bluuebunny/crossref_metadata_2025_split huggingface dataset:
Some of the out genes involved in the secretion of pectate lyases in Erwinia chrysanthemi are regulated by kdgR
Authors: Condemine Guy , Dorel Corinne , Hugouvieux‐Cotte‐Pattat Nicole , Robert‐Baudouy Janine 
Published: October 2006

SummaryThe out genes of Erwinia chrysanthemi are required for the translocation across the outer membrane of pectate lyases and cellulases. We present the characterization and the nucleotide sequence of five genes of the out cluster. The products of outS, B, C, D and E have significant homology with the Puts, B, C, D and E proteins necessary to the secretion of pullulanase in Klebsiella pneumoniae. An open reading frame, outT, located between outB and outC has no homology with the pul cluster but is involved in secretion. cute, outD and outE form an operon while outS, outB and outT constitute independent transcription units. outT and the outCDE operon are regulated by kdgR, the negative regulatory gene controlling pectinase production. outB and outS seem to be expressed constitutively.
The bgI1 gene encoding extracellular β‐glucosidase from Trichoderma reesei is required for rapid induction of the cellulase complex
Authors: Fowler Tim , Brown Ross D. 
Published: October 2006

SummaryWe have used a targeted gene deletion event to remove the coding region for the bgli gene encoding an extracellular β‐glucosidase from the genome of the cellulolytic fungus Trichoderma reesei. The bgli null mutants were used to investigate the role of [1‐glucosidase in the hydrolysis of cellulose and induction of the other cellulolytic enzyme components. In the absence of extracellular βglucosidase, growth of bgl1 null strains on several carbon sources was the same as that of the parent (as measured by mycelial dry weight). However, levels of extracellular protein and total endoglucanase production were seen to lag relative to those levels observed in the control strain. The mRNA levels of the CBHI, CBHII, EGI, and EGII cellulase genes (cbh1, cbh2, egli and egl3) showed a corresponding lag in induction, suggesting that the absence of extracellular β‐glucosidase has an effect on the co‐ordinate regulation of the other cellulase genes at the level of transcription. The addition of a potent inducer of the cellulase complex (sophorose) resulted in normal rates of cellulase gene mRNA production and extracellular protein release. This indicates that the absence of β‐glucosidase is not affecting some intrinsic cellular ability to produce mRNA or secrete protein. These data suggest that a functional β‐glucosidase is at feast partially responsible for the efficient induction of the depolymerase enzymes of the cellulase complex. The observation that the cellulase complex is induced, albeit after a lag, suggests that other enzymes are present that can substitute for the function of β‐glucosidase during induction.
Targeted gene replacements in a Streptomyces polyketide synthase gene cluster: role for the acyl carrier protein
Authors: Khosla Chaitan , Ebert‐Khosla Susanne , Hopwood David A. 
Published: October 2006

SummaryA methodology was developed to construct any desired chromosomal mutation in the gene cluster that encodes the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2). A positive selection marker (resistance gene) is first introduced by double crossing‐over into the chromosomal site of interest by use of an unstable delivery plasmid. This marker is subsequently replaced by the desired mutant allele via a second high‐frequency double recombination event. The technology has been used to: (i) explore the significance of translational coupling between two adjacent PKS genes; (ii) prove that the acyl carrier protein (ACP) encoded by a gene in the cluster is necessary for the function of the actinorhodin PKS; (iii) provide genetic evidence supporting the hypothesis that serine 42 is the site of phosphopantetheinylation in the ACP of the actinorhodin PKS; and (iv) demonstrate that this ACP can be replaced by a Saccharopolyspora fatty acid synthase ACP to generate an active hybrid PKS.
The HsdS polypeptide of the Type IC restriction enzyme Eco R124 is a sequence‐specific DNA‐binding protein
Authors: Kusiak Michelle , Price Clive , Rice David , Hornby David P. 
Published: October 2006

SummaryThe HsdS and HsdM polypeptides of the type IC restriction enzyme Eco R124 have been purified independently and used in a set of gel retardation experiments to determine the minimum requirements for sequence‐specific recognition of DNA by this enzyme. The HsdS polypeptide alone is able to bind to DNA in a sequence‐specific manner. In addition, whilst the presence of the HsdM polypeptide gives rise to a stimulation of DNA binding by the HsdS subunit it is not clear whether, under the conditions of the experiments reported here, the HsdS subunit maintains the same interactions with the HsdM subunits observed in the absence of DNA.
Peroxisome biogenesis in yeast
Authors: Aitchison John D. , Nuttley William M. , Szilard Rachel K. , Brade Anthony M. , Glover John R. , Rachubinski Richard A. 
Published: October 2006

SummaryEukaryotic cells have evolved a complex set of intracellular organelles, each of which possesses a specific complement of enzymes and performs unique metabolic functions. This compartmentalization of cellular functions provides a level of metabolic control not available to prokaryotes. However, it presents the eukaryotic cell with the problem of targeting proteins to their specific location (s). Proteins must be efficiently transported from their site of synthesis in the cytosol to their specific organelle (s). Such a process may require translocation across one or more hydrophobic membrane barriers and/or asymmetric integration into specific membranes.Proteins carry cis‐acting amino acid sequences that serve to act as recognition motifs for protein sorting and for the cellular translocation machinery. Sequences that target proteins to the endoplasmic reticulum/ secretory pathway, mitochondria, and chloroplasts are often present as cleavable amino‐terminal extensions. In contrast, most peroxisomal proteins are synthesized at their mature size and are translocated to the organelle without any post‐translational modification. This review will summarize what is known about how yeast solve the problem of specifically importing proteins into peroxisomes and will suggest future directions for investigations into peroxisome biogenesis in yeast.
Structure of selenocysteine synthase from Escherichia coli and location of tRNA in the seryl‐tRNAsec–enzyme complex
Authors: Engelhardt Harald , Forchhammer Karl , Müller Shirley , Goldie Kenneth N. , Böck August 
Published: October 2006

SummarySelenocysteine synthase of Escherichia coli catalyses the biosynthesis of selenocysteine in the form of the aminoacyl‐RNA complex, the reaction intermediate being aminoacrylyl‐tRNAsec covalently bound to the prosthetic group of the enzyme. Selenocysteine synthase and the specific aminoacrylyl‐tRNA sec‐enzyme complex as well as the isolated seryl‐tRNAsec were investigated in the electron microscope and analysed by means of image processing to a resolution of 2 nm in projection. The stoichiometric composition of the selenocysteine synthase molecule was elucidated by scanning transmission electron microscopic mass determination. The enzyme has a fivefold symmetric structure and consists of 10 monomers arranged in two rings. The tRNA is bound near the margin of the dimeric subunits. Principal component analysis of the tRNA‐enzyme complexes revealed that the selenocysteine synthase appears to bind only one seryl‐tRNAsec per dimer, which is consistent with the result of biochemical binding studies.
Identification of variable region differences in Neisseria meningitidis class 3 protein sequences among five group B serotypes
Authors: Zapata Gerardo A. , Vann Willie F. , Rubinstein Yaffa , Frasch Carl E. 
Published: October 2006

SummaryStrains of Neisseria meningitidis express one of two porin proteins. These porins have been identified as the class 2 and class 3 proteins, and express serotype‐specific epitopes. The gene for the class 3 protein was amplified by the polymerase chain reaction from the DNA of a serotype 4 strain as a 1025bp fragment. The nucleotide sequence of this gene was determined and compared with two recently published sequences. On the basis of this comparison we have identified two major variable regions in the translated protein sequence, VR1 and VR2, that may be associated with serotype specificity. Three other variable regions were also identified. The sequences in the VR1 and VR2 regions from five additional group B N. meningitidis strains of serotypes 1, 4, 8,12, and 15, all expressing class 3 proteins, were determined. The VR1 and VR2 regions were variable and were flanked by highly conserved regions among eight different class 3 sequences. These two variable regions of 15 and 9 amino acids are predicted to be in surface‐exposed loops.
Characterization of the minimal origin required for replication of the streptococcal plasmid plP501 in Bacillus subtilis
Authors: Brantl S. , Behnke D. 
Published: October 2006

SummaryBy using deletional analysis the origin of replication, oriR, of the streptococcal plasmid plP501 in Bacillus subtilis has been mapped at a position immediately downstream of the repR gene. Determination of both the right and left border of oriR allowed the definition of a sequence of a maximum of 52 nucleotides which theoretically constitutes the minimal origin of replication. Recently, the start point of leading‐strand synthesis of the closely related plasmid pAMβ1 has been mapped at a position which is located exactly in the middle of this sequence (Bruand et al., 1991). The function of ohR did not depend on its location downstream of the repR gene. Translocation of oriR containing fragments to other regions of the plasmid proved to be possible. The smallest translocated fragment that still reconstituted autonomous replication was 72bp in size. This fragment was also active in directing the replication of an Escherichia coli plasmid in B. subtilis when the RepR protein was supplied in trans from a repR gene integrated into the host chromosome. The transformation efficiency of plasmids carrying translocated oriR fragments showed a certain dependence on the fragment length and orientation. The DNA sequence of oriR included an inverted repeat, both branches of which appeared to be essential for oriR function. The repeats of oriR shared sequence similarity with a repeat located upstream of promoter pll, which has been suggested to be involved in autoregulation of repR expression.
Secretion of the STA3 heat‐stable enterotoxin of Escherichia coli: extracellular delivery of Pro‐STA is accomplished by either Pro or STA
Authors: Yang Yuan , Gao Zeren , Guzmán‐Verduzco Luz‐Maria , Tachias Kathy , Kupersztoch Yankel M. 
Published: October 2006

SummaryThe methanol‐soluble, heat‐stable enterotoxin of Escherichia coli is a protease‐resistant extracellular peptide which is synthesized as a 72‐amino‐acid precursor Pre‐Pro‐STA3. The specific roles of Fre (19 amino acids), Pro (34 amino acids) and STA3 (19 amino acids) in the secretion process were studied by functionally deleting each of the three domains. Deletion of the Pre signal sequence resulted in a short‐lived cell‐associated molecule with an Mr equivalent to that of Pro‐STA3. Deletion of Pro (i.e., Pre‐STA3) resulted in the rapid extracellular accumulation of STA3 the periplasmic intermediate found in the secretion of the wild‐type toxin was undetected. Deletion of the STA3 domain resulted in a cell‐associated Pre‐Pro peptide; with time this form converted to periplasmic Pro which later became extracellular. When DNA encoding either STA3, by itself, or Pro‐STA3 (lacking the signal peptide) was expressed, these peptides were degraded intracellularly, with no periplasmic or extracellular forms detected.The results presented demonstrate that the signal peptide (Pre) is essential even for the export of small peptides to the periplasm, and that its absence causes the STAS domain to become susceptible to intracellular proteases. The rapid degradation of intracetlular STA3 indicates that its proteolytic resistance is acquired in a compartment other than the cytoplasm. The results also show that after the Pre domain is proteolytically cleaved from Pre‐STA3 and Pre‐Pro, the STA3 and Pro peptides can exit to the culture supernatant. Since STA3 and Pro have neither sequence nor conformational homology, it appears
Structure and biosynthesis of unbranched multicopy single‐stranded DNA by reverse transcriptase in a clinical Eschehchia coli isolate
Authors: Lim Dongbin 
Published: October 2006

SummaryIt has been shown that retrons, retro‐elements in bacteria, produce a reverse transcriptase (RT) and multicopy single‐stranded DNA(msDNA) whose 5′ end is covalently linked to RNA (msdRNA) by a 2′‐5′ phosphodiester bond. Here, I show that a retron in clinical Escherichia coli strain 161 produces an msDNA unlinked to RNA. The msDNA produced by this retron is a 79‐nucleotlde‐long single‐stranded DNA with monophosphate on its 5′ terminus. When the retron in strain 161 is cloned into E. coli K‐12, the majority of msDNA produced in the clone is the same as the msDNA in the clinical strain. However, in the K‐12 clone, about 10% of the msDNA produced is present as a DNA covalently linked to RNA. The DNA part of this RNA‐DNA compound is an 83 nucleotides long with the same sequence as the unbranched msDNA, except for the presence of four additional nucleotides at the 5′ side. From the analysis of the RNA‐DNA compound and the results of in vitro synthesis, I show that the primary product of reverse transcription in this retron is an 83‐nucleotide‐tong DNA covalently linked to RNA. This RNA‐DNA compound is further processed to the final product, the 79‐nucleotide‐long msDNA with a terminal 5′ monophosphate, by an endonucleolytic cleavage between the fourth and fifth positions of the DNA component of the RNA‐DNA compound. The minimum region required for the production of such msDNA free of RNA contains only genes known to be required for the synthesis of branched msDNA‐RNA compound in other retrons (msd, msr and ret). This suggests that either the RT has an endonuclease activity or that the msDNA‐RNA compound is autoca‐talytically processed.
Streptomycetes possess peptidyl‐prolyl cis‐trans isomerases that strongly resemble cyclophilins from eukaryotic organisms
Authors: Pahl Andreas , Ühlein Monika , Bang Holger , Schlumbohm Wilhelm , Keller Ullrich 
Published: October 2006

SummaryA functionally active 17.5 kDa peptidyl‐prolyl cis‐trans isomerase was purified to homogeneity from Streptomyces chrysomallus, a Gram‐positive filamentous bacterium. Characterization of the enzyme revealed inhibition and binding characteristics, against the immunsuppressive drug cyclosporin A, which were similar to cyclophilins from eukaryotes such as mammals, plants, fungi and yeasts, but different from those of cyclophilins from enterobacteria such as Escherichia coli. The amino acid sequence of the S. chrysomallus cyclophilin, as deduced from the gene sequence, revealed a striking degree of amino acid sequence identity with the corresponding 17 kDa proteins of humans (66%), Neurospora (70%) and yeast (69%). Comparison with cyclophilin sequences from the Gram‐negative enterobacteria revealed much iess homoiogy (25% identity with E. coli b, 23% identity with E. coli a). Cyclophilin was detected in each of the four other Streptomyces species tested. The cyclophilins from the various streptomycetes differed in size, varying between 17 and 20.5 kDa. The cyclophilins were abundant in the Streptomyces cells, and present throughout growth.
Regulation of the Escherichia coli uncH gene by mRNA secondary structure and translational coupling
Authors: Pati Sushma , DiSilvestre Deborah , Brusilow William S. A. 
Published: October 2006

SummaryThe uncH gene is one of the most poorly‐expressed genes of the proton‐translocating ATPase (unc) operon of Escherichia coli. We constructed in‐frame lacZ fusions to uncH and used site‐directed mutagen‐esis to decrease the stability of the putative mRNA secondary structure in the Shine and Dalgarno region for this gene. These mutations significantly increased the expression of uncH. We also used the unc‐lac fusions to show that the insertion of stop codons and a frameshift mutation In uncF, the gene preceding uncH, caused a 10‐fold reduction in uncH expression. Hybridization of total cellular RNA with a lacZ‐specific probe indicated that transcriptional polarity could not account for the observed decrease in gene expression. These results demonstrate that uncH expression is controlled by mRNA sequences around the translational initiation region, and is translationally coupled to uncF, even in cases where the putative mRNA secondary structure is weakened or eliminated.
Identification of functional cis‐acting sequences involved in regulation of narK gene expression in Escherichia coli
Authors: Bonrtefoy Violaine , DeMoss John A. 
Published: October 2006

SummaryExpression of the narK gene of Escherichia coli, like the narGHJI operon, is positively regulated by two trans‐acting factors: Fnr, which is activated by anaerobic conditions, and NarL, which is activated by the presence of nitrate. Unlike the narGHJI operon, the 5′ untranslated region of the narK gene contains two putative Fnr‐binding‐site sequences and two putative NarL‐binding‐site sequences. To define the role of these putative cis‐acting regions, transcription start sites were identified and the effects of promoter region modifications on transcription were determined. Primer extension analysis identified several transcripts for the narK gene expressed from plasmids. Expression from the major promoter, P1, was induced by anaerobic growth conditions and further elevated in the presence of nitrate, while that from a weaker promoter, P2, appeared to be constitutive. The position of the major transcription start site placed one of the putative Fnr‐binding sites (Fnr1 box) and one of the NarL‐binding sites (NarL2 box) at positions analogous to those previously established for the narGHJI operon promoter region, while the other two binding sites were located in the non‐homologous 150 bp sequence which separates the Fnr1 and NarL2 boxes. Based on the effects of selective 5′ deletions and site‐directed modifications, Fnr‐dependent expression was dependent only on the Fnr1 box and nitrate stimulation was dependent on the presence of the NarL2 box. In the absence of the NarL2 box, the NarLI box did not promote stimulation by nitrate. The Fnr2 box was not required for anaerobic induction of expression but its modification appeared to reduce the level of stimulation by nitrate.
In vivo generation of linear plasmids with addition of telomeric sequences by Histoplasma capsulatum
Authors: Woods Jon P. , Goldman William E. 
Published: October 2006

Summary Histoplasma capsulatum is a dimorphic pathogenic fungus that is a major cause of respiratory and systemic mycosis. We previously developed a transformation system for Histoplasma and demonstrated chromosomal integration of transforming plasmid sequences. In this study, we describe another Histoplasma mechanism for maintaining transforming DNA i.e. the generation of modified, multicopy linear plasmids carrying DNA from the transforming Escherichia coli plasmid. Under selective conditions, these linear plasmids were stable and capable of retransforming Histoplasma without further modification. In vivo modification of the transforming DNA included duplication of plasmid sequence and telomeric addition at the termini of linear DNA. Apparently Histoplasma telomerase, like that of other organisms such as humans and Tetrahymena, is able to act on non‐telomeric substrates. The terminus of a Histoplasma linear plasmid was cloned and shown to contain multiple repeats of GGGTTA, the telomeric repeat unit also found in vertebrates, trypanosomes, and slime moulds.
The gene encoding the cellulase (Avicelase) Cell from Streptomyces reticuli and analysis of protein domains
Authors: Schlochtermeier André , Walter Stefan , Schröder Johannes , Moorman Michael , Schrempf Hildgund 
Published: October 2006

Summary Streptomyces reticuli produces an unusual cellulase (Avicelase), with an apparent molecular weight of 82 kDa, which is solely sufficient to degrade crystalline cellulose. During cultivation the processing of the Avicelase to a truncated enzyme (42 kDa) and an inactive protein (40 kDa) correlated with the occurrence of an extracellular protease. After its purification this 36 kDa protease cleaved the S. reticuli Avicelase in vitro in the same manner. Using antibodies raised against the Avicelase and its truncated form (42 kDa) and gene libraries of S. reticuli DNA in the Escherichia coli phage vectors lambda gt11 and Charon 35, the Avicelase gene (cel1) was identified. Further subcloning and DNA‐sequencing revealed a G+C rich (72%) reading frame of 2238 bp encoding a protein of 746 amino acids. The transcriptional start site was mapped about 180bp upstream from the GTG start codon. A signal sequence of 29 amino acids was identified by aligning the deduced amino acids with the characterized N‐terminus of the 82 kDa Avicelase. Comparison of the W‐terminal amino acids from the purified proteins with the amino acid sequence derived from the Avicelase gene revealed that the truncated enzyme (42 kDa) corresponds to the C‐terminal region whereas the inactive proteolytically derived protein (40 kDa) represents the N‐terminal part of the 82 kDa Avicelase.Comparisons with amino acid sequences deduced from known cellulase genes indicated the presence of three putative protein domains: (i) an N‐terminal part showing significant similarity with a repeat region of endoglucanase C from Cellulomonas fimi, recently shown to be a cellulose‐binding domain; (ii) an adjoining region sharing homology with the N‐terminal domains with unknown function of endoglucanase A from Pseudomonas fluoresoens, endoglucanase D from Clostridium thermocellum and cellodextrinase from Butyrivibrio fibrosolvens, and (iii) a C‐terminal catalytic domain belonging to cellulose family E.
Detergent‐shock response in enteric bacteria
Authors: Nickerson Kenneth W. , Aspedon Arden 
Published: October 2006

SummaryOur work on bacterial detergent resistance started with the realization that bacteria growing in a sink full of soap must be resistant to the detergents in that soap. We chose sodium dodecyl sulphate (SDS) as a model detergent and decided to see how much SDS the bacterium growing in the sink could tolerate. The research program thus initiated has shown that bacteria such as Enterobacter cloacae can grow in up to 25% SDS and that SDS‐shock proteins constitute c. 8% of the proteins synthesized by SDS‐grown Escherichia coli. It has also provided explanations why enteric bacteria are oxidase negative, and how pyrroloquinoline quinone (POQ) enters the periplasmic space. Finally, for E. coli, it has provided evidence for an alternate, phosphate‐limited, aquatic life style which places greater emphasis on the Entner‐Doudoroff pathway. Detergent resistance is important both medically and ecologically, e.g. entry of pathogens via bile‐salt‐containing intestinal tracts and biodegradation of detergent‐like pollutants such as those resulting from oil spills. Our current research is focused on SDS‐induced modifications of the cytoplasmic membrane and the presence of SDS in the periplasm.