bluuebunny/crossref_metadata_2025 huggingface dataset:
The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells
Authors: Molinari Gabriella , Rohde Manfred , Talay Susanne R. , Chhatwal Gursharan S. , Beckert Susanne , Podbielski Andreas
Published: March 2003

Group A streptococci (GAS) specifically attach to and internalize into human epithelial host cells. In some GAS isolates, fibronectin‐binding proteins were identified as being responsible for these virulence traits. In the present study, the previously identified global negative regulator Nra was shown to control the binding of soluble fibronectin probably via regulation of protein F2 and/or SfbII expression in the serotype M49 strain 591. According to results from a conventional invasion assay based on the recovery of viable intracellular bacteria, the increased fibronectin binding did not affect bacterial adherence to HEp‐2 epithelial cells, but was associated with a reduction in the internalization rates. However, when examined by confocal and electron microscopy techniques, the nra‐mutant bacteria were shown to exhibit higher adherence and internalization rates than the corresponding wild type. The mutant bacteria escaped from the phagocytic vacuoles much faster, promoting consistent morphological changes which resulted in severe host cell damage. The apoptotic and lytic processes observed in nra‐mutant infected host cells were correlated with an increased expression of the genes encoding superantigen SpeA, the cysteine protease SpeB, and streptolysin S in the nra‐mutant bacteria. Adherence and internalization rates of a nra/speB‐double mutant at wild‐type levels indicated that the altered speB expression in the nra mutant contributed to the observed changes in both processes. The Nra‐dependent effects on bacterial virulence were confined to infections carried out with stationary growth phase bacteria. In conclusion, the obtained results demonstrated that the global GAS regulator Nra modulates virulence genes, which are involved in host cell damage. Thus, by helping to achieve a critical balance of virulence factor expression that avoids the injury of target cells, Nra may facilitate GAS persistence in a safe intracellular niche.
Cold‐regulated genes under control of the cold sensor Hik33 in Synechocystis
Authors: Suzuki Iwane , Kanesaki Yu , Mikami Koji , Kanehisa Minoru , Murata Norio
Published: March 2003

A histidine kinase, Hik33, appears to sense decreases in temperature and to regulate the expression of certain cold‐inducible genes in the cyanobacterium Synechocystis sp. PCC6803. To examine the role of Hik33 in the regulation of gene expression, we analysed a ΔHik33 mutant using the DNA microarray technique. In wild‐type cells, genes that were strongly induced at low temperature encoded proteins that were predominantly subunits of the transcriptional and translational machinery. Most cold‐repressible genes encoded components of the photosynthetic machinery. Mutation of the hik33 gene suppressed the expression of some of these cold‐regulated genes, which could be divided into three groups according to the effect of the mutation of hik33. In the first group, regulation of gene expression by low temperature was totally abolished; in the second group, the extent of such regulation was reduced by half; and, in the third group, such regulation was totally unaffected. These results suggest that expression of the genes in the first group is regulated solely by Hik33, expression of genes in the third group is regulated by an as yet unidentified cold sensor, and expression of genes in the second group is regulated by both these cold sensors.
Analysis of temporal and spatial expression of the CcaR regulatory element in the cephamycin C biosynthetic pathway using green fluorescent protein
Authors: Kyung Yun Seung , Hu Wei‐Shou , Sherman David H.
Published: March 2003

The DNA‐binding capability of a key secondary metabolite regulatory element (CcaR) in the Streptomyces clavuligerus cephamycin C pathway was investigated by gel mobility retardation and DNase I footprinting analysis. These results revealed that CcaR specifically binds to the promoter region of the lysine‐ε‐aminotransferase gene (lat). Green fluorescent protein (GFP) was subsequently used as a reporter to analyse in vivo expression of CcaR. The corresponding isogenic strain containing ccaR::gfp in the chromosome produced cephamycin C at levels similar to those of wild‐type S. clavuligerus. Confocal laser scanning microscopy revealed that expression of CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. The highly fluorescent seed culture mycelia quickly lost fluorescence upon inoculation into fresh culture medium. The characteristic green colour reappeared in a small portion of mycelia during mid‐exponential growth phase. As the culture aged, the population expressing CcaR expanded, and the expression level increased. This was followed by a reduction in the CcaR‐expressing population towards the end of the culture period. During peak expression, CcaR was distributed uniformly in mycelia, but became localized distal to the chromosome when the culture entered stationary phase. In solid phase analysis, abundant CcaR expression was evident in the substrate mycelia, but was completely absent in aerial hyphae. These results show regulatory linkage between ccaR and lat, whose expression profile showed a similar spatial decoupling between morphogenesis and antibiotic production. In addition, visualizing CcaR within S. clavuligerus mycelia demonstrates a distinct pattern of localization over the course of physiological differentiation.
TheAspergillus nidulansGATA transcription factor geneareBencodes at least three proteins and features three classes of mutation
Authors: Conlon Helen , Zadra Ivo , Haas Hubertus , Arst Herbert N. , Jones Meriel G. , Caddick Mark X.
Published: March 2003

InAspergillus nidulans, the principal transcription factor regulating nitrogen metabolism, AREA, belongs to the GATA family of DNA‐binding proteins. In seeking additional GATA factors, we have clonedareB, which was originally identified via a genetic screen for suppressors ofareAloss‐of‐function mutations. Based on our analysis,areBis predicted to encode at least three distinct protein products. These arise from the use of two promoters, differential splicing and translation initiating at AUG and non‐AUG start codons. All the putative products include a GATA domain and a putative Leu zipper. These regions show strong sequence similarity to regulatory proteins fromSaccharomyces cerevisiae(Dal80p and Gzf3p),Penicillium chrysogenum(NREB) andNeurospora crassa(ASD4). We have characterized three classes of mutation inareB;the first are loss‐of‐function mutations that terminate the polypeptides within or before the GATA domain. The second class truncates the GATA factor either within or upstream of the putative Leu zipper but retains the GATA domain. The third class fuses novel gene sequences toareBwith the potential to produce putative chimeric polypeptides. These novel gene fusions transform the putative negative‐acting transcription factor into an activator that can partially replaceareA.
Sortase‐catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus
Authors: Mazmanian Sarkis K. , Ton‐That Hung , Schneewind Olaf
Published: March 2003

Many surface proteins of Gram‐positive bacteria are anchored to the cell wall envelope by a transpeptidation mechanism, requiring a C‐terminal sorting signal with a conserved LPXTG motif. Sortase, a membrane protein of Staphylococcus aureus, cleaves polypeptides between the threonine and the glycine of the LPXTG motif and catalyses the formation of an amide bond between the carboxyl‐group of threonine and the amino‐group of peptidoglycan cross‐bridges. S. aureus mutants lacking the srtA gene fail to anchor and display some surface proteins and are impaired in the ability to cause animal infections. Sortase acts on surface proteins that are initiated into the secretion (Sec) pathway and have their signal peptide removed by signal peptidase. The S. aureus genome encodes two sets of sortase and secretion genes. It is conceivable that S. aureus has evolved more than one pathway for the transport of 20 surface proteins to the cell wall envelope.
A connection between stress and development in the multicellular prokaryote Streptomyces coelicolor A3(2)
Authors: Kelemen Gabriella H. , Viollier Patrick H. , Tenor Jennifer , Marri Laura , Buttner Mark J. , Thompson Charles J.
Published: March 2003

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. σH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of σH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2–egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild‐type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and σH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2–egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter‐binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of σH is subject to temporal and spatial regulation during colony development, that this tissue‐specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis.
The Legionella pneumophila IcmR protein exhibits chaperone activity for IcmQ by preventing its participation in high‐molecular‐weight complexes
Authors: Duménil Guillaume , Isberg Ralph R.
Published: March 2003

A key event in legionellosis is the ability of Legionella pneumophila to survive and proliferate inside alveolar macrophages. The dot/icm genes, which are necessary for intracellular growth, show sequence similarity to genes encoding conjugative transfer systems, and it is believed that they are responsible for the formation of a secretion apparatus. Evidence is provided here that the IcmR and IcmQ proteins participate in a chaperone–substrate relationship similar to that observed for translocated proteins in type III and type IV secretion apparatuses. Immobilized IcmQ was found to bind IcmR from crude bacterial extracts efficiently. Furthermore, purified IcmR and IcmQ bind with high affinity. This interaction was also observed in vivo by co‐immunoprecipitation. The presence of IcmR was found to affect the physical state of IcmQ directly. In the absence of IcmR, IcmQ formed high‐molecular‐weight complexes both in vivo and in vitro, whereas IcmR prevented and reversed the formation of these complexes.
Molecular genetics of SaPI1 – a mobile pathogenicity island in Staphylococcus aureus
Authors: Ruzin Alexey , Lindsay Jodi , Novick Richard P.
Published: March 2003

The Staphylococcus aureus gene for toxic shock toxin (tst) is carried by a 15 kb mobile pathogenicity island, SaPI1, that has an intimate relationship with temperate staphylococcal phage 80α. During phage growth, SaPI1 is excised from its unique chromosomal site, attC, replicates autonomously, interferes with phage growth, and is efficiently encapsidated into special small phage heads commensurate with its size. Upon transfer to a recipient organism, SaPI1 integrates at attC by means of a self‐coded integrase. One or more phage functions are required for excision, autonomous replication and encapsidation of the element and, thus, the overall relationship between SaPI1 and 80α is similar to that between coliphages P4 and P2. Among other staphylococcal phages tested, only φ13 interacts with SaPI1, inducing excision but not replication or transfer of the element.
Natural transformation competence in Helicobacter pylori is mediated by the basic components of a type IV secretion system
Authors: Hofreuter Dirk , Odenbreit Stefan , Haas Rainer
Published: March 2003

Helicobacter pylori (Hp), a Gram‐negative bacterial pathogen and aetiologic agent of gastroduodenal disease in humans, is naturally competent for genetic transformation. Natural competence in bacteria is usually correlated with the presence of type IV pili or type IV pilin‐like proteins, which are absent in Hp. Instead, we recently identified the comB operon in Hp, carrying four genes tentatively designated as orf2, comB1, comB2 and comB3. We show here that all ComB proteins and the 37‐amino‐acid Orf2 peptide display significant primary sequence and structural homology/identity to the basic components of a type IV secretion apparatus. ComB1, ComB2 and ComB3, now renamed ComB8, ComB9 and ComB10, correspond to the Agrobacterium tumefaciens VirB8, VirB9 and VirB10 proteins respectively. The peptide Orf2 carries a lipoprotein motif and a second cysteine residue homologous to VirB7, and was thus designated ComB7. The putative ATPase ComB4, encoded by the open reading frame hp0017 of strain 26695, corresponds to virB4 of the A. tumefaciens type IV secretion system. A Hp comB4 transposon insertion mutant was totally defective in natural transformation. By complementation of a HpΔcomB deletion mutant, we demonstrate that each of the proteins from ComB8 to ComB10 is absolutely essential for the development of natural transformation competence. The putative lipoprotein ComB7 is not essential, but apparently stabilizes the apparatus and modulates the transformation efficiency. Thus, pathogenic type I Hp strains contain two functional independent type IV transport systems, one for protein translocation encoded by the cag pathogenicity island and one for uptake of DNA by natural transformation. The latter system indicates a possible novel mechanism for natural DNA transformation in bacteria.
Inactivation of a gene that is highly conserved in Gram‐positive bacteria stimulates degradation of non‐native proteins and concomitantly increases stress tolerance in Lactococcus lactis
Authors: Frees Dorte , Varmanen Pekka , Ingmer Hanne
Published: March 2003

Exposure of cells to elevated temperatures triggers the synthesis of chaperones and proteases including components of the conserved Clp protease complex. We demonstrated previously that the proteolytic subunit, ClpP, plays a major role in stress tolerance and in the degradation of non‐native proteins in the Gram‐positive bacterium Lactococcus lactis. Here, we used transposon mutagenesis to generate mutants in which the temperature‐ and puromycin‐sensitive phenotype of a lactococcal clpP null mutant was partly alleviated. In all mutants obtained, the transposon was inserted in the L. lactis trmA gene. When analysing a clpP, trmA double mutant, we found that the expression normally induced from the clpP and dnaK promoters in the clpP mutant was reduced to wild‐type level upon introduction of the trmA disruption. Additionally, the degradation of puromycyl‐containing polypeptides was increased, suggesting that inactivation of trmA compensates for the absence of ClpP by stimulating an as yet unidentified protease that degrades misfolded proteins. When trmA was disrupted in wild‐type cells, both stress tolerance and proteolysis of puromycyl peptides was enhanced above wild‐type level. Based on our results, we propose that TrmA, which is well conserved in several Gram‐positive bacteria, affects the degradation of non‐native proteins and thereby controls stress tolerance.
The cytosolic HinT protein of Mycoplasma hominis interacts with two membrane proteins
Authors: Kitzerow Annette , Henrich Birgit
Published: March 2003

Histidine triad nucleotide‐binding (HinT) proteins are dimeric proteins that bind to purines and are found in all three kingdoms: the eukarya, bacteria and archaea. In eukaryotes, HinT proteins have been detected intracellularly, but their function is unknown. Until now, knowledge about HinT proteins in prokaryotes was restricted to sequence similarities and nucleotide‐binding studies. In this study, we provide evidence that, in the cell wall‐less prokaryote, Mycoplasma hominis, the gene encoding the HinT protein forms an operon with two other genes. These genes encode the species‐specific membrane proteins, P60 and P80, which are associated within the mycoplasma membrane. The finding that HinT interacts with this complex by binding to P80 provides novel insight into the organization of bacterial HinT proteins.
Genome‐wide analysis of the general stress response in Bacillus subtilis
Authors: Price Chester W. , Fawcett Paul , Cérémonie Hélène , Su Nancy , Murphy Christopher K. , Youngman Philip
Published: March 2003

Bacteria respond to diverse growth‐limiting stresses by producing a large set of general stress proteins. In Bacillus subtilis and related Gram‐positive pathogens, this response is governed by the σB transcription factor. To establish the range of cellular functions associated with the general stress response, we compared the transcriptional profiles of wild and mutant strains under conditions that induce σB activity. Macroarrays representing more than 3900 annotated reading frames of the B. subtilis genome were hybridized to 33P‐labelled cDNA populations derived from (i) wild‐type and sigB mutant strains that had been subjected to ethanol stress; and (ii) a strain in which σB expression was controlled by an inducible promoter. On the basis of their significant σB‐dependent expression in three independent experiments, we identified 127 genes as prime candidates for members of the σB regulon. Of these genes, 30 were known previously or inferred to be σB dependent by other means. To assist in the analysis of the 97 new genes, we constructed hidden Markov models (HMM) that identified possible σB recognition sequences preceding 21 of them. To test the HMM and to provide an independent validation of the hybridization experiments, we mapped the σB‐dependent messages for seven representative genes. For all seven, the 5′ end of the message lay near typical σB recognition sequences, and these had been predicted correctly by the HMM for five of the seven examples. Lastly, all 127 gene products were assigned to functional groups by considering their similarity to known proteins. Notably, products with a direct protective function were in the minority. Instead, the general stress response increased relative message levels for known or predicted regulatory proteins, for transporters controlling solute influx and efflux, including potential drug efflux pumps, and for products implicated in carbon metabolism, envelope function and macromolecular turnover.
cDNA‐AFLP analysis unravels a genome‐wide hrpG‐regulon in the plant pathogen Xanthomonas campestris pv. vesicatoria
Authors: Noël Laurent , Thieme Frank , Nennstiel Dirk , Bonas Ulla
Published: March 2003

The Hrp type III protein secretion system is essential for pathogenicity of the Gram‐negative plant pathogen Xanthomonas campestris pv. vesicatoria. Expression of the hrp gene cluster is controlled by HrpG, a two‐component response regulator, and HrpX, an AraC‐type transcriptional activator. Using the cDNA‐AFLP technique, 30 hrpG‐induced (hgi) and five hrpG‐repressed (hgr) cDNA fragments were identified, defining a large hrpG‐regulon in X. campestris pv. vesicatoria. Expression of most genes in the hrpG‐regulon was dependent on hrpX. Seven cDNA fragments map to the known hrp gene cluster and flanking regions. All other genes appear to be scattered over the chromosome and endogenous plasmids. Sequence analysis identified genes encoding putative extracellular proteases, a putative transcriptional regulator and XopJ and XopB (Xanthomonas outer proteins), homologues of YopJ from Yersinia spp. and the avirulence protein AvrPphD of Pseudomonas syringae respectively. XopB is secreted by the Hrp type III secretion system. Analysis of deletion mutants in several hgi genes revealed a new virulence locus. This study demonstrates that cDNA‐AFLP is a powerful tool to study prokaryotic transcriptomes and to identify genes contributing to Xanthomonas virulence and putative effector proteins.
Topological and functional characterization of WbpM, an inner membrane UDP‐GlcNAc C6 dehydratase essential for lipopolysaccharide biosynthesis in Pseudomonas aeruginosa
Authors: Creuzenet Carole , Lam Joseph S.
Published: March 2003

WbpM is essential for the biosynthesis of B‐band lipopolysaccharide (LPS) in many serotypes of Pseudomonas aeruginosa. Homologues that can functionally complement a wbpM null mutant and that are also necessary for virulence have been identified in numerous pathogenic bacteria. WbpM and most of its homologues are large membrane proteins, which has long hampered the elucidation of their biochemical function. This paper describes the detailed characterization of WbpM using both in vivo and in vitro approaches. LacZ and PhoA fusion experiments showed that WbpM was anchored to the inner membrane via four N‐terminal transmembrane domains, whereas the C‐terminal catalytic domain resided in the cytoplasm. Although the membrane domains did not have any catalytic activity, complementation experiments suggested that they were important for the polymerization of high‐molecular‐weight B‐band LPS. The biochemical characterization of a soluble truncated form of WbpM, His‐S262, showed that WbpM was a C6 dehydratase specific for UDP‐GlcNAc. It exhibited unusual low temperature (25–30°C) and high pH (pH 10) optima. Although WbpM possessed an altered catalytic triad composed of SMK as opposed to SYK commonly found in other dehydratases, its catalysis was very efficient, with a kcat of 168 min−1 and a kcat/Km of 58 mM−1 min−1. These unusual physico‐kinetic properties suggested a potentially different mechanism of C6 dehydration for WbpM and its large homologues. His‐S262 is now a precious tool for further structure–function studies.
The protein kinase Pho85 is required for asymmetric accumulation of the Ash1 protein in Saccharomyces cerevisiae
Authors: McBride Helen J. , Sil Anita , Measday Vivien , Yu Yaxin , Moffat Jason , Maxon Mary E. , Herskowitz Ira , Andrews Brenda , Stillman David J.
Published: March 2003

The Ash1 protein is a daughter cell‐specific repressor of HO gene transcription in Saccharomyces cerevisiae. Both ASH1 mRNA and protein are localized to the incipient daughter cell at the end of mitosis; Ash1 then inhibits HO transcription in the daughter cell after cytokinesis. Mother cells, in contrast, contain little or no Ash1 and thus are able to transcribe HO. We show that deletion of PHO85, which encodes a cyclin‐dependent protein kinase, causes reduced transcription of HO and that this reduction is dependent on ASH1. In pho85 mutants, Ash1 protein is no longer asymmetrically localized and is present, instead, in both mother and daughter cells. Initially, it appears to be localized properly but then persists as daughter cells mature into mother cells. In contrast, ASH1 mRNA is localized appropriately to daughter cells in pho85 mutants. We observe that Ash1 protein is phosphorylated by Pho85 in vitro and that Ash1 stability increases in a pho85 mutant. These data suggest that phosphorylation of Ash1 by Pho85 governs stability of Ash1 protein.
Characterization of nitrogen metabolite signalling in Aspergillus via the regulated degradation of areA mRNA
Authors: Morozov Igor Y. , Galbis‐Martinez Marisa , Jones Meriel G. , Caddick Mark X.
Published: March 2003

AreA is the principal transcription factor involved in determining nitrogen utilization in Aspergillus nidulans. NH4+ and Gln are utilized preferentially but in their absence, AreA acts to facilitate the expression of genes involved in metabolizing alternative nitrogen sources. It is crucial to the function of AreA that its expression is tightly modulated by the quality and availability of nitrogen sources. One signalling mechanism involves regulated degradation of the areA transcript in response to NH4+ and Gln, which provides the first direct means of monitoring nitrogen signalling in this fungus. Here we assess the specificity of the transcript degradation response, determining that it responds qualitatively to a variety of additional nitrogen sources including Asn. Furthermore, the response to Gln and NH4+ requires the same discrete region of the areA 3′‐UTR but both NH4+ and Asn need to be metabolized to Gln before they are effective as a signal. However, NH4+ signalling is independent of AreA activity, unlike Gln and Asn signalling. A mutation in the structural gene for NADP‐linked glutamate dehydrogenase, gdhA, which disrupts metabolism of NH4+ to Glu, is additive with mutations in two distinct regions of areA that disrupt the previously identified signalling mechanisms. The triple mutant is both strongly derepressed and expresses very high levels of nitrate reductase activity. These data suggest nitrogen metabolism in A. nidulans is in part regulated in response to the intracellular levels of Gln via the regulated degradation of areA mRNA, but the intracellular Gln level is not the sole determinant of nitrogen metabolite repression.
Cas1p is a membrane protein necessary for the O‐acetylation of the Cryptococcus neoformans capsular polysaccharide
Authors: Janbon Guilhem , Himmelreich Uwe , Moyrand Frédérique , Improvisi Luce , Dromer Françoise
Published: March 2003

The capsule is certainly the most obvious virulence factor for Cryptococcus neoformans. The main capsule constituents are glucuronoxylomannans (GXM). Several studies have focused on the structure and chemistry of the GXM component of the capsule, yet little is known about the genetic basis of the capsule construction. Using a monoclonal antibody specific to a sugar epitope, we isolated a capsule‐structure mutant strain and cloned by complementation a gene named CAS1 that codes for a putative membrane protein. Although no sequence homology was found with any known protein in the different databases, protein analysis using the Propsearch software classified Cas1p as a putative glycosyltransferase. Cas1p is a well‐conserved evolutionary protein, as we identified one orthologue in the human genome, one in the drosophila genome and four in the plant Arabidopsis thaliana genome. Analysis of the capsule structure after CAS1 deletion showed that it is required for GXM O‐acetylation.
Feedback regulation of an Agrobacterium catalase gene katA involved in Agrobacterium–plant interaction
Authors: Xu Xiu Q. , Li Luo P. , Pan Shen Q.
Published: March 2003

Catalases are known to detoxify H2O2, a major component of oxidative stress imposed on a cell. An Agrobacterium tumefaciens catalase encoded by a chromosomal gene katA has been implicated as an important virulence factor as it is involved in detoxification of H2O2 released during Agrobacterium–plant interaction. In this paper, we report a feedback regulation pathway that controls the expression of katA in A. tumefaciens cells. We observed that katA could be induced by plant tissue sections and by acidic pH on a minimal medium, which resembles the plant environment that the bacteria encounter during the course of infection. This represents a new regulatory factor for catalase induction in bacteria. More importantly, a feedback regulation was observed when the katA–gfp expression was studied in different genetic backgrounds. We found that introduction of a wild‐type katA gene encoding a functional catalase into A. tumefaciens cells could repress the katA–gfp expression over 60‐fold. The katA gene could be induced by H2O2 and the encoded catalase could detoxify H2O2. In addition, the katA‐gfp expression of one bacterial cell could be repressed by other surrounding catalase‐proficient bacterial cells. Furthermore, mutation at katA caused a 10‐fold increase of the intracellular H2O2 concentration in the bacteria grown on an acidic pH medium. These results suggest that the endogenous H2O2 generated during A. tumefaciens cell growth could serve as the intracellular and intercellular inducer for the katA gene expression and that the acidic pH could pose an oxidative stress on the bacteria. Surprisingly, one mutated KatA protein, exhibiting no significant catalase activity as a result of the alteration of two important residues at the putative active site, could partially repress the katA–gfp expression. The feedback regulation of the katA gene by both catalase activity and KatA protein could presumably maintain an appropriated level of catalase activity and H2O2 inside A. tumefaciens cells.
PapB paralogues and their effect on the phase variation of type 1 fimbriae in Escherichia coli
Authors: Holden Nicola J. , Uhlin Bernt Eric , Gally David L.
Published: March 2003

Recent work has demonstrated that expression of type 1 fimbriae is repressed by PapB, a regulator of pyelonephritis‐associated pili (P‐pili). PapB belongs to family of related adhesin regulators, for which consensus residues required for DNA binding and oligomerization have been identified. Of the regulators tested in this study, PapB, SfaB (S‐fimbriae) and PefB (Salmonella enterica serovar Typhimurium –plasmid‐encoded fimbriae) repressed FimB‐promoted off‐to‐on inversion of the fim switch, although complete repression was only demonstrated by PapB. DaaA, FaeB, FanA, FanB and ClpB had no effect on fim switching. In addition, only PapB stimulated FimE‐promoted on‐to‐off inversion. Deletion analysis demonstrated that this specificity resides in the carboxy terminal of the protein, and not the amino terminal, with the central region being homologous among the family members. Exchange of Leu82 and Ile83 of PapB for the equivalent residues from the DaaA protein (Phe and Gln) within the carboxy terminal virtually abolished cross‐talk activity. Whereas PapB can bind to a region around the left inverted repeat of the fim switch, DaaA and the PapB double mutant were effectively unable to bind this region. A previously characterized PapB DNA binding mutant also failed to bind to this region and failed to inhibit FimB activity at the fim switch. Thus, repression of fim expression appears unique to PapB and SfaB within E. coli and requires DNA binding involving amino acid residues located both within the homologous core and in the heterogeneous carboxy terminus. The variation in the carboxy terminus between the PapB family members explains their differential effects on fim. This mechanism of cross‐talk seems restricted to the P and S family adhesins with type 1 fimbriae and may ensure variable and sequential expression of adhesins during urinary tract infections.
Binding of human C4BP to the hypervariable region of M protein: a molecular mechanism of phagocytosis resistance in Streptococcus pyogenes
Authors: Berggård Karin , Johnsson Eskil , Morfeldt Eva , Persson Jenny , Stålhammar‐Carlemalm Margaretha , Lindahl Gunnar
Published: March 2003

The amino‐terminal hypervariable region (HVR) of streptococcal M protein is required for the ability of this virulence factor to confer phagocytosis resistance. The function of the HVR has remained unknown, but the finding that many HVRs with extremely divergent sequences bind the human complement regulator C4b‐binding protein (C4BP) has suggested that this ligand may play a role in phagocytosis resistance. We used the M22 system to study the function of bound C4BP and provide several lines of evidence that C4BP indeed contributes to phagocytosis resistance. First, the ability of anti‐HVR antibodies to cause opsonization correlated with their ability to inhibit binding of C4BP. Secondly, a short deletion in the HVR eliminated C4BP binding and also reduced the ability of M22 to confer phagocytosis resistance. Thirdly, the addition of an excess of pure C4BP to a phagocytosis system almost completely blocked the effect of opsonizing anti‐HVR antibodies. Together, our data indicate that binding of C4BP to the HVR of M22 plays an important role in phagocytosis resistance, but other properties of M22 also contribute. This study provides the first molecular insight into the mechanisms by which the HVR of an M protein confers phagocytosis resistance.
Distribution of the Escherichia coli structural maintenance of chromosomes (SMC)‐like protein MukB in the cell
Authors: Den Blaauwen Tanneke , Lindqvist Arne , Löwe Jan , Nanninga Nanne
Published: March 2003

Fluorescent polyclonal antibodies specific for MukB have been used to study its localization in Escherichia coli. In wild‐type cells, the MukB protein appeared as a limited number of oblong shapes embracing the nucleoid. MukB remained associated with the nucleoid in the absence of DNA replication. The centre of gravity of the dispersed MukB signal initially localized near mid‐cell, but moved to approximately quarter positions well before the termination of DNA replication and its subsequent reinitiation. Because MukB had been reported to bind to FtsZ and to its eukaryotic homologue tubulin in vitro, cells were co‐labelled with MukB‐ and FtsZ‐specific fluorophores. No co‐localization of MukB with polymerized FtsZ (the FtsZ ring) was observed at any time during the cell cycle. A possible role for MukB in preventing premature FtsZ polymerization and in DNA folding that might assist DNA segregation is discussed.
The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis
Authors: Hamon Mélanie A. , Lazazzera Beth A.
Published: March 2003

Biofilms are structured communities of cells encased in a polymeric matrix and adherent to a surface, interface or each other. We report here that the soil bacterium Bacillus subtilis forms biofilms. By confocal scanning laser microscopy, we observed that B. subtilis adhered to abiotic surfaces and formed a three‐dimensional structure ≥ 30 µm in depth. These biofilms appeared to be at least partly encased in an extracellular polysaccharide matrix, as they could be stained with Calcofluor, a polysaccharide‐binding dye. To understand the molecular mechanism of biofilm formation, we screened previously characterized mutants for a defect in biofilm formation. We found that mutations in spo0A, which encodes the major early sporulation transcription factor, caused a defect in biofilm formation. spo0A mutant cells adhered to a surface in a monolayer of cells rather than a three‐dimensional biofilm. The requirement of Spo0A for biofilm development appears to result from its role in negatively regulating AbrB. Mutations in abrB suppressed the biofilm defect of a spo0A mutant, indicating that AbrB negatively regulates at least one gene that is required for the transition from a monolayer of attached cells to a mature biofilm. Implications of biofilm development for the ecology of B. subtilis are discussed.
Multiple virulence factors of Cryptococcus neoformans are dependent on VPH1
Authors: Erickson Todd , Liu Lide , Gueyikian Ara , Zhu Xudong , Gibbons Jack , Williamson Peter R.
Published: March 2003

Acidification of vesicular compartments plays an important role in a number of cellular transport processes, including protein secretion, metal cofactor insertion, glycosylation and pH stability. In the present study, we identify and characterize a component of the vesicular proton pump, Vph1p, to determine its role in the virulence of the AIDS‐related fungal pathogen Cryptococcus neoformans. Insertional mutagenesis and plasmid rescue were used to identify the VPH1 gene by screening for mutants defective in laccase activity. Disruption of VPH1 resulted in defects in three virulence factors (capsule production, laccase and urease expression), as well as a growth defect at 37°C, but only a small growth reduction at 30°C. These effects were duplicated by the vacuolar (H+)‐ATPase inhibitor bafilomycin A1. Furthermore, the vph1 insertional mutant was also avirulent in a mouse meningo‐encephalitis model. Complementation of the insertional mutant with wild‐type VPH1 resulted in a recovery of virulence factor expression, normal growth at 37°C and restoration of full virulence. These studies establish the importance of the VPH1 gene and vesicular acidification in the virulence of C. neoformans.
Mutations in FlbD that relieve the dependency on flagellum assembly alter the temporal and spatial pattern of developmental transcription in Caulobacter crescentus
Authors: Muir Rachel E. , Gober James W.
Published: March 2003

The transcription factor FlbD regulates the temporal and spatial transcription of flagellar genes in the bacterium Caulobacter crescentus. Activation of FlbD requires cell cycle progression and the assembly of an early (class II) flagellum structure. In this report, we identify 20 independent gain‐of‐function mutations in flbD that relieve regulation by flagellar assembly. One of these, flbD‐1204, contained a mutation in the receiver domain (V17M) and another, flbD‐1231, in the DNA binding domain (V451G). Both of these mutations resulted in an aberrant pattern of cell cycle transcription. The presence of the FlbD‐1204 allele also resulted in a loss of swarmer‐pole‐specific transcription. These results indicate that temporal and spatial transcription is influenced by the assembly of the nascent flagellar structure. The trans‐acting positive and negative regulatory factor, FliX, couples flagellar assembly to the activation of FlbD and, as we show here, also influences temporal transcription. Furthermore, we show that FliX can suppress the activity of FlbD mutants that cannot be phosphorylated, and that FliX is required for FlbD stability, and vice versa. These results indicate that FliX may interact directly with FlbD to regulate its activity.
An investigation into the compartmentalization of the sporulation transcription factor σE in Bacillus subtilis
Authors: Fujita Masaya , Losick Richard
Published: March 2003

Sporulation in Bacillus subtilis involves the formation of a polar septum, which divides the sporangium into a mother cell and a forespore. The σE factor, which is encoded within the spoIIG operon, is a cell‐specific regulatory protein that directs gene transcription in the mother cell. σE is synthesized as an inactive proprotein pro‐σE, which is converted to the mature factor by the putative processing enzyme SpoIIGA. Processing of pro‐σE does not commence until after asymmetric division when σE is largely confined to the mother cell. Processing depends on the signalling protein SpoIIR, which delays proteolysis until after polar septation, but the mechanism by which σE is confined to the mother cell is not understood. Previous work favoured a model in which pro‐σE localizes to the mother cell face of the polar septum, such that σE would be selectively released into mother cell cytoplasm. Based on the use of green fluorescent protein (GFP) fusions, we now report that pro‐σE is distributed approximately uniformly along all membrane surfaces and is not confined to the mother‐ cell face of the septum. Rather, our results are consistent with a model in which preferential and persistent transcription of the spoIIG operon in the mother cell and degradation of σE in the forespore contribute to the selective accumulation of σE in the mother cell. Persistent transcription of spoIIG after polar septation also contributes to the proper timing of pro‐σE processing.
Are the PE‐PGRS proteins of Mycobacterium tuberculosis variable surface antigens?
Authors: Banu Sayera , Honoré Nadine , Saint‐Joanis Brigitte , Philpott Dana , Prévost Marie‐Christine , Cole Stewart T.
Published: March 2003

Mycobacterium tuberculosis H37Rv contains 67 PE‐PGRS genes, with multiple
tandem repetitive sequences, encoding closely related proteins that are exceptionally
rich in glycine and alanine. As no functional information was available, 10 of these
genes were selected and shown to be expressed in vitro by reverse transcription–polymerase
chain reaction (RT–PCR). Antibodies against five PE‐PGRS proteins, raised
in mice by DNA vaccination, detected single proteins when the same plasmid constructs
used for immunization were expressed in epithelial cells or in reticulocyte extracts,
confirming that the PE‐PGRS proteins are antigenic. As expected from the conserved
repetitive structure, the antibodies cross‐reacted with more than one PE‐PGRS protein,
suggesting that different proteins share common epitopes. PE‐PGRS proteins were detected
by West‐ern blotting in five different mycobacterial species (M. tuberculosis,
M. bovis BCG, M. smegmatis, M. marinum and M. gordonae)
and 11 clinical isolates of M. tuberculosis. Whole‐genome comparisons of M.
tuberculosis predicted allelic diversity in the PE‐PGRS family, and this was
confirmed by immunoblot studies as size variants were detected in clinical strains.
Subcellular fractionation studies and immunoelectron microscopy localized many PE‐PGRS
proteins in the cell wall and cell membrane of M. tuberculosis. The data suggest that some PE‐PGRS proteins are variable surface antigens.
Engineering specificity of starter unit selection by the erythromycin‐producing polyketide synthase
Authors: Long Paul F. , Wilkinson Christopher J. , Bisang Christian P. , Cortés Jesús , Dunster Nicholas , Oliynyk Marko , McCormick Ellen , McArthur Hamish , Mendez Carmen , Salas José A. , Staunton James , Leadlay Peter F.
Published: March 2003

Chain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ
by KSQ, a ketosynthase‐like decarboxylase domain. Consistent with this, the acyltransferase
(AT) domains of all KSQ‐containing loading modules are shown here to contain a key
arginine residue at their active site. Site‐specific replacement of this arginine
residue in the oleandomycin (ole) loading AT domain effectively abolished
AT activity, consistent with its importance for catalysis. Substitution of the ole
PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery)
loading module gave polyketide products almost wholly either acetate derived or propionate
derived, respectively, instead of the mixture found normally. An authentic extension
module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately
when engineered in the place of the ole loading AT domain, and gave rise to
substantial amounts of C13‐methylerythromycins, as predicted. The role of direct
acylation of the ketosynthase domain of ex‐tension module 1 in chain initiation was
confirmed by demonstrating that a mutant of the triketide synthase DEBS1‐TE, in which
the 4′‐phosphopante‐theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro‐ducts in detectable amounts.
Transcription of multiple var genes by individual, trophozoite‐stage Plasmodium falciparum cells expressing a chondroitin sulphate A binding phenotype
Authors: Duffy Michael F. , Brown Graham V. , Basuki Wanny , Krejany Efrosinia O. , Noviyanti Rintis , Cowman Alan F. , Reeder John C.
Published: March 2003

In this study, we detected multiple var gene transcripts within single,
mature trophozoite‐infected red blood cells (iRBCs) bound to chondroitin sulphate
A (CSA). Several of the var detected had previously been demonstrated to encode
Plasmodium falciparum erythrocyte membrane protein‐1 (PfEMP‐1) variants with
domains that mediated iRBC adhesion to receptors other than CSA. Parasites expressing
the CSA‐adherent phenotype transcribed far more of one var than of all others,
but this gene was different from the two other var previously purported to
encode adhesion to CSA. Previous work suggesting that only single var are transcribed by mature trophozoites needs re‐examination in the light of these data from single, infected cells.
Two DNA‐binding domains of Mga are required for virulence gene activation in the group A streptococcus
Authors: McIver Kevin S. , Myles Rhonda L.
Published: March 2003

Mga is a DNA‐binding protein that activates expression of several important virulence genes in the group A streptococcus (GAS), including those encoding M protein (emm),

C5a peptidase (scpA) and Mga (mga). To determine the functionality

of four potential helix–turn–helix DNA‐binding motifs (HTH1–HTH4)

identified within the amino‐terminus of Mga, alanine substitutions were introduced

within each domain in a MBP–Mga fusion allele and purified proteins were assayed

for binding to Mga‐specific promoter fragments (Pmga, PscpA and Pemm)

in vitro. Although HTH‐1 and HTH‐2 mutations showed wild type DNA‐binding

activity, an altered HTH‐3 domain resulted in reduced binding to the three promoters

and an HTH‐4 mutant was devoid of detectable binding activity. Plasmid‐encoded expression

of the HTH‐3 and HTH‐4 alleles from a constitutive promoter (Pspac) in the

mga‐deleted GAS strain JRS519 demonstrated that Mga‐regulated emm expression

correlated directly to the DNA‐binding activity observed for each mutant protein

in vitro. Single‐copy expression of HTH‐3 and HTH‐4 from their native Pmga

resulted in a dramatic reduction in autoregulated mga expression in both mutant

strains. Thus, Mga appears to contain two DNA‐binding domains (HTH‐3 and HTH‐4) that

are required for direct activation of the Mga virulence regulon in vivo.
Expression of the ExeAB complex of Aeromonas hydrophila is required for the localization and assembly of the ExeD secretion port multimer
Authors: Ast Vivian M. , Schoenhofen Ian C. , Langen Geoffrey R. , Stratilo Chad W. , Chamberlain M. Dean , Howard S. Peter
Published: March 2003

Aeromonas hydrophila secretes protein toxins via the type II pathway, involving

the products of at least two operons, exeAB (gspAB) and exeC‐N

(gspC‐N). In the studies reported here, aerolysin secretion was restored to

C5.84, an exeA::Tn5–751 mutant, by overexpression of exeD

alone in trans. Expression studies indicated that these results did not reflect

a role of ExeAB in the regulation of the exeC‐N operon. Instead, immunoblot

analysis showed that ExeD did not multimerize in C5.84, and fractionation of the

membranes showed that the monomeric ExeD remained in the inner membrane. Expression

of ExeAB, but not either protein alone, from a plasmid in C5.84 resulted in increases

in the amount of multimeric ExeD, which correlated with increases in aerolysin secretion.

Pulse‐chase analysis also sug‐gested that the induction of ExeAB allowed multimerization

of previously accumulated monomer ExeD. In C5.84 cells overproducing ExeD, it multimerized

even in the absence of ExeAB and, although most remained in the inner membrane, an

amount similar to that in wild‐type outer membranes fractionated with the outer membrane

of the overproducing cells. These results indicate that the secretion defect of exeAB mutants is a result of an inability to assemble the ExeD secretin in the outer membrane. The localization and multimerization of overproduced ExeD in these mutants further suggests that the ExeAB complex plays either a direct or indirect role in the transport of ExeD into the outer membrane.