bluuebunny/crossref_metadata_2025 huggingface dataset:
A common drug‐responsive element mediates the upregulation of the Candida albicans ABC transporters CDR1 and CDR2, two genes involved in antifungal drug resistance
Authors: Micheli Michelle de , Bille Jacques , Schueller Christoph , Sanglard Dominique
Published: March 2003

Upregulation of the ATP‐binding cassette (ABC) transporter genes CDR1 and
CDR2 (Candidadrug resistance 1 and 2) is a common
mechanism observed in Candida albicans clinical isolates developing resistance
to the class of azole antifungals. In this work, the regulatory elements of both
genes were delimited using a reporter system in an azole‐susceptible strain exposed
to oestradiol, which allows transient induction of these genes. We found two regulatory
elements in the CDR1 promoter: one responsible for basal expression (basal
expression element; BEE) and the other required for oestradiol responsiveness (drug‐responsive
element I; DREI). In the CDR2 promoter, a single regulatory element
responsible for oestradiol responsiveness (DREII) was detected. Both DREs
shared a consensus of 21 bp with the sequence 5′‐CGGA(A/T)ATCGGATATTTTTTTT‐3′
having no equivalent to known eukaryotic regulatory sequence. Consistent with this
finding, two other C. albicans genes identified by a search for the presence
of DRE in the C. albicans genome sequence database were responsive to oestradiol.
Finally, the regulatory elements found in CDR1 and CDR2 were also functional
in an azole‐resistant strain with constitutive high expression of both transporters.
These results suggest that, although CDR1 and CDR2 upregulation can
be obtained by transient drug‐induced and constitutive upregulation, these two processes
converge to the same regulatory elements and probably mobilize the same trans‐acting factors.
Immediate GTP hydrolysis upon FtsZ polymerization
Authors: Scheffers Dirk‐Jan , Driessen Arnold J. M.
Published: March 2003

To understand the polymerization dynamics of FtsZ, a bacterial cell division protein similar to tubulin, insight is required into the nature of the nucleotide bound to the polymerized protein. In a previous study, we showed that the FtsZ polymers contain mostly GDP. A recent study challenged this result, suggesting that the polymerized FtsZ is in a GTP‐bound state. Here, we show that, when radiolabelled [γ‐32P]‐GTP is used to polymerize FtsZ, GTP is hydrolysed instantaneously. The FtsZ polymer contains both GDP and the radiolabelled inorganic phosphate.
Expression of mutant alanine tRNAs increases spontaneous mutagenesis in Escherichia coli
Authors: Dorazi Robert , Lingutla Josephine J. , Humayun M. Zafri
Published: March 2003

The expression of mutA, an allele of the glycine tRNA gene glyV,
can confer a novel mutator phenotype that correlates with its ability to promote
Asp→Gly mistranslation. Both activities are mediated by a single base
change within the anticodon such that the mutant tRNA can decode aspartate codons
(GAC/U) instead of the normal glycine codons (GCC/U). Here, we investigate whether
specific Asp→Gly mistranslation is required for the unexpected mutator
phenotype. To address this question, we created and expressed 18 individual alleles
of alaV, the gene encoding an alanine tRNA, in which the alanine anticodon
was replaced with those specifying other amino acids such that the mutant (alaVX)
tRNAs are expected to potentiate X→Ala mistranslation, where X is one
of the other amino acids. Almost all alaVX alleles proved to be
mutators in an assay that measured the frequency of rifampicin‐resistant mutants,
with one allele (alaVGlu) being a stronger mutator than mutA.
The alaVGlu mutator phenotype resembles that of mutA in
mutational specificity (predominantly transversions), as well as SOS independence,
but in a puzzling twist differs from mutA in that it does not require a functional
recA gene. Our results suggest that general mistranslation (as opposed to
Asp→Gly alone) can induce a mutator phenotype. Furthermore, these findings predict that a large number of conditions that increase translational errors, such as genetic defects in the translational apparatus, as well as environmental and physiological stimuli (such as amino acid starvation or exposure to antibiotics) are likely to activate a mutator response. Thus, both genetic and epigenetic mechanisms can accelerate the acquisition of mutations.
Functional complexity of the twin‐arginine translocase TatC component revealed by site‐directed mutagenesis
Authors: Buchanan Grant , Leeuw Erik de , Stanley Nicola R. , Wexler Margaret , Berks Ben C. , Sargent Frank , Palmer Tracy
Published: March 2003

The Escherichia coli Tat apparatus is a membrane‐bound protein translocase that serves to export folded proteins synthesized with N‐terminal twin‐arginine signal peptides. The essential TatC component of the Tat translocase is an integral membrane protein probably containing six transmembrane helices. Sequence analysis identified conserved TatC amino acid residues, and the role of these side‐chains was assessed by single alanine substitution. This approach identified three classes of TatC mutants. Class I mutants included F94A, E103A and D211A, which were completely devoid of Tat‐dependent protein export activity and thus represented residues essential for TatC function. Cross‐complementation experiments with class I mutants showed that co‐expression of D211A with either F94A or E103A regenerated an active Tat apparatus. These data suggest that different class I mutants may be blocked at different steps in protein transport and point to the co‐existence of at least two TatC molecules within each Tat translocon. Class II mutations identified residues important, but not essential, for Tat activity, the most severely affected being L99A and Y126A. Class III mutants showed no significant defects in protein export. All but three of the essential and important residues are predicted to cluster around the cytoplasmic N‐tail and first cytoplasmic loop regions of the TatC protein.
The Xanthomonas oryzae pv.◊oryzae raxP and raxQ genes encode an ATP sulphurylase and adenosine‐5′‐phosphosulphate kinase that are required for AvrXa21 avirulence activity
Authors: Shen Yuwei , Sharma Parveen , Silva Francisco G. da , Ronald Pamela
Published: March 2003

Xanthomonas oryzae pv. oryzae (Xoo) Philippine race 6 (PR6) is unable to cause bacterial blight disease on rice lines containing the rice resistance gene Xa21 but is virulent on non‐Xa21 rice lines, indicating that PR6 carries avirulence (avrXa21) determinants required for recognition by XA21. Here we show that two Xoo genes, raxP and raxQ, are required for AvrXa21 activity. raxP and raxQ, which reside in a genomic cluster of sulphur assimilation genes, encode an ATP sulphurylase and APS (adenosine‐5′‐phosphosulphate) kinase. These enzymes function together to produce activated forms of sulphate, APS and PAPS (3′‐phosphoadenosine‐5′‐phosphosulphate). Xoo PR6 strains carrying disruptions in either gene, PR6ΔraxP or PR6ΔraxQ, are unable to produce APS and PAPS and are virulent on Xa21‐containing rice lines. RaxP and RaxQ are similar to the bacterial symbiont Sinorhizobium meliloti host specificity proteins, NodP and NodQ and the Escherichia coli cysteine synthesis proteins CysD, CysN and CysC. The APS and PAPS produced by RaxP and RaxQ are used for both cysteine synthesis and sulphation of other molecules. Mutation in Xoo xcysI, a homologue of Escherichia coli cysI that is required for cysteine synthesis, blocked APS‐ or PAPS‐dependent cysteine synthesis but did not affect AvrXa21 activity, suggesting that AvrXa21 activity is related to sulphation rather than cysteine synthesis. Taken together, these results demonstrate that APS and PAPS production plays a critical role in determining avirulence of a phytopathogen and reveal a commonality between symbiotic and phytopathogenic bacteria.
The transcription termination factor Rho is required for oxidative stress survival in Caulobacter crescentus
Authors: Italiani Valéria C. S. , Zuleta Luiz Fernando G. , Marques Marilis V.
Published: March 2003

A transposon Tn5 mutagenesis library was generated from Caulobacter crescentus

strain NA1000, and clones with deficiency in survival in a high concentration of

NaCl were selected. One of these clones, 37G10, has the Tn5 integrated within

the coding region of the transcription termination factor Rho. Analysis of this mutant

phenotype showed that the cells are motile and present a normal cell cycle, but have

a longer generation time. This strain is sensitive to acidic pH, to the presence

of different salts and to heat shock, but it responds well to UV light and alkaline

pH. The most striking phenotype of the rho mutant is that it is extremely

sensitive to oxidative stress, in both exponential and stationary phases. Experiments

using a transcriptional fusion of the rho promoter region to the lacZ

gene showed that rho gene expression varies during the cell cycle, showing

very low expression levels at the swarmer cell stage and presenting maximum levels

in early predivisional cells. Transcription of the rho gene is increased in

the rho mutant strain, which is indicative of an autoregulatory circuit, and

there is a small variation in the cell cycle pattern of expression. Several peptides

have their synthesis altered in the mutant strain, as analysed by two‐dimensional

gel electrophoresis, most of which show a reduction in expression. These results

indicate that the Rho factor is essential for an efficient response to certain stresses

in Caulobacter.
A two‐component regulator induces the transmission phenotype of stationary‐phase Legionella pneumophila
Authors: Hammer Brian K. , Tateda Eiko S. , Swanson Michele S.
Published: March 2003

Pathogenic Legionella pneumophila evolved as a parasite of aquatic amoebae.
To persist in the environment, the microbe must be proficient at both replication
and transmission. In laboratory cultures, as nutrients become scarce a stringent
response‐like pathway coordinates exit from the exponential growth phase with induction
of traits correlated with virulence, including motility. A screen for mutants that
express the flagellin gene poorly identified five activators of virulence: LetA/LetS,
a two‐component regulator homologous to GacA/GacS of Pseudomonas and SirA/BarA
of Salmonella; the stationary‐phase sigma factor RpoS; the flagellar sigma
factor FliA; and a new locus, letE. Unlike wild type, post‐exponential‐phase
letA and letS mutants were not motile, cytotoxic, sodium sensitive
or proficient at infecting macrophages. L. pneumophila also required fliA
to become motile, cytotoxic and to infect macrophages efficiently and letE
to express sodium sensitivity and maximal motility and cytotoxicity. When induced
to express RelA, all of the strains exited the exponential phase, but only wild type
converted to the fully virulent form. In contrast, intracellular replication was
independent of letA, letS, letE or fliA. Together, the data indicate
that, as the nutrient supply wanes, ppGpp triggers a regulatory cascade mediated
by LetA/ LetS, RpoS, FliA and letE that coordinates differentiation of replicating
L. pneumophila to a transmissible form.
Genetics of the phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2)
Authors: Sumby Paul , Smith Margaret C. M.
Published: March 2003

The phage growth limitation (Pgl) system, encoded by Streptomyces coelicolor A3(2), confers protection against the temperate bacteriophage φC31 and its homoimmune relatives. The Pgl phenotype is characterized by the ability of Pgl+ hosts to support a phage burst on initial infection but subsequent cycles are severely attenuated. Previously, two adjacent genes pglY and pglZ were shown to be required for Pgl. It had been shown by Southern blotting that Streptomyces lividans, a close relative of S. coelicolor and naturally Pgl−, does not contain homologues of pglYZ and that introduction of pglYZ into S. lividans is not sufficient to confer a Pgl+ phenotype. Moreover, the mechanism of the Pgl+↔ Pgl− phase variation associated with this phenotype is also not understood. Here we describe two novel genes, pglW and pglX, that were shown to be part of this system by complementation of Pgl− mutants and by insertional mutagenesis. pglW encodes a 169 kDa protein that includes putative motifs for both serine/threonine protein kinase activity and DNA binding. pglX encodes a 136 kDa protein with putative adenine‐specific DNA methyltransferase activity. pglW and pglX have overlapping stop‐start codons suggesting transcriptional and translational coupling. S1 mapping of transcripts initiating up‐stream of pglW indicated that, like pglYZ, pglWX is expressed in uninfected cultures. A homologue of pglX with 76% amino acid identity was identified in S. coelicolor, and insertional mutagenesis indicated that this gene was not required for the Pgl+ phenotype. Southern blots indicated that S. lividans does not contain homologues of pglW or pglX. A plasmid encoding pglWXYZ was able to confer the Pgl+ phenotype to S. lividans implying that these four genes constitute the whole system.
Complementary activities of SseJ and SifA regulate dynamics of the Salmonella typhimurium vacuolar membrane
Authors: Ruiz‐Albert Javier , Yu Xiu‐Jun , Beuzón Carmen R. , Blakey Abigail N. , Galyov Edouard E. , Holden David W.
Published: March 2003

The Salmonella pathogenicity island 2 (SPI‐2) type III secretion system
(TTSS) of Salmonella typhimurium is required for bacterial replication within
host cells. It acts by translocating effector proteins across the membrane of the
Salmonella‐containing vacuole (SCV). The SifA effector is required to maintain
the integrity of the SCV membrane, and for the formation in epithelial cells of Salmonella‐induced
filaments (Sifs), which are tubular extensions of SCVs. We have investigated the
role in S. typhimurium virulence of the putative SPI‐2 effector genes sifB,
srfJ, sseJ and sseI. An S. typhimurium strain carrying a
mutation in sseJ was mildly attenuated for systemic virulence in mice, but
strains carrying mutations in either srfJ, sseI or sifB had
very little or no detectable virulence defect after intraperitoneal inoculation.
Expression of SseJ in HeLa cells resulted in the formation of globular membranous
compartments (GMCs), the composition of which appears to be similar to that of SCV
membranes and Sifs. The formation of GMCs was dependent on the serine residue of
the predicted acyltransferase/lipase active site of SseJ. Transiently expressed SseJ
also inhibited Sif formation by wild‐type bacteria, and was found to associate with
Sifs, SCV membranes and simultaneously expressed SifA. Intracellular vacuoles containing
sseJ mutant bacteria appeared normal but, in contrast to a sifA mutant,
a sifA sseJ double mutant strain did not lose its vacuolar membrane, indicating
that loss of vacuolar membrane around sifA mutant bacteria requires the action of SseJ. Collectively, these results suggest that the combined action of SseJ and SifA regulate dynamics of the SCV membrane in infected cells.
The Pneumocystis carinii drug target S‐adenosyl‐L‐methionine:sterol C‐24 methyl transferase has a unique substrate preference
Authors: Kaneshiro Edna S. , Rosenfeld Jill A. , Basselin‐Eiweida Mireille , Stringer James R. , Keely Scott P. , Smulian A. George , Giner José‐Luis
Published: March 2003

Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S‐adenosyl‐L‐methionine (SAM):C‐24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24‐methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24‐methylenelanosterol as substrates, the major reaction products were 24‐methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C‐24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C‐24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.
SecDFyajC forms a heterotetrameric complex with YidC
Authors: Nouwen Nico , Driessen Arnold J. M.
Published: March 2003

The Escherichia coli preprotein translocase is composed of a ‘preprotein conducting channel’ domain that consists of the peripherally bound translocation ATPase SecA and the heterotrimeric SecYEG membrane protein complex. SecD, SecF, and YajC form another heterotrimeric complex that can associate with the SecYEG complex. YidC is an essential membrane protein that plays a role in the integration of newly synthesized membrane proteins, and has been shown to co‐purify with SecYEG when all translocase components are overproduced. Here, we demonstrate that under conditions that YidC co‐purifies with overproduced SecDFyajC it does not co‐purify with overproduced SecYEG. Moreover, this interaction of YidC with the SecDFyajC complex is also found at chromosomal protein levels of SecD, SecF and YajC. Closer examination of the SecDFyajC–YidC complex showed that YidC binds to SecD and SecF, whereas YajC interacts only with SecF. As SecF and YajC have previously been shown to interact with SecY, we propose that these two proteins link the heterotetrameric SecDFyajC–YidC complex to the SecYEG complex.
Interaction surface of the Spo0A response regulator with the Spo0E phosphatase
Authors: Stephenson Sophie J. , Perego Marta
Published: March 2003

Spo0A~P is the essential response regulator and transcription factor for sporulation initiation in Bacillus subtilis. The phosphorylation level of Spo0A in the

cell is determined by the sensor kinase activity of the phosphorelay, donating phosphoryl

groups, and the antagonistic effects of dephosphorylation mediated by the Rap and

Spo0E families of phosphatases. In this study, spo0A mutations were generated

that encoded proteins less sensitive to the activity of Spo0E than the wild‐type

protein. The Spo0A substitutions N12K, P60S, L62P and F88L are surface exposed and

localize to the same face of the molecule as the active site and in its close proximity

on the β1–α1, β3–α3 and

β4–α4 loops. The corresponding surface in the Spo0F response regulator was shown previously to be involved in the interaction with the RapB phosphatase, as well as the KinA histidine kinase and the Spo0B phosphotransferase. Thus, residues occupying the same position (N12:Q12, F88:Y84) and the same loops in Spo0A or Spo0F are involved in the interaction with the structurally unrelated Spo0E and RapB phosphatases, respectively, in addition to kinases and phosphotransferase. The specificity in phosphatase target recognition must be the result of side‐chain variability within the response regulators and the interactions they promote. The residues involved in Spo0E interaction are identical in all Spo0A orthologues from spore‐forming Bacilli encoding Spo0E phosphatases.
Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus
Authors: Gérard‐Vincent Manon , Robert Viviane , Ball Geneviève , Bleves Sophie , Michel Gérard P. F. , Lazdunski Andrée , Filloux Alain
Published: March 2003

Gram‐negative bacteria have evolved several types of secretion mechanisms to release proteins into the extracellular medium. One such mechanism, the type II secretory system, is a widely conserved two‐step process. The first step is the translocation of signal peptide‐bearing exoproteins across the inner membrane. The second step, the translocation across the outer membrane, involves the type II secretory apparatus or secreton. The secretons are made up of 12–15 proteins (Gsp) depending on the organism. Even though the systems are conserved, hetero‐logous secretion is mostly species restricted. Moreover, components of the secreton are not systematically exchangeable, especially with distantly related microorganisms. In closely related species, two components, the GspC and GspD (secretin) family members, confer specificity for substrate recognition and/or secreton assembly. We used Pseudomonas aeruginosa as a

model organism to determine which domains of XcpP (GspC member) are involved in specificity.

By constructing hybrids between XcpP and OutC, the Erwinia chrysanthemi homologue, we identified a region of 35 residues that was not exchangeable. We showed that this region might influence the stability of the XcpYZ secreton subcomplex. Remarkably, XcpP and OutC have domains, coiled‐coil and PDZ, respectively, which exhibit the same function but that are structurally different. Those two domains are exchangeable and we provided evidence that they are involved in the formation of homomultimeric complexes of XcpP.
Direct and indirect roles of CcpA in regulation of Bacillus subtilis Krebs cycle genes
Authors: Kim Hyun‐Jin , Roux Agnes , Sonenshein Abraham L.
Published: March 2003

Carbon catabolite repression of the Bacillus subtilis citrate synthase (citZ) and aconitase (citB) genes, previously known to be regulated by CcpC, was shown to depend on CcpA as well. Transcription of the citZ gene was partially derepressed in ccpA and ccpC single mutants and fully derepressed in a ccpA ccpC double mutant. DNase I footprinting studies showed that CcpA binds to a catabolite‐responsive element (cre) site located at positions +80 to +97 with respect to the transcription start site, whereas CcpC binds at positions −14 to +6 and +16 to +36. Mutations in the citZ cre site greatly altered CcpA binding and repression. A ccpA null mutation also caused partial derepression of citB. Disruption of citrate synthase activity, however, suppressed the effect of the ccpA mutation, suggesting that increased citrate accumulation in a ccpA mutant partially inactivates CcpC and causes partial derepression of citB. Therefore, CcpA controls expression of Krebs cycle genes directly by regulating transcription of citZ and in‐directly by regulating availability of citrate, the inducer for CcpC.
Survival mechanisms for Streptomyces linear replicons after telomere damage
Authors: Qin Zhongjun , Cohen Stanley N.
Published: March 2003

The ability of linear replicons to propagate their DNA after telomere damage is essential for perpetuation of the genetic information they carry. We introduced deletions at specific locations within telomeres of streptomycete linear plasmids and investigated mechanisms that enable survival. Here, we report that rescue of such plasmids in Streptomyces lividans occurs by three distinct types of events: (i) repair of the damaged telomere by homologous recombination; (ii) circularization of the plasmid by non‐homologous end‐to‐end joining; and (iii) formation of long palindromic linear plasmids that duplicate the intact telomere by a non‐recombinational process. The relative frequency of use of these survival mechanisms depended on the location and length of the telomeric DNA deletion. Repair by intermolecular recombination between the telomeres of chromosomes and plasmids, deletion of additional DNA during plasmid circularization, and insertion of chromosomal DNA fragments into plasmids during end‐to‐end joining were observed. Our results show that damage to telomeres of Streptomyces linear replicons can promote major structural transformations in these replicons as well as genetic exchange between chromosomes and extrachromosomal DNA. Our findings also suggest that spontaneous circularization of linear Streptomyces chromosomes may be a biological response to instances of telomere damage that cannot be repaired by homologous recombination.
The Neurospora crassa pheromone precursor genes are regulated by the mating type locus and the circadian clock
Authors: Bobrowicz Piotr , Pawlak Rebecca , Correa Alejandro , Bell‐Pedersen Deborah , Ebbole Daniel J.
Published: March 2003

Pheromones play important roles in female and male behaviour in the filamentous ascomycete fungi. To begin to explore the role of pheromones in mating, we have identified the genes encoding the sex pheromones of the heterothallic species Neurospora crassa. One gene, expressed exclusively in mat A strains, encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by Kex2 processing sites. Strains of the opposite mating type, mat a, express a pheromone precursor gene whose polypeptide contains a C‐terminal CAAX motif predicted to produce a mature pheromone with a C‐terminal carboxy‐methyl isoprenylated cysteine. The predicted sequences of the pheromones are remarkably similar to those encoded by other filamentous ascomycetes. The expression of the pheromone precursor genes is mating type specific and is under the control of the mating type locus. Furthermore, the genes are highly expressed in conidia and under conditions that favour sexual development. Both pheromone precursor genes are also regulated by the endogenous circadian clock in a time‐of‐day‐specific fashion, supporting a role for the clock in mating.
Mutations in Bacillus subtilis glutamine synthetase that block its interaction with transcription factor TnrA
Authors: Fisher Susan H. , Brandenburg Jaclyn L. , Wray Lewis V.
Published: March 2003

In Bacillus subtilis, the activity of the nitrogen regulatory factor TnrA is regulated through a protein– protein interaction with glutamine synthetase. During growth with excess nitrogen, the feedback‐inhibited form of glutamine synthetase binds to TnrA and blocks DNA binding by TnrA. Missense mutations in glutamine synthetase that constitutively express the TnrA‐regulated amtB gene were characterized. Four mutant proteins were purified and shown to be defective in their ability to inhibit the in vitro DNA‐binding activity of TnrA. Two of the mutant proteins exhibited enzymatic properties similar to those of wild‐type glutamine synthetase. A model of B. subtilis glutamine synthetase was derived from a crystal structure of the Salmonella typhimurium enzyme. Using this model, all the mutated amino acid residues were found to be located close to the glutamate entrance of the active site. These results are consistent with the glutamine synthetase protein playing a direct role in regulating TnrA activity.
Donor strand complementation mechanism in the biogenesis of non‐pilus systems
Authors: Zavialov A. V. , Kersley J. , Korpela T. , Zav’yalov V. P. , MacIntyre S. , Knight S. D.
Published: March 2003

The F1 antigen of Yersinia pestis belongs to a class of non‐pilus adhesins assembled via a classical chaperone–usher pathway. Such pathways consist of PapD‐like chaperones that bind subunits and pilot them to the outer membrane usher, where they are assembled into surface structures. In a recombinant Escherichia coli model system, chaperone–subunit (Caf1M:Caf1n) complexes accumulate in the periplasm. Three inde‐pendent methods showed that these complexes are rod‐ or coil‐shaped linear arrays of Caf1 subunits capped at one end by a single copy of Caf1M chaperone. Deletion and point mutagenesis identified an N‐terminal donor strand region of Caf1 that was essential for polymerization in vitro, in the periplasm and at the cell surface, but not for chaperone–subunit interaction. Partial protease digestion of periplasmic complexes revealed that this region becomes buried upon formation of Caf1:Caf1 contacts. These results show that, despite the capsule‐like appearance of F1 antigen, the basic structure is assembled as a linear array of subunits held together by intersubunit donor strand complementation. This example shows that strikingly different architectures can be achieved by the same general principle of donor strand complementation and suggests that a similar basic polymer organization will be shared by all surface structures assembled by classical chaperone–usher pathways.
Stage‐specific gene expression during sexual development in Phytophthora infestans
Authors: Fabritius Anna‐Liisa , Cvitanich Cristina , Judelson Howard S.
Published: March 2003

Eight genes that are upregulated during sexual development in the heterothallic oomycete, Phytophthora infestans, were identified by suppression subtractive hybridization. Two genes showed very low but detectable expression in vegetative hyphae and became induced about 40‐ to >100‐fold early in mating, before gametangial initials appeared. The remaining six loci were not induced until later in mating, coincident with the formation of gametangia and oospores, with induction levels ranging from 60‐ to >100‐fold. Five genes were single copy, and three were members of families. Sequence analysis revealed that the predicted products of three of the genes had similarity to proteins that influence RNA stability, namely a ribonuclease activator, the pumilio family of RNA‐binding proteins and RNase H. The products of two other mating‐induced genes resembled two types of Phytophthora proteins previously shown to elicit plant defence responses. Each mating‐induced gene was also expressed in a self‐fertile strain, which was shown to be a heterokaryon. However, quantitative and qualitative differences existed in their expression in normal matings and in the self‐fertile heterokaryon. Besides the mating‐induced genes, two extrachromosomal RNA elements were identified.
The GlnD and GlnK homologues of Streptomyces coelicolor A3(2) are functionally dissimilar to their nitrogen regulatory system counterparts from enteric bacteria
Authors: Hesketh A. , Fink D. , Gust B. , Rexer H.‐U. , Scheel B. , Chater K. , Wohlleben W. , Engels A.
Published: March 2003

Glutamine synthetase I (GSI) enzyme activity in Streptomyces coelicolor is controlled post‐translationally by the adenylyltransferase (GlnE) as in enteric bacteria. Although other homologues of the Escherichia coli Ntr system (glnK, coding for a PII family protein; and glnD, coding for an uridylyltransferase) are found in the S. coelicolor genome, the regulation of the GSI activity was found to be different. The functions of glnK and glnD were analysed by specific mutants. Surprisingly, biochemical assay and two‐dimensional PAGE analysis showed that modification of GSI by GlnE occurs normally in all mutant strains, and neither GlnK nor GlnD are required for the regulation of GlnE in response to nitrogen stimuli. Analysis of the post‐translational regulation of GlnK in vivo by two‐dimensional PAGE and mass spectrometry indicated that it is subject to both a reversible and a non‐reversible modification in a direct response to nitrogen availability. The irreversible modification was identified as removal of the first three N‐terminal amino acid residues of the protein, and the reversible modification as adenylylation of the conserved tyro‐sine 51 residue that is known to be uridylylated in E. coli. The glnD insertion mutant expressing only the N‐terminal half of GlnD was capable of adenylylating GlnK, but was unable to perform the reverse deadenylylation reaction in response to excess ammonium. The glnD null mutant completely lacked the ability to adenylylate GlnK. This work provides the first example of a PII protein that is modified by adenylylation, and demonstrates that this reaction is performed by a homologue of GlnD, previously described only as a uridylyltransferase enzyme.
Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin
Authors: Garg Ram P. , Ma Yunqing , Hoyt Jeffrey C. , Parry Ronald J.
Published: March 2003

Streptomyces viridifaciens
MG456‐hF10 produces the antibiotic valanimycin, a naturally occurring azoxy compound. Valanimycin is known to be derived from valine and serine with the intermediacy of isobutylamine and isobutylhydroxylamine, but little is known about the stages in the pathway leading to the formation of the azoxy group. In previous studies, a cosmid containing
S. viridifaciens
DNA was isolated that conferred valanimycin production upon
Strepto‐myces lividans
TK24. Subcloning of DNA from the valanimycin‐producing cosmid has led to the identi‐fication of a 22 kb segment of DNA sufficient to allow valanimycin production in
S. lividans
TK24. Sequencing of this DNA segment and the surrounding DNA revealed the presence of 20 genes. Gene disruption experiments defined the boundaries of the valanimycin gene cluster, which appears to contain 14 genes. The cluster includes an amino acid decar‐boxylase gene (
vlmD
), a valanimycin resistance gene (
vlmF
), at least two regulatory genes (
vlmE
,
vlmI
), two genes encoding a flavin monooxygenase (
vlmH
,
vlmR
), a seryl tRNA synthetase gene (
vlmL
) and seven genes of unknown function. Overproduction and characterization of VlmD demonstrated that it catalyses the decarboxylation of
l ‐valine. An unusual feature of the valanimycin gene cluster is that four genes involved in branched amino acid biosynthesis are located near its 5′ end.
Characterization of interaction sites in the Saccharomyces cerevisiae ribosomal stalk components
Authors: Lalioti V. S. , Pérez‐Fernández J. , Remacha M. , Ballesta J. P. G.
Published: March 2003

The interactions among the yeast stalk components (P0, P1α, P1β, P2α and P2β) and with EF‐2 have been explored using immunoprecipitation, affinity chromatography and the two‐hybrid system. No stable association was detected between acidic proteins of the same type. In contrast, P1α and P1β were found to interact with P2β and P2α respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C‐terminal P0 fragments. Immunoprecipitation shows the association of EF‐2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two‐hybrid assay. This interaction improves using the 100‐amino‐acid‐long C‐end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1α with P2β and of P1β with P2α rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.
The UV‐B stimulon of the terrestrial cyanobacterium Nostoc commune comprises early shock proteins and late acclimation proteins
Authors: Ehling‐Schulz Monika , Schulz Stefan , Wait Robin , Görg Angelika , Scherer Siegfried
Published: March 2003

The UV‐B and desiccation‐tolerant terrestrial cyanobacterium Nostoc commune was grown under defined UV irradiation. Proteome changes were monitored in the membrane and the cytosolic and the extracellular fractions. Tools were developed to separate stress‐triggered from growth stage‐dependent changes. UV‐B changed the relative cellular concentration of 493 out of 1350 protein spots at least by a factor of three, rendering the UV‐B stimulon of N. commune the most complex one described so far. It comprises two different parts: an early shock response influencing 214 proteins and a late accli‐mation response involving 279 proteins. The shock response comprised many membrane or membrane‐associated proteins, whereas the acclimation re‐sponse mainly changed cytosolic proteins. Most of the shock‐induced changes were transient and did not overlap with the acclimation response. In the extracellular fraction, UV irradiation induced superoxide dismutase and the water stress protein. In total, 27 intracellular, UV‐B‐induced proteins were partially sequenced by electrospray ionization tandem mass spectrometry. Three functional classes were identified: proteins involved in lipid metabolism, in carbohydrate metabolism and in regulatory pathways. About 50% of the sequenced proteins were homologous to cyanobacterial database entries with un‐known function. Interestingly, all of these proteins belong to the UV‐B acclimation response. We conclude that the UV‐B shock response and the UV‐B acclimation response represent two completely different and remarkably complex strategies of N. commune to protect itself against UV‐B radiation in its natural environment.
The histone deacetylase Hda1 from Ustilago maydis is essential for teliospore development
Authors: Reichmann Michael , Jamnischek Alexander , Weinzierl Gerhard , Ladendorf Oliver , Huber Sieglinde , Kahmann Regine , Kämper Jörg
Published: March 2003

In the corn smut fungus Ustilago maydis, pathogenic development is controlled by the b mating type locus that encodes the two homeodomain proteins bE and bW. A heterodimer of bE and bW controls a large set of genes, either directly by binding to cis regulatory sequences or indirectly via a b‐dependent regulatory cascade. It is thought that several of the b‐regulated genes contribute to processes involved in pathogenicity. In a screen for components of the b‐dependent regulatory cascade we have isolated Hda1, a protein with homology to histone deacetylases of the RPD3 class. Hda1 can substitute for the histone deacetylase RPD3 in Saccharomyces cerevisiae, showing that it functions as a histone deacetylase. Deletion of hda1 results in the expression of several genes that are normally expressed only in the dikaryon, among these are several genes that are now expressed independently from their activation by the bE/bW heterodimer. hda1 mutant strains are capable to infect corn, and the proliferation of dikaryotic hyphae within the plant appears comparable to wild‐type strains during initial developmental stages. Upon karyogamy, however, the proliferation to mature teliospores is blocked. The block in sporogenesis in Δhda1 strains is probably a result of the deregulation of a specific set of genes whose temporal or spatial expression prevent the proper developmental progress.
Motility modes of Spiroplasma melliferum BC3: a helical, wall‐less bacterium driven by a linear motor
Authors: Gilad Rami , Porat Asher , Trachtenberg Shlomo
Published: March 2003

Spiroplasma
are members of the Mollicutes (
Mycoplasma, Acholeplasma
and
Spiroplasma
) – the simplest, minimal, free‐living and self‐replicating forms of life. The mollicutes are unique among bacteria in completely lacking cell walls and flagella and in having an internal, contractile cytoskeleton, which also functions as a linear motor.
Spiroplasma are
helical, chemotactic and viscotactic active swimmers. The Spiroplasmal cytoskeleton is a flat ribbon composed of seven pairs of fibrils. The ribbon is attached to the inner side of the cell membrane along its innermost (shortest) helical line. The cell's geometry and dynamic helical parameters, and consequently motility, can be controlled by changing differentially and in a co‐ordinated manner, the length of the fibrils. We identified several consistent modes of cell movements and motility originating, most likely, as a result of co‐operative or local molecular switching of fibrils: (i) regular extension and contraction within the limits of helical symmetry (this mode also includes straightening, beyond what is allowed by helical symmetry, and reversible change of helical sense); (ii) spontaneous and random change of helical sense originating at random sites along the cell (these changes propagate along the cell in either direction and hand switching is completed within ∼0.08 second); (iii) forming a deformation on one of the helical turns and propagating it along the cell (these helical deformations may travel along the cell at a speed of up to ∼40 µm s
−1
); (iv) random bending, flexing and twitching (equivalent to tumbling). In standard medium (viscosity = 1.147 centipoise) the cells run at ∼1.5 µm s
−1
, have a Reynolds number of ∼3.5 × 10
−6
and consume ∼30 ATP molecules s
−1 . Running velocity, duration, persistence and efficiency increase with viscosity upon adding ficoll, dextran and methylcellulose to standard media. Relative force measurements using optical tweezers confirm these findings.
Identification and characterization of a Cryptococcus neoformans ATP binding cassette (ABC) transporter‐encoding gene, CnAFR1, involved in the resistance to fluconazole
Authors: Posteraro Brunella , Sanguinetti Maurizio , Sanglard Dominique , La Sorda Marilena , Boccia Stefania , Romano Lucio , Morace Giulia , Fadda Giovanni
Published: March 2003

Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ‐cal meningitis, the most frequently encountered life‐threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole‐resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole‐resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter‐encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock‐out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.
Penicillin‐binding proteins in Streptococcus agalactiae: a novel mechanism for evasion of immune clearance
Authors: Jones Amanda L. , Needham Rachel H. V. , Clancy Anne , Knoll Katherine M. , Rubens Craig E.
Published: March 2003

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia and meningitis in the USA despite CDC‐recommended chemoprophylaxis strategies for preventing infection. To cause infection pathogens such as GBS must evade recognition and clearance by the host's immune system. Strategies for avoidance of opsonization and phagocytic killing include elaboration of antiopsonophagocytic capsules and surface proteins. During screening for mutants of GBS that were attenuated for virulence in a neonatal rat sepsis model, we identified a mutant with a transposon insertion in the ponA gene. ponA encodes an extra‐cytoplasmic penicillin‐binding protein PBP1a, a newly identified virulence trait for GBS that promotes resistance to phagocytic killing independent of capsular polysaccharide. Complementation analysis in vivo and in vitro confirmed that the altered phenotypes observed in the mutant were due to the transposon insertion in ponA. Deletion of PBP1a does not affect C3 deposition on GBS suggesting that mechanism by which PBP1a protects GBS from phagocytic killing is distinct from the antiopsonic activity of capsular polysaccharide. This is the first report describing expression of an antiphagocytic surface protein by GBS and represents a novel mechanism for evasion of immune recognition and clearance that may explain the decreased virulence observed in Gram‐positive bacterial species for penicillin‐binding protein mutants.
The essential role of the invariant GGQ motif in the function and stability in vivo of bacterial release factors RF1 and RF2
Authors: Mora Liliana , Heurgué‐Hamard Valérie , Champ Stéphanie , Ehrenberg Måns , Kisselev Lev L. , Buckingham Richard H.
Published: March 2003

Release factors RF1 and RF2 are required in bacteria for the cleavage of peptidyl‐tRNA. A single sequence motif, GGQ, is conserved in all eubacterial, archaebacterial and eukaryotic release factors and may mimic the CCA end of tRNA, although the position of the motif in the crystal structures of human eRF1 and Escherichia coli RF2 is strikingly different. Mutations have been introduced at each of the three conserved positions. Changing the Gln residue to Ala or Glu allowed the factors to retain about 22% of tetrapeptide release activity in vitro, but these mutants could not complement thermosensitive RF mutants in vivo. None of several mutants with altered Gly residues retained activity in vivo or in vitro. Many GGQ mutants were poorly expressed and are presumably unstable; many were also toxic to the cell. The toxic mutant factors or their degradation products may bind to ribosomes inhibiting the action of the normal factor. These data are consistent with a common role for the GGQ motif in bacterial and eukaryotic release factors, despite strong divergence in primary, secondary and tertiary structure, but are difficult to reconcile with the hypothesis that the amide nitrogen of the Gln plays a vital role in peptidyl‐tRNA hydrolysis.
The dnaAcos allele of Escherichia coli: hyperactive initiation is caused by substitution of A184V and Y271H, resulting in defective ATP binding and aberrant DNA replication control
Authors: Simmons Lyle A. , Kaguni Jon M.
Published: March 2003

Chromosomal DNA replication is regulated at the level of commitment to this biochemical pathway. In Escherichia coli, DnaA protein appears to regulate this process. A mutant form, DnaAcos, carrying four amino acid substitutions, is apparently defective in responding to regulatory signals, because it induces hyperactive initiation from the bacterial replication origin (oriC). In this report, the phenotype of hyperactive initiation is shown to be the result of two specific amino acid substitutions. One (A184V) immediately adjacent to a Walker A box (P loop motif) causes a defect in ATP binding (Carr and Kaguni, 1996, Mol Microbiol 20: 1307–1318). The second amino acid substitution (Y271H) appears to stabilize the activity of the mutant protein carrying the A184V substitution. The mutant protein carrying both amino acid substitutions (A184V + Y271H) is defective in modulating the frequency of initiation from oriC, as demonstrated by marker frequency analysis of oriC and a locus near the replication terminus. These results indicate that a defect in ATP binding results in aberrant control of DNA replication.