bluuebunny/crossref_metadata_2025_split huggingface dataset:
IS231 D, E and F, three new insertion sequences in Bacillus thuringiensis: extension of the IS231 family
Authors: Rezsöhazy René , Hallet Bernard , Delcour Jean 
Published: October 2006

SummaryIS231 constitutes a family of insertion sequences widespread among Bacillus thuringiensis subspecies. Three new IS231 variants have been isolated from B. thuringiensis subspecies finitimus (IS231 D and E) and israelensis (IS231 F). Like the previously described IS231 A, B and C, these 1.7 kb elements display single open reading frames encoding 477/478‐amino‐acid proteins which share between 72% and 88% identity with those of the other members of the family. Sequence comparisons also reveal that all the iso‐IS231 terminal inverted repeats are strongly conserved 20bp sequences. A region susceptible to forming a stable hairpin structure is found just upstream of the open reading frame. Nucleotide substitutions occurring on one strand of the hairpin stems are compensated for by complementary changes at facing positions, giving credence to the hypothesis that this secondary structure plays a role in the regulation of transposition. Examination of IS231 D, E and F flanking sequences reveals that IS231 F is bordered by a 12bp direct repeat. No direct repeats were found flanking IS231 D or IS231 E.
Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES
Authors: de Wit Rinke Tobias F. , Bekelie Siraj , Osland Arve , Miko Tivadar L. , Hermans Peter W. M. , van Soolingen Dick , Drijfhout Jan‐Wouter , Schöningh Ron , Janson Anneke A. M. , Thole Jelle E. R. 
Published: October 2006

SummaryIn contrast to other bacterial species, mycobacteria were thus far considered to contain groEL and groES genes that are present on separate loci on their chromosomes, Here, by screening a Mycobacterium leprae lambda gt11 expression library with serum from an Ethiopian lepromatous leprosy patient, two DNA clones were isolated that contain a groEL gene arranged in an operon with a groES gene. The complete DNA sequence of this groESL operon was determined. The predicted amino acid sequences of the GroES and GroEL proteins encoded by this operon are 85–90% and 59–61% homologous to the sequences from previously characterized mycobacterial GroES and GroEL proteins. Southern blotting analyses with M. leprae groES‐ and groEL‐specific probes demonstrate that similar groESL homologous DNA is present in the genomes of other mycobacteria, including Mycobacterium tuberculosis. This strongly suggests that mycobacteria contain a groESL operon in addition to a separately arranged second groEL gene. Using five T‐cell clones from two leprosy patients as probes, expression of the M. leprae GroES protein in Escherichia coli after heat shock was demonstrated. Four of these clones recognized the same M. leprae‐specific GroES‐derived peptide in a DR2‐restricted fashion. No expression of the groEL gene from this operon was detected in E. coli alter heat shock, as tested with a panel of T‐cell clones and monoclonal antibodies reactive to previously described GroEL proteins of mycobacteria.
Interaction between maltose‐binding protein and the membrane‐associated maltose transporter complex in Escherichia coli
Authors: Dean David A. , Hor Lien I. , Shuman Howard A. , Nikaido Hiroshi 
Published: October 2006

SummaryActive transport of maltose in Escherichia coli requires the presence of both maltose‐binding protein (MBP) in the periplasm and a complex of MalF, MalG, and MalK proteins (FGK2) located in the cytoplasmic membrane. Earlier, mutants in malF or MalG were isolated that are able to grow on maltose in the complete absence of MBP. When the wild‐type malE+ allele, coding for MBP, was introduced into these MBP‐independent mutants, they frequently lost their ability to grow on maltose. Furthermore, starting from these Mal‐ strains, Mal+ secondary mutants that contained suppressor mutations in malE were isolated. In this study, we examined the interaction of wild‐type and mutant MBPs with wild‐type and mutant FGK2 complexes by using right‐side‐out membrane vesicles. The vesicles from a MBP‐independent mutant (malG511) transported maltose in the absence of MBP, with Km and Vmax values similar to those found in intact cells. However, addition of wild‐type MBP to these mutant vesicles produced unexpected responses. Although male+ malG511 cells could not utilize maltose, wild‐type MBP at low concentrations stimulated the maltose uptake by malG511 vesicles. At higher concentrations of the wild‐type MBP and maltose, however, maltose transport into malG511 vesicles became severely inhibited. This behaviour of the vesicles was also reflected in the phenotype of male+ malG511 cells, which were found to be capable of transporting maltose from a low external concentration (1 μM), but apparently not from millimolar concentrations present in maltose minimal medium. We found that the mutant FGK2 complex, containing MalG511, had a much higher apparent affinity towards the wild‐type MBP than did the wild‐type FGK2 complex. We propose that the wild‐type FGK2 complex exists in at least two conformations, active and inactive, and that the binding of the liganded MBP converts the latter into the former. The mutant complex presumably exists predominantly in the active form that has a higher affinity toward liganded MBP, and the Inhibition of the mutant complex by an excess of maltose and wild‐type MBP may be explained as a form of inhibition by excess substrate.
Chromosomal and genetic analysis of the electrophoretic karyotype of Trichoderma reesei: mapping of the cellulase and xylanase genes
Authors: Carter Gina L. , Allison Dan , Rey Michael W. , Dunn‐Coleman Nigel S. 
Published: October 2006

SummaryAn electrophoretic karyotype has been established for Trichoderma reesei strain QM6a, and several of its derivatives, by pulsed‐field gel electrophoresis. All strains examined appear to have seven chromosomes with a total genome size of approximately 33 megabases (Mb). The sizes of the chromosomal bands in strain QM6a are approximately 6.2, 6.0, 5.1, 4.2 (doublet), 3.6 and 3.2 Mb. Genes encoding the cellulase complex and xylanases of T. reesei have been mapped, as have several other genes.
Decreasing transcription elongation rate in Escherichia Coli exposed to amino acid starvation
Authors: Vogel Ulla , Sørensen Michael , Pedersen Steen , Jensen Kaj Frank , Kilstrup Mogens 
Published: October 2006

SummaryThe time required for transcription of the lacZ gene in Escherichia coli was determined during exponential growth and under conditions, when the bacterium was exposed to partial isoleucine starvation. To do this, RNA was extracted from the cells at 10 s intervals following induction and quantified by Northern hybridization with probes complementary to either the beginning or the end of the lacZ mRNA. The time lag between inducer addition and the appearance of a hybridization signal at the ‘late’ probe represents the transit time for RNA polymerase on the lacZ gene, and this parameter and the known length of the transcribed sequence were used to calculate the lacZ mRNA chain growth‐rate. The transcription elongation rate was c. 43 nucleotides s‐1 during exponential growth and decreased abruptly to c. 20 nucleotides s‐1 in a relA+ strain after the onset of isoleucine starvation, when massive concentrations of guanosine tetraphosphate (ppGpp) accumulated in the cells. The starvation condition did not affect initiation of transcription at the lec‐promoter, but a substantial fraction of the initiated lacZ mRNA chains was never completed. For the rel+ strain the polarity was moderate, since c. 25% of the initiated lacZ mRNA’ chains were continued into full‐length mRNAs, but for the relA strain the polarity was so strong that no completed lacZ mRNA could be detected. The protein chain elongation rates decreased from 13 amino acids (aa) s‐1 in the unperturbed growth phase to approximately 6 aa s‐1, when the cells starved for isoleucine. In combination, these results suggest that ppGpp plays a major role in maintaining the coupling between transcription and translation during the downshift by inhibiting mRNA chain elongation. The implications of this result for the control of stable RNA synthesis during the stringent response are discussed.
Disulphide bridge formation in the periplasm of Escherichia coli: β‐lactamase::human lgG3 hinge fusions as a model system
Authors: De Sutter K. , Remaut E. , Fiers W. 
Published: October 2006

SummaryWe report the construction and the expression in Escherichia coli of three different fusion genes encoding the extended human lgG3 hinge region (Hi) fused in‐phase to the C‐terminal end of bacterial TEM1 β‐lactamase (Bla). In the first fusion gene blahi, the hinge sequence was directly coupled to the 3′ end of the β‐lactamase gene, whereas in the two other constructs, blal1hi and blal2hi, a linker encoding 14 and 10 amino acids, respectively, was inserted between the two subunits. After expression (24 h, 20° C) under control of the constitutive kanamycin phosphoribosyl transferase promoter, the fusion proteins, BlaHi, BlaL 1Hi and BlaL2Hi, respectively, were almost exclusively detected in the periplasmic fraction, and they conferred carbenicillin‐resistance to the cells. These results indicate that β‐lactamase can efficiently direct the export of proteins fused to its C‐ terminus, and moreover, at least some of the exported fusion proteins must carry the β‐lactamase moiety in a properly folded form. Analysis of their assembly, however, revealed that only a minor fraction was recovered as the expected F(ab′)2‐nke dimer. The presence in the periplasm of oxidized’monomers (with intrachain disulphide bonds) as well as of several high‐molecular‐mass proteins, probably resulting from the association between monomers and other cysteine‐rich proteins, strongly suggests that the conditions in the bacterial periplasm are insufficient to allow proper assembly of multimeric proteins with several interchain disulphide bonds.
(A)BC excinuclease: the Escherichia coli nucleotide excision repair enzyme
Authors: Lin Jing‐Jer , Sancar Aziz 
Published: October 2006

SummaryNucleotide excision repair is the major pathway for removing damage from DNA. (A)BC excinuclease is the nuclease activity which initiates nucleotide excision repair in Escherichia coli. in this review, we focus on current understanding of the structure‐function of the enzyme and the reaction mechanism of the repair pathway. In addition, recent biochemical studies on preferential repair of actively transcribed genes in E. coli are summarized.
Interrelated effects of DNA supercoiling, ppGpp, and low salt on melting within the Escherichia coli ribosomal RNA rrnB P1 promoter
Authors: Ohlsen Kari L. , Gralla Jay D. 
Published: October 2006

SummaryThe formation of complexes containing high levels of DNA melting at the ribosomal RNA rrnB P1 promoter in vitro is shown to be facilitated by DNA supercoiling or low salt. The effector nucleotide ppGpp is ineffective under these conditions. The loss of supercoils or addition of salt increases the effectiveness of ppGpp in inhibiting formation of these complexes. In vivo plasmid DNA supercoiling is shown to decrease during starvation protocols that also increase levels of ppGpp. The results suggest that ppGpp regulation may be affected by the state of DNA supercoiling in vivo.
Surface protein‐CAT reporter fusions demonstrate differential gene expression in the wr regulon of Streptococcus pyogenes
Authors: Podbielski A. , Peterson J. A. , Cleary P. 
Published: October 2006

Summary Streptococcus pyogenes expresses at least two virulence factors, the anti‐phagocytic M protein and an inhibitor of chemotaxis, the C5a peptidase (ScpA), under control of the virR locus. To facilitate studies of this regulatory unit, we constructed a new shuttle vector with a staphylococcal chloramphenicol acetyl transferase (CAT) reporter box which replicates in S. pyogenes. We cloned polymerase chain reaction (PCR)‐derived potential promoter regions of the virR, M protein (emm12), and ScpA (scpA) genes from an M type 12 5. pyogenes, strain CS24. Promoter activity was assessed by measurements of specific mRNAs, transacetylase activity, and minimum inhibitory concentrations (MICs) for chloramphenicol resistance. We demonstrated that VirR is a necessary but not always sufficient positive trans‐acting regulator of emm12 and scpA expression; however, virR is not autoregulated. A potential virR‐bindlng consensus sequence is postulated for emm12, scpA and other M‐like protein genes. Promoter activity of the structural genes was found to be dramatically influenced by growth conditions such as anaerobiosis. Levels of control, over and above the requirement for virR, are realized. The virR and scpA promoters were mapped for the first time using primer extension analysis. The observed mRNA start sites did not completely agree within the sequence predicted start sites. Data suggest that scpA could be subject to transcription attenuation.
A metalloprotease gene from Streptomyces coelicolor‘Müller’ and its transcriptional activator, a member of the LysR family
Authors: Dammann Tanja , Wohlleben Wolfgang 
Published: October 2006

SummaryA metalloprotease gene (mprA) and its regulatory gene (mprR) from Streptomyces coelicolor‘Müller’ DSM3030 were isolated and their DNA sequences determined. The protease secreted by the heterologous host Streptomyces lividans was characterized biochemically as a metalloprotease with a M, of 20000, which is in good agreement with data derived from DNA sequence analysis. The mprA gene is transcribed divergently from mprR, the deduced protein of which displays homology to prokaryotic transcriptional regulators of the LysR family. The regulatory protein (MprR) was shown to bind to the intergenic region between mprH and mprA. It was found to activate transcription of mprA in 5. lividans and also in Escherichia coli.
Polyphosphate‐selective porin OprO of Pseudomonas aeruginosa: expression, purification and sequence
Authors: Siehnel Richard J. , Egli Christine , Hancock Robert E. W. 
Published: October 2006

SummaryThe oprO gene of Pseudomonas aeruginosa codes for a polyphosphate‐specific porin and terminates 458 bp upstream of the start codon for the phosphate‐specific porin OprP. OprO was found to be expressed only under phosphate‐starvation conditions in both wild‐type and oprP::Tn501 mutant P. aeruginosa strains. However, unlike the rest of the genes of the Pho regulon, including oprP, expression of oprO required cells to be in the stationary growth phase in addition to phosphate starvation. Wild‐type P. aeruginosa cells were grown in fermentor culture under these conditions and fractionated by selective solubilization in octylpolyoxyethylene detergent solution. Solubilized OprO was separated from OprP by application to a Mono Q FPLC column and elution with a salt gradient and shown to be functionally identical to cloned OprO produced in Escherichia coli. DNA sequencing of oprO showed the gene product to be highly homologous to OprP, with 76% identity and 16% conserved substitutions. Most genes of the Pho regulon possess a modified ‐35 region called the Pho box. Two such elements, separated by 4 bp were found in oprO. DNA sequencing also revealed a second Pho box in the oprP gene with the same spacing.
Septal membrane fusion — a pivotal event in bacterial spore formation?
Authors: Higgins Michael L. , Piggot Patrick J. 
Published: October 2006

SummaryFormation of the asymmetrically located septum divides sporulating bacilli into two distinct cells: the mother cell and the prespore. The rigidifying wall material in the septum is subsequently removed by autolysis. Examination of published electron micrographs indicates that the two septal membranes then fuse to form a single membrane. Membrane fusion would be expected to have profound consequences for subsequent development. For example, it is suggested that fusion activates processing of pro‐σE to σE in the cytoplasm by exposing it to a membrane‐bound processing enzyme. Asymmetry of the fused membrane could restrict processing to one face of the membrane and hence explain why σEthe tanscription in the mother cell but not in the prespore. Asymmetry of the fused membrane might also provide a mechanism for restricting the activity of another factor, σF to the prespore. Attachment of the flexible fused septal membrane to the condensing prespore nucleoid could help drive the engulfment of the prespore by the mother cell.
Control of gene expression in the temperate coliphage 186. IX. B is the sole phage function needed to activate transcription of the phage late genes
Authors: Dibbens Justin A. , Egan J. Barry 
Published: October 2006

SummaryUsing plasmid clones we have determined that the late control function B is the only phage function that is needed to activate a late promoter of coliphage 186, and we predict that it functions as an auxiliary factor to RNA polymerase in the activation of late transcription. We have also shown that a high concentration of B will activate late transcription from a prophage, and we conclude that replicating DNA is not a template requirement for B to function. The original demonstration of a need for the replication gene A in late transcription can be explained by the fact that replication leads to an increase in B gene dosage, with the consequent increase in B concentration leading to the efficient activation of the late promoters.
Expression of bacterial cytotoxin genes in mammalian target cells
Authors: Wels W. , Baldrich M. , Chakraborty T. , Gross R. , Goebel W. 
Published: October 2006

SummaryWe have studied the expression of the gene fragments encoding the enzymatically active portion of three bacterial cytotoxins: exotoxin A (ETA) of Pseudomonas aeruginosa, and pertussis toxin (PT) and adenylate cyclase toxin (CYA) of Bordetella pertussis, in sensitive mammalian target cells. Expression of active ETA and CYA was lethal to the producing cells and stable transfectants of Cos‐1 cells containing the corresponding genes could not be obtained. The expression of the PTS1 subunit was tolerated by the producing mammalian cells. Since PT is cytotoxic because of ADP‐ribosylation of G‐proteins, we assume that the endogenously expressed PTS1 may not find the cellular target G proteins or PTS1 alone may not be sufficient for ADP‐ribosylation of these proteins in vivo.
Conservation of the photosynthesis gene cluster in Rhodospirillum centenum
Authors: Yildiz Fitnat H. , Gest Howard , Bauer Carl E. 
Published: October 2006

SummaryIntraspecies and intergenus complementation analysis were utilized to demonstrate that photosynthesis genes are clustered in distantly related purple photo‐synthetic bacteria. Specifically, we show that the linkage order for genes involved in bacteriochlorophyll and carotenoid biosynthesis in Rhodospirillum centenum are arranged essentially as in Rhodobacter capsulatus and Rhodobacter sphaeroides. In addition, the location and relative distance observed between the puf and puh operons which encode for light harvesting and reaction‐centre structural genes are also conserved between these species. Conservation of the photosynthesis gene cluster implies either that there are structural or regulatory constraints that limit rearrangement of the photosynthesis gene cluster or that there may have been lateral transfer of the photosynthesis gene cluster among different species of phototrophic bacteria.
flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP‐binding protein
Authors: Carpenter Phillip B. , Hanlon David W. , Ordal George W. 
Published: October 2006

Summarywe describe the sequence and characterization of the Bacillus subtilis flhF gene. flhF encodes a basic polypeptide of 41 kDa that contains a putative GTP‐binding motif. The sequence of FlhF reveals a structural relationship to two Escherichia coli proteins, Ffh and FtsY, as well as to other members of the SRP54 family, in a domain presumed to bind GTP. flhF is located in a large operon consisting of chemotaxis and flagellar genes. Cells deficient in flhF are non‐motile. Through the use of anti‐flagellar antibodies we have established that flhF is a flagellar (fla) gene. Thus, flhF is a unique flagellar gene in that it encodes a GTP‐binding protein with similarities to members of the SRP54 family of proteins. These data suggest that flagellar biosynthesis in B. subtilis requires GTP.
In vitro phosphorylation of AlgR, a regulator of mucoidy in Pseudomonas aeruginosa, by a histidine protein kinase and effects of small phospho‐donor molecules
Authors: Deretic V. , Leveau J. H. J. , Mohr C. D. , Hibler N. S. 
Published: October 2006

SummaryAlgR is a transcriptional regulator of mucoidy in Pseudomonas aeruginosa, a critical virulence factor expressed in cystic fibrosis. AlgR belongs to the superfamily of bacterial signal transduction systems, and has been shown to bind to the algD promoter, a critical point in the regulation of mucoidy. This protein, like other typical response regulators, contains highly conserved residues known to be critical for the phosphorylation and signal transduction processes. However, a typical second component Interacting with AlgR has not been identified. Here we demonstrate that AlgR undergoes phosphorylation in vitro when interacting with the well‐characterized histidine protein kinase CheA. These results Indicate that AlgR is capable of undergoing phosphorylation typical of other two‐component signal transduction systems. Moreover, the phosphotransfer reaction between CheA and AlgR was found to be affected by the presence of carbamoyl phosphate, acetyl phosphate, and salts of phosphoramidic acid, recently shown to act as small‐molecular‐weight phospho‐donors in the process of phosphorylation of several response regulators. These findings suggest that AlgR may react with intermediary metabolites such as carbamoyl phosphate and acetyl phosphate, and that these processes may play a role in the control of mucoidy in P. aeruginosa.