bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence variations in alleles of the avirulence gene avrPphE.R2 from Pseudomonas syringae pv. phaseolicola lead to loss of recognition of the AvrPphE protein within bean cells and a gain in cultivar‐specific virulence
Authors: Stevens Conrad , Bennett Mark A. , Athanassopoulos Evangelos , Tsiamis George , Taylor John D. , Mansfield John W.
Published: March 2003

The bean halo blight pathogen, Pseudomonas syringae pv. phaseolicola (Psph), is differentiated into nine races based on the presence or absence of five avirulence (avr ) genes in the bacterium, which interact with corresponding resistance genes, R1–R5, in Phaseolus vulgaris. The resistance gene R2 is matched by avrPphE, which is located adjacent to the cluster of hrp genes that are required for pathogenicity of Psph. Although only races 2, 4, 5 and 7 are avirulent on cultivars with R2 (inducing the hypersensitive response; HR), homologues of avrPphE are present in all races of Psph. DNA sequencing of avrPphE alleles from races of Psph has demonstrated two routes to virulence: via single basepair changes conferring amino acid substitutions in races 1, 3, 6 and 9 and an insertion of 104 bp in the allele in race 8. We have demonstrated that these base changes are responsible for the difference between virulence and avirulence by generating transconjugants of a virulent race harbouring plasmids expressing the various alleles of avrPphE. Agrobacterium tumefaciens‐directed expression of avrPphE from race 4 in bean leaves induced the HR in a resistance gene‐specific manner, suggesting that the AvrPphE protein is alone required for HR induction and is recognized within the plant cell. The allele from race 6, which is inactive if expressed in Psph, elicited a weak HR if expressed in planta, whereas the allele from race 1 did not. Our results suggest that the affinity of interaction between AvrPphE homologues and an unknown plant receptor mediates the severity of the plant's response. Mutation of avrPphE alleles did not affect the ability to colonize bean from a low level of inoculum. The avirulence gene avrPphB, which matches the R3 resistance gene, also caused a gene‐specific HR following expression in the plant after delivery by A. tumefaciens.
botR/A is a positive regulator of botulinum neurotoxin and associated non‐toxin protein genes in Clostridium botulinum A
Authors: Marvaud J. C. , Gibert M. , Inoue K. , Fujinaga Y. , Oguma K. , Popoff M. R.
Published: March 2003

The genes of the botulinum neurotoxin A (BoNT) complex are clustered in a locus consisting of two divergent polycistronic operons, one containing the non‐toxic, non‐haemagglutinin (NTNH) component and bontA genes, the other containing the haemagglutinin (HA) component genes. The two operons are separated by a gene (botR/A, previously called orf21) encoding a 21 kDa protein. A recombinant Clostridium botulinum A strain that overexpresses botR/A was constructed by electroporating strain 62 with the vector pAT19 containing botR/A under the control of its own promoter. The transformed strain produced more BoNT/A and associated non‐toxic proteins (ANTPs) and the corresponding mRNAs than the non‐transformed strain. Partial inhibition of botR/A by antisense mRNA resulted in lower levels of BoNT/A, NTNH and HA70 and the levels of the corresponding mRNAs. Gel mobility shift assays and immunoprecipitations showed that BotR/A bound to the DNA promoter region upstream from the two BoNT/A complex operons. These results show that botR/A activated transcription of the genes encoding BoNT/A and ANTPs in C. botulinum A by interacting directly with the region promoter, and that the homologous genes in C. botulinum B, C and D presumably have the same function.
Investigation of mycobacterial recA function: protein introns in the RecA of pathogenic mycobacteria do not affect competency for homologous recombination
Authors: Frischkorn Klaus , Sander Peter , Scholz Matthias , Teschner Kerstin , Prammananan Therdsak , Böttger Erik C.
Published: March 2003

The recA locus of pathogenic mycobacteria differs from that of non‐pathogenic species in that it contains large intervening sequences termed protein introns or inteins that are excised by an unusual protein‐splicing reaction. In addition, a high degree of illegitimate recombination has been observed in the pathogenic Mycobacterium tuberculosis complex. Homologous recombination is the main mechanism of integration of exogenous nucleic acids in M. smegmatis, a non‐pathogenic mycobacterium species that carries an inteinless RecA and is amenable to genetic manipulations. To investigate the function of recA in mycobacteria, recA− strains of M. smegmatis were generated by allelic exchange techniques. These strains are characterized (i) by increased sensitivity towards DNA‐damaging agents [ethylmethylsulphonate (EMS), mitomycin C, UV irradiation] and (ii) by the inability to integrate nucleic acids by homologous recombination. Transformation efficiencies using integrative or replicative vectors were not affected in recA− mutants, indicating that in mycobacteria RecA does not affect plasmid uptake or replication. Complementation of the recA− mutants with the recA from M. tuberculosis restored resistance towards EMS, mitomycin C and UV irradiation. Transformation of the complemented strains with suicide vectors targeting the pyrF gene resulted in numerous allelic exchange mutants. From these data, we conclude that the intein apparently does not interfere with RecA function, i.e. with respect to competency for homologous recombination, the RecAs from pathogenic and non‐pathogenic mycobacteria are indistinguishable.
Toxin binding site of the diphtheria toxin receptor: loss and gain of diphtheria toxin binding of monkey and mouse heparin‐binding, epidermal growth factor‐like growth factor precursors by reciprocal site‐directed mutagenesis
Authors: Cha Jeong‐Heon , Brooke Joanna S. , Eidels Leon
Published: March 2003

The transmembrane precursor of the monkey (Mk) heparin‐binding, epidermal growth factor‐like growth factor (proHB‐EGF) functions as a diphtheria toxin (DT) receptor, whereas the mouse (Ms) precursor does not. Previously, using chimeric Ms/Mk precursors, we have shown that DT resistance of cells bearing Ms proHB‐EGF may be accounted for by several amino acid substitutions between residues 122 and 148 within the EGF‐like domain and that Glu‐141 is an important amino acid residue for DT binding. In this study, reciprocal site‐directed mutagenesis was performed on the major non‐conserved residues in the region of 122–148, alone or in combination, between Mk and Ms precursors to identify more precisely which amino acid residues are important for DT binding. Two approaches were used. The first, more traditional approach was to destroy DT sensitivity and binding of Mk proHB‐EGF by substitution(s) with the corresponding Ms residue(s). From the single mutations, the greatest loss of DT sensitivity was observed with Mk/Glu‐141His (approximately 4000‐fold) and the next greatest with Mk/Ile‐133Lys (approximately fourfold). The double mutations Mk/Leu‐127Phe/Glu‐141His, Mk/Ile‐133Lys/Glu‐141His and Mk/His‐135Leu/Glu‐141His resulted in complete toxin resistance (> 100 000‐fold). The second approach, both novel and complementary, was to gain DT binding and sensitivity of Ms proHB‐EGF by substitution(s) with the corresponding Mk residue(s). Surprisingly, the single mutation Ms/His‐141Glu resulted in the gain of moderate DT sensitivity (> 260‐fold). The double mutation Ms/Lys‐133Ile/His‐141Glu and the triple mutation Ms/Lys‐133Ile/Leu‐135His/His‐141Glu resulted in a progressive gain in toxin sensitivity (> 4700‐fold and > 16 000‐fold respectively) and affinity. This triple mutant cell line is essentially as sensitive (IC50 = 3.1 ng ml−1) as the highly toxin‐sensitive monkey Vero cell line (IC50 = 4 ng ml−1), indicating that these three Mk residues enable the Ms proHB‐EGF to act as a fully functional DT receptor. Taken together, these results indicate that Glu‐141 plays the most critical role in DT binding and sensitivity and that two additional amino acid residues, Ile‐133 and His‐135, also play significant roles.
The identification of exported proteins with gene fusions to invasin
Authors: Worley Micah J. , Stojiljkovic Igor , Heffron Fred
Published: March 2003

Exported proteins are integral to understanding the biology of bacterial organisms. They have special significance in pathogenesis research because they can mediate critical interactions between pathogens and eukaryotic cell surfaces. Further, they frequently serve as targets for vaccines and diagnostic tests. The commonly used genetic assays for identifying exported proteins use fusions to alkaline phosphatase or beta‐lactamase. These systems are not ideal for identifying outer membrane proteins because they identify a large number of inner membrane proteins as well. We addressed this problem by developing a gene fusion system that preferentially identifies proteins that contain cleavable signal sequences and are released from the inner membrane. This system selects fusions that restore outer membrane localization to an amino terminal‐truncated Yersinia pseudotuberculosis invasin derivative. In the present study, a variety of Salmonella typhimurium proteins that localize beyond the inner membrane were identified with gene fusions to this invasin derivative. Previously undescribed proteins identified include ones that share homology with components of fimbrial operons, multiple drug resistance efflux pumps and a haemolysin. All of the positive clones analysed contain cleavable signal sequences. Moreover, over 40% of the genes identified encode putative outer membrane proteins. This system has several features that may make it especially useful in the study of genetically intractable organisms.
Adaptation of Haemophilus influenzae to acquired and innate humoral immunity based on phase variation of lipopolysaccharide
Authors: Weiser Jeffrey N. , Pan Nina
Published: March 2003

Phase variation in colony morphology has been associated with the pathogenesis of infection caused by Haemophilus influenzae. This study shows that differences in colony opacity in non‐typeable H. influenzae (NTHi) strain H233 involve phase changes in the lipopolysaccharide (LPS) and depend on the expression of lic1 and lic2, which contain translational switches based on intragenic tandem repeats of 5′‐CAAT‐3′. Genetic analysis showed that opaque organisms have an out‐of‐frame number of repeats in both lic1, required for the expression of phosphorylcholine (ChoP), and lic2, a putative galactosyl transferase that adds the terminal galactose on Galα1‐4Gal. Defined variants in these loci were used to examine the contribution of individual LPS structures to resistance to serum bactericidal activity mediated by antibody and C‐reactive protein (CRP). The addition of ChoP by lic1 was the only factor in serum killing involving CRP and complement. The terminal galactose moiety, in contrast, conferred resistance to killing by naturally acquired antibody and complement present in human serum. As Galα1‐4Gal is also found on human glycolipids, it appears that decoration of the cell surface with this host‐like antigen blocks antibody‐mediated serum bactericidal activity. Genetic analysis of NTHi within the human respiratory tract demonstrated that Galα1‐4Gal may not be expressed during carriage but may be advantageous for the organism in inflammatory states such as pneumonia.
Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA
Authors: Luesink Evert J. , Van Herpen René E. M. A , Grossiord Benoît P. , Kuipers Oscar P. , De Vos Willem M.
Published: March 2003

The Lactococcus lactis ccpA gene, encoding the global regulatory protein CcpA, was identified and characterized. Northern blot and primer extension analyses showed that the L. lactis ccpA gene is constitutively transcribed from a promoter that does not contain a cre sequence. Inactivation of the ccpA gene resulted in a twofold reduction in the growth rate compared with the wild type on glucose, sucrose and fructose, while growth on galactose was almost completely abolished. The observed growth defects could be complemented by the expression of either the L. lactis or the Bacillus subtilis ccpA gene. The disruption of the ccpA gene reduced the catabolite repression of the gal operon, which contains a cre site at the transcription start site and encodes enzymes involved in galactose catabolism. In contrast, CcpA activates the transcription of the cre‐containing promoter of the las operon, encoding the glycolytic enzymes phosphofructokinase, pyruvate kinase and L‐lactate dehydrogenase, because its transcription level was fourfold reduced in the ccpA mutant strain compared with the wild‐type strain. The lower activities of pyruvate kinase and L‐lactate dehydrogenase in the ccpA mutant strain resulted in the production of metabolites characteristic of a mixed‐acid fermentation, whereas the fermentation pattern of the wild‐type strain was essentially homolactic.
Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium
Authors: Björkman Johanna , Samuelsson Patrik , Andersson Dan I. , Hughes Diarmaid
Published: March 2003

Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation. Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE. Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors. We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium. To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium. We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type. Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL, being resistant to streptomycin and restrictive (hyperaccurate) in translation. These phenotypes have not been previously associated with the ribosomal protein S4. Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL. This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity (ram) phenotype.
Carbon catabolite repression of the Aspergillus nidulans xlnA gene
Authors: Orejas Margarita , MacCabe Andrew Peter , Pérez González José Antonio , Kumar Sudeep , Ramón Daniel
Published: March 2003

Expression of the Aspergillus nidulans 22 kDa endoxylanase gene, xlnA, is controlled by at least three mechanisms: specific induction by xylan or xylose; carbon catabolite repression (CCR); and regulation by ambient pH. Deletion analysis of xlnA upstream sequences has identified two positively acting regions: one that mediates specific induction by xylose; and another that mediates the influence of ambient pH and contains two PacC consensus binding sites. The extreme derepressed mutation creAd30 results in considerable, although not total, loss of xlnA glucose repressibility, indicating a major role for CreA in its CCR. Three consensus CreA binding sites are present upstream of the structural gene. Point mutational analysis using reporter constructs has identified a single site, xlnA.C1, that is responsible for direct CreA repression in vivo. Using the creAd30 derepressed mutant background, our results indicate the existence of indirect repression by CreA.
Ecs, an ABC transporter of Bacillus subtilis: dual signal transduction functions affecting expression of secreted proteins as well as their secretion
Authors: Leskelä Soile , Wahlström Eva , Hyyryläinen Hanne‐Leena , Jacobs Myra , Palva Airi , Sarvas Matti , Kontinen Vesa P.
Published: March 2003

ecs is a three‐cistron operon of Bacillus subtilis, encoding proteins with similarity to the ATPase (EcsA) and hydrophobic components (EcsB) of ABC transporters. The ecsA26 point mutation was shown to cause a strong processing defect of a secreted α‐amylase precursor (preAmyQ) and of three other exoproteins. Northern analysis of the level of amyQ mRNA showed that ecsA26 also decreases amyQ transcription. This effect too was pleiotropic, as judged by a drastic decrease in the expression from an exoprotease promoter of a reporter protein. A knockout mutation of the ecsB cistron caused a processing defect similar to ecsA26 but, unlike ecsA26, did not affect amyQ transcription. There was also no defect in transcription in the ecsA ecsB double mutant. Thus, an intact ecsB product was required for the downregulation of amyQ by the mutant ecsA. These results suggest a dual regulatory function for Ecs, in which Ecs, possibly as part of a signal transduction mechanism, regulates some component(s) of the protein secretion apparatus as well as secretory protein transcription in a co‐ordinated fashion.
Regulation of excision of the conjugative transposon Tn916
Authors: Marra Diana , Scott June R.
Published: March 2003

Excision from the donor DNA molecule is the first step in conjugative transposition of Tn916 and is followed by circularization of the transposon and its transfer to a new host. We have demonstrated that, in Gram‐positive hosts, the Xis protein, as well as the site‐specific recombinase Int, is required for the excision of Tn916. Using assays for closure of the excised covalently closed transposon and for repair of the donor DNA molecule, we found that neither protein alone is rate limiting for excision, but overexpression of Int and Xis together results in increased excision. After excision, the frequency of Tn916 circle formation was found to be the same as the frequency of repair of the donor DNA molecule. This suggests that a single reaction results in the closure of both molecules. We have also identified two transcripts that encode Int, one of which also encodes Xis and one of which does not, suggesting that there are steps in conjugative transposition of Tn916 that require Int without Xis.
The hxB gene, necessary for the post‐translational activation of purine hydroxylases in Aspergillus nidulans, is independently controlled by the purine utilization and the nicotinate utilization transcriptional activating systems
Authors: Amrani Laıamp;;uml;la , Cecchetto Gianna , Scazzocchio Claudio , Glatigny Annie
Published: March 2003

Molybdenum‐containing enzymes of the hydroxylase class (such as xanthine dehydrogenase, aldehyde oxidase and nicotinate dehydrogenase) require a terminal sulphur atom attached to the molybdenum to hydroxylate their specific substrates. The transulphurylation reaction is carried out in Drosophila melanogaster by the product of the ma‐l gene. In Aspergillus nidulans, the activity of the isofunctional and homologous HxB protein is needed in at least two different metabolic contexts, when the organism grows on purines and when it grows on nicotinate as nitrogen sources. We show here that the expression of the hxB gene is not constitutive. It is induced independently and additively by the inducers of the purine and of the nicotinate utilization pathways. Each of these induction pathways is affected independently by mutations in their cognate genes, uric acid induction by mutations in the UaY protein and nicotinate and 6‐nicotinate induction by those in the hxnR/aplA complex. It is, in both metabolic contexts, exquisitely sensitive to nitrogen metabolite repression and highly dependent on the AreA GATA factor.
Molecular characterization of the pH‐inducible and growth phase‐dependent promoter P170 of Lactococcus lactis
Authors: Madsen Søren M. , Arnau José , Vrang Astrid , Givskov Michael , Israelsen Hans
Published: March 2003

In a previous study, we described the use of transposon Tn917‐LTV1 for identification of environmentally regulated promoters in Lactococcus lactis. Here, we report the molecular analysis of one of these promoters, P170, that is upregulated at low pH during the transition to stationary phase. The minimal DNA region required for both promoter activity and pH regulation was mapped to a 51 bp fragment located 7 bp upstream of the transcriptional start site. This fragment lacked the consensus −35 promoter region, but it contained an ‘extended’−10 promoter region. When a 28 bp segment, containing the consensus −35 region and 22 bp upstream of this in a constitutive promoter, was replaced with the corresponding sequence of P170, the hybrid promoter became regulated by pH and growth phase. This demonstrates that the P170 segment contains a cis‐acting sequence involved in the control of promoter regulation. Transcriptional analysis showed that P170 is responsible for the transcription of a monocistronic gene orfX encoding a polypeptide homologous to a hypothetical protein from Bacillus subtilis. Analysis of total RNA from L. lactis grown at constant pH confirmed that transcription from P170 was induced between pH 6.5 and pH 6.0, but only when the culture entered stationary phase. Deletion analysis and chemical mutagenesis of P170 defined a specific region within the untranslated mRNA leader that is able to modulate the expression level directed by the P170 promoter. Deletion of a 72 bp HaeIII fragment from this leader region resulted in a 150‐ to 200‐fold increase in the level of gene expression, without affecting the regulation. The functionality was confirmed by introducing this modulating element downstream of other lactococcal promoters.
Cell cycle‐dependent degradation of a flagellar motor component requires a novel‐type response regulator
Authors: Aldridge Phillip , Jenal Urs
Published: March 2003

The poles of each Caulobacter crescentus cell undergo morphological development as a function of the cell cycle. A single flagellum assembled at one pole during the asymmetric cell division is later ejected and replaced by a newly synthesized stalk when the motile swarmer progeny differentiates into a sessile stalked cell. The removal of the flagellum during the swarmer‐to‐stalked cell transition coincides with the degradation of the FliF flagellar anchor protein. We report here that the cell cycle‐dependent turnover of FliF does not require the structural components of the flagellum itself, arguing that it is the initial event leading to the ejection of the flagellum. Analysis of a polar development mutant, pleD, revealed that the pleD gene was required for efficient removal of FliF and for ejection of the flagellar structure during the swarmer‐to‐stalked cell transition. The PleD requirement for FliF degradation was also not dependent on the presence of any part of the flagellar structure. In addition, only 25% of the cells were able to synthesize a stalk during cell differentiation when PleD was absent. The pleD gene codes for a member of the response regulator family with a novel C‐terminal regulatory domain. Mutational analysis confirmed that a highly conserved motif in the PleD C‐terminal domain is essential to promote both FliF degradation and stalk biogenesis during cell differentiation. Signalling through the C‐terminal domain of PleD is thus required for C. crescentus polar development. A second gene, fliL, was shown to be required for efficient turnover of FliF, but not for stalk biogenesis. The possible roles of PleD and FliL in C. crescentus polar development are discussed.
Microbial genomes
Authors: Pallen Mark J.
Published: March 2003

Microbial genome sequencing is driven by the need to understand and control pathogens and to exploit extremophiles and their enzymes in bioremediation and industry. It is hard for the traditional bacteriologist to grasp the scale and pace of the venture. Around two dozen microbial genomes have now been completed and, within a decade, genomes from every significant species of bacterial pathogen of humans, animals and plants will have been sequenced. Indeed, we will often have more than one sequence from a species or genus — for example, we already have sequences from two strains of Helicobacter pylori, from two strains of Mycobacterium tuberculosis and from three species of Pyrococcus. However, genome sequencing risks becoming expensive molecular stamp‐collecting without the tools to mine the data and fuel hypothesis‐driven laboratory‐based research. Bioinformatics, twinned with the new experimental approaches forming ‘functional genomics’, provides some of the needed tools. Nonetheless, there will be an increasing need for us to explore the detailed implications of genomic findings. Microbial genome sequencing thus represents not a threat, but an exciting opportunity for molecular microbiologists.
Identification and characterization of a stress‐responsive promoter in the macromolecular synthesis operon of Bacillus subtilis
Authors: Liao Chao‐Tsai , Wen Yu‐Der , Wang Wen‐Horng , Chang Ban‐Yang
Published: March 2003

Bacillus subtilis DB1005 is a temperature‐sensitive (Ts) sigA mutant. Induction of σA has been observed exclusively in this mutant harbouring extra copies of the plasmid‐borne Ts sigA gene transcriptionally controlled by the P1P2 promoters of the B. subtilis macromolecular synthesis (MMS; rpoD or sigA) operon. Investigation of the mechanisms leading to the induction has allowed us to identify a σB‐type promoter, P7, in the MMS operon for the first time. Therefore, at least seven promoters in total are responsible for the regulation of the B. subtilis MMS operon, including the four known σA‐ and σH‐type promoters, as well as two incompletely defined promoters. The P7 promoter was activated in B. subtilis after the imposition of heat, ethanol and salt stresses, indicating that the MMS operon of B. subtilis is subjected to the control of general stress. The significant heat induction of P7 in B. subtilis DB1005 harbouring a plasmid‐borne Ts sigA gene can be explained by a model of competition between σA and σB for core binding; very probably, the σB factor binds more efficiently to core RNA polymerase under heat shock. This mechanism may provide a means for the expression of the B. subtilis MMS operon when σA becomes defective in core binding.
Sialic acid in the lipopolysaccharide of Haemophilus influenzae: strain distribution, influence on serum resistance and structural characterization
Authors: Hood Derek W. , Makepeace Katherine , Deadman Mary E. , Rest Richard F. , Thibault Pierre , Martin Adele , Richards James C. , Moxon E. Richard
Published: March 2003

A survey of Haemophilus influenzae strains indicated that around one‐third of capsular strains and over two‐thirds of non‐typeable strains included sialic acid in their lipopolysaccharides (LPS). Mutation of the CMP‐Neu5Ac synthetase gene (siaB) resulted in a sialylation‐deficient phenotype. Isogenic pairs, wild type and siaB mutant of two non‐typeable strains were used to demonstrate that sialic acid influences resistance to the killing effect of normal human serum but has little effect on attachment to, or invasion of, cultured human epithelial cells or neutrophils. We determine for the first time the site of attachment of sialic acid in the LPS of a non‐typeable strain and report that a small proportion of glycoforms include two sialic acid residues in a disaccharide unit.
Identification of CesT, a chaperone for the type III secretion of Tir in enteropathogenicEscherichia coli
Authors: Elliott Simon J. , Hutcheson Steven W. , Dubois Maria S. , Mellies Jay L. , Wainwright Leslie A. , Batchelor Miranda , Frankel Gad , Knutton Stuart , Kaper James B.
Published: March 2003

The locus of enterocyte effacement of enteropathogenicEscherichia coliencodes a type III secretion system, an outer membrane protein adhesin (intimin, the product ofeae ) and Tir, a translocated protein that becomes a host cell receptor for intimin. Many type III secreted proteins require chaperones, which function to stabilize proteins, prevent inappropriate protein–protein interactions and aid in secretion. An open reading frame located betweentir and eae, previously namedorfU, was predicted to encode a protein with partial similarity to theYersiniaSycH chaperone. We examined the potential of theorfUgene product to serve as a chaperone for Tir. TheorfUgene encoded a 15 kDa cytoplasmic protein that specifically interacted with Tir as demonstrated by the yeast two‐hybrid assay, column binding and coimmunoprecipitation experiments. AnorfUmutant was defective in attaching–effacing lesion formation and Tir secretion, but was unaffected in expression of other virulence factors. OrfU appeared to stabilize Tir levels in the cytoplasm, but was not absolutely necessary for secretion of Tir. Based upon the physical similarities, phenotypic characteristics and the demonstrated interaction with Tir,orfUis redesignated ascesTfor thechaperone forE. colisecretion ofT ir.
Mitogen‐activated protein kinase Mkp1 of Pneumocystis carinii complements the slt2Δ defect in the cell integrity pathway of Saccharomyces cerevisiae
Authors: Fox Deborah , Smulian A. George
Published: March 2003

Signal transduction pathways are important in the adaptive response of microbes to their environment. A Pneumocystis carinii extracellular signal‐regulated protein kinase (MAPK) homologue, Mkp1, has been isolated by sequence similarity screening of P. carinii genomic DNA. The Mkp1 of P. carinii shows closest homology to other fungal MAP kinases involved in cell integrity signal transduction cascades, including Slt2p/Mpk1p of Saccharomyces cerevisiae, Mkc1 of Candida albicans and Mps1 of Magnaporthe grisea. Defects of Slt2p in S. cerevisiae result in phenotypes of slow growth, and temperature sensitivity in the absence of an osmostabilizer. Overexpression of mkp1 in a strain with the slt2Δ defect fully restored the normal growth rate, and partially reduced lysis at elevated temperatures. Complementation of the slt2Δ defect by Mkp1 demonstrates that Mkp1 is a functional MAP kinase, and that it may be the MAP kinase component of a similar signal transduction cascade within P. carinii. Furthermore, Mkp1 is activated in vitro upon the exposure of P. carinii to conditions of oxidative stress. The investigation of a MAP kinase signal transduction pathway of P. carinii will result in both a better understanding of the mechanism the organism utilizes to respond to environmental changes, and a system to assay responses to these changes.
Identification of a fourth cheY gene in Rhodobacter sphaeroides and interspecies interaction within the bacterial chemotaxis signal transduction pathway
Authors: Shah Deepan S. H. , Porter Steven L. , Harris Daniel C. , Wadhams George H. , Hamblin Paul A. , Armitage Judith P.
Published: March 2003

The Escherichia coli chemotaxis signal transduction pathway has: CheA, a histidine protein kinase; CheW, a linker between CheA and sensory proteins; CheY, the effector; and CheZ, a signal terminator. Rhodobacter sphaeroides has multiple copies of these proteins (2 × CheA, 3 × CheW and 3 × CheY, but no CheZ). In this study, we found a fourth cheY and expressed these R. sphaeroides proteins in E. coli. CheA2 (but not CheA1) restored swarming to an E. coli cheA mutant (RP9535). CheW3 (but not CheW2) restored swarming to a cheW mutant of E. coli (RP4606). R. sphaeroides CheYs did not affect E. coli lacking CheY, but restored swarming to a cheZ strain (RP1616), indicating that they can act as signal terminators in E. coli. An E. coli CheY, which is phosphorylated but cannot bind the motor (CheY109KR), was expressed in RP1616 but had no effect. Overexpression of CheA2, CheW2, CheW3, CheY1, CheY3 and CheY4 inhibited chemotaxis of wild‐type E. coli (RP437) by increasing its smooth‐swimming bias. While some R. sphaeroides proteins restore tumbling to smooth‐swimming E. coli mutants, their activity is not controlled by the chemosensory receptors. R. sphaeroides possesses a phosphorelay cascade compatible with that of E. coli, but has additional incompatible homologues.
Identification of a novel genetic locus that is required for in vitro adhesion of a clinical isolate of enterohaemorrhagic Escherichia coli to epithelial cells
Authors: Nicholls Larissa , Grant Travis H. , Robins‐Browne Roy M.
Published: March 2003

Enterohaemorrhagic Escherichia coli (EHEC) are food‐borne intestinal pathogens with a low infectious dose. Adhesion of some EHEC strains to epithelial cells is attributed, in part, to intimin, but other factors may be required for the intestinal colonizing ability of these bacteria. In order to identify additional adherence factors of EHEC, we generated transposon mutants of a clinical EHEC isolate of serotype O111:H–, which displayed high levels of adherence to cultured Chinese hamster ovary (CHO) cells. One mutant was markedly deficient in CHO cell adherence, human red blood cell agglutination and autoaggregation. Sequence analysis of the gene disrupted in this mutant revealed a 9669 bp novel chromosomal open reading frame (ORF), which was designated efa1, for EHEC factor for adherence. efa1 displayed 28% amino acid identity with the predicted product of a recently described ORF from the haemolysin‐encoding plasmid of EHEC O157:H7. The amino termini of the putative products of these two genes exhibit up to 38% amino acid similarity to Clostridium difficile toxins A and B. efa1 occurred within a novel genetic locus, at least 15 kb in length, which featured a low G+C content, several insertion sequence homologues and a homologue of the Shigella flexneri enterotoxin ShET2. DNA probes prepared from different regions of efa1 hybridized with all of 116 strains of attaching–effacing E. coli (AEEC) of a variety of serotypes, including enteropathogenic E. coli (EPEC) and EHEC, but with none of 91 non‐AEEC strains. Nevertheless, efa1 was not required for the attachment–effacement phenotype, and the efa1 locus was not physically linked to the locus for enterocyte effacement (LEE) pathogenicity island, which is responsible for this phenotype in EPEC. These findings suggest that efa1 encodes a novel virulence‐associated determinant of AEEC, which contributes to the adhesive capacity of these bacteria.
The position of phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae affects binding and sensitivity to C‐reactive protein‐mediated killing
Authors: Lysenko Elena , Richards James C. , Cox Andrew D. , Stewart Andrea , Martin Adele , Kapoor Miki , Weiser Jeffrey N.
Published: March 2003

The lic1 locus of Haemophilus influenzae controls the incorporation of environmental choline into lipopolysaccharide (LPS) as phosphorylcholine (ChoP) as well as the phase variation of this structure. ChoP is the target of an acute phase reactant in serum, C‐reactive protein (CRP), which mediates killing through the activation of complement when bound to the organism. Structural analysis of the oligosaccharide region of the H. influenzae LPS showed that ChoP is linked to different hexose residues on different chain extensions in strains Rd and Eagan. Differences in the molecular environment of ChoP affect the epitope defined by monoclonal antibody 12D9 and were associated with polymorphisms within LicD, a putative diphosphonucleoside choline transferase. Exchanging the licD genes between the two strains with ChoP on different chain extensions was sufficient to switch its position. Allelic variants with ChoP on a hexose on heptose III rather than heptose I were sensitive to CRP‐mediated serum bactericidal activity regardless of the genetic background. Differences in CRP‐mediated killing correlated with differences in the binding of CRP from human serum to whole bacteria. This suggests that, in addition to the mechanism involving phase variation, the structural rearrangements within the oligosaccharide contribute to evasion of innate and acquired immunity.
Mutational analysis of MarR, the negative regulator of marRAB expression in Escherichia coli, suggests the presence of two regions required for DNA binding
Authors: Alekshun Michael N. , Kim Yang Soo , Levy Stuart B.
Published: March 2003

MarR, the negative regulator of the Escherichia coli multiple antibiotic resistance (marRAB) operon, is a member of a newly recognized family of regulatory proteins. The amino acid sequences of these proteins do not display any apparent homologies to the DNA binding domains of prokaryotic transcription regulators and a DNA binding motif for any one of the MarR homologues is currently unknown. In order to define regions of MarR required for DNA binding, mutant repressors, selected based on their ability to interfere with (negatively complement) the activity of wild‐type MarR, were isolated. As determined using gel mobility shift assays, 13 out of 14 negative complementing mutants tested were unable to bind DNA in vitro. Three negative complementing alleles presumably specify truncated repressors and one of these proteins, a 120 residue MarR, can bind DNA in vitro. Most of the negative complementing mutations were clustered within two areas of MarR with features related to a helix–turn–helix DNA binding motif. These regions are presumed to be required for the DNA binding activity of the repressor.
PEST sequences in cAMP‐dependent protein kinase subunits of the aquatic fungus Blastocladiella emersonii are necessary for in vitro degradation by endogenous proteases
Authors: Borges Ana Claudia Cantisani , Gomes Suely Lopes
Published: March 2003

During Blastocladiella emersonii germination, the regulatory (R) and the catalytic (C) subunits of the cAMP‐dependent protein kinase (PKA) are rapidly and concurrently degraded, after PKA activation in response to a transient increase in intracellular cAMP levels. The possibility that PEST sequences could be acting as proteolytic recognition signals in this process was investigated, and high score PEST sequences were found in both B. emersonii R and C subunits. Deletions in the PEST sequences were obtained by site‐directed mutagenesis and the different PKA subunits were independently expressed in Escherichia coli. Proteolysis assays of the various R and C recombinant forms, using B. emersonii cell extracts as the source of proteases, showed a strong correlation between the presence of high score PEST sequences and susceptibility to degradation. Furthermore, the amino‐terminal sequence of the proteolytic fragments indicated that the cleavage sites in both subunits are located at or near the PEST regions. The PEST sequence in B. emersonii C subunit, which when deleted or disrupted leads to resistance to proteolysis, is entirely contained in the 72‐amino‐acid extension located in the N‐terminus of the protein. C subunit mutants carrying deletions in this region displayed little difference in their kinetic properties or enzyme thermostability. These results suggest that the N‐terminal extension may only play a role in C subunit degradation.
A role for lipopolysaccharide in turkey tracheal colonization byBordetella aviumas demonstratedin vivoandin vitro
Authors: Spears Patricia A. , Temple Louise M. , Orndorff Paul E.
Published: March 2003

We isolated two insertion mutants ofBordetella aviumthat exhibited a peculiar clumped‐growth phenotype and found them to be attenuated in turkey tracheal colonization. The mutants contained transposon insertions in homologues of thewlbAandwlbLgenes ofBordetella pertussis. Thewlbgenetic locus ofB. pertussishas been previously described as containing 12 genes involved in lipopolysaccharide (LPS) biosynthesis. Polyacrylamide gel analysis of LPS fromB. avium wlbAandwlbLinsertion mutants confirmed an alteration in the LPS profile. Subsequent cloning and complementation of thewlbAandwlbLmutantsin transwith a recombinant plasmid containing the homologouswlblocus fromB. aviumeliminated the clumped‐growth phenotype and restored the LPS profile to that of wild‐typeB. avium. Also, a parental level of tracheal colonization was restored to both mutants by the recombinant plasmid. Interestingly, complementation of thewlbAandwlbLmutants with a recombinant plasmid containing the heterologouswlblocus fromB. pertussis,B. bronchiseptica,orBordetella parapertussiseliminated the clumped‐growth phenotype and resulted in a change in the LPS profile, although not to that of wild‐typeB. avium. The mutants also acquired resistance to a newly identifiedB. avium‐specific bacteriophage, Ba1. Complementation of bothwlbAandwlbLmutants with the homologouswlblocus ofB. avium, but not the heterologousB. pertussislocus, restored sensitivity to Ba1. Complementation of thewlbLmutant, but not thewlbAmutant, with the heterologouswlblocus ofBordetella bronchisepticaorB. parapertussisrestored partial sensitivity to Ba1. Comparisons of the LPS profile and phage sensitivity of the mutants upon complementation bywlbloci from the heterologous species and byB. aviumsuggested that phage sensitivity required the presence of O‐antigen. At the mechanistic level, both mutants showed a dramatic decrease in serum resistance and a decrease in binding to turkey tracheal ringsin vitro. In the case of serum resistance, complementation of both mutants with the homologouswlblocus ofB. aviumrestored serum resistance to wild‐type levels. However, in the case of epithelial cell binding, only complementation of thewlbAmutant completely restored binding to wild‐type levels (binding was only partially restored in thewlbLmutant). This is the first characterization of LPS mutants ofB. aviumat the genetic level and the first report of virulence changes by bothin vivoandin vitromeasurements
Antisense RNA regulation of the pAD1 par post‐segregational killing system requires interaction at the 5′ and 3′ ends of the RNAs
Authors: Greenfield Tony J. , Weaver Keith E.
Published: March 2003

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA‐regulated post‐segregational killing system (PSK) identified in a Gram‐positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA I encodes an open reading frame for a 33‐amino‐acid toxin called Fst. Expression of fst is regulated post‐transcriptionally by RNA II. In this paper, RNA II is shown to interact with RNA I by a unique antisense RNA mechanism. RNA I and RNA II contain complementary direct repeats at their 5′ ends and a complementary transcriptional terminator stem–loop at their 3′ ends. Deletion of the terminator or mutations within the terminator loop of RNA II severely reduced the rate of interaction in vitro. Mutations in the 5′ direct repeats of RNA II prevented the RNAs from interacting in vitro. For these mutations in RNA II, complementary mutations in RNA I were shown to restore interaction. The reduced binding efficiency of the RNA II mutants was paralleled by the failure of these mutants to suppress par‐mediated killing in vivo. These results indicate that regions at both the 5′ and the 3′ ends of the par transcripts are important for RNA I–RNA II interaction.
Helicobacter pylori inhibits phagocytosis by professional phagocytes involving type IV secretion components
Authors: Ramarao Nalini , Gray‐Owen Scott D. , Backert Steffen , Meyer Thomas F.
Published: March 2003

Gastric infections by Helicobacter pylori are characteristically associated with an intense inflammation and infiltration of mainly polymorphonuclear lymphocytes (PMNs) and monocytes. The inflammatory response by infiltrated immune cells appears to be a primary cause of the damage to surface epithelial layers and may eventually result in gastritis, peptic ulcer, gastric cancer and/or MALT‐associated gastric lymphoma. Our analysis of the interaction between H. pylori and PMNs and monocytes revealed that H. pylori inhibits its own uptake by these professional phagocytes. To some degree, this effect resembles antiphagocytosis by Yersinia enterocolitica. Increasing numbers of bacteria associated per cell are more efficient at blocking their own engulfment. In H. pylori, bacterial protein synthesis is necessary to block phagocytic uptake, as shown by the time and concentration dependence of the bacteriostatic protein synthesis inhibitor chloramphenicol. Furthermore, H. pylori appears broadly to inhibit the phagocytic function of monocytes and PMNs, as infection with H. pylori abrogates the phagocytes' ability to engulf latex beads or adherent Neisseria gonorrhoeae cells. This antiphagocytic phenotype depends on distinct virulence (vir) genes, such as virB7 and virB11, encoding core components of a putative type IV secretion apparatus. Our data indicate that H. pylori exhibits an antiphagocytic activity that may play an essential role in the immune escape of this persistent pathogen.
Analysis of mutants of tetanus toxin HC fragment: ganglioside binding, cell binding and retrograde axonal transport properties
Authors: Sinha Katharine , Box Michael , Lalli Giovanna , Schiavo Giampietro , Schneider Holm , Groves Michael , Siligardi Giuliano , Fairweather Neil
Published: March 2003

Tetanus toxin binds neuronal tissue prior to internalization and trafficking to the central nervous system. Binding of the carboxy‐terminal 50 kDa HC fragment of tetanus toxin to polysialogangliosides is important for this initial cell binding step. Using the three‐dimensional structure of HC, mutants were designed to investigate the role of individual residues in ganglioside binding. Mutant proteins were tested for binding to GT1b gangliosides, to primary motoneurons and for their ability to undergo retrograde transport in mice. Two classes of mutant were obtained: (i) those containing deletions in loop regions within the C‐terminal β‐trefoil domain which showed greatly reduced ganglioside and cell binding and did not undergo retrograde transport and (ii) those that showed reduced ganglioside binding, but retained primary neuronal cell binding and retrograde transport. The second class included point mutants of Histidine‐1293, previously implicated in GT1b binding. Our deletion analysis is entirely consistent with recent structural studies which have identified sugar‐binding sites in the immediate vicinity of the residues identified by mutagenesis. These results demonstrate that ganglioside binding can be severely impaired without abolishing cell binding and intracellular trafficking of tetanus toxin.
bgs2+, a sporulation‐specific glucan synthase homologue is required for proper ascospore wall maturation in fission yeast
Authors: Martín Victoria , Ribas Juan Carlos , Carnero Elena , Durán Angel , Sánchez Yolanda
Published: March 2003

The formation of the ascospore cell wall of Schizosaccharomyces pombe requires the co‐ordinated activity of enzymes involved in the biosynthesis of its components, such as glucans. We have cloned the bgs2+ gene. bgs2+ belongs to the glucan synthase family of S. pombe and is homologous to the Saccharomyces cerevisiae FKS1 and FKS2 genes. Deletion or overexpression of this gene does not lead to any apparent defect during vegetative growth, but homozygous bgs2Δ diploids do show a sporulation defect. Although meiosis takes place normally, ascospores are unable to mature, and their wall differs from that of wild‐type ascospores. Moreover, bgs2Δ zygotes were not able to release ascospores spontaneously, and the ascospores were unable to germinate. We show that expression of bgs2+ is restricted to sporulation and that a bgs2–green fluorescent protein (GFP) fusion protein localizes to the ascospore envelope. The glucan synthase activity in sporulating diploids bearing a bgs2 deletion was diminished in comparison with that of the wild‐type diploids, a fact that underscores the importance of the bgs2+ gene and glucan synthesis for the proper formation and maturation of the ascospore wall.