bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of a cis‐acting mutation to study the role of FLP‐mediated recombination in the maintenance of native yeast 2μm plasmids
Authors: Morrissey Julie A. , Cashmore Annette M.
Published: October 2006

The 2μm plasmid encodes a mechanism that ensures the partitioning of the plasmid at cell division. Little is known about the detailed mechanism of this partitioning system; for example, is there equal or unequal distribution of the plasmid molecules at mitosis? The plasmid also encodes a site‐specific recombination system that is thought to be involved in plasmid copy‐number amplification, although to date there has been no direct evidence that the recombination process itself is important for maintenance. We have identified a natural 2μm variant that has a cis‐acting mutation in the FLP‐mediated recombination system. We show that this plasmid is unable to amplify in vivo. Our results demonstrate that the average copy number per cell is not affected for the mutant but there is a large clonal variation. This is a direct demonstration that plasmid partitioning results in an unequal distribution of plasmids and that FLP‐mediated amplification compensates for this and therefore has an important role in maintenance.
Molecular and symbiotic characterization of exopolysaccharide‐deficient mutants of Rhizobium tropici strain CIAT899
Authors: Milner Jocelyn L. , Araujo Ricardo S. , Handelsman Jo
Published: October 2006

We studied the symbiotic behaviour of 20 independent Tn5 mutants of Rhizobium tropici strain CIAT899 that were deficient in exopolysaccharide (EPS) production. The mutants produced non‐mucoid colonies, were motile, grew in broth cultures at rates similar to those of the parent, and produced significantly less EPS than did CIAT899 in broth culture. A genomic library of strain CIAT899, constructed in pLA2917, was mobilized into all of the mutants, and cosmids that restored EPS production were identified. EcoRI restriction digests of the cosmids revealed nine unique inserts. Mutant complementation and hybridization analysis showed that the mutations affecting EPS production fell into six functional and physical linkage groups. On bean, the mutants were as efficient in nodulation and as effective in acetylene reduction as strain CIAT899, induced a severe interveinal chlorosis, and all but one were less competitive than CIAT899. On siratro, CIAT899 induced nodules that were ineffective in acetylene reduction, whereas the EPS‐deficient mutants induced effective nodules. Microscopic examination of thin sections showed that nodules from both siratro and bean plants inoculated with either CIAT899 or an EPS‐deficient mutant contained infected cells. These data indicate that EPS is not required for normal nodulation of bean by R tropici, that it may contribute to competitiveness of R. tropici on bean, and that the loss of EPS production is accompanied by acquisition of the ability to reduce acetylene on siratro.
A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium: an anti‐sigma factor inhibits the activity of the flagellum‐specific Sigma factor, σF
Authors: Ohnishi K. , Kutsukake K. , Suzuki H. , Lino T.
Published: October 2006

We have studied the molecular mechanism of the negative regulation by flgM of the late operons of the flagellar regulon of Salmonella typhimurium. A 7.8 kDa protein that was identified as the flgM gene product was purified to homogeneity; its amino‐terminal sequence was identical to the deduced sequence except for the lack of the initiating methionine. The purified FlgM repressed transcription from the fliC promoter, one that is activated by the sigma factor, FliA (σF). No DNA‐binding activity was detected in FlgM. Chemical cross‐linking experiments showed that the purified FlgM bound to σF and disturbed Its ability to form a complex with RNA polymerase core enzyme. These results indicate that FlgM is a novel type of negative regulator that probably inactivates the flagellum‐specific sigma factor through direct interaction, i.e. it is an anti‐sigma factor.
A putative anaerobic coproporphyrinogen III oxidase in Rhodobacter sphaeroides. I. Molecular cloning, transposon mutagenesis and sequence analysis of the gene
Authors: Coomber S. A. , Jones R. M. , Jordan P. M. , Hunter C. N.
Published: October 2006

A mutant of Rhodobacter sphaeroides, N1, has been isolated which is incapable of photosynthetic growth and, instead of synthesizing bacteriochlorophyll, N1 excretes coproporphyrin III into the growth medium. Using conjugative gene transfer, several clones were isolated from a R. sphaeroides gene library which restored normal pigment synthesis and photosynthetic growth to N1. Using transposon Tn5 mutagenesis, the gene was located to a 1.05 kb Eco RI fragment. Sequence and transcription analysis defined the position and expression of an open reading frame of approximately 920 bp, which is proposed as the anaerobic coproporphyrinogen III oxidase dedicated to bacteriochlorophyll biosynthesis.
A putative anaerobic coproporphyrinogen III oxidase in Rhodobacter sphaeroides. II. Analysis of a region of the genome encoding hemF and the puc operon
Authors: Gibson L.C.D. , McGlynn P. , Chaudhri M. , Hunter C.N.
Published: October 2006

The puc operon of Rhodobacter sphaeroides encoding polypeptides of the major light‐harvesting complex, LH2, has been found to be linked to hemF, a gene encoding a putative anaerobic coproporphyrinogen III oxidase. The puc‐hemF region of the R. sphaeroides genome has been investigated by insertional mutagenesis, complementation analysis of these insertional mutants and DNA sequencing. A third gene, designated pucC, has been found immediately downstream of pucA and has been shown to be essential for LH2 expression. pucC is cotranscribed with pucB and pucA; however, hemF and the pucBAC operon were found not to be transcriptionally linked. Ultrastructural studies indicated that the morphology of the intracytoplasmic membrane may depend upon expression of pucC as well as pucBA.
Cloning, mapping and nucleotide sequencing of a gene encoding a universal stress protein in Eschericha coli
Authors: Nyström Thomas , Neidhardt Frederick C.
Published: October 2006

The response of non‐differentiating bacteria to nutrient starvation is complex and includes the sequential synthesis of starvation‐inducible proteins. Although starvation for different individual nutrients generally provokes unique and individual patterns of protein expression, some starvation stimulons share member proteins. Two‐dimensional polyacrylamide gel electrophoresis revealed that the synthesis of a small (13.5 kDa) cytoplasmic protein in Escherichia coli was greatly increased during growth inhibition caused by the exhaustion of any of a variety of nutrients (carbon, nitrogen, phosphate, sulphate, required amino acid) or by the presence of a variety of toxic agents including heavy metals, oxidants, acids and antibiotics. To determine further the mode of regulation of the protein designated UspA (üniversal stress pUrotein A) we cloned the gene encoding the protein by the technique of reverse genetics. We isolated the protein from a preparative two‐dimensional polyacrylamide gel, determined its N‐terminal amino acid sequence, and used this sequence to construct a degenerate oligonucleotide probe. Two phages of the Kohara library were found to contain the gene which then was subcloned from the DNA in the overlapping region of these two clones. The amino acid sequence, deduced from the nucleotide sequence of the uspA gene, shows no significant homology with any other known protein. The uspA gene maps at 77 min on the E. coli W3110 chromosome, and is transcribed in a clockwise direction. The increase in the level of UspA during growth arrest was found to be primarily a result of transcriptional activation of the corresponding gene. The induction was independent of the RelA/SpoT, RpoH, KatF, OmpR, AppY, Lrp, PhoB and H‐NS proteins during stress conditions that are known to induce or activate these global regulators. The ‐10 and ‐35 regions upstream of the transcriptional start site of the uspA gene are characteristic of a σ70‐dependent promoter.
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

The 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

We 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

A 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

The 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

Eukaryotic 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

Selenocysteine 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

Strains 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

By 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

The 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

It 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

A 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

The 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

Expression 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

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

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

Our 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.
Global changes in gene expression related to antibiotic synthesis in Streptomyces hygroscopicus
Authors: Holt T. G. , Chang C. , Laurent‐Winter C. , Murakami T. , Garrels J. I. , Davies J. E. , Thompson C. J.
Published: October 2006

Two‐dimensional gel electrophoresis was used to follow low changes in gene expression associated with antibiotic (bialaphos) biosynthesis in Streptomyces hygroscopicus. Cultures were pulse‐labelled with [35S]‐methionine before, during, and after the switch from primary to secondary metabolism in order to compare kinetic profiles of bialaphos (antibiotic) production (bap) genes during this metabolic transition. Separation of gene products on two‐dimensional gels revealed that 27 were dependent on brpA for optimal expression and were activated as the culture approached stationary phase. Genes which encoded 10 brp4‐dependent proteins were mapped to a 10 kb Sstl fragment of the 35 kb bap gene cluster by expressing them in Streptomyces lividans using the thiostrepton‐inducible tipA promoter. N‐terminal amino acid sequences of two brpA‐dependent proteins, obtained by direct microsequencing of protein spots excised from two‐dimensional gels, identified them as gene products mapping to the same region and involved in secondary metabolic conversions of the bap pathway. The kinetics of synthesis of 16 brpA‐dependent gene products were characterized using QUEST computer software. Cluster analysis performed on the kinetics of synthesis of 346 of the most highly expressed gene products of HP5‐29, including 16 brpA‐dependent ones, identified 75 families having distinct patterns of expression. Many brpA‐dependent proteins were clustered together; 10 were found in one kinetic family. These kinetic families also included brpA‐independent gene products perhaps subject to similar regulatory mechanisms and thus possibly involved in bialaphos biosynthesis. The activation/derepression of bap expression took place as cultures approached stationary phase and was temporally related to synthesis of ppGpp.
Secretion of the Escherichia coli outer membrane proteins OmpA and OmpF in Bacillus subtilis is blocked at an early intracellular step
Authors: Puohiniemi Ritvaleena , Simonen Marjo , Muttilainen Susanna , Himanen Juha‐Pekka , Sarvas Matti
Published: October 2006

When the genes coding for the outer membrane (OM) proteins OmpA and OmpF of Escherichia coli are fused to a signal sequence of a bacillar exoenzyme and expressed in Bacillus subtilis they remain cell‐bound and the signal sequence is not cleaved. To identify the step of arrest in the export of these proteins we studied their accessibility to protease applied to intact protoplasts; they remained resistant indicating fully intracellular localization. Both proteins appeared associated with the cell membranes in sedimentation and flotation centrifugation experiments. However, OmpA and OmpF proteins synthesized in B. subtilis without a signal sequence were similarly associated with membranes in centrifugation experiments whereas electron microscopy showed the presence of intracytoplasmic inclusion bodies not obviously attached to the cytoplasmic membrane. We conclude that OmpA and OmpF proteins even when provided with a functional signal sequence do not enter the export pathway in B. subtilis, probably owing to lack of a specific export component in B. subtilis.
Cloning and sequence of a β‐tubulin cDNA from Pneumocystis carinii: possible implications for drug therapy
Authors: Dyer M. , Volpe F. , Delves C. J. , Somia N. , Burns S. , Scaife J. G.
Published: October 2006

This work describes the isolation and characterization of a full‐length cDNA clone encoding β‐tubulin from the pathogen Pneumocystis carinii, P. carinii contains a single gene encoding β‐tubulin. The complete sequence of this cDNA has been determined and its inferred amino acid sequence compared with the β‐tubulins from other organisms. This analysis augments the data indicating that P. carinii should be classified as a fungal organism. Further comparisons between the P. cariniiβ‐tubulin and those of fungal β‐tubulins resistant to benomyl, a β‐tubulin‐binding drug, indicate a difference which may be exploited in the development of a new drug therapy for P. carinii pneumonitis. These results suggest that, theoretically, a drug presently administered for treatment of nematode worm infections may be an effective agent against P. carinii, without being toxic to the mammalian host. This possibility is currently being investigated.
Elongation factor 3 (EF‐3) from Candida albicans shows both structural and functional similarity to EF‐3 from Saccharomyces cerevisiae
Authors: Colthurst D. R. , Schauder B. S. , Hayes M. V. , Tuite M.F.
Published: October 2006

As with many other fungi, including the budding yeast Saccharomyces cerevisiae, the dimorphic fungus Candida albicans encodes the novel translation factor, elongation factor 3 (EF‐3). Using a rapid affinity chromatography protocol, EF‐3 was purified to homogeneity from C. albicans and shown to have an apparent molecular mass of 128 kDa. A polyclonal antibody raised against C. albicans EF‐3 also showed cross‐reactivity with EF‐3 from S. cerevisiae. Similariy, the S. cerevisiae TEF3 gene (encoding EF‐3) showed cross‐hybridization with genomic DNA from C. albicans in Southern hybridization anaiysis, demonstrating the existence of a single gene closely related to TEF3 in the C. albicans genome. This gene was cloned by using a 0.7 kb polymerase chain reaction‐amplified DNA fragment to screen a C. albicans gene library. DNA sequence analysis of 200 bp of the cloned fragment demonstrated an open reading frame showing 51% predicted amino acid identity between the putative C. albicans EF‐3 gene and its S. cerevisiae counterpart over the encoded 65‐aminoacid stretch. That the cloned C. aibicans sequence did indeed encode EF‐3 was confirmed by demonstrating its ability to rescue an otherwise non‐viable S. cerevisiae tef3:HIS3 null mutant. Thus EF‐3 from C. albicans shows both structural and functional similarlity to EF‐3 from S. cerevisiae.
Mapping and characterization of the promoter elements of the regulatory nif genes rpoN, nifA1 and nifA2 in Rhodobacter capsulatus
Authors: Preker Pascal , Hübner Philipp , Schmehl Manfred , Klipp Werner , Bickle Thomas A.
Published: October 2006

The promoter elements responsible for the expression of the regulatory nif genes rpoN, nifA1 and nifA2 of Rhodobacter capsulatus were mapped by exonuclease‐III‐mediated deletions and by primer extension analysis. The rpoN promoter maps 600 bp upstream of rpoN and has the characteristic features of a –24/–12 promoter. The upstream activator sequence (UAS) displays two mismatches with the NIFA consensus sequence and is located 37 bp upstream of a perfect –24/–12 promoter element. The spacing and/or the helical phasing of these two promoter elements was found to be important for promoter function. In addition, an UAS half‐site may contribute to optimal promoter function. The rpoN UAS can partially substitute for the UAS of the nifE promoter. An open reading frame with homology to Klebsiella pneumoniae NIFU was identified between the rpoN promoter and rpoN and termed nifU2 since another nifU‐like gene (nifUl) is located in a conventional nifUSVW operon in nif region A. Thus, rpoN, encoding an alternative sigma factor for RNA polymerase, is cotranscribed with a nifU analogous gene from an rpoN‐dependent promoter. Mapping of the promoter elements involved in the expression of nif A copy 1 and copy 2 identified a novel promoter type. A conserved distal promoter element is likely to represent the binding site of NTRC in R. capsulatus. The DNA region preceding the mapped 5′ ends of the nif A transcripts displays much less homology. The distance between the distal and proximal elements is about 100 bp.
Three‐stranded DNA structure; is this the secret of DNA homologous recognition?
Authors: Stasiak Andrzej
Published: October 2006

A novel type of triple‐stranded DNA structure was proposed by several groups to play a crucial role in homologous recognition between single‐ and double‐stranded DNA molecules. In this still putative structure a duplex DNA was proposed to co‐ordinate a homologous single strand in its major groove side. In contrast to the well‐characterized pyrimidine‐purine‐pyrimidine triplexes in which the two like strands are antiparallel and which are restricted to poly‐pyrimidine‐containing stretches, the homology‐specific triplexes would have like strands in parallel orientation and would not be restricted to any particular sequence provided that there is a homology between interacting DNA molecules. For many years the stereo‐chemical possibility of forming homology‐dependent three‐ or four‐stranded DNA structures during the pairing stage of recombination reactions was seriously considered in published papers. However, only recently has there been a marked increase in the number of papers that have directly tested the formation of triple‐stranded DNA structures during the actual pairing stage of the recombination reaction. Unfortunately the results of these tests are not totally clear cut; while some laboratories presented experimental evidence consistent with the formation of triplexes, others studying the same or very similar systems offered alternative explanations. The aim of this review is to present the current state of the central question in the mechanism of homologous recombination, namely, what kind of DNA structure is responsible for DNA homologous recognition. Is it a novel triplex structure or just a classical duplex?
How bacterial protein toxins enter cells: the role of partial unfolding in membrane translocation
Authors: London Erwin
Published: October 2006

Bacterial protein toxins translocate across membranes by processes that are still mysterious. Studies on diphtheria toxin have shown that partial unfolding processes play a major role in toxin membrane insertion and translocation. Similar unfolding behaviour is seen with other bacterial toxins. The lessons gained from this behaviour allow us to propose novel mechanisms for toxin translocation.
Subtelomeric expression regions of Borrelia hermsii linear plasmids are highly polymorphic
Authors: Restrepo B. I. , Kitten T. , Carter C.J. , Infante D. , Barbour A. G.
Published: October 2006

Borrelia hermsii, a relapsing fever agent, undergoes multiphasic antigenic variation to evade its host's immune response. Serotype specificity is determined by variable membrane lipoproteins, Vmps, which are expressed from genes located near the end of a linear plasmid. Using the polymerase chain reaction and primers representing the promoter of the active vmp and a conserved telomeric sequence, we characterized the subtelomeric expression regions of the 25 known serotypes of strain HS1. The distance from the promoter to the telomere fell into three size classes of approximately 1.0, 1.5, and 2.5 kilobases. In the sequenced serotypes the size differences were accounted for by variable lengths of the vmp genes and intervening sequences between 3’end of the vmp gene and the start of a downstream homology block. The degree of nucleotide identity between different vmp genes, or between the different 3’flanking DNA varied from 39‐78%. Thus, there is length and sequence variability not only between vmp genes themselves but also between the 3’flanking regions of vmp genes.
Expression of Escherichia coliβ‐galactosidase in Mycobacterium bovis BCG using an expression system isolated from Mycobacterium paratuberculosis which induced humoral and cellular immune responses
Authors: Murray A. , Winter N. , Lagranderie M. , Hill D. F. , Rauzier J. , Timm J. , Leclerc C. , Moriarty K. M. , Gheorghiu M. , Gicquel B.
Published: October 2006

A promoter sequence, Pan', was isolated from Mycobacterium paratuberculosis and characterized. This promoter lies adjacent to, and outside, the 3’end of an IS900 insertion element. IS900 contains an open reading frame, ORF2, on the complementary strand which codes for the putative transposase of this insertion sequence. A DNA fragment containing PAN and part of ORF2 was fused to the lacZ gene and inserted into the replicative shuttle vector pRR3. Mycobacterium smegmatis and Mycobacterium bovis BCG (BCG) transformed with this plasmid exhibited β‐galactosidase activity. However, lacZ was only expressed in Escherichia coli under the control of Pan, when ORF2 was deleted. Immunization of mice with the recombinant M. bovis BCG expressing lacZ resulted in the induction of a high humoral and cellular response directed against β‐galactosidase. The Pan‐ORF2 expression system may prove to be particularly useful for cloning and expression of heterologous genes in the BCG vaccine strain.
Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences
Authors: Romano Nicoletta , Macino Giuseppe
Published: October 2006

Up to 36% of Neurospora crassa transformants showing an albino phenotype were recovered by transforming a wild‐type strain with different portions of the carotenogenic albino‐3 (al‐3) and albino‐1 (al‐1) genes. The presence of the exogenous sequences (which were randomly integrated in ectopic locations) provoked a severe impairment in the expression of the endogenous al‐1 or al‐3 genes. This phenomenon, which we have termed‘quelling’, was found to be spontaneously and progressively reversible, leading to wild‐type or intermediate phenotypes. The phenotypic reversion is characterized by a progressive release of the transcriptional inhibition and seems to correlate with a reduction of the number of the ectopic integrated sequences. Moreover, quelling appears to be monodirectional, as, once relieved, it cannot take place again, despite the continuing presence of some of the ectopic sequences in the genome.
Purification of Aeromonas hydrophila major outer‐membrane proteins: N‐terminal sequence analysis and channel‐forming properties
Authors: Jeanteur Denis , Gletsu Nana , Pattus Franc , Buckley J. Thomas
Published: October 2006

Four outer‐membrane proteins of Aeromonas hydrophila were purified and their N‐terminal sequences and channel‐forming properties were determined. Three could be matched with proteins from other species. One was a maltoporin, as its level increased when cells were grown in maltose‐containing media, and the channel it formed was blocked by maltose. Another was like OmpF and OmpC of Escherichia coli, except that its channel fluctuated much more rapidly. The third protein, which was produced in low‐phosphate medium, exhibited several properties of the general anion porin PhoE. The fourth showed no similarity to any known proteins. It had a unique N‐terminus and it formed small sharply‐defined cation‐selective channels. Two other proteins which corresponded to OmpW of Vibrio cholerae and E. coli OmpA were partly characterized.
The β‐tubulin gene from rat and human isolates of Pneumocystis carinii
Authors: Edlind T. D. , Bartlett M. S. , Weinberg G. A. , Prah G. N. , Smith J. W.
Published: October 2006

The development of new drugs for treating Pneumocystis carinii infections in AIDS patients is hampered by the lack of long‐term culture systems, and by our generally limited knowledge of this organism. Recently, however, we observed significant activity of various benzimidazoles against growth of this organism in short‐term cultures. Benzimidazoles inhibit microtubule polymerization; there is strong evidence that the primary target is the β‐tubulin subunit. To understand the basis for benzimidazole activity against P. carinii, and to examine the apparent relatedness of this organism to fungi, we have cloned and sequenced the single β‐tubulin gene from a rat P. carinii isolate. There was 89‐91% identity at the amino acid level to β‐tubulins from filamentous fungi, but only 79‐82% identity to yeast and protozoal β‐tubulins. Also, eight introns were distributed throughout the P. cariniiβ‐tubulin gene in a pattern characteristic of filamentous fungi. Specific residues previously implicated in benzimidazole sensitivity were conserved in P. cariniiβ‐tubulin. The polymerase chain reaction was used to amplify a segment of P. cariniiβ‐tubulin DNA from bronchoalveolar lavages obtained from two patients with AIDS. There was considerable divergence at the DNA level between the human and rat sequences, but 100% identity at the ammo‐acid level.