bluuebunny/crossref_metadata_2025 huggingface dataset:
A 10.3 kbp Segment from nprB to argJ at the 102 Region of the Bacillus Subtilis Chromosome
Authors: Levine A. , Vannier F. , Roche B. , Autret S. , Mavel D. , Sror S. J.
Published: June 2009

The approximately 10 kbp region encompassing nprB and argJ at 102 on the Bacillus subtilis chromosome was sequenced, revealing 12 ORFs, four known genes (argJ, argC, ipi and nprB) and two genes, yitY and yitS, whose products respectively display significant homology with L-gulono-?-lactone oxidase of rat and dihydrofolate reductase of Staphylococcus aureus. The data also indicated that nprB mapped to a different position than previously published.
Peptides 14VIDLL18 and 96FEAAAL101 defined as epitopes of antibodies raised against amino acid sequences of enterotoxigenic Escherichia coli colonization factor antigen I fused to Salmonella flagellin
Authors: Luna Maria G. , Ferreira Luis C. S. , Almeida Darcy F. , Rudin Anna
Published: June 2009

Antibodies raised against four hybrid Salmonella flagellins carrying amino acid sequences derived from the fimbrial subunit of the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC), i.e. hybrid flagellins Fla I (aa 1-15), Fla II (aa 11-25), Fla III (aa 32-45) and Fla IV (aa 88-102), were not able to inhibit the in vitro binding of CFA/I-expressing ETEC bacteria to enterocyte-like Caco-2 cells. However, one of the hybrid flagellins (Fla II) was recognized by a previously described anti-CFA/I subunit mAb (S-CFA/I 17:8) which was able to block adhesion of CFA/l-expressing bacteria to Caco-2 cells and to bind to the amino acid sequence 15IDLLQ19 of the CFA/I fimbrial subunit. Pepscan analysis of antibodies raised against the hybrid flagellins Fla II and Fla IV showed that they were specific for the sequences 14VIDLL18 and 96FEAAAL101, respectively, of the CFA/I fimbrial subunit. Thus, the discrepancy in the abilities of the anti-Fla II serum and the mAb S-CFA/I 17:8 to block binding might be ascribed to their slightly different fine specificity for epitopes.
Sequencing of regions downstream of addA (98°) and citG (28°) in Bacillus subtilis
Authors: Médina N. , Vannier F. , Roche B. , Autret S. , Levine A. , Séror S. J.
Published: June 2009

The nucleotide sequence of 17.3 kbp downstream of addA (98°) on the Bacillus subtilis chromosome was determined. Twenty putative ORFs were identified. Three of them coincided with known B. subtilis genes, addA, sbcD and wprA. The product of four other ORFs showed similarity to SbcC of Clostridium perfringens, CotH of B. subtilis, 2-hydroxyhepta-2,4-diene-1,7-diodate isomerase of Methanococcus jannaschi and a putative ORF of Pseudomonas syringae. In addition, a sequence of 7.6 kbp downstream of citG (189°) was analysed. Among 10 putative ORFs identified, two coincided with known genes, citG and mrgA, whilst three showed homology with X86780, a sensory protein kinase of Streptomyces hygroscopicus, an alkaline phosphatase regulatory protein and a hypothetical protease, YyxA, of B. subtilis.
A Bacillus subtilis chromosome segment at the 100° to 102° position encoding 11 membrane proteins
Authors: Roche B. , Autret S. , Levine A. , Vannier F. , Medina N. , Séror S. J.
Published: June 2009

The 25·9 kbp region upstream of nprB at 100°-102° on the Bacillus subtilis chromosome was sequenced. This revealed a known gene, degA, which was previously mislocated on the genetic map. A total of 29 putative ORFs were identified including a cluster of three ORFs whose products show clear homology with sulphate adenylyl pathway enzymes and, in addition, 11 ORFs whose products have one or more membrane domains, as indicated by their hydropathy profiles.
Expression of the Streptomyces aureofaciens glyceraldehyde-3-phosphate dehydrogenase gene (gap) is developmentally regulated and induced by glucose
Authors: Kormanec J. , Lempel'ov� A. , Nov�kov� R. , Re�uchov� B. , Hom�rov� D.
Published: June 2009

In previous experiments, the Streptomyces aureofaciens gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified. To investigate expression of the gene, S1 nuclease mapping and Northern blot hybridization were performed using RNA prepared from S. aureofaciens cultivated under various conditions. These studies suggested monocistronic organization and developmental regulation of the gene. A single promoter, gap-P, was identified upstream of the gap coding region. In cultures grown on solid medium in the absence of glucose, its transcription was induced at the time of aerial mycelium formation. In addition, gap transcription was also induced in substrate mycelium by glucose. A promoter-bearing DNA fragment was inserted into two promoter-probe vectors, to give expression patterns consistent with the results of direct RNA analysis.
Natural kirromycin resistance of elongation factor Tu from the kirrothricin producer Streptomyces cinnamoneus
Authors: Cappellano Carmela , Monti Federica , Sosio Margherita , Donadio Stefano , Sarubbi Edoardo
Published: June 2009

The antibiotic kirromycin (Kr) inhibits bacterial protein synthesis by binding to elongation factor Tu (EF-Tu). Streptomyces cinnamoneus and Nocardia lactamdurans, producers of antibiotics of the Kr class, are known to possess an EF-Tu resistant to Kr. Both micro-organisms appear to possess a single tuf gene and we have characterized the one from S. cinnamoneus, which belongs to the tuf
1 family. To assess the molecular determinants of Kr resistance, the S. cinnamoneus tuf gene was expressed in Escherichia coli as a translational fusion to malE, which enabled the recovery by affinity chromatography of the recombinant protein uncontaminated by the host factor. The recombinant EF-Tu was able to catalyse polyU-directed polyPhe synthesis in two heterologous cell-free systems, even as an uncleaved fusion. When tested for antibiotic sensitivity it behaved like the natural S. cinnamoneus protein, showing equivalent resistance to Kr but sensitivity to the antibiotic GE2270, indicating that all determinants for Kr resistance are intrinsic to the EF-Tu sequence. Multiple sequence analysis of EF-Tu proteins, together with knowledge of mutations conferring Kr resistance, allowed the identification of key residues as likely candidates for the natural Kr resistance of the S. cinnamoneus EF-Tu. One of these, Thr378, was mutated to the consensus Ala and the resulting mutant protein was sensitive to Kr. Interestingly, it retained some activity (30% of the control) even at. high Kr concentrations.
Analysis of a Rhizobium leguminosarum gene encoding a protein homologous to glutathione S-transferases
Authors: Alkafaf Najláa K. Tawfiq , Yeoman Kay H. , Wexler Margaret , Hussain Haitham , Johnston Andrew W. B.
Published: June 2009

A novel Rhizobium leguminosarum gene, gstA, the sequence of which indicated that it was a member of the gene family of glutathione S-transferases (GSTs), was identified. The homology was greatest to the GST enzymes of higher plants. The Rhizobium gstA gene was normally expressed at a very low level. The product of gstA was over-expressed and purified from Escherichia coli. It was shown to bind to the affinity matrix glutathione-Sepharose, but no enzymic GST activity with 1-chloro-2,4-dinitrobenzene as substrate was detected. gstA encoded a polypeptide of 203 amino acid residues with a calculated molecular mass of 21990 Da. Transcribed divergently from gstA is another gene, gstR, which was similar in sequence to the LysR family of bacterial transcriptional regulators. A mutation in gstR had no effect on the transcription of itself or gstA under the growth conditions used here. Mutations in gstA and gstR caused no obvious phenotypic defect and the biological functions of these genes remain to be determined.
Structure and gene-polypeptide relationships of the region encoding glycerol diffusion facilitator (glpF) and glycerol kinase (glpk) of Pseudomonas aeruginosa
Authors: Schweizer Herbert P. , Jump Robin , Po Cecilia
Published: June 2009

The glycerol facilitator is one of the few known examples of bacterial solute transport proteins that catalyse facilitated diffusion across the cytoplasmic membrane. A second protein, glycerol kinase, is involved in entry of external glycerol into cellular metabolism by trapping glycerol in the cytoplasm assn-glycerol 3-phosphate. Evidence is presented that glycerol transport inPseudomonas aeruginosais mediated by a similar transport system. The genes encoding the glycerol facilitator,glpF,and glycerol kinase,glpK,were isolated on a 4.5 kbEcoRIfragment from a chromosomal mini-library by functional complementation of anEscherichia coli glpKmutant after establishing a map of the chromosomalglpFKregion with the help of a PCR-amplifiedglpKsegment. The nucleotide sequence revealed thatglpFis the promoter-proximal gene of theglpFKoperon. The glycerol facilitator and glycerol kinase were identified in a T7 expression system as proteins with apparent molecular masses of 25 and 56 kDa, respectively. The identities of the glycerol facilitator and glycerol kinase amino acid sequences with their counterparts fromEscherichia coliwere 70 and 81%, respectively; this similarity extended to two homologues in the genome sequence ofHaemophilus influenzae.A chromosomal δglpFKmutant was isolated by gene replacement. This mutant no longer transported glycerol and could no longer utilize it as sole carbon and energy source. Two ORFs,orfXandorfY,encoding a putative regulatory protein and a carbohydrate kinase of unknown function, were located upstream of theglpFKoperon.
Identification by PCR of genes encoding multiple response regulators
Authors: Morel-Deville Francoise , Ehrlich S. Dusko , Morel Patrice
Published: June 2009

Environmental sensing in bacteria often involves the concerted action of sensor kinases and response regulators. Degenerate oligonucleotide primers were designed on the basis of amino acid similarity in the response regulators of these two-component sytems. The primers were used in PCR to specifically amplify an internal DNA segment corresponding to the receiver module domain from genes encoding response regulators. Amplification products of the expected size were obtained from 12 different Gram-positive and Gram-negative bacteria. Sequence analysis revealed that 22 DNA fragments, which clearly originated from response regulator genes, were amplified from Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis and Lactobacillus bulgaricus. In each of these four species the receiver module of putative response regulator genes, which do not seem to be related to any of the already characterized genes, was identified. This simple and powerful method is therefore particularly useful for discovering new signal transduction systems which cannot be revealed by usual genetic studies.
Analysis of a new dimeric extradiol dioxygenase from a naphthalenesulfonate-degrading sphingomonad
Authors: Heiss Gesche , Muller Claudia , Altenbuchner Josef , Stolz Andreas
Published: June 2009

A new extradiol dioxygenase was cloned by screening a gene bank from the naphthalenesulfonate-degrading bacterial strain BN6 for colonies with 2,3-dihydroxybiphenyl dioxygenase (DHBPDO) activity. A 16 kb DNA fragment was sequenced and an ORF of 954 bp identified. Comparison of the deduced amino acid sequence of DHBPDO II from strain BN6 with previously published sequences showed the closest relationship to a metapyrocatechase (Mpcll) from Alcaligenes eutrophus JMP 222. Thus, the enzyme was only distantly related to the main groups of catechol 2,3-dioxygenases or DHBPDOs. The dioxygenase was expressed using a T7 expression vector and the enzymic characteristics of the protein were examined. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3-methylcatechol, 4-fluorocatechol and 1,2-dihydroxynaphthalene. Comparison of the UV/visible spectrum of the product formed from 3,5-dichlorocatechol with previous reports suggested that this substrate is oxidized by different extradiol dioxygenases either by proximal or distal ring cleavage. The enzyme required Fe2+for maximal activity. In contrast to most other extradiol dioxygenases, the enzyme consisted of only two identical subunits.
A spectinomyciin resistance determinant from the spectinomycin producerStreptomyces flavopersicus
Authors: Lyutzkanova Dimitrina , Distler Jurgen , Altenbuchner Josef
Published: June 2009

Summary: The spectinomycin (Sp) resistance determinant from Streptomyces flavopersicus was cloned into Streptomyces lividans using the plasmid vector pIJ699. A plasmid, pDGL15, with a 3.65 kb insert from S. flavopersicus conferring resistance to Sp was isolated. DNA sequence analysis of the 3651 bp DNA insert revealed four open reading frames (ORFs). The amino acid sequence deduced from one ORF (SpcN) showed a high degree of similarity to an aminoglycoside phosphotransferase (StrN) and from a second one (SpcR) to a regulatory protein (StrR) of the streptomycin biosynthesis gene cluster from S. griseus. The two other ORFs were incomplete and the deduced amino acid sequences showed similarities to an amidinotransferase encoded in the streptomycin biosynthesis gene cluster of S. griseus and to the transposase of IS112, respectively. Expression of the spcN gene in E. coli under the control of tac promoter conferred Sp resistance to the cells. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent aminoglycoside phosphotransferase which phosphoryiates Sp and actinamine, the aminocyclitol moiety of Sp.
Spatial interactions between subsurface bacterial colonies in a model system: a territory model describing the inhibition of Listeria monocytogenes by a nisin-producing lactic acid bacterium
Authors: Thomas Linda V. , Wimpenny Julian W. T. , Barker Gary C.
Published: June 2009

Summary: The effect of spatial separation on interactions between subsurface bacterial colonies was tested using a model system: the inhibition of Listeria monocytogenes by nisin-producing and nisin-non-producing Lactococcus lactis subsp. lactis. Separation distance was controlled by altering the number of inoculum organisms within the agar. Mean separation distance was calculated by determining the mean volume available to each cell at the start of the experiment. Inhibition was assessed by comparing the growth of L. monocytogenes in pure culture with its growth in the presence of Lac. lactis subsp. lactis. Increasing the distance between colonies resulted in an exponential decrease in inhibition. When L. monocytogenes and Lac. lactis subsp. lactis colonies were within 100 μm of each other, the increase in cell numbers per L. monocytogenes colony was only 0.6 c.f.u. (which indicated some cells had become non-viable). This was a log reduction of 3.5 compared to the pure culture control. A separation distance of 1000 μm resulted in a L. monocytogenes colony growth increment of 2.5 × 102 c.f.u. per colony, a log reduction of 3.0 compared to the control. Increasing the separation distance to 3000 μm resulted in a L. monocytogenes colony growth increment of 1.3 × 106 c.f.u. per colony, a log reduction of 0.9 compared to the control. The effects of nisin and acidity were investigated by using a nisin-non-producing strain of Lac. lactis subsp. lactis and by buffering the medium. Data were obtained for the effect of separation on inhibition, as well as competition between colonies of the same species. The inhibition was mathematically described in terms of a simplified ‘territory’ model of immobilized bacterial growth. There was a strong qualitative agreement between the mathematical model and the experimental data. It was concluded that the phenomenon of propinquity is of important consideration when modelling and predicting microbial growth within solid food systems.
Genomic rearrangements during evolution of the obligate intracellular parasite Rickettsia prowazekii as inferred from an analysis of 52015 bp nucleotide sequence
Authors: Andersson Jan O. , Andersson Siv G. E.
Published: June 2009

Summary: In this study a description is given of the sequence and analysis of 52 kb from the 1.1 Mb genome ofRickettsia prowazekii, a member of the α-Proteobacteria. An investigation was made of nucleotide frequencies and amino acid composition patterns of 41 coding sequences, distributed in 10 genomic contigs, of which 32 were found to have putative homologues in the public databases. Overall, the coding content of the individual contigs ranged from 59 to 97%, with a mean of 81%. The genes putatively identified included genes involved in the biosynthesis of nucleotides, macromolecules and cell wall structures as well as citric acid cycle component genes. In addition, a putative identification was made of a member of the regulatory response family of two-component signal transduction systems as well as a gene encoding haemolysin. For one gene, the homologue ofmetK, an internal stop codon was discovered within a region that is otherwise highly conserved. Comparisons with the genomic structures ofEscherichia coli, Haemophilus influenzaeandBacillus subtilishave revealed several atypical gene organization patterns in theR. prowazekiigenome. For example,R. prowazekiiwas found to have a unique arrangement of genes upstream ofdnaAin a region that is highly conserved among other microbial genomes and thought to represent the origin of replication of a primordial replicon. The results presented in this paper support the hypothesis that theR. prowazekiigenome is a highly derived genome and provide examples of gene order structures that are unique for theRickettsia.
Regulation of the ndh gene of Escherichia coli by integration host factor and a novel regulator, Arr
Authors: Green Jeffrey , Anjum Muna F. , Guest John R.
Published: June 2009

Summary: The ndh gene of Escherichia coli encodes the non-proton-translocating NADH dehydrogenase II. Expression of the ndh gene is subject to a complex network of regulatory controls at the transcriptional level. Under anaerobic conditions ndh is repressed by the regulator of fumarate and nitrate reduction (FNR). However, in the absence of FNR, ndh expression is activated by the amino acid response regulator (Arr) during anaerobic growth in rich medium. Expression of the ndh gene varies during the growth cycle in response to the intracellular concentration of the heat-stable DNA-binding protein, Fis. In this work two additional heat-stable proteins, integration host factor (IHF) and the histone-like protein HU were found to interact with the ndh promoter. IHF was shown to bind at three sites centred at +26, -17 and -58 in the ndh promoter (K
d = 10−8 M), to prevent open-complex formation and to repress ndh transcription in vitro. Studies with an ndh-lacZ fusion confirmed that IHF represses ndh expression in vivo. Two putative binding sites for Arr, which overlap the two FNR boxes in the ndh promoter, were identified. Studies with the FNR-activated and amino-acid-inducible asparaginase II gene (ansB) showed that IHF and a component of the Arr-containing fraction (but not HU) interact with the corresponding ansB promoter.
Sequence of the Bacillus subtilis genome region in the vicinity of the lev operon reveals two new extracytoplasmic function RNA polymerase sigma factors SigV and SigZ
Authors: Sorokin Alexei , Bolotin Alexandre , Purnelle Helmut , Hilbert Helmut , Lauber Juergen , Düsterhöft Andreas , Ehrlich S. Dusko
Published: June 2009

Summary: Two regions with sizes 18 900 and 25400 bp, which join previously known contigs containing levRDEFG, aadK and bit genes near 235δ of the Bacillus subtilis chromosome, were sequenced. Among others, two genes, which encode proteins homologous to RNA polymerase σ-factors, were identified within this region. The gene products designated SigV and SigZ, show the highest homology with σ-factors encoded by the gene carQ of Myxococcus xanthus and sigX (formerly orfX20) of B. subtilis, correspondingly. All σ-factors which show statistically significant homology to SigV and SigZ, belong to the ECF (extracytoplasmic functions) subfamily. SigV and SigZ do not have N-terminal sequence which prevents such proteins from binding to DNA without RNA polymerase core enzyme.
Isolation of carbon- and nitrogen-deprivation-induced loci of Sinorhizobium meliloti 1021 by Tn5-luxAB mutagenesis
Authors: Milcamps Anne , Ragatz Daniel M. , Lim PyungOk , Berger Kelly A. , de Bruijin Frans J.
Published: June 2009

SUMMARY: Soil bacteria, such as Sinorhizobium meliloti, are subject to variation in environmental conditions, including carbon- and nitrogen-deprivation. The ability of bacteria to sense changes in their environment and respond accordingly is of vital importance to their survival and persistence in the soil and rhizosphere. A derivative of Tn5 which creates transcriptional fusions to the promoterless luxAB genes was used to mutagenize 5. meliloti 1021 and 5000 insertion mutants were subsequently screened for gene fusions induced by selected environmental stresses. The isolation of 21 gene fusions induced
by nitrogen-deprivation and 12 induced by carbon-deprivation is described. Cloning and partial DNA sequence analysis of the transposon-tagged loci revealed a variety of novel genes, as well as S. meliloti genes with significant similarity to known bacterial loci. In addition, nodule occupancy studies were carried out with selected TnSluxAB insertion mutants to examine the role of the tagged genes in competition.
Determination of a 15437 bp nucleotide sequence around the inhA gene of Mycobacterium avium and similarity analysis of the products of putative ORFs
Authors: Labó Monica , Gusberti Laura , De Rossi Edda , Speziale Pietro , Riccardi Giovanna
Published: June 2009

A 15437 bp region encompassing the inhA locus from the Mycobacterium avium chromosome was cloned and sequenced. From the sequencing data generated and the results of homology searches, the primary structure of this region was determined. This region contains four known genes (acnA, fabG, inhA and hemH) and two genes, invA and invB, whose products display homology with p60 invasion protein of Listeria monocytogenes. Six proteins encoded by putative ORFs contained an RGD motif (often involved in binding to macrophage integrins), while ORF1 and MoxR are probably transcriptional regulators. The rest of the putative products encoded by ORFs in the sequenced region showed little homology with the proteins contained in the databases and were considered to be unknown proteins.
The Staphylococcus aureus and Staphylococcus epidermidis transferrin-binding proteins are expressed in vivo during infection
Authors: Modun Belinda J. , Cockayne Alan , Finch Roger , Williams Paul
Published: June 2009

Staphylococci express a 42 kDa cell-wall-associated protein which functions as a receptor for the mammalian iron-binding glycoprotein transferrin. To determine whether this transferrin-binding protein (TBP) is expressed during infection, Staphylococcus aureus and Staphylococcus epidermidis were grown in vivo in chambers implanted intraperitoneally in rats. SDS-PAGE and Western blotting of cell wall proteins prepared from staphylococci recovered directly from the chambers revealed the presence of both the TBP and bacterial-surface-associated rat transferrin. To obtain evidence for the in vivo expression of the staphylococcal TBPs in humans, sera and human peritoneal dialysate (HPD) from non-infected patients undergoing continuous ambulatory peritoneal dialysis (CAPD) and sera from healthy human volunteers were screened for anti-TBP antibodies. Western immunoblots revealed that three out of ten samples from the latter group, seven out of ten HPD samples and ten of ten CAPD patient serum samples contained antibodies to the TBP of both S. aureus and S. epidermidis. To gain further insights into the appearance of TBP antibodies, HPD samples were collected over time from CAPD patients whose HPD samples taken immediately after catheter insertion lacked anti-TBP antibodies. In two of these patients, each of whom experienced an episode of peritonitis due to S. epidermidis or Staphylococcus hominis, antibodies to the TBP appeared in the HPD collected immediately post-infection. To determine whether such TBP antibodies were capable of blocking interactions between transferrin and its staphylococcal receptor, HPD immunoglobulin fractions were purified using protein A-Sepharose beads. In competition assays, these immunoglobulins blocked the binding of 125l-labelled transferrin both to whole bacteria and to the isolated 42 kDa TBPs of S. aureus and S. epidermidis. These provide evidence to show that staphylococcal TBPs are expressed in vivo during infection.
Phytophthora cactorum can synthesize substances needed for sexual reproduction but requires a stress factor to trigger the process
Authors: Jee Hyeong-Jin , Ko Wen-Hsiung
Published: June 2009

Phytophthora cactorum grown on basal agarose medium or in liquid basal medium produced oospores after being transferred to water agarose. The numbers of oospores produced under such conditions depended on the age of the culture prior to exposure to nutrient deprivation. When the concentration of basal medium used for cultivation of P. cactorum was increased, the numbers of oospores produced after being transferred to water agarose was also increased. P. cactorum grown on basal agarose medium also produced oospores when its mycelial growth was restricted after reaching the edge of Petri plates. In 5 cm plates oospore formation occurred in the third week, whereas in 9 cm and 14 cm plates oospores appeared in the fourth week. Most oospores were formed near the edge of the plates. The non-saponifiables extracted from mycelia of P. cactorum grown in liquid basal medium were stimulatory to oospore formation by P. cactorum and Phytophthora parasitica, whereas the saponifiables were stimulatory to P. cactorum only. Extracts from culture filtrate and basal medium were not stimulatory to oospore formation by either fungus. When the non-saponifiables were fractionated by Florisil columm chromatography, only fraction 1 was not active. Fractions 2, 3 and 4 were stimulatory to oospore formation by both P. cactorum and P. parasitica. These results support the hypothesis that P. cactorum, and possibly other pythiaceous fungi as well, can synthesize substances needed for sexual reproduction but requires a stress factor to trigger the process.
Characterization of novel immunodominant antigens of Mycobacterium tuberculosis
Authors: Amara Rama Rao , Shanti S. , Satchidanandam Vijaya
Published: June 2009

Seven novel antigens of Mycobacterium tuberculosis, which had previously been identified based on reactivity to sera from patients with tuberculosis, were characterized. Nucleotide sequence analysis of the genes encoding these seven antigens identified one of them as the FtsH and a second as the aminoimidazole ribotide synthase of M. tuberculosis. Antisera raised to the recombinant forms of each of these seven antigens were used to study the distribution of these proteins within mycobacterial species as well as to determine their subcellular localization and hydrophobicity. Four of the seven antigens were conserved only among pathogenic strains of mycobacteria. Of the seven proteins studied, FtsH and a second protein of unknown identity were localized in membranes. Two were cytosolic, while two others, which had a high proline content, weretightly associated with the cell wall. One protein was secreted. This secreted protein could be identified by serum from a majority of tuberculosis patients but not BCG-vaccinated individuals, suggesting its potential use in the immunodiagnosis of tuberculosis.
Spacing and orientation requirements of GcvA-binding sites 3 and 2 and the Lrp-binding region for gcvT::lacZ expression in Escherichia coli
Authors: Stauffer Lorraine T. , Stauffer George V.
Published: June 2009

Both GcvA and Lrp are required for normal regulation of the gcv operon. Moving the GcvA-binding sites 3 and 2 and the Lrp-binding region either closer to, or further away from, the gcv promoter by approximately one helical turn of DNA resulted in a less than twofold decrease in glycine-mediated activation or inosine-mediated repression of a gcvT::lacZ fusion. Moving these sites approximately two helical turns of DNA away from the gcv promoter resulted in a further loss of both activation and repression; moving these sites approximately three helical turns of DNA from the gcv promoter resulted in an essentially complete loss of both glycine-mediated activation and inosinemediated repression. However, when these sites were moved by approximately 1.5 and 2.5 helical turns of DNA away from the gcv promoter, there was a complete loss of both glycine-mediated activation and inosine-mediated repression of the gcvT::lacZ fusion. The flexibility in the absolute distance of the GcvA- and Lrp-binding sites relative to the gcv promoter, but strict orientation dependence of these sites is consistent with a possible protein-protein interaction of either GcvA, Lrp, or both of these proteins with RNA polymerase. Because of the location of these target sites relative to the gcv promoter, it is also likely that DNA looping is required for this mechanism of regulation.
Identification of Mycobacterium tuberculosis signal sequences that direct the export of a leaderless β-lactamase gene product in Escherichia coli
Authors: Chubb Anthony J. , Woodman Zenda L. , da Silva Tatley Fernanda M. P. R. , Hoffmann Hans Jürgen , Scholle Renate R. , Ehlers Mario R. W.
Published: June 2009

Proteins secreted by Mycobacterium tuberculosis may play a key role in virulence and may also constitute antigens that elicit the host immune response. However, the M. tuberculosis protein export machinery has not been characterized. A library of M. tuberculosis H37Rv genomic DNA fragments ligated into a signal sequence selection vector that contained a leaderless β-lactamase gene and an upstream Tac promoter was constructed. Transformation of Escherichia coli with the M. tuberculosis DNA library and selection on plates containing 50-100 μg ampicillin ml-1 resulted in the identification of 15 Ampr clones out of a total of 14000 transformants. Twelve of the β-lactamase gene fusions conferred high levels of Ampr (up to 1 mg ampicillin ml-1); insert sizes ranged from 350 to 3000 bp. Of ten inserts that were completely sequenced, two were identified as fragments of the genes for M. tuberculosis antigens 85A and 85C, which are the major secreted proteins of this pathogen. Seven of the remaining inserts were ≥97% identical to hypothetical ORFs in the M. tuberculosis genome, one of which encoded a protein with 35% identity to a low-affinity penicillin-binding protein (PBP) from Streptomyces clavuligerus. Four of the seven hypothetical ORFs encoded putative exported proteins with one or more membrane interaction elements, including lipoprotein attachment sites and type I and II transmembrane (TM) segments. All of the inserts encoded typical signal sequences, with the exception of a possible type II membrane protein. It is concluded that expression of β-lactamase gene fusions in E. coli provides a useful system for the identification and analysis of M. tuberculosis signal-sequence-encoding genes.
Changes in fatty acid composition of Neurospora crassa accompany sexual development and ascospore germination
Authors: Goodrich-Tanrikulu Marta , Howe Kelly , Stafford Allan , Nelson Mary Anne
Published: June 2009

Fatty acid composition was determined during several stages of sexual development in Neurospora crassa. Triacylglycerol was the predominant acyl lipid in cultures undergoing sexual development. The absolute amounts of triacylglycerol in fertilized cultures varied over time, in contrast to control (unfertilized or mock-fertilized) cultures, in which the amount of triacylglycerol decreased linearly with age. In cultures competent to undergo sexual development, -linoleate was the predominant fatty acid, ranging from 53 to 65% of the total fatty acid mass. -Linolenate was 3% or less of the total fatty acid, in marked contrast to the much higher levels (10--35%) typically reported for vegetative cultures. In fertilized cultures, a slightly higher mass ratio of oleate was also observed. This difference was due to the developing asci: in developing asci and mature ascospores, oleate replaced α-linoleate as the predominant fatty acid (45 to 50% of the total). In germinating ascospores, the fatty acid composition approached that of vegetative cultures 6 h after inducing germination by heat activation. These results show that the fatty acid composition of sexual tissues of Neurospora differs substantially from the composition of asexual tissues, and that extensive changes in fatty acid composition correlate with several events in the sexual stage of development.
Antioxidant vitamins C and E affect the superoxide-mediated induction of the soxRS regulon of Escherichia coli
Authors: Fuentes Ana M. , Ambile-Cuevas Carlos F.
Published: June 2009

The mechanism of activation of Escherichia coli redox sensory protein SoxR still unclear: a [2Fe--25] cluster contained in a SoxR dimer is potentially redo sensitive, but the nature of the signal is unknown. Antioxidant vitamins C (ascorbate) and E (a-tocopherol) were used to explore the mechanism of activation of the SoxR protein in vivo. Treating E. coli cells with ascorbate o a-tocopherol increased their tolerance to paraquat (PQ, a redox-cycling compound), even in the absence of the soxRS locus, suggesting a radical-quenching activity. When using a soxS:: lacZ fusion, whose expression is governed by activated SoxR, ascorbate and a-tocopherol also prevented the expression of a-galactosidase after PQ treatment. A secondary activity was observed in cells carrying soxR101, a mutation resulting in the constitutive expression of the sox regulon, where the overexpression of soxS::lacZ was also reduced by ascorbate or a-tocopherol treatment. Additionally, different mechanisms of action were revealed as a-tocopherol was capable of preventing both PQ and menadione (MD) lethality, whilst ascorbate prevent PQ lethality but increased MD-mediated cell death. It is proposed that a-tocopherol, positioned in membranes, can prevent superoxide-dependent membrane damage; however, water-soluble ascorbate is unable to do so an can even increase the concentration of oxygen radicals reacting with release membrane-associated Fe(ll).
A novel system with positive selection for the chromosomal integration of replicative plasmid DNA in Corynebacterium glutamicum
Authors: Ikeda Masato , Katsumata Ryoichi
Published: June 2009

A simple system has been developed for generating Corynebacterium glutamicum strains containing stable replicative plasmids integrated into the chromosome via homologous recombination. The system is based upon extremely strong incompatibility between two plasmids, which cannot be comaintained even under antibiotic selective pressure. Integration of the resident plasmid that contained the trpD gene of C. glutamicum was achieved by introduction of a second plasmid and subsequent selection for the maintenance of both plasmids. Plasmid integrates positive for both plasmid markers were obtained at a frequency about 10-3 of the normal transformation frequency with selection for the maintenance of only the second plasmid. Southern analysis revealed that the integration had occurred through a single-crossover homologous recombination between the trpD regions of the host genome and the plasmid. On the basis of the Campbell-type integration, chromosome walking was attempted by using Escherichia coli replication origins that were also present in the integrated plasmid. The chromosomal DNA was digested, ligated, and used to transform E. coli, which enabled recovery of the expected adjacent genomic DNA regions. The plasmid integrate was stably maintained for 30 generations under non-selective culture conditions, suggesting that the integrated sequences carrying a replicon active in the host were maintained as a stable chromosomal insert in C. glutamicum.
A swarming-defective mutant of Proteus mirabilis lacking a putative cation-transporting membrane P-type ATPase
Authors: Lai Hsin-Chih , Gygi Daniel , Fraser Gillian M. , Hughes Colin
Published: June 2009

The motile TnphoA mutant IC24 of Proteus mirabilis U6450 generates an aberrant swarming colony, and was shown to be impaired in swarm cell differentiation, i.e. cell elongation and hyperflagellation, causing delayed and slower population migration across a solid growth medium. Levels of transcript from the flagellin filament gene fliC, the flagellar master operon flhDC, and the leucine-responsive regulatory protein gene Irp, a regulator of swarming differentiation, were reduced in IC24 mutant swarm cells. The transposon had inserted into a gene encoding a putative P-type ATPase closely related to those transporting cations across bacterial membranes. This ppa gene (Proteus P-type ATPase) was maximally expressed in differentiated swarm cells. The data suggest an effect of ion homeostasis on swarm cell differentiation, possibly mediated via the Irp--flhDC pathway.
Regulation of an anthranilate synthase gene in Streptomyces venezuelae by a trp attenuator
Authors: Lin Cong , Paradkar Ashish S. , Vining Leo C.
Published: June 2009

The nucleotide sequence of a 2·4 kb BamHI--Sall fragment of Streptomyces venezuelae ISP5230 DNA that complements trpE and trpG mutations in Escherichia coli contains two ORFs. The larger of these (ORF2) encodes a 624 amino acid sequence similar to the overall sequence of the two subunits of anthranilate synthase. The two-thirds nearest the amino terminus resembles the aminase subunit; the remaining one-third resembles the glutamine amidotransferase subunit. Upstream of ORF2 is a small ORF encoding 18 amino acids that include three adjacent Trp residues; in addition the ORF contains inverted repeats with sequence and positional similarity to the products of attenuator (trpL) regions that regulate tryptophan biosynthesis in other bacteria. In cultures of a trpC mutant of S. venezuelae, increasing the concentration of exogenous tryptophan decreased the formation of anthranilate synthase; similar evidence of endproduct repression was obtained in a trpCER mutant of E. coli transformed with a vector containing the cloned DNA fragment from S. venezuelae. The anthranilate synthase activity in S. venezuelae cell extracts was inhibited by tryptophan, although only at high concentrations of the amino acid. A two-base deletion introduced into the cloned S. venezuelae DNA fragment prevented complementation of a trpE mutation in E. coli. However, S. venezuelae transformants in which the two-base deletion had been introduced by replacement of homologous chromosomal DNA did not exhibit a Trp- phenotype. The result implies that S. venezuelae has one or more additional genes for anthranilate synthase. In alignments with anthranilate synthase genes from other organisms, ORF2 from S. venezuelae most closely resembled genes for phenazine biosynthesis in Pseudomonas. The results bear on the function of the gene in S. venezuelae.
Disruption studies of a Candida albicans gene, ELF1: a member of the ATP-binding cassette family
Authors: Sturtevant Joy , Cihlar Ronald , Calderone Richard
Published: June 2009

A 3.6 kb gene (ELF1) with homology to the ATP-binding cassette (ABC) gene family has been isolated from genomic libraries of Candida albicans. Members of this gene family include both membrane transport proteins which confer a drug-resistance phenotype, and proteins whose functions are associated with protein translation. ELF1 (Elongation Like Factor) showed greatest homology with a Saccharomyces cerevisiae ORF (YPL226W), whose function is unknown, and lower homology with fungal elongation factor 3 (EF-3) genes. In comparison, homology with a gene conferring a drug-resistant phenotype (CDR1) was low. To understand the function of ELF1 in C. albicans, gene-knockout experiments were conducted using the hisG-URA3-hisG disruption cassette. Both single-copy (heterozygote) and double-disrupted strains in ELF1 were isolated. Phenotypically, the disrupted strains grew more slowly than wild-type and produced a mixture of large, irregular cells and apparently normal cells.
Genetic engineering of an industrial strain of Saccharopolyspora erythraea for stable expression of the Vitreoscilla haemoglobin gene (vhb)
Authors: Brünker Peter , Minas Wolfgang , Kallio Pauli T. , Baile James E.
Published: June 2009

Several Actinomycetes/Streptomycetes expression vectors are described for expression of the Vitreoscilla haemoglobin gene (vhb) in an industrial erythromycin-producing strain of Saccharopolyspora erythraea. Cloning of vhb under the control of either the thiostrepton-inducible PtipA promoter or the constitutive PermE*
promoter led to the production of chemically active haemoglobin (VHb) in Streptomyces lividans TK24 transformed with these constructs. However, the plasmids could not be transformed into Sac. erythraea. Transformants of Sac. erythraea and/or exconjugants were obtained using a novel Escherichia coli/Streptomyces shuttle vector comprised of vhb under the control of the PermE*
promoter, the Streptomyces plasmid pIJ350 origin of replication, the thiostrepton-resistance gene (tsr) for selection, and the oriT region which is necessary for conjugal transfer. Increased plasmid stability in Sac. erythraea was obtained by construction of a vector for chromosomal integration. This vector contained the Streptomyces phage øC31 attachment site for chromosomal integration and vhb expressed under the PmerR promoter and was stably maintained in the chromosome of Sac. erythraea. Shake-flask cultivations of the transformed Sac. erythraea strain with the chromosomally integrated vhb gene show that vhb is expressed in an active form. The corresponding amount of erythromycin produced in the vhb-expressing strain was approximately 60% higher relative to the original VHb-negative strain.
An in silico evaluation of Tn916 as a tool for generalized mutagenesis in Haemophilus influenzae Rd
Authors: Hosking Sarah L. , Deadman Mary E. , Moxon E. Richard , Peden John F. , Saunders Nigel J. , High Nicola J.
Published: June 2009

The transposon Tn916 was evaluated as a tool for generalized mutagenesis of the genome of Haemophilus influenzae. This was achieved in silico by searching the genome sequence of H. influenzae Rd for the published Tn916 target site consensus sequence 5′ TT/ATTTT(N)6AAAAAA/TA. This search identified 16 putative target sites. In subsequent experiments, integration of Tn916 did not occur at any of these sites. Using the nucleotide sequences of these observed integration sites, a new consensus sequence, 5′ TTTTT(N)xAAAAA (4x7), was derived. This sequence reflects the curve-twist-curve DNA topology which is a feature common to all Tn916 integration sites. A search of the H. influenzae Rd genome using the new consensus sequence identified 167 potential target sites, representing approximately 1% of the total genome. Only 80 of these sites were located within ORFs. The presence of such a limited number of target sites places severe constraints on the use of Tn916 as a tool for generalized mutagenesis of the genome of H. influenzae.
Biosynthesis of triacylglycerol in the filamentous fungus Mucor circinelloides
Authors: Jackson Frances M. , Michaelson Louise , Fraser Thomas C. M , Stobart A. Keith , Griffiths Gareth
Published: June 2009

Lipid metabolism was studied in 2-d-old liquid cultures of Mucor circinelloides grown at 25 C. Under these conditions, oil accumulated to 0.5 g I-1 with a ?-linolenic acid content (?18:3) of 60 mg I-1. The major labelled lipids in cultures incubated with [14C]acetate were triacylglycerol (TAG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The proportion of label declined in the phospholipids and increased in TAG with time. [14C]18:1 and [14C]18:2 rapidly appeared in PC and PE and later accumulated in [14C]?18:3. TAG-synthesizing capacity was greatest in the microsomal membrane fraction, which accumulated high levels of phosphatidic acid in the presence of glycerol 3-phosphate and acyl-CoA substrates at pH 7.0. Further metabolism of phosphatidic acid to diacylglycerol and TAG was achieved by increasing the pH to 8.0. Lysophosphatidic acid:acyl-CoA acyltransferase (LPAAT) activity was particularly high and may have accounted for the rapid accumulation of phosphatidic acid in the membranes. The glycerol-3-phosphate:acyl-CoA acyltransferase (GPAAT) and LPAAT were non-specific for a range of saturated and unsaturated species of acyl-CoA although the GPAAT showed a marked selectivity for palmitoyl-CoA and the LPAAT for oleoyl- and linoleoyl-CoA. ?-Linolenic acid was detected at all three positions of sn-TAG and was particularly enriched at the sn-3 position. The preparation of active in vitro systems (microsomal membranes) capable of the complete biosynthetic pathway for TAG assembly may be valuable in understanding the assembly of oils in future transgenic applications.
Ammonia-dependent synthesis and metabolic channelling of carbamoyl phosphate in the hyperthermophilic archaeon Pyrococcus furiosus
Authors: Legrain Christianne , Demarez Marc , Glansdorff Nicolas , Piérard André
Published: June 2009

The biosynthesis of carbamoyl phosphate (CP), a metabolic precursor of arginine and the pyrimidines was investigated in the hyperthermophilic archaeon Pyrococcus furiosus. The half-life of CP was found to be less than 2 s in the optimum temperature range of this organism (100-102 °C). The carbamoyl-phosphate synthase (CPSase) of P. furiosus uses ammonia as the nitrogen donor, and not glutamine like all micro-organisms investigated so far. The Mr of the enzyme, which is devoid of regulatory properties, is 70000, at variance with that of known CPSases. The possible significance of these findings with regard to hyperthermophilic nitrogen metabolism is discussed. Competition experiments with P. furiosus crude extracts indicated a marked preference of ornithine carbamoyltransferase (OTCase) for CP synthesized by CPSase rather than for CP added to the reaction mixture. In addition, the bisubstrate analogue -N-phosphonoacetyl-L-ornithine inhibits the formation of citrulline from bicarbonate, ammonia, ATP and ornithine much less than its synthesis from ornithine and CP in the presence of free OTCase. Such results suggest that, in vivo, CPSase and OTCase associate in a complex able to channel CP. Such a channelling may confer protection to CP, thus avoiding the accumulation of toxic amounts of cyanate arising from its decomposition as well as the waste of the two molecules of ATP required for its synthesis.
Nucleotide sequences of two fimbrial major subunit genes, pmpA and ucaA, from canine-uropathogenic Proteus mirabilis strains
Authors: Bijlsma Isaac G. W. , Dijk Linda van , Kusters Johannes G. , Gaastra Wim
Published: June 2009

Proteus mirabilis strains were isolated from dogs with urinary tract infection (UTI) and fimbriae were prepared from two strains. The N-terminal amino acid sequences of the major fimbrial subunits were determined and both sequences appeared identical to the N-terminal amino acid sequence of a urinary cell adhesin (UCA) (Wray, S. K., Hull, S. I., Cook, R. G., Barrish, J. Hull, R. A., 1986, Infect Immun 54, 43-49). The genes of two different major fimbrial subunits were cloned using oligonucleotide probes that were designed on the basis of the N-terminal UCA sequence. Nucleotide sequencing revealed the complete ucaA gene of 540 bp (from strain IVB247) encoding a polypeptide of 180 amino acids, including a 22 amino acid signal sequence peptide, and the pmpA (P. mirabilis P-like pili) gene of 549 bp (from strain IVB219) encoding a polypeptide of 183 amino acids, including a 23 amino acid signal sequence. Hybridization experiments gave clear indications of the presence of both kinds of fimbriae in many UTI-related canine P. mirabilis isolates. However, the presence of these fimbriae could not be demonstrated in P. vulgaris or other Proteus-related species. Database analysis of amino acid sequences of major subunit proteins revealed that the UcaA protein shares about 56% amino acid identity with the F17A and F111A major fimbrial subunits from bovine enterotoxigenic Escherichia coli. In turn, the PmpA protein more closely resembled the pyelonephritis-associated pili (Pap)-like major subunit protein from UTI-related E. coli. The evolutionary relationship of UcaA, PmpA and various other fimbrial subunit proteins is presented in a phylogenetic tree.