bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA‐independent transport of plasmid primase protein between bacteria by the I1 conjugation system
Authors: Wilkins Brian M. , Thomas Angela T.
Published: March 2003

The ColIb‐P9 (IncI1)‐encoded conjugation system supports transfer of the plasmid T‐strand plus hundreds of molecules of the Sog polypeptides determined by the plasmid primase gene. Here, we report that Sog primase is abundantly donated to the recipient cell from cells carrying a non‐transferable ColIb plasmid deleted of the nic site essential for DNA export. Such DNA‐independent secretion of Sog primase is typical of authentic conjugation, both in being blocked when the recipient cell specifies the entry exclusion function of ColIb and in requiring the thin I1 pilus encoded by the ColIb pil system under the mating conditions used. It is proposed that Sog polypeptides form a complex with the ColIb T‐strand during conjugation and aid DNA transport through processive secretion of the proteins into the recipient cell. Functional and genetic relationships between the ColIb conjugation system and other type IV secretion pathways are discussed.
Phage conversion of exfoliative toxin A production in Staphylococcus aureus
Authors: Yamaguchi Takayuki , Hayashi Tetsuya , Takami Hideto , Nakasone Kaoru , Ohnishi Makoto , Nakayama Keisuke , Yamada Sakuo , Komatsuzawa Hitoshi , Sugai Motoyuki
Published: March 2003

The staphylococcal exfoliative toxins (ETs) are extracellular proteins that cause splitting of human skin at the epidermal layer during infection in infants. Two antigenically distinct toxins possessing identical activity have been isolated from Staphylococcus aureus, ETA and ETB. The gene for ETA (eta) is located on the chromosome, whereas that for ETB is located on a large plasmid. The observation that relatively few clinical isolates produce ETA suggests that the eta gene is acquired by horizontal gene transfer. In this study, we isolated a temperate phage (φETA) that encodes ETA and determined the complete nucleotide sequence of the φETA genome. φETA has a head with a hexagonal outline and a non‐contractile and flexible tail. The genome of φETA is a circularly permuted linear double‐stranded DNA, and the genome size is 43 081 bp. Sixty‐six open reading frames (ORFs) were identified on the φETA genome, including eta, which was found to be located very close to a putative attachment site (attP). φETA converted ETA non‐producing strains into ETA producers. Southern blot analysis of chromosomal DNA from clinical isolates suggested that φETA or related phages are responsible for the acquisition of eta genes in S. aureus.
ASalmonellainositol polyphosphatase acts in conjunction with other bacterial effectors to promote host cell actin cytoskeleton rearrangements and bacterial internalization
Authors: Zhou Daoguo , Chen Li‐Mei , Hernandez Lorraine , Shears Stephen B. , Galán Jorge E.
Published: March 2003

A central feature ofSalmonellapathogenicity is the bacterium's ability to enter into non‐phagocytic cells. Bacterial internalization is the consequence of cellular responses characterized by Cdc42‐ and Rac‐dependent actin cytoskeleton rearrangements. These responses are triggered by the co‐ordinated function of bacterial proteins delivered into the host cell by a specialized protein secretion system termed type III. We report here that SopB, aSalmonellainositol polyphosphatase delivered to the host cell by this secretion system, mediates actin cytoskeleton rearrangements and bacterial entry in a Cdc42‐dependent manner. SopB exhibits overlapping functions with two other effectors of bacterial entry, the Rho family GTPase exchange factors SopE and SopE2. Thus,Salmonellastrains deficient in any one of these proteins can enter into cells at high efficiency, whereas a strain lacking all three effectors is completely defective for entry. Consistent with an important role for inositol phosphate metabolism inSalmonella‐induced cellular responses, a catalytically defective mutant of SopB failed to stimulate actin cytoskeleton rearrangements and bacterial entry. Furthermore, bacterial infection of intestinal cells resulted in a marked increase in Ins(1,4,5,6)P4, a consumption of InsP5and the activation of phospholipase C. In agreement with thein vivofindings, purified SopB specifically dephosphorylated InsP5to Ins(1,4,5,6)P4in vitro. Surprisingly, the inositol phosphate fluxes induced bySalmonellawere not caused exclusively by SopB. We show that the SopB‐independent inositol phosphate fluxes are the consequence of the SopE‐dependent activation of an endogenous inositol phosphatase. The ability ofSalmonellato stimulate Rho GTPases signalling and inositol phosphate metabolism through alternative mechanisms is an example of the remarkable ability of this bacterial pathogen to manipulate host cellular functions.
Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm ofEscherichia coli
Authors: Arié Jean‐Philippe , Sassoon Nathalie , Betton Jean‐Michel
Published: March 2003

The nature of molecular chaperones in the periplasm ofEscherichia colithat assist newly translocated proteins to reach their native state has remained poorly defined. Here, we show that FkpA, a heat shock periplasmic peptidyl‐prolylcis/transisomerase (PPIase), suppresses the formation of inclusion bodies from a defective‐folding variant of the maltose‐binding protein, MalE31. This chaperone‐like activity of FkpA, which is independent of its PPIase activity, requires a full‐length structure of the protein.In vitro, FkpA does not catalyse a slow rate‐limiting step in the refolding of MalE31, but prevents its aggregation at stoichiometric amounts and promotes the reactivation of denaturated citrate synthase. We propose that FkpA functions as a chaperone for envelope proteins in the bacterial periplasm.
Blue light adaptation and desensitization of light signal transduction in Neurospora crassa
Authors: Schwerdtfeger Carsten , Linden Hartmut
Published: March 2003

The ascomycete Neurospora crassa has the capacity of adapting to a given light quantity, leading to transient blue light responses under continuous light conditions. Here, we present an investigation of this photoadaptation phenomenon. We demonstrated previously that two proteins of the Neurospora blue light signal transduction chain, WC1 and WC2, are subject to light‐dependent phosphorylation. WC1 was phosphorylated in parallel with the transient increase in transcript levels of light‐regulated genes. Using the light‐dependent phosphorylation of WC1 as a marker for an active signalling state of WC1, we show that the transiency of Neurospora blue light responses results from desensitization of the photoreceptor and/or the signalling cascade. Furthermore, a Neurospora mutant was characterized that revealed a specific defect in photoadaptation. In this mutant, the transient expression of light‐regulated genes under continuous light, the temporary insensitivity after a light pulse and the capability of differentiating between and adapting to low and high light intensities were abolished. The corresponding protein seems to represent a central component of a negative feedback desensitization mechanism. This negative feedback regulation requires continuous and light‐dependent protein de novo biosynthesis.
The Neurospora circadian clock regulates a transcription factor that controls rhythmic expression of the output eas(ccg‐2) gene
Authors: Bell‐Pedersen Deborah , Lewis Zachary A. , Loros Jennifer J. , Dunlap Jay C.
Published: March 2003

The circadian clock provides a link between an organism's environment and its behaviour, temporally phasing the expression of genes in anticipation of daily environmental changes. Input pathways sense environmental information and interact with the clock to synchronize it to external cycles, and output pathways read out from the clock to impart temporal control on downstream targets. Very little is known about the regulation of outputs from the clock. In Neurospora crassa, the circadian clock transcriptionally regulates expression of the clock‐controlled genes, including the well‐characterized eas(ccg‐2) gene. Dissection of the eas(ccg‐2) gene promoter previously localized a 68 bp sequence containing an activating clock element (ACE) that is both necessary and sufficient for rhythmic activation of transcription by the circadian clock. Using electrophoretic mobility shift assays (EMSAs), we have identified light‐regulated nuclear protein factors that bind specifically to the ACE in a time‐of‐day‐dependent fashion, consistent with their role in circadian regulation of expression of eas(ccg‐2). Nucleotides in the ACE that interact with the protein factors were determined using interference binding assays, and deletion of the core interacting sequences affected, but did not completely eliminate, rhythmic accumulation of eas(ccg‐2) mRNA in vivo, whereas deletion of the entire ACE abolished the rhythm. These data indicate that redundant binding sites for the protein factors that promote eas(ccg‐2) rhythms exist within the 68 bp ACE. The ACE binding complexes formed using protein extracts from cells with lesions in central components of the Neurospora circadian clock were identical to those formed with extracts from wild‐type cells, indicating that other proteins directly control eas(ccg‐2) rhythmic expression. These data suggest that the Neurospora crassa circadian clock regulates an unknown transcription factor, which in turn activates the expression of eas(ccg‐2) at specific times of the day.
KIN241: a gene involved in cell morphogenesis in Paramecium tetraurelia reveals a novel protein family of cyclophilin–RNA interacting proteins (CRIPs) conserved from fission yeast to man
Authors: Krzywicka Anna , Beisson Janine , Keller Anne‐Marie , Cohen Jean , Jerka‐Dziadosz Maria , Klotz Catherine
Published: March 2003

In this study, we report cloning, by functional complementation of the KIN241 gene involved in Paramecium cell morphogenesis, cortical organization and nuclear reorganization. This gene is predicted to encode a protein of a novel type, comprising a cyclophilin‐type, peptidyl‐prolyl isomerase domain, an RNA recognition motif, followed by a region rich in glutamate and lysine (EK domain) and a C‐terminal string of serines. As homologues of this protein are present in the genomes of Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Homo sapiens, the Kin241p predicted sequence defines a new family of proteins that we propose to call ‘CRIP’, for cyclophilin–RNA interacting protein. We demonstrate that, in Paramecium, Kin241p is localized in the nucleus and that deletion of some nuclear localization signals (NLSs) decreases transport of the protein into the nucleus. No Kin241‐1 protein is present in mutant cells, suggesting that the C‐terminal serine‐rich region is responsible for protein stability.
Extracellular superoxide production by Enterococcus faecalis requires demethylmenaquinone and is attenuated by functional terminal quinol oxidases
Authors: Huycke Mark M. , Moore Danny , Joyce Wendy , Wise Phillip , Shepard Laura , Kotake Yashige , Gilmore Michael S.
Published: March 2003

The intestinal commensal bacterium, Enterococcus faecalis, is unusual among prokaryotic organisms in its ability to produce substantial extracellular superoxide. Transposon mutagenesis, allelic replacement, and electron spin resonance (ESR)‐spin trapping showed that superoxide production and generation of derivative hydroxyl radical were dependent on membrane‐associated demethylmenaquinone. Extracellular superoxide was generated through univalent reduction of oxygen by reduced demethylmenaquinone. Moreover, extracellular superoxide production was inhibited by exogenous haematin, an essential cofactor for cytochrome bd, and by fumarate, a substrate for fumarate reductase. As integral membrane quinol oxidases, cytochrome bd and fumarate reductase redox cycle demethylmenaquinone, and are necessary for aerobic and anaerobic respiration respectively. A rat model of intestinal colonization demonstrated that conditions exist in the mammalian intestinal tract that permit a mode of respiration for E. faecalis that results in the formation of hydroxyl radical. These results identify and characterize the mechanism by which E. faecalis generates extracellular free radicals.
The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages
Authors: Gold Benjamin , Rodriguez G. Marcela , Marras Salvatore A. E. , Pentecost Mickey , Smith Issar
Published: March 2003

In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR (iron‐dependent regulator), an iron‐responsive DNA‐binding protein of the DtxR family that has been shown to regulate iron acquisition in Mycobacterium smegmatis. To identify some of the genes that constitute the IdeR regulon, we searched the M. tuberculosis genome for promoter regions containing the consensus IdeR/DxR binding sequence. Genes preceded by IdeR boxes included a set encoding proteins necessary for iron acquisition, such as the biosynthesis of siderophores (mbtA, mbtB, mbtI), aromatic amino acids (pheA, hisE, hisB‐like) and others annotated to be involved in the synthesis of iron‐storage proteins (bfrA, bfrB). Some putative IdeR‐regulated genes identified in this search encoded proteins predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)‐like molecules (rv3402c), lipids (acpP) and peptidoglycan (murB). We analysed four promoter regions containing putative IdeR boxes, mbtA–mbtB, mbI, rv3402c and bfrA–bfd, for interaction with IdeR and for iron‐dependent expression. Gel retardation experiments and DNase footprinting analyses with purified IdeR showed that IdeR binds to these IdeR boxes in vitro. Analysis of the promoters by primer extension indicated that the IdeR boxes are located near the −10 position of each promoter, suggesting that IdeR acts as a transcriptional repressor by blocking RNA polymerase binding. Using quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) coupled to molecular beacons, we showed that mRNA levels of mbtA, mbtB, mbtI, rv3402c and bfd are induced 14‐ to 49‐fold in cultures of M. tuberculosis starved for iron, whereas mRNA levels of bfrA decreased about threefold. We present evidence that IdeR not only acts as a transcriptional repressor but also functions as an activator of bfrA. Three of the IdeR‐ and iron‐repressed genes, mbtB, mbtI and rv3402c, were induced during M. tuberculosis infection of human THP‐1 macrophages.
The Fur repressor controls transcription of iron‐activated and ‐repressed genes in Helicobacter pylori
Authors: Delany Isabel , Spohn Gunther , Rappuoli Rino , Scarlato Vincenzo
Published: March 2003

The ferric uptake regulator (Fur) protein is known to act as a Fe2+‐dependent transcriptional repressor of bacterial promoters. Here, we show that, in Helicobacter pylori, Fur can mediate the regulation of iron‐activated genes in contrast to classical Fur regulation, in which iron acts as a co‐repressor. Inactivation of the fur gene in the chromosome of H. pylori resulted in the derepression of a 19 kDa protein that was identified by N‐terminal sequencing as the non‐haem‐containing ferritin (Pfr). Growth of the wild‐type H. pylori strain on media treated with increasing concentrations of FeSO4 resulted in induction of transcription from the Ppfr promoter and, conversely, depletion of iron resulted in repression of Ppfr, indicating that this promoter is iron activated. In the fur mutant, the Ppfr promoter is constitutively highly expressed and no longer responds to iron, indicating that the Fur protein mediates this type of iron regulation. Footprinting analysis revealed that Fur binds to the Ppfr promoter region and that Fe2+ decreases the efficiency of binding. In contrast, Fe2+ increased the affinity of Fur for a classical Fur‐regulated promoter, the iron‐repressed frpB gene promoter. To our knowledge, this is the first evidence of direct interaction between the Fur protein and the promoter of an iron‐activated (‐derepressed) gene. Our results support a model in which the iron status of the Fur protein differentially alters its affinity for operators in either iron‐repressed or iron‐activated genes.
Identification of genes in the bW/bE regulatory cascade in Ustilago maydis
Authors: Brachmann Andreas , Weinzierl Gerhard , Kämper Jörg , Kahmann Regine
Published: March 2003

In the phytopathogenic fungus Ustilago maydis, the switch to filamentous growth and pathogenic development is controlled by a heterodimeric transcription factor consisting of the bW and bE homeodomain proteins. To identify genes in the regulatory cascade triggered by the bW/bE heterodimer, we have constructed strains in which transcription of the b genes is inducible by either arabinose or nitrate. At different time‐points after induction, genes that are switched on or off were identified through a modified, non‐radioactive RNA fingerprint procedure. From 348 gene fragments isolated initially, 48 fragments representing 34 different genes were characterized in more detail. After eliminating known genes, false positives and genes influenced in their expression profile by media conditions, 10 new b‐regulated genes were identified. Of these, five are upregulated and five are downregulated in presence of the b heterodimer. Two do not share significant similarity to database entries, whereas the other eight show similarity to disulphide isomerases, exochitinases, cation antiporters, plasma membrane (H+)‐ATPases, acyl transferases, a capsular associated protein of Cryptococcus neoformans, DNA polymerases X, as well as to a potential protein of Neurospora crassa. We demonstrate that in one of the early upregulated genes, the promoter can be bound by a bW/bE fusion protein in vitro. Interestingly, three out of the four genes that are downregulated by the b heterodimer appear upregulated after pheromone stimulation, suggesting a connection to the mating process.
The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins
Authors: Yang Chien‐Chin , Huang Chih‐Hung , Li Chien‐Yi , Tsay Yeou‐Guang , Lee Sheng‐Chung , Chen Carton W.
Published: March 2003

The chromosomes of the soil bacteria Streptomyces, unlike those of most other bacteria, are linear DNA molecules. Their telomeres contain long‐terminal inverted repeats and covalently bound terminal proteins (TPs). These bacteria also harbour linear plasmids that share the same structural features. In this study, we demonstrated that the TP was covalently bound to the 5′ ends as proposed previously. A linear plasmid with chromosomal telomeres was constructed and used to purify the TPs of the Strep‐tomyces coelicolor A3(2) chromosome. A 20 kDa protein and its 10 kDa degradation product were isolated and their sequences determined by mass spectrometry. The coding sequence (tpgC) was about 100 kb from the right end of the chromosome. Two tpg homologues were identified by sequencing the 50 kb linear plasmid SLP2 of Streptomyces lividans: tpgSLP2 at 6 kb from the left end and a putative tpg pseudogene at 8 kb from the right. The latter was in a terminal repeat shared by the right end of SLP2 and both ends of the S. lividans chromosome. The lack of the typical Streptomyces codon preference in this open reading frame suggests that it is a pseudogene. The close physical linkage between the tpg genes and their cognate telomeres would favour their co‐segregation and co‐evolution. All the Tpg polypeptides are similar in length (184–185 amino acids) and sequences, which include a putative helix domain that is homologous to part of the DNA‐binding ‘thumb’ domain of HIV reverse transcriptase, and a putative amphiphilic beta‐sheet that may be involved in the observed self‐aggregation of the TP and/or the proposed membrane binding.
Control of the AcrAB multidrug efflux pump by quorum‐sensing regulator SdiA
Authors: Rahmati Sonia , Yang Shirley , Davidson Amy L. , Zechiedrich E. Lynn
Published: March 2003

SdiA is an Escherichia coli protein that regulates cell division in a cell
density‐dependent, or quorum‐sensing, manner. We report that SdiA also controls multidrug
resistance by positively regulating the multidrug resistance pump AcrAB. Overproduction
of SdiA confers multidrug resistance and increased levels of AcrAB. Conversely, sdiA null mutants are hypersensitive to drugs and have decreased levels of AcrB protein. Our findings provide a link between quorum sensing and multidrug efflux. Combined with previously published reports, our data support a model in which a role of drug efflux pumps is to mediate cell–cell communication in response to cell density. Xenobiotics expelled by pumps may resemble the communication molecules that they normally efflux.
Phage P4 origin‐binding domain structure reveals a mechanism for regulation of DNA‐binding activity by homo‐ and heterodimerization of winged helix proteins
Authors: Yeo Hye‐Jeong , Ziegelin Günter , Korolev Sergey , Calendar Richard , Lanka Erich , Waksman Gabriel
Published: March 2003

The origin‐binding domain of the gpα protein of phage P4 (P4‐OBD) mediates

origin recognition and regulation of gpα activity by the protein Cnr.

We have determined the crystal structure of P4‐OBD at 2.95 Å resolution. The

structure of P4‐OBD is that of a dimer with pseudo twofold symmetry. Each subunit

has a winged helix topology with a unique structure among initiator proteins. The

only structural homologue of the P4‐OBD subunit is the DNA‐binding domain of the

eukaryotic transcriptional activator Rfx1. Based on this structural alignment, a

model for origin recognition by the P4‐OBD dimer is suggested. P4‐OBD mutations that

interfere with Cnr binding locate to the dimer interface, indicating that Cnr acts

by disrupting the gpα dimer. P4‐OBD dimerization is mediated by helices

α1 and α3 in both subunits, a mode of winged helix protein dimerization that is reminiscent of that of the eukaryotic transcription factors E2F and DP. This, in turn, suggests that Cnr is also a winged helix protein, a possibility that is supported by previously unreported sequence homologies between Cnr and Rfx1 and homology modelling. Hence, in a mechanism that appears to be conserved from phage to man, the DNA‐binding activity of winged helix proteins can be regulated by other winged helix proteins via the versatile use of the winged helix motif as a homo‐ or heterodimerization scaffold.
The fle1 gene encoding a C2H2 zinc finger protein co‐ordinates male and female sexual differentiation in Podospora anserina
Authors: Coppin Evelyne
Published: March 2003

The flexuosa (fle1‐1) mutant, isolated in Podospora anserina,

displays vegetative defects and two antagonistic sexual phenotypes: it produces several

1000‐fold fewer microconidia (male gametes) than the wild‐type strain and, conversely,

more abundant protoperithecia (female organs). Cloning and sequencing of the fle1

gene and of cDNA identified an open reading frame encoding a 382‐amino‐acid polypeptide

with two C2H2 zinc finger motifs. The predicted FLE1 protein shares 46% identity

with the FlbC protein of Aspergillus nidulans and 68% identity

with a putative protein identified by a search in the Neurospora crassa database.

The nuclear localization of FLE1 was demonstrated by fusion with the green fluorescent

protein. Sequencing of the fle1‐1 mutant allele revealed a frameshift mutation

upstream of the zinc finger domain. The fle1‐1 mutant was a null mutant, as

targeted disruption of fle1 sequence led to the same pleiotropic phenotype.

When fle1 was overexpressed by introduction of a transgenic copy of the native

fle1 gene or a fusion with a strong promoter, formation of protoperithecia

was impaired, leading to partial or complete female sterility. We propose that fle1 acts as a repressor of female sexual differentiation in order to maintain the balance between male and female sexual pathways.
The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants
Authors: Oliver Antonio , Baquero Fernando , Blázquez Jesús
Published: March 2003

We have recently described the presence of a high proportion of Pseudomonas

aeruginosa isolates (20%) with an increased mutation frequency (mutators)

in the lungs of cystic fibrosis (CF) patients. In four out of 11 independent P.

aeruginosa strains, the high mutation frequency was found to be complemented

with the wild‐type mutS gene from P. aeruginosa PAO1. Here, we report

the cloning and sequencing of two additional P. aeruginosa mismatch repair

genes and the characterization, by complementation of deficient strains, of these

two putative P. aeruginosa mismatch repair genes (mutL and uvrD).

We also describe the alterations in the mutS, mutL and uvrD

genes responsible for the mutator phenotype of hypermutable P. aeruginosa

strains isolated from CF patients. Seven out of the 11 mutator strains were found

to be defective in the MMR system (four mutS, two mutL and one uvrD).

In four cases (three mutS and one mutL), the genes contained frameshift

mutations. The fourth mutS strain showed a 3.3 kb insertion after the 10th

nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight

nucleotide direct repeats. This deletion, involving domain II of MutS, was found

to be the main one responsible for mutS inactivation. The second mutL

strain presented a K310M mutation, equivalent to K307 in Escherichia coli

MutL, a residue known to be essential for its ATPase activity. Finally, the uvrD

strain had three amino acid substitutions within the conserved ATP binding site of

the deduced UvrD polypeptide, showing defective mismatch repair activity. Interestingly,

cells carrying this mutant allele exhibited a fully active UvrABC‐mediated excision

repair. The results shown here indicate that the putative P. aeruginosa mutS,

mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype.
Sin recombinase fromStaphylococcus aureus: synaptic complex architecture and transposon targeting
Authors: Rowland Sally‐J. , Stark W. Marshall , Boocock Martin R.
Published: March 2003

The Sin recombinase fromStaphylococcus aureusbuilds a distinctive DNA‐protein synaptic complex to regulate strand exchange. Sin binds at two sites within an 86 basepair (bp) recombination site,resH. We propose that inverted motifs at the crossover site, and tandem motifs at the regulatory site, are recognized by structurally disparate Sin dimers. An essential architectural protein, Hbsu, binds at a discrete central site inresH. Positions of Hbsu‐induced DNA deformation coincide with natural targets for Tn552integration. Remarkably, Sin has the same topological selectivity as Tn3and γδ resolvases. Our model for the recombination synapse has at its core an assembly of four Sin dimers; Hbsu plays an architectural role that is taken by two resolvase dimers in models of the Tn3/γδ synapse.
Overexpression of White Collar‐1 (WC‐1) activates circadian clock‐associated genes, but is not sufficient to induce most light‐regulated gene expression in Neurospora crassa
Authors: Lewis Z. A. , Correa A. , Schwerdtfeger C. , Link K. L. , Xie X. , Gomer R. H. , Thomas T. , Ebbole D. J. , Bell‐Pedersen D.
Published: March 2003

Many processes in fungi are regulated by light, but the molecular mechanisms are not well understood. The White Collar‐1 (WC‐1) protein is required for all known blue‐light responses in Neurospora crassa. In response to light, WC‐1 levels increase, and the protein is transiently phosphorylated. To test the hypothesis that the increase in WC‐1 levels after light treatment is sufficient to activate light‐regulated gene expression, we used microarrays to identify genes that respond to light treatment. We then overexpressed WC‐1 in dark‐grown tissue and used the microarrays to identify genes regulated by an increase in WC‐1 levels. We found that 3% of the genes were responsive to light, whereas 7% of the genes were responsive to WC‐1 overexpression in the dark. However, only four out of 22 light‐induced genes were also induced by WC‐1 overexpression, demonstrating that changes in the levels of WC‐1 are not sufficient to activate all light‐responsive genes. The WC proteins are also required for circadian rhythms in dark‐grown cultures and for light entrainment of the circadian clock, and WC‐1 protein levels show a circadian rhythm in the dark. We found that representative samples of the mRNAs induced by over‐expression of WC‐1 show circadian fluctuations in their levels. These data suggest that WC‐1 can mediate both light and circadian responses, with an increase in WC‐1 levels affecting circadian clock‐responsive gene regulation and other features of WC‐1, possibly its phosphorylation, affecting light‐responsive gene regulation.
Sequence‐specific interaction of nascent antiterminator RNA with the zinc‐finger motif of Escherichia coli RNA polymerase
Authors: Sen Ranjan , King Rodney A. , Mzhavia Nino , Madsen Peter L. , Weisberg Robert A.
Published: March 2003

The N‐terminal Zn‐finger motif of the β′ subunit of RNA polymerase contains two pairs of invariant cysteines flanking a moderately well‐conserved segment of 13 amino acids that is rich in basic residues. Previous work showed that replacement of certain Zn‐finger residues prevented transcription antitermination in response to phage HK022 put sites. Nascent put RNA binds to and modifies transcribing polymerase, so that it becomes resistant to termination. To characterize the Zn finger further, we replaced each of the basic residues with alanine and determined the effects of the substitutions on termination, antitermination and cell viability. All the mutants were defective in put‐mediated antitermination. The severity of the defect depended on the mutant and on the sequence of the upstream stem–loop of put RNA. Some, but not all, mutants distinguished between put variants that differed in this region. This suggests that the Zn‐finger motif interacts directly and specifically with put RNA. All the mutants in the basic residues complemented a temperature‐sensitive β′ mutant for cell growth at a non‐permissive temperature, and those mutant enzymes that were tested transcribed and terminated normally in vitro on a template that lacked a put site.
Positive role of peptidoglycan breaks in lactococcal biofilm formation
Authors: Mercier Carine , Durrieu Christele , Briandet Romain , Domakova Elena , Tremblay Josselyne , Buist Girbe , Kulakauskas Saulius
Published: March 2003

Bacterial attachment to solid matrices depends on adhesive molecules present on the cell surface. Here we establish a positive correlation between peptidoglycan (PG) breaks, rather than particular molecules, and biofilm‐forming capacity in the Gram‐positive bacterium Lactococcus lactis. The L. lactis acmA strain, which is defective in PG hydrolase, adhered less efficiently than the wild‐type (wt) strain to different solid surfaces and was unable to form biofilms. These phenotypes were abolished by addition of lysozyme, a PG hydrolytic enzyme. Thus, the presence of PG breaks introduced by PG hydrolase, and not the AcmA protein itself, appears to be responsible for biofilm formation. Two different genetic screens confirmed the importance of PG breaks in L. lactis biofilm formation. Using the chain‐forming ability of the acmA strain as a phenotypic indicator of PG integrity, we selected for insertional mutants generating short chains. Five independent mutants were all mapped to ponA, which encodes the PG synthesis enzyme PBP1A. Double acmA ponA mutants displayed increased adhesion and biofilm‐forming capacity. Direct selection for strains with increased biofilm‐forming capacity resulted in the isolation of another five mutations in ponA. Based on these results, we conclude that PG breaks are important for both adhesion and biofilm formation in L. lactis.
Antifungal activity in Saccharomyces cerevisiae is modulated by calcium signalling
Authors: Edlind Thomas , Smith Lamar , Henry Karl , Katiyar Santosh , Nickels Joseph
Published: March 2003

The most important group of antifungals is the azoles (e.g. miconazole), which act by inhibiting lanosterol demethylase in the sterol biosynthesis pathway. Azole activity can be modulated through structural changes in lanosterol demethylase, altered expression of its gene ERG11, alterations in other sterol biosynthesis enzymes or altered expression of multidrug transporters. We present evidence that azole activity versus Saccharomyces cerevisiae is also modulated by Ca2+‐regulated signalling. (i) Azole activity was reduced by the addition of Ca2+. Conversely, azole activity was enhanced by the addition of Ca2+ chelator EGTA. (ii) Three structurally distinct inhibitors (fluphenazine, calmidazolium and a W‐7 analogue) of the Ca2+‐binding regulatory protein cal‐modulin enhanced azole activity. (iii) Two structurally distinct inhibitors (cyclosporin and FK506) of the Ca2+‐calmodulin‐regulated phosphatase calcineurin enhanced azole activity. (iv) Strains in which the Ca2+ binding sites of calmodulin were eliminated and strains in which the calcineurin subunit genes were disrupted demonstrated enhanced azole sensitivity; conversely, a mutant with constitutively activated calcineurin phosphatase demonstrated decreased azole sensitivity. (v) CRZ1/TCN1 encodes a transcription factor regulated by calcineurin phosphatase; its disruption enhanced azole sensitivity, whereas its overexpression decreased azole sensitivity. All the above treatments had comparable effects on the activity of terbinafine, an inhibitor of squalene epoxidase within the sterol biosynthesis pathway, but had little or no effect on the activity of drugs with unrelated targets. (vi) Treatment of S. cerevisiae with azole or terbinafine resulted in transcriptional upregulation of genes FKS2 and PMR1 known to be Ca2+ regulated. A model to explain the role of Ca2+‐regulated signalling in azole/terbinafine tolerance is proposed.
Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter
Authors: Kovacikova Gabriela , Skorupski Karen
Published: March 2003

Quorum sensing negatively influences virulence gene expression in certain toxigenic Vibrio cholerae strains. At high cell densities, the response regulator LuxO fails to reduce the expression of HapR, which, in turn, represses the expression of the virulence cascade. A critical regulatory step in the cascade is activation of tcpPH expression by AphA and AphB. We show here that HapR influences the virulence cascade by directly repressing aphA expression. In strain C6706, aphA expression was increased in a ΔhapR mutant and decreased in a ΔluxO mutant, indicating a negative and positive influence, respectively, of these gene products on the promoter. Overexpression of HapR also reduced aphA expression in both C6706 and Escherichia coli. DNase I footprinting showed that purified HapR binds to the aphA promoter between −85 and −58. Although it appears that quorum sensing does not influence virulence gene expression in strain O395 solely because of a frameshift in hapR, overproduced HapR did not repress expression from the O395 aphA promoter in either Vibrio or E. coli, nor did the protein bind to the promoter. Two basepair differences from C6706 are present in the O395 HapR binding site at −85 and −77. Introducing the −77 change into C6706 prevented HapR binding and repression of aphA expression. This mutation also eliminated the repression of toxin‐co‐regulated pilus (TCP) and cholera toxin (CT) that occurs in a ΔluxO mutant, indicating that HapR function at aphA is critical for density‐dependent regulation of virulence genes.
Formation of the stoichiometric complex of EnvZ, a histidine kinase, with its response regulator, OmpR
Authors: Yoshida Takeshi , Qin Ling , Inouye Masayori
Published: March 2003

EnvZ, a histidine kinase, and its cognate response regulator OmpR of Escherichia coli are responsible for adaptation to external osmotic changes by regulating the levels of the outer membrane porin proteins, OmpF and OmpC. The osmosensor, EnvZ, has dual enzymatic functions with OmpR kinase and OmpR‐P phosphatase. Here, we demonstrate that the cytoplasmic kinase domain of EnvZ (EnvZc) and OmpR are able to form a 1:1 complex detected by native PAGE. This indicates that two OmpR molecules can bind to one EnvZc dimer. As this 1:1 EnvZc/OmpR complex is formed even in the presence of a large excess of EnvZc, OmpR binding to EnvZc is co‐operative. The complex formation is also observed between EnvZc and phosphorylated OmpR for the phosphatase reaction. OmpR‐P bound to EnvZc was readily released upon the addition of OmpR, indicating that OmpR and OmpR‐P can compete for the binding to EnvZ. On the basis of these results, a model is discussed to explain how cellular OmpR‐P concentrations are regulated in response to medium osmolarity.
The expression level determines the surface distribution of the transferrin receptor in Trypanosoma brucei
Authors: Mußmann Rainer , Janssen Hans , Calafat Jero , Engstler Markus , Ansorge Iris , Clayton Christine , Borst Piet
Published: March 2003

The transferrin receptor (TfR) of Trypanosoma brucei is a heterodimer attached to the surface membrane by a glycosylphosphatidylinositol (GPI) anchor. The TfR is restricted to the flagellar pocket, a deep invagination of the plasma membrane. The membrane of the flagellar pocket and the rest of the cell surface are continuous, and the mechanism that selectively retains the TfR in the pocket is unknown. Here, we report that the TfR is retained in the flagellar pocket by a specific and saturable mechanism. In bloodstream‐form trypanosomes transfected with the TfR genes, TfR molecules escaped flagellar pocket retention and accumulated on the entire surface, even at modest (threefold) overproduction levels. Similar surface accumulation was observed when the TfR levels were physiologically upregulated threefold when trypanosomes were starved for transferrin. These results suggest that the TfR flagellar pocket retention mechanism is easily saturated and that control of the expression level is critical to maintain the restricted surface distribution of the receptor.
Modulation of phage Mu repressor DNA binding and degradation by distinct determinants in its C‐terminal domain
Authors: Mukhopadhyay Bani , Marshall‐Batty Kimberly R. , Kim Benjamin D. , O'Handley Diane , Nakai Hiroshi
Published: March 2003

Rapid degradation of the bacteriophage Mu immunity repressor can be induced in trans by mutant, protease‐hypersensitive repressors (Vir) with an altered C‐terminal domain (CTD). Genetic and biochemical analysis established that distinct yet overlapping determinants in the wild‐type repressor CTD modulate Vir‐induced degradation by Escherichia coli ClpXP protease and DNA binding by the N‐terminal DNA‐binding domain (DBD). Although deletions of the repressor C‐terminus resulted in both resistance to ClpXP protease and suppression of a temperature‐sensitive DBD mutation (cts62), some cysteine‐replacement mutations in the CTD elicited only one of the two phenotypes. Some CTD mutations prevented degradation induced by Vir and resulted in the loss of intrinsic ClpXP protease sensitivity, characteristic of wild‐type repressor, and at least two mutant repressors protected Vir from proteolysis. One protease‐resistant mutant became susceptible to Vir‐induced degradation when it also contained the cts62 mutation, which weakens DNA binding but apparently facilitates conversion to a protease‐sensitive conformation. Conversely, this CTD mutation was able to suppress temperature sensitivity of DNA binding by the cts62 repressor. The results suggest that determinants in the CTD not only provide a cryptic ClpX recognition motif but also direct CTD movement that exposes the motif and modulates DNA binding.
Specialized osmotic stress response systems involve multiple SigB‐like sigma factors in Streptomyces coelicolor
Authors: Viollier Patrick H. , Kelemen Gabriella H. , Dale Glenn E. , Nguyen Kien T. , Buttner Mark J. , Thompson Charles J.
Published: March 2003

Whereas in Bacillus subtilis, a general stress response stimulon under the control of a single sigma factor (SigB) is induced by different physiological and environmental stresses (heat, salt or ethanol shock), in Streptomyces coelicolor, these environmental stresses induce independent sets of proteins, and its genome encodes nine SigB paralogues. To investigate possible functions of multiple sigB‐like genes in S. coelicolor, Pctc, a promoter routinely used to assay SigB activity in vivo, was analysed as a heterologous reporter. The fact that Pctc was activated by osmotic shock, but not by heat or ethanol, confirmed that stress response system(s) could operate independently in S. coelicolor. Pctc was also induced transiently during growth of liquid cultures, presumably by nutritional signals. We purified an RNA polymerase holoenzyme from crude extracts that catalysed specific transcription of Pctc in vitro. Its sigma subunit was identified as a product of the sigH gene, which is co‐transcribed downstream of a putative antisigma factor gene (prsH). Although the sigH function was not needed for normal colony morphology, prsH was conditionally required for both aerial hyphae formation and regulation of antibiotic biosynthesis. Levels of two different sigH‐encoded proteins were growth phase dependent but not significantly changed by osmotic stress, implying the important roles of post‐translational regulatory elements such as PrsH. In addition, synthesis of three other SigH‐like proteins was induced by osmotic stress, but not by ethanol or heat. Two of them were genetically assigned to sigH homologous loci sigI and sigJ and shown to be independently regulated. This family of SigH‐like proteins displayed different osmotic response kinetics. Thus, in contrast to many other bacteria, S. coelicolor uses an osmotic sensory system that can co‐ordinate the activity of multiple paralogues to control the relative activity of promoters within the same stress stimulon. Such specialized stress response systems may reflect adaptive functions needed for colonial differentiation.
The product of the UTH1 gene, required for Bax‐induced cell death in yeast, is involved in the response to rapamycin
Authors: Camougrand Nadine , Grelaud‐Coq Angela , Marza Esther , Priault Muriel , Bessoule Jean‐Jacques , Manon Stéphen
Published: March 2003

A yeast mutant was isolated that was resistant to Bax‐induced cell death. It supports a mutation leading to decreased amounts of the protein Uth1p. A strain in which the UTH1 gene is disrupted also exhibits resistance to Bax expression. The absence of Uth1p does not change the mitochondrial localization of Bax, its insertion in the mitochondrial outer membrane or its cytochrome c‐releasing activity. On the other hand, the absence of Uth1p does prevent the appearance of other hallmarks related to Bax expression in yeast, such as oxidation of mitochondrial lipid, production of reactive oxygen species and maintenance of plasma membrane properties after ethanol stress. The absence of Uth1p was also found to induce resistance to rapamycin, a specific inducer of autophagy. This resistance only appears when cells are grown under respiratory conditions, but not under fermentative conditions, suggesting that Uth1p acts in an autophagic pathway involving mitochondria, in accordance with its main localization in the outer mitochondrial membrane. Taken together, these data show that Bax is able to activate a death pathway related to autophagy in yeast, which also exhibits typical hallmarks of apoptosis, revealing a possible dual function of Bax in both types of death. This hypothesis is discussed in the light of observations suggesting a co‐regulation of apoptosis and autophagy in mammalian cells.
Ssn6, an important factor of morphological conversion and virulence in Candida albicans
Authors: Hwang Cheol‐Sang , Oh Jang‐Hyun , Huh Won‐Ki , Yim Hyung‐Soon , Kang Sa‐Ouk
Published: March 2003

Candida albicans
, the major fungal pathogen in humans, undergoes morphological conversion from yeasts to filamentous growth forms depending upon various environmental conditions. Here, we have identified a
C. albicans
gene, namely
SSN6
, encoding a putative global transcriptional co‐repressor that is highly homologous to the
Saccharomyces cerevisiae
Ssn6. The isolated
C. albicans SSN6
complemented the pleiotropic phenotypes of
S. cerevisiae ssn6
mutation, and its expression levels declined significantly in response to a strong true hyphal inducer, serum. The mutant lacking
C. albicans
Ssn6 displayed a stubby pseudohyphal growth pattern, derepressed filament‐specific genes in response to elevated temperature 37°C and failed to develop true hyphae, extensive filamentation and virulence. Such morphological defects of
ssn6
/
ssn6
mutant were not rescued by overexpression of Tup1, Cph1 or Efg1. Moreover, epistatic analysis showed that, as far as cell morphology was concerned, Ssn6 was epistatic to Tup1 at the higher temperature but that, at the lower temperature, the
ssn6
/
ssn6 tup1
/
tup1
double mutant grew in a stubby form of pseudohyphae distinct from the phenotypes of either single mutant. Furthermore, overexpression of
SSN6
in
C. albicans led to enhanced filamentous growth and attenuated virulence. These findings suggest that Ssn6 may function as an activator as well as a repressor of filamentous growth and be a target for candidacidal drugs, as its excess or deficiency resulted in impaired virulence.
Iron and contact with host cells induce expression of adhesins on surface of Trichomonas vaginalis
Authors: Garcia Ana F. , Chang Te‐Hung , Benchimol Marlene , Klumpp David Jichael , Lehker Michael W. , Alderete John F.
Published: March 2003

The proteins AP65, AP51, AP33 and AP23 synthesized by Trichomonas vaginalis organisms in high iron play a role in adherence. Multigene families encode enzymes of the hydrogenosome organelles, which have identity to adhesins. This fact raises questions regarding the compartmentalization of the proteins outside the organelle and about the interactions of adhesins with host cells. Data here demonstrate the presence of the proteins outside the organelle under high‐iron conditions. Fluorescence and immuno‐cytochemical experiments show that high‐iron‐grown organisms coexpressed adhesins on the surface and intracellularly in contrast with low‐iron parasites. Furthermore, the AP65 epitopes seen by rabbit anti‐AP65 serum that blocks adherence and detects surface proteins were identified, and a mAb reacting to those epitopes recognized the trichomonal surface. Two‐dimensional electrophoresis and immunoblot of adhesins from surface‐labelled parasites provided evidence that all members of the multigene family were co‐ordinately expressed and placed on the trichomonal surface. Similar two‐dimensional analysis of proteins from purified hydrogenosomes obtained from iodinated trichomonads confirmed the specific surface labelling of proteins. Contact of trichomonads with vaginal epithelial cells increased the amount of surface‐expressed adhesins. Moreover, we found a direct relationship between the levels of adherence and amount of adhesins bound to immortalized vaginal and ureter epithelial cells, further reinforcing specific associations. Finally, trichomonads of MR100, a drug‐resistant isolate absent in hydrogenosome proteins and adhesins, were non‐adherent. Overall, the results confirm an important role for iron and contact in the surface expression of adhesins of T. vaginalis organisms.