bluuebunny/crossref_metadata_2025 huggingface dataset:
A variable genetic island specific for Neisseria gonorrhoeae is involved in providing DNA for natural transformation and is found more often in disseminated infection isolates
Authors: Dillard Joseph P. , Seifert H. Steven
Published: March 2003

Neisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually transmitted disease gonorrhoea. Most gonococcal infections remain localized to the genital tract but, in a small proportion of untreated cases, the bacterium becomes systemic to produce the serious complication of disseminated gonococcal infection (DGI). We have identified a large region of chromosomal DNA in N. gonorrhoeae that is not found in a subset of gonococcal isolates (a genetic island), in the closely related pathogen, Neisseria meningitidis or in commensal Neisseria that do not usually cause disease. Certain versions of the island carry a serum resistance locus and a gene for the production of a cytotoxin; these versions of the island are found preferentially in DGI isolates. All versions of the genetic island encode homologues of F factor conjugation proteins, suggesting that, like some other pathogenicity islands, this region encodes a conjugation‐like secretion system. Consistent with this hypothesis, a wild‐type strain released large amounts of DNA into the medium during exponential growth without cell lysis, whereas an isogenic strain mutated in a peptidoglycan hydrolase gene (atlA) was drastically reduced in its ability to donate DNA for transformation during growth. This genetic island constitutes the first major discriminating factor between the gonococcus and the other Neisseria and carries genes for providing DNA for genetic transformation.
Haemophore‐mediated bacterial haem transport: evidence for a common or overlapping site for haem‐free and haem‐loaded haemophore on its specific outer membrane receptor
Authors: Létoffé Sylvie , Deniau Clarisse , Wolff Nicolas , Dassa Emmanuel , Delepelaire Philippe , Lecroisey Anne , Wandersman Cécile
Published: March 2003

Bacterial extracellular haemophores also named HasA for haem acquisition system form an independent family of haemoproteins that take up haem from host haeme carriers and shuttle it to specific receptors (HasR). Haemophore receptors are required for the haemophore‐dependent haem acquisition pathway and alone allow free or haemoglobin‐bound haem uptake, but the synergy between the haemophore and its receptor greatly facilitates this uptake. The three‐dimensional structure of the Serratia marcescens holo‐haemophore (HasASM) has been determined previously and revealed that the haem iron atom is ligated by tyrosine 75 and histidine 32. The phenolate of tyrosine 75 is also tightly hydrogen bonded to the Nδ atom of histidine 83. Alanine mutagenesis of these three HasASM residues was performed, and haem‐binding constants of the wild‐type protein, the three single mutant proteins, the three double mutant proteins and the triple mutant protein were compared by absorption spectrometry to probe the roles of H32, Y75 and H83 in haem binding. We show that one axial iron ligand is sufficient to ligate haem efficiently and that H83 may become an alternative iron ligand in the absence of Y75 or both H32 and Y75. All the single mutant proteins retained the ability to stimulate haemophore‐dependent haem uptake in vivo. Thus, the residues H32, Y75 and H83 are not individually necessary for haem delivery to the receptor. The binding of haem‐free and haem‐loaded HasASM proteins to HasRSM‐producing strains was studied. Both proteins bind to HasRSM with similar apparent Kd. The double mutant H32A‐Y75A competitively inhibits binding to the receptor of both holo‐HasASM and apo‐HasASM, showing that there is a unique or overlapping site on HasRSM for the apo‐ and holo‐haemophores. Thus, we propose a new mechanism for haem uptake, in which haem is exchanged between haem‐loaded haemophores and unloaded haemophores bound to the receptor without swapping of haemophores on the receptor.
The Rhizobium leguminosarum tonB gene is required for the uptake of siderophore and haem as sources of iron
Authors: Wexler Margaret , Yeoman Kay H. , Stevens James B. , De Luca Nicola G. , Sawers Gary , Johnston Andrew W. B.
Published: March 2003

In the N2‐fixing bacterium Rhizobium leguminosarum, mutations in a homologue of tonB (tonBRl) block the import of vicibactin and haem as iron sources in free‐living bacteria. TonBRl mutants were normal for growth with ferric dicitrate and slightly reduced for growth with haemoglobin as sole iron sources. The deduced TonBRl product is larger than that of (for example) Escherichia coli, on account of an extended N‐terminal domain. Transcription of tonBRl was enhanced in low‐Fe growth conditions; this was not controlled by Fur, nor RpoI, an Fe‐regulated extracytoplasmic σ factor. Upstream of tonBRl and transcribed divergently is an operon, hmuPSTUV, whose products are homologous to ABC transporters involved in haem uptake in pathogenic bacteria. Expression of hmuPSTUV was enhanced in low‐Fe conditions, and hmu mutants show slightly diminished growth on haem as sole Fe source, suggesting that there is more than one system for the uptake of this molecule. hmuPSTUV expression appears to be from three closely linked promoters. Downstream of hmuPSTUV, a gene that may encode an extracytoplasmic σ factor was identified, but this gene, rpoZ, did not affect the transcription of tonBRl or hmuPSTUV. Mutations in tonBRl, hmu genes and rpoZ did not affect symbiotic N2 fixation in peas.
Determinants of segregational stability of the linear plasmid‐prophage N15 of Escherichia coli
Authors: Grigoriev Petr S. , Łobocka Małgorzata B.
Published: March 2003

N15 is a bacteriophage of Escherichia coli that resembles λ, but, unlike λ, it lysogenizes as a linear plasmid. We show that stable maintenance of this unusual plasmid‐prophage depends on the parA and parB genes, relatives of the partition genes of F and P1 plasmids. ParB of N15, like its F‐ and P1‐encoded homologues, destabilizes plasmids carrying its target centromere, when present in excess. Within the genome of N15, we identified four unlinked, palindromic sequences that can promote the ParB‐mediated destabilization of a moderate‐copy vector in cis. They are distant from the parAB operon, unlike the centromeric sites of F and P1. Each of these palindromes could interact in vivo and in vitro with ParB. Each, when cloned separately, had properties characteristic of centromeric sites: exerted incompatibility against the N15 prophage and mini‐N15 plasmids, and stabilized a mini‐P1 plasmid depleted of its own partition genes when ParA and ParB of N15 were supplied. A pair of sites was more effective than a single site. Two of the centromeric sites are located in the proximity of promoters of phage genes, suggesting that, in addition to their function in partitioning of N15 prophage, they may control expression of N15 lytic functions.
Involvement of FIS in the H‐NS‐mediated regulation of virF gene of Shigella and enteroinvasive Escherichia coli ‡
Authors: Falconi Maurizio , Prosseda Gianni , Giangrossi Mara , Beghetto Elisa , Colonna Bianca
Published: March 2003

The mechanism of pathogenicity in Shigella and enteroinvasive Escherichia coli (EIEC) requires the co‐ordinated expression of several genes located on both the virulence plasmid and the chromosome. We found that cells lacking a functional FIS protein (factor for inversion stimulation) are partially impaired in expressing the virulence genes and that full expression is totally restored when Shigella wild‐type fis gene is offered in trans. We also identified virF, among the virulence genes, as a target of FIS‐mediated activation and showed that FIS binds to four specific sites in the promoter region of virF. Previous studies have demonstrated that the expression of VirF, the first positive activator of a multistep regulatory cascade, is subject to temperature‐dependent regulation by H‐NS, one of the main nucleoid‐associated proteins. We now demonstrate that two of the four FIS sites overlap one of the two H‐NS sites responsible for thermoregulation (H‐NS site I). FIS was found to exercise a direct positive transcriptional control at permissive temperature (37°C), when H‐NS fails to repress virF, as well as an indirect effect by partially counteracting H‐NS inhibition at the transition temperature (32°C). Our data indicate that FIS may be relevant for the rapid increase in virF expression after penetration of bacteria into the host.
Promoter recognition and discrimination by EσS RNA polymerase
Authors: Gaal Tamas , Ross Wilma , Estrem Shawn T. , Nguyen Lam H. , Burgess Richard R. , Gourse Richard L.
Published: March 2003

Although more than 30 Escherichia coli promoters utilize the RNA polymerase holoenzyme containing σS (EσS), and it is known that there is some overlap between the promoters recognized by EσS and by the major E. coli holoenzyme (Eσ70), the sequence elements responsible for promoter recognition by EσS are not well understood. To define the DNA sequences recognized best by EσSin vitro, we started with random DNA and enriched for EσS promoter sequences by multiple cycles of binding and selection. Surprisingly, the sequences selected by EσS contained the known consensus elements (−10 and −35 hexamers) for recognition by Eσ70. Using genetic and biochemical approaches, we show that EσS and Eσ70 do not achieve specificity through ‘best fit’ to different consensus promoter hexamers, the way that other forms of holoenzyme limit transcription to discrete sets of promoters. Rather, we suggest that EσS‐specific promoters have sequences that differ significantly from the consensus in at least one of the recognition hexamers, and that promoter discrimination against Eσ70 is achieved, at least in part, by the two enzymes tolerating different deviations from consensus. DNA recognition by EσS versus Eσ70 thus presents an alternative solution to the problem of promoter selectivity.
Transcript profiling in Candida albicans reveals new cellular functions for the transcriptional repressors CaTup1, CaMig1 and CaNrg1
Authors: Murad A. Munir A. , D'Enfert Christophe , Gaillardin Claude , Tournu Helene , Tekaia Fredj , Talibi Driss , Marechal Daniel , Marchais Véronique , Cottin Jane , Brown Alistair J.P.
Published: March 2003

The pathogenic fungus, Candida albicans contains homologues of the transcriptional repressors ScTup1, ScMig1 and ScNrg1 found in budding yeast. In Saccharomyces cerevisiae, ScMig1 targets the ScTup1/ScSsn6 complex to the promoters of glucose repressed genes to repress their transcription. ScNrg1 is thought to act in a similar manner at other promoters. We have examined the roles of their homologues in C. albicans by transcript profiling with an array containing 2002 genes, representing about one quarter of the predicted number of open reading frames (ORFs) in C. albicans. The data revealed that CaNrg1 and CaTup1 regulate a different set of C. albicans genes from CaMig1 and CaTup1. This is consistent with the idea that CaMig1 and CaNrg1 target the CaTup1 repressor to specific subsets of C. albicans genes. However, CaMig1 and CaNrg1 repress other C. albicans genes in a CaTup1‐independent fashion. The targets of CaMig1 and CaNrg1 repression, and phenotypic analyses of nrg1/nrg1 and mig1/mig1 mutants, indicate that these factors play differential roles in the regulation of metabolism, cellular morphogenesis and stress responses. Hence, the data provide important information both about the modes of action of these transcriptional regulators and their cellular roles. The transcript profiling data are available at http://www.pasteur.fr/recherche/unites/RIF/transcriptdata/.
Identification of loci critical for replication and compatibility of a Borrelia burgdorferi cp32 plasmid and use of a cp32‐based shuttle vector for the expression of fluorescent reporters in the Lyme disease spirochaete
Authors: Eggers Christian H. , Caimano Melissa J. , Clawson Michael L. , Miller William G. , Samuels D. Scott , Radolf Justin D.
Published: March 2003

The 32 kb circular plasmid (cp32) family of Borrelia burgdorferi has been the subject of intensive investigation because its members encode numerous differentially expressed lipoproteins. As many as nine different cp32s appear to be capable of stable replication within a single spirochaete. Here, we show that a construct (pCE310) containing a 4 kb fragment from the putative maintenance region of a B. burgdorferi CA‐11.2A cp32 was capable of autonomous replication in both high‐passage B. burgdorferi B31 and virulent B. burgdorferi 297. Deletion analysis revealed that only the member of paralogous family 57 and the adjacent non‐coding segment were essential for replication. The PF32 ParA orthologue encoded by the pCE310 insert was almost identical to the PF32 orthologues encoded on the B31 and 297 cp32‐3 plasmids. The finding that cp32‐3 was selectively deleted in both B31 and 297 transformants carrying pCE310 demonstrated the importance of the PF32 protein for cp32 compatibility and confirmed the prediction that cp32 plasmids expressing identical PF32 paralogues are incompatible. A shuttle vector containing the CA‐11.2A cp32 plasmid maintenance region was used to introduce green, yellow and cyan fluorescent protein reporters into B. burgdorferi. Flow cytometry revealed that the green fluorescent protein was well expressed by almost 90% of both avirulent and infectious transformants. In addition to enhancing our understanding of B. burgdorferi plasmid biology, our results further the development of genetic systems for dissecting pathogenic mechanisms in Lyme disease.
Functional analysis of a vacuolar ABC transporter in wild‐type Candida albicans reveals its involvement in virulence
Authors: Theiss Stephanie , Kretschmar Marianne , Nichterlein Thomas , Hof Herbert , Agabian Nina , Hacker Jörg , Köhler Gerwald A.
Published: March 2003

ATP‐driven transport proteins belonging to the ATP‐binding cassette (ABC) superfamily perform important functions in cell metabolism and detoxification. Compounds can be actively transported across membranes, including the plasma membrane or organellar membranes. The vacuole is an important organelle in fungal cells required for compartmentalization of metabolites as well as toxic substances. Sequestration into the vacuole is often energy‐dependent. We present the first isolation and molecular analysis of a vacuolar ABC transporter gene in the opportunistic fungal pathogen Candida albicans.
The protein encoded by the MLT1 gene is highly similar to Multiple Drug Resistance‐associated
Protein (MRP)‐like transporters of yeast and higher organisms that form the Cystic
Fibrosis Transmembrane Conductance Regulator (CFTR)/MRP subfamily of ABC transporters,
a class of proteins so far not characterized in C. albicans. MLT1 expression
is extensively growth phase‐regulated, and gene transcripts are inducible by metabolic
poisons. Gene replacement mutants generated in wild‐type C. albicans with
the dominant selection marker MPAR showed a profound reduction
in virulence in a mouse peritonitis model that was reversed by complementation with
an intact MLT1 gene. Hence, this report provides primary evidence for the involvement of vacuolar ABC transporters in fungal virulence.
Characterization of a bi‐directional promoter for divergent transcription of a PHD‐zinc finger protein gene and a ran gene in the protozoan pathogen Giardia lamblia
Authors: Ong Shiou‐Jeng , Huang Li‐Chin , Liu Hsing‐Wei , Chang Shih‐Chia , Yang Yen‐Ching , Bessarab Irina , Tai Jung‐Hsiang
Published: March 2003

We showed previously that transcription of the ran gene in Giardia lamblia is regulated by an AT‐rich initiator. In the present study, the ran initiator was found to regulate transcription of a neighbouring PHD zinc‐finger protein gene. Deletion and scanning mutagenesis of the phd promoter in a firefly luciferase reporter system showed that the promoter activity is determined by multiple single‐stranded T‐tract DNA elements distributed into a distal domain spanning the ran initiator (–134/–103) and a proximal domain (–88/–48) spanning phd messenger RNA (mRNA) start sites (–74, –55 and –53 relative to the first ATG). The promoter activity is repressed by the single T‐tract element on a non‐template strand of the ran initiator, and is activated by closely spaced T‐tract elements on the opposite strand. The T‐tract elements in the phd and ran initiators compete for similar ssDNA binding proteins. Mutation of –47/–42 resulted in dramatic reduction of luciferase activity without changing luciferase mRNA levels, indicating the potential involvement of a regulatory mechanism in PHD protein translation. These findings suggest that G. lamblia uses multiple copies of a T‐tract element as both core and distal elements in regulating transcription initiation, and that expression of the phd gene is regulated at multiple levels.
Salmonella effectors within a single pathogenicity island are differentially expressed and translocated by separate type III secretion systems
Authors: Knodler Leigh A. , Celli Jean , Hardt Wolf‐Dietrich , Vallance Bruce A. , Yip Calvin , Finlay B. Brett
Published: March 2003

Pathogenicity islands (PAIs) are large DNA segments in the genomes of bacterial pathogens that encode virulence factors. Five PAIs have been identified in the Gram‐negative bacterium Salmonella enterica. Two of these PAIs, Salmonella pathogenicity
island (SPI)‐1 and SPI‐2, encode type III secretion systems (TTSS), which are essential
virulence determinants. These ‘molecular syringes’ inject effectors
directly into the host cell, whereupon they manipulate host cell functions. These
effectors are either encoded with their respective TTSS or scattered elsewhere on
the Salmonella chromosome. Importantly, SPI‐1 and SPI‐2 are expressed under
distinct environmental conditions: SPI‐1 is induced upon initial contact with the
host cell, whereas SPI‐2 is induced intracellularly. Here, we demonstrate that a
single PAI, in this case SPI‐5, can encode effectors that are induced by distinct
regulatory cues and targeted to different TTSS. SPI‐5 encodes the SPI‐1 TTSS translocated
effector, SigD/SopB. In contrast, we report that the adjacently encoded effector
PipB is part of the SPI‐2 regulon. PipB is translocated by the SPI‐2 TTSS to the
Salmonella‐containing vacuole and Salmonella‐induced filaments. We
also show that regions of SPI‐5 are not conserved in all Salmonella spp. Although
sigD/sopB is present in all Salmonella spp., pipB is not found
in Salmonella bongori, which also lacks a functional SPI‐2 TTSS. Thus, we demonstrate a functional and regulatory cross‐talk between three chromosomal PAIs, SPI‐1, SPI‐2 and SPI‐5, which has significant implications for the evolution and role of PAIs in bacterial pathogenesis.
Staphylococcus and biofilms
Authors: Götz Friedrich
Published: March 2003

The genetic and molecular basis of biofilm formation in staphylococci is multifaceted. The ability to form a biofilm affords at least two properties: the adherence of cells to a surface and accumulation to form multilayered cell clusters. A trademark is the production of the slime substance PIA, a polysaccharide composed of β‐1,6‐linked
N‐acetylglucosamines with partly deacetylated residues, in which the cells
are embedded and protected against the host’s immune defence and antibiotic
treatment. Mutations in the corresponding biosynthesis genes (ica operon)
lead to a pleiotropic phenotype; the cells are biofilm and haemagglutination negative,
less virulent and less adhesive on hydrophilic surfaces. ica expression is
modulated by various environmental conditions, appears to be controlled by SigB and
can be turned on and off by insertion sequence (IS) elements. A number of biofilm‐negative
mutants have been isolated in which polysaccharide intercellular adhesin (PIA) production
appears to be unaffected. Two of the characterized mutants are affected in the major
autolysin (atlE) and in D‐alanine esterification of teichoic
acids (dltA). Proteins have been identified that are also involved in biofilm formation, such as the accumulation‐associated protein (AAP), the clumping factor A (ClfA), the staphylococcal surface protein (SSP1) and the biofilm‐associated protein (Bap). Concepts for the prevention of obstinate polymer‐associated infections include the search for new anti‐infectives active in biofilms and new biocompatible materials that complicate biofilm formation and the development of vaccines.
Microarray analysis of the Bacillus subtilis K‐state: genome‐wide expression changes dependent on ComK
Authors: Berka Randy M. , Hahn Jeanette , Albano Mark , Draskovic Irena , Persuh Marjan , Cui Xianju , Sloma Alan , Widner William , Dubnau David
Published: March 2003

In Bacillus subtilis, the competence transcription factor ComK activates
its own transcription as well as the transcription of genes that encode DNA transport
proteins. ComK is expressed in about 10% of the cells in a culture grown
to competence. Using DNA microarrays representing ≈ 95% of the
protein‐coding open reading frames in B. subtilis, we compared the expression
profiles of wild‐type and comK strains, as well as of a mecA mutant
(which produces active ComK in all the cells of the population) and a comK mecA double mutant. In these comparisons, we identified at least 165 genes that are upregulated by ComK and relatively few that are downregulated. The use of reporter fusions has confirmed these results for several genes. Many of the ComK‐regulated genes are organized in clusters or operons, and 23 of these clusters are preceded by apparent ComK‐box promoter motifs. In addition to those required for DNA uptake, other genes that are upregulated in the presence of ComK are probably involved in DNA repair and in the uptake and utilization of nutritional sources. From this and previous work, we conclude that the ComK regulon defines a growth‐arrested state, distinct from sporulation, of which competence for genetic transformation is but one notable feature. We suggest that this is a unique adaptation to stress and that it be termed the ‘K‐state’.
The p21‐activated kinase, Shk1, is required for proper regulation of microtubule dynamics in the fission yeast, Schizosaccharomyces pombe
Authors: Qyang Yibing , Yang Peirong , Du Hongyan , Lai Hong , Kim HyeWon , Marcus Stevan
Published: March 2003

The p21‐activated kinase, Shk1, is required for the proper establishment of cell polarity in the fission yeast, Schizosaccharomyces pombe. We showed recently that loss of the essential Shk1 inhibitor, Skb15, causes significant spindle defects in fission yeast, thus implicating Shk1 as a potential regulator of microtubule dynamics. Here, we show that cells deficient in Shk1 function have malformed interphase microtubules and mitotic microtubule spindles, are hypersensitive to the microtubule‐destabilizing drug thiabendazole (TBZ) and cold sensitive for growth. TBZ treatment causes a downregulation of Shk1 kinase activity, which increases rapidly after release of cells from the drug, thus providing a correlation between Shk1 kinase function and active microtubule polymerization. Consistent with a role for Shk1 as a regulator of microtubule dynamics, green fluorescent protein (GFP)–Shk1 fusion proteins localize to interphase microtubules and mitotic microtubule spindles, as well as to cell ends and septum‐forming regions of fission yeast cells. We show that loss of Tea1, a cell end‐ and microtubule‐localized protein previously implicated as a regulator of microtubule dynamics in fission yeast, exacerbates the growth and microtubule defects resulting from partial loss of Shk1 and that Shk1 localizes to illicit growth tips produced by tea1 mutant cells. Our results demonstrate that Shk1 is required for the proper regulation of microtubule dynamics in fission yeast and implicate Tea1 as a potential Shk1 regulator.
Identification and characterization of differentiation mutants in the protozoan parasite Toxoplasma gondii
Authors: Matrajt Mariana , Donald Robert G. K. , Singh Upinder , Roos David S.
Published: March 2003

Two forms of the protozoan parasite Toxoplasma gondii are associated with
intermediate hosts such as humans: rapidly growing tachyzoites are responsible for
acute illness, whereas slowly dividing encysted bradyzoites can remain latent within
the tissues for the life of the host. In order to identify genetic factors associated
with parasite differentiation, we have used a strong bradyzoite‐specific promoter
(identified by promoter trapping) to drive the expression of T. gondii hypoxanthine–xanthine–guanine
phosphoribosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage‐specific
positive/negative selectable marker. Insertional mutagenesis has been carried out
on this parental line, followed by bradyzoite induction in vitro and selection
in 6‐thioxanthine to identify misregulation mutants. Two different mutants fail to
induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants
are also defective in other aspects of differentiation: they replicate well under
bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachyzoites.
Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos
biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under bradyzoite differentiation conditions.
Dam‐dependent phase variation of Ag43 in Escherichia coli is altered in a seqA mutant
Authors: Correnti Jason , Munster Vincent , Chan Teresa , Woude Marjan van der
Published: March 2003

In Escherichia coli, phase variation of the outer membrane protein Ag43 encoded by the agn43 gene is mediated by DNA methylation and the global regu‐lator OxyR. Transcription of agn43 occurs (ON phase) when three Dam target sequences in the agn43 regulatory region are methylated, which prevents the repressor OxyR from binding. Conversely, transcription is repressed (OFF) when these Dam target sequences are unmethylated and OxyR binds. A change in expression phase requires a concomitant change in the DNA methylation state of these Dam target sequences. To gain insight into the process of inheritance of the expression phase and the DNA methylation state, protein–DNA interactions at agn43 were examined. Binding of OxyR at agn43 was sufficient to protect the three GATC sequences contained within its binding site from Dam‐dependent methylation in vitro, suggesting that no other factors are required to maintain the unmethylated state and OFF phase. To maintain the methylated state of the ON phase, however, Dam must access the hemimethylated agn43 region after DNA replication, and OxyR binding must not occur. OxyR bound hemimethylated agn43 DNA, but the affinity was severalfold lower than for unmethylated DNA. This presumably contributes to the maintenance of the methylated state but, at the same time, may allow for infrequent OxyR binding and a switch to the OFF phase. Hemimethylated agn43 DNA was also a binding substrate for the sequestration protein SeqA. Thus, SeqA, OxyR and Dam may compete for the same hemimethylated agn43 DNA that is formed after DNA replication in an ON phase cell. In isolates with a mutant seqA allele, agn43 phase variation rates were altered and resulted in a bias to the OFF phase. In part, this can be attributed to the observed decrease in the level of DNA methylation in the seqA mutant.
Regulation of yeast protein kinase C activity by interaction with the small GTPase Rho1p through its amino‐terminal HR1 domain
Authors: Schmitz Hans‐Peter , Lorberg Anja , Heinisch Jürgen J.
Published: March 2003

Protein kinase C from Saccharomyces cerevisiae (Pkc1p) constitutes a prototypic

member of the protein kinase C superfamily, as it shares all the conserved regions

scattered among the isoenzymes of higher eukaryotes. The functional significance

of some of the conserved domains in the yeast enzyme has not yet been investigated.

We examined strains carrying a partial deletion in the amino‐terminal region of the

enzyme, which is homologous to the HR1 of the protein kinase C‐related kinases. This

strain was sensitive to the presence of caffeine, Calcofluor white and Congo red,

all drugs known to affect mutants defective in the signal transduction pathway ensuring

cellular integrity in which Pkc1p is a central component. Isolation of a single point

mutation in HR1A, which shares the sensitivity to the drugs mentioned, confirmed

the importance of this region for proper regulation of protein kinase C activity

in vivo. Two‐hybrid analysis provided evidence for an interaction of the small GTPase Rho1p with the HR1A region, in addition to the reported interaction of this protein with the C1 region of Pkc1p. MAP kinase phosphorylation assays indicate that this Rho1p–Pkc1p/HR1A interaction does not result in an activation of the kinase cascade. The intragenic lethality of mutants affected in both HR1A and the C1 domain reported in this work implies an essential role for Rho1p–Pkc1p interaction in yeast.
Hexose phosphorylation and the putative calcium channel component Mid1p are required for the hexose‐induced transient elevation of cytosolic calcium response in Saccharomyces cerevisiae
Authors: Tökés‐Füzesi Margit , Bedwell David M. , Repa Imre , Sipos Katalin , Sümegi Balázs , Rab András , Miseta Attila
Published: March 2003

Saccharomyces cerevisiae responds to environ‐mental stimuli such as an exposure
to pheromone or to hexoses after carbon source limitation with a transient elevation
of cytosolic calcium (TECC) response. In this study, we examined whether hexose transport
and phosphorylation are necessary for the TECC response. We found that a mutant strain
lacking most of the known hexose transporters was unable to carry out the TECC response
when exposed to glucose. A mutant strain that lacked the ability to phosphorylate
glucose was unable to respond to glucose addition, but displayed a normal TECC response
after the addition of galactose. These results indicate that hexose uptake and phosphorylation
are required to trigger the hexose‐induced TECC response. We also found that the
TECC response was significantly smaller than normal when the level of environmental
calcium was reduced, and was abolished in a mid1 mutant that lacked a subunit of the high‐affinity calcium channel of the yeast plasma membrane. These results indicate that most or all of the TECC response is mediated by an influx of calcium from the extracellular space. Our results indicate that this transient increase in plasma membrane calcium permeability may be linked to the accumulation of Glc‐1‐P (or a related glucose metabolite) in yeast.
Lon protease functions as a negative regulator of type III protein secretion in Pseudomonas syringae
Authors: Bretz James , Losada Liliana , Lisboa Keilla , Hutcheson Steven W.
Published: March 2003

The central conserved region of the Pseudomonas syringae hrp pathogenicity island encodes a type III protein secretion system (TTSS) that is required for pathogenicity in plants. Expression of the hrp TTSS is controlled by the alternative sigma factor, HrpL, whose expression, in turn, is positively controlled by two truncated enhancer binding proteins, HrpR and HrpS. Although a number of environmental conditions are known to modulate hrp TTSS expression, such as stringent conditions and pathogenesis, the mechanism by which the activities of these transcriptional factors are modulated had not been established. In this study, HrpR and HrpS were found to be constitutively expressed under conditions in which the hrpL promoter was inactive. To identify a postulated negative regulator of hrpL expression, transposome (Tz) mutagenesis was used to isolate hrp constitutive mutants. P. syringae Pss61 and DC3000 hrp constitutive mutants were identified that carried lon::Tz insertions and exhibited increased cell length and UV sensitivity typical of Δlon mutants. The P. syringae Lon protease retained structural features of its homologues found in other bacteria and was capable of complementing an Escherichia coliΔlon mutant. P. syringae lon::Tz mutants exhibited enhanced expression of the hrpL promoter, suggesting an effect on HrpR and/or HrpS. HrpR was observed to be unstable in wild‐type P. syringae strains grown in non‐inductive media. However, the apparent half‐life increased more than 10‐fold in the P. syringae lon::Tz mutants or upon transfer to an inductive medium. The P. syringae lon mutants elicited rapidly developing plant responses and were shown to hypersecrete effector proteins, such as AvrPto. These results indicate that expression of the hrp regulon and type III secretion are negatively regulated by Lon‐mediated degradation of HrpR.
Protein interactions within Saccharomyces cerevisiae Elongator, a complex essential for Kluyveromyces lactis zymocicity
Authors: Fichtner Lars , Frohloff Frank , Jablonowski Daniel , Stark Michael J. R. , Schaffrath Raffael
Published: March 2003

mTn3‐tagging identified Kluyveromyces lactis zymocin target genes from Saccharomyces cerevisiae as TOT1–3/ELP1–3 coding for the RNA polymerase II (pol II) Elongator histone acetyltransferase (HAT) complex. tot phenotypes resulting from mTn3 tagging were similar to totΔ null alleles, suggesting loss of Elongator's integrity. Consistently, the Tot1–3/Elp1–3 proteins expressed from the mTn3‐tagged genes were all predicted to be C‐terminally truncated, lacking ≈ 80% of Tot1p, five WD40 Tot2p repeats and two HAT motifs of Tot3p. Besides its role as a HAT, Tot3p assists subunit communication within Elongator by mediating Tot2–Tot4, Tot2–Tot5, Tot2–Tot1 and Tot4–Tot5 protein–protein interactions. TOT1 and TOT2 are essential for Tot4–Tot2 and Tot4–Tot3 interactions respectively. The latter was lost with a C‐terminal Tot2p truncation; the former was affected by progressively truncating TOT1. Despite being dispensable for Tot4–Tot2 interaction, the extreme C‐terminus of Tot1p may play a role in TOT/Elongator function, as its truncation confers zymocin resistance. Tot4p/Kti12p, an Elongator‐associated factor, also interacted with pol II and could be immunoprecipitated while being bound to the ADH1 promoter. Two‐hybrid analysis showed that Tot4p also interacts with Cdc19p, suggesting that Tot4p plays an additional role in concert with Cdc19p, perhaps co‐ordinating cell growth with carbon source metabolism.
Polyadenylation of Escherichia coli transcripts plays an integral role in regulating intracellular levels of polynucleotide phosphorylase and RNase E
Authors: Mohanty Bijoy K. , Kushner Sidney R.
Published: March 2003

Polyadenylation in Escherichia coli has been implicated in the destabilization of a variety of transcripts. However, transiently increasing intracellular poly(A) levels has also been shown to stabilize the pnp and rne transcripts, leading to increased polynucleotide phosphorylase (PNPase) and RNase E levels respectively. Here, we show that the half‐lives of both the pnp and rne transcripts are dependent on the intracellular level of polyadenylated transcripts. In addition, experiments using pnp–lacZ and rne–lacZ translational fusions demonstrate that the variations in transcript stability and protein levels arise from alterations in the autoregulation of both genes. Further support for this conclusion is provided by the fact that, in an rne mutant in which autoregulation is inactivated by deletion of most of the 5′ untranslated region, variations in the level of polyadenylated transcripts no longer affect RNase E protein expression. Of even more interest is the fact that the presence of a functional degradosome is essential for RNase E to detect increased levels of poly(A). Thus, it appears that polyadenylation of transcripts in E. coli serves as a sensing mechanism by which the cell adjusts the levels of both RNase E and PNPase.
Multiple bacteria encode metallothioneins and SmtA‐like zinc fingers
Authors: Blindauer Claudia A. , Harrison Mark D. , Robinson Andrea K. , Parkinson John A. , Bowness Peter W. , Sadler Peter J. , Robinson Nigel J.
Published: March 2003

Zinc is essential but toxic in excess. Bacterial metallothionein, SmtA from Synechococcus PCC 7942, sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA. The dearth of other reported bacterial metallothioneins has been surprising. Here we describe related bacterial metallothioneins (BmtA) from Anabaena PCC 7120, Pseudomonas aeruginosa and Pseudomonas putida that bind multiple zinc ions with high stability towards protons. Thiol modification demonstrates that cysteine coordinates zinc in all of these proteins. Additionally, 111Cd‐NMR, and 111Cd‐edited 1H‐NMR, identified histidine ligands in Anabaena PCC 7120 BmtA, analogous to SmtA. A related Escherichia coli protein bound only a single zinc ion, via four cysteine residues, with low stability towards protons; 111Cd‐NMR and 111Cd‐edited 1H‐NMR confirmed exclusive cysteine‐coordination, and these cysteine residues reacted rapidly with 5,5′‐dithiobis‐(2‐nitrobenzoic acid). 1H‐NMR of proteins from P. aeruginosa, Anabaena PCC 7120 and E. coli generated fingerprints diagnostic for the GATA‐like zinc finger fold of SmtA. These studies reveal first the existence of multiple bacterial metallothioneins, and second proteins with SmtA‐like lone zinc fingers, devoid of a cluster, and designated GatA. We have identified 12 smtA‐like genes in sequence databases including four of the gatA type.
Novel Mycobacterium tuberculosis anti‐σ factor antagonists control σF activity by distinct mechanisms
Authors: Beaucher Jocelyn , Rodrigue Sébastien , Jacques Pierre‐Étienne , Smith Issar , Brzezinski Ryszard , Gaudreau Luc
Published: March 2003

The aetiological agent of tuberculosis, Mycobacterium tuberculosis, encodes 13 σ factors, as well as several putative anti‐, and anti‐anti‐ σ factors. Here we show that a σ factor that has been previously shown to be involved in virulence and persistence processes, σF, can be specifically inhibited by the anti‐σ factor UsfX. Importantly, the inhibitory activity of UsfX, in turn, can be negatively regulated by two novel anti‐anti‐σ factors. The first anti‐anti‐σ factor seems to be regulated by redox potential, and the second may be regulated by phosphorylation as it is rendered non‐functional by the introduction of a mutation that is believed to mimic phosphorylation of the anti‐anti‐σ factor. These results suggest that σF activity might be post‐translationally modulated by at least two distinct pathways in response to different possible physiological cues, the outcome being consistent with the bacteria's ability to adapt to diverse host environments during disease progression, latency and reactivation.
Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity
Authors: DiGiandomenico Antonio , Matewish Mauricia J. , Bisaillon Amy , Stehle John R. , Lam Joseph S. , Castric Peter
Published: March 2003

The structural similarity between the pilin glycan and the O‐antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O‐antigen and produced only non‐glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O‐antigen and glycosylated pilin. Expression of a cosmid clone containing the O‐antigen biosynthetic gene cluster from P. aeruginosa PA103 (LPS serotype O11) in P. aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens. The presence of the O11 repeating unit was confirmed by matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Expression of the O‐antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O‐antigens. A role for pilO in the glycosylation of pilin in P. aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P. aeruginosa strains. Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin. These results show that the pilin glycan of P. aeruginosa 1244 is a product of the O‐antigen biosynthetic pathway. In addition, the structural diversity of the O‐antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.
Domains in the S‐layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self‐assembly
Authors: Antikainen Jenni , Anton Lena , Sillanpää Jouko , Korhonen Timo K.
Published: March 2003

The protein regions in the S‐layer protein CbsA of Lactobacillus crispatus JCM 5810, needed for binding to collagens and laminin, anchoring to bacterial cell wall, as well as self‐assembly, were mapped by deletion analysis of His‐tagged peptides isolated from Escherichia coli and by heterologous expression on Lactobacillus casei. Mature CbsA is 410 amino acids long, and stepwise genetic truncation at both termini revealed that the region 32–271 carries the infor‐mation for self‐assembly of CbsA into a periodic structure. The lactobacillar S‐layer proteins exhibit sequence variation in their assembly domain, but the border regions 30–34 and 269–274 in CbsA are conserved in valine‐rich short sequences. Short deletions or substitutions at these regions affected the morphology of His‐CbsA polymers, which varied from sheet‐like to cylindrical tubular polymers, and further truncation beyond the DNA encoding residues 32 and 271 leads to a non‐periodic aggregation. The self‐assembly of the truncated peptides, as seen by electron microscopy, was correlated with their behaviour in a cross‐linking study. The shorter peptides not forming a regular polymer were observed by the cross‐linking study and mass spectrometry to form dimers, trimers and tetramers, whereas the other peptides were cross‐linked to large multimers only. Binding of solubilized type I and IV collagens was observed with the His‐CbsA peptides 1–274 and 31–287, but not with the smaller peptides regardless of their ability to form regular polymers. Strain JCM 5810 also adheres to immobilized laminin and, in order to analyse the possible laminin binding by CbsA, cbsA and its fragments were expressed on the surface of L. casei. Expression of the CbsA peptides 1–274, 1–287, 28–287 and 31–287 on L. casei conferred adhesiveness to both laminin and collagen immobilized on glass as well as to laminin‐ and collagen‐containing regions in chicken colon and ileum. The C‐terminal peptides 251–410 and 288–410 bound to L. crispatus JCM 5810 cells from which the S‐layer had been depleted by chemical extraction, whereas no binding was seen with the His‐CbsA peptides 1–250 or 1–269 or to cells with an intact S‐layer. The His‐CbsA peptides 251–410 and 288–410 bound to teichoic acids of several bacterial species. The results show that CbsA is an adhesive complex with an N‐terminal assembly domain exhibiting affinity for pericellular tissue components and a cationic C‐terminal domain binding to negatively charged cell wall components.
The rpf gene of Micrococcus luteus encodes an essential secreted growth factor
Authors: Mukamolova Galina V. , Turapov Obolbek A. , Kazarian Konstantin , Telkov Miroslav , Kaprelyants Arseny S. , Kell Douglas B. , Young Michael
Published: March 2003

Micrococcus luteus secretes a small protein called Rpf, which has autocrine and paracrine signalling functions and is required for the resuscitation of dormant cells. Originally isolated from the supernatant of actively growing cultures, Rpf was also detected on the surface of actively growing bacteria. Most molecules may be sequestered non‐productively at the cell surface, as a truncated form of the protein, encompassing only the ‘Rpf domain’ is fully active. The C‐terminal LysM module, which probably mediates binding to the cell envelope, is not required for biological activity. Rpf was essential for growth of M. luteus. Washed cells, inoculated at low density into a minimal medium, could not grow in its absence. Moreover, the incorporation of anti‐Rpf antibodies into the culture medium at the time of inoculation also prevented bacterial growth. We were unable to inactivate rpf using a disrupted form of the gene, in which most of the coding sequence was replaced with a selectable thiostrepton resistance marker. Gene disruption was possible in the presence of a second, functional, plasmid‐located copy of rpf, but not in the presence of a rpf derivative whose protein product lacked the secretory signal sequence. As far as we are aware, Rpf is the first example of a truly secreted protein that is essential for bacterial growth. If the Rpf‐like proteins elaborated by Mycobacterium tuberculosis and other mycobacteria prove similarly essential, interference with their proper functioning may offer novel opportunities for protecting against, and treating, tuberculosis and other mycobacterial disease.
The N‐terminus of enteropathogenic Escherichia coli (EPEC) Tir mediates transport across bacterial and eukaryotic cell membranes
Authors: Crawford J. Adam , Kaper James B.
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to translocate into host cells several effector molecules that are required for virulence. One of these, the translocated intimin receptor, Tir, inserts into the host cell cytoplasmic membrane, where it functions as the receptor for intimin, an outer membrane adhesin expressed by EPEC. A chaperone for Tir, called CesT, is required for stability of Tir in the EPEC cytoplasm. In this study, the cyaA gene reporter system was used to identify domains in Tir that mediate secretion into the culture supernatant and translocation into host cells. A Tir–CyaA fusion containing the first 15 N‐terminal residues of Tir was secreted and translocated into HeLa cells by a ΔtirΔcesT mutant; however, maximal secretion and translocation was observed with the first 26 N‐terminal residues of Tir. Fusions containing progressively larger N‐terminal sequences of Tir were also efficiently secreted and translocated into HeLa cells by the ΔtirΔcesT strain. However, in a Δtir mutant that expresses CesT, Tir26–CyaA and an additional fusion containing the first 69 N‐terminal residues of Tir were not secreted or translocated, but fusions containing larger N‐terminal Tir sequences were secreted and translocated by the Δtir mutant. Wild‐type EPEC secreted and translocated the Tir15–CyaA fusion, whereas longer fusions, such as Tir26–CyaA and Tir69–CyaA, were translocated to higher levels, similar to what was observed with the ΔtirΔcesT mutant. A Tir–CyaA fusion containing the CesT binding domain was translocated into HeLa cells more rapidly in the presence of CesT compared with translocation in the absence of CesT. Collectively, these results suggest that an N‐terminal domain of 26 amino acids functions as a CesT‐independent signal that is capable of delivering Tir into both the culture supernatant and the cytosol of host cells. Furthermore, in addition to its role in the stability of Tir, CesT may function in translocation by mediating rapid delivery of Tir into host cells.