bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular characterization of the proteinase‐encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum
Authors: Geremia Roberto A. , Goldman Gustavo H. , Jacobs Dirk , Ardrtes W. , Vila Silvia B. , Van Montagu Marc , Herrera‐Estrella Alfredo 
Published: October 2006

SummaryThe soil fungus Trichoderma harzianum is a mycoparasitic fungus known for its use as a biocontrol agent of phytopathogenic fungi. Among other factors, Trichoderma produces a series of antibiotics and fungal cell wall‐degrading enzymes. These enzymes are believed to play an important role in mycoparasitism. Among the hydrolytic enzymes, we have identified a basic proteinase (Prb1) which is induced by either autoclaved mycelia, fungal cell wall preparation or chitin; however, the induction does not occur in the presence of glucose. The proteinase was purified and biochemically characterized as a serine proteinase of 31 kDa and pl 9.2. Based on the sequence of three internal peptides, synthetic oligonudeotide probes were designed. These probes allowed subsequent isolation of a cDNA and its corresponding genomic clone. The deduced amino acid sequence indicates that the proteinase is synthesized as a pre‐proenzyme and allows its classification as a serine proteinase. Northen analysis shows that the induction of this enzyme is due to an increase in the corresponding mRNA level.
The arsD gene encodes a second trans‐acting regulatory protein of the plasmid‐encoded arsenical resistance operon
Authors: Wu Jianhua , Rosen Barry P. 
Published: October 2006

SummaryThe plasmid‐encoded arsenical resistance (ars) operon produces resistance to trivalent and pentavalent salts of arsenic and antimony. The first gene in the operon, arsR, was previously shown to encode a repressor protein. A newly identified gene, arsD, is shown here to encode a regulatory protein, the ArsD protein. The gene was identified by construction of an in‐frame fusion between the C‐terminally truncated arsD gene and the coding region for the mature form of β‐lactamase (blaM). The native arsD gene product was overexpressed and radioactively labelled as a 13kDa polypeptide. A frameshift mutation within the arsD gene resulted in elevated levels of expression of downstream ars genes. Co‐expression of a wild‐type arsD gene in trans with the operon containing the mutated arsD gene reduced expression of the downstream genes to wild‐type levels. The presence of the arsD gene had no effect on the basal level of operon expression set by the arsR gene product, and the repression produced by the arsD gene product was not affected by inducers of the operon. The results indicate that the ArsD protein is an inducer‐independent trans‐acting regulatory protein.
Multiple copies of a DNA sequence from Pseudomonas syringae pathovar phaseolicola abolish thermoregulation of phaseolotoxin production
Authors: Rowley Karla B. , Clements David E. , Mandel Morton , Humphreys Tom , Patil Suresh S. 
Published: October 2006

SummaryPhaseolotoxin, a phytotoxin of Pseudomonas syringae pv. phaseolicola, is produced at 18°C but not at 28°C. Here we report that a fragment (24.4 kb) cloned from the wild‐type strain, which does not harbour a gene(s) involved in phaseolotoxin biosynthesis, abolishes this thermoregulation in the wild type and suppresses a Tox− mutant at both temperatures. A subclone harbouring a 465bp fragment contains motifs that are characteristic of DNA‐binding sites. In mobility shift assays we have detected a protein(s) from the wild‐type and the mutant strains, grown at appropriate temperatures, that specifically binds to the fragment containing the DNA‐binding motifs. We propose that the binding protein is a repressor which is ‘titrated’ by this fragment when it is present in the cell on a multiple copy plasmid, thus allowing expression of phaseolotoxin genes.
Identification of a gene cluster encoding three high‐molecular‐weight proteins, which is required for synthesis of tolaasin by the mushroom pathogen Pseudomonas tolaasii
Authors: Rainey Paul B. , Brodey Catherine L. , Johnstone Keith 
Published: October 2006

SummaryThe extracellular lipodepsipeptide toxin tolaasin is the primary disease determinant of pathogenicity of Pseudomonas tolaasii on the cultivated mushroom, Agaricus bisporus. Transposon mutagenesis of P. tolaasii NCPPB 1116 with Tn5‐generated 5000 chromosomal insertions of which 35 (0.7%) were tolaasin negative and 12 (0.25%) produced a reduced amount of tolaasin. In addition, TnphoA mutagenesis yielded a single tolaasin‐negative mutant which was phoA active. Restriction enzyme mapping of mutant DNAs by Southern hybridization analysis revealed that the majority of Tn5 insertions were confined to a single genetic locus of approximately 65kbp. Pulsed‐field gel electrophoresis of representative Tn5 mutant DNAs showed that this region is at one end of a 640kbp Pacl chromosomal fragment and that the P. tolaasii genome is 6.7 Mbp. SDS‐PAGE analysis of protein extracts from wild‐type P. tolaasii demonstrated the presence of three high‐molecular‐Weight proteins (designated TL1, TL2 and TL3). Alterations in the presence of these proteins, as well as apparently truncated forms of the 465 kDa (TL1), 440 kDa (TL2) and 435 kDa (TL3) proteins were observed in some mutants, enabling the direction and order of the transcriptional units to be determined. Two other Tn5 mutations were also identified which resulted in a tolaasin‐negative phenotype, but which did not affect the expression of TL1, TL2, or TL3. One of these mutants is linked to the TL‐cluster, but the other is located outside this region. It is concluded that at least five genetic loci, including those encoding TL1, TL2 and TL3, are required for tolaasin synthesis.
Forespore‐specific disappearance of the sigma‐factor antagonist SpollAB: implications for its role in determination of cell fate in Bacillus subtilis
Authors: Kirchman Paul A. , DeGrazia Henry , Kellner Ellen M. , Moran Charles P. 
Published: October 2006

SummaryEndospore formation in Bacillus subtilis is a morphologically complex process in which the bacterium divides into two compartments (forespore and mother cell) that follow different developmental paths. Compartment‐specific transcription in the forespore is initiated by RNA polymerase containing σ;F, and results in the forespore‐specific production of σ;G, which directs most of the subsequent forespore‐specific transcription. The activity of σ;F is thought to be restricted to the forespore by the sigma factor antagonist SpollAB. We used antibodies against SpollAB to monitor its accumulation during sporulation. We found that SpollAB accumulates early after the initiation of sporulation, and that it was present in the mother‐cell compartment 2h after σ;F became active in the forespore. SpollAB disappeared preferentially from the forespore during development, and its disappearance from the forespore compartment correlated with the activation of σ;G in that compartment, raising the possibility that SpollAB may be involved regulating σ;G activity. We tested whether SpollAB could antagonize σ;G activity by replacing the σ;F‐dependent promoter that drives expression of spolllG, the structural gene for σ;G, with a σ;H‐dependent promoter. This resulted in a lytic phenotype that was supressed by the simultaneous expression of a plasmid‐borne copy of spollAB. This suggests that SpollAB can suppress this effect of σ;G expression. Moreover, these cells formed spores efficiently. Since σ;G synthesis in these cells was not restricted to the forespore by the σ;F‐dependent transcription of its structural gene that normally occurs in wild‐type cells, the forespore‐specific activity of σ;G required for Sporulation appears to have resulted from expression of SpollAB.
Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus
Authors: Schüddekopf Kerstin , Hennecke Silke , Liese Ute , Kutsche Michael , Klipp Werner 
Published: October 2006

SummaryTo identify Rhodobacter capsulatus nif genes necessary for the alternative nitrogenase, strains carrying defined mutations in 32 genes and open reading frames of nif region A, B or C were constructed. The ability of these mutants to grow on nitrogen‐free medium with molybdenum (Nif phenotype) or in a nifHDK deletion background on medium without molybdenum (Anf phenotype) was tested. Nine nif genes and nif‐associated coding regions are absolutely essential for the alternative nitrogenase. These genes comprise nifV and nifB, the nif‐specific ntr system (nifR1, R2, R4) and four open reading frames, which exhibit no homology to known genes. In addition, a significantly reduced activity of both the alternative nitrogenase and the molybdenum‐dependent nitrogenase was found for fdxN mutants. By random Tn5 mutagenesis of a nifHDK deletion strain 42 Anf− mutants were isolated. Southern hybridization experiments demonstrated that 17 of these Tn5 mutants were localized in at least 13 different restriction fragments outside of known nif regions. Ten different Anf− Tn5 mutations are clustered on a 6 kb DNA fragment of the chromosome designated anf region A. DNA sequence analysis revealed that this region contained the structural genes of the alternative nitrogenase (anfHDGK). The identification of several Tn5 insertions mapping outside of anf region A indicated that at least 10 genes specific for the alternative nitrogenase are present in R. capsulatus.
Characterization of the nucM gene coding for a nuclease of the phytopathogenic bacteria Erwinia chrysanthemi
Authors: Moulard M. , Condemine G. , Robert‐Baudouy J. 
Published: October 2006

SummaryThe gene nucM encoding a nuclease was cloned from a genomic library of Erwinia chrysanthemi. The nucM gene was subcloned, and mutagenized by insertion of a uidA‐KanR cartridge. This mutation was introduced by recombination into the Erwinia chrysanthemi chromosome. The nucM mutant lost NucM activity when tested on a DNA plate after 24 hours, but still possessed secondary weak nuclease activity. The nucleotide sequence of nucM was determined. It presents a 798 bp open reading frame, coding for a 266‐amino‐acid protein, with a predicted molecular mass of 29 910 Da. The deduced NucM protein shows 59% sequence identity with the DNasel precursor from Vibrio cholerae. It contains a typical leader sequence. Experiments of cell fractionation showed that NucM is periplasmic In E. chrysanthemi. The transcription start has been determined by S1 mapping. The −10 and −35 regions do not show homology with consensus sequence of the promoters recognized by σ;70. In fact, The promoter seems to be dependent on the σ;70, but the first transcription nucleotide is unusually far from the −10 region. nucM seems to be expressed constitutively.
Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells
Authors: Nassif Xavier , Lowy Jonathan , Stenberg Paula , O'Gaora Peadar , Ganji Amir , So Magdalene 
Published: October 2006

SummaryPili have been shown to play an essential role in the adhesion of Neisseria meningitidis to epithelial cells. However, among piliated strains, both inter‐ and intrastrain variability exist with respect to their degree of adhesion to epithelial cells in vitro (Virji et al., 1992). This suggests that factors other than the presence of pili per se are involved in this process. The N. meningitidis pilin subunit undergoes extensive antigenic variation. Piliated low‐ and high‐adhesive derivatives of the same N. meningitidis strain were selected and the nucleotide sequence of the pilin gene expressed in each was determined. The highly adhesive derivatives had the same pilin sequence. The alleles encoding the pilin subunit of the low‐adhesive derivatives were completely different from the one found in the high‐adhesive isolates. Using polyclonal antibodies raised against one hyperadhesive variant, it was confirmed that the low‐adhesive piliated derivatives expressed pilin variants antigenically different from the highly adhesive strains. The role of antigenic variation in the adhesive process of N. meningitidis was confirmed by performing allelic exchanges of the pilE locus between low‐and high‐adhesive isolates. Antigenic variation has been considered a means by which virulent bacteria evade the host immune system. This work provides genetic proof that a bacterial pathogen, N. meningitidis, can use antigenic variation to modulate their degree of virulence.
The PrsA lipoprotein is essential for protein secretion in Bacillus subtilis and sets a limit for high‐level secretion
Authors: Kontinen Vesa P. , Sarvas Matti 
Published: October 2006

SummaryMutations of the prsA gene of Bacillus subtilis have indicated that the gene is involved in protein secretion and it encodes a novel component of the cellular secretion machinery. We now demonstrate that the gene product is a membrane‐associated lipoprotein, presumably bound to the outer face of the cytoplasmic membrane. Experiments to inactivate the prsA gene with insertions indicated that it is indispensable for viability. The cellular level of PrsA protein was shown to be decreased in prsA mutants with decreased level of exoproteins, consistent with an essential function in protein secretion. An increased amount of cellular PrsA protein was introduced by Increasing the copy number of prsA in B. subtilis. This enhanced, from six‐ to twofold, the secretion of α‐amylases and a protease in strains, which expressed high levels of these exoenzymes. This suggests that PrsA protein is the rate‐limiting component of the secretion machinery, a finding that is of considerable biotechnological interest.
Molecular analysis of the hsp (groE) operon of Leptospira interrogans serovar copenhageni
Authors: Ballard S. A. , Segers R. P. A. M. , Bleumink‐Pluym N. , Fyfe J. , Faine S. , Adler B. 
Published: October 2006

SummaryA chromosomal gene library of Leptospira interrogans serovar copenhageni strain Wijnberg was constructed in phage lambda gt11. Plaque immunoassay with RαP64 antiserum identified one clone expressing a putative groEL homologue. DNA sequence analysis of the 2.4kb EcoRI‐BamHI cloned fragment from strain Wijnberg revealed two open reading frames encoding polypeptides of 10.5kDa (Hsp10) and 58kDa (Hsp58). Sequence comparison of the deduced amino acid sequences of these ORFs confirmed the operon as the groE equivalent of Leptospira. Transcriptional analysis suggested that this operon is primarily under the control of an Eσ70 promoter element. However, both Hsp10 and Hsp58 were overexpressed under heat‐shock conditions as determined by [35S]‐methionine pulse labelling experiments. As no functional heat‐shock promoter could be identified, a 9bp inverted repeat, located between the transcription and translation start sites, may play a role in the upregulation of this operon under heat‐shock conditions, similar to mechanisms described for several Gram‐positive organisms.
Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis
Authors: Haas Rainer , Meyer Thomas F. , van Putten Jos P. M. 
Published: October 2006

SummaryThree out of 10 Helicobacter pylori clinical isolates were found to be naturally competent for genetic transformation to streptomycin resistance by chromosomal DNA extracted from a spontaneous streptomycin‐resistant H. pylori mutant. The frequency of transformation varied between 5 × 10−4 and 4 × 10−6, depending on the H. pylori isolate used. Transposon shuttle mutagenesis based on this natural competence was established using the flagellin gene flaA as the target. The cloned flaA gene was interrupted by insertion of TnMax1, a mini‐Tn1721 transposon carrying a modified chloramphenicol‐acetyltransferase gene, the catGC cassette. Natural transformation of competent H. pylori strains with plasmid constructs harbouring a catGC‐inactivated flaA gene resulted in chloramphenicol‐resistant transformants at an average frequency of 4 × 10−5. Southern hybridization experiments confirmed the replacement of the chromosomal H. pylori flaA gene by the cat‐inactivated cloned gene copy via homologous recombination resulting in allelic exchange. Phenotypic characterization of the mutants demonstrated the absence of flagella under the electron microscope and the loss of bacterial motility. Immunoblots of cell lysates of the H. pylori mutants with an antiserum raised against the C‐terminal portion of recombinant H. pylori major flagellin (FlaA) confirmed the absence of the 54kDa FlaA protein. This efficient transposon shuttle mutagenesis procedure for H. pylori based on natural competence opens up new possibilities for the genetic assessment of putative H. pylori virulence determinants.
Chromosomal mapping, expression and synthesis of lipopolysaccharide in Pseudomonas aeruginosa: a role for guanosine diphospho (GDP)‐D‐mannose
Authors: Lightfoot J. , Lam J. S. 
Published: October 2006

Summary Pseudomonas aeruginosa can express two distinct forms of lipopolysaccharide (LPS), called A‐band and B‐band. As an attempt to understand the molecular biology of the synthesis and regulation of these LPS antigens, a recombinant plasmid, pFV3, containing genes for A‐band expression was isolated previously. In the present study, P. aeruginosa strain PAO1 was mutagenized with transposon Tn5‐751 and yielded a B‐band‐deficient mutant, called ge6. This mutant was mated with a PAO1 genomic library, and transconjugants were screened for complementation of B‐band using B‐band‐specific monoclonal antibody MF15‐4. Recombinant plasmid pFV100 was subsequently isolated by its ability to complement B‐band expression in ge6. SDS‐PAGE analysis of LPS from ge6 and ge6(pFV100) revealed that ge6 was deficient in expression of B‐band, while ge6(pFV100) had an LPS profile similar to that of the parent strain PA01. With A‐band and B‐band genes cloned in separate plasmids, pFV3 and pFV100 respectively, we were able to determine the map location of these LPS genes on the P. aeruginosa PAO1 chromosome using pulsed‐field gel electrophoresis. A‐band genes mapped at 5.75 to 5.89 Mbp (Spel fragment SpK; Dpnl fragment DpF2), while genes involved with expression of B‐band LPS mapped at 1.9 Mbp (Spel fragments SpC, Spl and SpAl; Dpnl fragment DpD) on the 5.9 Mbp chromosome. We also performed initial characterization of a gene involved with synthesis of A‐band present on pFV3. We previously reported that recombinant plasmid pFV3 and subcloned plasmid pFV36 complemented A‐band synthesis in rd7513, an A− mutant derived from A+ strain AK1401. pFV36 was mutagenized with transposon Tn1000 to reveal a one‐kilobase region capable of complementing the expression of A‐band in the A− strain rd7513. This region was subcloned as a 1.6 kb Kpnl fragment into plasmid vector pAK1900 and the resulting clone named pFV39. Labelling of proteins encoded by pAK1900 and pFV39 in Escherichia coli maxicells revealed a single unique polypeptide of approximately 37kDa expressed by pFV39. Supernatants from disrupted cells of rd7513(pFV39) and AK1401 converted 14C‐labelled‐guanosine diphospho (GDP)‐D‐mannose to GDP‐rhamnose, while supernatants from rd7513 did not show synthesis of GDP‐rhamnose. The data therefore suggest that conversion of GDP‐D‐mannose to GDP‐rhamnose is required for synthesis of A‐band LPS, and that a 37kDa protein is involved in this conversion.
Rolling circle‐replicating plasmids from Gram‐positive and Gram‐negative bacteria: a wall falls
Authors: del Solar Gloria , Moscoso Miriam , Espinosa Manuel 
Published: October 2006

SummaryRolling circle‐replicating plasmids constitute a group of small, promiscuous multicopy replicons spread among eubacteria. Until recently, rolling circle replication seemed to be limited to small plasmids from Gram‐positive hosts and to single‐stranded bacteriophages from Gram‐negative bacteria. However, characterization of two small plasmids from Gram‐negative hosts has shown that this replication mechanism is general among eubacteria. This review focuses on a family of highly related promiscuous plasmids that replicate by the rolling circle mechanism, and that have been isolated from various Gram‐positive bacteria and from the Gram‐negative bacterium Helicobacter. They all share homologies at the leading‐strand origins and at the initiator of replication proteins. The plasmids of this family have directly repeated sequences at their plus origin of replication, which is located 5′ from the start point of the mRNA for the initiation of replication protein. Replication is controlled by an antisense RNA and by a transcriptional repressor protein. The features and regulatory circuits of replication of this plasmid family seem to be unique among rolling circle‐replicating plasmids. Members of this family replicate autonomously in Gram‐positive and‐negative hosts.
Electrophoretic karyotyping of the lignin‐degrading basidiomycete Phanerochaete chrysosporium
Authors: D'Souza Trevor M. , Dass S. Balachandra , Rasooly Avraham , Reddy C. Adinarayana 
Published: October 2006

SummaryElectrophoretic karyotyping of the two most widely studied strains of Phanerochaete chrysosporium, BKMF‐1767 and ME‐446, has been determined using transverse alternating field etectrophoresis. The genomic DNA of BKMF‐1767 was resolved into 10 chromosomes ranging in size from 1.8–5.0 Mb, amounting to a total genome size of about 29 Mb. The genomic DNA of strain ME‐446, on the other hand, was resolved into 11 chromosomes, amounting to a total genome size of about 32Mb. Lignin peroxidase genes have been localized to five chromosomes in strain BKMF‐1767 and to four chromosomes in strain ME‐446.
An M protein with a single C repeat prevents phagocytosis of Streptococcus pyogenes: use of a temperature‐sensitive shuttle vector to deliver homologous sequences to the chromosome of S. pyogenes
Authors: Perez‐Casal José , Price Jennifer Ayres , Maguin Emmanuelle , Scott June R. 
Published: October 2006

SummaryThe major virulence factor of the important human pathogen Streptococcus pyogenes is the M protein, which prevents phagocytosis of the bacterium. In different strains of streptococci, there are over 80 serologically different M proteins and there are additional M‐like proteins, some of which bind immunoglobulins. Although the sequence of the M molecules differs among different S. pyogenes strains, all M proteins, and some of the immunogiobulin‐binding molecules, have at least two copies of the C repeat region. We describe construction of a deletion mutation in S. pyogenes, which has only one C repeat copy, and show that the mutant strain is still resistant to phagocytosis. The mutation was constructed in vitro and used to replace the resident emm allele in an S. pyogenes strain. To facilitate homologous recombination into the streptococcal chromosome, we adapted a shuttle vector which is temperature sensitive for replication in Gram‐positive bacteria but not in Gram‐negative hosts. This new method for delivery of a homologous DNA fragment to the S. pyogenes chromosome is efficient and reproducible and should be of general use.
Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis
Authors: Cosmina Paola , Rodriguez Francesco , de Ferra Francesco , Grandi Guido , Perego Marta , Venema Gerard , van Sinderen Douwe 
Published: October 2006

SummaryThe chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilo‐bases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.