bluuebunny/crossref_metadata_2025 huggingface dataset:
Metal ion homeostasis and intracellular parasitism
Authors: Agranoff Daniel D. , Krishna Sanjeev
Published: March 2003

Bacteria possess multiple mechanisms for the transport of metal ions. While many of these systems may have evolved in the first instance to resist the detrimental effects of toxic environmental heavy metals, they have since become adapted to a variety of important homeostatic functions. The ‘P’‐type ATPases playa key role in metal ion transport in bacteria. A Cu+‐ATPase from the intracellular bacterium Listeria monocytogenes is implicated in pathogenesis, and similar pumps in Mycobacterium tuberculosis and M. leprae may play a comparable role. Intracellular bacteria require transition metal cations for the synthesis of superoxide dismutases and catalases, which constitute an important line of defence against macrophage‐killing mechanisms. The macrophage protein Nramp1, which confers resistance to a variety of intracellular pathogens, has also been shown recently to be a divalent amphoteric cation transporter. Mycobacterial homologues have recently been identified by genomic analysis. These findings suggest a model in which competition for divalent cations plays a pivotal role in the interaction between host and parasite.
The hexA gene of Erwinia carotovora encodes a LysR homologue and regulates motility and the expression of multiple virulence determinants
Authors: Harris Stephen J. , Shih Yu‐Ling , Bentley Stephen D. , Salmond George P. C.
Published: March 2003

We have identified a gene important for the regulation of exoenzyme virulence factor synthesis in the plant pathogen Erwinia carotovora ssp. carotovora (Ecc) and virulence and motility in Erwinia carotovora ssp. atroseptica (Eca). This gene, hexA (hyperproduction of exoenzymes), is a close relative of the Erwinia chrysanthemi (Echr ) gene pecT and encodes a member of the LysR family of transcriptional regulators. hexA mutants in both Ecc and Eca produce abnormally high levels of the exoenzyme virulence factors pectate lyase, cellulase and protease. In addition, Eca hexA mutants show increased expression of the fliA and fliC genes and hypermotility. Consistent with a role as a global regulator, expression of hexA from even a low‐copy plasmid can suppress exoenzyme production in Ecc and Eca and motility in Eca. Production of the quorum‐sensing pheromone OHHL in Ecc hexA is higher throughout the growth curve compared with the wild‐type strain. Overexpression of Ecc hexA also caused widespread effects in several strains of the opportunistic human pathogen, Serratia. Low‐copy hexA expression resulted in repression of exoenzyme, pigment and antibiotic production and repression of the spreading phenotype. Finally, mutations in hexA were shown to increase Ecc or Eca virulence in planta.
Pilus‐mediated adhesion of Neisseria meningitidis: the essential role of cell contact‐dependent transcriptional upregulation of the PilC1 protein
Authors: Taha Muhamed‐Kheir , Morand Philippe C. , Pereira Yannick , Eugène Emmanuel , Giorgini Dario , Larribe Mireille , Nassif Xavier
Published: March 2003

Pilus‐mediated adherence makes an essential contribution to the pathogenesis of Neisseria meningitidis by allowing the initial localized adherence. Pili are assembled from a protein subunit called pilin. Two proteins, PilC1 and PilC2, are also key elements in the formation of pili as the production of at least one PilC protein is required for pilus assembly. In addition, PilC1 but not PilC2 modulates adhesiveness, most probably by being the adhesin. Recently, both genes have been demonstrated to be controlled by different promoters, pilC2 is expressed from a single transcription starting point (TSP), whereas pilC1 has three TSPs. One of these, PC1.1, corresponds to the unique TSP of pilC2, and two others, PC1.2 and PC1.3, are located in a region upstream of pilC1 but not pilC2. This suggests that both genes may be under the control of separate regulatory pathways. In this work, by engineering pilC1–lacZ and pilC2–lacZ transcriptional fusions, we provide evidence that expression of pilC1, but not that of pilC2, is transiently induced by bacterial cell contact. This induction required viable cells, did not need the presence of pili and relied on the expression of pilC1 from PC1.3. Destruction of this TSP by site‐directed mutagenesis did not significantly diminish the piliation level or the basal expression of PilC1, but led to the loss of cell contact‐dependent upregulation of pilC1 and to a dramatic decrease in bacterial adhesiveness. Taken together, these data demonstrate that cell contact‐dependent upregulation of the transcription of pilC1 at PC1.3 is essential for meningococcal pilus‐mediated adhesion.
A two‐component regulatory system playing a critical role in plant pathogens and endosymbionts is present in Brucella abortus and controls cell invasion and virulence
Authors: Sola‐Landa Alberto , Pizarro‐Cerdá Javier , Grilló María‐Jesús , Moreno Edgardo , Moriyón Ignacio , Blasco José‐María , Gorvel Jean‐Pierre , López‐Goñi Ignacio
Published: March 2003

Two mutants showing increased sensitivity to polycations and surfactants were obtained by transposon mutagenesis of virulent Brucella abortus 2308 Nalr. These mutants showed no obvious in vitro growth defects and produced smooth‐type lipopolysaccharides. However, they hardly multiplied or persisted in mouse spleens, displayed reduced invasiveness in macrophages and HeLa cells, lost the ability to inhibit lysosome fusion and were unable to replicate intracellularly. Subsequent DNA analyses identified a two‐component regulatory system [Brucella virulence related (Bvr)] with a regulatory (BvrR) and sensory (BvrS) protein. Cloning of bvrR in the BvrR‐deficient mutant restored the resistance to polycations and, in part, the invasiveness and the ability to multiply intracellularly. BvrR and BvrS were highly similar (87–89% and 70–80% respectively) to the regulatory and sensory proteins of the chromosomally encoded Rhizobium meliloti ChvI–ExoS and Agrobacterium tumefaciens ChvI–ChvG systems previously shown to be critical for endosymbiosis and pathogenicity in plants. Divergence among the three sensory proteins was located mostly within a periplasmic domain probably involved in stimulus sensing. As B. abortus, R. meliloti and A. tumefaciens are phylogenetically related, these observations suggest that these systems have a common ancestor that has evolved to sense stimuli in plant and animal microbial environments.
A Bacillus subtilis gene‐encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition
Authors: Moriya Shigeki , Tsujikawa Eitoku , Hassan Anwarul K. M. , Asai Kei , Kodama Takeko , Ogasawara Naotake
Published: March 2003

We have analysed the function of a gene of Bacillus subtilis, the product of which shows significant homology with eukaryotic SMC proteins essential for chromosome condensation and segregation. Two mutant strains were constructed; in one, the expression was under the control of the inducible spac promoter (conditional null) and, in the other, the gene was disrupted by insertion (disrupted null). Both could form colonies at 23°C but not at 37°C in the absence of the expression of the Smc protein, indicating that the B. subtilis smc gene was essential for cell growth at higher temperatures. Microscopic examination revealed the formation of anucleate and elongated cells and diffusion of nucleoids within the elongated cells in the disrupted null mutant grown at 23°C and in the conditional null mutant grown in low concentrations of IPTG at 37°C. In addition, immunofluorescence microscopy showed that subcellular localization of the Spo0J partition protein was irregular in the smc disrupted null mutant, compared with bipolar localization in wild‐type cells. These results indicate that the B. subtilis smc gene is essential for chromosome partition. The role of B. subtilis Smc protein in chromosome partition is discussed.
PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae
Authors: Wolfgang Matthew , Lauer Peter , Park Hae‐Sun , Brossay Laurent , Hébert Jacques , Koomey Michael
Published: March 2003

Neisseria gonorrhoeae, the Gram‐negative aetiological agent of gonorrhoea, is one of many mucosal pathogens of man that expresses competence for natural transformation. Expression of this phenotype by gonococci appears to rely on the expression of type IV pili (Tfp), but the mechanistic basis for this relationship remains unknown. During studies of gonococcal pilus biogenesis, a homologue of the PilT family of proteins, required for Tfp‐dependent twitching motility in Pseudomonas aeruginosa and social gliding motility in Myxococcus xanthus, was discovered. Like the findings in these other species, we show here that gonococcal pilT mutants constructed in vitro no longer display twitching motility. In addition, we demonstrate that they have concurrently lost the ability to undergo natural transformation, despite the expression of structurally and morphologically normal Tfp. These results were confirmed by the findings that two classes of spontaneous mutants that failed to express twitching motility and transformability carried mutations in pilT. Piliated pilT mutants and a panel of pilus assembly mutants were found to be deficient in sequence‐specific DNA uptake into the cell, the earliest demonstrable step in neisserial competence. The PilT‐deficient strains represent the first genetically defined mutants that are defective in DNA uptake but retain Tfp expression.
Exochelin genes in Mycobacterium smegmatis: identification of an ABC transporter and two non‐ribosomal peptide synthetase genes
Authors: Zhu Wenming , Arceneaux Jean E. L. , Beggs Marjorie L. , Byers B. Rowe , Eisenach Kathleen D. , Lundrigan Michael D.
Published: March 2003

Many strains of mycobacteria produce two ferric chelating substances that are termed exochelin (an excreted product) and mycobactin (a cell‐associated product). These agents may function as iron acquisition siderophores. To examine the genetics of the iron acquisition system in mycobacteria, ultraviolet (UV) and transposon (Tn611 ) mutagenesis techniques were used to generate exochelin‐deficient mutants of Mycobacterium smegmatis strains ATCC 607 and LR222 respectively. Mutants were identified on CAS siderophore detection agar plates. Comparisons of the amounts of CAS‐reactive material excreted by the possible mutant strains with that of the wild‐type strains verified that seven UV mutant strains and two confirmed transposition mutant strains were deficient in exochelin production. Cell‐associated mycobactin production in the mutants appeared to be normal. From the two transposon mutants, the mutated gene regions were cloned and identified by colony hybridization with an IS6100 probe, and the DNA regions flanking the transposon insertion sites were then used as probes to clone the wild‐type loci from M. smegmatis LR222 genomic DNA. Complementation assays showed that an 8 kb Pst I fragment and a 4.8 kb Pst I/SacI subclone of this fragment complemented one transposon mutant (LUN2) and one UV mutant (R92). A 10.1 kb SacI fragment restored exochelin production to the other transposon mutant (LUN1). The nucleotide sequence of the 15.3 kb DNA region that spanned the two transposon insertion sites overlapped the 5′ region of the previously reported exochelin biosynthetic gene fxbA and contained three open reading frames that were transcribed in the opposite orientation to fxbA. The corresponding genes were designated exiT, fxbB and fxbC. The deduced amino acid sequence of ExiT suggested that it was a member of the ABC transporter superfamily, while FxbB and FxbC displayed significant homology with many enzymes (including pristinamycin I synthetase) that catalyse non‐ribosomal peptide synthesis. We propose that the peptide backbone of the siderophore exochelin is synthesized in part by enzymes resembling non‐ribosomal peptide synthetases and that the ABC transporter ExiT is responsible for exochelin excretion.
Analysis of the effect of ppGpp on the ftsQAZ operon in Escherichia coli
Authors: Navarro Francisco , Robin Aline , D'Ari Richard , Joseleau‐Petit Danièle
Published: March 2003

Escherichia coli loses its rod shape by inactivation of PBP2 (penicillin‐binding protein 2), target of the β‐lactam mecillinam. Under these conditions, cell division is blocked in rich medium. Division in the absence of PBP2 activity is restored (and resistance to mecillinam is conferred) when the three cell division proteins FtsQ, FtsA and FtsZ are overproduced, but not when only one or two of them are overproduced. Division in the absence of PBP2 activity is also restored by a doubling in the ppGpp pool, as in the argS201 mutant. However, the nucleotide ppGpp, a transcriptional regulator of many operons, does not govern any of the five promoters of the ftsQAZ operon, as shown by S1 mapping of ftsQAZ mRNA 5′ ends in exponentially growing wild‐type cells in the mecillinam‐resistant argS201 mutant (intermediate ppGpp level) or during the stringent response elicited by isoleucine starvation (high ppGpp level). Furthermore, the concentration of FtsZ protein is not increased in exponentially growing mecillinam‐resistant argS201 cells. These results show that the ftsQAZ operon is not the ppGpp target responsible for mecillinam resistance. We are currently trying to identify those targets that, at intermediate ppGpp levels, allow cells to divide as spheres in the absence of PBP2.
Identification of a pathogenicity island required for Salmonella enteropathogenicity
Authors: Wood Michael W. , Jones Michael A. , Watson Patricia R. , Hedges Samantha , Wallis Tim S. , Galyov Edouard E.
Published: March 2003

Salmonella spp. interact with ileal mucosa and disrupt normal intestinal function, which results in an acute inflammatory cell influx, fluid secretion and enteritis. We have recently characterized SopB, a novel secreted effector protein of Salmonella dublin, and presented evidence that SopB is translocated into eukaryotic cells via a sip‐dependent pathway to promote fluid secretion and inflammatory responses. Here, we show that sopB is located on a large DNA fragment unique to the Salmonella chromosome. This locus is conserved in Salmonella and maps at approximately 20 centisome of the S. typhimurium chromosome. Sequence analysis revealed that this Salmonella‐specific DNA fragment is flanked by DNA sequences with significant sequence similarity to the Escherichia coli K‐12 genes, tRNA1Ser (serT ) on one side and copS/copR on the other. Thus, this Salmonella‐specific DNA fragment has features characteristic of ‘pathogenicity islands’ and, therefore, it was denoted SPI‐5 (Salmonella pathogenicity island‐5). SPI‐5 was sequenced and was found to contain five novel genes, pipA, pipB, pipC, pipD (pathogenicity island‐encoded proteins) and orfX, in addition to sopB. The effect of mutations in pipA, pipB and pipD on the induction of fluid secretion and an acute inflammatory cell influx was assessed in bovine ligated ileal loops. The effect of mutations in SPI‐5‐encoded genes on systemic salmonellosis was assessed in mice. The results of these experiments suggest that SPI‐5‐encoded genes contribute to enteric but not to systemic salmonellosis.
An ExeAB complex in the type II secretion pathway of Aeromonas hydrophila: effect of ATP‐binding cassette mutations on complex formation and function
Authors: Schoenhofen Ian C. , Stratilo Chad , Howard S. Peter
Published: March 2003

The energy‐dependent secretion of aerolysin by Aeromonas hydrophila requires the ExeA and ExeB proteins. An 85 kDa complex containing the two proteins was identified in wild‐type cells but not in cells producing either protein alone. Radiolabelling followed by cross‐linking, immunoprecipitation and then reduction of the cross‐links confirmed the presence of the two proteins in the same complex. The complex could also be extracted intact from cell membranes with non‐ionic detergents. A G229D substitution in the kinase‐3a motif of ExeA strongly reduced the level of aerolysin secretion, as did the replacement of the invariant Lys of the kinase‐1a motif (K56) with Arg. The G229D mutant contained very little of the ExeA–ExeB complex, but overexpression of the mutant complex until wild‐type levels were achieved allowed normal secretion. In contrast, the K56R mutation had no effect on complex formation, but normal secretion levels occurred only when there was a far greater amount of the complex present. These results are consistent with a model in which binding of ATP by ExeA is required for ExeA–ExeB complex formation, while hydrolysis is required for its function in secretion once established.
The pilH gene encodes an ABC transporter homologue required for type IV pilus biogenesis and social gliding motility in Myxococcus xanthus
Authors: Wu Samuel S. , Wu Jie , Cheng Yvonne L. , Kaiser Dale
Published: March 2003

Type IV pilus genes have been shown to be required for social gliding motility in Myxococcus xanthus. We report the discovery of four additional pil genes: pilD, a homologue of type IV prepilin leader peptidases; and pilG, pilH and pilI, which have no known homologues in other type IV pilus systems. pilH encodes an ATP‐binding cassette (ABC) transporter homologue, the first such homologue to be required for the biogenesis of any bacterial pilus type. pilG and pilI are co‐transcribed with pilH and appear to be functionally related to pilH. Null mutants of pilG, pilH and pilI all lack social motility, are deficient in pilus production, have elevated sporulation efficiencies and display similar developmental abnormalities. In addition, all three mutations reduced the amount of PilA found in the supernatant after cells were sedimented from liquid culture. We suggest that the products of these three genes form a single ABC exporter complex, in which pilI is an integral membrane protein with membrane‐spanning domains, and pilG is an accessory factor. The complex may participate in pilus assembly and/or the export of PilA pilin.
Sphingomyelin, glycosphingolipids and ceramide signalling in cells exposed to P‐fimbriated Escherichia coli
Authors: Hedlund Maria , Duan Rui‐Dong , Nilsson Åke , Svanborg Catharina
Published: March 2003

Uropathogenic Escherichia coli attach to epithelial cells through P fimbriae that bind Galα1‐4Galβ‐oligosaccharide sequences in cell surface glycosphingolipids. The binding of P‐fimbriated E. coli to uroepithelial cells causes the release of ceramide, activation of the ceramide signalling pathway and a cytokine response in the epithelial cells. The present study examined the molecular source of ceramide in human kidney A498 cells exposed to P‐fimbriated E. coli. Agonists such as TNF‐α and IL‐1β released ceramide from sphingomyelin by the activation of endogenous sphingomyelinases and hydrolysis of sphingomyelin, and triggered an IL‐6 response. P‐fimbriated E. coli caused a slight increase in endogenous sphingomyelinase activity, but there was no associated sphingomyelin hydrolysis. Instead, the concentration of galactose‐containing glycolipids decreased. We propose that P‐fimbriated E. coli differ from other activators of the ceramide pathway, in that release of ceramide is from receptor glycolipids and not from sphingomyelin. Receptor breakdown may be an efficient host defence strategy, as it reduces the concentration of cell surface receptors, releases soluble receptor analogues and activates an inflammatory response.
Secretion and properties of the large and small lobes of the channel‐forming toxin aerolysin
Authors: Diep Dzung B. , Lawrence Tracy S. , Ausio Juan , Howard S. Peter , Buckley James Thomas
Published: March 2003

Aerolysin is a dimeric protein secreted by Aeromonas spp. that binds to glycosylphosphatidylinositol‐anchored receptors on target cells and becomes insertion competent by oligomerizing. The protein comprises two lobes joined by a short arm. The large lobe is thought to be responsible for channel formation, whereas the small lobe is believed to stabilize the dimer, and it may also contain the receptor binding site. We cloned and expressed the DNA for both lobes of the toxin separately and together in A. salmonicida. The large lobe produced alone was secreted, although more poorly than native protein. The small lobe with the arm produced by itself was not secreted. When the large lobe without the arm was co‐produced with the small lobe with the arm, both were secreted, and they co‐purified as a stoichiometric complex. Analytical ultracentrifugation showed that they form a heterotetramer corresponding to the native dimer. The purified product was nearly as active as aerolysin, but lost activity and became trypsin sensitive above 25°C. The large lobe with the arm was also purified. It was shown to be monomeric, confirming that the small lobe is responsible for dimer stabilization. The large lobe had very low channel‐forming activity, although it was correctly processed by trypsin, and it could form stable oligomers. Surprisingly, the large lobe was found to bind to several glycosylphosphatidylinositol‐anchored proteins, indicating that it contains at least part of the receptor‐binding domain.
Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs
Authors: Tedin Karsten , Moll Isabella , Grill Sonja , Resch Armin , Graschopf Anton , Gualerzi Claudio O. , Bläsi Udo
Published: March 2003

In this study, we have examined the influence of initiation factors on translation initiation of leaderless mRNAs whose 5′‐terminal residues are the A of the AUG initiating codon. A 1:1 ratio of initiation factors to ribosomes abolished ternary complex formation at the authentic start codon of different leaderless mRNAs. Supporting this observation, in vitro translation assays using limiting ribosome concentrations with competing leaderless λcI and Escherichia coli ompA mRNAs, the latter containing a canonical ribosome binding site, revealed reduced cI synthesis relative to OmpA in the presence of added initiation factors. Using in vitro toeprinting and in vitro translation assays, we show that this effect can be attributed to IF3. Moreover, in vivo studies revealed that the translational efficiency of a leaderless reporter gene is decreased with increased IF3 levels. These studies are corroborated by the observed increased translational efficiency of a leaderless reporter construct in an infC mutant strain unable to discriminate against non‐standard start codons. These results suggest that, in the absence of a leader or a Shine–Dalgarno sequence, the function(s) of IF3 limits stable 30S ternary complex formation.
Mannopinic acid and agropinic acid catabolism region of the octopine‐type Ti plasmid pTi15955
Authors: Lyi Sangbom M. , Jafri Samina , Winans Stephen C.
Published: March 2003

Octopine‐type Ti plasmids such as pTi15955, pTiA6 and pTiR10 direct the catabolism of at least eight compounds called opines that are released from crown gall tumours. Four of these compounds are denoted mannityl opines, each of which possesses a D‐mannityl substituent on the nitrogen atom of either glutamate or glutamine. We have analysed a 20 kb region of the Ti plasmid pTi15955 that is required for the catabolism of two such opines, mannopinic acid and agropinic acid. A total of 12 genes in four operons were identified by DNA sequence analysis. Transposons Tn5lacZ and MudK were used to mutagenize these genes and to create aga–lacZ and moa–lacZ translational fusions. The expression of all fusions was induced by agropinic acid and by mannopinic acid. One of these four operons encodes an agropinic acid permease, whereas a second one encodes a mannopinic acid permease. A third operon contains three genes encoding probable catabolic enzymes, two of which (AgaF and AgaG) are thought to convert agropinic acid to mannopinic acid, while the third (AgaE) probably converts mannopinic acid to mannose and glutamate. AgaE resembles a bacterial amino acid deaminase, whereas AgaF and AgaG resemble two bacterial proteins that together catabolize substituted hydantoins, whose chemical structure resembles that of agropinic acid. The remaining operon encoded the MoaR protein, a negative regulator of itself and of the other three operons.
The mitochondrial dicarboxylate carrier is essential for the growth of Saccharomyces cerevisiae on ethanol or acetate as the sole carbon source
Authors: Palmieri Luigi , Vozza Angelo , Hönlinger Angelika , Dietmeier Klaus , Palmisano Annamaria , Zara Vincenzo , Palmieri Ferdinando
Published: March 2003

The dicarboxylate carrier (DIC) is an integral membrane protein that catalyses a dicarboxylate–phosphate exchange across the inner mitochondrial membrane. We generated a yeast mutant lacking the gene for the DIC. The deletion mutant failed to grow on acetate or ethanol as sole carbon source but was viable on glucose, galactose, pyruvate, lactate and glycerol. The growth on ethanol or acetate was largely restored by the addition of low concentrations of aspartate, glutamate, fumarate, citrate, oxoglutarate, oxaloacetate and glucose, but not of succinate, leucine and lysine. The expression of the DIC gene in wild‐type yeast was repressed in media containing ethanol or acetate with or without glycerol. These results indicate that the primary function of DIC is to transport cytoplasmic dicarboxylates into the mitochondrial matrix rather than to direct carbon flux to gluconeogenesis by exporting malate from the mitochondria. The ΔDIC mutant may serve as a convenient host for overexpression of DIC and for the demonstration of its correct targeting and assembly.
The Spo0A sof mutations reveal regions of the regulatory domain that interact with a sensor kinase and RNA polymerase
Authors: Cervin Marguerite A. , Spiegelman George B.
Published: March 2003

Spo0A is a two‐domain response regulator required for the initiation of sporulation in Bacillus subtilis. Spo0A is activated by phosphorylation of its regulatory domain by a multicomponent phosphorelay. To define the role of the regulatory domain in the activation of Spo0A, we have characterized four of the sof mutations in vitro. The sof mutations were identified previously as suppressors of the sporulation‐negative phenotype resulting from a deletion of the gene for one of the phosphorelay components, spo0F. Like wild‐type Spo0A, the transcription stimulation properties of all of the Sof proteins were dependent upon phosphorylation. Sof mutants from two classes were improved substrates for direct phosphorylation by the KinA sensor kinase, providing an explanation for their suppression properties. Two other Sof proteins showed a phosphorylation‐dependent enhancement of the stability of the Sof∼P–RNA polymerase–DNA complex. One of these mutants, Sof114, increased the stability of the Sof114∼P–RNAP–DNA complex without increasing its own affinity for the spoIIG promoter. A comparison of the location of the sof mutations with mutations in CheY suggests that phosphorylation of Spo0A results in the exposure of a region in the regulatory domain that interacts with RNA polymerase, thereby contributing to the signal transduction mechanism.
A downstream CA repeat sequence increases translation from leadered and unleadered mRNA in Escherichia coli
Authors: Martin‐Farmer Julie , Janssen Gary R.
Published: March 2003

When placed downstream of the start codon, multimers of the dinucleotide CA stimulated translation from lacZ, gusA and neo mRNAs in the presence or absence of an untranslated leader sequence. Enhanced expression in the absence of a leader and Shine–Dalgarno sequence indicated that stimulation by CA multimers was independent of translation signals contained within the untranslated leader. Multimers of CA stimulated a significantly higher level of lacZ expression than multimers of individual C or A nucleotides. Translation levels increased as the number of CA repeats increased; fewer multimers were required for enhanced expression from leadered mRNA than from mRNA that was deleted for its leader sequence. Addition of downstream CA multimers increased the ribosome binding strength of mRNA in vitro and the amount of full‐length mRNA in vivo, suggesting that the enhanced expression resulted from translation of a more abundant functional message containing a stronger ribosome binding site. The presence of downstream CA‐rich sequences, occurring naturally in several Escherichia coli genes, might contribute to translation of other mRNAs. Addition of CA multimers might represent a general mechanism for increasing expression from genes of interest.
The molecular basis for the specificity of fimE in the phase variation of type 1 fimbriae of Escherichia coli K‐12
Authors: Kulasekara Hemantha Don , Blomfield Ian C.
Published: March 2003

The expression of type 1 fimbriae in Escherichia coli is phase variable, with cells switching between fimbriate (ON) and afimbriate (OFF) phases. The phase variation is dependent on the orientation of a 314 bp DNA element (the switch) that undergoes DNA inversion. DNA inversion requires either fimB or fimE, site‐specific recombinases that differ in both specificity and activity. Whereas fimB promotes recombination with little orientational bias, fimE promotes recombination in the ON‐to‐OFF direction exclusively. In wild‐type cells, fimE activity predominates and, hence, most bacteria are afimbriate. Here, it is shown that fimE specificity is caused by two different, but complementary, mechanisms. First, FimE shows a strong preference for the switch in the ON orientation as a substrate for recombination. Differences in the nucleotide sequence of the recombinase binding sites is a key factor in determining FimE specificity, although one or more additional cis‐active sites that flank the fim switch also appear to be involved. Secondly, the orientation of the switch controls fimE in cis, most probably to control recombinase expression.
The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation
Authors: Wolfgang Matthew , Van Putten Jos P. M. , Hayes Stanley F. , Koomey Michael
Published: March 2003

The expression of type IV pili (Tfp) by Neisseria gonorrhoeae has been shown to be essential for natural genetic transformation at the level of sequence‐specific uptake of DNA. All previously characterized mutants defective in this step of transformation either lack Tfp or are altered in the expression of Tfp‐associated properties, such as twitching motility, autoagglutination and the ability to bind to human epithelial cells. To examine the basis for this relationship, we identified potential genes encoding polypeptides sharing structural similarities to PilE, the Tfp subunit, within the N. gonorrhoeae genome sequence database. We found that disruption of one such gene, designated comP (for competence‐associated prepilin), leads to a severe defect in the capacity to take up DNA in a sequence‐specific manner, but does not alter Tfp biogenesis or expression of the Tfp‐associated properties of autoagglutination, twitching motility and human epithelial cell adherence. Indirect evidence based on immunodetection suggests that ComP is expressed at very low levels relative to that of PilE. The process of DNA uptake in gonococci, therefore, is now known to require the expression of at least three distinct components: Tfp, the recently identified PilT protein and ComP.
Sequence‐specific cleavage by bacteriophage T4 endonuclease II in vitro
Authors: Carlson Karin , Kosturko Linda D. , Nyström Anna‐Chey
Published: March 2003

The 136 codon (408 bp) denA gene encoding endonuclease II (EndoII) of bacteriophage T4 was unambiguously identified through sequencing and subsequent cloning. EndoII prepared from cloned DNA through coupled in vitro transcription–translation nicked and cut DNA in vitro in a sequence‐specific manner. In vitro (and in vivo ), the bottom strand was nicked between the first and second base pair to the right of a top‐strand CCGC motif shared by favoured in vitro and in vivo cleavage sites; top‐strand cleavage positions varied. To the right of the cleavage position, favoured in vitro sites lacked a sequence element conserved at favoured in vivo sites. In pBR322 DNA, the sites cleaved in vivo as previously described were also cleaved in vitro, but in vitro additional sites were nicked or cleaved and the preference for individual sites was different. Also, different from the in vivo reaction, nicking was more frequent than ds cutting; in many copies of a ds cleavage site, only the bottom strand was nicked in vitro. A model is discussed in which sequential nicking of the two strands, and different factors influencing bottom‐strand nicking and top‐strand nicking, can explain the differences between the in vitro and the in vivo reaction.
Cell surface protease PRT1 identified in the fungal pathogen Pneumocystis carinii
Authors: Lugli Elena B. , Bampton Edward T. W. , Ferguson David J. P. , Wakefield Ann E.
Published: March 2003

The subtelomeric regions of the chromosomes of many organisms contain gene families that allow adaptation to a changing environment. In a number of parasites, these subtelomeric gene families encode cell surface proteins that undergo antigenic variation. Proteases are another important virulence determinant in pathogenic microorganisms. We report the localization of the PRT1 protease of the pathogenic fungus Pneumocystis carinii sp. f. carinii, encoded by a subtelomeric gene family, to the cell surface of both the trophozoite and the cyst phase of the organism. Using anti‐PRT1 antiserum, we demonstrated specificity to P. carinii sp. f. carinii in sections of infected rat lungs and, using immunofluorescence, we showed that the PRT1 protease has the characteristic distribution of a surface protein. The anti‐PRT1 antiserum showed cross‐reactivity with a number of P. carinii sp. f. carinii proteins migrating between 185 kDa and 28 kDa, the majority migrating between 42 kDa and 52 kDa, a region that has been shown by serological studies to contain important immunodominant P. carinii proteins. Cross‐reactivity was also observed with P. carinii sp. f. hominis proteins. We have also cloned a portion of the catalytic domain of PRT1 from P. carinii sp. f. hominis, P. carinii sp. f. muris and P. carinii sp. f. rattus. Our data suggest that the PRT1 protease plays an important role in the pathogenicity of P. carinii.
ClpE, a novel type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis
Authors: Derré Isabelle , Rapoport Georges , Devine Kevin , Rose Matthias , Msadek Tarek
Published: March 2003

Clp ATPases, which include the ubiquitous HSP100 family, are classified according to their structural features and sequence similarities. During the course of the Bacillus subtilis genome sequencing project, we identified a gene encoding a new member of the HSP100 family. We designated this protein ClpE, as it is the prototype of a novel subfamily among the Clp ATPases, and have identified homologues in several bacteria, including Listeria monocytogenes, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, Lactobacillus sakei and Clostridium acetobutylicum. A unique feature of these Hsp100‐type Clp ATPases is their amino‐terminal zinc finger motif. Unlike the other class III genes of B. subtilis (clpC and clpP ), clpE does not appear to be required for stress tolerance. Transcriptional analysis revealed two σA‐type promoters, expression from which was shown to be inducible by heat shock and puromycin treatment. Investigation of the regulatory mechanism controlling clpE expression indicates that this gene is controlled by CtsR and is thus a member of the class III heat shock genes of B. subtilis. CtsR negatively regulates clpE expression by binding to the promoter region, in which five CtsR binding sites were identified through DNase I footprinting and sequence analysis.
The role of the trehalose system in regulating the maltose regulon of Escherichia coli
Authors: Decker Katja , Gerhardt Friederike , Boos Winfried
Published: March 2003

The maltose regulon consists of 10 genes encoding an ABC transporter for maltose and maltodextrins as well as enzymes necessary for their degradation. MalK, the energy‐transducing subunit of the transport system, acts phenotypically as a repressor of MalT, the transcriptional activator of the mal genes. Using MacConkey maltose indicator plates we isolated an insertion mutation that strongly reduced the repressing effect of overproduced MalK. The insertion had occurred in treR encoding the repressor of the trehalose system. The loss of TreR function led to derepression of treB encoding an enzymeIITre of the PTS for trehalose and of treC encoding TreC, the cytoplasmic trehalose‐6‐phosphate hydrolase. Further analysis revealed that maltose can enter the cell by facilitated diffusion through enzymeIITre, thus causing induction of the maltose system. In addition, derepression of TreC by itself caused induction of the maltose system, and a mutant lacking TreC was reduced in the uninduced level of mal gene expression indicating synthesis of endogenous inducer by TreC. Extracts containing TreC transformed [14C]‐maltose into another 14C‐labelled compound (preliminarily identified as maltose 1‐phosphate) that is likely to be an alternative inducer of the maltose system.
Intramolecular transposition of insertion sequence IS91 results in second‐site simple insertions
Authors: Bernales Irantzu , Mendiola M. Victoria , De La Cruz Fernando
Published: March 2003

A series of plasmids carrying an IRL‐kan‐IRR transposable cassette, in which IRL and IRR are the left‐ and right‐terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co‐integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100‐fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL‐kan‐IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling‐circle transposition.
The selC‐associated SHI‐2 pathogenicity island of Shigella flexneri
Authors: Moss Jeremy E. , Cardozo Timothy J. , Zychlinsky Arturo , Groisman Eduardo A.
Published: March 2003

Pathogenicity islands are chromosomal gene clusters, often located adjacent to tRNA genes, that encode virulence factors present in pathogenic organisms but absent or sporadically found in related non‐pathogenic species. The selC tRNA locus is the site of integration of different pathogenicity islands in uropathogenic Escherichia coli, enterohaemorrhagic E. coli and Salmonella enterica. We show here that the selC locus of Shigella flexneri, the aetiological agent of bacterial dysentery, also contains a pathogenicity island. This pathogenicity island, designated SHI‐2 (Shigellaisland 2), occupies 23.8 kb downstream of selC and contains genes encoding the aerobactin iron acquisition siderophore system, colicin V immunity and several novel proteins. Remnants of multiple mobile genetic elements are present in SHI‐2. SHI‐2‐hybridizing sequences were detected in all S. flexneri strains tested and parts of the island were also found in other Shigella species. SHI‐2 may allow Shigella survival in stressful environments, such as those encountered during infection.
Molecular function of the dual‐start motif in the λ S holin
Authors: Graschopf Anton , Bläsi Udo
Published: March 2003

The λS gene represents the prototype of holin genes with a dual‐start motif, which leads to the synthesis of two polypeptides, S105 and S107. They differ at their N‐terminus by only two amino acids, Met‐1 and Lys‐2, at the beginning of the longer product. Despite the minor difference, the two proteins have opposing functions in lysis, with protein S107 being an inhibitor and protein S105 being an effector of ‘hole formation’ in the inner membrane. Here, we have studied the molecular mechanism underlying the ‘lysis clock’ contributed by the dual‐start motif. We have used protein fusions in which the secretory signal sequence of the M13 procoat protein VIII has been abutted to the N‐terminal Met residues of S105 and S107 respectively. S‐dependent ‘hole formation’ required removal of the signal sequence in both fusion proteins, as both the VIII–S105 and the VIII–S107 fusion proteins were non‐functional when leader peptidase cleavage was inhibited. These results strongly supported the hypothesis that functional assembly of S proteins requires translocation of their N‐terminus to the periplasm. Using signal sequence cleavage as a measure of translocation, we observed that the translocation kinetics of the N‐terminus of the S107 moiety was reduced about threefold when compared with the N‐terminus of the S105 moiety. Moreover, depolarization of the membrane resulted in an immediate cleavage of the signal sequence and ‘hole formation’ exerted by the S107 moiety of the VIII–S107 fusion protein. A model is presented in which S107 with a reversed topology of its N‐terminus interacts with S105 and poisons ‘hole formation’. Upon depolarization of the membrane, translocation of the N‐terminus of S107 to the periplasm results in the functional assembly of S proteins, i.e. ‘hole formation’.
Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases
Authors: Minakhina S. , Kholodii G. , Mindlin S. , Yurieva O. , Nikiforov V.
Published: March 2003

DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase‐like proteins. Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044. Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans. To our knowledge, this is the first case when a site‐specific recombination system outside a transposon has been shown to be involved in transposition.
Effect of growth rate and incC mutation on symmetric plasmid distribution by the IncP‐1 partitioning apparatus
Authors: Bignell Colin R. , Haines Anthony S. , Khare Dheeraj , Thomas Christopher M.
Published: March 2003

The incC and korB genes of IncP‐1 plasmid RK2 encode homologues of ubiquitous ParA and ParB partitioning proteins of bacterial plasmids and chromosomes. Using immunofluorescence microscopy, we found that KorB, which binds to 12 widely distributed sites on the genome, is located in symmetrically placed foci in cells containing IncP‐1 plasmids. When maintained by the low‐copy‐number P7 replicon, an RK2 segment including incC, korB and the kla, kle and korC regions encodes an efficient partitioning system that gives a pattern of foci similar to RK2 itself. Symmetrical distribution of KorB foci correlates with segregational stability conferred by either the IncP‐1 or P7 partitioning systems; KorB distribution follows plasmid distribution. In the absence of a second partitioning system, incC inactivation resulted in paired or clumped foci that were not symmetrically distributed. At a slow growth rate, position analysis of foci showed a cycle from one central focus to two foci (at one‐ and three‐quarter positions) and back, and at a high growth rate it showed a cycle from two foci to four and back. This pattern fits with the plasmid being coupled to the replication zones in the cell and being moved to successively younger zones by active partitioning, indicating a tight association between replication and partitioning.
Identification of a peptide synthetase involved in the biosynthesis of glycopeptidolipids of Mycobacterium smegmatis
Authors: Billman‐Jacobe Helen , McConville Malcolm J. , Haites Ruth E. , Kovacevic Svetozar , Coppel Ross L.
Published: March 2003

Five rough colony mutants of Mycobacterium smegmatis mc2155 were produced by transposon mutagenesis. The mutants were unable to synthesize glycopeptidolipids that are normally abundant in the cell wall of wild‐type M. smegmatis. The glycopeptidolipids have a lipopeptide core comprising a fatty acid amide linked to a tetrapeptide that is modified with O‐methylated rhamnose and O‐acylated 6‐deoxy talose. Compositional analysis of lipids extracted from the mutants indicated that the defect in glycopeptidolipid synthesis occurred in the assembly of the lipopeptide core. No other defects or compensatory changes in cell wall structure were detected in the mutants. All five mutants had transposon insertions in a gene encoding an enzyme belonging to the peptide synthetase family. Targeted disruption of the gene in the wild‐type strain gave a phenotype identical to that of the five transposon mutants. The M. smegmatis peptide synthetase gene is predicted to encode four modules that each contain domains for cofactor binding and for amino acid recognition and adenylation. Three modules also have amino acid racemase domains. These data suggest that the common lipopeptide core of these important cell wall glycolipids is synthesized by a peptide synthetase.