bluuebunny/crossref_metadata_2025_split huggingface dataset:
Substrate induction and catabolite repression of the Streptomyces coelicoior glycerol operon are mediated through the GyIR protein
Authors: Hindle Zoë , Smith Colin P. 
Published: October 2006

SummaryThe pathway for glycerol catabolism In Streptomyces coelicolor is determined by the gylCABX operon, which is transcribed from two closely spaced glycerol‐inducible, glucose‐repressible promoters. Glucose (or catabolite) repression of gyl is known to be exerted by a general catabolite repression system In which the soluble glucose kinase plays a central role. The gylR gene is contained in a separate glycerol‐inducible, weakly glucose‐repressible transcription unit immediately upstream from the gyl operon. The role of gylR in the regulation of gyl transcription was assessed by introducing specific null mutations into the chromosomal gylR gene. Direct quantification of gyl transcripts from the gylR null mutants grown on different carbon sources demonstrated that GylR is the repressor of the gylCABX operon and also revealed that GylR functions as a negative autoregulator. Moreover, the transcriptional analysis revealed that the gylR null mutants were relieved of glucose repression of both gylCABX and gylR. We conclude that both substrate induction and catabolite repression of gyl are mediated through the GylR protein. This is the first direct evidence that catabolite repression In Streptomyces Is not exerted at the transcriptional level by a general ‘catabolite repressor protein’. Models for catabolite repression are discussed.
The efficiency of processing and secretion of the thermolysin‐like neutral protease from Bacillus cereus does not require the whole prosequence, but does depend on the nature of the amino acid sequence in the region of the cleavage site
Authors: Wetmore Diana R. , Wong Sui‐Lam , Roche Rodney S. 
Published: October 2006

SummaryUsing deletion mutants, It is shown that part of the prosequence, the Ω‐peptide (‐4, ‐24), of the thermolysin‐like neutral protease (TNP) from Bacillus cereus, Cnp, is not required for efficient processing and secretion of fully functional mature protease. It is demonstrated that the rate and selectivity of pro‐protein processing is dependent on both the flexibility and primary sequence of the processing site. Processing is found to be particularly sensitive to the nature of the amino acid three residues upstream from the site of cleavage. A consensus sequence for TNP pro‐protein processing has been identified, which provides further Insights. Finally, a larger deletion of a portion of the Cnp prosequence upstream from the Ω‐peptide that includes amino acids conserved among TNPs reduces the rate of processing and secretion of Cnp and results in the accumulation of export‐incompetent pre‐proprotein in the cell fraction.
Streptomyces lividans 66 contains a gene for phage resistance which is similar to the phage λea59 endonuclease gene
Authors: Zhou X. , Deng Z. , Hopwood D. A. , Kieser T. 
Published: October 2006

SummaryThe DNA of wild‐type Streptomyces lividans 66 is degraded during electrophoresis in buffers containing traces of ferrous iron. S. lividans ZX1, a mutant selected for resistance to DNA degradation, simuiltaneously became sensitive to φHAU3, a wide‐host‐range temperate bacteriophage. A DNA fragment conferring φHAU3 resistance was cloned; it contains a phage resistance gene whose deduced amino acid sequence is similar to the phage λ Ea59 endonuclease. The S. lividansφHAU3 resistance does not seem to be a classical restriction‐modification system, because no host‐modified phages able to propagate on the wild‐type strain could be isolated. The cloned fragment did not make the host DNA prone to degradation during electrophoresis, indicating that the two phenotypes are controlled by different genes which were deleted together from the chromosome of ZX1.
Identification of a novel sugar‐H+ symport protein, FucP, for transport of L‐fucose into Escherichia coli
Authors: Gunn Frank J. , Tate Christopher G. , Henderson Peter J. F. 
Published: October 2006

Summary l‐Fucose (6‐deoxy‐l‐galactose) is used as sole carbon source by many microorganisms, and its transport into Escherichia coli is mediated by An l‐fucose‐H+ symport activity, in order to determine the nature of a putative transporter encoded by the E. coli fucP gene and Identify its protein product it was cloned downstream of the inducible T7 RNA polymerase and lambda Ol Pl promoters, induction of the T7 promoter resulted in the expression of [14C]‐l‐fucose uptake activity and the concomitant expression of a [35S]‐Met‐labelled 32 kDa protein at levels too tow for detection by staining with Coomassie briiiiant blue or for protein sequencing, induction of the lambda Ol Pl promoter caused the appearance of l‐fucose‐H+ symport activity and of a Coomassie brilliant blue‐stained 32 kDa membrane protein expressed at high levels sufficient for identification as FucP by N‐terminal protein sequencing. The FucP protein is, therefore, a sugar‐H+ symporter different in amino acid sequence from any other known transporter. These and other results illustrate the general unpredictability of cloning strategies for attempting the amplified expression of membrane transport proteins.
Mutational analysis of Agrobacterium tumefaciens pTiA6 virD1: identification of functionally important residues
Authors: Vogel Ann M. , Das Anath 
Published: October 2006

SummaryMutagenesis experiments were used to identify functionally important regions of Agrobacterium tumefaciens pTiA6 VirD1. Random mutations were introduced by using Taq polymerase in a mutagenic reaction buffer containing manganese and altered nucleotide ratios to increase errors during the polymerase chain reaction (PCR). The mutants were assayed for VirD1‐, VirD2‐dependent border‐nicking activity in Escherichia coli harbouring a border‐containing substrate plasmid. Analysis of the mutants led to the identification of a region from amino acids 45–60 that is important for VirD1 activity. This region corresponds to a previously postulated potential DNA‐binding domain. Deletion mutagenesis indicated that amino acids 2–16 could be deleted without affecting VirD1 function, whereas a larger deletion, amino acids 5–27, completely inactivated VirD1.
The glucose‐starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival
Authors: Nyström Thomas 
Published: October 2006

SummaryProteins of the glucose‐starvation stimulon were identified by using two‐dimensional gel electrophoresis and the gene–protein database of Escherichia coli. Members of this stimulon Included enzymes of the Embden–Meyerhof–Parnas (EMP) pathway, phosphotransacetylase (Pta) and acetate kinase (AckA) of the acetyl phosphate/acetate production pathway, and formate transacetytase. The synthesis of these enzymes was found to be Induced concomitantly with the decreased synthesis of enzymes of the Krebs cycle. Thus, the modulation in the synthesis of specific proteins during aerobic glucose starvation is, In part, similar to the response of cells shifted to anaerobiosis. These modulations suggest that the glucose‐starved cell increases the relative flow of carbon through the Pta–AckA pathway. Indeed, the ability to synthesize acetyl phosphate, an intermediate of the pathway, appears to be indispensable for glucose‐starved cells as pta and pta–ackA double mutants were found to be impaired in their ability to survive glucose starvation. The survival characteristics of ackA mutants and the wild‐type parent were indistinguishable. Moreover, the pta mutant failed to induce several proteins of the glucose‐starvation stimulon.
Dual promoters of the Listeria monocytogenes prfA transcriptional activator appear essential in vitro but are redundant in vivo
Authors: Freitag Nancy E. , Portnoy Daniel A. 
Published: October 2006

SummaryThe PrfA transcriptional activator is an essential determinant of Listeria monocytogenes pathogenesis. prfA expression is governed by three differentially regulated promoters: prfAP1 and prfAP2, which are located immediately upstream of prfA coding sequences, and the adjacent plcA promoter via the generation of a plcA–prfA read‐through transcript. A series of promoter deletion mutants were constructed to assess the roles of prfAP1 and prfAP2. Elimination of either prfAP1 or prfAP2 resulted in altered regulation of PrfA‐regulated genes after in vitro growth. However, these mutants were fully virulent both in an animal model and in tissue culture models of infection, suggesting that the two prfA promoters are functionally redundant in vivo. In contrast, a mutant lacking both prfAP1 and prfAP2 was 100‐fold less virulent and was delayed In escape from the host vacuole. Once in the host cytoplasm, however, the double mutant was apparently normal in cell‐to‐cell spread.
A‐factor as a microbial hormone that controls cellular differentiation and secondary metabolism in Streptomyces griseus
Authors: Horinouchi Sueharu , Beppu Teruhiko 
Published: October 2006

SummaryA‐factor, containing a γ‐butyrolactone in its structure, is an autoregulatory factor or a‘microbiai hormone’controlling secondary metabolism and cellular differentiation in Streptomyces griseus. A‐factor exerts its regulatory role by binding to a specific receptor protein which, in the absence of A‐factor, acts as a repressor‐type regulator for morphological and physiological differentiation, in the signal relay leading to streptomycin production in S. griseus, the A‐factor signal is transferred from the A‐factor receptor to the upstream activation sequence of a regulatory gene, strR, in the streptomycin biosynthetic gene cluster via an A‐factor‐dependent protein that serves as a transcription factor for strR. The StrR protein thus Induced appears to activate the transcription of other streptomycin‐production genes. The presence of A‐factor homologues in a wide variety of Streptomyces species and distantly related bacteria implies the generality of γ‐butyrolactones as chemical cellular signalling molecules in microorganisms.
Rhs elements of Escherichia coli: a family of genetic composites each encoding a large mosaic protein
Authors: Hill Charles W. , Sandt Carol H. , Vlazny Donald A. 
Published: October 2006

SummaryThe Rhs family comprises a set of composite elements found in the chromosomes of many natural Escherichia coli strains. Five Rhs elements occur in strain K‐12. The most prominent Rhs component is a giant core open reading frame (core ORF) whose features are suggestive of a cell surface ligand‐binding protein. This hypothetical protein contains a peptide motff, xxGxxxRYxYDxxGRL(I or T)xxxx, that is repeated 28 times. A similar repeated motif is found in a Bacillus subtilis wall‐associated protein. The Rhs core ORFs consist of two distinct parts: a large N‐terminal core that is conserved in all Rhs elements, and a smaller C‐terminus that Is highly variable. Distinctive G+C contents of Rhs components indicate that the elements have a recent origin outside the E. coli species, and that they are composites assembled from segments with very different evolutionary histories. The Rhs cores fail into three sub‐families that are mutually more than 20% divergent Downstream of the core ORF is a second, much shorter ORF. Like the adjacent core extension, these are highly variable. In most examples, the hypothetical product of this ORF has a candidate signal sequence for transport across the cytoplasmic membrane. Another Rhs component, the 1.3 kb H‐rpt, has features typical of insertion sequences. Structures homologous to H‐rpt have been detected in other bacterial genera, such as Vibrio and Salmonella, where they are associated with loci that determine O‐antigen variation.
Unique fimbriae‐like structures encoded by sefD of the SEF14 fimbrial gene cluster of Salmonella enteritidis
Authors: Clouthler S. C. , Collinson S. K. , Kay W. W. 
Published: October 2006

SummaryThe SEF14 gene cluster of Salmonella enteritidis was recently shown to contain three genes, sefABC, encoding a unique fimbrin, and proteins homologous to fimbrial chaperones and outer membrane proteins (ushers), respectively. A fourth open reading frame, designated sefD, was found immediately downstream of sefABC and overlapping sefC. The translated protein sequence of sefD was unique, but the composition was similar to that of other bacterial fimbriae. SefD was produced in abundance by wild‐type S. enteritidis as shown by Western blot analysis using antibodies raised to affinity‐purified, recombinant SefD. Furthermore, unusually long, thin, fimbriae‐like structures were evident on S. enteritidis and Escherichia coli by immunoelectron microscopy, but in other bacterial species SefD was expressed as amorphous material. Therefore, in S. enteritidis and E. coli, SefD is the predominant structural subunit of SEF18. The SEF18 fimbriae‐like structures were shown to be serologically distinct from the three known S. enteritidis fimbriae SEF14, SEF17 and SEF21. Furthermore, SEF18 was still produced in set A insertion mutants, indicating that SEF14 and SEF18 were structurally distinct. Thus, the SEF14 gene cluster is the first example in the Enterobacteriaceae of a gene cluster that encodes two fimbrin‐like proteins, which are assembled into two distinct cell‐surface structures, SEF14 and SEF1B. DNA hybridization and Western blot analyses showed that SefD was widely distributed among the Enterobacteriaceae and was present in E. coli, Shigella, Enterobacter, Citrobacter, Erwinia, Hafnia, Klebsiella, Providencia, and Proteus but not in the non‐Enterobacteriaceae Gram‐negative bacteria Pseudomonas and Aeromonas, or in Gram‐positive bacteria Bacillus or Staphylococcus. Immunoelectron microscopy revealed that sefD was also present on the surface of Providencia and Klebsiella but did not appear filamentous. This is the first instance of highly conserved, thin fimbriaelike structers which are ubiquitous among the Enterobacteriaceae.
The functional units of a peptostreptococcal protein L
Authors: Murphy Jonathan P. , Duggleby Clive J. , Atkinson Max A. , Trowern Angus R. , Atkinson Tony , Goward Christopher R. 
Published: October 2006

SummaryProtein L is a cell‐surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin‐binding units have high amino acid sequence identity with the albumin‐binding units of streptococcal cell‐surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units.
A Reb1p‐binding site is required for efficient activation of the yeast RAP1 gene, but multiple binding sites for Rap1p are not essential
Authors: Graham Ian R. , Chambers Alistair 
Published: October 2006

SummaryThe Saccharomyces cersvislae RAP1 protein (Rap1p) is a key multifunctional transcription factor. Using gel retardation analysis, four binding sites for RAP1p have been identified within the promoter of the RAP1 gene. These sites are located downstream of a binding site for the transcription factor Reb1p. The Reb1p site and an associated AT‐rich region are important for transcriptional activation, but deletion of three of the RAP1 p‐binding sites had little effect on promoter activity. The activity of the RAP1 promoter has been analysed in a yeast strain (YDS410) that contains a temperature‐sensitive mutation In the RAP1 gene. This mutation renders the DNA‐binding activity of Rapip temperature dependent. When YDS410 was grown at a semi‐permissive temperature (30°C), the activity of the RAP1 promoter increased by approximately 170%, compared with the same strain grown at the permissive temperature (25°C). A RAP1 promoter in which three of the four RAP1 p‐binding sites had been deleted, showed only a small increase in activity in the same experiment. These data confirm that Rap1p is not required for activation of the RAP1 gene, and suggest a role for Rap1p In negative auto‐regulation.
Genetic and molecular characterization of capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 3
Authors: Dillard Joseph P. , Yother Janet 
Published: October 2006

SummaryTo achieve a better understanding of the genetics of capsular polysaccharide synthesis in Streptococcus pneumoniae, we have identified and characterized mutants deficient in type 3 capsule production. We Identified a clone that restored encapsulation in one of our mutants and in a mutant deficient in UDP‐glucose dehydrogenase. By hybridization, we developed a chromosomal map of the type 3‐specific region and identified a flanking region containing DNA common to all capsule types examined. Insertion mutations were used to identify chromosomal loci required for capsule synthesis, and to map transcription within the region. Using non‐destructive insertions linked to type‐specific genes of type 2, 3, or 5, we were able to select for the transformation of all necessary genes specific for capsule type. Our data provide molecular evidence to show that all the type‐specific genes are linked in a cassette and can be transferred as a unit during transformation.
Bacterial binding protein‐dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins
Authors: Saurin William , Köster Wolfgang , Dassai Elle 
Published: October 2006

SummaryBacterial binding protein‐dependent transport systems belong to the superfamily of ABC transporters, which is widely distributed among living organisms. Their hydrophobic membrane proteins are the least characterized components. The primary structures of 61 integral membrane proteins from 35 uptake systems were compared in order to characterize a short conserved hydrophilic segment, with a consensus EAA … G ………‐I ‐ LP, located approximately 100 residues from the C‐terminus. Secondary structure predictions indicated that this conserved region might be formed by two amphipathic α‐helices connected by a loop containing the invariant G residue. We classified the conserved motifs and found that membrane proteins from systems transporting structurally related substrates specifically display a greater number of identical residues in the conserved region. We determined a consensus for each class of membrane protein and showed that these can be considered as signatures.
Light‐regulated promoters from Synechocystis PCC6803 share a consensus motif involved in photoregulation
Authors: Marraccini Pierre , Cassier‐Chauvat Corinne , Bulteau Stéphane , Chavez Sebastian , Chauvat Franck 
Published: October 2006

SummaryA library of Synechocystis PCC6803 (S.6803) DNA cioned in front of the promoterless cat reporter gene of the plasmid pFF11 was used to transform S.6803 to high light‐dependent resistance to chloramphenicol. In five clones harbouring a stably replicating pFF11‐derived plasmid, this phenotype occurred independently of the photosystem II electron transport and resulted from the correlated increase of CAT activity level and cat mRNA accumulation. The five promoter inserts contained no Escherichia collω70 promoter element, in agreement with their lack of activity in this organism, but shared two conserved motifs. Two secondary mutations, which restored light‐regulated promoter activity to an inactive mutant of the smallest insert, mapped within one of the common motifs, emphasizing the probable involvement of this element in photoregulation.
A topological model for the high‐affinity nickel transporter of Alcaligenes eutrophus
Authors: Eitinger Thomas , Friedrich Bärbel 
Published: October 2006

SummaryThe gene hoxN of Alcaligenes eutrophus encodes a membrane protein with a molecular mass of 33.1 kDa that mediates energy‐dependent uptake of nickel ions. Based on the hydrophobicity of the HoxN protein five, six, or seven transmembrane segments were predicted, depending on the algorithm used for computer analysis. To distinguish between these possibilities varying segments of the amino‐terminal end of the transporter were fused to the Escherichia coli enzymes aikaline phosphatase (PhoA) or β‐galactosidase (LacZ). The enzymatic activity of 16 HoxN‐PhoA and 15 HoxN‐LacZ fusions was determined. On the assumption that PhoA fusions only exhibit high activity when fused to periplasmic domains of the target, while LacZ fusions are only active when oriented towards the cytoplasm, a two‐dimensional model for the nickel transporter was developed. This model proposes that HoxN contains four periplasmic and four cytoplasmic regions, and seven transmembrane helices. The amino terminus is located in the cytoplasm, and the carboxyl terminus faces the periplasm.
Transcriptional control of toxT, a regulatory gene in the ToxR regulon of Vibrio cholerae
Authors: Higgins Dairen E. , DiRita Victor J. 
Published: October 2006

SummaryCo‐ordinate expression of many virulence genes in Vibrio cholerae is under the control of the ToxR and ToxT proteins. These proteins function in a regulatory cascade in which ToxR is required to activate toxT, and ToxT activates virulence genes. The precise mechanism for ToxR activation of toxT is unknown, but data presented in this report suggest a direct involvement of ToxR. Primer extension and gene fusion analyses identified a ToxR‐regulated promoter directly upstream of toxT, immediately following a region of inverted repeats capable of terminating transcription. Gel mobility shift studies indicate that ToxR binds DNA within the inverted repeat region, yet preliminary evidence suggests that ToxR binding alone is not sufficient for activation of toxT. Possible mechanisms of ToxR‐dependent toxT expression are discussed.
Mutational analysis of the pseudoknot structure of the S15 translational operator from Escherichia coli
Authors: Bénard L. , Philippe C. , Dondon L. , Grunberg‐Manago M. , Ehresmann B. , Ehresmann C. , Portier C. 
Published: October 2006

SummaryExpression of rpsO, the gene encoding the small ribosomal protein S15, is autoregulated at the translational level by S15, which binds to its mRNA in a region overlapping the ribosome‐binding site. By measuring the effect of mutations on the expression of a translational rpsO‐lacZ fusion and the S15 binding affinity for the translational operator, the formation of a pseudoknot in the operator site in vivo is fully demonstrated and appears to be a prerequisite for S15 binding. The mutational analysis suggests also that specific determinants for S15 binding are located in very limited regions of the structure formed by the pseudoknot. It is deduced that a specific pseudoknot conformation is a key element for autoregulation.