bluuebunny/crossref_metadata_2025 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

The 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

The 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

Phaseolotoxin, 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

The 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

Endospore 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

To 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

The 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

Pili 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

Mutations 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

A 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

Three 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

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

Rolling 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

Electrophoretic 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

The 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

The 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.
Identification of cpsD, a gene essential for type III capsule expression in group B streptococci
Authors: Rubens Craig E. , Heggen Laura M. , Haft Rachel F. , Wessels Michael R.
Published: October 2006

We showed previously that a mutant strain of group B Streptococcus (GBS) defective in capsule production was avirulent. This study describes the derivation of an unencapsulated mutant from a highly encapsulated wild‐type strain of type III GBS, COH1, by transposon mutagenesis with Tn916ΔE. The mutant, COH1‐13, was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat sepsis model compared with the wild‐type strain. No capsular polysaccharide was evident in the cytoplasm or on the cell surface of the mutant strain. The Tn916ΔE insertion site in COH1‐13 was mapped to the same chromosomal location as the Tn916 insertion site in the unencapsulated type III mutant COH31‐15 reported previously. Nucleotide sequencing of DNA flanking the insertion site in COH1‐13 revealed an open reading frame, designated cpsD, with significant homology to the rfbP gene of Salmonella typhimurium. RfbP encodes a galactosyl transferase enzyme that catalyses the transfer of galactose to undecaprenol phosphate, the initial step in O‐polysaccharide synthesis. A particulate fraction of a lysate of wild‐type strain GBS COH1 mediated the transfer of galactose from UDP‐galactose to an endogenous acceptor. The galactose–acceptor complex partitioned into organic solvents, suggesting it is lipid in nature or membrane‐associated. Galactosyl transferase activity was significantly reduced in the unencapsulated mutant strain COH1‐13. These results, together with the similarity in deduced amino acid sequence between cpsD and rfbP suggest that cpsD encodes a galactosyl transferase essential for assembly of the GBS type III capsular polysaccharide.
Proteins essential for expression of the Hms+ phenotype of Yersinia pestis
Authors: Pendrak Michael L. , Perry Robert D.
Published: October 2006

One characteristic of pigmented (Pgm+) cells of Yersinia pestis is the adsorption of sufficient quantities of exogenous haemin during growth at 26°C to form dark‐brown colonies. Carriage of the cloned haemin‐storage (hms) locus in pHMS1 restores this phenotype to spontaneous Pgm− chromosomal deletion mutants of Y. pestis. We have mapped the location of the structural genes for four proteins encoded on pHMS1 using minicell, in vitro transcription/translation, and complementation analysis. The hmsH and hmsF genes encode 90kDa and 72 kDa protein precursors processed to surface‐exposed, outer membrane proteins of 86kDa and 70kDa, respectively. Beta‐galactosidase positive MudII1734 insertions in hmsR suggest that it encodes a protein that is also essential for haemin storage. Finally, the structural gene for a 41 kDa protein lies distal to the hmsH gene but, unlike hmsH, hmsF, and hmsR, its expression is not essential for the Hms+ phenotype in Y. pestis.
Synthesis of the Escherichia coli K‐12 nucleoid‐associated DNA‐binding protein H‐NS is subjected to growth‐phase control and autoregulation
Authors: Dersch Petra , Schmidt Keratin , Bremer Erhard
Published: October 2006

Mutations in the structural gene (hns) for the Escherichia coli nucleoid‐associated DNA‐binding protein H‐NS cause highly pleiotropic effects on gene expression, site‐specific recombination, transposition of phage Mu, the stability of the genetic material and the topological state of the DNA. We have investigated the regulation of hns expression and found that hns transcription is subjected to stationary phase induction and negative autoregulation. A set of hns–lacZ protein and operon fusions was constructed in vitro and integrated in single copy into the attB site of the bacterial genome. Quantification of β‐galactosidase activity along the bacterial growth curve showed that hns expression increases approximately 10‐fold in stationary phase compared with exponentially growing cells. Immunological detection of the H‐NS protein in growing and stationary phase cells supported the genetic data and showed that H‐NS synthesis varies with growth phase. In addition, primer extension experiments demonstrated that the amount of hns mRNA is elevated in stationary phase cultures and that hns transcription is directed by a unique promoter functioning in both log and stationary phase. Disruption of the hns gene by an insertion mutation led to the derepression (approximately fourfold) of the expression of an hns–lacZ operon fusion integrated at the attB site, showing that hns transcription is subjected to negative regulation by its own gene product. Autoregulation of hns expression is particularly pronounced in log phase. Both stationary phase control and autoregulation of hns transcription are associated with a 130 bp fragment that contains the hns promoter. In order to study the interaction of H‐NS with its own regulatory region, we developed an efficient overproduction procedure and a simple purification scheme for H‐NS. DNA gel retardation assays showed that the H‐NS protein can preferentially interact with a restriction fragment carrying the hnspromoter. This restriction fragment showed features of curved DNA as judged by two‐dimensional polyacrylamide gel electrophoresis performed at 4°C and 60°C.
The role of galE in the biosynthesis and function of gonococcal lipopolysaccharide
Authors: Robertson Brian D. , Frosch Matthias , van Putten Jos P. M.
Published: October 2006

Lipopolysaccharide is an essential component of the outer membrane of Gram‐negative bacteria and an important virulence factor of many pathogens, such as Neisseria gonorrhoeae. We have cloned the gonococcal galE gene which was found to be located in the gonococcal homologue of the meningococcal capsule gene complex region D. Sequence alignment indicated extensive homology with the Escherichia coli and Salmonella GalE proteins. Mutants with insertions in the galE gene were used as a tool to characterize the structure and function of gonococcal lipopolysaccharide. They displayed deep rough phenotypes, and chemical analysis confirmed the loss of galactose from the mutant lipopolysaccharide. Functional analysis indicated that the terminal oligosaccharides contain galactose and that these are lost in galE mutants. The importance of these oligosaccharides in gonococcal biology is clear from the fact that they contain the epitopes that are the targets for killing by normal human serum, and the acceptor site for sialic acid, which acts to protect the gonococcus from this killing. Furthermore, infection experiments in vitro indicate that the galE mutants exhibit unaltered intergonococcal adhesion as well as adhesion to, and invasion of, epithelial cells.
Sequence, genetic, and lacZ fusion analyses of a nifR3–ntrB–ntrC operon in Rhodobacter capsulatus
Authors: Foster‐Hartnett Dawn , Cullen Paul J. , Gabbert Karen K. , Kranz Robert G.
Published: October 2006

Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. Regulatory genes required to sense and relay the nitrogen status of the cell are gInB, ntrB (nifR2), and ntrC (nifR1). R. capsulatus nifA1 and nifA2 require ntrC for activation when fixed nitrogen is limiting. The polypeptides encoded by nifA1 and nif42 along with the alternate Sigma factor RpoN activate nifHDK and the remaining nif genes in the absence of both fixed nitrogen and oxygen. In this study we report the sequence and genetic analysis of the previously Identified nifR3–ntrB–ntrC regulatory locus. nifR3 is predicted to encode a 324‐amino‐acid protein with significant homology to an upstream open reading frame cotranscribed with the Escherichia coli fregulatory gene, fis. Analysis of ntrC–lacZ fusions and complementation data indicate that nifR3ntrBC constitute a single operon. nifR3–lacZfusions are expressed only when lacZ is in the proper reading frame with the predicted nifR3 gene product. Tn5, a kanamycin‐resistance cassette, and miniMu insertions in nifR3 are polar on nfrBC (required for nif transcription). This gene organization suggests that the nifR3gene product may be involved in nitrogen regulation, although nifR3 is not stringently required for nitrogen fixation when ntrBC are present on a multicopy plasmid. In addition, a R. capsulatus strain with a 22‐nucleotide insert in the chromosomal nifR3 gene was constructed. This nifR3 strain is able to fix nitrogen and activate nifA1 and nifA2 genes, again supporting the hypothesis that nifR3 is not stringently required for nfrC‐dependent gene activation in R. capsulatus.
Formation of a putative relaxation intermediate during T‐DNA processing directed by the Agrobacterium tumefaciens VirD1, D2 endonuclease
Authors: Filichkin Sergei A. , Gelvin Stanton B.
Published: October 2006

During the initial stages of crown gall tumorigenesis, the T‐DNA region of the Agrobacterium tumefaciens Ti‐plasmid is processed, resulting in the production of T‐DNA molecules that are subsequently transferred to the plant cell. Processing of the T‐DNA in the bacterium involves the nicking of T‐DNA border sequences by an endonuclease encoded by the virD locus, and the subsequent tight (possibly covalent) association of the VirD2 protein with the 5′ end of the processed single‐stranded or double‐stranded T‐DNA molecule. To investigate the interaction of the VirD1,D2 endonuclease with a right T‐DNA border, a set of plasmids containing both the border and virD sequences on the same high‐copy‐number replicon has been constructed and introduced into Escherichia coli. In this model system a tight nucleoprotein complex is formed between the relaxed double‐stranded substrate plasmid and the VirD2 protein. This putative T‐DNA processing complex may be analogous to the covalent relaxation complex formed between the pilot protein and plasmid DNA during bacterial conjugation. VirD2 attachment to the relaxed substrate plasmid was resistant to denaturing agents but sensitive to S1 nuclease digestion, indicating a single‐stranded region near the site of protein attachment. We speculate that this structure may be an intermediate formed prior to T‐strand unwinding from the substrate plasmid in a host bacterium.
Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae
Authors: Van Aelst Linda , Hohmann Stefan , Bulaya Botchaka , de Koning Wim , Sierkstra Laurens , Neves Maria José , Luyten Kattie , Alijo Rafael , Ramos José , Coccetti Paola , Martegani Enzo , de Magalhães‐Rocha Neuza Maria , Brandão Rogelio Lopes , Van Dijck Patrick , Vanhalewyn Mieke , Durnez Peter , Arnold W. H. Jans  , Thevelein Johan M.
Published: October 2006

Cells of the yeast Saccharomyces cerevisiae display a wide range of glucose‐induced regulatory phenomena, including glucose‐induced activation of the RAS‐adenylate cyclase pathway and phosphatidylinositol turnover, rapid post‐translational effects on the activity of different enzymes as well as long‐term effects at the transcriptional level. A gene called GGS1 (for General Glucose Sensor) that is apparently required for the glucose‐induced regulatory effects and several ggs1 alleles (fdp1, byp1 and cif1) has been cloned and characterized. A GGS1 homologue is present in Methanobacterium thermoautotrophicum. Yeast ggs1 mutants are unable to grow on glucose or related readily fermentable sugars, apparently owing to unrestricted influx of sugar into glycolysis, resulting in its rapid deregulation. Levels of intracellular free glucose and metabolites measured over a period of a few minutes after addition of glucose to cells of a ggsi1Δ strain are consistent with our previous suggestion of a functional interaction between a sugar transporter, a sugar kinase and the GGS1 gene product. Such a glucose‐sensing system might both restrict the influx of glucose and activate several signal transduction pathways, leading to the wide range of glucose‐induced regulatory phenomena. Deregulation of these pathways in ggs1 mutants might explain phenotypic defects observed in the absence of glucose, e.g. the inability of ggs1 diploids to sporulate.
The importance of morphological events and intercellular interactions in the regulation of prespore‐specific gene expression during sporulation in Bacillus subtilis
Authors: Partridge S. R. , Errington J.
Published: October 2006

We have established a time course for the early morphological events of sporulation in Bacillus subtilis and related this to changes in gene expression, particularly those occurring in the prespore compartment. We have also systematically studied the effects of mutations in various regulatory (spo) genes on prespore‐specific gene expression. On the basis of these results, and those of other laboratories, at least four distinct temporal classes of prespore‐specific gene expression can now be distinguished. The first class begins within 15min of the formation of the spore septum, and requires the σ;F form of RNA polymerase. The second class, also directed by RNA polymerase containing σ;F, begins soon after the completion of prespore engulfment, and depends on an intercellular signal from the mother cell. This transcription results in synthesis of σ;G. However, σ;G activity, directing the third class of gene expression, appears only about 30min later and is dependent on the completion of prespore engulfment and on further interactions with the mother cell. The fourth class of gene expression has been described. The results demonstrate that the prespore programme of gene expression incorporates a series of control points modulated by information from the mother cell and on progress through the morphogenetic process.
Linear chromosomal physical and genetic map of Borrelia burgdorferi, the Lyme disease agent
Authors: Casjens Sherwood , Huang Wai Mun
Published: October 2006

A physical map of the 952kbp chromosome of Borrelia burgdorferi Sh‐2‐82 has been constructed. Eighty‐three intervals on the chromosome, defined by the cleavage sites of 15 restriction enzymes, are delineated. The intervals vary in size from 96kbp to a few hundred bp, with an average size of 11.5 kbp. A striking feature of the map is its linearity; no other bacterial groups are known to have linear chromosomes. The two ends of the chromosome do not hybridize with one another, indicating that there are no large common terminal regions. The chromosome of this strain was found to be stable in culture; passage 6, 165 and 320 cultures have identical chromosomal restriction maps. We have positioned all previously known Borrelia burgdorferi chromosomal genes and several newly identified ones on this map. These include the gyrA/gyrB/dnaA/dnaN gene cluster, the rRNA gene cluster, fla, flgE, groEL (hsp60), recA, the rho/hip cluster, the dnaK (hsp70)/dnaJ/grpE cluster, the pheT/pheS cluster, and the genes which encode the potent immunogen proteins p22A, p39 and p83. Our electrophoretic analysis detects five linear and at least two circular plasmids in B. burgdorferi Sh‐2‐82. We have constructed a physical map of the 53 kbp linear plasmid and located the operon that encodes the two major outer surface proteins ospA and ospB on this plasmid. Because of the absence of functional genetic tools for this organism, these maps will serve as a basis for future mapping, cloning and sequencing studies of B. burgdorferi.
Nucleotide substitutions and small‐scale insertion produce size and antigenic variation in group A streptococcal M1 protein
Authors: Harbaugh M. P. , Podbielski A. , Hügl S. , Cleary P. P.
Published: October 2006

The presence of M protein on the surface of group A streptococci (GAS) confers the ability of the cell to resist phagocytosis in the absence of type‐specific antibodies. It undergoes antigenic variation with more than 80 different serotypes having been defined. We have sequenced the M protein gene (emm1.1) from strain CS190 and present evidence that individual nucleotide substitutions are responsible for sequence variation in the N‐terminal non‐repeat region of emm1.1 and these substitutions have altered antibody recognition of opsonic epitopes. The N‐terminal non‐repeat domains of two other closely related strains, 71‐155 and 76‐088, were found to have sequence identical to emm1.1 with the addition of a 21 bp insert. This study provides the first evidence that nucleotide substitutions and small insertions are responsible for size and antigenic variation in the N terminal non‐repeat domain of the M protein of GAS.
Cloning and nucleotide sequence of anaerobically induced porin protein E1 (OprE) of Pseudomonas aeruginosa PAO1
Authors: Yamano Yoshinori , Nishikawa Tohru , Komatsu Yoshihide
Published: October 2006

The porin oprE gene of Pseudomonas aeruginosa PAO1 was isolated. Its nucleotide sequence indicated that the structural gene of 1383 nucleotide residues encodes a precursor consisting of 460 amino acid residues with a signal peptide of 29 amino acid residues, which was confirmed by the N‐terminal 23‐amino‐acid sequence and the reaction with anti‐OprE polyclonal antiserum. Anaerobiosis induced OprE production at the transcription level. The transcription start site was determined to be 40 nucleotides upstream from the ATG initiation codon. The control region contained an appropriately situated Eσ54 recognition site and the putative second half of an ANR box. The amino acid sequence of OprE had some clusters of sequence homologous with that of OprD of P. aeruginosa, which might be responsible for the outer membrane permeability of imipenem and basic amino acids.
Regulation by Ca2+ in the Yersinia low‐Ca2+ response
Authors: Straley Susan C. , Plano Gregory V. , Skrzypek Elźbieta , Haddix Pryce L. , Fields Kenneth A.
Published: October 2006

The Yersinia low‐Ca2+ response (LCR) is a regulatory response in which a set of plasmid‐borne operons is transcriptionally regulated at 37°C in response to the presence or absence of mM concentrations of Ca2+. LCR‐regulated operons encode secreted proteins with regulatory and virulence roles as well as non‐secreted regulatory proteins and components of the secretion machinery. Downregulation by Ca2+ is imposed by a signalling cascade that includes secreted proteins and possibly also components of the secretion system and is hypothesized to act on membrane‐bound inductive components. An important rote in LCR induction is played by LcrD, an inner‐membrane protein with homologues in several virulence‐associated and flagella assembly‐related systems in diverse bacterial species. The mechanism of signal transduction in response to Ca2+ is not known, and the proteins that bind DNA to downregulate transcription have not been identified.
Multicopy plasmid instability: the dimer catastrophe hypothesis
Authors: Summers David K. , Beton Christopher W. H. , Withers Helen L.
Published: October 2006

Multimer formation reduces plasmid copy number and is an established cause of segregational instability. Nevertheless, it is difficult to rationalize observations that low levels of dimers can cause severe instability, if we assume they are distributed evenly in cell populations. We report here that dimer distribution is in fact heterogeneous in recombination‐proficient strains. Most cells in the population contain only monomers; dimers are confined to a small sub‐population from which plasmid‐free daughters arise at high frequency. In a rec+ culture where 4% of pBR322 molecules are dimers, more than half are in dimer‐only cells. We show that this situation is inevitable because dimers replicate at twice the rate of monomers. Runaway multimerization is avoided because dimer‐containing cells grow more slowly than their monomer‐containing counterparts. A computer simulation is used to show how dimers proliferate after formation by homologous recombination. The equilibrium concentration of dimers is proportional to the inter‐plasmid recombination rate and is essentially independent of the rate at which homologous recombination converts dimers to monomers.
The alkane oxidation system of Pseudomonas oleovorans: induction of the alk genes in Escherichia coli W3110(pGEc47) affects membrane biogenesis and results in overexpression of alkane hydroxylase in a distinct cytoplasmic membrane subtraction
Authors: Nieboer Maarten , Kingma Jaap , Witholt Bernard
Published: October 2006

The alkane hydroxylase system of Pseudomonas oleovorans, which catalyses the initial oxidation of aliphatic substrates, is encoded by three genes. One of the gene products, the alkane hydroxyiase AlkB, is an integral cytoplasmic membrane protein. Induction leads to the synthesis of 1.5–2% AlkB relative to the total cell protein, both in P. oleovorans and in recombinant Escherichia coli DH1. We present a study on the Induction and localization of the alkane hydroxylase in E. coli W3110, which appears to be an interesting host strain because it permits expression levels of AlkB of up to 10–15% of the total cell protein. This expression level had negative effects on cell growth. The phospholipid content of such cells was about threefold higher than that of wild‐type W3110. Freeze‐fracture electron microscopy showed that induction of the alk genes led to the appearance of membrane vesicles in the cytoplasm; these occurred much more frequently in cells expressing alkB than in the negative control, which contained all of the alk genes except for alkB. Isolation and separation of the membranes of cells expressing alkB by density gradient centrifugation showed the customary cytoplasmic and outer membranes, as well as a low‐density membrane fraction. This additional fraction was highly enriched in AlkB, as shown both by SDS‐PAGE and enzyme activity measurements. A typical cytoplasmic membrane protein, NADH oxidase, was absent from the low‐density membrane fraction, alkB expression in W3110 changed the composition of the phospholipid headgroup in the membrane, as well as the fatty acid composition of the membrane. The major changes occurred in the unsaturated fatty acids: C16:1 and C18:1 increased at the expense of C17:0cyc and C19:0cyc*
Genetic stability and xanthan gum production in Xanthomonas campestris pv. campestris NRRL B1459
Authors: Martínez‐Salazar Jaime M. , Palacios Alma Nirvana , Sánchez Rosalba , Caro Alma Delia , Soberón‐Chavez Gloria
Published: October 2006

A transposon (Tn5‐SC) was constructed that can be used to quantify genetic deletions or amplifications. This transposon was used to evaluate the genomic stability of Xanthomonas campestris pv. campestris NRRL B1459 and we found that the genome of this bacterium is as stable as other Gram‐negative bacteria or even more stable. Homologous recombination between plasmid sequences was determined in strain NRRL B1459 and was found to occur at a similar level to that reported for other Gram‐negative bacteria. We report here that in X.c.c. NRRL B1459 there is no straightforward correlation between the occurrence of genetic rearrangements and frequency of homologous recombination. These data are discussed with respect to the reported instability of strain NRRL B1459 for xanthan gum production.