bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selective expression of the virulence factor BAD1 upon morphogenesis to the pathogenic yeast form of Blastomyces dermatitidis: evidence for transcriptional regulation by a conserved mechanism
Authors: Rooney Peggy J. , Sullivan Thomas D. , Klein Bruce S. 
Published: March 2003

Most dimorphic fungal pathogens grow as non‐pathogenic moulds in soil and convert to pathogenic yeast in the host, suggesting that virulence factors are upregulated during phase transition. Such factors have been difficult to identify. We analysed BAD1 (formerly WI‐1), a virulence factor in the dimorphic fungus Blastomyces dermatitidis, for expression in yeast and mycelial morphotypes. BAD1 was expressed in yeast but not in mycelia of North American strains of B. dermatitidis, and this expression pattern was confirmed for BAD1 transcript. BAD1 under the control of its promoter was transferred into African B. dermatitidis lacking a native BAD1 locus, and phase‐specific expression was conserved. Sequence similarity was identified between the BAD1 promoter and the promoters of two yeast phase‐specific genes in Histoplasma capsulatum. In H. capsulatum BAD1 transformants, yeast phase‐specific expression of BAD1 was conserved, and no transcript was detected in mycelia. BAD1β‐galactosidase reporter fusions analysed in B. dermatitidis and H. capsulatum confirmed that BAD1 is transcriptionally regulated in both fungi. BAD1 promoter activity and surface BAD1 expression were detected 6 h after shifting mycelia to 37°C. Thus, BAD1 is expressed after transition to the pathogenic yeast morphotype and is regulated by a mechanism for phase‐specific gene expression that appears to be conserved.
Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis
Authors: Feucht Andrea , Lucet Isabelle , Yudkin Michael D. , Errington Jeffery 
Published: March 2003

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

SummaryThe 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

SummaryFamily 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

SummaryThe 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

SummaryMotA 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

SummaryThe 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

SummaryThe 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

SummaryThe 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

SummaryIn 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

SummaryLipopolysaccharide (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

SummaryThe 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

SummaryIn 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

SummaryWe 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

SummaryUrea 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.