bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and characterization of BipA, a Bordetella Bvg‐intermediate phase protein
Authors: Stockbauer Kathryn E. , Fuchslocher Bryna , Miller Jeff F. , Cotter Peggy A. 
Published: March 2003

The Bordetella BvgAS sensory transduction system has traditionally been viewed as controlling a transition between two distinct phenotypic phases: the Bvg+ or virulent phase and the Bvg− or avirulent phase. Recently, we identified a phenotypic phase of Bordetella bronchiseptica that displays reduced virulence in a rat model of respiratory infection concomitant with increased ability to survive nutrient deprivation. Characterization of this phase, designated Bvg‐intermediate (Bvgi), indicated the presence of antigens that are maximally, if not exclusively, expressed in this phase and therefore suggested the existence of a previously unidentified class of Bvg‐regulated genes. We now report the identification and characterization of a Bvgi phase protein, BipA (Bvg‐intermediate phase protein A), and its structural gene, bipA. Reverse transcriptase–polymerase chain reaction (RT–PCR) analysis indicates that bipA is expressed maximally under Bvgi phase conditions and thus represents the first identified Bvgi phase gene. bipA encodes a 1578‐amino‐acid protein that shares amino acid sequence similarity at its N‐terminus with the proposed outer membrane localization domains of intimin (Int) of enteropathogenic and enterohaemorrhagic Escherichia coli and invasin (Inv) of Yersinia spp. Although not apparent at the amino acid level, BipA is also similar to Int and Inv in that the proposed membrane‐spanning domain is followed by several 90‐amino‐acid repeats and a distinct C‐terminal domain. Localization studies using an antibody directed against the C‐terminus of BipA indicated that its C‐terminus is exposed on the bacterial cell surface. Western blot analysis with this same antibody indicated that BipA homologues are expressed in Bvgi phase Bordetella pertussis and Bordetella parapertussis. Comparison of a ΔbipA strain with wild‐type B. bronchiseptica indicated that BipA is not required for Bvgi phase‐specific aggregative adherence to rat lung epithelial cells in vitro or for persistent colonization of the rabbit respiratory tract in vivo. However, our data are consistent with the hypothesis that BipA, and the Bvgi phase in general, play an important role in the Bordetella infectious cycle, perhaps by contributing to aerosol transmission.
Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi
Authors: Stewart Philip E. , Thalken Rebecca , Bono James L. , Rosa Patricia 
Published: March 2003

Borrelia burgdorferi contains abundant circular and linear plasmids, but the mechanism of replication of these extrachromosomal elements is unknown. A B. burgdorferi 9 kb circular plasmid (cp9) was amplified in its entirety by the polymerase chain reaction and used to construct a shuttle vector that replicates in Escherichia coli and B. burgdorferi. A 3.3 kb region of cp9 containing three open reading frames was used to construct a smaller shuttle vector, designated pBSV2. This vector was stably maintained in B. burgdorferi, indicating that all elements necessary for autonomous replication are probably located on this 3.3 kb fragment. A non‐infectious B. burgdorferi strain was efficiently transformed by pBSV2. Additionally, infectious B. burgdorferi was also successfully transformed by pBSV2, indicating that infectious strains of this important human pathogen can now be genetically manipulated.
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.
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

SummaryThe 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

SummaryATP‐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

SummaryWe 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

SummaryPathogenicity 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

SummaryThe 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

SummaryIn 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

SummaryThe 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

SummaryTwo 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

SummaryIn 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

SummaryProtein 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

Summary 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.