bluuebunny/crossref_metadata_2025 huggingface dataset:
The genetic element pSSVx of the extremely thermophilic crenarchaeon Sulfolobus is a hybrid between a plasmid and a virus
Authors: Arnold Hans Peter , She Qunxin , Phan Hien , Stedman Kenneth , Prangishvili David , Holz Ingelore , Kristjansson Jakob K. , Garrett Roger , Zillig Wolfram
Published: March 2003

A new Sulfolobus islandicus strain, REY15/4, harboured both a novel fusellovirus, SSV2, and a small plasmid, pSSVx. The plasmid spread in S. solfataricus P1 together with the virus after infection with either the supernatant of a culture of REY15/4 or purified virus. Spreading of the plasmid required co‐transfection with either SSV2 or the related SSV1 as helpers. Virus purified from REY15/4 constituted a mixture of two sizes of particles, one with the dimensions of a normal fusellovirus and the other smaller. Cloned SSV2 produced only the larger particles and only SSV2 DNA, indicating that the smaller particles contained pSSVx packaged into capsids made up of SSV2 components. The 5.7 kb genome of pSSVx revealed regions of high sequence similarity to the cryptic Sulfolobales plasmids pRN1, pRN2 and pDL10. Thus, pSSVx belongs to the family of pRN plasmids that share a highly conserved region, which probably constitutes the minimal replicon. They also contain a variable region showing no sequence similarity. In pSSVx, this region contains three open reading frames (ORFs), two of which are juxtapositioned and show high sequence similarity to a tandem of ORFs in fusellovirus genomes. Neither pRN1 nor pRN2, which lack this tandem, spread in the presence of the fuselloviruses, which implies that the sequences of these ORFs enable pSSVx to use the packaging system of the viral helpers for spreading.
Phosphorylation‐induced dimerization of the FixJ receiver domain
Authors: Da Re Sandra , Schumacher Jörg , Rousseau Philippe , Fourment Joëlle , Ebel Christine , Kahn Daniel
Published: March 2003

The ‘two‐component’ transcriptional activator FixJ controls nitrogen fixation in Sinorhizobium meliloti. Phosphorylation of FixJ induces its dimerization, as evidenced by gel permeation chromatography and equilibrium sedimentation analysis. Phosphorylation‐induced dimerization is an intrinsic property of the isolated receiver domain FixJN. Accordingly, chemical phosphorylation of both FixJ and FixJN are second‐order reactions with respect to protein concentration. However, the second‐order phosphorylation constant is 44‐fold higher for FixJN than for FixJ. Therefore, the C‐terminal transcriptional activator domain FixJC inhibits the chemical phosphorylation of the receiver domain FixJN. Conversely, FixJN has been shown previously to inhibit FixJC activity ≈ 40‐fold, reflecting the interaction between FixJN and FixJC. Therefore, we propose that modulation of FixJ activity involves both its dimerization and the disruption of the interface between FixJN and FixJC, resulting in the opening of the protein structure. Alanine scanning mutagenesis of FixJN indicated that the FixJ~P dimerization interface involves Val‐91 and Lys‐95 in helix α4. Dimerization was required for high‐affinity binding to fixK promoter DNA.
Cis‐acting signals controlling translational initiation in the thermophilic archaeon Sulfolobus solfataricus
Authors: Condò Ivano , Ciammaruconi Andrea , Benelli Dario , Ruggero Davide , Londei Paola
Published: March 2003

In this work, we have studied the in vitro translational features of a bicistronic mRNA of the extremely thermophilic Archaeon Sulfolobus solfataricus, with the aim of determining the nature of the cis‐acting signals controlling the recognition of the translation initiation sites in the Archaea. We found that the most important feature for efficient initiation was the presence of a Shine–Dalgarno (SD)‐like ribosome‐binding motif, whose disruption entirely abolished the translation of the corresponding cistron. The influence of other features, such as the type of initiation codon, was variable and depended upon the gene and its position in the mRNA. However, the translational block caused by the disruption of the SD sequences could be removed by deleting the 5′ untranslated region altogether, thereby creating a ‘leaderless’ mRNA. This suggests that ‘leaderless’ initiation operates by a default mechanism that does not require a specific mRNA–rRNA interaction and may be common to all three primary domains of life.
Transposition genes of theBacteroidesmobilizable transposon Tn4555: role of a novel targeting gene
Authors: Tribble Gena D. , Parker Anita C. , Smith C. Jeffrey
Published: March 2003

Conjugative transposons have been identified in several bacterial species, most notably the Gram‐positiveEnterococciand the Gram‐negativeBacteroides. InBacteroidesspecies, these elements encode a complete conjugative machinery, which mediates their own intercellular transfer, and they can mobilizein transco‐resident elements. One such mobilizable element is the antibiotic resistance transposon, Tn4555, which was previously found to integrate into a specific genome target site via a site‐specific recombination mechanism. In this work, we demonstrate that three Tn4555genes were involved in integration of the element. These wereintencoding a lambda‐type integrase, which was absolutely required for integration of the transposon, and two accessory genes, which increased the frequency of integration. Interestingly, one of these accessory gene products, TnpA, directed the insertion of Tn4555into the genome target site; in the absence oftnpA, the insertion pattern was essentially random. This is the first example of a site‐specific recombinase that uses a specific targeting protein.
Topological analysis of the MraY protein catalysing the first membrane step of peptidoglycan synthesis
Authors: Bouhss Ahmed , Mengin‐Lecreulx Dominique , Le Beller Dominique , Van Heijenoort Jean
Published: March 2003

The two‐dimensional membrane topology of the Escherichia coli and Staphylococcus aureus MraY transferases, which catalyse the formation of the first lipid intermediate of peptidoglycan synthesis, was established using the β‐lactamase fusion system. All 28 constructed mraY–blaM fusions produced hybrid proteins. Analysis of the ampicillin resistance of the strains with hybrids led to a common topological model possessing 10 transmembrane segments, five cytoplasmic domains and six periplasmic domains including the N‐ and C‐terminal ends. The agreement between the topologies of E. coli and S. aureus, their agreement to a fair extent with predicted models and a number of features arising from the comparative analysis of 25 orthologue sequences strongly suggested the validity of the model for all eubacterial MraYs. The primary structure of the 10 transmembrane segments diverged among orthologues, but they retained their hydrophobicity, number and size. The similarity of the sequences and distribution of the five cytoplasmic domains in both models, as well as their conservation among the MraY orthologues, clearly suggested their possible involvement in substrate recognition and in the catalytic process. Complementation tests showed that only fusions with untruncated mraY restored growth. It was noteworthy that S. aureus MraY was functional in E. coli. An increased MraY transferase activity was observed only with the untruncated hybrids from both organisms.
Sporulation timing in Myxococcus xanthus is controlled by the espAB locus
Authors: Cho Kyungyun , Zusman David R.
Published: March 2003

The fruiting body development of Myxococcus xanthus consists of two separate but interacting pathways: one for aggregation of many cells to form raised mounds and the other for sporulation of individual cells into myxospores. Sporulation of individual cells normally occurs after mound formation, and is delayed at least 30 h after starvation under our laboratory conditions. This suggests that M. xanthus has a mechanism that monitors progress towards aggregation prior to triggering sporulation. A null mutation in a newly identified gene, espA (early sporulation), causes sporulation to occur much earlier compared with the wild type (16 h earlier). In contrast, a null mutation in an adjacent gene, espB, delays sporulation by about 16 h compared with the wild type. Interestingly, it appears that the espA mutant does not require raised mounds for sporulation. Many mutant cells sporulate outside the fruiting bodies. In addition, the mutant can sporulate, without aggregation into raised mounds, under some conditions in which cells normally do not form fruiting bodies. Based on these observations, it is hypothesized that EspA functions as an inhibitor of sporulation during early fruiting body development while cells are aggregating into raised mounds. The aggregation‐independent sporulation of the espA mutant still requires starvation and high cell density. The espA and espB genes are expressed as an operon and their translations appear to be coupled. Expression occurs only under developmental conditions and does not occur during vegetative growth or during glycerol‐induced sporulation. Sequence analysis of EspA indicates that it is a histidine protein kinase with a fork head‐associated (FHA) domain at the N‐terminus and a receiver domain at the C‐terminus. This suggests that EspA is part of a two‐component signal transduction system that regulates the timing of sporulation initiation.
Expression of the Escherichia coli NRZ nitrate reductase is highly growth phase dependent and is controlled by RpoS, the alternative vegetative sigma factor
Authors: Chang Lily , Wei Linda I.‐C. , Audia Jonathon P. , Morton Richard A. , Schellhorn Herb E.
Published: March 2003

In the absence of oxygen, many bacteria preferentially use nitrate as a terminal electron acceptor for anaerobic respiration. In Escherichia coli, there are two membrane‐bound, differentially regulated nitrate reductases. While the physiological basis for this metabolic redundancy is not completely understood, during exponential growth, synthesis of NRA is greatly induced by anaerobiosis plus nitrate, whereas NRZ is expressed at a low level that is not influenced by anaerobiosis or nitrate. In the course of identifying genes controlled by the stationary phase regulatory factor RpoS (σs), we found that the expression of NRZ is induced during entry into stationary phase and highly dependent on this alternative sigma factor. Expression studies, using operon fusions and nitrate reductase assays, revealed that the NRZ operon is controlled mainly at the level of transcription and is induced 10‐fold at the onset of stationary phase in rich media. Consistent with previous reports of RpoS expression, the RpoS dependency of NRZ in minimal media was very high (several hundredfold). We also observed a fivefold stationary phase induction of NRZ in an rpoS background, indicating that other regulatory factors, besides RpoS, are probably involved in transcriptional control of NRZ. The RpoS dependence of NRZ expression was confirmed by Northern analyses using RNA extracted from wild‐type and rpoS− strains sampled in exponential and stationary phase. In toto, these data indicate that RpoS‐mediated regulation of NRZ may be an important physiological adaptation that allows the cell to use nitrate under stress‐associated conditions.
Phosphorylation of the flagellar regulatory protein FlrC is necessary for Vibrio cholerae motility and enhanced colonization
Authors: Correa Nidia E. , Lauriano Crystal M. , McGee Raynia , Klose Karl E.
Published: March 2003

The human pathogen Vibrio cholerae specifically expresses virulence factors within the host, including cholera toxin (CT) and the toxin co‐regulated pilus (TCP), which allow it to colonize the intestine and cause disease. V. cholerae is a highly motile organism by virtue of a polar flagellum, and motility has been inferred to be an important aspect of virulence, yet the exact role of motility in pathogenesis has remained undefined. The two‐component regulatory system FlrB/FlrC is required for polar flagellar synthesis; FlrC is a σ54‐dependent transcriptional activator. We demonstrate that the transcriptional activity of FlrC affects both motility and colonization of V. cholerae. In a purified in vitro reaction, FlrB transfers phosphate to the wild‐type FlrC protein, but not to a mutant form in which the aspartate residue at amino acid position 54 has been changed to alanine (D54A), consistent with this being the site of phosphorylation of FlrC. The wild‐type FlrC protein, but not the D54A protein, activates σ54‐dependent transcription in a heterologous system, demonstrating that phospho‐FlrC is the transcriptionally active form. A V. cholerae strain containing a chromosomal flrCD54A allele did not synthesize a flagellum and had no detectable levels of transcription of the critical σ54‐dependent flagellin gene flaA. The V. cholerae flrCD54A mutant strain was also defective in its ability to colonize the infant mouse small intestine, approximately 50‐fold worse than an isogenic wild‐type strain. Another mutation of FlrC (methionine 114 to isoleucine; M114I) confers constitutive transcriptional activity in the absence of phosphorylation, but a V. cholerae flrCM114I mutant strain, although flagellated and motile, was also defective in its ability to colonize. The strains carrying D54A or M114I mutant FlrC proteins expressed normal levels of CT and TCP under in vitro inducing conditions. Our results show that FlrC ‘locked’ into either an inactive (D54A) or an active (M114I) state results in colonization defects, thereby demonstrating a requirement for modulation of FlrC activity during V. cholerae pathogenesis. Thus, the σ54‐dependent transcriptional activity of the flagellar regulatory protein FlrC contributes not only to motility, but also to colonization of V. cholerae.
The asgE locus is required for cell–cell signalling during Myxococcus xanthus development
Authors: Garza Anthony G. , Harris Baruch Z. , Pollack Jeffrey S. , Singer Mitchell
Published: March 2003

In response to starvation, Myxococcus xanthus undergoes a multicellular developmental process that produces a dome‐shaped fruiting body structure filled with differentiated cells called myxospores. Two insertion mutants that block the final stages of fruiting body morphogenesis and reduce sporulation efficiency were isolated and characterized. DNA sequence analysis revealed that the chromosomal insertions are located in open reading frames ORF2 and asgE, which are separated by 68 bp. The sporulation defect of cells carrying the asgE insertion can be rescued phenotypically when co‐developed with wild‐type cells, whereas the sporulation efficiency of cells carrying the ORF2 insertion was not improved when mixed with wild‐type cells. Thus, the asgE insertion mutant appears to belong to a class of developmental mutants that are unable to produce cell–cell signals required for M. xanthus development, but they retain the ability to respond to them when they are provided by wild‐type cells. Several lines of evidence indicate that asgE cells fail to produce normal levels of A‐factor, a cell density signal. A‐factor consists of a mixture of heat‐stable amino acids and peptides, and at least two heat‐labile extracellular proteases. The asgE mutant yielded about 10‐fold less heat‐labile A‐factor and about twofold less heat‐stable A‐factor than wild‐type cells, suggesting that the primary defect of asgE cells is in the production or release of heat‐labile A‐factor.
Rapid inactivation of the Escherichia coli Kdp K+ uptake system by high potassium concentrations
Authors: Roe Andrew J. , McLaggan Debra , O’Byrne Conor P. , Booth Ian R.
Published: March 2003

The Kdp K+ uptake system of Escherichia coli is induced by limitation for K+ and/or high osmolarity. In the present study, the regulation of the activity of the Kdp system has been investigated in E. coli mutants possessing only the Kdp system as the mechanism of K+ accumulation. Cells grown in the presence of low K+ (0.1–1 mM) exhibit normal growth. However, growth inhibition results from exposure of cells to moderate levels of external K+ (> 5 mM). Measurement of the cytoplasmic pH, of K+ pools and of transport via the Kdp system demonstrates that the Kdp system is rapidly and irreversibly inhibited by moderate external K+. Concentrations of K+ greater than 2 mM are sufficient to cause inhibition of Kdp. At pH 6, this results in rapid lowering of the capacity for pH homeostasis, but at pH 7 the intracellular pH is unaffected. Parallel analysis of the expression of the Kdp system in a Kdp+/kdpFABC–lacZ strain shows that levels of K+ that are sufficient to inhibit Kdp activity also repress expression. As a result, growth inhibition of strains solely possessing Kdp arises jointly from inhibition of Kdp activity and repression of Kdp gene expression. These data identify an important aspect of the regulation of potassium transport via the Kdp system and also provide support for a model of regulation of Kdp expression via at least two mechanisms: sensing of both turgor and external K+ concentration.
Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins
Authors: Que Qiang , Helmann John D.
Published: March 2003

The Bacillus subtilis yqhN gene encodes a metalloregulatory protein distantly related to the Corynebacterium diphtheriae diphtheria toxin repressor (DtxR). While DtxR mediates the iron‐dependent repression of iron uptake, we demonstrate that yqhN (herein renamed mntR) encodes a manganese modulated regulator of manganese transport. An mntR mutant strain is sensitive to both manganese and cadmium, suggesting that the transport of these metals is derepressed. We selected Tn10 insertions that suppress the Mn(II) sensitivity of the mntR mutant or that increase the Cd(II) tolerance of wild‐type cells, and in both cases we recovered insertions in mntH (formerly ydaR). MntH is a member of the NRAMP family of proton‐coupled, metal ion transporters. MntR also regulates expression of a Mn(II) ABC transporter (MntABCD). The MntH and MntABCD transporters are both selectively repressed by Mn(II) and this regulation requires MntR. In high Mn(II) conditions, MntR functions as a Mn(II)‐dependent repressor of mntH transcription. In contrast, MntR acts as a positive regulator of the mntABCD operon under low Mn(II) growth conditions. Biochemical studies demonstrate that MntR binding to the mntH control region requires Mn(II), while interaction with the mntABCD control region does not depend on Mn(II).
Phosphorylcholine decoration of lipopolysaccharide differentiates commensal Neisseriae from pathogenic strains: identification of licA‐type genes in commensal Neisseriae
Authors: Serino Laura , Virji Mumtaz
Published: March 2003

Phosphorylcholine (ChoP) is a potential candidate for a plurispecific vaccine, because it is present on surface components of many mucosal organisms, including Haemophilus influenzae, Streptococcus pneumoniae and Pseudomonas aeruginosa. In addition, ChoP has been detected on pili of Neisseria meningitidis and Neisseria gonorrhoeae. In this study, we demonstrate the presence of the phosphorylcholine epitope on the lipopolysaccharides (LPSs) of several species of commensal Neisseriae (Cn), a property that differentiates commensal from the pathogenic strains of Neisseriae. In an extended survey of 78 strains, we confirmed the exclusive expression of the ChoP epitope on pili of pathogenic Neisseriae. Despite the presence of pili on Cn, which are homologous to Class II pili of N. meningitidis, they did not react with anti‐ChoP antibody. This observation was further supported by the fact that 14C‐labelled choline was incorporated only in the LPSs of Cn. Analysis of the LPS of N. lactamica strain NL4 revealed two distinct and interconvertible molecular species of LPS with high and low levels of reactivity with anti‐ChoP antibody. In addition, on/off phase variation gave rise to frequent modulation in the levels of antibody reactivity. A concurrent modulation was also observed in the binding of C‐reactive protein, CRP, a ChoP‐binding reactant that is implicated in bacterial clearance. Genetic analysis showed the presence of a gene in several Cn spp. with significant sequence identity to H. influenzae licA. This gene encodes choline kinase and is also involved in phase variation of the LPS‐associated ChoP in H. influenzae. In contrast, licA‐like genes were not identified in the pathogenic Neisseria strains tested. They are absent from N. meningitidis strain Z2491 genome database. These data suggest that the genetic basis for ChoP incorporation in Cn LPS resembles that in H. influenzae spp. and may be distinct from that generating the ChoP epitope on pili of pathogenic Neisseriae. Further, the modulation of ChoP expression on Cn LPS, and corresponding modulation of CRP binding, has the potential to confer the property of immune avoidance and thus of persistence on mucosa.
Macrolide–ketolide inhibition of MLS‐resistant ribosomes is improved by alternative drug interaction with domain II of 23S rRNA
Authors: Douthwaite Stephen , Hansen Lykke Haastrup , Mauvais Pascale
Published: March 2003

The macrolide antibiotic erythromycin and its 6‐O‐methyl derivative (clarithromycin) bind to bacterial ribosomes primarily through interactions with nucleotides in domains II and V of 23S rRNA. The domain II interaction occurs between nucleotide A752 and the macrolide 3‐cladinose moiety. Removal of the cladinose, and substitution of a 3‐keto group (forming the ketolide RU 56006), results in loss of the A752 interaction and an ≈ 100‐fold drop in drug binding affinity. Within domain V, the key determinant of drug binding is nucleotide A2058 and substitution of G at this position is the major cause of drug resistance in some clinical pathogens. The 2058G mutation disrupts the drug‐domain V contact and leads to a further > 25 000‐fold decrease in the binding of RU 56006. Drug binding to resistant ribosomes can be improved over 3000‐fold by forming an alternative and more effective contact to A752 via alkyl–aryl groups linked to a carbamate at the drug 11/12 position (in the ketolide antibiotics HMR 3647 and HMR 3004). The data indicate that simultaneous drug interactions with domains II and V strengthen binding and that the domain II contact is of particular importance to achieve binding to the ribosomes of resistant pathogens in which the domain V interaction is perturbed.
Subcellular localization of the Agrobacterium tumefaciens T‐DNA transport pore proteins: VirB8 is essential for the assembly of the transport pore
Authors: Kumar Renu B. , Xie Yong‐Hong , Das Anath
Published: March 2003

Agrobacterium tumefaciens transforms plants by transferring DNA to the plant cell nucleus. The VirB membrane proteins are postulated to form a pore for the transport of the DNA across the bacterial membranes. Immunofluorescence and immunoelectron microscopy were used to study the transport pore complex. Three likely components of the transport pore, VirB8, VirB9 and VirB10, localized primarily to the inner membrane, outer membrane and periplasm respectively. A significant amount of VirB10 was also found associated with the outer membrane. When expressed alone VirB9 and VirB10 were randomly distributed along the cell membrane. Subcellular location of both proteins changed dramatically in the presence of the other VirB proteins. Both proteins localized to fewer sites and most of the gold particles representing protein molecules were found in clusters suggesting that the two proteins are in a protein complex. VirB8, on the other hand, localized to clusters even in the absence of the other VirB proteins. To investigate the role of VirB8 in the formation of VirB9 and VirB10 protein complexes, we studied the effect of deletion of virB8 on the subcellular location of VirB9 and VirB10. In a virB8 deletion mutant both proteins were distributed randomly on the cell membrane indicating that VirB8 is essential for complex assembly.
Carcinoembryonic antigens are targeted by diverse strains of typable and non‐typable Haemophilus influenzae
Authors: Virji Mumtaz , Evans Debbie , Griffith Jo , Hill Darryl , Serino Laura , Hadfield Andrea , Watt Suzanne M.
Published: March 2003

Haemophilus influenzae (Hi), a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We show that distinct strains belonging to typable (THi) and non‐typable (NTHi) H. influenzae target human carcinoembryonic antigens (the membrane associated CEA family of cell adhesion molecules, are now termed CEACAMs). All strains of H. influenzae biogroup aegyptius (Hi‐aeg) and more than 70% of THi and NTHi strains tested specifically recognize CEACAMI‐Fc soluble constructs. Furthermore, transfection of Chinese hamster ovary cells with human CEACAM1 cDNA alone was sufficient for promoting Hi interactions with the transfected cells. The majority of the Hi‐aeg strains tested interacted with soluble constructs containing only the N‐terminal domain. In contrast, several THi and NTHi strains reacted with soluble constructs only when additional extracellular A and B domains of the receptor were present. The use of monoclonal antibodies confirmed that THi and NTHi strains also interact primarily at the N‐domain. We used site‐directed mutants of CEACAM1 that contained substitutions at surface exposed amino acids and a molecular model of the N‐domain to identify the residues involved in interactions with Hi ligands. The studies show that a common region exposed at the CFG face of the molecule is targeted by diverse Hi strains. However, mutation at distinct sites within this area affected the interactions of distinct strains signifying the potential for tissue tropism via this receptor. Analyses of the molecular basis of interaction with human cell lines and purified CEA show that Hi strains, especially those belonging to Hi‐aeg, interact with multiple CEACAMs. Because Neisseria meningitidis (Nm) strains are also known to bind at the CFG face of the receptor, we used Nm and Hi strains in co‐infection experiments and demonstrate competition between these mucosal pathogens in colonization of target cells via CEACAMs.
Polynucleotide phosphorylase, RNase II and RNase E play different roles in the in vivo modulation of polyadenylation in Escherichia coli
Authors: Mohanty Bijoy K. , Kushner Sidney R.
Published: March 2003

Poly(A) tails in Escherichia coli are hypothesized to provide unstructured single‐stranded substrates that facilitate the degradation of mRNAs by ribonucleases. Here, we have investigated the role that such nucleases play in modulating polyadenylation in vivo by measuring total poly(A) levels, polyadenylation of specific transcripts, growth rates and cell viabilities in strains containing various amounts of poly(A) polymerase I (PAP I), polynucleotide phosphorylase (PNPase), RNase II and RNase E. The results demonstrate that both PNPase and RNase II are directly involved in regulating total in vivo poly(A) levels. RNase II is primarily responsible for degrading poly(A) tails associated with 23S rRNA, whereas PNPase is more effective in modulating the polyadenylation of the lpp and 16S rRNA transcripts. In contrast, RNase E appears to affect poly(A) levels indirectly through the generation of new 3′ termini that serve as substrates for PAP I. In addition, whereas excess PNPase suppresses polyadenylation by more than 70%, the toxicity associated with increased poly(A) levels is not reduced. Conversely, toxicity is significantly reduced in the presence of excess RNase II. Overproduction of RNase E leads to increased polyadenylation and no reduction in toxicity.
Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions
Authors: Sourjik Victor , Berg Howard C.
Published: March 2003

We prepared fusions of yellow fluorescent protein [the YFP variant of green fluorescent protein (GFP)] with the cytoplasmic chemotaxis proteins CheY, CheZ and CheA and the flagellar motor protein FliM, and studied their localization in wild‐type and mutant cells of Escherichia coli. All but the CheA fusions were functional. The cytoplasmic proteins CheY, CheZ and CheA tended to cluster at the cell poles in a manner similar to that observed earlier for methyl‐accepting chemotaxis proteins (MCPs), but only if MCPs were present. Co‐localization of CheY and CheZ with MCPs was CheA dependent, and co‐localization of CheA with MCPs was CheW dependent, as expected. Co‐localization with MCPs was confirmed by immunofluorescence using an anti‐MCP primary antibody. The motor protein FliM appeared as discrete spots on the sides of the cell. These were seen in wild‐type cells and in a fliN mutant, but not in flhC or fliG mutants. Co‐localization with flagellar structures was confirmed by immunofluorescence using an antihook primary antibody. Surprisingly, we did not observe co‐localization of CheY with motors, even under conditions in which cells tumbled.
New insights into the role of CcmC, CcmD and CcmE in the haem delivery pathway during cytochrome c maturation by a complete mutational analysis of the conserved tryptophan‐rich motif of CcmC
Authors: Schulz Henk , Pellicioli Erica C. , Thöny‐Meyer Linda
Published: March 2003

Maturation of c‐type cytochromes in Escherichia coli is a complex process requiring eight membrane proteins encoded by the ccmABCDEFGH operon. CcmE is a mediator of haem delivery. It binds haem transiently at a conserved histidine residue and releases it for directed transfer to apocytochrome c. CcmC, an integral membrane protein with six transmembrane helices, is necessary and sufficient to incorporate haem covalently into CcmE. CcmC contains a highly conserved tryptophan‐rich motif, WGXXWXWD, in its second periplasmic loop. Here, we present the results of a systematic mutational analysis of this motif. Changes of the non‐conserved T121 and W122 to A resulted in wild‐type CcmC activity. Changes of the single amino acids W119A, G120A, W123A, W125I and D126A or of the spacing within the motif by deleting V124 (ΔV124) inhibited the covalent haem incorporation into CcmE. Enhanced expression of ccmD suppressed this mutant phenotype by increasing the amounts of CcmC and CcmE polypeptides in the membrane. The ΔV124 mutant showed the strongest defect of all single mutants. Mutants in which six residues of the tryptophan‐rich motif were changed showed no residual CcmC activity. This phenotype was independent of the level of ccmD expression. Our results demonstrate the functional importance of the tryptophan‐rich motif for haem transfer to CcmE. We propose that the three membrane proteins CcmC, CcmD and CcmE interact directly with each other, establishing a cytoplasm to periplasm haem delivery pathway for cytochrome c maturation.
Mapping of IS6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticity
Authors: Warren R. M. , Sampson S. L. , Richardson M. , Van Der Spuy G. D. , Lombard C. J. , Victor T. C. , Van Helden P. D.
Published: March 2003

Southern hybridization was used in combination with IS6110 insertion‐locus‐specific probes in a comparative study to determine the structure of chromosomal domains flanking IS6110 elements in clinical isolates of Mycobacterium tuberculosis. The resulting restriction fragment length polymorphism (RFLP) data demonstrated three mutational mechanisms responsible for the polymorphisms observed: IS6110 insertion, chromosomal mutation and deletion. The frequency of IS6110 insertion within many of the chromosomal regions demonstrates that preferential integration regions are common in M. tuberculosis. Mapping the IS6110 insertion positions and chromosomal deletions in relation to the M. tuberculosis H37Rv and M. bovis BCG genome sequences reveals numerous disruptions of predicted open reading frames (ORFs). A phylogenetic tree, based on the mutational data, showed a number of independently evolving lineages of M. tuberculosis, while analysis of the mutational events occurring at each branch point suggests both divergent and convergent evolution. A significant positive correlation was demonstrated between the mutation rate and the frequency of occurrence of different isolates in families of strains, suggesting that evolution may impact on strain ‘fitness’ or that strain proliferation may increase the chance of mutation. We conclude that the genome of clinical isolates of M. tuberculosis continues to evolve.
A pot‐pourri of plasmid paradoxes: effects of a second copy
Authors: Yarmolinsky Michael B.
Published: March 2003

Bacterial plasmids are exemplary subjects for study, being conveniently isolated, dissected, reassembled, and introduced into various hosts. Their versatility and power make them eminently worthy of our attention. In what follows I consider some consequences of simply doubling the dosage of particular plasmid genes or of forming a plasmid dimer. These consequences can be perverse, paradoxical, or informative. They bear on questions of cell viability, copy number limitation, clonal homogeneity, check‐point control, and the recovery of mutants. They have relevance to biotechnology, evolution and medicine. In reviewing these effects, my motivation is largely to share my enthusiasm for certain kinds of biological narratives, the nature of which is best left for the reader to discern.
The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa
Authors: Middleton A. M. , Chadwick M. V. , Nicholson A. G. , Dewar A. , Groger R. K. , Brown E. J. , Wilson R.
Published: March 2003

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non‐immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M. avium FAP in a concentration‐dependant manner. The number of bacteria adhering to fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.
In vivo N‐glycosylation of the Mep2 high‐affinity ammonium transporter of Saccharomyces cerevisiae reveals an extracytosolic N‐terminus
Authors: Marini Anne‐Marie , André Bruno
Published: March 2003

Saccharomyces cerevisiae possesses three related ammonium transporters, Mep1, Mep2 and Mep3, differing in their kinetic properties and in the level and regulation of their gene expression. The three Mep proteins belong to a family conserved in bacteria, plants and animals, which also includes proteins of the rhesus blood group family. In addition to its role in scavenging extracellular ammonium, the Mep2 protein has been proposed to act as an ammonium sensor, essential to pseudohyphal differentiation in response to ammonium limitation. To pursue the biochemical study of the Mep transporters, we raised polyclonal antibodies against the C‐terminal tail of each Mep protein. When electrophoresed on SDS–polyacrylamide gel, the Mep1 and Mep3 proteins migrate as expected from their predicted size, whereas the Mep2 protein migrates as a high‐molecular‐weight smear. Protein deglycosylation with peptide‐N‐glycosidase F (PNGase F) indicates that, in contrast to Mep1 and Mep3, Mep2 is an asparagine‐linked glycoprotein. Site‐directed mutagenesis of the four potential N‐glycosylation sites of Mep2 shows that Asn‐4 of the protein's N‐terminal tail is the only site that binds oligosaccharides. This provides evidence for the extracytosolic location of the Mep2 N‐terminus. Consistently, treatment of intact protoplasts with proteinase K leads to specific proteolysis of the N‐terminal tail of Mep2. The protein's C‐terminus, on the other hand, is protected against protease degradation under these conditions, but digested after protoplast permeabilization, suggesting a cytoplasmic location for this part of the protein. Mep2 glycosylation is not required for pseudohyphal differentiation in response to ammonium starvation, and its absence causes only a slight reduction in the affinity of the transporter for its substrate.
Single‐stranded DNA intermediates in IS91 rolling‐circle transposition
Authors: Del Pilar Garcillán‐Barcia M. , Bernales Irantzu , Mendiola M. Victoria , De La Cruz Fernando
Published: March 2003

IS91 displays a number of characteristics unique among insertion sequence (IS) elements, suggesting that it transposes by a novel mechanism called rolling‐circle (RC) transposition. We reported previously that IS91 transposase (TnpA) amino acid sequence shares a series of five conserved signatures with A proteins of RC replicating phages, including a pair of invariant tyrosines that catalyse two successive transesterification reactions during replication initiation and termination. To analyse their role in IS91 transposition, we constructed a series of TnpA derivatives in which the invariant Tyr‐249 and/or Tyr‐253 were mutated to either phenylalanine or serine. Mutation of either tyrosine resulted in complete loss of transposition activity in vivo. This result was taken as a first new line of evidence that TnpA is a functional analogue of φX174 phage A protein. Secondly, RC replication plasmids and phages accumulate single‐stranded DNA (ssDNA) intermediates as a result of uncoupled leading and lagging DNA strand synthesis. Using a plasmid carrying an IS91‐derived IRLkan‐IRR transposable cassette, in which the left (IRL)‐ and right (IRR)‐terminal sequences of IS91 flank a kanamycin resistance gene (kan), we demonstrated the in vivo formation of two new DNA species after induction of transposase expression. The first was a circular ssDNA that contained the transposable cassette covalently joined at its exact termini, whereas the second was a double‐stranded circle of the same element. When this experiment was repeated using the mutant transposases described above, the ssDNA and dsDNA intermediates could not be observed, indicating that the integrity of both Y249 and Y253 was essential for their appearance. The presence of ssDNA intermediate products is the first biochemical evidence for a RC mechanism of IS91 transposition.
Regulation of immunity to the two‐component lantibiotic, lacticin 3147, by the transcriptional repressor LtnR
Authors: McAuliffe Olivia , O'Keeffe Triona , Hill Colin , Ross R. Paul
Published: March 2003

Lacticin 3147 is a membrane‐active, two‐component lantibiotic produced by Lactococcus lactis ssp. lactis DPC3147. In this study, the promoters of the lacticin 3147 gene cluster were mapped to the intergenic region between ltnR and ltnA1 (the genes encoding the regulatory protein LtnR and the first structural gene, LtnA1), and Northern analyses revealed that the biosynthetic and immunity genes are divergently transcribed in two operons, ltnA1A2M1TM2D and ltnRIFE respectively. Although the promoter controlling biosynthesis (Pbac) appears to be constitutive, characterization of a downstream β‐galactosidase (β‐gal) fusion beyond an intragenic stem–loop structure in ltnM1 confirmed that this putative transcriptional attenuator allows limited readthrough to the downstream biosynthetic genes, thus maintaining the correct stoichiometry between structural peptides and biosynthetic machinery. The promoter of the ltnRIFE operon (Pimm) was shown to be regulated by the transcriptional repressor LtnR. A mutant with a truncated ltnR gene exhibited a hyperimmune phenotype, whereas overexpression of ltnR resulted in cells with increased sensitivity to lacticin 3147. Gel mobility shift analysis indicated that LtnR binds to the Pimm promoter region, and fusion of this promoter to the β‐gal gene of pAK80 revealed that expression from Pimm is significantly reduced in the presence of LtnR. Thus, we have demonstrated that lacticin 3147 uses a regulatory mechanism not previously identified in lantibiotic systems.
Selective expression of the virulence factor BAD1 upon morphogenesis to the pathogenic yeast form of Blastomyces dermatitidis: evidence for transcriptional regulation by a conserved mechanism
Authors: Rooney Peggy J. , Sullivan Thomas D. , Klein Bruce S.
Published: March 2003

Most dimorphic fungal pathogens grow as non‐pathogenic moulds in soil and convert to pathogenic yeast in the host, suggesting that virulence factors are upregulated during phase transition. Such factors have been difficult to identify. We analysed BAD1 (formerly WI‐1), a virulence factor in the dimorphic fungus Blastomyces dermatitidis, for expression in yeast and mycelial morphotypes. BAD1 was expressed in yeast but not in mycelia of North American strains of B. dermatitidis, and this expression pattern was confirmed for BAD1 transcript. BAD1 under the control of its promoter was transferred into African B. dermatitidis lacking a native BAD1 locus, and phase‐specific expression was conserved. Sequence similarity was identified between the BAD1 promoter and the promoters of two yeast phase‐specific genes in Histoplasma capsulatum. In H. capsulatum BAD1 transformants, yeast phase‐specific expression of BAD1 was conserved, and no transcript was detected in mycelia. BAD1β‐galactosidase reporter fusions analysed in B. dermatitidis and H. capsulatum confirmed that BAD1 is transcriptionally regulated in both fungi. BAD1 promoter activity and surface BAD1 expression were detected 6 h after shifting mycelia to 37°C. Thus, BAD1 is expressed after transition to the pathogenic yeast morphotype and is regulated by a mechanism for phase‐specific gene expression that appears to be conserved.
The phenylacetyl‐CoA catabolon: a complex catabolic unit with broad biotechnological applications
Authors: Luengo José M. , García José L. , Olivera Elías R.
Published: March 2003

The term catabolon was introduced to define a complex functional unit integrated by different catabolic pathways, which are, or could be, co‐ordinately regulated, and that catalyses the transformation of structurally related compounds into a common catabolite. The phenylacetyl‐CoA catabolon encompasses all the routes involved in the transformation of styrene, 2‐phenylethylamine, trans‐styrylacetic acid, phenylacetaldehyde, phenylacetic acid, phenylacetyl amides, phenylacetyl esters and n‐phenylalkanoic acids containing an even number of carbon atoms, into phenylacetyl‐CoA. This common intermediate is subsequently catabolized through a route of convergence, the phenylacetyl‐CoA catabolon core, into general metabolites. The genetic organization of this central route, the biochemical significance of the whole functional unit and its broad biotechnological applications are discussed.
The novel σ54‐ and σ28‐dependent flagellar gene transcription hierarchy of Vibrio cholerae
Authors: Prouty Michael G. , Correa Nidia E. , Klose Karl E.
Published: March 2003

The human pathogen Vibrio cholerae is a highly motile organism by virtue of a polar flagellum. Flagellar transcriptional regulatory factors have been demonstrated to contribute to V. cholerae virulence, but the role these factors play in the transcription hierarchy controlling flagellar synthesis has been unclear. The flagellar genes revealed by the V. cholerae genome sequence are located in three large clusters, with the exception of the motor genes, which are found in three additional locations. It had previously been demonstrated that the alternative sigma factor σ54 and the σ54‐dependent activators FlrA and FlrC are necessary for flagellar synthesis. The V. cholerae genome sequence revealed the presence of a fliA gene, which is predicted to encode the alternative flagellar sigma factor σ28. A V. choleraeΔfliA mutant strain is non‐motile, and synthesizes a truncated flagellum. Vibrio cholerae FliA complements both V. cholerae and Salmonella typhimurium fliA mutants for motility, consistent with its function as an alternative flagellar sigma factor. Analysis of lacZ transcriptional fusions of the V. cholerae flagellar promoters in both V. cholerae and S. typhimurium identified σ28‐, σ54‐, FlrA‐ and FlrC‐dependent promoters, as well as promoters that were independent of all these factors. Our results support a model of V. cholerae flagellar gene transcription as a novel hierarchy composed of four classes of genes. Class I is composed solely of the gene encoding the σ54‐dependent activator FlrA, which along with the σ54‐holoenzyme form of RNA polymerase activates expression of Class II genes. These genes include structural components of the MS ring, switch and export apparatus, as well as the genes encoding both FliA and FlrC. FlrC, along with σ54‐holoenzyme, activates expression of Class III genes, which include basal body, hook and filament genes. Finally, σ28‐holoenzyme activates expression of Class IV genes, which include additional filament genes as well as motor genes. Thus, this novel V. cholerae flagellar hierarchy has incorporated elements from both the σ54‐dependent Caulobacter crescentus polar flagellar hierarchy and the σ28‐dependent S. typhimurium peritrichous flagellar hierarchy.
Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis
Authors: Feucht Andrea , Lucet Isabelle , Yudkin Michael D. , Errington Jeffery
Published: March 2003

The actin‐like protein FtsA is present in many eubacteria, and genetic experiments have shown that it plays an important, sometimes essential, role in cell division. Here, we show that Bacillus subtilis FtsA is targeted to division sites in both vegetative and sporulating cells. As in other organisms FtsA is probably recruited immediately after FtsZ. In sporulating cells of B. subtilis FtsZ is recruited to potential division sites at both poles of the cell, but asymmetric division occurs at only one pole. We have now found that FtsA is recruited to only one cell pole, suggesting that it may play an important role in the generation of asymmetry in this system. FtsA is present in much higher quantities in B. subtilis than in Escherichia coli, with approximately one molecule of FtsA for five of FtsZ. This means that there is sufficient FtsA to form a complete circumferential ring at the division site. Therefore, FtsA may have a direct structural role in cell division. We have purified FtsA and shown that it behaves as a dimer and that it has both ATP‐binding and ATP‐hydrolysis activities. This suggests that ATP hydrolysis by FtsA is required, together with GTP hydrolysis by FtsZ, for cell division in B. subtilis (and possibly in most eubacteria).