bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular characterization of thymidine kinase from Ureaplasma urealyticum: nucleoside analogues as potent inhibitors of mycoplasma growth
Authors: Carnrot Cecilia , Wehelie Rahma , Eriksson Staffan , Bölske Göran , Wang Liya 
Published: October 2003

Summary Ureaplasma urealyticum (U. urealyticum), belonging to the class Mollicutes, is a human pathogen colonizing the urogenital tract and causes among other things respiratory diseases in premature infants. We have studied the salvage of pyrimidine deoxynucleosides in U. urealyticum and cloned a key salvage enzyme, thymidine kinase (TK) from U. urealyticum. Recombinant Uu‐TK was expressed in E. coli, purified and characterized with regards to substrate specificity and feedback inhibition. Uu‐TK efficiently phosphorylated thymidine (dThd) and deoxyuridine (dUrd) as well as a number of pyrimidine nucleoside analogues. All natural ribonucleoside/deoxyribonucleoside triphosphates, except dTTP, served as phosphate donors, while dTTP was a feedback inhibitor. The level of Uu‐TK activity in U. urealyticum extracts increased upon addition of dUrd to the growth medium. Fluoropyrimidine nucleosides inhibited U. urealyticum and M. pneumoniae growth and this inhibitory effect could be reversed by addition of dThd, dUrd or deoxytetrahydrouridine to the growth medium. Thus, the mechanism of inhibition was most likely the depletion of dTTP, either via a blocked thymidine kinase reaction and/or thymidylate synthesis step and these metabolic reactions should be suitable targets for antimycoplasma chemotherapy.
CotB is essential for complete activation of green light‐induced genes during complementary chromatic adaptation in Fremyella diplosiphon
Authors: Balabas Barbara E. , Montgomery Beronda L. , Ong Laura E. , Kehoe David M. 
Published: October 2003

SummaryThe dramatic modifications of photosynthetic light harvesting antennae called phycobilisomes that occur during complementary chromatic adaptation in cyanobacteria are controlled by two separate photosensory systems. The first system involves the signal transduction components RcaE, RcaF and RcaC, which appear to make up a complex multistep phosphorelay. This system controls the light responsive expression of the cpcB2A2H2I2D2, cpeBA and cpeCDE operons, which encode phycobilisome proteins. The second system, which is not yet characterized, acts in concert with the first but only regulates the light responses of cpeBA and cpeCDE. We have generated and characterized a new mutant class, named the Tan mutants. In at least one member of this class, light‐regulated RNA accumulation patterns are altered for cpeBA and cpeCDE, but not for cpcB2A2H2I2D2. Thus this mutant contains a lesion that may impair the operation of the second system. We demonstrate that several Tan mutants are the result of improper expression of the gene cotB. CotB has limited similarity to lyase class proteins, particularly those related to NblB, which is required for degradation of phycobilisomes in other cyanobacteria. Possible roles of CotB in the biogenesis of phycobilisomes are discussed.
Dispersed mode of Staphylococcus aureus cell wall synthesis in the absence of the division machinery
Authors: Pinho Mariana G. , Errington Jeff 
Published: October 2003

SummaryWe have developed several new fluorescent staining procedures that enabled us to study the synthesis of cell wall material in the spherical Gram‐positive bacterium Staphylococcus aureus. The results obtained support previous proposals that these cells synthesize new wall material specifically at cell division sites, in the form of a flat circular plate that is subsequently cleaved and remodelled to produce the new hemispherical poles of the daughter cells. We have shown that formation of the septal peptidoglycan is dependent on the key cell division protein FtsZ, which recruits penicillin‐binding protein (PBP) 2. Unexpectedly, in FtsZ‐depleted cells, the cell wall synthetic machinery becomes dispersed and new wall material is made in dispersed patches over the entire surface of the cells, which increase in volume by up to eightfold before lysing. The results have implications for understanding the nature of S. aureus morphogenesis and for inhibitors of cell division proteins as drug targets.
The ω‐site sequence of glycosylphosphatidylinositol‐anchored proteins in Saccharomyces cerevisiae can determine distribution between the membrane and the cell wall
Authors: Frieman Matthew B. , Cormack Brendan P. 
Published: October 2003

SummaryGlycosylphosphatidylinositol (GPI)‐anchored cell wall proteins play an important role in the structure and function of the cell wall in yeast and other fungi. Although the majority of characterized fungal GPI‐anchored proteins do in fact localize to the cell wall, some are believed to reside at the plasma membrane and not to traffic significantly to the cell wall. There is evidence suggesting that the amino acids immediately upstream of the site of GPI anchor addition (the ω site) serve as the signal determining whether a GPI protein localizes to the cell wall or to the plasma membrane, although this remains controversial. Here, we examine in detail the functional and biochemical differences between the GPI anchor addition signals of putative cell wall (CW) and plasma membrane (PM) GPI proteins. We find strong evidence for the existence of PM‐class and CW‐class GPI proteins. We show that the biological function of a GPI‐CWP is strongly compromised by changing the GPI anchor signal from a CW‐class signal to a PM‐class signal. Biochemically, this abrogation of function corresponds to a change in the protein from a cell wall form to a membrane form. To understand better the basis for the difference between the two classes of proteins, we mutated the amino acids upstream of the ω site in a GPI‐PM protein and selected mutant proteins that were now localized to the cell wall. We were also able to design simple amino acid mutations in a GPI‐CW protein that efficiently redirected the protein to the plasma membrane. These studies make clear that different GPI anchor sequences can have dramatic effects on localization of the proteins and help to define the GPI anchor addition signal sequences that distinguish the PM‐class and CW‐class GPI proteins.
Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene
Authors: Kim Amy I. , Ghosh Pallavi , Aaron Michelle A. , Bibb Lori A. , Jain Shruti , Hatfull Graham F. 
Published: October 2003

SummaryMycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis and forms stable lysogens in which the Bxb1 genome is integrated into the host chromosome. Bxb1 encodes an integrase of the large serine recombinase family that catalyses integration and excision of the Bxb1 genome. We show here that Bxb1 integrates into a chromosomal attB site located within the 3′ end of the groEL1 gene such that integration results in alteration of the C‐terminal 21 amino acid residues. An integration‐proficient plasmid vector containing the Bxb1 integrase gene and flanking DNA sequences efficiently transforms M. smegmatis via integration at attB. Bxb1‐integrated recombinants are stable and fully compatible with L5 integration vectors. Strand exchange occurs within an 8 bp common core sequence present in attB and within an attP site situated immediately upstream of the phage integrase gene. Establishment of a defined in vitro system for Bxb1 integration shows that recombination occurs efficiently without requirement for high‐energy cofactors, divalent metals, DNA supercoiling or additional proteins.
Pseudaminic acid, the major modification on Campylobacter flagellin, is synthesized via the Cj1293 gene
Authors: Goon Scarlett , Kelly John F. , Logan Susan M. , Ewing Cheryl P. , Guerry Patricia 
Published: October 2003

SummaryFlagellins from Campylobacter jejuni 81‐176 and Campylobacter coli VC167 are heavily glycosylated. The major modifications on both flagellins are pseudaminic  acid (Pse5Ac7Ac), a  nine  carbon sugar that is similar to sialic acid, and an acetamidino‐substituted analogue of pseudaminic acid (PseAm). Previous data have indicated that PseAm is synthesized via Pse5Ac7Ac in C. jejuni 81‐176, but that the two sugars are synthesized using independent pathways in C. coli VC167. The Cj1293 gene of C. jejuni encodes a putative UDP‐GlcNAc C6‐dehydratase/C4‐reductase that is similar to a protein required for glycosylation of Caulobacter crescentus flagellin. The Cj1293 gene is expressed either under the control of a σ54 promoter that overlaps the coding region of Cj1292 or as a polycistronic message under the control of a σ70 promoter upstream of Cj1292. A mutant in gene Cj1293 in C. jejuni 81‐176 was non‐motile and non‐flagellated and accumulated unglycosylated flagellin intracellularly. This mutant was complemented in trans with the homologous C. jejuni gene, as well as the Helicobacter pylori homologue, HP0840, which has been shown to encode a protein with UDP‐GlcNAc C6‐dehydratase/C4‐reductase activity. Mutation of Cj1293 in C. coli VC167 resulted in a fully motile strain that synthesized a flagella filament composed of flagellin in which Pse5Ac7Ac was replaced by PseAm. The filament from the C. coli Cj1293 mutant displayed increased solubility in SDS compared with the wild‐type filament. A double mutant in C. coli VC167, defective in both Cj1293 and ptmD, encoding part of the independent PseAm pathway, was also non‐motile and non‐flagellated and accumulated unglycosylated flagellin intracellularly. Collectively, the data indicate that Cj1293 is essential for Pse5Ac7Ac biosynthesis from UDP‐GlcNAc, and that glycosylation is required for flagella biogenesis in campylobacters.
Interaction of the RNA chaperone Hfq with mRNAs: direct and indirect roles of Hfq in iron metabolism of Escherichia coli
Authors: Večerek Branislav , Moll Isabella , Afonyushkin Taras , Kaberdin Vladimir , Bläsi Udo 
Published: October 2003

SummaryThe Escherichia coli Sm‐like host factor I (Hfq) is thought to play direct and indirect roles in post‐transcriptional regulation by targeting small regulatory RNAs and mRNAs. In this study, we have used proteomics to identify new mRNA targets of Hfq. We have identified 11 candidate proteins, synthesis of which was differentially affected in a hfq– background. The effect of Hfq on some of the corresponding mRNAs including fur, gapA, metF, ppiB and sodB mRNA was assessed, using different in vitro and in vivo methods. This allowed us to distinguish between direct and indirect effects of Hfq in modulating the translational activities of these mRNAs. From the collection of mRNAs tested, only fur and sodB mRNA, encoding the master regulator of iron metabolism and the iron superoxide dismutase, respectively, were found to be regulated by Hfq. Fur is known to be a negative regulator of transcription of the small RNA RyhB. Mutations in the sodB leader and compensating mutations in RyhB revealed that RyhB in turn represses translation of sodB mRNA, explaining the previously reported positive control of sodB by Fur. These data assign a role to Hfq in regulation of iron uptake and in switching off of iron scavenger genes.
A rare leucine codon in adpA is implicated in the morphological defect of bldA mutants of Streptomyces coelicolor
Authors: Takano E. , Tao M. , Long F. , Bibb Maureen J. , Wang L. , Li W. , Buttner M. J. , Bibb Mervyn J. , Deng Z. X. , Chater K. F. 
Published: October 2003

SummaryStreptomycetes are mycelial bacteria that produce sporulating aerial hyphae on solid media. Bald (bld) mutants fail to form aerial mycelium under at least some conditions. bldA encodes the only tRNA species able to read the leucine codon UUA efficiently, implying the involvement of a TTA‐containing gene in initiating aerial growth. One candidate for such a gene was bldH, because the bldH109 mutant of Streptomyces coelicolor resembles bldA mutants in some aspects. In the work reported here, adpAc, an S. coelicolor gene similar to the Streptomyces griseus A factor‐regulated adpAg, was found to complement the bldH109 mutant partially at both single and multiple copies. The sequence of adpAc from the bldH109 mutant revealed a frameshift. A constructed in frame deletion of adpAc conferred a bald colony phenotype, and the mutant behaved like bldA mutants and bldH109 in its pattern of extracellular signal exchange. Both adpAc and adpAg contain a TTA codon. A TTA‐free version of adpAc was engineered by replacing the TTA leucine codon with a cognate TTG leucine codon. The adpA(TTA→TTG) gene could partially restore aerial mycelium formation to a bldA mutant when it was followed in cis by the gene ornA, as in the natural chromosomal arrangement. This indicated that the UUA codon in adpAc mRNA is the principal target through which bldA influences morphological differentiation. It also implied that translational arrest at the UUA codon in adpAc mRNA caused a polar effect on the downstream ornA, and that the poor translation of both genes contributes extensively to the deficiency of aerial mycelium formation in bldA mutants. Unlike the situation in S. griseus, adpAc transcription does not depend on the host's γ‐butyrolactone signalling system, at least in liquid cultures. In addition, sigma factor BldN, which is the homologue of an S. griseus sigma factor AdsA that is absent from adpAg mutants of S. griseus, was present in the constructed adpAc null mutant of S. coelicolor.
The PmrA‐PmrB two‐component system responding to acidic pH and iron controls virulence in the plant pathogen Erwinia carotovora ssp. carotovora
Authors: Hyytiäinen Heidi , Sjöblom Solveig , Palomäki Tiina , Tuikkala Anne , Tapio Palva E. 
Published: October 2003

SummaryEfficient response to environmental cues is crucial to successful infection by plant‐pathogenic bacteria such as Erwinia carotovora ssp. carotovora. The expression of the main virulence genes of this pathogen, encoding extracellular enzymes that degrade the plant‐cell wall, is subject to complex regulatory machinery where two‐component systems play an important role. In this paper, we describe for the first time the involvement of the PmrA‐PmrB two‐component system in regulation of virulence in a plant‐pathogenic bacterium. Disruption of pmrB resulted in reduced virulence both in potato and in Arabidopsis. This is apparently due to reduced production of the extracellular enzymes. In contrast, a pmrA mutant exhibited increased levels of these enzymes implying negative regulation of the corresponding genes by PmrA. Furthermore, the pmrB but not pmrA mutant exhibited highly increased resistance to the cationic antimicrobial peptide polymyxin B suggesting alterations in cell surface properties of the mutant. A similar increase of polymyxin resistance was detected in the wild type at mildly acidic pH with low Mg2+. Functional pmrA is essential for bacterial survival on excess iron at acidic pH, regardless of the Mg2+ concentration. We propose that PmrA‐PmrB TCS is involved in controlling of bacterial response to external pH and iron and is crucial for bacterial virulence and survival in planta.
The Gap1 general amino acid permease acts as an amino acid sensor for activation of protein kinase A targets in the yeast Saccharomyces cerevisiae
Authors: Donaton Monica C. V. , Holsbeeks Inge , Lagatie Ole , Van Zeebroeck Griet , Crauwels Marion , Winderickx Joris , Thevelein Johan M. 
Published: October 2003

SummaryAddition of a nitrogen source to yeast (Saccharomyces cerevisiae) cells starved for nitrogen on a glucose‐containing medium triggers activation of protein kinase A (PKA) targets through a pathway that requires for sustained activation both a fermentable carbon source and a complete growth medium (fermentable growth medium induced or FGM pathway). Trehalase is activated, trehalose and glycogen content as well as heat resistance drop rapidly, STRE‐controlled genes are repressed, and ribosomal protein genes are induced. We show that the rapid effect of amino acids on these targets specifically requires the general amino acid permease Gap1. In the gap1Δ strain, transport of high concentrations of l‐citrulline occurs at a high rate but without activation of trehalase. Metabolism of the amino acids is not required. Point mutants in Gap1 with reduced or deficient transport also showed reduced or deficient signalling. However, two mutations, S391A and S397A, were identified with a differential effect on transport and signalling for l‐glutamate and l‐citrulline. Specific truncations of the C‐terminus of Gap1 (e.g. last 14 or 26 amino acids) did not reduce transport activity but caused the same phenotype as in strains with constitutively high PKA activity also during growth with ammonium as sole nitrogen source. The overactive PKA phenotype was abolished by mutations in the Tpk1 or Tpk2 catalytic subunits. We conclude that Gap1 acts as an amino acid sensor for rapid activation of the FGM signalling pathway which controls the PKA targets, that transport through Gap1 is connected to signalling and that specific truncations of the C‐terminus result in permanently activating Gap1 alleles.
The vanG glycopeptide resistance operon from Enterococcus faecalis revisited
Authors: Depardieu Florence , Bonora Maria Grazia , Reynolds Peter E. , Courvalin Patrice 
Published: October 2003

SummaryAcquired VanG‐type resistance to vancomycin (MIC = 16 µg ml−1) but susceptibility to teicoplanin in Enterococcus faecalis BM4518 and WCH9 is due to the inducible synthesis of peptidoglycan precursors ending in d‐alanine‐d‐serine. The vanG cluster, assigned to a chromosomal location, was composed of genes recruited from various van operons. The 3′ end encoded VanG, a d‐Ala:d‐Ser ligase, VanXYG, a putative bifunctional d,d‐peptidase and VanTG, a serine racemase: VanG and VanTG were implicated in the synthesis of d‐Ala:d‐Ser as in VanC‐ and VanE‐type strains. Upstream from the structural genes for these proteins were vanWG with unknown function and vanYG containing a frameshift mutation which resulted in premature termination of the encoded protein and accounted for the lack of UDP‐MurNAc‐tetrapeptide in the cytoplasm. Without the frameshift mutation, VanYG had homology with Zn2+ dependent d,d‐carboxypeptidases. The 5′ end of the gene cluster contained three genes vanUG, vanRG and vanSG encoding a putative regulatory system, which were co‐transcribed constitutively from the PYG promoter, whereas transcription of vanYG,WG,G,XYG,TG was inducible and initiated from the PYG promoter. Transfer of VanG‐type glycopeptide resistance to E. faecalis JH2‐2 was associated with the movement, from chromosome to chromosome, of genetic elements of c. 240 kb carrying also ermB‐encoded erythromycin resistance. Sequence determination of the flanking regions of the vanG cluster in donor and transconjugants revealed the same 4 bp direct repeats and 22 bp imperfect inverted repeats that delineated the large element.
RshA, an anti‐sigma factor that regulates the activity of the mycobacterial stress response sigma factor SigH
Authors: Song Taeksun , Dove Simon L. , Lee Kon Ho , Husson Robert N. 
Published: October 2003

SummarySigH, an alternative sigma factor of Mycobacterium tuberculosis,  is  a  central regulator  of  the  response to oxidative and heat stress. Exposure to these stresses results in increased expression of sigH itself, and of genes encoding additional regulators and effectors of the bacterial response to these stresses. In this work we show that RshA, a protein encoded by a gene in the sigH operon, is an anti‐sigma factor of SigH. We demonstrate that RshA binds to SigH in vitro and in vivo. This protein–protein interaction, as  well  as the  ability  of RshA to inhibit SigH‐dependent transcription, is redox‐dependent, with RshA functioning as a negative regulator of SigH activity only under reducing conditions. The interaction of SigH and RshA is also disrupted in vitro by elevated temperature. RshA, a protein of 101 amino acids, contains five conserved cysteine residues of which two appear to be essential for RshA to inhibit SigH activity, suggesting that these cysteines may be important for the redox state dependence of RshA function. Our results indicate that RshA is a sensor that responds to oxidative stress, and also to heat stress, resulting in activation of SigH and expression of the SigH‐dependent genes that allow M. tuberculosis to adapt to these stresses.
A four‐tiered transcriptional regulatory circuit controls flagellar biogenesis in Pseudomonas aeruginosa
Authors: Dasgupta Nandini , Wolfgang Matthew C. , Goodman Andrew L. , Arora Shiwani K. , Jyot Jeevan , Lory Stephen , Ramphal Reuben 
Published: October 2003

SummaryThe single polar flagellum of Pseudomonas aeruginosa is an important virulence and colonization factor of this opportunistic pathogen. In this study, the annotation of the genes belonging to the fla regulon was updated and their organization was analysed in strains PAK and PAO1, representative type‐a and type‐b strains of P. aeruginosa respectively. The flagellar genes are clustered in three non‐contiguous regions of the chromosome. A polymorphic locus flanked by flgJ and fleQ in Region I contains a glycosylation island in PAK. The expression and ordered assembly of the complex multicomponent flagellum is intricately regulated. Dedicated flagellar genes fleQ, fleS, fleR, fliA, flgM and fleN encode proteins that participate in the regulation of the flagellar transcriptional circuit. In addition, expression of the flagellum is coordinately regulated with other P. aeruginosa virulence factors by the alternative sigma factor σ54, encoded by rpoN. In order to gain insight into the hierarchical regulation of flagellar genes, deletion mutations were constructed in fleQ, fleR, fliA and rpoN. The transcriptional impact of these mutations was examined by transcriptional profiling using a P. aeruginosa whole genome microarray. Analysis of the transcriptomes generated for each of these mutants indicates a four‐tiered (Classes I‐IV) hierarchy of transcriptional regulation. Class I genes are constitutively expressed and include the transcriptional regulator fleQ and the alternative sigma factor fliA (σ28). Class II genes including fleSR, encoding a two‐component regulatory system require FleQ and RpoN (σ54) for their transcriptional activation. Class III genes are positively regulated by the activated response regulator FleR in concert with RpoN. The transcription of Class IV genes is dependent on the availability of free FliA following the export of the FliA specific antisigma factor FlgM through the basal body rod‐hook structure (assembled from Class II and III gene products). Two previously uncharacterized genes, which are coordinately regulated with known flagellar genes have been identified by genome‐wide analysis and their role in flagellar biogenesis was analysed.