bluuebunny/crossref_metadata_2025_split huggingface dataset:
Global changes in gene expression related to antibiotic synthesis in Streptomyces hygroscopicus
Authors: Holt T. G. , Chang C. , Laurent‐Winter C. , Murakami T. , Garrels J. I. , Davies J. E. , Thompson C. J. 
Published: October 2006

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

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

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

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

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

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

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

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

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

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

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

SummaryThe development of new drugs for treating Pneumocystis carinii infections in AIDS patients is hampered by the lack of long‐term culture systems, and by our generally limited knowledge of this organism. Recently, however, we observed significant activity of various benzimidazoles against growth of this organism in short‐term cultures. Benzimidazoles inhibit microtubule polymerization; there is strong evidence that the primary target is the β‐tubulin subunit. To understand the basis for benzimidazole activity against P. carinii, and to examine the apparent relatedness of this organism to fungi, we have cloned and sequenced the single β‐tubulin gene from a rat P. carinii isolate. There was 89‐91% identity at the amino acid level to β‐tubulins from filamentous fungi, but only 79‐82% identity to yeast and protozoal β‐tubulins. Also, eight introns were distributed throughout the P. cariniiβ‐tubulin gene in a pattern characteristic of filamentous fungi. Specific residues previously implicated in benzimidazole sensitivity were conserved in P. cariniiβ‐tubulin. The polymerase chain reaction was used to amplify a segment of P. cariniiβ‐tubulin DNA from bronchoalveolar lavages obtained from two patients with AIDS. There was considerable divergence at the DNA level between the human and rat sequences, but 100% identity at the ammo‐acid level.
The Rhizobium leguminosarum FnrN protein is functionally similar to Escherichia coli Fnr and promotes heterologous oxygen‐dependent activation of transcription
Authors: Schlüter A. , Patschkowski T. , Unden G. , Priefer U. B. 
Published: October 2006

SummaryAn open reading frame from Rhizobium leguminosarum bv. viciae strain VF39, previously identified and found to be similar to Escherichia coli fnr and Rhizobium meliloti fixK (orf240, thereafter called fnrN), was further analysed. Analysis of the expression of an fnrN‐lacZ transcriptional fusion revealed that fnrN is preferentially expressed under oxygen limitation. Using R. meliloti fixN‐lacZ fusions it was shown that the fnrN gene product only mediates transcriptional activation under microaerobiosis, indicating that the FnrN protein responds, directly or indirectly, to oxygen. Plasmids which expressed fnrN under the control of an E. coli promoter were able to complement an E. coli fnr mutant with respect to anaerobic growth on nitrate but not fumarate, and to promote anaerobic but not aerobic activation of the Fnr‐dependent E. coli genes narGHJI, nirB and fdnGHI coding for nitrate reductase, NADH‐dependent nitrite reductase and formate dehydrogenase‐N, respectively. Fumarate and DMSO reductase activities were not induced by FnrN. The E. coli fnr gene substituted for fnrN in oxygen‐regulated transcription of nirB‐ and fixN‐lacZ fusions in R leguminosarum. The results indicate that Fnr and FnrN are functionally very similar and share a common mode of oxygen‐dependent transcriptional activation. From hybridization studies, it appeared that fnrN‐like genes are present in a number of different R. leguminosarum strains.
Genetic analysis of the immunity region of phage‐plasmid P4
Authors: Ghisotti Daniela , Chiaramonte Raffaelia , Forti Francesca , Zangrossi Sandro , Sironi Gianpiero , Deho Gianni 
Published: October 2006

SummaryIn the prophage P4, expression of the early genes is prevented by premature termination of transcription from the constitutive promoter Ple. In order to identify the region coding for the immunity determinant, we cloned several fragments of P4 DNA and tested their ability to confer immunity to P4 superinfection. A 357 bp long fragment (P4 8418‐8774) is sufficient to confer immunity to an infecting P4 phage and to complement the immunity‐defective P4 cl405 mutant, both in the presence and in the absence of the helper phage P2.The immunity region covers PLE and the cl locus. We were unable to obtain evidence of translation of the region, thus we suggest that P4 immunity is not elicited by a protein but by a transcript (or transcripts) encoded by the region downstream of the promoter PLE, The promoter PLE appears to be necessary for the expression of P4 immunity: fragments in which the PLE region is deleted did not complement P4 cl405 for lysogenization, although they still interfered with P4 growth. Two complementary sequences downstream of PLE(seqA and seqB) at the 5’and 3’ends of the immunity region play an essential role in the control of P4 immunity.
A plasmid‐encoded type IV fimbrial gene of enteropathogenic Escherichia coli associated with localized adherence
Authors: Donnenberg M. S. , Girón J. A. , Nataro J. P. , Kaper J. B. 
Published: October 2006

SummaryEnteropathogenic Escherichia coli (EPEC) form adherent microcolonies on the surface of tissue culture cells in a pattern termed localized adherence. Localized adherence requires the presence of a large EPEC adherence factor (EAF) plasmid. Recently a bundle‐forming pilus has been described in EPEC possessing the EAF plasmid. An analysis of 22 non‐invasive EPEC TnphoA mutants revealed that seven have insertions in the EAF plasmid and are incapable of localized adherence. We report here the mapping of the TnphoA insertions in these mutants. The nucleotide sequence of the gene interrupted in these TnphoA mutants (bfpA) was determined and found to correspond to the N‐terminal amino acid sequence of the major structural protein of the bundle‐forming pilus. The bfpA gene bears sequence similarities to members of the type IV fimbrial gene family and encodes a potential site for processing by a prepilin peptidase. A plasmid containing bfpA as the only open reading frame directs the synthesis of a protein recognized by antiserum raised against the bundle‐forming pilus. TnphoA mutants at this locus are unable to synthesize BfpA, but synthesis is restored by introduction of a plasmid containing the cloned gene. The minimum fragment of DNA required to restore localized adherence is considerably greater than that required to restore BfpA synthesis. BfpA expression, as assessed by alkaline phophatase activity in bfpA::TnphoA mutants, is affected by temperature and growth medium. These studies describe an EPEC plasmid‐encoded fimbrial gene, a candidate for the elusive EPEC adherence factor responsible for localized adherence.
Interaction of two variable proteins (PilE and PilC) required for pilus‐mediated adherence of Neisseria gonorrhoeae to human epithelial cells
Authors: Rudel Thomas , van Putten Jos P. M. , Gibbs Carol P. , Haas Rainer , Meyer Thomas F. 
Published: October 2006

SummaryPili confer the initial ability of Neisseria gonorrhoeae to bind to epithelial cells. Pilin (PilE), the major pilus subunit, and a minor protein termed PilC, reportedly essential for pilus biogenesis, undergo intra‐strain phase and structural variation. We demonstrate here that at least two different adherence properties are associated with the gonococcal pili: one is specific for erythrocytes, which is virtually unaffected by PilE variation, and another is specific for epithelial cells, and is modulated in response to the variation of PilE. Based on this finding, mutants of a recA ‐ strain were selected that had lost the ability to bind to human cornea epithelial cells (A‐) but retained the ability to form pili (P+) and to agglutinate human erythrocytes (H+). The adherence‐negative mutants failed to produce detectable levels of PMC1 or PilC2 proteins, representing pilC phase variants generated in the absence of RecA. The A‐pilC phase variants were indistinguishable from their A+parents and spontaneous A+ revertants with regard to the amount of PilE produced and its electrophoretic mobility, the degrees of piliation and haemagglutination, and the pilE nucleotide sequence. These data demonstrate a central role for PilC in pilus‐mediated adherence of N. gonorrhoeae to human epithelial cells and further indicate that neither PNC1 nor PilC2 is obligatory for the assembly of gonococcal pili.