bluuebunny/crossref_metadata_2025_split huggingface dataset:
Parallel pathways for oxidation of 14‐membered polyketide macrolactones in Saccharopolyspora erythraea
Authors: Gaisser Sabine , Lill Rachel , Staunton James , Méndez Carmen , Salas José , Leadlay Peter F. 
Published: March 2003

SummaryThe glycosyltransferases OleG1 and OleG2 and the cytochrome P450 oxidase OleP from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus have
been expressed, either separately or from artificial gene cassettes, in strains of
Saccharopolyspora erythraea blocked in erythromycin biosynthesis, to investigate
their potential for the production of diverse novel macrolides from erythronolide
precursors. OleP was found to oxidize 6‐deoxyerythronolide B, but not erythronolide
B. However, OleP did oxidize derivatives of erythronolide B in which a neutral sugar
is attached at C‐3. The oxidized products 3‐O‐mycarosyl‐8a‐hydroxyerythronolide
B, 3‐O‐mycarosyl‐8,8a‐epoxyerythronolide B, 6‐deoxy‐8‐hydroxyerythronolide
B and the olefin 6‐deoxy‐8,8a‐dehydroerythronolide B were all isolated and their
structures determined. When oleP and the mycarosyltransferase eryBV
were co‐expressed in a gene cassette, 3‐O‐mycarosyl‐6‐deoxy‐8,8a‐dihydroxyerythronolide
B was directly obtained. When oleG2 was co‐expressed in a gene cassette together
with oleP, 6‐deoxyerythronolide B was converted into a mixture of 3‐O‐rhamnosyl‐6‐deoxy‐8,8a‐dehydroerythronolide
B and 3‐O‐rhamnosyl‐6‐deoxy‐8,8a‐dihydroxyerythronolide B, confirming previous
reports that OleG2 can transfer rhamnose, and confirming that oxidation by OleP and
attachment of the neutral sugar to the aglycone can occur in either order. Similarly,
four different 3‐O‐mycarosylerythronolides were found to be substrates for the desosaminyltransferase OleG1. These results provide additional insight into the nature of the intermediates in OleP‐mediated oxidation, and suggest that oleandomycin biosynthesis might follow parallel pathways in which epoxidation either precedes or follows attachment of the neutral sugar.
Spx (YjbD), a negative effector of competence in Bacillus subtilis, enhances ClpC–MecA–ComK interaction
Authors: Nakano Michiko M. , Nakano Shunji , Zuber Peter 
Published: March 2003

SummaryComK, a key transcriptional regulator in the development of competence in Bacillus subtilis, is required for its own transcription as well as that of the late competence genes encoding proteins involved in DNA uptake. ComK is sequestered in a complex with ClpC and MecA until a peptide, ComS, accumulates in cells. ComS releases ComK from the inhibitory complex, thus allowing ComK to carry out its function as a transcriptional activator. Spx (formerly YjbD), a negative effector of competence, accumulates in clpP mutants. High concentrations of Spx may be responsible for the inability of clpP mutants to become competent because spx mutations are able to restore competence in the clpP mutant. In this paper, we showed, based on in vitro experiments, that Spx forms a quaternary complex with ClpC, MecA and ComK and enhances ComK binding to ClpC–MecA. Two ComS alanine substitution mutants (I13A and W43A), previously shown to be defective in vivo, were less efficient in releasing ComK from ClpC–MecA. The I13A mutant with a weaker binding affinity to MecA was inefficient in releasing ComK regardless of whether Spx was present. In contrast, the defect of the W43A mutant in dissociating ComK was more readily observed in the presence of Spx. Spx is a highly conserved protein among Gram‐positive bacteria, in which it may function closely with the protease adaptor protein, MecA.
Molecular analysis of dimethyl sulphide dehydrogenase from Rhodovulum sulfidophilum: its place in the dimethyl sulphoxide reductase family of microbial molybdopterin‐containing enzymes
Authors: McDevitt Christopher A. , Hugenholtz Philip , Hanson Graeme R. , McEwan Alastair G. 
Published: March 2003

SummaryDimethyl sulphide dehydrogenase catalyses the oxidation of dimethyl sulphide to dimethyl sulphoxide (DMSO) during photoautotrophic growth of Rhodovulum sulfidophilum. Dimethyl sulphide dehydrogenase was shown to contain bis(molybdopterin guanine dinucleotide)Mo, the form of the pterin molybdenum cofactor unique to enzymes of the DMSO reductase family. Sequence analysis of the ddh gene cluster showed that the ddhA gene encodes a polypeptide with highest sequence similarity to the molybdop‐terin‐containing subunits of selenate reductase, ethylbenzene dehydrogenase. These polypeptides form a distinct clade within the DMSO reductase family. Further sequence analysis of the ddh gene cluster identified three genes, ddhB, ddhD and ddhC. DdhB showed sequence homology to NarH, suggesting that it contains multiple iron–sulphur clusters. Analysis of the N‐terminal signal sequence of DdhA suggests that it is secreted via the Tat secretory system in complex with DdhB, whereas DdhC is probably secreted via a Sec‐dependent mechanism. Analysis of a ddhA mutant showed that dimethyl sulphide dehydrogenase was essential for photolithotrophic growth of Rv. sulfidophilum on dimethyl sulphide but not for chemo‐trophic growth on the same substrate. Mutational analysis showed that cytochrome c2 mediated photosynthetic electron transfer from dimethyl sulphide dehydrogenase to the photochemical reaction centre, although this cytochrome was not essential for photoheterotrophic growth of the bacterium.
Cyclin‐dependent kinase TPK2 is a critical cell cycle regulator in Toxoplasma gondii
Authors: Khan Farzana , Tang Jianzhong , Qin Chang‐le , Kim Kami 
Published: March 2003

SummaryThe Apicomplexan parasite Toxoplasma gondii replicates by endodyogeny, an unusual form of binary fission. We tested the role of TPK2, a homologue of the CDC2 cyclin‐dependent kinases, in cell cycle regulation. TPK2 tagged with HA epitope (TPK2‐HA‐wt) was expressed in mammalian cells as confirmed by Western blot analysis using HA tag and PSTAIRE antibodies. TPK2‐HA‐wt phosphorylated a peptide from Histone H1, proving that TPK2 is a functional kinase. TPK2‐HA‐wt coimmunoprecipitated with mammalian cyclins A, B1, D3 and E. Despite being a functional kinase, TPK2 did not rescue Schizosaccharomyces pombe cdc2 and Saccharomyces cerevisiae cdc28 mutant strains. Overexpression of a dominant‐negative mutant of TPK2 (TPK2‐HA‐dn) in T. gondii tachyzoites arrested replication. FACS analysis of tachyzoites expressing TPK2‐HA‐dn revealed an increase in the fraction of cells in S‐phase when compared with TPK2‐HA‐wt transfected parasites. Expression of TPK2‐HA‐wt did not arrest tachyzoite replication. No discernable G2 cell cycle block was evident suggesting that cell cycle checkpoints differ in T. gondii from most other eukaryotic cells. These data suggest that TPK2 executes an essential function in T. gondii cell cycle and is likely to be the T. gondii CDC2 orthologue.
Regulation of tylosin production and morphological differentiation in Streptomyces fradiae by TylP, a deduced γ‐butyrolactone receptor
Authors: Stratigopoulos George , Gandecha Atul R. , Cundliffe Eric 
Published: March 2003

SummaryDuring promoter‐probe analysis carried out in Streptomyces lividans, the TylP protein powerfully inhibited reporter gene expression from the tylP promoter, raising the likelihood that tylP is autoregulated in its native host, Streptomyces fradiae. Also in S. lividans, TylP negatively controlled the tylQ promoter, even though tylQ could still be switched off in S. fradiae strains specifically disrupted in tylP. Under the latter conditions, tylosin production was brought forward and enhanced, whereas overexpression of tylP re‐sulted in reduced levels of the antibiotic, accompanied by barely detectable transcription from multiple genes of the tylosin biosynthetic cluster. Unexpectedly, overexpression of tylP reduced transcription of tylS, a transcriptional activator essential for tylosin production. This was probably a direct effect, as TylP also reduced expression from the tylS promoter in S. lividans. For these several reasons, we conclude that TylP acts as a repressor during tylosin biosynthesis. In addition, TylP influences morphological differentiation in S. fradiae. On solid media, strains in which tylP was disrupted sporulated significantly earlier than wild type and, in liquid culture, displayed hyperfragmentation.
Adaptation of protein secretion to extremely high‐salt conditions by extensive use of the twin‐arginine translocation pathway
Authors: Rose R. Wesley , Brüser Thomas , Kissinger Jessica C. , Pohlschröder Mechthild 
Published: March 2003

SummaryHalophilic archaea thrive in environments with salt concentrations approaching saturation. However, little is known about the way in which these organisms stabilize their secreted proteins in such ‘hostile’ conditions. Here, we present data suggesting that the utilization of protein translocation pathways for protein secretion by the Halobacteriaceae differs significantly from that of non‐haloarchaea, and most probably represents an adaptation to the high‐salt environment. Although most proteins are secreted via the general secretion (Sec) machinery, the twin‐arginine translocation (Tat) pathway is mainly used for the secretion of redox proteins and is distinct from the Sec pathway, in that it allows cytoplasmic folding of secreted proteins. tatfind (developed in this study) was used for systematic whole‐genome analysis of Halobacterium sp. NRC‐1 and several other prokaryotes to identify putative Tat substrates. Our analyses revealed that the vast majority of haloarchaeal secreted proteins were predicted substrates of the Tat pathway. Strikingly, most of these putative Tat substrates were non‐redox proteins, the homologues of which in non‐haloarchaea were identified as putative Sec substrates. We confirmed experimentally that the secretion of one such putative Tat substrate depended on the twin‐arginine motif in its signal sequence. This extensive utilization of the Tat pathway in haloarchaea suggests an evolutionary adaptation to high‐salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion.
Chromosome replication patterns in the hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii
Authors: Maisnier‐Patin Sophie , Malandrin Laurence , Birkeland Nils‐Kåre , Bernander Rolf 
Published: March 2003

SummaryWe analysed chromosome replication patterns in the two hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii by marker frequency analysis (MFA). For A. fulgidus, the central region of the chromosomal physical map displayed a higher relative abundance in gene dosage during exponential growth, with two continuous gradients to a region of lower abundance at the diametrically opposite side of the genome map. This suggests bidirectional replication of the A. fulgidus chromosome from a single origin. The organization of the putative replication origin region relative to the cdc6, mcm and DNA polymerase genes differed from that reported for Pyrococcus species. No single replication origin or termination regions could be identified for M. jannaschii, adding to the list of unusual properties of this organism. The organization of the A. fulgidus cell cycle was characterized by flow cytometry analysis of the samples from which genomic DNA was extracted for MFA. The relative lengths of the cell cycle periods were found to be similar to those of crenarchaea.
Suppression of a nuclear frameshift mutation by a mitochondrial tRNA in the yeast Kluyveromyces lactis‡
Authors: Fiori Alessandro , Francisci Silvia , Falcone Claudio 
Published: March 2003

SummaryA fragment of mitochondrial DNA containing the Kluyveromyces lactis gene for valine‐tRNA (tRNAVAL) was isolated as a multicopy suppressor of a respiratory‐deficient mutant of this yeast. The mutant produced a truncated Cox14p because of a +1 frameshift mutation in COX14, a nuclear gene encoding a protein imported into mitochondria which is necessary for respiration (Fiori et al. 2000 Yeast 16: 307–314). We report here that the mitochondrial tRNAVAL gene, when transformed into K. lactis cells, is transcribed outside mitochondria and suppresses the frameshift mutation in COX14 restoring the correct reading frame during translation of its mRNA. In fact, using histidine tagging, the existence of a suppressed Cox14p of normal length was demonstrated in mutants expressing the mt‐tRNAVAL from the nucleus. Suppression could occur through a non‐canonical four base pairing between the tRNAVAL and the mutated mRNA or through slippage of ribosomes during translation. This is a new case of informational suppression in that the suppression of a chromosomal mutation is not casused by a second mutation but to a mislocalization/expression of a mt‐tRNA.
DNA damage induction of recA in Mycobacterium tuberculosis independently of RecA and LexA
Authors: Davis Elaine O. , Springer Burkhard , Gopaul Krishna K. , Papavinasasundaram K. G. , Sander Peter , Böttger Erik C. 
Published: March 2003

SummaryThe ubiquitous and highly conserved RecA protein is generally expressed from a single promoter, which is regulated by LexA in conjunction with RecA. We show here using transcriptional fusions to a reporter gene that the Mycobacterium tuberculosis recA gene is expressed from two promoters. Although one promoter is clearly regulated in the classical way, the other remains DNA damage inducible in the absence of RecA or when LexA binding is prevented. These observations demonstrate convincingly for the first time that there is a novel mechanism of DNA damage induction in M. tuberculosis that is independent of LexA and RecA.
Saccharomyces cerevisiae C1D is implicated in both non‐homologous DNA end joining and homologous recombination
Authors: Erdemir Tuba , Bilican Bilada , Cagatay Tolga , Goding Colin R. , Yavuzer Ugur 
Published: March 2003

SummaryC1D is a gamma‐irradiation inducible nuclear matrix protein that interacts with and activates the DNA‐dependent protein kinase (DNA‐PK) that is essential for the repair of the DNA double‐strand breaks and V(D)J recombination. Recently, it was demonstrated that C1D can also interact with TRAX and prevent the association of TRAX with Translin, a factor known to bind DNA break‐point junctions, and that over expression of C1D can induce p53‐dependent apoptosis. Taken together, these findings suggest that mammalian C1D could be involved in maintenance of genome integrity by regulating the activity of proteins involved in DNA repair and recombination. To obtain direct evidence for the biological function of C1D that we show is highly conserved between diverse species, we have analysed the Saccharomyces cerevisiae C1D homologue. We report that the disruption of the YC1D gene results in a temperature sensitivity and that yc1d mutant strains exhibit defects in non‐homologous DNA end joining (NHEJ) and accurate DNA repair. In addition, using a novel plasmid‐based in vivo recombination assay, we show that yc1d mutant strains are also defective in homologous recombination. These results indicate that YC1D is implicated in both homologous recombination and NHEJ pathways for the repair of DNA double‐strand breaks.
The Neisseria meningitidis adhesion regulatory protein CrgA acts through oligomerization and interaction with RNA polymerase
Authors: Deghmane Ala‐Eddine , Taha Muhamed‐Kheir 
Published: March 2003

SummaryCrgA is a LysR‐type transcriptional regulator involved in the intimate adhesion of Neisseria meningitidis to target human epithelial cells. It is negatively autoregulated, and its expression is transiently induced upon contact with target cells. We analysed the functional organization of CrgA using in frame deleted proteins. Four truncated proteins were constructed and purified. They were deleted between residues 20 and 40, 121 and 154, 111 and 181 and 268 and 291. Meningococcal mutants harbouring the corresponding deleted crgA alleles were also constructed. All mutants showed a reduced ability to adhere to epithelial cells. β‐Galactosidase activity assays using a crgA–lacZ transcriptional fusion showed that all the mutations except the 268–291 deletion resulted in loss of induction upon contact with target cells. Gel mobility shift assays and cross‐linking assays showed that the oligomerization of CrgA is required for DNA binding and that the N‐terminal part of CrgA is directly involved in DNA binding through a helix–turn–helix motif. The C‐terminal region is also involved in DNA binding, probably by permitting the oligomerization of CrgA. The C‐terminal region also seemed to interact with RNA polymerase. Therefore, the binding of CrgA and its interaction with RNA polymerase may inhibit the clearance of meningococcal promoters that are repressed by CrgA during the intimate adhesion of N. meningitidis to target cells.
Localization of Mycoplasma pneumoniae cytadherence‐associated protein HMW2 by fusion with green fluorescent protein: implications for attachment organelle structure
Authors: Balish Mitchell F. , Santurri Ryan T. , Ricci Alessondra M. , Lee Kyungok K. , Krause Duncan C. 
Published: March 2003

SummaryThe terminal organelle of the cell wall‐less pathogenic bacterium Mycoplasma pneumoniae is a complex structure involved in adherence, gliding motility and cell division. This membrane‐bound extension of the mycoplasma cell possesses a characteristic electron‐dense core. A number of proteins having direct or indirect roles in M. pneumoniae cytadherence have been previously localized to the terminal organelle. However, the cytadherence‐accessory protein HMW2, which is required for the stabilization of several terminal organelle components, has been refractory to antibody‐based approaches to subcellular localization. In the current study, we constructed a sandwich fusion of HMW2 and enhanced green fluorescent protein (EGFP) and expressed this fusion in wild‐type M. pneumoniae and the hmw2– mutant I‐2. The fusion protein was produced in both backgrounds at wild‐type levels and supported stabilization of proteins HMW1, HMW3 and P65, and haemadsorption function in mutant I‐2. Furthermore, the fusion protein was fluorescent and localized specifically to the terminal organelle. However, the EGFP moiety appeared to interfere partially with processes related to cell division, as transformant cells exhibited an increased incidence of bifurcated attachment organelles. These data together with structural predictions suggest that HMW2 is the defining component of the electron‐dense core of the terminal organelle.
Phosphate availability regulates biosynthesis of two antibiotics, prodigiosin and carbapenem, in Serratia via both quorum‐sensing‐dependent and ‐independent pathways
Authors: Slater Holly , Crow Matthew , Everson Lee , Salmond George P. C. 
Published: March 2003

Summary Serratia sp. ATCC 39006 produces two secondary metabolite antibiotics, 1‐carbapen‐2‐em‐3‐carboxylic acid (Car) and the red pigment, prodigiosin (Pig). We have previously reported that production of Pig and Car is controlled by N‐acyl homoserine lactone (N‐AHL) quorum sensing, with synthesis of N‐AHLs directed by the LuxI homologue SmaI, and is also regulated by Rap, a member of the SlyA family. We now describe further characterization of the SmaI quorum‐sensing system and its connection with other regulatory mechanisms. We show that the genes responsible for biosynthesis of Pig, pigA–O, are transcribed as a single polycistronic message in an N‐AHL‐dependent manner. The smaR gene, transcribed convergently with smaI and predicted to encode the LuxR homologue partner of SmaI, was shown to possess a negative regulatory function, which is uncommon among the LuxR‐type transcriptional regulators. SmaR represses transcription of both the pig and car gene clusters in the absence of N‐AHLs. Specifically, we show that SmaIR exerts its effect on car gene expression via transcriptional control of carR, encoding a pheromone‐independent LuxR homologue. Transcriptional activation of the pig and car gene clusters also requires a functional Rap protein, but Rap dependency can be bypassed by secondary mutations. Transduction of these suppressor mutations into wild‐type backgrounds confers a hyper‐Pig phenotype. Multiple mutations cluster in a region upstream of the pigA gene, suggesting this region may represent a repressor target site. Two mutations mapped to genes encoding pstS and pstA homologues, which are parts of a high‐affinity phosphate transport system (Pst) in Escherichia coli. Disruption of pstS mimicked phosphate limitation and caused concomitant hyper‐production of Pig and Car, which was mediated, in part, through increased transcription of the smaI gene. The Pst and SmaIR systems define distinct, yet overlapping, regulatory circuits which form part of a complex regulatory network controlling the production of secondary metabolites in Serratia ATCC 39006.
Characterization of a novel inhibitory feedback of the anti‐anti‐sigma SpoIIAA on Spo0A activation during development in Bacillus subtilis
Authors: Arabolaza Ana L. , Nakamura Akira , Pedrido María E. , Martelotto Luciano , Orsaria Lelia , Grau Roberto R. 
Published: March 2003

SummaryCompartmentalized gene expression during sporulation is initiated after asymmetric division by cell‐specific activation of the transcription factors σF and σE. Synthesis of these σ factors, and their regulatory proteins, requires the activation (phosphorylation) of Spo0A by the phosphorelay signalling system. We report here a novel regulatory function of the anti‐anti‐σF SpoIIAA as inhibitor of Spo0A activation. This effect did not require σF activity, and it was abolished by expression of the phosphorelay‐independent form Spo0A‐Sad67 indicating that SpoIIAA directly interfered with Spo0A∼P generation. IPTG‐directed synthesis of the SpoIIE phosphatase in a strain carrying a multicopy plasmid coding for SpoIIAA and its specific inhibitory kinase SpoIIAB blocked Spo0A activation suggesting that the active form of the inhibitor was SpoIIAA and not SpoIIAA‐P. Furthermore, expression of the non‐phosphorylatable mutant SpoIIAAS58A (SpoIIAA‐like), but not SpoIIAAS58D (SpoIIAA‐P‐like), completely blocked Spo0A‐dependent gene expression. Importantly, SpoIIAA expressed from the chromosome under the control of its normal spoIIA promoter showed the same negative effect regulated not only by SpoIIAB and SpoIIE but also by septum morphogenesis. These findings are discussed in relation to the potential contribution of this novel inhibitory feedback with the proper activation of σF and σE during development.