designated RcaD was found by gel retardation experiments to specifically bind to the cpc2 promoter region and to be present only in red‐light‐grown cells. This protein was partially purified and its binding activity was shown to be sensitive to an alkaline phosphatase treatment. RcaO can protect two regions of the cpc2 promoter sequence against degradation by DNase I. Because its activity is detected only under the conditions required for cpc2 expression, we propose that RcaD is a positive effector of transcription.
Specific initiation of transcription at a cyanobacterial promoter with RNA polymerase purified from Calothrix sp. PCC 7601
Authors: Schyns Ghislain , Sobczyk André , de Marsac Nicole Tandeau , Houmard Jean 
Published: October 2006

SummaryAlthough in cyanobacteria many genes have been shown to be transcriptionally controlled by specific stimuli, little is known about promoter structure and the form of RNA polymerase that recognizes individual promoters. RNA polymerase holoenzyme has been purified from Calothrix sp. PCC 7601. its polypeptide composition resembles that of the plant chloroplast enzymes. To study transcription in cyanobacteria further, we have analysed the promoter‐recognition properties of the purified enzyme. In vitro transcription was assayed with the promoter of the phycocyanin gene (cpc1) that is expressed whatever the incident light conditions. Transcription initiation at the same start point as in vivo was obtained with the Calothrix sp. PCC 7601 purified enzyme and the Escherichia coli core enzyme supplemented with a Calothrix sp. PCC 7601 sigma factor, but not with the E. coli holoenzyme.
ppGpp concentration, growth without PBP2 activity, and growth‐rate control in Escherichia coli
Authors: Joseleau‐Petit Danièle , Thévenet Danielle , D'Arl Richard 
Published: October 2006

Summary Escherichia coli strains partially induced for the stringent response are resistant to mecillinam, a β‐lactam antibiotic which specifically inactivates penicillin‐binding protein 2, the key enzyme determining cell shape. We present evidence that mecillinam resistance occurs whenever the intracellular concentration of the nucleotide ppGpp (guanosine 3’‐diphosphate 5’‐diphosphate), the effector of the stringent response, exceeds a threshold level. First, the ppGpp concentration was higher in a mecillinam‐resistant mutant than in closely related sensitive strains. Second, the ppGpp pool was controlled by means of a plasmid carrying a ptac‐relA′ gene coding for a hyperactive (p)ppGpp synthetase, ReiA′; increasing the ppGpp pool by varying the concentration of lac operon inducer IPTG resulted in a sharp threshold ppGpp concentration, above which cells were mecillinam resistant. Third, the ppGpp pool was increased by using poor media; again, at the lowest growth rate studied, the cells were mecillinam resistant, in all experiments, cells with a ppGpp concentration above 140pmoles/A600 were mecillinam resistant whereas those with lower concentrations were sensitive. We discuss a possible role for ppGpp as transcriptional activator of cell division genes whose products seem to become limiting in the presence of mecillinam, when cells form large spheres. We confirmed the well‐known inverse correlation between growth rate and ppGpp concentration but, surprisingly, for a given growth rate, the ppGpp concentration was lower in poor medium than in richer medium in which RelA is induced. We conclude that, for E. coli growing in poor media, the concentration of the nucleotide ppGpp is not the major growth rate determinant.
The opaH locus of Neisseria gonorrhoeae MS11A is involved in epithelial cell invasion
Authors: Waldbeser Lillian S. , Ajioka Richard S. , Merz Alexey J. , Puaoi Deidre , Lin Lan , Thomas Mary , So Magdalene 
Published: October 2006

SummaryIn order to produce a successful infection, Neisseria gonorrhoeae (GC) must attach to and invade mucosal epithelial cells. To identify GC gene products involved in this early interaction with host cells we constructed a gene bank derived from a clinical isolate of GC, and isolated a clone which had the capacity to adhere to the human endometrial adenocarcinoma tissue‐culture line HEC‐1‐B. The cloned sequence was identified as a member of the opa gene family whose protein products have been associated with virulence. The GC chromosome contains numerous variant opa genes which, in MS11, are designated opaA‐K. Previous work showed that expression of opaC confers a highly invasive phenotype upon strain MS11. When our cloned opa gene was mutated and returned to the GC MS11A chromosome by transformation and homologous recombination, we isolated one transformation that was significantly reduced in its invasive capacity. The locus mutated in this transformant was identified as opaH. Our resuits indicate that invasive‐ness of GC for human epithelail cells can be determined by more than one opa gene in strain MS11 A.
The role of the pilus in recipient cell recognition during bacterial conjugation mediated by F‐like plasmids
Authors: Anthony Karen G. , Sherburne Craig , Sherburne Richard , Frost Laura S. 
Published: October 2006

SummaryThe effects of defined mutations In the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating‐pair formation in liquid media by the transfer systems of the F‐Iike plasmids pOX38 (F), ColB2 and R100‐1 were investigated. Transfer of all three plasmids was affected differently by mutations in the rfa (LPS) locus of the recipient cell, the F plasmid being most sensitive to mutations that affected rfaP gene expression which is responslbie for the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS. CoIB2 transfer was more strongly affected by mutations in the heptose II‐heptose III region of the LPS (rfaF) whereas R100‐1 was not strongly affected by any of the rfa mutations tested. ompA but not rfa mutations further decreased the mating efficiency of an F plasmid carrying a mutation in the mating‐pair stabilization protein TraN. An F derivative with a chloramphenicol acetyltransferase (CAT) cassette interrupting the traA pilin gene was constructed and pilin genes from F‐like plasmids (F, ColB2, R100‐1) were used to complement this mutation. Unexpectediy, the results suggested that the differences in the pilin sequences were not responsible for recognizing specific groups in the LPS, OmpA or the TraT surface exclusion protein. Other corroborating evidence is presented suggesting the presence of an adhesin at the F pilus tip.
Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis‐acting function dispensable for chromosomal gene transfer
Authors: Pettis Gregg S. , Cohen Stanley N. 
Published: October 2006

SummaryThe tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101‐induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid‐borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer‐associated pocking phenomenon, and interfered with the ability of tra+ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis‐acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt‐like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.
Synergism of mosquitocidal toxicity between CytA and CrylVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis
Authors: Wu Dong , Johnson Jeffrey J. , Federici Brian A. 
Published: October 2006

SummaryThe toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG‐14 isolate of B. thuringiensis subsp. morrisoni is at least 20‐fold greater than any of the four mosquitocidal proteins of which It is composed (CytA, CrylVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CrylVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bio‐assays against first instars of Aedes aegypti. The LC50 for the CytA inclusion was 60 ng ml−1, whereas the LC50 for the CryIVD was 85ng ml−1 In comparison, the LC50s for different combinations of CytA and CrylVD inclusions ranged from 12–15 ng ml−1, 4–5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild‐type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni Is due to synergism among three or four of their major proteins.
Regulation of R100 conjugation requires traM in cis to traJ
Authors: Dempsey Walter B. 
Published: October 2006

SummaryDeletion mutants of R100‐1 were constructed by classical methods to remove various segments of the traM open reading frame, pTraM‐binding sites and the traM promoters. Complementation tests showed that traM was efficiently complemented only when the frans‐acting fragment contained both the complete traM gene and the adjacent traJ promoter and leader sequences. The conclusion is that traM and traJ constitute a complex operon. A deletion mutant lacking all of the fraJ gene, and one containing a frameshifting traM deletion, retained the ability to transfer at a low level, thereby showing that neither pTraM nor pTraJ is absolutely essential for transfer.
Mutants of Escherichia coli Trp repressor with changes of conserved, helix‐turn‐helix residue threonine 81 have altered DNA‐binding specificities
Authors: Pfau James , Arvidson Dennis N. , Youderian Philip 
Published: October 2006

SummaryThreonine is found at the third position of the second α‐helix in the helix‐turn‐helix motifs of most bacterial DNA‐binding proteins. To investigate the role of this conserved residue in Escherichia coli Trp repressor function, plasmids encoding mutant Trp repressers with each of the 19 amino acid changes of Thr‐81 were made by site‐directed mutagenesis. All 19 changes decrease the activity of Trp holorepressor, indicating that the Thr‐81 side‐chain is critical for TrpR function. Three mutant repressors, Ser‐81, Lys‐81 and Arg‐81, retain partial DNA‐binding activity and inhibit transcription from the wild‐type trp promoter/operator complex; challenge‐phage assays show that Ser‐81 and Lys‐81 holorepressors have altered DNA‐binding specificities. The side‐chain of Thr‐81 may make direct contacts with base pairs 4 and 3 of the trp operator, consistent with the nuclear magnetic resonance solution structures of the holorepressoroperator complex.
σS‐dependent growth‐phase induction of the csgBA promoter in Escherichia coli can be achieved in vivo by σ70 in the absence of the nucleoid‐associated protein H‐NS
Authors: Arnqvist Anna , Olsén Arne , Normark Staffan 
Published: October 2006

SummaryThe stationary‐phase‐specific sigma factor σS (RpoS/KatF) is required for Escherichia coli to induce expression of fibronectin‐binding curli organelles upon reaching stationary phase. We show that the csgA gene which encodes the curlin subunit protein belongs to a dicistronic operon, csgBA. The transcriptional start site of csgBA was determined and an AT‐rich upstream activating sequence (UAS) required for transcriptional activation was identified. The pcsgBA promoter is not specific for σS since the same promoter sequence can be used by Eσ70in vivo in a strain lacking nucleoid‐associated protein H‐NS and σS. Transcription remained growth‐phase induced and dependent upon the UAS in such a double mutant. Furthermore, we demonstrate that an additional operon, hdeAB, which is also dependent upon σS for transcription, can be transcribed by Eσ70in vivo in the absence of H‐NS by utilizing the phdeAB promoter. Two other genes known to be under the control of σS for expression, bolA and katE, remained transcriptionally silent in the absence of H‐NS. It is suggested that a subset of E. coli promoters can be recognized by both EσS and Eσ70in vivo but H‐NS interacting with these sequences prevents formation of succesful transcription‐initiation complexes with Eσ70.
Molecular characterization and transcription of the histone H2B gene from the protozoan parasite Trypanosoma cruzi
Authors: García‐Salcedo José A. , Oliver José L. , Stock Roberto P. , González Antonio 
Published: October 2006

SummaryThe structure, genomic organization and transcription of the gene encoding histone H2B in the protozoan parasite Trypanosoma cruzi have been studied. This gene consists of a 746‐nucleotlde unit, tandemly repeated at least 18 times in each of two clusters. DNA probes corresponding to histones H2B and H3 hybridized to different chromosomes revealing that the genes coding for these two histones are not physically linked in the genome of T. cruzi. The primary transcription product of the H2B gene is processed by trans‐splicing and polyadenylation. Inhibition of DNA synthesis with aphidicolin resulted in the reduction of histone H2B mRNA to undetectable levels in about two hours, suggesting that its abundance is regulated throughout the cell cycle as it occurs in other eukaryotes. in addition, a concomitant inhibition of translation by cycloheximide reverted this effect indicating that de novo protein synthesis is required for RNA instability. Histone mRNA abundance was dependent on the life‐cycle stage of T. cruzi: abundant in amastigotes and epimastigotes, the dividing forms in the host cell and the insect vector, respectively, white undetected in trypomastigotes, the parasite's non‐dividing life stage.
Gene expression in mycobacteria: transcriptional fusions based on xylE and analysis of the promoter region of the response regulator mtrA from Mycobacterium tuberculosis
Authors: Curcic R. , Dhandayuthapani S. , Oeretic V. 
Published: October 2006

SummaryUnderstanding promoter regulation and signal‐transduction systems in pathogenic mycobacteria is critical for uncovering the processes that govern interactions of these bacteria with the human host. In order to develop additional genetic tools for analysis of mycobacterial promoters, the xylE gene from Pseudomonas was tested as a transcriptional fusion reporter in fast‐ and slow‐growing mycobacteria. initially, its utility was demonstrated by expression behind the hsp60 promoter in Mycobacterium smegmatis and Mycobacterium bovis BCG. The presence of an active promoter in front of the promoterless xylE cassette on a plasmid was scored by development of a bright yellow colour upon spraying of mycobacterial colonies on plates with a solution of catechol. The gene product of xylE, catechol 2,3 dioxygenase, was measurable in sonic extracts and whole cells, permitting quantitative determination of promoter activity in both fast‐ and slow‐growing mycobacteria. The xylE‐based mycobacterial transcriptional fusion plasmid pRCX3 was constructed and used to assess promoter activity within the sequences located upstream of the newly characterized Mycobacterium tuberculosis H37Rv response regulator mtrA, a member of the superfamily of bacterial signal‐transduction systems.