bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential role of the Mu B protein in phage Mu integration vs. replication: mechanistic insights into two transposition pathways
Authors: Roldan Leslie Ann S. , Baker Tania A.
Published: March 2003

The Mu B protein is an ATP‐dependent DNA‐binding protein and an allosteric activator of the Mu transposase. As a result of these activities, Mu B is instrumental in efficient transposition and target‐site choice. We analysed in vivo the role of Mu B in the two different recombination reactions performed by phage Mu: non‐replicative transposition, the pathway used during integration, and replicative transposition, the pathway used during lytic growth. Utilizing a sensitive PCR‐based assay for Mu transposition, we found that Mu B is not required for integration, but enhances the rate and extent of the process. Furthermore, three different mutant versions of Mu B, Mu BC99Y, Mu BK106A, and Mu B1−294, stimulate integration to a similar level as the wild‐type protein. In contrast, these mutant proteins fail to support Mu growth. This deficiency is attributable to a defect in formation of an essential intermediate for replicative transposition. Biochemical analysis of the Mu B mutant proteins reveals common features: the mutants retain the ability to stimulate transposase, but are defective in DNA binding and target DNA delivery. These data indicate that activation of transposase by Mu B is sufficient for robust non‐replicative transposition. Efficient replicative transposition, however, demands that the Mu B protein not only activate transposase, but also bind and deliver the target DNA.
Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility
Authors: Hendrixson David R. , Akerley Brian J. , DiRita Victor J.
Published: March 2003

Campylobacter jejuni constitutes the leading cause of bacterial gastroenteritis in the United States and a major cause of diarrhoea worldwide. Little is known about virulence mechanisms in this organism because of the scarcity of suitable genetic tools. We have developed an efficient system of in vitro transposon mutagenesis using a mariner‐based transposon and purified mariner transposase. Through in vitro transposition of C. jejuni chromosomal DNA followed by natural transformation of the transposed DNA, large random transposon mutant libraries consisting of ≈ 16 000 individual mutants were generated. The first genetic screen of C. jejuni using a transposon‐generated mutant library identified 28 mutants defective for flagellar motility, one of the few known virulence determinants of this pathogen. We developed a second genetic system, which allows for the construction of defined chromosomal deletions in C. jejuni, and demonstrated the requirement of σ28 and σ54 for motility. In addition, we show that σ28 is involved in the transcription of flaA and that σ54 is required for transcription of three other flagellar genes, flaB and flgDE. We also identified two previously uncharacterized genes required for motility encoding proteins that we call CetA and CetB, which mediate energy taxis responses. Through our analysis of the Cet proteins, we propose a unique mechanism for sensing energy levels and mediating energy taxis in C. jejuni.
Biology of type II secretion
Authors: Sandkvist Maria
Published: March 2003

The type II secretion pathway or the main terminal branch of the general secretion pathway, as it has also been referred to, is widely distributed among Proteobacteria, in which it is responsible for the extracellular secretion of toxins and hydrolytic enzymes, many of which contribute to pathogenesis in both plants and animals. Secretion through this pathway differs from most other membrane transport systems, in that its substrates consist of folded proteins. The type II secretion apparatus is composed of at least 12 different gene products that are thought to form a multiprotein complex, which spans the periplasmic compartment and is specifically required for translocation of the secreted proteins across the outer membrane. This pathway shares many features with the type IV pilus biogenesis system, including the ability to assemble a pilus‐like structure. This review discusses recent findings on the organization of the secretion apparatus and the role of its various components in secretion. Different models for pilus‐mediated secretion through the gated pore in the outer membrane are also presented, as are the possible properties that determine whether a protein is recognized and secreted by the type II pathway.
Regulation of catalase–peroxidase (KatG) expression, isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosis
Authors: Pym Alexander S. , Domenech Pilar , Honoré Nadine , Song Jian , Deretic Vojo , Cole Stewart T.
Published: March 2003

Mycobacterium tuberculosis has two genes for ferric uptake regulator orthologues, one of which, furA, is situated immediately upstream of katG encoding catalase–peroxidase, a major virulence factor that also activates the prodrug isoniazid. This association suggested that furA might regulate katG and other genes involved in pathogenesis. Transcript mapping showed katG to be expressed from a strong promoter, with consensus −10 and −35 elements, preceding furA. No promoter activity was demonstrated downstream of the furA start codon, using different gene reporter systems, indicating that furA and katG are co‐transcribed from a common regulatory region. The respective roles of these two genes in the isoniazid susceptibility and virulence of M. tuberculosis were assessed by combinatorial complementation of a Δ(furA–katG) strain that is heavily attenuated in a mouse model of tuberculosis. In the absence of furA, katG was upregulated, cells became hypersensitive to isoniazid, and full virulence was restored, indicating that furA regulates the transcription of both genes. When furA alone was introduced into the Δ(furA–katG) mutant, survival in mouse lungs was moderately increased, suggesting that FurA could regulate genes, other than katG, that are involved in pathogenesis. These do not include the oxidative stress genes ahpC and sodA, or those for siderophore production.
Co‐ordinate regulation of the Escherichia coli cell cycle or The cloud of unknowing
Authors: Donachie William D.
Published: March 2003

A discussion of some aspects of the regulation of chromosome replication, segregation and cell division in Escherichia coli.
Mutational analysis of the high‐affinity BvgA binding site in the fha promoter of Bordetella pertussis
Authors: Boucher Philip E. , Yang Mei‐Shin , Stibitz Scott
Published: March 2003

In order to define a consensus binding sequence for the response regulator BvgA, we have undertaken a systematic analysis of contributions made by each nucleotide within the heptad half‐sites that are present in an inverted orientation at the promoter for the fha operon. Using in vitro binding assays, we examined the full complement of 21 single point mutations symmetrically arranged in this heptad repeat. Both gel shift and nitrocellulose filter‐binding assays provided evidence that nucleotides at positions 3 (thymidine), 4 (cytosine) and 7 (adenine) in the binding heptad contribute substantially to sequence‐specific recognition by BvgA. Furthermore, a T to A conversion at position 6 reduced binding. Selected binding site mutations were introduced into a modified fha promoter and examined for their effects on BvgA activation of promoter activity in vivo. Only those substitutions most severely affecting binding in vitro affected promoter activity in vivo. The in vivo effects of substitutions that had a significant effect on binding in vitro but did not severely affect in vivo promoter activity under standard culture conditions could be detected in vivo either in combination with additional substitutions or from their effect on the sensitivity of the mutant promoters to modulation by magnesium sulphate.
Signalling substitutions in the periplasmic domain of chemoreceptor Trg induce or reduce helical sliding in the transmembrane domain
Authors: Beel Bryan D. , Hazelbauer Gerald L.
Published: March 2003

We used in vivo oxidative cross‐linking of engineered cysteine pairs to assess conformational changes in the four‐helix transmembrane domain of chemoreceptor Trg. Extending previous work, we searched for and found a fourth cross‐linking pair that spanned the intrasubunit interface between transmembrane helix 1 (TM1) and its partner TM2. We determined the effects of ligand occupancy on cross‐linking rate constants for all four TM1–TM2 diagnostic pairs in conditions that allowed the formation of receptor–kinase complexes for the entire cellular complement of Trg. Occupancy altered all four rates in a pattern that implicated sliding of TM2 relative to TM1 towards the cytoplasm as the transmembrane signalling movement in receptor–kinase complexes. Transmembrane signalling can be reduced or induced by single amino acid substitutions in the ligand‐binding region of the periplasmic domain of Trg. We determined the effects of these substitutions on conformation in the transmembrane domain and on ligand‐induced changes using the diagnostic TM1–TM2 cysteine pairs. Effects on rates of in vivo cross‐linking showed that induced signalling substitutions altered the relative positions of TM1 and TM2 in the same way as ligand binding, and reduced signalling substitutions blocked or attenuated the ligand‐induced shift. These results provide strong support for the helical sliding model of transmembrane signalling.
degS (hhoB) is an essential Escherichia coli gene whose indispensable function is to provide σE activity
Authors: Alba Benjamin M. , Zhong Hong Ji , Pelayo Juan Carlos , Gross Carol A.
Published: March 2003

DegS (HhoB), a putative serine protease related to DegP/HtrA, regulates the basal and induced activity of the essential Escherichia coli sigma factor σE, which is involved in the cellular response to extracytoplasmic stress. DegS promotes the destabilization of the σE‐specific anti‐sigma factor RseA, thereby releasing σE to direct gene expression. We demonstrate that degS is an essential E. coli gene and show that the essential function of DegS is to provide the cell with σE activity. We also show that the putative active site of DegS is periplasmic and that DegS requires its N‐terminal transmembrane domain for its σE‐related function.
Environmental control of invasin expression in Yersinia pseudotuberculosis is mediated by regulation of RovA, a transcriptional activator of the SlyA/Hor family
Authors: Nagel Geraldine , Lahrz Angela , Dersch Petra
Published: March 2003

Invasin is the primary invasive factor of Yersinia pseudotuberculosis that allows efficient internalization into eukaryotic cells. We investigated invasin expression and found that the inv gene is regulated in response to a variety of environmental signals, such as temperature, growth phase, nutrients, osmolarity and pH, and requires the product of rovA, a member of the SlyA/Hor transcriptional activator family. The rovA gene was found by a genetic complementation strategy that restores temperature regulation of an unexpressed inv–phoA fusion in Escherichia coli K‐12. RovA plays a role in the invasion of Y. pseudotuberculosis into mammalian cells and mediates the regulation of invasin in response to all environmental signals analysed. Deletion analysis of the inv promoter region revealed a DNA segment extending 207 bp upstream of the transcriptional start site, which is required for maximal RovA‐induced inv transcription. Gel retardation assays showed that RovA interacts preferentially with this promoter fragment and suggested two potential RovA binding sites. Studies with chromosomal gene fusions also demonstrated that rovA follows the same pattern of regulation as invasin, indicating that environmental control of inv expression is mainly mediated by the control of RovA synthesis. Furthermore, we showed that a rovA–lacZ fusion is only slightly expressed in a rovA mutant strain, indicating that a positive autoregulatory mechanism is also involved in rovA expression.
A bacterial linear motor: cellular and molecular organization of the contractile cytoskeleton of the helical bacterium Spiroplasma melliferum BC3
Authors: Trachtenberg Shlomo , Gilad Rami
Published: March 2003

The Mollicutes (Mycoplasma, Acholeplasma, and Spiroplasma) are the smallest, simplest and most primitive free‐living and self‐replicating known cells. These bacteria have evolved from Clostridia by regressive evolution and genome reduction to the range of 5.8 × 105−2.2 × 106 basepairs (bp). Structurally, the Mollicutes completely lack cell walls and are enveloped by only a cholesterol containing cell membrane. The Mollicutes contain what can be defined as a bacterial cytoskeleton. The Spiroplasmas are unique in having a well‐defined, dynamic, helical cell geometry and a flat, monolayered, membrane‐bound cytoskeleton, which follows, intracellularly, the shortest helical line on the cellular coil. By applying cryo‐electron‐microscopy to whole cells, isolated cytoskeletons and cytoskeletal fibrils and subunits, as well as by selective extraction of cellular components, we determined, at a resolution of ∼25 Å, the cellular and molecular organization of the cytoskeleton. The cytoskeleton is assembled from a 59 kDa protein. The 59 kDa protein, has an equivalent sphere diameter of ∼50 Å. Given the ∼100 Å axial and lateral spacings in the cytoskeletal ribbons and the near‐circular shape of the subunit, we suggest that the subunit is a tetramer of 59 kDa monomers; the tetramers assemble further into flat fibrils, seven of which form a flat, monolayered, well‐ordered ribbon. The cytoskeleton may function as a linear motor by differential and coordinated length‐changes of the fibrils driven by conformational changes of the tetrameric subunits, the shape of which changes from near circular to elliptical. The cytoskeleton controls both the dynamic helical shape and the consequent motility of the cell. A stable cluster of proteins co‐purifies with the cytoskeleton. These apparent membrane and membrane‐associated proteins may function as anchor proteins.
Identification of regions of Ail required for the invasion and serum resistance phenotypes
Authors: Miller Virginia L. , Beer Karen B. , Heusipp Gerhard , Young Briana M. , Wachtel Marian R.
Published: March 2003

Yersinia enterocolitica is an enteric pathogen that has served as a model system for the study of microbial pathogenesis. Numerous virulence gene have been identified both on the virulence plasmid and on the chromosome. One of the chromosomal genes that is highly correlated with virulence is ail, a gene identified along with inv in a screen for Y. enterocolitica genes that could confer an invasive phenotype to Escherichia coli. Ail also promotes serum resistance in both E. coli and Y. enterocolitica. Several virulence factors homologous to Ail have been identified in other pathogens, yet very little is known about what constitutes the functional domain(s) of these proteins. Proteins in this family are predicted to consist of eight transmembrane β‐sheets and four cell surface‐exposed loops. We constructed and characterized a number of insertion, deletion and point mutations in the regions of ail predicted to encode the cell surface loops. The results from the analysis of these mutants indicate that cell surface loops one and four do not directly promote invasion or serum resistance, whereas mutations in loop three appear to modulate both phenotypes. Analysis of mutations in loop 2 suggests that this surface‐exposed loop contains sequences required for serum resistance and invasion. In addition, a peptide derived from the sequence of loop 2 was able specifically to inhibit Ail‐mediated invasion in a dose‐dependent manner. These results suggest that Ail directly promotes invasion and that loop 2 contains an active site, perhaps a receptor‐binding domain. Analyses of the mutations also suggest that the serum resistance and invasion phenotypes may be separable, because there are numerous mutations that affect one phenotype but not the other.
A chimeric activator of transcription that uses two  DNA‐binding domains to make simultaneous contact with pairs of recognition sites
Authors: Langdon Robert C. , Burr Tom , Pagan‐Westphal Sylvia , Hochschild Ann
Published: March 2003

Many well‐known transcriptional regulatory proteins are composed of at least two independently folding domains and, typically, only one of these is a DNA‐binding domain. However, some transcriptional regulators have been described that have more than one DNA‐binding domain. Regulators with a single DNA‐binding domain often bind co‐operatively to the DNA in homotypic or heterotypic combinations, and two or more DNA‐binding domains of a single regulatory protein can also bind co‐operatively to suitably positioned recognition sequences. Here, we examine the behaviour of a chimeric activator of transcription with two different DNA‐binding domains, that of the bacteriophage λ cI protein and that of the Escherichia coli cyclic AMP receptor protein. We show that these two DNA‐binding moieties, when present in the same molecule, can bind co‐operatively to a pair of cognate recognition sites located upstream of a test promoter, thereby permitting the chimera to function as a particularly strong activator of transcription from this promoter. Our results show how such a bivalent DNA‐binding protein can be used to regulate transcription differentially from promoters that bear either one or both recognition sites.
Glycine betaine loses its osmoprotective activity in a bspA strain of Erwinia chrysanthemi
Authors: Touzé Thierry , Gouesbet Gwenola , Boiangiu Clara , Jebbar Mohamed , Bonnassie Sylvie , Blanco Carlos
Published: March 2003

Erwinia chrysanthemi insertion mutants were isolated that grew poorly specifically in the presence of glycine betaine (GB) or its analogues in high‐salt media. Transposon insertions were found to affect the bspA gene, which forms an operon including the psd locus coding for phosphatidylserine decarboxylase. Initial GB uptake is not affected by the bspA mutation. However, in high‐salt medium, its initial accumulation is followed by a reduced glucose uptake and a release of GB but not a loss of viability. BspA is homologous to the widespread MscS channel, YggB, but does not seem to constitute a mechanosensitive channel. We suggest that BspA is a protein sensing both intracellular GB and the extracellular salt content of the medium, the hypothesis being built on the observation that BspA is necessary to maintain the GB pool during osmoadaptation in high‐salt media containing this osmoprotectant.
Genetic evidence for a microtubule‐destabilizing  effect of conventional kinesin and analysis of its consequences for the control of nuclear distribution  in Aspergillus nidulans
Authors: Requena Natalia , Alberti‐Segui Cristina , Winzenburg Else , Horn Christian , Schliwa Manfred , Philippsen Peter , Liese Ralf , Fischer Reinhard
Published: March 2003

Conventional kinesin is a microtubule‐dependent motor protein believed to be involved in a variety of intracellular transport processes. In filamentous fungi, conventional kinesin has been implicated in different processes, such as vesicle migration, polarized growth, nuclear distribution, mitochondrial movement and vacuole formation. To gain further insights into the functions of this kinesin motor, we identified and characterized the conventional kinesin gene, kinA, of the established model organism Aspergillus nidulans. Disruption of the gene leads to a reduced growth rate and a nuclear positioning defect, resulting in nuclear cluster formation. These clusters are mobile and display a dynamic behaviour. The mutant phenotypes are pronounced at 37°C, but rescued at 25°C. The hyphal growth rate at 25°C was even higher than that of the wild type at the same temperature. In addition, kinesin‐deficient strains were less sensitive to the microtubule destabilizing drug benomyl, and disruption of conventional kinesin suppressed the cold sensitivity of an α‐tubulin mutation (tubA4). These results suggest that conventional kinesin of A. nidulans plays a role in cytoskeletal dynamics, by destabilizing microtubules. This new role of conventional kinesin in microtubule stability could explain the various phenotypes observed in different fungi.
The integrative element pSAM2 from Streptomyces: kinetics and mode of conjugal transfer
Authors: Possoz Christophe , Ribard Carin , Gagnat Josette , Pernodet Jean‐Luc , Guérineau Michel
Published: March 2003

pSAM2 is an 11 kb integrative element from Streptomyces ambofaciens that is capable of conjugal transfer. A system based on differential DNA modification by SalI methyltransferase was used to localize pSAM2 in the donor or recipient strain, and thus to determine the various steps associated with transfer. Initiation (i.e. excision and replication of pSAM2 in the donor) occurs a few hours after mating with a recipient strain. pSAM2 replicates in the recipient strain, spreads within the mycelium and then integrates into the chromosome. Transfer generally involves single‐stranded DNA. In Streptomyces, only a few genes, such as traSA for pSAM2, are required for conjugal transfer. Using the differential sensitivity to the SalI restriction–modification system of transfers involving single‐ and double‐stranded DNA, we found that pSAM2 was probably transferred to the recipient as double‐stranded DNA. This provides the first experimental evidence for the transfer of double‐stranded DNA during bacterial conjugation. Thus, TraSA, involved in pSAM2 transfer, and SpoIIIE, which is involved in chromosome partitioning in Bacillus subtilis, display similarities in both sequence and function: both seem to transport double‐stranded DNA actively, either from donor to recipient or from mother cell to prespore.
DnaB, DnaD and DnaI proteins are components of the Bacillus subtilis replication restart primosome
Authors: Bruand Claude , Farache Michaël , McGovern Stephen , Ehrlich S. Dusko , Polard Patrice
Published: March 2003

Phenotypes of Bacillus subtilis priA mutants suggest that they are deficient in the restart of stalled chromosomal replication forks. The presumed activity of PriA in the restart process is to promote the assembly of a multiprotein complex, the primosome, which functions to recruit the replication fork helicase onto the DNA. We have proposed previously that three proteins involved in the initiation of replication at oriC in B. subtilis, DnaB, DnaD and DnaI, are components of the PriA primosome in this bacterium. However, the involvement of these proteins in replication restart has not yet been studied. Here, we describe dnaB mutations that suppress the phenotypes of B. subtilis priA mutants. In a representative mutant, the DnaC helicase is loaded onto single‐stranded DNA in a PriA‐independent, DnaD‐ and DnaI‐dependent manner. These observations confirm that DnaB, DnaD and DnaI are primosomal proteins in B. subtilis. Moreover, their involvement in the suppression of priA phenotypes shows that they participate in replication fork restart in B. subtilis.
Protected environments allow parallel evolution of a bacterial pathogen in a patient subjected to long‐term antibiotic therapy
Authors: Low Alison S. , MacKenzie Fiona M. , Gould Ian M. , Booth Ian R.
Published: March 2003

Long‐term antibiotic treatment offers a rare opportunity to study the evolution of bacteria within the same individual. The appearance of new variants has been suggested to take place via the selection of enhanced resistance in compartments of the body in which the antibiotic concentration is low. Laboratory models of protected compartments have elegantly demonstrated their potential in selecting novel variants. However, comparable data from patients have been rare. In this study, extended antibiotic therapy in a single patient suffering from multiple infected liver cysts has provided the opportunity to observe and analyse the molecular evolution of antibiotic resistance. Each isolate has the same basic ompC gene sequence that is distinct from other Escherichia coli isolates, which suggests that they derive from the same founder population. However, the isolates differ in their auxotrophic markers, in the pI values of their dominant β‐lactamase activities and in the mutations in the promoter region of the ampC gene leading to increased expression of the AmpC enzyme. The data provide strong evidence for a single focal infection expanding via parallel pathways of evolution to give a range of antibiotic‐resistant isolates. These data suggest that the infected cysts provide numerous protected environments that are the foci for the separate development of distinct variants.
Arylamine N‐acetyltransferase of Mycobacterium tuberculosis is a polymorphic enzyme and a site of isoniazid metabolism
Authors: Upton A. M. , Mushtaq A. , Victor T. C. , Sampson S. L. , Sandy J. , Smith D‐M. , Van Helden P. V. , Sim E.
Published: March 2003

Arylamine N‐acetyltransferases (NATs; E.C 2.3.1.5) N‐acetylate arylhydralazine and arylamine substrates using acetyl coenzyme A. Human NAT2 acetylates and inactivates the antituberculosis drug, isoniazid (INH), and is polymorphic. We previously demonstrated that there is a homologue of human NAT2 in Mycobacterium tuberculosis, whose product N‐acetylates INH in vitro. We now demonstrate that the nat gene is expressed in M. tuberculosis and M. bovis Bacille Calmette‐Guerin (BCG), using reverse transcription–polymerase chain reaction and Western blotting. The NAT protein is active in M. bovis BCG in vivo, as detected by the presence of N‐acetyl INH in M. bovis BCG lysates grown in INH. Sequence analysis of the M. tuberculosis nat coding region reveals a single nucleotide polymorphism in 18% of a random cohort of M. tuberculosis clinical isolates, conferring a G to R change. The recombinant mutant protein appears less stable than the wild type, and has an apparent affinity for INH of 10‐fold less than the wild type. Modelling the change in M. tuberculosis NAT shows that the G to R change is close to the active site, and supports the experimental findings. Minimum inhibitory concentration data suggest that this polymorphism in nat is linked to low‐level changes in the INH susceptibility of M. tuberculosis clinical isolates.
Ras links cellular morphogenesis to virulence by regulation of the MAP kinase and cAMP signalling pathways in the pathogenic fungus Candida albicans
Authors: Leberer Ekkehard , Harcus Doreen , Dignard Daniel , Johnson Lyne , Ushinsky Sophia , Thomas David Y. , Schröppel Klaus
Published: March 2003

The pathogenic fungus Candida albicans is capable of responding to a wide variety of environmental cues with a morphological transition from a budding yeast to a polarized filamentous form. We demonstrate that the Ras homologue of C. albicans, CaRas1p, is required for this morphological transition and thereby contributes to the development of pathogenicity. However, CaRas1p is not required for cellular viability. Deletion of both alleles of the CaRAS1 gene caused in vitro defects in morphological transition that were reversed by either supplementing the growth media with cAMP or overexpressing components of the filament‐inducing mitogen‐activated protein (MAP) kinase cascade. The induction of filament‐specific secreted aspartyl proteinases encoded by the SAP4–6 genes was blocked in the mutant cells. The defects in filament formation were also observed in situ after phagocytosis of C. albicans cells in a macrophage cell culture assay and, in vivo, after infection of kidneys in a mouse model for systemic candidiasis. In the macrophage assay, the mutant cells were less resistant to phagocytosis. Moreover, the defects in filament formation were associated with reduced virulence in the mouse model. These results indicate that, in response to environmental cues, CaRas1p is required for the regulation of both a MAP kinase signalling pathway and a cAMP signalling pathway. CaRas1p‐dependent activation of these pathways contributes to the pathogenicity of C. albicans cells through the induction of polarized morphogenesis. These findings elucidate a new medically relevant role for Ras in cellular morphogenesis and virulence in an important human infectious disease.
Pairing of P1 plasmid partition sites by ParB
Authors: Edgar Rotem , Chattoraj Dhruba K. , Yarmolinsky Michael
Published: March 2003

The mechanisms by which bacterial plasmids and chromosomes are partitioned are largely obscure, but it has long been assumed that the molecules to be separated are initially paired, as are sister chromatids in mitosis. We offer in vivo evidence that the partition protein ParB encoded by the bacterial plasmid P1 can pair cis‐acting partition sites of P1 inserted in a small, multicopy plasmid. ParB was shown previously to be capable of extensive spreading along DNA flanking the partition sites. Experiments in which ParB spreading was constrained by physical roadblocks suggest that extensive spreading is not required for the pairing process.
The polyphosphate kinase plays a negative role in the control of antibiotic production in Streptomyces lividans
Authors: Chouayekh Hichem , Virolle Marie‐Joelle
Published: March 2003

The polyphosphate kinase gene (ppk) from Streptomyces lividans, which encodes a 774‐amino‐acid protein (86.4 kDa) showing extensive homology to other bacterial polyphosphate kinases, was cloned by polymerase chain reaction (PCR) using oligonucleotides derived from the putative ppk gene from the closely related species, Streptomyces coelicolor. In vitro, the purified Ppk was shown to be able to synthesize the polyphosphate [poly(P)] from ATP (forward reaction) as well as to regenerate ATP from the poly(P) in the presence of an excess of ADP (reverse reaction). In conditions of poly(P) synthesis, a phosphoenzyme intermediate was detected, indicating an autophosphorylation of the enzyme in the presence of ATP. The ppk gene was shown to be transcribed as a monocistronic mRNA from a unique promoter. Its transcription was only detectable during the late stages of growth in liquid minimal medium. A mutant strain interrupted for ppk was characterized by increased production of the antibiotic actinorhodin on rich R2YE solid medium (0.37 mM KH2PO4 added). This production was enhanced on the same medium with no KH2PO4 added but was completely abolished by the addition of 1.48 mM KH2PO4. In the ppk mutant strain, this increased production correlated with enhanced transcription of actII‐ORF4 encoding the specific activator of the actinorhodin pathway. In that strain, the transcription of redD and cdaR, encoding the specific activators of the undecylprodigiosin and calcium‐dependent antibiotic biosynthetic pathways, respectively, was also increased but to a lesser extent. The enhanced expression of these regulators did not seem to be related to increased relA‐dependent ppGpp synthesis, as no obvious increase in relA expression was observed in the ppk mutant strain. These results suggested that the negative regulatory effect exerted by Ppk on antibiotic biosynthesis was most probably caused by the repression exerted by the endogenous Pi, resulting from the hydrolysis of the poly(P) synthesized by Ppk, on the expression of the specific activators of the antibiotic biosynthetic pathways.
The Fap1 fimbrial adhesin is a glycoprotein: antibodies specific for the glycan moiety block the adhesion of Streptococcus parasanguis in an in vitro tooth model
Authors: Stephenson Aimee E. , Wu Hui , Novak Jan , Tomana Milan , Mintz Keith , Fives‐Taylor Paula
Published: March 2003

Streptococcus parasanguis is a primary colonizer of the tooth surface and
plays a pivotal role in the formation of dental plaque. The fimbriae of S. parasanguis
are important in mediating adhesion to saliva‐coated hydroxylapatite (SHA), an in
vitro tooth adhesion model. The Fap1 adhesin has been identified as the major
fimbrial subunit, and recent studies suggest that Fap1 is a glycoprotein. Monosaccharide
analysis of Fap1 purified from the culture supernatant of S. parasanguis indicated
the presence of rhamnose, glucose, galactose, N‐acetylglucosamine and N‐acetylgalactosamine.
A glycopeptide moiety was isolated from a pronase digest of Fap1 and purified by
immunoaffinity chromatography. The monosaccharide composition of the purified glycopeptide
was similar to that of the intact molecule. The functionality of the glycan moiety
was determined using monoclonal antibodies (MAbs) specific for the intact Fap1 glycoprotein.
These antibodies were grouped into two categories based on their ability to block
adhesion of S. parasanguis to SHA and their corresponding specificity for
either protein or glycan epitopes of the Fap1 protein. ‘Non‐blocking’
MAb epitopes were mapped to unique protein sequences in the N‐terminus of the Fap1
protein using non‐glycosylated recombinant Fap1 proteins (rFap1 and drFap1) expressed
in Escherichia coli. In contrast, the ‘blocking’ antibodies
did not bind to the recombinant Fap1 proteins, and were effectively competed by the
binding to the purified glycopeptide. These data suggest that the ‘blocking’
antibodies are specific for the glycan moiety and that the adhesion of S. parasanguis is mediated by sugar residues associated with Fap1.
Regulation of the Bacillus subtilis ccpC gene by CcpA and CcpC
Authors: Kim Hyun‐Jin , Jourlin‐Castelli Cécile , Kim Sam‐In , Sonenshein Abraham L.
Published: March 2003

Bacillus subtilis CcpC, a LysR‐type transcriptional regulator, represses the transcription of genes for citrate synthase (citZ) and aconitase (citB) in response to citrate availability. Transcription of ccpC was shown to initiate at two promoters, P1, located just upstream of the ccpC gene, and P2, located within or upstream of the neighbouring ykuL gene. Expression from the ccpC‐specific promoter (P1) was negatively regulated by CcpC but independent of the carbon source in the medium. Gel shift and DNase I footprinting experiments revealed that CcpC binds to an interrupted dyad sequence that surrounds the ccpC transcriptional start point. Transcription of ccpC from the upstream promoter (P2) was repressed by glucose in a CcpA‐dependent manner. A putative CcpA binding site (cre) was identified upstream of the –35 region of the P1 promoter. Transcriptional fusion studies demonstrated that glucose repression of ccpC expression from the P2 promoter depends on this cre site. In addition, DNase I footprinting experiments showed that CcpA specifically binds to this cre site and that the introduction of mutations (cre*) into this site abolished the binding. These results suggest that CcpA may control CcpC syn‐thesis by acting as a road‐block to readthrough transcription from the P2 promoter.
Differential roles of two SARP‐encoding regulatory genes during tylosin biosynthesis
Authors: Bate Neil , Stratigopoulos George , Cundliffe Eric
Published: March 2003

The tylosin biosynthetic gene cluster of Streptomyces fradiae is remarkable in harbouring at least five regulatory genes, two of which (tylS and tylT) encode proteins of the Streptomyces antibiotic regulatory protein (SARP) family. The aim of the present work was to assess the respective contributions of TylS and TylT to tylosin production. A combination of targeted gene disruption, fermentation studies and gene expression analysis via reverse transcriptase–polymerase chain reaction (RT–PCR) suggests that tylS is essential for tylosin production and controls the expression of tylR (previously shown to be a global activator of the biosynthetic pathway) plus at least one other gene involved in polyketide metabolism or regulation thereof. This is the first demonstration of a SARP acting to control another regulatory gene during antibiotic biosynthesis. In contrast, tylT is not essential for tylosin production.
A novel type II secretion system in Pseudomonas aeruginosa
Authors: Ball Geneviève , Durand Éric , Lazdunski Andrée , Filloux Alain
Published: March 2003

The genome sequence of Pseudomonas aeruginosa strain PAO1 has been determined to facilitate post‐genomic studies aimed at understanding the capacity of adaptation of this ubiquitous opportunistic pathogen. P. aeruginosa produces toxins and hydrolytic enzymes that are secreted via the type II secretory pathway using the Xcp machinery or ‘secreton’. In this study, we characterized a novel gene cluster, called hxc for homologous to xcp. Characterization of an hxcR mutant, grown in phosphate‐limiting medium, revealed the absence of a 40 kDa protein found in the culture supernatant of wild‐type or xcp derivative mutant strains. The protein corresponded to the alkaline phosphatase L‐AP, renamed LapA, which is secreted in an xcp‐independent but hxc‐dependent manner. Finally, we showed that expression of the hxc gene cluster is under phosphate regulation. This is the first report of the exist‐ence of two functional type II secretory pathways within the same organism, which could be related to the high adaptation potential of P. aeruginosa.
OmpR‐dependent and OmpR‐independent responses of Escherichia coli to sublethal attack by the neutrophil bactericidal/permeability increasing protein
Authors: Prohinar Polonca , Forst Steve A. , Reed Deoine , Mandic‐Mulec Ines , Weiss Jerrold
Published: March 2003

Bactericidal/permeability‐increasing protein (BPI) of neutrophils is a lipopolysaccharide (LPS)‐binding antibacterial protein with specificity for Gramnegative bacteria. BPI binding to the bacterial surface rapidly triggers potentially reversible bacterial growth inhibition and alterations of the outer membrane and, later, disruption of the inner membrane and lethal injury. Initial effects include selective OmpR‐dependent changes in the synthesis of outer membrane porins (OmpF and OmpC). Because OmpR is a global transcriptional regulator, we have examined its possible role in responses of E. coli to sublethal injury caused by BPI. Early (<15 min) reversible

effects of BPI on bacterial colony‐forming ability and outer membrane permeability

were virtually identical in isogenic wild‐type (wt) and ompR−E. coli. Both strains could repair the outer membrane permeability barrier

after Mg2+‐induced displacement of bound BPI. However, OmpR was

essential for the ability of E. coli to tolerate low doses of BPI and escape

the progression of sublethal to lethal damage. Scanning electron microscopy revealed

that BPI treatment produced greater membrane perturbations in the ompR−

strain, apparent even before lethal injury. These findings suggest that the fate

of E. coli exposed to BPI depends on both OmpR‐independent mechanisms engaged in outer membrane repair and OmpR‐ dependent processes that modulate porin synthesis and retard progression of injury from the outer to the inner membrane.
Transcytosis of iota‐toxin across polarized CaCo‐2 cells
Authors: Richard Jean François , Mainguy Gael , Gibert Maryse , Marvaud Jean Christophe , Stiles Bradley G. , Popoff Michel R.
Published: March 2003

Iota‐toxin from Clostridium perfringens type E is a binary toxin consisting of two independent proteins, an enzymatic Ia and binding Ib component. Ia catalyses ADP‐ribosylation of actin monomers, thus disrupting the actin cytoskeleton. In this report, we show that Ia plus Ib applied apically or basolaterally induce a rapid decrease in the transepithelial resistance (TER) of CaCo‐2 cell monolayers and disorganization of actin filaments as well as the tight and adherens junctions. Ib alone, on the apical or basolateral side, slowly decreased the TER without affecting the actin cytoskeleton, possibly via pore formation. Interestingly, the two iota‐toxin components inoculated separately on each cell surface induced cytopathic effects and a TER decrease. Anti‐Ib sera, raised against the whole molecule or the Ia docking domain and applied to the opposite cell side versus Ib, neutralized the TER decrease. In addition, radioactive Ib incubated in the basolateral compartment was detected on the apical side by selective cell surface biotinylation. This argues for a transcytotic routing of Ib to mediate internalization of Ia from the opposite cell surface. Bafilomycin A1 also prevented the cytopathic effects of Ia and Ib applied separately to each cell side, possibly by blocking translocation of Ia into the cytosol and/or the intracellular transport of Ib. Ib is either routed into the cell independently of Ia, trans‐cytosed and permanently exposed on the opposite cell surface or continuously recycled between an endosomal compartment and the cell surface.
Control of chitin synthesis through Shc1p, a functional homologue of Chs4p specifically induced during sporulation
Authors: Sanz Maria , Trilla Ose Angel , Duran Ngel , Roncero Cesar
Published: March 2003

The Saccharomyces cerevisiae SHC1 gene encodes a protein with a high homology

to Chs4p, a positive regulator of chitin synthase III (CSIII) during vegetative growth.

SHC1 is not expressed during vegetative growth but is strongly induced during

sporulation as a mid‐late gene. shc1/shc1 mutants do not show any defect in

the total rate of sporulation and meiosis occurs normally. However, shc1/shc1

ascospores be‐come highly permeable to DAPI, much more sensitive to glusulase treatment,

and have very low levels of chitosan in their cell walls. All these observations

indicate that Shc1p is required for proper maturation of the ascospore through its

participation in the synthesis of the chitosan layer. Lack of SHC1 during

sporulation can be partially compensated by over‐expression of the CHS4 gene.

During vegetative growth, SHC1 has no apparent function but, when ectopically

overexpressed, it can substitute Chs4p as an activator of the CSIII activity; however,

Shc1p fails to localize it properly, as Chs4p does. In conclusion, S. cerevisiae

contains two functionally redundant genes in the control of CSIII activity: CHS4,

whose function is restricted to vegetative growth because Chs4p is rapidly degraded

during sporulation, and SHC1, whose function in cell wall ascospore assembly is transcriptionally restricted to the sporulation process.
Polar location and functional domains of the Agrobacterium tumefaciens DNA transfer protein VirD4
Authors: Kumar Renu B. , Das Anath
Published: March 2003

Agrobacterium tumefaciens VirD4 is essential for DNA transfer to plants.

VirD4 presumably functions as a coupling factor that facilitates communication between

a substrate and the transport pore. To serve as a coupling protein, VirD4 may be

required to localize near the transport apparatus. In a previous study, we observed

that several constituents of the transport apparatus localize to the cell membranes.

In this study, we demonstrate that VirD4 has a unique cellular location. In immunofluorescence

microscopy, cells probed with anti‐VirD4 antibodies had foci of fluorescence primarily

at the cell poles, indicating that VirD4 localizes to the cell pole. Polar location

of VirD4 was not dependent on T‐DNA processing, the formation of the transport apparatus

and the presence of other Vir proteins. VirD4 is an integral membrane protein with

one periplasmic domain. The large cytoplasmic region contains a nucleotide‐binding

domain. To investigate the role of these domains in DNA transfer, we introduced mutations

in virD4 and studied the effect of a mutation on substrate transfer. A deletion

of most of the periplasmic domain as well as the alterations of glycine 151 to serine

and lysine 152 to alanine led to the complete loss of DNA transfer, indicating that

both domains are essential for substrate transfer. Subcellular localization of the

mutant proteins indicated that both the periplasmic and the nucleotide‐binding domains

are required for polar localization of VirD4. The periplasmic domain mutant VirD4Δ36–61

was distributed throughout the cell membrane, whereas the nucleotide binding site

mutant VirD4G151S localized to sites other than the cell poles. Polar location of VirD4 suggests a role for the cell pole in DNA transfer.
The flagella of enteropathogenicEscherichia colimediate adherence to epithelial cells
Authors: Girón Jorge A. , Torres Alfredo G. , Freer Enrique , Kaper James B.
Published: March 2003

EnteropathogenicEscherichia coli(EPEC) utilizes a type III protein secretion system to target effector molecules into the host cell leading to effacement of the intestinal mucosa. This secretion apparatus shares many structural features of the flagellar type III export system involved in flagella assembly and motility. We report here thatfliCinsertional mutants constructed in two wild‐type EPEC strains were markedly impaired in adherence and microcolony formation on cultured cells. AnE. coliK‐12 strain harbouring the EPEC H6fliCgene on a plasmid showed discrete adhering clusters on HeLa cells, albeit to less extent than the wild‐type EPEC strain. Flagella purified from EPEC bound to cultured epithelial cells and antiflagella antibodies blocked adherence of several EPEC serotypes. We determined that eukaryotic cells in culture stimulate expression of flagella by motile and non‐motile EPEC. Isogenic strains mutated inperA(a transcriptional activator),bfpA(a type IV pilin),luxS(a quorum‐sensing autoinducer gene) and in the type III secretion genes were reduced for motility in Dulbecco’s modified Eagle medium (DMEM) motility agar and produced none or few flagella when associated with epithelial cells. Growth of these mutants in preconditioned tissue culture medium restored motility and their ability to produce flagella, suggesting the influence of a signal provided by mammalian cells that triggers flagella production. This study shows for the first time that the flagella of EPEC are directly involved in the adherence of these bacteria and supports the existence of a molecular relationship between the two existing type III secretion pathways of EPEC, the EPEC adherence factor (EAF) plasmid‐encoded regulator, quorum sensing and epithelial cells.
A NarX–Tar chimera mediates repellent chemotaxis to nitrate and nitrite
Authors: Ward Scott M. , Delgado Asuncion , Gunsalus Robert P. , Manson Michael D.
Published: March 2003

Membrane receptors communicate between the external world and the cell interior. In bacteria, these receptors include the transmembrane sensor kinases, which control gene expression via their cognate response regulators, and chemoreceptors, which control the direction of flagellar rotation via the CheA kinase and CheY response regulator. Here, we show that a chimeric protein that joins the ligand‐binding, transmembrane and linker domains of the NarX sensor kinase to the signalling and adaptation domains of the Tar chemoreceptor of Escherichia coli mediates repellent responses to nitrate and nitrite. Nitrate induces a stronger response than nitrite and is effective at lower concentrations, mirroring the relative sensitivity to these ligands exhibited by NarX itself. We conclude that the NarX–Tar hybrid functions as a bona fide chemoreceptor whose activity can be predicted from its component parts. This observation implies that ligand‐dependent activation of a sensor kinase and repellent‐initiated activation of receptor‐coupled CheA kinase involve a similar transmembrane signal.