bluuebunny/crossref_metadata_2025 huggingface dataset:
Dispersed mode of Staphylococcus aureus cell wall synthesis in the absence of the division machinery
Authors: Pinho Mariana G. , Errington Jeff
Published: October 2003

We 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

Glycosylphosphatidylinositol (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

Mycobacteriophage 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

Flagellins 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

The 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

Streptomycetes 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

Efficient 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

Addition 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

Acquired 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

SigH, 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

The 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.
The first committed step in the biosynthesis of sialic acid by Escherichia coli K1 does not involve a phosphorylated N‐acetylmannosamine intermediate
Authors: Ringenberg Michael A. , Steenbergen Susan M. , Vimr Eric R.
Published: October 2003

A variety of pathogens or commensals use at least one of four distinct mechanisms for decorating their surfaces with sialic acid as a strategy to avoid, subvert or inhibit host innate immunity. The metabolism of sialic acid thus is central to a range of host–pathogen interactions. The first committed step in this process, the production of free N‐acetylmannosamine (ManNAc), has not been defined. Here we show that ManNAc‐6‐phosphate (ManNAc‐6‐P) is not an obligate sialate precursor in Escherichia coli K1. This conclusion was supported by 31P NMR spectroscopy of E. coli K1 derivatives engineered with different combinations of mutations in nanA (sialate aldolase or lyase), nanK (ManNAc kinase), nanE (ManNAc‐6‐P 2‐epimerase), neuS (polysialyltransferase) and neuB (sialate synthase). The product specificities for purified NanK and NanE were determined by chromatographic analyses. Direct biochemical analysis showed that ManNAc‐6‐P was stable in a nanE mutant extract. The combined results indicate that neither ManNAc‐6‐P nor specific or non‐specific phosphatase are necessary to generate the requisite ManNAc for sialate biosynthesis. Our results imply that the neuC gene product encodes an UDP‐N‐acetylglucosamine 2‐epimerase that generates ManNAc directly from the dinucleotide‐sugar precursor despite detection of only this enzyme's UDP‐GlcNAc hydrolase activity. This study describes the first use of NMR for analysing intermediate flux within the sialate biosynthetic pathway.
Upstream short sequence repeats regulate expression of the alpha C protein of group B Streptococcus
Authors: Puopolo Karen M. , Madoff Lawrence C.
Published: October 2003

Group B streptococci (GBS) express a family of repeat‐containing surface proteins, the prototype of which is the alpha C protein expressed in type Ia/C strain A909. We have isolated a series of mutant GBS strains by mouse‐passage of A909 that do not produce normal levels of the alpha C protein. Polymerase chain reaction amplification and sequencing of the gene encoding the alpha C protein, bca, from four mutant strains revealed the presence of a full‐length gene in each strain. However, Northern and RT‐PCR analysis revealed greatly reduced levels of RNA encoding the alpha C protein. Sequence analysis of the mutant genes found the coding region unchanged from the wild‐type gene in each case, but variation was observed in a specific locus located 110 bp upstream of the start codon. The presence of a 5‐nucleotide repeat, AGATT, and a string of adenine residues mark this locus. Both deletion and expansion of the AGATT motif were associated with the complete null phenotype. Deletions in the string of adenine residues were associated with both a decreased‐production phenotype and a complete null phenotype. Cloning of this upstream region into a green‐fluorescent protein (GFP) reporter system in GBS demonstrated promoter activity that was completely abolished by changes in the pentanucleotide repeat or adenine string. Primer extension studies of the wild‐type strain revealed one dominant and two minor transcription start sites. Primer extension studies of the null and low‐expression mutant strains revealed that the dominant transcript is completely absent in each mutant. The short sequence repeat locus is located at position − 55 to − 78 relative to the start site of the dominant transcript. We have demonstrated in vitro phase variation in expression of the alpha C protein associated with variation at the pentanucleotide repeat locus. We conclude that this short sequence repeat motif is located upstream of the dominant promoter for the alpha C protein and represents a regulatory site for alpha C protein expression. This is the first evidence of transcriptional regulation by short‐sequence repeats in a Gram‐positive organism.
Segregation of the Escherichia coli chromosome terminus
Authors: Li Yongfang , Youngren Brenda , Sergueev Kirill , Austin Stuart
Published: October 2003

We studied the segregation of the replication terminus of the Escherichia coli chromosome by time‐lapse and still photomicroscopy. The replicated termini lie together at the cell centre. They rapidly segregate away from each other immediately before cell division. At fast growth rate, the copies move progressively and quickly toward the centres of the new‐born cells. At slow growth rate, the termini usually remain near the inner cell pole and migrate to the cell centre in the middle of the cell cycle. A terminus domain of about 160kb, roughly centred on the dif recombination site, segregated as a unit at cell division. Sequences outside this domain segregated before division, giving two separate foci in predivision cells. Resolution of chromosome dimers via the terminus dif site requires the XerC recombinase and an activity of the FtsK protein that is thought to align the dif sequences at the cell centre. We found that anchoring of the termini at the cell centre and proper segregation at cell division occurred normally in the absence of recombination via the XerC recombinase. Anchoring and proper segregation were, however, frequently disrupted when the C‐terminal domain of FtsK was truncated.
Nitrogen metabolism and virulence of Candida albicans require the GATA‐type transcriptional activator encoded by GAT1
Authors: Limjindaporn Thawornchai , Khalaf Roy A. , Fonzi William A.
Published: October 2003

Nitrogen acquisition and metabolism is central to microbial growth. A conserved family of zinc‐finger containing transcriptional regulators known as GATA‐factors ensures efficient utilization of available nitrogen sources by fungi. GATA factors activate expression of nitrogen catabolic pathways when preferred nitrogen sources are absent or limiting, a phenomenon known as nitrogen catabolite repression. GAT1 of Candida albicans encodes a GATA‐factor homologous to the AREA protein of Aspergillus nidulans and related transcription factors involved in nitrogen regulation. Two observations implicated GAT1 in nitrogen regulation. The growth of mutants lacking GAT1 was reduced when isoleucine, tyrosine or tryptophan were the sole source of nitrogen. Secondly, when cultured on a secondary nitrogen source, gat1Δ mutants were unable to activate expression of GAP1, UGA4 or DAL5, which were shown to be nitrogen regulated in C. albicans. This regulatory defect did not prevent filamentation of gat1Δ mutants in nitrogen repressing or non‐repressing conditions, demonstrating that nitrogen catabolite repression does not influence dimorphism. The mutants were, however, highly attenuated in a murine model of disseminated candidiasis. Attenuation was not associated with any diminution of growth in serum or ability to utilize serum amino acids. The results indicate an important role for nitrogen regulation in the virulence of C. albicans.
Mapping the binding domains on meningococcal Opa proteins for CEACAM1 and CEA receptors
Authors: De Jonge Marien I. , Hamstra Hendrik Jan , Van Alphen Loek , Dankert Jacob , Van Der Ley Peter
Published: October 2003

The opacity (Opa) proteins of pathogenic Neisseria spp. are adhesins, which play an important role in adhesion and invasion of host cells. Most members of this highly variable family of outer membrane proteins can bind to the human carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs). Several studies have identified the Opa‐binding region on the CEACAM receptors; however, not much is known about the binding sites on the Opa proteins for the corresponding CEACAM‐receptors. The high degree of sequence variation in the surface‐exposed loops of Opa proteins raises the question how the binding sites for the CEACAM receptors are conserved. Neisseria meningitidis strain H44/76 possesses four different Opa proteins, of which OpaA and OpaJ bind to CEACAM1, while OpaB and OpaD bind to CEACAM1 and CEA. A sequence motif involved in binding to CEACAM1 was identified by alanine scanning mutagenesis of those amino acid residues conserved within the hypervariable (HV) regions of all four Opa proteins. Hybrid Opa variants with different combinations of HV‐1 and HV‐2 derived from OpaB and OpaJ showed a reduced binding to CEACAM1 and CEA, indicating that particular combinations of HV‐1 and HV‐2 are required for the Opa binding capacity. Homologue scanning mutagenesis was used to generate more refined hybrids containing novel combinations of OpaB and OpaJ sequences within HV‐1 and HV‐2. They could be used to identify residues determining the specificity for CEA binding. The combined results obtained with mutants and hybrids strongly suggest the existence of a conserved binding site for CEACAM receptors by the interaction of HV‐1 and HV‐2 regions.
The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA
Authors: Rand Lucinda , Hinds Jason , Springer Burkhard , Sander Peter , Buxton Roger S. , Davis Elaine O.
Published: October 2003

In many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction. We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis. Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA. Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild‐type M. tuberculosis and a recA deletion strain of M. tuberculosis using microarrays. This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain. Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction. Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination. Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M. tuberculosis.
Growth and division of Streptococcus pneumoniae: localization of the high molecular weight penicillin‐binding proteins during the cell cycle
Authors: Morlot Cécile , Zapun André , Dideberg Otto , Vernet Thierry
Published: October 2003

The bacterial peptidoglycan, the main component of the cell wall, is synthesized by the penicillin‐binding proteins (PBPs). We used immunofluorescence microscopy to determine the cellular localization of all the high molecular weight PBPs of the human pathogen Streptococcus pneumoniae, for a wild type and for several PBP‐deficient strains. Progression through the cell cycle was investigated by the simultaneous labelling of DNA and the FtsZ protein. Our main findings are: (i) the temporal dissociation of cell wall synthesis, inferred by the localization of PBP2x and PBP1a, from the constriction of the FtsZ‐ring; (ii) the localization of PBP2b and PBP2a at duplicated equatorial sites indicating the existence of peripheral peptidoglycan synthesis, which implies a similarity between the mechanism of cell division in bacilli and streptococci; (iii) the abnormal localization of some class A PBPs in PBP‐defective mutants which may explain the apparent redundancy of these proteins in S. pneumoniae.
The NblAI protein from the filamentous cyanobacterium Tolypothrix PCC 7601: regulation of its expression and interactions with phycobilisome components
Authors: Luque Ignacio , Ochoa de Alda Jesús A. G. , Richaud Catherine , Zabulon Gérald , Thomas Jean‐Claude , Houmard Jean
Published: October 2003

Cyanobacteria respond to changes in light or nutrient availability by modifications in their photosynthetic light harvesting antenna. In unicellular cyanobacteria a small polypeptide (NblA) is required for phycobilisome degradation following environmental stresses. In the filamentous strain Tolypothrix sp. PCC 7601 the nblAI gene, encoding a NblA homologue, is located upstream of the operon coding for phycoerythrin (cpeBA). The nblAI transcripts all originate from a single transcription start point; their intracellular levels vary according to nitrogen regimes but not with light spectral quality. Using recombinant His‐tagged NblAI protein, we found that in vitro NblAI has affinity for both phycocyanin and phycoerythrin subunits from Tolypothrix sp. PCC 7601, but not for allophycocyanin from this cyanobacterium or for phycobiliproteins from other cyanobacterial species. We also observed that although nblAI is mainly expressed under nitrogen starvation, NblAI polypeptides are always present in the cell; a significant portion of them co‐purify with phycobilisome preparations but only if cells were grown under red light. Our data indicate that NblAI attaches to the phycobilisomes even under non‐inducing conditions and suggest a preferential affinity of NblAI for phycocyanin.
Internalization of OspA in rsCD14 complex and aggregated forms
Authors: Welty Diane M. , Snyder D. Scott
Published: October 2003

Although the spirochetal protein OspA is capable of stimulating immune cells in a CD14‐ and TLR2‐dependent manner, little is known about how TLR2 receptor complex ligands, such as OspA, are handled by the cell once delivered. We examine here the internalization of the fluorescently derivatized forms of both the full length OspA lipoprotein delivered as a recombinant soluble CD14 (rsCD14) complex and the corresponding lipohexapeptide given to the cells as an aggregate. Both forms of OspA are internalized in a similar manner to acetylated low density lipoprotein (AcLDL), a scavenger receptor ligand. Acetylated low density lipoprotein is capable of competing for internalization with OspA even when OspA is delivered as a rsCD14 complex. We observe co‐localization of OspA with lysosomes but not with the Golgi complex. These phenomena are similar between RAW264.7 macrophages and endothelial cells but change drastically when the cells are deprived of serum. Upon serum starvation, OspA shows some localization to the Golgi apparatus whereas the lipohexapeptide remains on the cell surface. Inhibition of internalization of OspA via treatment with cytochalasin D or of the lipohexapeptide via serum starvation does not interfere with TNF induction activity, consistent with signalling from the cell surface.
NarE: a novel ADP‐ribosyltransferase from Neisseria meningitidis
Authors: Masignani Vega , Balducci Enrico , Di Marcello Federica , Savino Silvana , Serruto Davide , Veggi Daniele , Bambini Stefania , Scarselli Maria , Aricò Beatrice , Comanducci Maurizio , Adu‐Bobie Jeannette , Giuliani Marzia M. , Rappuoli Rino , Pizza Mariagrazia
Published: October 2003

Mono ADP‐ribosyltransferases (ADPRTs) are a class of functionally conserved enzymes present in prokaryotic and eukaryotic organisms. In bacteria, these enzymes often act as potent toxins and play an important role in pathogenesis. Here we report a profile‐based computational approach that, assisted by secondary structure predictions, has allowed the identification of a previously undiscovered ADP‐ribosyltransferase in Neisseria meningitidis (NarE). NarE shows structural homologies with E. coli heat‐labile enterotoxin (LT) and cholera toxin (CT) and possesses ADP‐ribosylating and NAD‐glycohydrolase activities. As in the case of LT and CT, NarE catalyses the transfer of the ADP‐ribose moiety to arginine residues. Despite the absence of a signal peptide, the protein is efficiently exported into the periplasm of Neisseria. The narE gene is present in 25 out of 43 strains analysed, is always present in ET‐5 and Lineage 3 but absent in ET‐37 and Cluster A4 hypervirulent lineages. When present, the gene is 100% conserved in sequence and is inserted upstream of and co‐transcribed with the lipoamide dehydrogenase E3 gene. Possible roles in the pathogenesis of N. meningitidis are discussed.
A six amino acid deletion, partially overlapping the VanSB G2 ATP‐binding motif, leads to constitutive glycopeptide resistance in VanB‐type Enterococcus faecium
Authors: Depardieu Florence , Courvalin Patrice , Msadek Tarek
Published: October 2003

Enterococcus faecium
clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal
vanB
cluster, including the
vanS
B
/
vanR
B
two‐component system regulatory genes.
Enterococcus faecium
strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The
ddl
gene of BM4525 had a 2 bp insertion leading to an impaired
d
‐alanine:
d
‐alanine ligase. Sequencing of the
vanB
operon in BM4525 also revealed an 18 bp deletion in the
vanS

B

gene designated
vanS

BΔ

. The resulting six amino acid deletion partially overlapped the G2 ATP‐binding domain of the VanS
BΔ
histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base‐pairs. The VanS
B
, VanS
BΔ
and VanR
B
proteins were overproduced in
Escherichia coli
and purified.
In vitro
autophosphorylation of the VanS
B
and VanS
BΔ
histidine kinases and phosphotransfer to the VanR
B
response regulator did not differ significantly. However, VanS
BΔ
was deficient in VanR
B
phosphatase activity leading to accumulation of phosphorylated VanR
B
. Increased glycopeptide resistance in
E. faecium
BM4525 was therefore a result of the lack of production of
d
‐alanyl‐
d
‐alanine ending pentapeptide and to constitutive synthesis of
d
‐alanyl‐
d
‐lactate terminating peptidoglycan precursors, following loss of
d
‐alanine:
d
‐alanine ligase and of VanS
B
phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in
vanS

B
may favour the appearance of high level constitutively expressed vancomycin resistance through a ‘slippage’ type of genetic rearrangement in VanB‐type strains.
A Neisserial autotransporter NalP modulating the processing of other autotransporters
Authors: Van Ulsen Peter , Van Alphen Loek , Ten Hove Jan , Fransen Floris , Van Der Ley Peter , Tommassen Jan
Published: October 2003

Autotransporters constitute a relatively simple secretion system in Gram‐negative bacteria, depending for their translocation across the outer membrane only on a C‐terminal translocator domain. We have studied a novel autotransporter serine protease, designated NalP, from Neisseria meningitidis strain H44/76, featuring a lipoprotein motif at the signal sequence cleavage site. Indeed, lipidation of NalP could be demonstrated, but the secreted 70 kDa domain of NalP lacked the lipid‐moiety as a result of additional N‐terminal processing. A nalP mutant showed a drastically altered profile of secreted proteins. Mass‐spectrometric analysis of tryptic fragments identified the autotransporters IgA protease and App, a homologue of the adhesin Hap of Haemophilus influenzae, as the major secreted proteins. Two forms of both of these proteins were found in the culture supernatant of the wild‐type strain, whereas only the lower molecular‐weight forms predominated in the culture supernatant of the nalP mutant. The serine‐protease active site of NalP was required for the modulation of the processing of these autotransporters. We propose that, apart from the autoproteolytic processing, NalP can process App and IgA protease and hypothesize that this function of NalP could contribute to the virulence of the organism.
Lateral gene transfer: when will adolescence end?
Authors: Lawrence Jeffrey G. , Hendrickson Heather
Published: October 2003

The scope and impact of horizontal gene transfer (HGT) in Bacteria and Archaea has grown from a topic largely ignored by the microbiological community to a hot‐button issue gaining staunch supporters (on particular points of view) at a seemingly ever‐increasing rate. Opinions range from HGT being a phenomenon with minor impact on overall microbial evolution and diversification to HGT being so rampant as to obfuscate any opportunities for elucidating microbial evolution – especially organismal phylogeny – from sequence comparisons. This contentious issue has been fuelled by the influx of complete genome sequences, which has allowed for a more detailed examination of this question than previously afforded. We propose that the lack of common ground upon which to formulate consensus viewpoints probably stems from the absence of answers to four critical questions. If addressed, they could clarify concepts, reject tenuous speculation and solidify a robust foundation for the integration of HGT into a framework for long‐term microbial evolution, regardless of the intellectual camp in which you reside. Here, we examine these issues, why their answers shape the outcome of this debate and the progress being made to address them.
Death's toolbox: examining the molecular components of bacterial programmed cell death
Authors: Rice Kelly C. , Bayles Kenneth W.
Published: October 2003

Programmed cell death (PCD) is a genetically determined process of cellular suicide that is activated in response to cellular stress or damage, as well as in response to the developmental signals in multicellular organisms. Although historically studied in eukaryotes, it has been proposed that PCD also functions in prokaryotes, either during the developmental life cycle of certain bacteria or to remove damaged cells from a population in response to a wide variety of stresses. This review will examine several putative examples of bacterial PCD and summarize what is known about the molecular components of these systems.
Three rice nicotianamine synthase genes, OsNAS1, OsNAS2, and OsNAS3 are expressed in cells involved in long‐distance transport of iron and differentially regulated by iron
Authors: Inoue Haruhiko , Higuchi Kyoko , Takahashi Michiko , Nakanishi Hiromi , Mori Satoshi , Nishizawa Naoko K.
Published: October 2003

Nicotianamine (NA), a chelator of metals, is ubiquitously present in higher plants. In graminaceous plants, NA is a biosynthetic precursor of phytosiderophores and is thus a crucial component for iron (Fe) acquisition. Here, we show that three rice NA synthase (NAS) genes, OsNAS1, OsNAS2, and OsNAS3 are expressed in cells involved in long‐distance transport of Fe and that the three genes are differentially regulated by Fe. OsNAS1 and OsNAS2 transcripts were detected in Fe‐sufficient roots but not in leaves, and levels of both increased markedly in both roots and leaves in response to Fe deficiency. In contrast, the OsNAS3 transcript was present in leaves but was very low in roots of Fe‐sufficient plants. Further, OsNAS3 expression was induced in roots but was suppressed in leaves in response to Fe deficiency. Promoter–GUS analysis revealed that OsNAS1 and OsNAS2 were expressed in Fe‐sufficient roots in companion cells and pericycle cells adjacent to the protoxylem. With Fe deficiency, OsNAS1 and OsNAS2 expression extended to all root cells along with an increase in phytosiderophore secretion. In Fe‐deficient plants, OsNAS1 and OsNAS2 were expressed in the vascular bundles of green leaves and in all cells of leaves showing severe chlorosis. OsNAS3 expression was restricted to the pericycle and companion cells of the roots, and in companion cells of leaves irrespective of Fe status. These results strongly suggested that NAS and NA play an important role in long‐distance transport of Fe in rice plants, in addition to their roles in phytosiderophore secretion from roots.
Overexpression of a cell wall glycoprotein in Fusarium oxysporum increases virulence and resistance to a plant PR‐5 protein
Authors: Narasimhan Meena L. , Lee Hyeseung , Damsz Barbara , Singh Narendra K. , Ibeas José I. , Matsumoto Tracie K. , Woloshuk Charles P. , Bressan Ray A.
Published: October 2003

Fusarium oxysporum f. sp. nicotianae is a causal agent for vascular wilt disease in tobacco. It is sensitive to osmotin, a tobacco pathogenesis‐related protein (PR‐5) that is implicated in plant defense against phytopathogenic fungi. We show that osmotin susceptibility of F. oxysporum f. sp. nicotianae was reduced by overexpression of the heterologous cell wall glycoprotein Saccharomyces cerevisiae protein containing inverted repeats (PIR2), a member of the PIR family of fungal cell wall glycoproteins that protect S. cerevisiae from the toxic action of osmotin. S. cerevisiae PIR2 was targeted to the cell wall of F. oxysporum. Disease severity and fungal growth were increased in tobacco seedlings inoculated with F. oxysporum transformed with PIR2 compared to seedlings infected with untransformed F. oxysporum or that transformed with vector, although accumulation of transcript and protein of defense genes was similar. The results show that fungal cell wall components can increase resistance to plant defense proteins and affect virulence.
The phenotype of the Arabidopsis cue1 mutant is not simply caused by a general restriction of the shikimate pathway
Authors: Voll Lars , Häusler Rainer E. , Hecker Rolf , Weber Andreas , Weissenböck Gottfried , Fiene Gabriele , Waffenschmidt Sabine , Flügge Ulf‐Ingo
Published: October 2003

The Arabidopsis thalianachlorophyll a/b binding protein underexpressed (cue1) mutant, which has been isolated in a screen for chlorophyll a/b binding protein (CAB) underexpressors, exhibits a reticulate leaf phenotype combined with delayed chloroplast development and aberrant shape of the palisade parenchyma cells. The affected gene in cue1 is a phosphoenolpyruvate (PEP)/phosphate translocator (PPT) of the plastid inner envelope membrane. The proposed function of the PPT in C3‐plants is the import of PEP into the stroma as one of the substrates for the shikimate pathway, from which aromatic amino acids and a variety of secondary plant products derive. The mutant phenotype could be: (i) complemented by constitutive overexpression of a heterologous PPT from cauliflower; and (ii) rescued by overexpression of a C4‐type pyruvate,orthophosphate dikinase (PPDK). The latter approach indicates that PEP deficiency within plastids triggers developmental constraints in cue1. The impact of the mutation on aspects of primary and secondary metabolism was assessed in cue1 as well as in the individual transformant lines. The majority of the data obtained in this and an accompanying paper suggest that the mutant phenotype is not simply caused by a general restriction of the shikimate pathway because of a defect in a PPT.
Living related renal transplantation for end‐stage renal disease after liver transplantation from a brain‐dead donor
Authors: KOBAYASHI TAKASHI , OKUNO HIROSHI , TACHIBANA MITSUHIRO , MORI NORIKO , YOSHIDA HIROSHI , YAMAMOTO SHINGO , KAMOTO TOSHIYUKI , TERAI AKITO , OGAWA OSAMU
Published: October 2003

Abstract  We report a case in which a living related renal transplantation was successfully performed for end‐stage renal disease that had progressed after a liver transplantation from a brain‐dead donor for liver cirrhosis associated with type C hepatitis. Because the transplanted liver function had been excellent with the use of tacrolimus and mycophenolate mofetil, the same immunosuppressive agents with prednisolone were employed for the renal transplantation. Both grafts are functioning well without recurrence of hepatitis at 10 months after the renal transplantation. From our experience, renal transplantation should not be contraindicated even if the patient has undergone liver transplantation or has hepatitis C viral infection.
Acute eosinophilic pneumonia associated with intravesical bacillus Calmette‐Guérin therapy of carcinoma in situ of the bladder
Authors: ORIKASA KAZUHIKO , NAMIMA TAKASHIGE , OTA SHOZO , MIURA MOTOHIKO , HAMA HIKARU , KIMURA NORIKO , OHNUMA TETSUTARO
Published: October 2003

Abstract  A 71‐year‐old man with a history of rheumatoid arthritis was treated with intravesical bacillus Calmette‐Guérin (BCG) instillation of 80 mg once‐a‐week for carcinoma in situ. He developed low‐grade fever followed by dyspnea and severe hypoxemia. Radiological and laboratory studies revealed bilateral diffuse reticulonodular infiltrates and hypereosinophilia. A lymphocyte stimulation test for BCG was strongly positive. From these findings, a pulmonary hypersensitivity reaction to immunotherapy was suspected, and therefore, methylprednisolone (500 mg per day) was started. After that, the fever and dyspnea disappeared, the hypereosinophilia was normalised and chest radiography results were clear. The present case is the first reported case of eosinophilic pneumonia following intravesical BCG therapy.