bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction
Authors: Singh Upinder , Brewer Jeremy L. , Boothroyd John C.
Published: March 2003

Developmental switching in Toxoplasma gondii, from the virulent tachyzoite

to the relatively quiescent bradyzoite stage, is responsible for disease propagation

and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd−)

mutants in T. gondii and used these in combination with a cDNA microarray

to identify developmental pathways in bradyzoite formation. Four independently generated

Tbd− mutants were analysed and had defects in bradyzoite development

in response to multiple bradyzoite‐inducing conditions, a stable phenotype after

in vivo passages and a markedly reduced brain cyst burden in a murine model

of chronic infection. Transcriptional profiles of mutant and wild‐type parasites,

growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated

genes, including many bradyzoite‐induced genes whose transcripts were reduced in

all mutants. A set of non‐developmentally regulated genes whose transcripts were

less abundant in Tbd− mutants were also identified. These may represent

genes that mediate downstream effects and/or whose expression is dependent on the

same transcription factors as the bradyzoite‐induced set. Using these data, we have

generated a model of transcription regulation during bradyzoite development in T.

gondii. Our approach shows the utility of this system as a model to study developmental biology in single‐celled eukaryotes including protozoa and fungi.
Two kinds of archaeal group II chaperonin subunits with different thermostability in Thermococcus strain KS‐1
Authors: Yoshida Takao , Ideno Akira , Suzuki Rintaro , Yohda Masafumi , Maruyama Tadashi
Published: March 2003

The thermostability of the recombinant α‐ and β‐subunit homo‐oligomers (α16mer and β16mer) and of natural chaperonins purified from cultured Thermococcus strain KS‐1 cells was measured to understand the mechanism for the thermal acclimatization of T. KS‐1. The β‐subunit content of the natural chaperonin from cells grown at 90°C was higher than that at 80°C. The optimum temperature for ATPase activity of the natural chaperonins was 80–90°C, whereas that for α16mer and β16mer was 60°C and over 90°C respectively. Judging from the ATPase activity, β16mer was more thermostable than α16mer. The thermostabilities of the natural chaperonins were intermediate between α16mer and β16mer, whereas the natural chaperonin with a higher β‐subunit content was more stable than that with a lower β‐subunit content. Native polyacrylamide gel electrophoresis (PAGE) revealed that the chaperonin oligomers thermally dissociated to their ATPase‐inactive monomers. The thermal denaturation process monitored by circular dichroism showed that the free β‐subunit was more stable than the free α‐subunit, and that the secondary structure of the chaperonin monomer in the oligomer was more stable than that in the free monomer. These results suggest that the structure of these subunits was stabilized in the oligomer, and that an increase in the β‐subunit content conferred higher thermostability to the natural hetero‐oligomeric chaperonin.
Mg2+ homeostasis and avoidance of metal toxicity
Authors: Chamnongpol Sangpen , Groisman Eduardo A.
Published: March 2003

Because Mg2+ is required for a wide variety of cellular functions, its intracellular levels must be tightly regulated. In the Gram‐negative bacterium Salmonella enterica, three transporters mediate Mg2+ uptake: the P‐type ATPases MgtA and MgtB, whose expression is transcriptionally induced in low Mg2+ by the Mg2+‐regulated PhoP/PhoQ two‐component system; and CorA, whose transcription is regulated neither by the levels of Mg2+ nor by the PhoP/PhoQ system. We now report that mutants defective in phoP or in both mgtA and mgtB are hypersensitive to oxidative stress‐dependent Fe (II)‐mediated killing. These mutants display increased iron accumulation and heightened Ni2+ uptake. Inactivation of the corA gene restored Fe(II) resistance to the phoP mutant and eliminated uptake of Ni2+. Neither corA transcription nor CorA protein levels were altered in the phoP mutant, suggesting that CorA alters its activity in response to the presence of PhoP‐regulated determinants. Downregulation of CorA activity in low Mg2+ environments may enable Salmonella to avoid the uncontrolled influx of toxic metals.
The vbs genes that direct synthesis of the siderophore vicibactin in Rhizobium leguminosarum: their expression in other genera requires ECF σ factor RpoI
Authors: Carter R. A. , Worsley P. S. , Sawers G. , Challis G. L. , Dilworth M. J. , Carson K. C. , Lawrence J. A. , Wexler M. , Johnston A. W. B. , Yeoman K. H.
Published: March 2003

A cluster of eight genes, vbsGSO, vbsADL, vbsC and vbsP,
are involved in the synthesis of vicibactin, a cyclic, trihydroxamate siderophore
made by the symbiotic bacterium Rhizobium leguminosarum. None of these vbs
genes was required for symbiotic N2 fixation on peas or Vicia.
Transcription of vbsC, vbsGSO and vbsADL (but not vbsP)
was enhanced by growth in low levels of Fe. Transcription of vbsGSO and vbsADL,
but not vbsP or vbsC, required the closely linked gene rpoI,
which encodes an ECF σ factor of RNA polymerase. Transfer of the cloned
vbs genes, plus rpoI, to Rhodobacter, Paracoccus and
Sinorhizobium conferred the ability to make vicibactin on these other genera.
We present a biochemical genetic model of vicibactin synthesis, which accommodates
the phenotypes of different vbs mutants and the homologies of the vbs
gene products. In this model, VbsS, which is similar to many non‐ribosomal peptide
synthetase multienzymes, has a central role. It is proposed that VbsS activates L‐N5‐hydroxyornithine via covalent attachment as an acyl thioester to a peptidyl carrier protein domain. Subsequent VbsA‐catalysed acylation of the hydroxyornithine, followed by VbsL‐mediated epimerization and acetylation catalysed by VbsC, yields the vicibactin subunit, which is then trimerized and cyclized by the thioesterase domain of VbsS to give the completed siderophore.
Regulation of cell separation in the dimorphic fungus Ustilago maydis
Authors: Weinzierl Gerhard , Leveleki Leonora , Hassel Annette , Kost Gerhard , Wanner Gerhard , Bölker Michael
Published: March 2003

During its haploid phase the dimorphic fungus Ustilago maydis grows vegetatively by budding. We have identified two genes, don1 and don3, which control the separation of mother and daughter cells. Mutant cells form tree‐like clusters in liquid culture and grow as ring‐like (donut‐shaped) colonies on solid medium. In wild‐type U. maydis cells, two distinct septa are formed during cytokinesis and delimit a fragmentation zone. Cells defective for either don1 or don3 display only a single septum and fail to complete cell separation. don1 encodes a guanine nucleotide exchange factor (GEF) of the Dbl family specific for Rho/Rac GTPases. Don3 belongs to the germinal‐centre‐kinase (GC) subfamily of Ste20‐like protein kinases. We have isolated the U. maydis homologues of the small GTP binding proteins Rho2, Rho3, Rac1 and Cdc42. Out of these, only Cdc42 interacts specifically with Don1 and Don3 in the yeast two‐hybrid system. We propose that Don1 and Don3 regulate the initiation of the secondary septum, which is required for proper cell separation.
Novel mode of transcription regulation of divergently overlapping promoters by PhoP, the regulator of two‐component system sensing external magnesium availability
Authors: Yamamoto Kaneyoshi , Ogasawara Hiroshi , Fujita Nobuyuki , Utsumi Ryutaro , Ishihama Akira
Published: March 2003

PhoP is a response regulator of the PhoQ‐PhoP two‐component system controlling a set of the Mg(II)‐response genes in Escherichia coli. Here we demonstrate the mode of transcription regulation by phosphorylated PhoP of divergently transcribed mgtA and treR genes, each encoding a putative Mg(II) transporter and a repressor for the trehalose utilization operon respectively. Under Mg(II)‐limiting conditions in vivo, two promoters, the upstream constitutive P2 and the downstream inducible P1, were detected for the mgtA gene. Gel‐shift analysis in vitro using purified PhoP indicates its binding to a single DNA target, centred between –43 and –24 of the mgtAP1 promoter. This region includes the PhoP box, which consists of a direct repeat of the heptanucleotide sequence (T)G(T)TT(AA). Site‐directed mutagenesis studies indicate the critical roles for T (position 3), T (position 4) and A (position 6) for PhoP‐dependent transcription from mgtAP1. DNase I footprinting assays reveal weak binding of PhoP to this PhoP box, but the binding becomes stronger in the simultaneous presence of RNA polymerase. Likewise the RNA polymerase binding to the P1 promoter becomes stronger in the presence of PhoP. For the PhoP‐assisted formation of open complex at the mgtAP1 promoter, however, the carboxy‐terminal domain of α subunit (αCTD) is not needed. For transcription in vivo of the treR gene, four promoters were identified. The most upstream promoter treRP4 divergently overlaps with the mgtAP1 promoter, sharing the same sequence as the respective –10 signal in the opposite direction. In vitro transcription using mutant promoters support this prediction. In the presence of PhoP, transcription from the promoter treRP3 was repressed with concomitant activation of mgtAP1 transcription. The PhoP box is located between −46 and –30 with respect to treRP3, and the αCTD is needed for this repression.
DNA replication initiation is required for mid‐cell positioning of FtsZ rings in Caulobacter crescentus
Authors: Quardokus Ellen M. , Brun Yves V.
Published: March 2003

Polymerization of the GTPase FtsZ to form a structure called the Z‐ring is the earliest known step in bac‐terial cell division. Mid‐cell Z‐ring assembly coincides with the beginning of the replication cycle in the differentiating bacterium Caulobacter crescentus. Z‐ring disassembly occurs at the end of the division cycle, resulting in the complete degradation of FtsZ from both stalked and swarmer progeny cells. New Z‐rings can only form in the replicative stalked cell. Conditional mutants in DNA replication were used to determine what role DNA replication events play in the process of Z‐ring assembly at different stages in the cell cycle. Z‐ring assembly occurred even when early stages of DNA replication were blocked; however, the Z‐rings were localized at a subpolar region of the cell. Z‐rings only assembled at the proper mid‐cell location if DNA replication had initiated. Z‐ring assembly coincided with areas containing little or no DNA, and Z‐rings could not form over an unreplicated chromosome. Overexpressed FtsZ in the absence of DNA replication did not stimulate productive mid‐cell Z‐ring assembly but, instead, caused the ends of cells to constrict over an extended area away from the nucleoid. These results indicate that the state of chromosome replication is a major determinant of Z‐ring localization in Caulobacter.
Characterization of a novel phenazine antibiotic gene cluster in Erwinia herbicola Eh1087
Authors: Giddens Stephen R. , Feng Yunjiang , Mahanty H. Khris
Published: March 2003

Erwinia herbicola
strain Eh1087 produces the broad‐spectrum phenazine antibiotic
D
‐alanylgriseoluteic acid (AGA). In this report, a cluster of 16
ehp
(

E
rwinia
h
erbicola

p
henazine) plasmid genes required for the production of AGA by Eh1087 is described. The extent of the gene cluster was revealed by the isolation of 82 different Eh1087 AGA

−

mutants, all found to possess single mini‐Tn
5lacZ2
insertions within a 14 kbp DNA region. Additional transposon insertions that did not affect antibiotic production by Eh1087 were created to define the boundaries of the gene cluster. The size and location of genes between these boundaries were derived from a combination of DNA sequence analyses, minicell protein analyses and the correlation between mutation position and the production of coloured AGA intermediates by many
ehp
mutants. Precursor‐feeding and complementation experiments resulted in 15
ehp
genes being assigned to one of four functional groups according to their role in the synthesis of AGA. Group 1 is required for the synthesis of the phenazine nucleus in the form of antibiotic precursor one (AP1, phenazine‐1,6‐dicarboxylic acid). Group 2 is responsible for conversion of AP1 to AP2, which is subsequently modified to AP3 (griseoluteic acid) and exported by the group 3 gene products. Group 4 catalyses the addition of
D
‐alanine to AP3 to create AGA, independently of groups 1, 2 and 3. A gene that is divergently transcribed from the 15 AGA synthesis
ehp genes confers resistance to AGA.
The ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of parS sequences within the origin‐proximal region of the linear chromosome
Authors: Jakimowicz Dagmara , Chater Keith , Zakrzewska‐Czerwi´nska Jolanta
Published: March 2003

Summary The mycelial prokaryote Streptomyces coelicolor A3(2) possesses a large linear chromosome (8.67 Mb) with a centrally located origin of replication (oriC). Recently, chromosome partitioning genes (parA and parB) and putative ParB binding sites (parS sequences) were identified in its genome. The S. coelicolor chromosome contains more parS sequ­ences than any other bacterial chromosome characterized so far. Twenty of the 24 parS sequences are densely packed within a relatively short distance (≈ 200 kb) around oriC. A series of in vitro and in vivo experiments showed that S. coelicolor ParB protein interacts specifically with the parS sequences, albeit with a rather low affinity. Our results suggested that the binding of ParB is not only determined by the parS sequence, but also by the location of target DNA close to oriC. The unusually high number and close proximity to each other of the parS sites, together with in vivo and in vitro evidence that multiple ParB molecules may assemble along the DNA from an initial ParB–parS complex, suggest that a large DNA segment around the replication origin may form a massive nucleoprotein complex as part of the replication‐partitioning cycle.
Amplification of Hot DNA segments in Escherichia coli
Authors: Kodama Ken‐ichi , Kobayashi Takehiko , Niki Hironori , Hiraga Sota , Oshima Taku , Mori Hirotada , Horiuchi Takashi
Published: March 2003

In Escherichia coli, a replication fork blocking event at a DNA replication terminus (Ter) enhances homologous recombination at the nearby sister chromosomal region, converting the region into a recombination hotspot, Hot, site. Using a RNaseH negative (rnhA–) mutant, we identified eight kinds of Hot DNAs (HotA–H). Among these, enhanced recombination of three kinds of Hot DNAs (HotA–C) was dependent on fork blocking events at Ter sites. In the present study, we examined whether HotA DNAs are amplified when circular DNA (HotA plus a drug‐resistance DNA) is inserted into the homologous region on the chromosome of a rnhA– mutant. The resulting HotA DNA transformants were analysed using pulsed‐field gel electrophoresis, fluorescence in situ hybridization and DNA microarray technique. The following results were obtained: (i) HotA DNA is amplified by about 40‐fold on average; (ii) whereas 90% of the cells contain about 6–10 copies of HotA DNA, the remaining 10% of cells have as many as several hundred HotA copies; and (iii) amplification is detected in all other Hot DNAs, among which HotB and HotG DNAs are amplified to the same level as HotA. Furthermore, HotL DNA, which is activated by blocking the clockwise oriC‐starting replication fork at the artificially inserted TerL site in the fork‐blocked strain with a rnhA+ background, is also amplified, but is not amplified in the non‐blocked strain. From these data, we propose a model that can explain production of three distinct forms of Hot DNA molecules by the following three recombination pathways: (i) unequal intersister recombination; (ii) intrasister recombination, followed by rolling‐circle replication; and (iii) intrasister recombination, producing circular DNA molecules.
The functions of OmpATb, a pore‐forming protein of Mycobacterium tuberculosis
Authors: Raynaud Catherine , Papavinasasundaram K. G. , Speight Richard A. , Springer Burkhard , Sander Peter , Böttger Erik C. , Colston M. Joseph , Draper Philip
Published: March 2003

The functions of OmpATb, the product of the ompATb gene of Mycobacterium tuberculosis and a putative porin, were investigated by studying a mutant with a targeted deletion of the gene, and by observing expression of the gene in wild‐type M. tuberculosis H37Rv by real‐time polymerase chain reaction (PCR) and immunoblotting. The loss of ompATb had no effect on growth under normal conditions, but caused a major reduction in ability to grow at reduced pH. The gene was substantially upregulated in wild‐type bacteria exposed to these conditions. The mutant was impaired in its ability to grow in macrophages and in normal mice, although it was as virulent as the wild type in mice that lack T cells. Deletion of the ompATb gene reduced permeability to several small water‐soluble substances. This was particularly evident at pH 5.5; at this pH, uptake of serine was minimal, suggesting that, at this pH, OmpATb might be the only functioning porin. These data indicate that OmpATb has two functions: as a pore‐forming protein with properties of a porin, and in enabling M. tuberculosis to respond to reduced environmental pH. It is not known whether this second function is related to the porin‐like activity at low pH or involves a completely separate role for OmpATB. The involvement with pH is likely to contribute to the ability of M. tuberculosis to overcome host defence mechanisms and grow in a mammalian host.
Intracellular translation initiation factor levels in Saccharomyces cerevisiae and their role in cap‐complex function
Authors: von der Haar Tobias , McCarthy John E. G.
Published: March 2003

Knowledge of the balance of activities of eukaryotic initiation factors (eIFs) is critical to our understanding of the mechanisms underlying translational control. We have therefore estimated the intracellular levels of 11 eIFs in logarithmically growing cells of Saccharomyces cerevisiae using polyclonal antibodies raised in rabbits against recombinant proteins. Those factors involved in 43S complex formation occur at levels comparable (i.e. within a 0.5‐ to 2.0‐fold range) to those published for ribosomes. In contrast, the subunits of the cap‐binding complex eIF4F showed considerable variation in their abundance. The helicase eIF4A was the most abundant eIF of the yeast cell, followed by eIF4E at multiple copies per ribosome, and eIF4B at approximately one copy per ribosome. The adaptor protein eIF4G was the least abundant of the eIF4 factors, with a copy number per cell that is substoichiometric to the ribosome and similar to the abundance of mRNA. The observed excess of eIF4E over its functional partner eIF4G is not strictly required during exponential growth: at eIF4E levels artificially reduced to 30% of those in wild‐type yeast, growth rates and the capacity for general protein synthesis are only minimally affected. This demonstrates that eIF4E does not exercise a higher level of rate control over translation than other eIFs. However, other features of the yeast life cycle, such as the control of cell size, are more sensitive to changes in eIF4E abundance. Overall, these data constitute an important basis for developing a quantitative model of the workings of the eukaryotic translation apparatus.
A fibrinogen receptor from group B Streptococcus interacts with fibrinogen by repetitive units with novel ligand binding sites
Authors: Schubert Axel , Zakikhany Katherina , Schreiner Mark , Frank Ronald , Spellerberg Barbara , Eikmanns Bernhard J. , Reinscheid Dieter J.
Published: March 2003

Group B Streptococcus (GBS) is a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection, GBS colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present report, we describe the isolation of the fbsA gene, which encodes a fibrinogen receptor from GBS. The deduced FbsA protein is characterized by repetitive units, each 16 amino acids in length. Sequencing of the fbsA gene from five different GBS strains revealed significant variation in the number of repeat‐encoding units. The deletion of the fbsA gene in the genome of GBS 6313 completely abolished fibrinogen binding, suggesting that FbsA is the major fibrinogen receptor in this strain. Growth of the fbsA deletion mutant in human blood was significantly impaired, indicating that FbsA protects GBS from opsonophagocytosis. In Western blot experiments with truncated FbsA ­proteins, the repeat region of FbsA was identified as mediating fibrinogen binding. Using synthetic peptides, even a single repeat unit of FbsA was demonstrated to bind to fibrinogen. Spot membrane analysis and competitive binding experiments with peptides carrying single amino acid substitutions allowed the prediction of a fibrinogen‐binding motif with the consensus sequence G‐N/S/T‐V‐L‐A/E/M/Q‐R‐R‐X‐K/R/W‐A/D/E/N/Q‐A/F/I/L/V/Y‐X‐X‐K/R‐X‐X.
Regulation of the yeast Rlm1 transcription factor by the Mpk1 cell wall integrity MAP kinase
Authors: Jung Un Sung , Sobering Andrew K. , Romeo Martin J. , Levin David E.
Published: March 2003

The Mpk1 MAP kinase of the Saccharomyces cerevisiae cell wall integrity signalling pathway phosphorylates and activates the Rlm1 transcription factor in response to cell wall stress. Rlm1 is related to mammalian MEF2 isoforms, and shares a similar DNA‐binding specificity. Signalling through Rlm1 regulates the expression of at least 25 genes, most of which have been implicated in cell wall biogenesis. We report here the transcriptional induction by agents of cell wall stress of a set of lacZ reporter plasmids derived from several Rlm1‐responsive genes. Analysis of substitution mutations at putative Mpk1 phosphorylation sites within Rlm1 revealed that Ser427 and Thr439 are important for its stress‐induced transcriptional activation of these reporter plasmids. Assessment of Rlm1 activation potency when fused to a heterologous DNA‐binding domain showed that the identified seryl and threonyl residues are necessary for the Rlm1 transcriptional activation function independently of its DNA binding. We also demonstrate that a MAP kinase docking site, shown recently to mediate activation of MEF2A and MEF2C, is conserved in Rlm1 and is required for its ability to mediate transcriptional activation in response to agents that induce cell wall stress. Finally, intracellular localization analyses show that Rlm1 resides in the nucleus regardless of its activation and phosphorylation status. Together these observations support the inference that Mpk1 regulates the Rlm1 transcriptional activation function by phosphorylation of Ser427 and Thr439.
The third chemotaxis locus of Rhodobacter sphaeroides is essential for chemotaxis
Authors: Porter Steven L. , Warren Anna V. , Martin Angela C. , Armitage Judith P.
Published: March 2003

The purple photosynthetic bacterium Rhodobacter sphaeroides has three loci encoding multiple homologues of the bacterial chemosensory proteins: 13 putative chemoreceptors, four CheW, four CheA, six CheY, two CheB and three CheR. Previously, studies have shown that, although deletion of cheOp1 led to only minor changes in behaviour, deletion of cheOp2 led to a loss of taxis. The third locus encodes two CheA, one CheR, one CheB, one CheW, one CheY, a putative cytoplasmic chemoreceptor (TlpT) and a protein showing homology to the chromosomal partitioning factor Soj (designated Slp). Here, we show that every protein encoded by this locus is essential for normal chemotaxis. Phototaxis is also dependent upon all the components of this locus, except CheB2 and Slp. The two putative CheA proteins encoded in this locus are unusual. CheA3 has only the P1 domain and the P5 regulatory domain linked by a large internal domain, whereas CheA4 lacks the P1 and P2 domains required for phosphorylation and response regulator binding. These data indicate that the minimal set of proteins required for normal chemotaxis in R. sphaeroides is all the proteins encoded by cheOp2 and the third chemotaxis locus, and that the multiple chemosensory protein homologues found in R. sphaeroides are not redundant.
Mutants in flaI and flaJ of the archaeon Methanococcus voltae are deficient in flagellum assembly
Authors: Thomas Nikhil A. , Mueller Steffen , Klein Albrecht , Jarrell Ken F.
Published: March 2003

The fla gene locus of Methanococcus voltae encodes the major structural components of the flagellum as well as other flagellar‐related proteins. The flaHIJ genes have been found in all flagellated archaea, suggesting a central role in flagella biogenesis. FlaI shares similarity with the type II and type IV secretion NTPases (such as PilB, VirB11 and TadA), and FlaJ exhibits similarity to putative bacterial integral membrane proteins involved in type IV pilus biogenesis such as TadB. In this study, reverse transcription polymerase chain reaction (RT‐PCR) and Northern blotting data revealed that flaHIJ are co‐transcribed with the upstream structural flagellin genes, thus demonstrating the expression of the entire fla gene cluster in vivo. Non‐polar mutants in flaI and flaJ of M. voltae were isolated using insertional inactivation via a novel mutagenic vector. These mutants were non‐motile and non‐flagellated by microscopy, demonstrating the involvement of FlaI and FlaJ in flagella biogenesis. Interestingly, all the mutants maintained the ability to produce and localize flagellins to the cytoplasmic membrane. Amino‐terminal sequencing of flagellins produced by the flaJ mutant strain revealed that the flagellins did not have their cognate leader peptides, thus indicating that preflagellin processing had occurred in vivo. This result was confirmed using an in vitro processing assay. The fla− phenotype and protein secretion characteristics of the flaI and flaJ mutants therefore implicate these respective genes in archaeal flagellin secretion and assembly. These findings further support a model describing the archaeal flagellum as a novel prokaryotic motility structure.
Autoregulation of Helicobacter pylori Fur revealed by functional analysis of the iron‐binding site
Authors: Delany Isabel , Spohn Gunther , Pacheco Ana‐Beatriz F. , Ieva Raffaele , Alaimo Cristina , Rappuoli Rino , Scarlato Vincenzo
Published: March 2003

The ferric uptake regulator protein Fur regulates iron‐dependent gene expression in bacteria. In Helicobacter pylori it has been shown to regulate iron‐activated and iron‐repressed genes. In this study, we show that H. pylori Fur protein regulates transcription from its own σ80 promoter Pfur in response to iron. Footprinting analysis shows that Fur binds at three distinct operators at Pfur overlapping and proximal to the promoter elements. Site‐directed mutagenesis of the proposed iron‐binding site of the protein results in derepression of Pfur and the loss of iron regulation. In vivo oligomerization assays reveals that the C‐terminus of Fur is necessary for multimerization of the protein and that the mutations do not affect this activity. Molecular and phenotypic analysis of the mutant proteins provides evidence that the iron‐binding site controls the specific affinity of Fur for the operators at Pfur and hence its repressive ability. In summary, the data presented are consistent with a model in which Fur acts as a rheostat of transcription to autoregulate its own expression in response to iron, which in turn controls expression of iron‐induced and iron‐repressed genes, providing maintenance of homeostasis.
Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane
Authors: Grahn A. Marika , Daugelavičius Rimantas , Bamford Dennis H.
Published: March 2003

DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double‐stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build‐up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.
The yeast zinc finger regulators Pdr1p and Pdr3p control pleiotropic drug resistance (PDR) as homo‐ and heterodimers in vivo
Authors: Mamnun Yasmine M. , Pandjaitan Rudy , Mahé Yannick , Delahodde Agnés , Kuchler Karl
Published: March 2003

The transcription factors Pdr1p and Pdr3p from Saccharomyces cerevisiae mediate pleiotropic drug resistance (PDR) by controlling expression of ATP‐binding cassette (ABC) transporters such as Pdr5p, Snq2p and Yor1p. Previous in vitro studies demonstrated that Pdr1p and Pdr3p recognize so‐called pleiotropic drug resistance elements (PDREs) in the promoters of target genes. In this study, we show that both Pdr1p and Pdr3p are phosphoproteins; Pdr3p isoforms migrate as two bands in gel electrophoresis, reflecting two distinct phosphorylation states. Most importantly, native co‐immunoprecipitation experiments, using functional epitope‐tagged Pdr1p/Pdr3p variants, demonstrate that Pdr1p and Pdr3p can form both homo‐ and heterodimers in vivo. Furthermore, in vivo footprinting of PDRE‐containing promoters demonstrate that Pdr1p/Pdr3p constitutively occupy both perfect and degenerate PDREs in vivo. Thus, in addition to interaction with other regulators, differential dimerization provides a plausible explanation for the observation that Pdr3p and Pdr1p can both positively and negatively control PDR promoters with different combinations of perfect and degenerate PDREs.
Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina
Authors: Pinan‐Lucarré Bérangère , Paoletti Mathieu , Dementhon Karine , Coulary‐Salin Bénédicte , Clavé Corinne
Published: March 2003

In filamentous fungi, a cell death reaction occurs when cells of unlike genotype fuse. This cell death reaction, known as incompatibility reaction, is genetically controlled by a set of loci termed het loci (for heterokaryon incompatibility loci). In Podospora anserina, genes induced during this cell death reaction (idi genes) have been identified. The idi‐6/pspA gene encodes a serine protease that is the orthologue of the vacuolar protease B of Saccharomyces cerevisiae involved in autophagy. We report here that the PSPA protease participates in the degradative autophagic pathway in Podospora. We have identified the Podospora orthologue of the AUT7 gene of S. cerevisiae involved in the early steps of autophagy in yeast. This gene is induced during the development of the incompatibility reaction and was designated idi‐7. We have used a GFP–IDI7 fusion protein as a cytological marker of the induction of autophagy. Relocalization of this fusion protein and detection of autophagic bodies inside the vacuoles during the development of the incompatibility reaction provide cytological evidence of induction of autophagy during this cell death reaction. Therefore, cell death by incompatibility in fungi appears to be related to type II programmed cell death in metazoans. In addition, we found that pspA and idi‐7 null mutations confer differentiation defects such as the absence of female reproductive structures, indicating that autophagy is required for differentiation in Podospora.
From pre‐cells to Eukarya – a tale of two lipids
Authors: Wächtershäuser G.
Published: March 2003

A mechanistic hypothesis for the origin of the three domains of life is proposed. A population of evolving pre‐cells is suggested to have had a membrane of a racemate of chiral lipids that continuously underwent spontaneous symmetry breaking by spatial phase segregation into two enantiomerically enriched membrane domains. By frequent pre‐cell fusions and fissions these membrane domains became partitioned between two pre‐cell subpopulations having predominantly one lipid enantiomer or the other. The origin of the Bacteria and Archaea is explained by divergence of first a population of proto‐bacteria and later a population of proto‐archaea from the evolving pre‐cells, each by the emergence of an enantio‐selective lipid biosynthesis within the corresponding pre‐cell subtype. The origin of the Eukarya is explained by symbiosis between a population of Bacteria and a subpopulation of pre‐cells with a predominance of the bacteria‐type lipid enantiomers.
Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine‐rich sites
Authors: Hendrixson D. R. , Qiu J. , Shewry S. C. , Fink D. L. , Petty S. , Baker E. N. , Plaut A. G. , St. Geme J. W.
Published: March 2003

Lactoferrin is a member of the lactotransferrin family of non‐haem, iron‐binding glycoproteins and is found at high concentrations in all human secretions, where it plays a major role in mucosal defence. In recent work, we observed that lactoferrin has proteolytic activity and attenuates the pathogenic potential of Haemophilus influenzae by cleaving and removing two putative colonization factors, namely the IgA1 protease protein and the Hap adhesin. Experiments with protease inhibitors further suggested that lactoferrin may belong to a serine protease family. In the present study we explored the mechanism of lactoferrin protease activity and discovered that mutation of either Ser259 or Lys73 results in a dramatic decrease in proteolysis. Examination of the crystal structure revealed that these two residues are located in the N‐terminal lobe of the protein, adjacent to a 12–15 Å cleft that separates the N‐lobe and the C‐lobe and that can readily accommodate large polypeptide substrates. In additional work, we found that lactoferrin cleaves IgA1 protease at an arginine‐rich region defined by amino acids 1379–1386 (RRSRRSVR) and digests Hap at an arginine‐rich sequence between amino acids 1016 and 1023 (VRSRRAAR). Based on our results, we conclude that lactoferrin is a serine protease capable of cleaving arginine‐rich sequences. We speculate that Ser259 and Lys73 form a catalytic dyad, reminiscent of a number of bacterial serine proteases. In addition, we speculate that lactoferrin may cleave arginine‐rich sequences in a variety of microbial virulence proteins, contributing to its long‐recognized antimicrobial properties.
EspH, a new cytoskeleton‐modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli
Authors: Tu Xuanlin , Nisan Israel , Yona Chen , Hanski Emanuel , Rosenshine Ilan
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. During infection, EPEC and EHEC use a type III secretion system (TTSS) to translocate effector proteins into the infected cells and thereby modify specific host functions. These include transient filopodium formation which is Cdc42‐dependent. Filopodia formation is followed by assembly of actin pedestals, the process enhanced by inhibition of Cdc42. We discovered that orf 18 of the enterocyte effacement locus encodes a new effector, which we termed EspH. We show that EspH is translocated efficiently into the infected cells by the TTSS and localizes beneath the EPEC microcolonies. Inactivation of espH resulted in enhanced formation of filopodia and attenuated the pedestals formation. Furthermore, overexpression of EspH resulted in strong repression of filopodium formation and heightened pedestal formation. We also demonstrate that overexpression of EspH by EHEC induces marked elongation of the typically flat pedestals. Similar pedestal elongation was seen upon infection of COS cells overexpressing EspH. EspH transiently expressed by the COS cells was localized to the membrane and disrupted the actin cytoskeletal structure. Our findings indicate that EspH is a modulator of the host actin cytoskeleton structure.
Identification of a novel Gsp‐related pathway required for secretion of the manganese‐oxidizing factor of Pseudomonas putida strain GB‐1
Authors: De Vrind Johannes , De Groot Arjan , Brouwers Geert Jan , Tommassen Jan , De Vrind‐de Jong Elisabeth
Published: March 2003

The manganese‐oxidizing factor of Pseudomonas putida strain GB‐1 is associated with the outer membrane. One of the systems of protein transport across the outer membrane is the general secretory pathway (Gsp). The gsp genes are called xcp in Pseudomonas species. In a previous study, it was shown that mutation of the prepilin peptidase XcpA and of a homologue of the pseudopilin XcpT inhibited transport of the factor. In the present study, we describe the genomic region flanking the xcpT homologue (designated xcmT1). We show that xcmT1 is part of a two‐gene operon that includes an xcpS homologue (designated xcmS). No other xcp‐like genes are present in the regions flanking the xcmT1/xcmS cluster. We also characterized the site of transposon insertion of another transport mutant of P. putida GB‐1. This insertion appeared to be located in a gene (designated xcmX) possibly encoding another pseudopilin‐related protein. This xcmX is clustered with two other xcpT‐related genes (designated xcmT2 and xcmT3) on one side and homologues of three csg genes (designated csmE, csmF and csmG) on the other side. The csg genes are involved in production of aggregative fibres in Escherichia coli and Salmonella typhimurium. A search for XcmX homologues revealed that the recently published genome of Ralstonia solanacearum and the unannotated genome of P. putida KT2440 contain comparable gene clusters with xcmX and xcp homologues that are different from the well‐described ‘regular’xcp/gsp clusters. They do contain xcpR and xcpQ homologues but, for example, homologues of xcpP, Y and Z are lacking. The results suggest a novel Xcp‐related system for the transport of manganese‐oxidizing enzymes to the cell surface.
Interaction of the sensor module of Mycobacterium tuberculosis H37Rv KdpD with members of the Lpr family
Authors: Steyn Adrie J. C. , Joseph Joan , Bloom Barry R.
Published: March 2003

The genetic and biochemical mechanisms by which Mycobacterium tuberculosis senses and responds to the complex environment that it encounters during infection and persistence within the host remain unknown. In a number of bacterial species, the Kdp signal transduction pathway appears to be the primary response to environmental osmotic stress, which is primarily mediated by K+ concentration in bacteria. We show that kdp encodes for components of a mycobacterial signalling pathway by demonstrating the K+ dependence of kdpFABC expression in both M. tuberculosis H37Rv and Mycobacterium smegmatis. To identify proteins of M. tuberculosis that participate in this signalling pathway, we used the N‐terminal sensing module of the histidine kinase KdpD as bait in a yeast two‐hybrid screen. We show that the sensing domain of KdpD interacts specifically with two membrane lipoproteins, LprJ (Rv1690) and LprF (Rv1368). Overexpression of lprF and lprJ alleles in mycobacterial kdpF–lacZ reporter strains enabled us to identify alleles that modulate kdpFABC expression. By exploiting the yeast three‐hybrid system, we have found that the histidine kinase domain of KdpD forms ternary complexes with LprF and LprJ and the sensing module of KdpD. Our results establish a role for membrane proteins in the Kdp signalling pathway and suggest that LprF and LprJ function as accessory or ligand‐binding proteins that communicate directly with the sensing domain of KdpD to modulate kdp expression.
Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase A pathway in the yeast Saccharomyces cerevisiae
Authors: Giots Frank , Donaton Monica C. V. , Thevelein Johan M.
Published: March 2003

Yeast cells starved for inorganic phosphate on a glucose‐containing medium arrest growth and enter the resting phase G0. We show that re‐addition of phosphate rapidly affects well known protein kinase A targets: trehalase activation, trehalose mobilization, loss of heat resistance, repression of STRE‐controlled genes and induction of ribosomal protein genes. Phosphate‐induced activation of trehalase is independent of protein synthesis and of an increase in ATP. It is dependent on the presence of glucose, which can be detected independently by the G‐protein coupled receptor Gpr1 and by the glucose‐phosphorylation dependent system. Addition of phosphate does not trigger a cAMP signal. Despite this, lowering of protein kinase A activity by mutations in the TPK genes strongly reduces trehalase activation. Inactivation of phosphate transport by deletion of PHO84 abolishes phosphate signalling at standard concentrations, arguing against the existence of a transport‐independent receptor. The non‐metabolizable phosphate analogue arsenate also triggered signalling. Constitutive expression of the Pho84, Pho87, Pho89, Pho90 and Pho91 phosphate carriers indicated pronounced differences in their transport and signalling capacities in phosphate‐starved cells. Pho90 and Pho91 sustained highest phosphate transport but did not sustain trehalase activation. Pho84 sustained both transport and rapid signalling, whereas Pho87 was poor in transport but positive for signalling. Pho89 displayed very low phosphate transport and was negative for signalling. Although the results confirmed that rapid signalling is independent of growth recovery, long‐term mobilization of trehalose was much better correlated with growth recovery than with trehalase activation. These results demonstrate that phosphate acts as a nutrient signal for activation of the protein kinase A pathway in yeast in a glucose‐dependent way and they indicate that the Pho84 and Pho87 carriers act as specific phosphate sensors for rapid phosphate signalling.
Clinical isolates of Enterococcus faecium exhibit strain‐specific collagen binding mediated by Acm, a new member of the MSCRAMM family
Authors: Nallapareddy Sreedhar R. , Weinstock George M. , Murray Barbara E.
Published: March 2003

A collagen‐binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen‐binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin‐resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI‐binding vancomycin‐resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non‐CI‐binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration‐dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm‐specific IgGs purified from anti‐recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen‐binding clinical isolates of E. faecium tested, but in none of the strains with a non‐functional pseudo acm gene. Introduction of a functional acm gene into two non‐CI‐binding natural acm mutant strains conferred a CI‐binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection‐derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.
Action potential generation, Kit receptor immunohistochemistry and morphology of steel‐Dickie (Sl/Sld) mutant mouse small intestine
Authors: MIKKELSEN  , MALYSZ  , HUIZINGA  , THUNEBERG
Published: March 2003

In contrast to wild‐type mice, homozygotes with mutations of the W locus do not express the functional Kit receptor and are severely deficient in the Auerbach's plexus (AP)‐associated subtype of interstitial cells of Cajal (ICC‐AP). With a morphologically intact neural and muscular structure, the absence in these mutants of both small‐intestinal slow waves and ICC‐AP constitutes strong evidence for a key role of ICC‐AP as pacemaker cells. In steel‐Dickie mutant mice (Sl/Sld), the gene coding for the Kit ligand (stem cell factor) is defective. We examined Sl/Sld mutants and controls with intracellular microelectrode techniques, combined with light and electron microscopy. The absence of the normal Kit ligand (Sl/Sld mice) had very similar effects as the absence of the Kit receptor in viable mice, mutated at the White spotting, W, locus (W/Wv mice), in that neither slow waves, nor Kit receptor immunoreactivity in the region of Auerbach's plexus nor ICC‐AP were present in the small intestine. In the Sl/Sld mouse, the smooth muscle cells generated action potentials at variable frequencies from a depolarized cell membrane of – 40 to – 55 mV. Increasing excitability by K channel blockers created many different patterns of action potential generation and the frequency increased from ∼ 16 cpm to 66 cpm. This was in sharp contrast to control mice where action potentials were always restricted to the plateau phase of the slow waves and the slow wave frequency remained constant at ∼ 39 cpm. Our data provide further strong support for the identification of ICC‐AP as small‐intestinal pacemaker cells. In addition, they provide a basis for the understanding of intestinal motor function without pacemaker activity.