bluuebunny/crossref_metadata_2025 huggingface dataset:
Pore‐forming activity of type III system‐secreted proteins leads to oncosis of Pseudomonas aeruginosa‐infected macrophages
Authors: Dacheux Denis , Goure Julien , Chabert Jacqueline , Usson Yves , Attree Ina
Published: March 2003

The Pseudomonas aeruginosa cystic fibrosis isolate CHA induces type III secretion system‐dependent but ExoU‐independent oncosis of neutrophils and macrophages. Time‐lapse microscopy of the infection process revealed the rapid accumulation of motile bacteria around infected cells undergoing the process of oncosis, a phenomenon we termed pack swarming. Characterization of the non‐chemotactic CHAcheZ mutant showed that pack swarming is a bacterial chemotactic response to infected macrophages. A non‐cytotoxic mutant, lacking the type III‐secreted proteins PcrV, PopB and PopD, was able to pack swarm only in the presence of the parental strain CHA or when macrophages were pretreated with the pore‐forming toxin streptolysin O. Interaction of P. aeruginosa with red blood cells (RBCs) showed that the contact‐dependent haemolysis provoked by CHA requires secretion via the type III system and the PcrV, PopB/PopD proteins. The pore inserted into RBC membrane was estimated from osmoprotection experiments to be between 2.8 and 3.5 nm. CHA‐infected macrophages could be protected from cell lysis with PEG3350, indicating that the pore introduced into RBC and macrophage membranes is of similar size. The time course uptake of the vital fluorescent dye, Yo‐Pro‐1, into infected macrophages confirmed that the formation of transmembrane pores by CHA precedes cellular oncosis. Therefore, CHA‐induced macrophage death results from a pore‐forming activity that is dependent on the intact pcrGVHpopBD operon.
RpoS co‐operates with other factors to induce Legionella pneumophila virulence in the stationary phase
Authors: Bachman Michael A. , Swanson Michele S.
Published: March 2003

Legionella pneumophila replicates within amoebae and macrophages and causes the severe pneumonia Legionnaires' disease. When broth cultures enter the post‐exponential growth (PE) phase or experience amino acid limitation, L. pneumophila accumulates the stringent response signal (p)ppGpp and expresses traits likely to promote transmission to a new phagocyte. The hypothesis that a stringent response mechanism regulates L. pneumophila virulence was bolstered by our finding that the avirulent mutant Lp120 contains an internal deletion in the gene encoding the stationary phase sigma factor RpoS. To test directly whether RpoS co‐ordinates virulence with stationary phase, isogenic wild‐type, rpoS‐120 and rpoS null mutant strains were constructed and analysed. PE phase L. pneumophila became cytotoxic by an RpoS‐independent pathway, but their sodium sensitivity and maximal expression of flagellin required RpoS. Likewise, full induction of sodium sensitivity by experimentally induced (p)ppGpp synthesis required RpoS. To replicate efficiently in macrophages, L. pneumophila used both RpoS‐dependent and ‐independent pathways. Like those containing the dotA type IV secretory apparatus mutant, phagosomes harbouring either rpoS or dotA rpoS mutants rapidly acquired the late endosomal protein LAMP‐1, but not the lysosomal marker Texas red–ovalbumin. Together, the data support a model in which RpoS co‐operates with other regulators to induce L. pneumophila virulence in the PE phase.
The highly conserved domain of the Caulobacter McpA chemoreceptor is required for its polar localization
Authors: Alley M. R. K.
Published: March 2003

We have fused GFP to the C‐terminus of McpA to study chemoreceptor polar localization in Caulobacter crescentus. The full‐length McpA–GFP fusion is polarly localized and methylated. The methylation is dependent on the chemoreceptor methyltransferase (cheR) and chemoreceptor methylesterase (cheB) genes present in the mcpA operon. C‐terminal and internal deletions of McpA were constructed and fused to the N‐terminus of GFP to identify the domains required for polar localization. When the R1 methylation domain was deleted, the McpA–GFP fusion was still polarly localized, suggesting that this domain is dispensable for polar localization. However, when the highly conserved domain (HCD), which is involved in interacting with CheW, was deleted either by an internal deletion or C‐terminal deletion, the resulting McpA–GFP fusions were completely delocalized. When the mcpA operon, which contains the cheW and cheA homologues, was deleted, the full‐length McpA–GFP fusion was delocalized. Although additional chemotaxis genes are required for the polar localization of McpA–GFP, the presence of the single polar flagellum is not required. However, in filamentous cells, which are frequently found in C. crescentus fliF mutants, the McpA–GFP fusion was observed at mid‐cell positions.
Characterization of integration host factor (IHF) binding upstream of the cysteine‐rich protein operon (omcAB) promoter of Chlamydia trachomatis LGV serovar L2
Authors: Zhong Jianmin , Douglas Annemarie L. , Hatch Thomas P.
Published: March 2003

Chlamydiae are bacterial parasites that carry out a distinct developmental cycle within host cells; however, the mechanisms by which these organisms regulate stage‐specific gene expression are not known. We identified a DNA element located between nucleotide (nt) −135 and −90 upstream from the transcription start point of the late stage‐specific CRP operon (omcAB) of Chlamydia trachomatis, to which a protein in extracts of chlamydiae harvested at 23 h after infection binds. A recombinant protein of C. trachomatis open reading frame (ORF) CT267, which is homologous to bacterial integration host factor (IHF) and the heat‐unstable nucleoid protein (HU), bound to the same element and produced the same DNase I footprint as the protein in chlamydial extracts. Recombinant ORF CT267 protein bound with high affinity to the DNA element and induced a sharp bend in a DNA fragment containing the binding site, suggesting that ORF CT267 encodes a protein with IHF‐like activity, and recombinant protein had a positive effect on in vitro transcription of the CRP operon. IHF‐binding activity and IHF protein were detected in extracts of C. trachomatis during the early to intermediate phases of the late stage of the developmental cycle (between 17 and 30 h after infection), but were absent in the extreme late phase of the cycle and in the infectious form of chlamydiae. The presence of an IHF binding site upstream of the CRP operon and the presence of chlamydial IHF‐like protein when late stage genes are transcribed suggests that the chlamydial IHF may play a role in stage‐specific gene expression.
Evidence for dual functionality of the operon plnABCD in the regulation of bacteriocin production in Lactobacillus plantarum
Authors: Diep Dzung B. , Johnsborg Ola , Risøen Per Arne , Nes Ingolf F.
Published: March 2003

The regulatory operon (plnABCD) involved in bacteriocin production in Lactobacillus plantarum C11 encodes four different proteins: a cationic prepeptide (PlnA); a histidine protein kinase (PlnB); and two highly homologous response regulators (PlnC and PlnD; over 75% sequence similarity). The mature product of PlnA, plantaricin A, serves as an extracellular pheromone that induces bacteriocin production. The exact roles of plnBCD in bacteriocin production have not been established experimentally. A reporter system containing the gusA gene fused with the plnA promoter was used to study plnABCD. We demonstrated that the plnABCD operon codes for an autoregulatory unit capable of activating its own promoter. Deletion analyses, performed in a heterologous expression host to define the roles of the individual genes, confirmed that both the inducer gene (plnA) and the kinase gene (plnB) are required for autoactivation. Apparently, the latter gene encodes a protein that serves as a receptor for the pheromone peptide. It was also demonstrated conclusively that the two regulators PlnC and PlnD, which have been shown previously to bind specifically to the DNA regulatory repeats of the plnA promoter, possess differential activities on the plnA promoter, with PlnC being much more active than PlnD. The functions of the response regulators were investigated further in the bacteriocin producer strain C11 in order to reveal their roles in bacteriocin production. Surprisingly, the two response regulators display totally opposite functions: although overexpression of plnC activated transcription and bacteriocin production, the overexpression of plnD repressed both processes, thus strongly suggesting that PlnD plays a role in the downregulation of bacteriocin synthesis. To our knowledge, this is the first evidence for a protein involved directly in negative regulation of bacteriocin production, and also it was shown for the first time that two highly homologous response regulators, with opposite functions, are encoded by genes located on the same operon.
Escherichia coli DNA glycosylase Mug: a growth‐regulated enzyme required for mutation avoidance in stationary‐phase cells
Authors: Mokkapati Sanath K. , Fernández de Henestrosa A. R. , Bhagwat Ashok S.
Published: March 2003

The Escherichia coli DNA glycosylase Mug excises 3,N4‐ethenocytosines (εC) and uracils from DNA, but its biological function is obscure. This is because εC is not found in E. coli DNA, and uracil‐DNA glycosylase (Ung), a distinct enzyme, is much more efficient at removing uracils from DNA than Mug. We find that Mug is overexpressed as cells enter stationary phase, and it is maintained at a fairly high level in resting cells. This is true of cells grown in rich or minimal media, and the principal regulation of mug is at the level of mRNA. Although the expression of mug is strongly dependent on the stationary‐phase sigma factor, σS, when cells are grown in minimal media, it shows only a modest dependence on σS when cells are grown in rich media. When mug cells are maintained in stationary phase for several days, they acquire many more mutations than their mug+ counterparts. This is true in ung as well as ung+ cells, and a majority of new mutations may not be C to T. Our results show that the biological role of Mug parallels its expression in cells. It is expressed poorly in exponentially growing cells and has no apparent role in mutation avoidance in these cells. In contrast, Mug is fairly abundant in stationary‐phase cells and has an important anti‐mutator role at this stage of cell growth. Thus, Mug joins a very small coterie of DNA repair enzymes whose principal function is to avoid mutations in stationary‐phase cells.
A novel two‐component regulatory system in Bacillus subtilis for the survival of severe secretion stress
Authors: Hyyryläinen Hanne‐Leena , Bolhuis Albert , Darmon Elise , Muukkonen Leila , Koski Pertti , Vitikainen Marika , Sarvas Matti , Prágai Zoltán , Bron Sierd , Van Dijl Jan Maarten , Kontinen Vesa P.
Published: March 2003

The Gram‐positive eubacterium Bacillus subtilis is well known for its high capacity to secrete proteins into the environment. Even though high‐level secretion of proteins is an efficient process, it imposes stress on the cell. The present studies were aimed at the identification of systems required to combat this so‐called secretion stress. A two‐component regulatory system, named CssR–CssS, was identified, which bears resemblance to the CpxR–CpxA system of Escherichia coli. The results show that the CssR/S system is required for the cell to survive the severe secretion stress caused by a combination of high‐level production of the α‐amylase AmyQ and reduced levels of the extracytoplasmic folding factor PrsA. As shown with a prsA3 mutation, the Css system is required to degrade misfolded exported proteins at the membrane–cell wall interface. This view is supported by the observation that transcription of the htrA gene, encoding a predicted membrane‐bound protease of B. subtilis, is strictly controlled by CssS. Notably, CssS represents the first identified sensor for extracytoplasmic protein misfolding in a Gram‐positive eubacterium. In conclusion, the results show that quality control systems for extracytoplasmic protein folding are not exclusively present in the periplasm of Gram‐negative eubacteria, but also in the Gram‐positive cell envelope.
Fba, a novel fibronectin‐binding protein from Streptococcus pyogenes, promotes bacterial entry into epithelial cells, and the fba gene is positively transcribed under the Mga regulator
Authors: Terao Yutaka , Kawabata Shigetada , Kunitomo Eiji , Murakami Jumpei , Nakagawa Ichiro , Hamada Shigeyuki
Published: March 2003

In infection by Streptococcus pyogenes, fibronectin (Fn)‐binding proteins play important roles as adhesins and invasins. Here, we present a novel Fn‐binding protein of S. pyogenes that exhibits a low similarity to other Fn‐binding proteins reported. After searching the Oklahoma Streptococcal Genome Sequencing Database for open reading frames (ORFs) with an LPXTG motif, nine ORFs were found among those recognized as putative surface proteins, and one of them was designated as Fba. The fba gene was found in M types 1, 2, 4, 22, 28 and 49 of S. pyogenes, but not in other serotypes or groups of streptococci. Fba, a 37.8 kDa protein, possesses three or four proline‐rich repeat domains and exhibits a high homology to FnBPA, the Fn‐binding protein of Staphylococcus aureus. Recombinant Fba exhibited a strong binding ability to Fn. In addition, Fba‐deficient mutants showed diminished invasive capabilities to HEp‐2 cells and low mortality in mice following skin infection. The fba gene was located downstream of the mga regulon and analysis using an mga‐inactivated mutant revealed that it was transcribed under the control of the Mga regulator. These results indicate that Fba is a novel protein and one of the important virulence factors of S. pyogenes.
Feedback regulation of the Bradyrhizobium japonicum nodulation genes
Authors: Loh John T. , Stacey Gary
Published: March 2003

Lipochitin Nod signals are produced by rhizobia and are required for the establishment of a nitrogen‐fixing symbiosis with a legume host. The nodulation genes encode products required for the synthesis of this signal and are induced in response to plant‐produced flavonoid compounds. The addition of chitin and lipo‐chitin oligomers to Bradyrhizobium japonicum cultures resulted in a significant reduction in the expression of a nod–lacZ fusion. Intracellular expression of NodC, encoding a chitin synthase, also reduced nod gene expression. In contrast, expression of the ChiB chitinase increased nod gene expression. The chain length of the oligosaccharide was important in feedback regulation, with chitotetraose molecules the best modulators of nod gene expression. Feedback regulation is mediated by the induction of nolA by chitin, resulting in elevated levels of the repressor protein, NodD2.
À la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer‐binding protein XylR to non‐natural effectors
Authors: Garmendia Junkal , Devos Damien , Valencia Alfonso , De Lorenzo Víctor
Published: March 2003

To investigate the activation mechanism of the enhancer‐binding protein XylR encoded by the TOL plasmid of Pseudomonas putida mt‐2, a combinatorial library was generated composed of shuffled N‐terminal A domains of the homologous regulators DmpR, XylR and TbuT, reassembled within the XylR structure. When the library was screened in vivo for responsiveness to non‐effectors bulkier than one aromatic ring (such as biphenyl) or bearing an entirely different distribution of electronegative groups (e.g. nitrotoluenes), protein variants were found that displayed an expanded inducer range including the new effectors. Although the phenotypes endowed with the corresponding changes were largely similar, the modifications involved different sites within the A domain. The positions of the mutations within a structural model of the A domain suggest that expansion of the inducer profile can be brought about not only by changes in the effector pocket of the protein but also by unlocking steps of the signal transmission mechanism that follows effector binding. These results provide a rationale for evolving in vitro regulators à la carte that are responsive to predetermined, natural or xenobiotic chemical species.
Isolation and characterization of mutations in region 1.2 of Escherichia coliσ70
Authors: Baldwin Nicole E. , Dombroski Alicia J.
Published: March 2003

Eubacterial RNA polymerase uses the sigma (σ) subunit for recognition of and transcription initiation from promoter DNA sequences. One family of σ factors includes those related to the primary σ factor from Escherichia coli, σ70. Members of the σ70 family have four highly conserved domains, of which regions 2 to 4 are present in all members. Region 1 can be subdivided into regions 1.1 and 1.2. Region 1.1 affects DNA binding by σ70 alone, as well as transcription initiation by holoenzyme. Region 1.2, present and highly conserved in most sigma factors, has not yet been assigned a putative function, although previous work has demonstrated that it is not required for either association with the core subunits of RNA polymerase or promoter‐specific binding by holoenzyme. We generated random single amino acid substitutions targeted to region 1.2 of E. coliσ70 as well as a deletion of region 1.2, and characterized the behaviour of the mutant σ factors both in vivo and in vitro to investigate the function of region 1.2 during transcription initiation. In this study, we show that mutations in region 1.2 can affect promoter binding, open complex and initiated complex formation and the transition from abortive transcription to elongation.
Requirement for the molecular adapter function of StpA at the Escherichia coli bgl promoter depends upon the level of truncated H‐NS protein
Authors: Free Andrew , Porter Megan E. , Deighan Padraig , Dorman Charles J.
Published: March 2003

Truncated derivatives of the Escherichia coli nucleoid‐associated protein H‐NS that lack the DNA‐binding domain remain competent for silencing of the cryptic bgl operon in vivo. Previous studies have provided evidence for the involvement of either the homologous nucleoid protein StpA or the alternative sigma factor RpoS in this unusual silencing mechanism. Here, we rationalize this apparent discrepancy. We show that two hns alleles (hns‐205::Tn10 and hns60), which produce virtually identical amino‐terminal fragments of H‐NS, have very different requirements for StpA to mediate bgl silencing. The hns60 allele produces a high level of truncated H‐NS, which can overcome the absence of StpA, whereas the lower level expressed by hns‐205::Tn10 requires StpA for silencing. Reversing the relative levels of the two H‐NS fragments reverses their requirement for StpA to silence bgl transcription. This suggests that the amino‐terminal fragment of H‐NS can be targeted to DNA to mediate silencing by multiple protein–protein interactions. The high‐specificity interaction with StpA can function at low levels of truncated H‐NS, whereas an alternative mechanism, perhaps involving lower specificity interactions with another protein(s), is only functional when truncated H‐NS is abundant. These findings have important implications for the involvement of other proteins in H‐NS‐dependent transcriptional repression.
Rok (YkuW) regulates genetic competence in Bacillus subtilis by directly repressing comK
Authors: Hoa Tran Thu , Tortosa Pablo , Albano Mark , Dubnau David
Published: March 2003

The Rok (YkuW) protein acts as a negative regulator of comK, which encodes
the competence transcription factor of Bacillus subtilis. In the absence of
Rok, ComK is overproduced, and when excess Rok is present comK transcription
is inhibited. Rok acts transcriptionally to repress comK expression but does
not affect ComK stability, which is controlled by the MecA switch. Gel‐shift assays
show that Rok binds directly to a DNA fragment that contains the comK promoter.
SinR and AbrB act negatively on rok transcription, and the inactivation of
rok bypasses the positive requirements for sinR and abrB for
the expression of comK. Therefore, the dependence of comK expression
on SinR and AbrB may be a result of their repression of rok transcription.
It has also been shown in vivo that Rok and ComK can indivi‐dually repress
rok transcription, and that Rok and ComK bind to the rok promoter in
vitro. These interactions establish feedback loops, and the roles of these circuits are discussed.
Co‐operative binding of triplicate carbohydrate‐binding modules from a thermophilic xylanase
Authors: Boraston Alisdair B. , McLean Bradley W. , Chen Grace , Li Anson , Warren R. Antony J. , Kilburn Douglas G.
Published: March 2003

Family 6 carbohydrate‐binding modules were am‐plified by polymerase chain reaction (PCR) from Clostridium stercorarium strain NCIB11754 genomic DNA as a triplet. Individually, these modules bound to xylooligosaccharides and cellooligosaccharides with affinities varying from ≈ 3 × 103 M–1 to ≈ 1 × 105 M–1. Tandem and triplet combinations of these modules bound co‐operatively to soluble xylan and insoluble cellulose to give ≈ 20‐ to ≈ 40‐fold increases in affinity relative to the individual modules. This co‐operativity was an avidity effect resulting from the modules within the tandems and triplet interacting simul‐ taneously with proximal binding sites on the polysac‐charides. This occurred by both intrachain and interchain interactions. The duplication or triplication of modules appears to be linked to the growth temperature of the organism; co‐operativity in these multiplets may compensate for the loss of affinity at higher temperatures.
Resveratrol acts as a natural profungicide and induces self‐intoxication by a specific laccase
Authors: Schouten Alexander , Wagemakers Lia , Stefanato Francesca L. , Kaaij Rachel M. van der , Kan Jan A. L. van
Published: March 2003

The grapevine (Vitis) secondary metabolite resveratrol is considered a phytoalexin, which protects the plant from Botrytis cinerea infection. Laccase activity displayed by the fungus is assumed to detoxify resveratrol and to facilitate colonization of grape. We initiated a functional molecular genetic analysis of B. cinerea laccases by characterizing laccase genes and evaluating the phenotype of targeted gene replacement mutants. Two different laccase genes from B. cinerea were characterized, Bclcc1 and Bclcc2. Only Bclcc2 was strongly expressed in liquid cultures in the presence of either resveratrol or tannins. This suggested that Bclcc2, but not Bclcc1, plays an active role in the oxidation of both resveratrol and tannins. Gene replacement mutants in the Bclcc1 and Bclcc2 gene were made to perform a functional analysis. Only Bclcc2 replacement mutants were incapable of converting both resveratrol and tannins. When grown on resveratrol, both the wild type and the Bclcc1 replacement mutant showed inhibited growth, whereas Bclcc2 replacement mutants were unaffected. Thus, contrary to the current theory, BcLCC2 does not detoxify resveratrol but, rather, converts it into compounds that are more toxic for the fungus itself. The Bclcc2 gene was expressed during infection of B. cinerea on a resveratrol‐producing host plant, but Bclcc2 replacement mutants were as virulent as the wild‐type strain on various hosts. The activation of a plant secondary metabolite by a pathogen introduces a new dimension to plant–pathogen interactions and the phytoalexin concept.
The MotA transcription factor from bacteriophage T4 contains a novel DNA‐binding domain: the ‘double wing’ motif
Authors: Li Ning , Sickmier E. Allen , Zhang Rongguang , Joachimiak Andrzej , White Stephen W.
Published: March 2003

MotA is a transcription factor from bacteriophage T4 that helps adapt the host Escherichia coli transcription apparatus to T4 middle promoters. We have determined the crystal structure of the C‐terminal DNA‐binding domain of MotA (MotCF) to 1.6 Å resolution using multiwavelength, anomalous diffraction methods. The structure reveals a novel DNA‐binding α/β motif that contains an exposed β‐sheet surface that mediates interactions with the DNA. Independent biochemical experiments have shown that MotCF binds to one surface of a single turn of DNA through interactions in adjacent major and minor grooves. We present a model of the interaction in which β‐ribbons at opposite corners of the six‐stranded β‐sheet pen‐etrate the DNA grooves, and call the motif a ‘double wing’ to emphasize similarities to the ‘winged‐helix’ motif. The model is consistent with data on how MotA functions at middle promoters, and provides an explanation for why MotA can form non‐specific multimers on DNA.
Dual control of subtilin biosynthesis and immunity in Bacillus subtilis
Authors: Stein Torsten , Borchert Stefan , Kiesau Peter , Heinzmann Stefan , Klöss Stephan , Klein Cora , Helfrich Markus , Entian Karl‐Dieter
Published: March 2003

The production of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 is highly regulated. Transcriptional organization and regulation of the subtilin gene cluster encompassing 11 genes was characterized. Two polycistronic mRNAs encoding transcript spaBTC (6.8 kb) and encoding transcript spaIFEG (3.5 kb) as well as the monocistronic spaS (0.3 kb) mRNA were shown by Northern hybridization. Primer extension experiments and β‐galactosidase fusions confirmed three independent promoter sites preceding genes spaB, spaS and spaI. β‐Galactosidase expression of spaB, spaS and spaI promoter lacZ fusions initiated in mid‐exponential growth. Maximal activities were reached at the transition to stationary growth and were collinear with subtilin production. The lacZ activity was dependent on co‐expression with the two‐component regulatory system spaRK. The presence of subtilin was needed for efficient expression of all three promoter lacZ fusions. This suggests a transcriptional autoregulation according to a quorum‐sensing mechanism with subtilin as autoinducer and signal transduction via SpaRK. Additionally, spaR expression was found to be under positive control of the alternative sigma factor H. Deletion of sigma H strongly decreased subtilin production. Full subtilin production could be restored after in‐trans complementation of spaR. Deletion of the major B. subtilis transition state regulator AbrB strongly increased subtilin production. These results show that the spaRK two‐component regulatory system, and hence subtilin biosynthesis and immunity, is under dual control of two independent regulatory systems: autoinduction via subtilin and transcriptional regulation via sigma factor H.
Structural basis of tropism of Escherichia coli to the bladder during urinary tract infection
Authors: Hung Chia‐Suei , Bouckaert Julie , Hung Danielle , Pinkner Jerome , Widberg Charlotte , DeFusco Anthony , Auguste C. Gale , Strouse Robert , Langermann Solomon , Waksman Gabriel , Hultgren Scott J.
Published: March 2003

The first step in the colonization of the human urinary tract by pathogenic Escherichia coli is the mannose‐sensitive binding of FimH, the adhesin present at the tip of type 1 pili, to the bladder epithelium. We elucidated crystallographically the interactions of FimH with D‐mannose. The unique site binding pocket occupied by D‐mannose was probed using site‐directed mutagenesis. All but one of the mutants examined had greatly diminished mannose‐binding activity and had also lost the ability to bind human bladder cells. The binding activity of the mono‐saccharide D‐mannose was delineated from this of mannotriose (Man(α1–3)[Man(α1–6)]Man) by gener‐ating mutants that abolished D‐mannose binding but retained mannotriose binding activity. Our structure/function analysis demonstrated that the binding of the monosaccharide α‐D‐mannose is the primary bladder cell receptor for uropathogenic E. coli and that this event requires a highly conserved FimH binding pocket. The residues in the FimH mannose‐binding pocket were sequenced and found to be invariant in over 200 uropathogenic strains of E. coli. Only enterohaemorrhagic E. coli (EHEC) possess a sequence variation within the mannose‐binding pocket of FimH, suggesting a naturally occurring mechanism of attenuation in EHEC bacteria that would prevent them from being targeted to the urinary tract.
cAMP and ras signalling independently control spore germination in the filamentous fungus Aspergillus nidulans
Authors: Fillinger Sabine , Chaveroche Marie‐Kim , Shimizu Kiminori , Keller Nancy , D’Enfert Christophe
Published: March 2003

The role of cAMP signalling during germination of asexual spores (conidia) of the filamentous fungus Aspergillus nidulans was investigated. A. nidulans
strains defective for adenylate cyclase (CyaA) or for the functionally overlapping
cAMP‐dependent protein kinase (PkaA) and newly characterized SchA protein kinase,
homologous to Saccharomyces cerevisiae Sch9, show altered trehalose mobilization
and kinetics of germ tube outgrowth, in addition to other defects in colony formation.
cAMP‐dependent trehalose breakdown is triggered by the addition of a carbon source
independently of further catabolism, suggesting that cAMP signalling controls early
events of conidial germination in response to carbon source sensing. Additional results
suggest that cAMP has targets other than PkaA and SchA and that PkaA retains activity
in the absence of cAMP. Conversely, PkaA regulates cAMP levels in A. nidulans
because these are elevated by ≈ 250‐fold in a strain that lacks PkaA. Furthermore,
analysis of mutant strains impaired in both adenylate cyclase and RasA GTPase previously
implicated in the control of A. nidulans spore germination suggested that
RasA and cAMP signalling proceed independently during germination in A. nidulans.
The effect of genomic position on reversion of a lac frameshift mutation (lacIZ33) during non‐lethal selection (adaptive mutation)
Authors: Slechta E. Susan , Harold Jennifer , Andersson Dan I. , Roth John R.
Published: March 2003

In a system described by Cairns and Foster, starvation of a particular leaky lac mutant (lacIZ33) in the presence of lactose appears to direct mutation in non‐growing cells to sites that allow growth (adaptive mutation). This behaviour requires that the lac operon be located on an F′ plasmid. This position effect was investigated by placing the mutant lac operon at many sites in the genome of Salmonella enterica (Typhimurium; LT2) and testing reversion behaviour. Genomic position did not affect reversion during non‐selective growth. When lac was at any of 550 chromosomal sites, starvation caused little or no enhancement of reversion. In the 28 strains with the lac on Salmonella’s conjugative plasmid (pSLT), selection enhanced reversion strongly, just as seen for strains with lac on an F′ plasmid. In 46 strains, the lac operon was inserted within a small chromosomal duplication, and selection stimulated RecA‐dependent partial reversion by simple amplification (about 8×) of the mutant lac region. The position of lac on a conjugative plasmid is important to reversion because it allows more frequent gene duplication and amplification. These events are central to growth and reversion under selection because they increase the number of replicating lac alleles within each developing revertant clone.
Regulatory network of lipopolysaccharide O‐antigen biosynthesis in Yersinia enterocolitica includes cell envelope‐dependent signals
Authors: Bengoechea José Antonio , Zhang Lijuan , Toivanen Paavo , Skurnik Mikael
Published: March 2003

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane of all Gram‐negative bacteria, and it is one of the signature molecules recognized by the receptors of the innate immune system. In addition to its lipid A portion (the endotoxin), its O‐chain polysaccharide (the O‐antigen) plays a critical role in the bacterium–host interplay and, in a number of bacterial pathogens, it is a virulence factor. We present evidence that, in Yersinia enterocolitica serotype O:8, a complex signalling network regulates O‐antigen expression in response to temperature. Northern blotting and reporter fusion analyses indicated that temperature regulates the O‐antigen expression at the transcriptional level. Promoter cloning showed that the O‐antigen gene cluster contains two transcriptional units under the control of promoters Pwb1 and Pwb2. The activity of both promoters is under temperature regulation and is repressed in bacteria grown at 37°C. We demonstrate that the RosA/RosB efflux pump/potassium antiporter system and Wzz, the O‐antigen chain length determinant, are indirectly involved in the regulation mainly affecting the activity of promoter Pwb2. The rosAB transcription, under the control of Pros, is activated at 37°C, and Pwb2 is repressed through the signals generated by the RosAB system activation, i.e. decreased [K+] and increased [H+]. The wzz transcription is under the control of Pwb2, and we show that, at 37°C, overexpression of Wzz downregulates slightly the Pwb1 and Pwb2 activities and more strongly the Pros activity, with the net result that more O‐antigen is produced. Finally, we demonstrate that overexpression of Wzz causes membrane stress that activates the CpxAR two‐component signal transduction system.
Transfer origins in the conjugative Enterococcus faecalis plasmids pAD1 and pAM373: identification of the pAD1 nic site, a specific relaxase and a possible TraG‐like protein
Authors: Francia M. Victoria , Clewell Don B.
Published: March 2003

The Enterococcus faecalis conjugative plasmids pAD1 and pAM373 encode a mating response to the peptide sex pheromones cAD1 and cAM373 respectively. Sequence determination of both plasmids has recently been completed with strong similarity evident over many of the structural genes related to conjugation. pAD1 has two origins of transfer, with oriT1 being located within the repA determinant, whereas the more efficiently utilized oriT2 is located between orf53 and orf57, two genes found in the present study to be essential for conjugation. We have found a similarly located oriT to be present in pAM373. oriT2 corresponds to about 285 bp based on its ability to facilitate mobilization by pAD1 when ligated to the shuttle vector pAM401; however, it was not mobilized by pAM373. In contrast, a similarly ligated fragment containing the oriT of pAM373 did not facilitate mobilization by pAD1 but was efficiently mobilized by pAM373. The oriT sites of the two plasmids each contained a homologous large inverted repeat (spanning about 140 bp) adjacent to a series of non‐homologous short (6 bp) direct repeats. A hybrid construction containing the inverted repeat of pAM373 and direct repeats of pAD1 was mobilized efficiently by pAD1 but not by pAM373, indicating a significantly greater degree of specificity is associated with the direct repeats. Mutational (deletion) analyses of the pAD1 oriT2 inverted repeat structure suggested its importance in facilitating transfer or perhaps ligation of the ends of the newly transferred DNA strand. Analyses showed that Orf57 (to be called TraX) is the relaxase, which was found to induce a specific nick in the large inverted repeat inside oriT; the protein also facilitated site‐specific recombination between two oriT2 sites. Orf53 (to be called TraW) exhibits certain structural similarities to TraG‐like proteins, although there is little overall homology.
Uptake of transforming DNA in Gram‐positive bacteria: a view from Streptococcus pneumoniae
Authors: Bergé Mathieu , Moscoso Miriam , Prudhomme Marc , Martin Bernard , Claverys Jean‐Pierre
Published: March 2003

In a working model for the uptake of transforming DNA based on evidence taken from both Bacillus subtilis and Streptococcus pneumoniae, the ComG proteins are proposed to form a structure that provides access for DNA to the ComEA receptor through the peptidoglycan. DNA would then be delivered to the ComEC–ComFA transport complex. A DNA strand would be degraded by a nuclease, while its complement is pulled into the cell by ComFA through an aqueous pore formed by ComEC. The nuclease is known in S. pneumoniae only as EndA. We have examined the processing (i.e. binding, degradation and internalization) of DNA in S. pneumoniae strains lacking candidate uptake proteins. Mutants were generated by transposon insertion in endA, comEA/C, comFA/C, comGA and dprA. Processing of DNA was abolished only in a comGA mutant. As significant binding was measured in comEA mutants, we suggest the existence of two stages in binding: surface attachment (abolished in a comGA mutant) required for and preceding deep binding (by ComEA). Abolition of degradation in comGA and comEA mutants indicated that, despite its membrane location, EndA cannot access donor DNA by itself. We propose that ComEA is required to deliver DNA to EndA. DNA was still bound and degraded in comEC and comFA mutants. We conclude that recruitment of EndA can occur in the absence of ComEC or ComFA and that EndA is active even when the single strands it produces are not pulled into the cell. Finally, inactivation of dprA had no effect on the internalization of DNA, indicating that DprA is required at a later stage in transformation.
The unfolding story of the Escherichia coli Hsp70 DnaK: is DnaK a holdase or an unfoldase?
Authors: Slepenkov Sergey V. , Witt Stephan N.
Published: March 2003

We discuss recent experiments that have illuminated individual steps in the reaction cycle of the Escherichia coli Hsp70 molecular chaperone DnaK. Using this new information, we comparetwo distinctly different global mechanisms of action – holding versus unfolding – and argue that the available evidencesuggests that DnaK is an unfoldase.
The Yersinia pseudotuberculosis Yut protein, a new type of urea transporter homologous to eukaryotic channels and functionally interchangeable in vitro with the Helicobacter pylori UreI protein
Authors: Sebbane Florent , Bury‐Moné Stéphanie , Cailliau Katia , Browaeys‐Poly Edith , De Reuse Hilde , Simonet Michel
Published: March 2003

Urea uptake in eukaryotes and prokaryotes occurs via diffusion or active transport across the cell membrane. Facilitated diffusion of urea in both types of organisms requires a single‐component channel. In bacteria, these transport systems allow rapid access of urease to its substrate, resulting in ammonia production, which is needed either for resistance to acidity or as a nitrogen source. In Yersinia pseudotuberculosis, a ureolytic enteropathogenic bacterium, a gene of unknown function (yut) located near the urease locus was found to encode a putative membrane protein with weak homology to single‐component eukaryotic urea transporters. When expressed in Xenopus oocytes, Yut greatly increases cellular permeability to urea. Inactivation of yut in Y. pseudotuberculosis results in diminished apparent urease activity and reduced resistance to acidity in vitro when urea is present in the medium. In the mouse model, bacterial colonization of the intestine mucosa is delayed with the Yut‐deficient mutant. Although structurally unrelated, Yut and the Helicobacter pylori UreI urea channel were shown to be functionally interchangeable in vitro and are sufficient to allow urea uptake in both bacteria, thereby confirming their function in the respective parent organisms. Homologues of Yut were found in other yersiniae, Actinobacillus pleuropneumoniae, Brucella melitensis, Pseudomonas aeruginosa and Staphylococcus aureus. The Y. pseudotuberculosis Yut protein is therefore the first member of a novel class of bacterial urea permeases related to eukaryotic transporters.
Conservation of dynamic localization among MinD and MinE orthologues: oscillation of Neisseria gonorrhoeae proteins in Escherichia coli
Authors: Ramirez‐Arcos Sandra , Szeto Jason , Dillon Jo‐Anne R. , Margolin William
Published: March 2003

Min proteins are involved in the correct placement of division septa in many bacterial species. In Escherichia coli (Ec) cells, these proteins oscillate from pole to pole, ostensibly to prevent unwanted polar septation. Here, we show that Min proteins from the coccus Neisseria gonorrhoeae (Ng) also oscillate in E. coli. Green fluorescent protein (GFP) fusions to gonococcal MinD and MinE localized dynamically in different E. coli backgrounds. GFP–MinDNg moved from pole to pole in rod‐shaped E. coli cells with a 70 ± 25 s localization cycle when MinENg was expressed in cis. The oscillation time of GFP–MinDNg was reduced when wild‐type MinENg was replaced with MinENg carrying a R30D mutation, but lengthened by 15 s when activated by MinEEc. Several mutations in the N‐terminal domain of MinDNg, including K16Q and 4‐ and 19‐amino acid truncations, prevented oscillation; these MinDNg mutants showed decreased or lost interaction with themselves and MinENg. Like MinEEc–GFP, MinENg–GFP formed MinE rings and oscillated in E. coli cells when MinDEc was expressed in cis. Finally, in round E. coli cells, GFP–MinDNg appeared to move in a plane parallel to completed septa. This pattern of movement is predicted to be similar in gonococcal cells, which also divide in alternating perpendicular planes.
Evolution of the paralogous hap and iga genes in Haemophilus influenzae: evidence for a conserved hap pseudogene associated with microcolony formation in the recently diverged Haemophilus aegyptius and H. influenzae biogroup aegyptius
Authors: Kilian Mogens , Poulsen Knud , Lomholt Hans
Published: March 2003

Certain non‐capsulate strains belonging to the Haemophilus influenzae/Haemophilus aegyptius complex show unusually high pathogenicity, but the evolutionary origin of these virulent phenotypes, termed H. influenzae biogroup aegyptius, is as yet unknown. The aim of the present study was to elucidate the mechanisms of evolution of two paralogous genes, hap and iga, which encode the adhesion and penetration Hap protein and the IgA1 protease respectively. Partial sequencing of hap and iga genes in a comprehensive collection of strains belonging to the H. influenzae/H. aegyptius complex revealed considerable genetic polymorphism and pronounced mosaic‐like patterns in both genes, but no evidence of intrastrain recombination between the two genes. A conserved hap pseudogene was present in all strains of H. aegyptius and H. influenzae biogroup aegyptius, each of which constituted distinct subpopulations as revealed by phylogenetic analysis. There was no evidence for a second, functional copy of the hap gene in these strains. The perturbed expression of the Hap serine protease appears to be associated with the formation of elongated bacterial cells growing in chains and a distinct colonization pattern on conjunctival cells, previously termed microcolony formation. The fact that individual hap pseudogenes differed from the ancestral sequence by zero to two positions within a 1.5 kb stretch suggests that the silencing event happened ≈ 2000–11 000 years ago. Divergence of H. aegyptius and H. influenzae biogroup aegyptius occurred subsequent to this genetic event. The loss of Hap protein expression may be one of the genetic events that facilitated exploitation of the conjunctivae as a new niche.
Linkage disequilibrium between minisatellite loci supports clonal evolution of Mycobacterium tuberculosis in a high tuberculosis incidence area
Authors: Supply Philip , Warren Robin M. , Bañuls Anne‐Laure , Lesjean Sarah , Van Der Spuy Gian D. , Lewis Lee‐Anne , Tibayrenc Michel , Van Helden Paul D. , Locht Camille
Published: March 2003

Deciphering the structure of pathogen populations is instrumental for the understanding of the epidemiology and history of infectious diseases and for their control. Although Mycobacterium tuberculosis is the most widespread infectious agent in humans, its actual population structure has remained hypothetical until now because: (i) its structural genes are poorly polymorphic; (ii) adequate samples and appropriate statistics for population genetic analysis have not been considered. To investigate this structure, we analysed the statistical associations (linkage disequilibrium) between 12 independent M. tuberculosis minisatellite‐like loci by high‐throughput genotyping within a model population of 209 isolates representative of the genetic diversity in an area with a very high incidence of tuberculosis. These loci contain variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs). Highly significant linkage disequilibrium was detected among the MIRU‐VNTR loci in this model. This linkage disequilibrium was also evident when the MIRU‐VNTR types were compared with the IS6110 restriction fragment length polymorphism types. These results support a predominant clonal evolution of M. tuberculosis.
The spa‐box for transcriptional activation of subtilin biosynthesis and immunity in Bacillus subtilis
Authors: Stein Torsten , Heinzmann Stefan , Kiesau Peter , Himmel Bettina , Entian Karl‐Dieter
Published: March 2003

The subtilin gene cluster (spa) of Bacillus subtilis ATCC 6633 is organized in transcriptional units spaBTC, spaS, spaIFEG and spaRK. Specific binding of the response regulator protein SpaR to spaB, spaS and spaI DNA promoter fragments was shown by means of electromobility shift assays. A repeated pentanucleotide sequence spaced by six nucleotides was identified as SpaR binding motif (spa‐box). Saturating mutational analysis of the spa‐box by single‐ and multiple‐base‐pair substitutions revealed the consensus motif (A/T)TGAT for optimal SpaR binding with the second, third and fifth position being absolutely conservative. Variations in the spacer size between the two pentanucleotide repeats revealed a strong conservation of their relative location. Only DNA with a proximal arrangement of two pentanucleotide repeats showed affinity to SpaR. A 2:1 stoichiometry between SpaR and DNA was obtained by optical biosensor analyses, which corresponds to the binding of two SpaR proteins per spa‐box.
Axe–Txe, a broad‐spectrum proteic toxin–antitoxin system specified by a multidrug‐resistant, clinical isolate of Enterococcus faecium
Authors: Grady Ruth , Hayes Finbarr
Published: March 2003

Enterococcal species of bacteria are now acknowledged as leading causes of bacteraemia and other serious nosocomial infections. However, surprisingly little is known about the molecular mechanisms that promote the segregational stability of antibiotic resistance and other plasmids in these bacteria. Plasmid pRUM (24 873 bp) is a multidrug resistance plasmid identified in a clinical isolate of Enterococcus faecium. A novel proteic‐based toxin–antitoxin cassette identified on pRUM was demonstrated to be a functional segregational stability module in both its native host and evolutionarily diverse bacterial species. Induced expression of the toxin protein (Txe) of this system resulted in growth inhibition in Escherichia coli. The toxic effect of Txe was alleviated by co‐expression of the antitoxin protein, Axe. Homologues of the axe and txe genes are present in the genomes of a diversity of Eubacteria. These homologues (yefM–yoeB) present in the E. coli chromosome function as a toxin–antitoxin mechanism, although the Axe and YefM antitoxin components demonstrate specificity for their cognate toxin proteins in vivo. Axe–Txe is one of the first functional proteic toxin–antitoxin systems to be accurately described for Gram‐positive bacteria.