bluuebunny/crossref_metadata_2025 huggingface dataset:
The making of a gradient: IcsA (VirG) polarity in Shigella flexneri
Authors: Robbins Jennifer R. , Monack Denise , McCallum Sandra J. , Vegas Arturo , Pham Estella , Goldberg Marcia B. , Theriot Julie A.
Published: March 2003

The generation and maintenance of subcellular organization in bacteria is critical for many cell processes and properties, including growth, structural integrity and, in pathogens, virulence. Here, we investigate the mechanisms by which the virulence protein IcsA (VirG) is distributed on the bacterial surface to promote efficient transmission of the bacterium Shigella flexneri from one host cell to another. The outer membrane protein IcsA recruits host factors that result in actin filament nucleation and, when concentrated at one bacterial pole, promote unidirectional actin‐based motility of the pathogen. We show here that the focused polar gradient of IcsA is generated by its delivery exclusively to one pole followed by lateral diffusion through the outer membrane. The resulting gradient can be modified by altering the composition of the outer membrane either genetically or pharmacologically. The gradient can be reshaped further by the action of the protease IcsP (SopA), whose activity we show to be near uniform on the bacterial surface. Further, we report polar delivery of IcsA in Escherichia coli and Yersinia pseudotuberculosis, suggesting that the mechanism for polar delivery of some outer membrane proteins is conserved across species and that the virulence function of IcsA capitalizes on a more global mechanism for subcellular organization.
Discovery of a haem uptake system in the soil bacterium Bradyrhizobium japonicum
Authors: Nienaber Andrea , Hennecke Hauke , Fischer Hans‐Martin
Published: March 2003

In Bradyrhizobium japonicum, the nitrogen‐fixing symbiont of soybeans, we have identified a haem uptake system, Hmu, that comprises a cluster of nine open reading frames. Predicted products of these genes include: HmuR, a TonB‐dependent haem receptor in the outer membrane; HmuT, a periplasmic haem‐binding protein; and HmuUV, an ABC transporter in the inner membrane. Furthermore, we identified homologues of ExbBD and TonB, that are required for energy transduction from the inner to the outer membrane. Mutant analysis and complementation tests indicated that HmuR and the ExbBD–TonB system, but not the HmuTUV transporter, are essential for haem uptake or haem acquisition from haemoglobin and leghaemoglobin. The TonB system seems to be specific for haem uptake as it is dispensable for siderophore uptake. Therefore, we propose the existence of a second TonB homologue functioning in the uptake of Fe‐chelates. When tested on soybean host plants, hmuT‐hmuR and exbD‐tonB mutants exhibited wild‐type symbiotic properties. Thus, haem uptake is not essential for symbiotic nitrogen fixation but it may enable B. japonicum to have access to alternative iron sources in its non‐symbiotic state. Transcript analysis and expression studies with lacZ fusions showed that expression of hmuT and hmuR is induced under low iron supply. The same was observed in fur and irr mutant backgrounds although maximal induction levels were decreased. We conclude either that both regulators, Fur and Irr, independently mediate transcriptional control by iron or that a yet unknown iron regulatory system activates gene expression under iron deprivation. An A/T‐rich cis‐acting element, located in the promoter region of the divergently transcribed hmuTUV and hmuR genes, is possibly required for this type of iron control.
SREA is involved in regulation of siderophore biosynthesis, utilization and uptake in Aspergillus nidulans
Authors: Oberegger Harald , Schoeser Michelle , Zadra Ivo , Abt Beate , Haas Hubertus
Published: March 2003

Under conditions of low iron availability, most fungi excrete siderophores in order to mobilize extracellular iron. We show that lack of the GATA‐type transcription factor SREA in Aspergillus nidulans not only leads to derepression of siderophore biosynthesis but also to deregulation of siderophore‐bound iron uptake and ornithine esterase expression. Furthermore, SREA deficiency causes increased accumulation of ferricrocin, the siderophore responsible for intracellular iron storage. In sreA deletion strains, extracellular siderophore production is derepressed but still regulated negatively by iron availability, indicating the presence of an additional iron‐regulatory mechanism. In contrast, iron affects ferricrocin accumulation in a positive way, suggesting a protective role for this siderophore in detoxification of intracellular iron excess. The harmfulness of deregulated iron uptake in this mutant is demonstrated by increased expression of genes encoding the antioxidative enzymes catalase CATB and the superoxide dismutases SODA and SODB. It is noteworthy that iron starvation was found to repress catB expression in wild‐type (wt) and SREA‐deficient strains, consistent with catB being subject to SREA‐independent iron regulation. Differential display led to the identification of putative SREA target genes amcA and mirA. The deduced MIRA amino acid sequence displays significant similarity to recently characterized siderophore permeases of Saccharomyces cerevisiae. amcA encodes a putative mitochondrial carrier for the siderophore precursor ornithine, indicating cross‐regulation of siderophore and ornithine metabolism.
Analysis of type I restriction modification systems in the Neisseriaceae: genetic organization and properties of the gene products
Authors: Piekarowicz Andrzej , Kłyż Aneta , Kwiatek Agnieszka , Stein Daniel C.
Published: March 2003

The hsd locus (host specificity of DNA) was identified in the Neisseria gonorrhoeae genome. The DNA fragment encoding this locus produced an active restriction and modification (R/M) system when cloned into Escherichia coli. This R/M system was designated NgoAV. The cloned genomic fragment (7800 bp) has the potential to encode seven open reading frames (ORFs). Several of these ORFs had significant homology with other proteins found in the databases: ORF1, the hsdM, a methylase subunit (HsdM); ORF2, a homologue of dinD; ORF3, a homologue of hsdS; ORF4, a homologue of hsdS; and ORF5, an endonuclease subunit hsdR. The endonuclease and methylase subunits possessed strongest protein sequence homology to the EcoR124II R/M system, indicating that NgoAV belongs to the type IC R/M family. Deletion analysis showed that only ORF3 imparted the sequence specificity of the RM.NgoAV system, which recognizes an interrupted palindrome sequence (GCAN8TGC). The genetic structure of ORF3 (208 amino acids) is almost identical to the structure of the 5′ truncated hsdS genes of EcoDXXI or EcoR124II R/M systems obtained by in vitro manipulation. Genomic sequence analysis allowed us to identify hsd loci with a very high homology to RM.NgoAV in two strains of Neisseria meningitidis. However, significant differences in the organization and structure of the hsdS genes in both these systems suggests that, if functional, they would possess recognition sites that differ from the gonococcus and from themselves.
SEPH, a Cdc7p orthologue fromAspergillus nidulans, functions upstream of actin ring formation during cytokinesis
Authors: Bruno Kenneth S. , Morrell Jennifer L. , Hamer John E. , Staiger Christopher J.
Published: March 2003

In the filamentous fungus,Aspergillus nidulans, multiple rounds of nuclear division occur before cytokinesis, allowing an unambiguous identification of genes required specifically for cytokinesis. As in animal cells, both an intact microtubule cytoskeleton and progression through mitosis are required for actin ring formation and contraction. ThesepHgene fromA. nidulanswas discovered in a screen for temperature‐sensitive cytokinesis mutants. Sequence analysis showed that SEPH is 42% identical to the serine–threonine kinase Cdc7p from fission yeast. Signalling through theSeptationInitiationNetwork (SIN), which includes Cdc7p and the GTPase Spg1p, is emerging as a primary regulatory pathway used by fission yeast to control cytokinesis. A similar group of proteins comprise theMitoticExitNetwork (MEN) in budding yeast. This is the first direct evidence for the existence of a functional SIN–MEN pathway outside budding and fission yeast. In addition to SEPH, potential homologues were also identified in other fungi and plants but not in animal cells. Deletion ofsepHresulted in a viable strain that failed to septate at any temperature. Interestingly, quantitative analysis of the actin cytoskeleton revealed thatsepHis required for construction of the actin ring. Therefore, SEPH is distinct from its counterpart in fission yeast, in which SIN components operate downstream of actin ring formation and are necessary for ring contraction and later events of septation. We conclude thatA. nidulanshas components of a SIN–MEN pathway, one of which, SEPH, is required for early events during cytokinesis.
Characterization of the expression site of the major surface glycoprotein of human‐derived Pneumocystis carinii
Authors: Kutty Geetha , Ma Liang , Kovacs Joseph A.
Published: March 2003

The major surface glycoprotein (MSG) of Pneumocystis carinii, a pathogen responsible for pulmonary infection in AIDS and other immunocompromised patients, is an abundant surface protein that potentially allows the organism to evade host defences by antigenic variation. MSG is encoded by a multicopy gene family; in two specific forms of rat‐derived P. carinii, regulation of MSG expression uses a single expression site, termed the upstream conserved sequence (UCS), through two related but distinct mechanisms. In the current study, the UCS of the MSG from human‐derived P. carinii was obtained using an RNA ligase‐mediated rapid amplification of cDNA ends technique. Southern blot analysis demonstrated that the UCS was present in a single copy per genome, whereas multiple copies of the downstream MSG gene were present. Sequencing and restriction fragment length polymorphism analysis of polymerase chain reaction products amplified from pulmonary samples of patients with P. carinii pneumonia demonstrated that multiple MSG genes were expressed in a given host, and that different patterns of MSG expression were seen among different patients. Tandem repeats present in the single intron occurred with varying frequency in different patient isolates, potentially providing a new method for typing human isolates. Thus, human‐derived P. carinii regulates MSG expression in a manner similar to P. carinii f. sp. carinii and, in immunosuppressed patients, in whom immune pressures that probably drive antigenic variation are functioning inadequately, P. carinii can express a broad repertoire of MSG variants.
The chromosome partitioning protein, ParB, is required for cytokinesis in Caulobacter crescentus
Authors: Mohl Dane A. , Easter Jesse , Gober James W.
Published: March 2003

In Caulobacter crescentus, the genes encoding the chromosome partitioning proteins, ParA and ParB, are essential. Depletion of ParB resulted in smooth filamentous cells in which DNA replication continued. Immunofluorescence microscopy revealed that the formation of FtsZ rings at the mid‐cell, the earliest molecular event in the initiation of bacterial cell division, was blocked in cells lacking ParB. ParB binds sequences near the C. crescentus origin of replication. Cell cycle experiments show that the formation of bipolarly localized ParB foci, and presumably localization of the origin of replication to the cell poles, preceded the formation of FtsZ rings at the mid‐cell by 20 min. These results suggest that one major function of ParA and ParB may be to regulate the initiation of cytokinesis in C. crescentus.
Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin‐8
Authors: Fischer Wolfgang , Püls Jürgen , Buhrdorf Renate , Gebert Bettina , Odenbreit Stefan , Haas Rainer
Published: March 2003

Helicobacter pylori (Hp) carries a type IV secretion system encoded by the cag pathogenicity island (cag‐PAI), which is used to: (i) translocate the bacterial effector protein CagA into different types of eukaryotic cells; and (ii) induce the synthesis and secretion of chemokines, such as interleukin‐8 (IL‐8). The cag‐PAI in Hp 26695 consists of 27 putative genes, six of which were identified as homologues to the basic type IV secretion system represented by the Agrobacterium tumefaciens virB operon. To define the role and contribution of each of the 27 genes, we applied a precise deletion/insertion mutagenesis procedure to knock out each individual gene without causing polar effects on the expression of downstream genes. Seventeen out of 27 genes were found to be absolutely essential for translocation of CagA into host cells and 14 out of 27 for the ability of Hp fully to induce transcription of IL‐8. The products of hp0524 (virD4 homologue), hp0526 and hp0540 are absolutely essential for the translocation of CagA, but not for the induction of IL‐8. In contrast, the products of hp0520, hp0521, hp0534, hp0535, hp0536 and hp0543 are not necessary for either translocation of CagA or for IL‐8 induction. Our data argue against a translocated IL‐8‐inducing effector protein encoded by the cag‐PAI. We isolated a variant of Hp 26695, which spontaneously switched off its capacity for IL‐8 induction and translocation of CagA, but retained the complete cag‐PAI. We identified a point mutation in gene hp0532, causing a premature translational stop in the corresponding polypeptide chain, providing a putative explanation for the defect in the type IV secretion system of the spontaneous mutant.
Plasmid transfer from Streptomyces to Mycobacterium smegmatis by spontaneous transformation
Authors: Bhatt Apoorva , Melton Rachel E. , Kieser Tobias
Published: March 2003

Hybrids of the Streptomyces coelicolor conjugative plasmid SCP2* and the
Mycobacterium plasmid pAL5000 were transferred from Streptomyces coelicolor
or Streptomyces lividans to Mycobacterium smegmatis mc2155
in plate crosses. Inactivation of the SCP2* transfer function did not prevent or
reduce plasmid transfer. This transfer was DNase I sensitive and thus involved release
of DNA from Streptomyces, followed by transformation of M. smegmatis.
M. smegmatis growing on specific solid media was also transformed by pure
CCC and linear plasmid DNA. Small plasmids were taken up intact but large plasmids
suffered deletions. Competence developed within 24 h of incubation at 30°C or
37°C, and up to 400 transformants were obtained per μg of CCC plasmid
DNA. Transformation frequencies were higher when M. smegmatis was co‐cultivated
with plasmid‐free Streptomyces, but unaffected by resident homologous sequences
or inactivation of recA in M. smegmatis. Spontaneous transformation
was also observed with a circular Streptomyces transposable element which
inserted into chromosomal sites. Transformative plasmid transfer was also shown to
occur between M. smegmatis strains. This is the first report of non‐artificially
induced, spontaneous plasmid transformation in Mycobacterium.
RNase E levels in Escherichia coli are controlled by a complex regulatory system that involves transcription of the rne gene from three promoters
Authors: Ow Maria C. , Liu Qi , Mohanty Bijoy K. , Andrew Margaret E. , Maples Valerie F. , Kushner Sidney R.
Published: March 2003

The rne gene of Escherichia coli encodes RNase E, an essential endoribonuclease
that is involved in both mRNA decay and rRNA processing. Here we present evidence
that the gene is transcribed from three promoters: p1, p2 and p3. The p2 and p3 promoters
map 34 and 145 nt upstream from the previously characterized rne promoter,
p1, generating unusually long 5′ UTRs of 395 and 506 nt respectively.
Based on promoter–lacZ transcriptional fusions, p1 is a more efficient
promoter than either p2 or p3. Low copy number or single copy number vectors carrying
rne transcribed from either p1, p2 or p3 alone complement the rneΔ1018::bla
deletion mutation at 30°C, 37°C and 44°C. However,
normal autoregulation requires the presence of all three promoters. A comparison
among intracellular levels of RNase E, the half‐lives of the rpsO, rpsT
and rne mRNAs, and growth rates, indicates that the cell contains a considerable
excess of RNase E protein. In addition, when the rne transcript is stabilized at low RNase E levels, it is not efficiently translated.
Interactions of haemoglobin with the Neisseria meningitidis receptor HpuAB: the role of TonB and an intact proton motive force
Authors: Rohde K. H. , Gillaspy A. F. , Hatfield M. D. , Lewis L. A. , Dyer D. W.
Published: March 2003

We have characterized the interaction of the Neis‐seria meningitidis TonB‐dependent receptor HpuAB with haemoglobin (Hb). Protease accessibility assays indicated that HpuA and HpuB are surface exposed, HpuB interacts physically with HpuA, and TonB energization affects the conformation of HpuAB. Binding assays using [125I]‐Hb revealed that the bipartite receptor has a single binding site for Hb (Kd≈ 150 nM). Competitive binding assays using heterologous Hbs revealed that HpuAB Hb recognition was not species specific. The binding kinetics of Hb to HpuAB were dramatically altered in a TonB– mutant and in wild‐type meningococci treated with the protonophore carbonylcyanide m‐chlorophenylhydrazone (CCCP), indicating that TonB and an intact proton motive force are required for normal Hb binding and release from HpuAB. Our results support a model in which both HpuA and HpuB are required to form a receptor complex in the outer membrane with a single binding site, whose structure and ligand interactions are significantly affected by the TonB‐mediated energy state of the receptor.
Identification and characterization of phage‐resistance genes in temperate lactococcal bacteriophages
Authors: McGrath Stephen , Fitzgerald Gerald F. , Sinderen Douwe van
Published: March 2003

The sie2009 gene, which is situated between the genes encoding the repressor and integrase, on the lysogeny module of the temperate lactococcal bacteriophage Tuc2009, was shown to mediate a phage‐resistance phenotype in Lactococcus lactis against a number of bacteriophages. The Sie2009 protein is associated with the cell membrane and its expression leaves phage adsorption, transfection and plasmid transformation unaffected, but interferes with plasmid transduction, as well as phage replication. These observations indicate that this resistance is as a result of DNA injection blocking, thus representing a novel superinfection exclusion system. A polymerase chain reaction (PCR)‐based strategy was used to screen a number of lactococcal strains for the presence of other prophage‐encoded phage‐resistance systems. This screening resulted in the identification of two such systems, without homology to sie2009, which were shown to mediate a phage‐resistance phenotype similar to that conferred by sie2009. To our knowledge, this is the first description of a phage‐encoded superinfection exclusion/injection blocking mechanism in the genus Lactococcus.
Molecular analysis of KTI12/TOT4, a Saccharomyces cerevisiae gene required for Kluyveromyces lactis zymocin action
Authors: Fichtner Lars , Frohloff Frank , Bürkner Konstanze , Larsen Michael , Breunig Karin D. , Schaffrath Raffael
Published: March 2003

TOT, the putative Kluyveromyces lactis zymocin target complex from Saccharomyces cerevisiae, is encoded by TOT1–7, six loci of which are isoallelic to RNA polymerase II (RNAPII) Elongator genes (ELP1–6). Unlike TOT1–3 (ELP1–3) and TOT5–7 (ELP5, ELP6 and ELP4 respectively), which display zymocin resistance when deleted, TOT4 (KTI12) also renders cells refractory to zymocin when maintained in multicopy or overexpressed from the GAL10 promoter. Elevated TOT4 copy number results in an intermediate tot phenotype, which includes mild sensitivities towards caffeine, Calcofluor white and elevated growth temperature, suggesting that TOT4 influences TOT/Elongator function. Tot4p interacts with Elongator, as shown by co‐immunoprecipitation, and cell fractionation studies demonstrate partial co‐migration with RNAPII and Elongator. As Elongator subunit interaction is not affected by either deletion of TOT4 or multicopy TOT4, Tot4p may not be a structural Elongator subunit but, rather, may regulate TOT/Elongator in a fashion that requires transient physical contact with TOT/Elongator. Consistent with a regulatory role, the presence of a potential P‐loop motif conserved between yeast and human TOT4 homologues suggests capability of ATP or GTP binding and P‐loop deletion renders Tot4p biologically inactive.
Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose
Authors: Solano Cristina , García Begoña , Valle Jaione , Berasain Carmen , Ghigo Jean‐Marc , Gamazo Carlos , Lasa Iñigo
Published: March 2003

We report here a new screening method based on the fluorescence of colonies on calcofluor agar plates to identify transposon insertion mutants of Salmonella enteritidis that are defective in biofilm development. The results not only confirmed the requirement of genes already described for the modulation of multicellular behaviour in Salmonella typhimurium and other species, but also revealed new aspects of the biofilm formation process, such as two new genetic elements, named as bcsABZC and bcsEFG operons, required for the synthesis of an exopolysaccharide, digestible with cellulase. Non‐polar mutations of bcsC and bcsE genes and complementation experiments demonstrated that both operons are respon‐sible for cellulose biosynthesis in both S. enteritidis and S. typhimurium. Using two different growth media, ATM and LB, we showed that the biofilm produced by S. enteritidis is made of different constituents, suggesting that biofilm composition and regulation depends on environmental conditions. Bacterial adherence and invasion assays of eukaryotic cells and in vivo virulence studies of cellulose‐deficient mutants indicated that, at least under our experimental conditions, the production of cellulose is not involved in the virulence of S. enteritidis. However, cellulose‐deficient mutants were more sensitive to chlorine treatments, suggesting that cellulose production and biofilm formation may be an important factor for the survival of S. enteritidis on surface environments.
An intrinsic control element for translational initiation in class 1 integrons
Authors: Hanau‐Berçot Béatrice , Podglajen Isabelle , Casin Isabelle , Collatz Ekkehard
Published: March 2003

Integrons are genetic elements able to capture anti‐biotic resistance and other genes and to promote their transcription. Here, we have investigated integron‐dependent translation of an aminoglycoside 6′‐N‐acetyltransferase gene (aac(6′)‐Ib7)
inserted at the attI1 site. N‐terminal sequencing revealed that translation
of this gene was initiated at a GTG codon, which is not part of a plausible translation
initiation region (TIR). A short open reading frame (called ORF‐11) overlapping the
attI1 site was probed by site‐directed mutagenesis for its contribution to
aac(6′)‐Ib7 translation. When ORF‐11 and its
TIR were deleted en bloc, translational efficiency dropped by over 80%,
as determined with an acetyltransferase– luciferase fusion product. Invalidation
of the ATG start codon of ORF‐11 or its putative Shine–Dalgarno sequence resulted
in a decrease of over 60%, whereas the decrease was much less pronounced
when the amino acid sequence of the putative ORF‐11‐encoded peptide was altered or
when the distance between ORF‐11 and aac(6′)‐Ib7
was doubled. This demonstrates that aac(6′)‐Ib7
translation is dependent upon the translation of ORF‐11, but almost certainly not
upon the corresponding peptide. These results lead us to conclude that an intrinsic
short ORF present in the 5′‐conserved segment of many class 1 integrons may substantially enhance expression at the translational level of captured TIR‐deficient anti‐biotic resistance genes.
Methylation at nucleotide G745 or G748 in 23S rRNA distinguishes Gram‐negative from Gram‐positive bacteria
Authors: Liu Mingfu , Douthwaite Stephen
Published: March 2003

Bacteria tune the function of their ribosomes by methylating specific rRNA nucleotides. Nucleotide G745 in Escherichia coli 23S rRNA is methylated by the methyltransferase
enzyme RrmA, whereas in Streptomyces fradiae, the neighbouring nucleotide
G748 is methylated by the enzyme TlrB. Both nucleotides line the peptide exit channel
of the ribosome at the binding site of macrolide, lincosamide and streptogramin B
antibiotics. Resistance to the macrolide tylosin, which is produced by S. fradiae,
is conferred by methylation of G748. RrmA and TlrB are homologues (29% identical),
and a database search against all presently available sequences revealed a further
two dozen homologues from a wide variety of Bacteria. No homologues were found among
the Archaea or Eukarya. The bacterial sequences adhere to the species phylogeny and
segregate into two groups, in which the Gramnegative sequences align with RrmA and
the Gram‐positives with TlrB. Consistently, in more than 20 species tested, the distribution
of methylation in the Gram‐negative rRNAs (methylated at G745) and the Gram‐positives
(methylated at G748) perfectly matches the bacterial phylogeny. Cloning and ex‐pression
of representative methyltransferase genes showed that this specificity of methylation
is determined solely by the methyltransferase enzyme and is independent of the origin
of the rRNA substrate. This is the first case in which the position of an RNA methylation
defines a sharp division between the Gram‐negative and Gram‐positive bacteria. Given
the specificities and distribution of these methyltransferases, we propose a change
in the nomenclature of RrmA to RlmAI (rRNA large subunit
methyltransferase) and of TlrB to RlmAII.
Candida albicans Als1p: an adhesin that is a downstream effector of the EFG1 filamentation pathway
Authors: Fu Yue , Ibrahim Ashraf S. , Sheppard Donald C. , Chen Yee‐Chun , French Samuel W. , Cutler Jim E. , Filler Scott G. , Edwards John E.
Published: March 2003

Filamentation and adherence to host cells are critical virulence factors of Candida

albicans. Multiple filamentation regulatory pathways have been discovered in

C. albicans using Saccharomyces cerevisiae as a model. In S. cerevisiae,

these pathways converge on Flo11p, which functions as a downstream effector of filamentation

and also mediates cell–cell adherence (flocculation). In C. albicans,

such effector(s) have not yet been identified. Here, we demonstrate that the cell

surface protein Als1p is an effector of filamentation in C. albicans. We show

that Als1p expression is controlled by the transcription factor Efg1p, which is known

to be a key regulator of filamentation in C. albicans. Further, disruption

of ALS1 inhibited filamentation, and autonomous expression of Als1p restored

filamentation in an efg1 homozygous null mutant. Thus, Als1p functions as

a downstream effector of the EFG1 filamentation pathway. In addition, we found

that Als1p mediates both flocculation and adherence of C. albicans to endothelial

cells in vitro. As a cell surface glycoprotein that mediates filamentation

and adherence, Als1p has both structural and functional similarity to S. cerevisiae

Flo11p. Consistent with our in vitro results, Als1p was required for both normal filamentation and virulence in the mouse model of haematogenously disseminated candidiasis.
RusA proteins from the extreme thermophile Aquifex aeolicus and lactococcal phage r1t resolve Holliday junctions
Authors: Sharples Gary J. , Bolt Edward L. , Lloyd Robert G.
Published: March 2003

The RusA protein of Escherichia coli is a DNA structure‐specific endonuclease that resolves Holliday junction intermediates formed during DNA replication, recombination and repair by introducing symmetrically paired incisions 5′ to CC dinucleotides. It is encoded by the defective prophage DLP12, which raises the possibility that it may be of bacteriophage origin. We show that rusA‐like sequences are indeed often associated with prophage sequences in the genomes of several bacterial species. They are also found in many bacteriophages, including Lactococcus lactis phage r1t. However, rusA is also present in the chromosome of the hyperthermophilic bacterium Aquifex aeolicus. In this case, there is no obvious association of rusA with prophage‐like sequences. Given the ancient lineage of Aquifex aeolicus, this observation provides the first indication that RusA may be of bacterial origin. The RusA proteins of A. aeolicus and bacteriophage r1t were purified and shown to resolve Holliday junctions. The r1t enzyme also promotes DNA repair in strains lacking the RuvABC resolvase. Both enzymes cleave junctions in a sequence‐dependent manner, but the A. aeolicus RusA shows a different sequence preference (3′ to TG) from the E. coli protein (5′ to CC), and the r1t RusA has relaxed sequence dependence, requiring only a single cytosine.
Genetic dissection of the Ralstonia solanacearum hrp gene cluster reveals that the HrpV and HrpX proteins are required for Hrp pilus assembly
Authors: Gijsegem Frédérique Van , Vasse Jacques , Rycke Riet De , Castello Philippe , Boucher Christian
Published: March 2003

In both plant and mammalian Gram‐negative pathogenic bacteria, type III secretion systems (TTSSs) play a crucial role in interactions with the host. All these systems share conserved proteins (called Hrc in plant pathogens), but each bacterium also produces a variable number of additional type III proteins either unique or with counterparts only in a limited number of related systems. In order to investigate the role of the different proteins encoded by the hrp gene cluster of the
phytopathogenic bacterium Ralstonia solanacearum, non‐polar mutants in all
hrp genes (except for hrcQ) were analysed for their interactions with
plants, their ability to secrete the PopA protein and their production of the Hrp
pilus. In addition to Hrc proteins and the HrpY major component of the Hrp pilus,
four additional Hrp proteins are indispensable for type III secretion and for interactions
with plants. We also provide evidence that hrpV and hrpX mutants can still target the HrpY pilin outside the bacterial cell but are impaired in the production of Hrp pili, indicating that HrpV and HrpX proteins are involved in the assembly of this appendage.
Host versus in vitro signals and intrastrain allelic differences in the expression of a Candida albicans virulence gene
Authors: Staib Peter , Kretschmar Marianne , Nichterlein Thomas , Hof Herbert , Morschhäuser Joachim
Published: March 2003

The yeast Candida albicans is a harmless colonizer of mucosal surfaces in healthy people but can become a serious pathogen in immunocompromised patients, causing superficial as well as systemic infections. The evolution of gene families encoding pathogenicity‐related functions, like adhesins and secreted aspartic proteinases (Saps), which are differentially induced by host signals at various stages of colonization and infection, may have allowed C. albicans an optimal adaptation to many different host niches. We found that even the two alleles of a single gene can be differentially regulated in the diploid C. albicans. In the model strain SC5314, the in vitro expression of one of the two SAP2 alleles, SAP2‐1, depended on the presence of a functional SAP2‐2 allele. In contrast, inactivation of SAP2‐1 did not in‐fluence the expression of SAP2‐2. The proteinase encoded by the SAP2‐2 allele serves as a signal sensor and amplifier to enhance its own expression as well as to induce the SAP2‐1 allele to achieve maximal proteolytic activity under appropriate conditions. Using in vivo expression technology, we could demonstrate that the SAP2‐1 allele is significantly activated only in the late stages of systemic candidiasis in mice, whereas the SAP2‐2 allele is induced much earlier. The differential regulation of the two SAP2 alleles was due to differences in their pro‐moters, which contained a variable number of two pentameric nucleotide repeats. Mutations that re‐duced or increased the copy number of these repeats diminished the inducibility of the SAP2 promoter during infection but not in vitro, suggesting that the mutations affected interactions of regulatory factors that are necessary for SAP2 activation in vivo but dispensable for its induction in vitro. Therefore, the signals and signal transduction pathways that mediate SAP2 expression within certain host niches may differ from those that activate the gene in vitro. In addition to the generation of gene families whose members exhibit functional and regulatory diversification, C. albicans seems to use its diploid genome to create further variability and host adaptation by differential evolution of even the two alleles of a single gene.
The role of co‐transcriptional translation and protein translocation (transertion) in bacterial chromosome segregation
Authors: Woldringh Conrad L.
Published: March 2003

Many recent reviews in the field of bacterial chromosome segregation propose that newly replicated DNA is actively separated by the functioning of specific proteins. This view is primarily based on an interpretation of the position of fluorescently labelled DNA regions and proteins in analogy to the active segregation mechanism in eukaryotic cells, i.e. to mitosis. So far, physical aspects of DNA organization such as the diffusional movement of DNA supercoil segments and their interaction with soluble proteins, leading to a phase separation between cytoplasm and nucleoid, have received relatively little attention. Here, a quite different view is described taking into account DNA–protein interactions, the large variation in the cellular position of fluorescent foci and the compaction and fusion of segregated nucleoids upon inhibition of RNA or protein synthesis. It is proposed that the random diffusion of DNA supercoil segments is transiently constrained by the process of co‐ transcriptional translation and translocation (transertion) of membrane proteins. After initiation of DNA replication, a bias in the positioning of transertion areas creates a bidirectionality in chromosome segre‐gation that becomes self‐enhanced when neigh‐bouring genes on the same daughter chromosome are expressed. This transertion‐mediated segregation model is applicable to multifork replication during rapid growth and to multiple chromosomes and plasmids that occur in many bacteria.
Diethylmaleate activates the transcription factor Pap1 by covalent modification of critical cysteine residues
Authors: Castillo Esther A. , Ayté José , Chiva Cristina , Moldón Alberto , Carrascal Montse , Abián Joaquín , Jones Nic , Hidalgo Elena
Published: March 2003

During the last decade, much has been learnt about the mechanisms by which oxidative stress is perceived by aerobic organisms. The Schizosaccharomyces pombe Pap1 protein is a transcription factor localized at the cytoplasm, which accumulates in the nucleus in response to different inducers, such as the pro‐oxidant hydrogen peroxide (H2O2) or the glutathione‐depleting agent diethylmaleate (DEM). As described for other H2O2 sensors, our genetic data indicates that H2O2 reversibly oxidizes two cysteine residues in Pap1 (Cys278 and Cys501). Surprisingly, our studies demonstrate that DEM generates a non‐reversible modification of at least two cysteine residues located in or close to the nuclear export signal of Pap1 (Cys523 and Cys532). This modification impedes the interaction of the nuclear exporter Crm1 with the nuclear export signal located at the carboxy‐terminal domain of Pap1. Mass spectrometry data suggest that DEM binds to the thiol groups of the target cysteine residues through the formation of a thioether. Here we show that DEM triggers Pap1 nuclear accumulation by a novel molecular mechanism.
The plastid DNA of the malaria parasite Plasmodium falciparum is replicated by two mechanisms
Authors: Williamson D. H. , Preiser P. R. , Moore P. W. , McCready S. , Strath M. , Wilson R. J. M. (Iain)
Published: March 2003

In common with other apicomplexan parasites, Plasmodium falciparum, a causative organism of human malaria, harbours a residual plastid derived from an ancient secondary endosymbiotic acquisition of an alga. The function of the 35 kb plastid genome is unknown, but its evolutionary origin and genetic content make it a likely target for chemotherapy. Pulsed field gel electrophoresis and ionizing radiation have shown that essentially all the plastid DNA comprises covalently closed circular monomers, together with a tiny minority of linear 35 kb molecules. Using two‐dimensional gels and electron microscopy, two replication mechanisms have been revealed. One, sensitive to the topoisomerase inhibitor ciprofloxacin, appears to initiate at twin D‐loops located in a large inverted repeat carrying duplicated rRNA and tRNA genes, whereas the second, less drug sensitive, probably involves rolling circles that initiate outside the inverted repeat.
The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition
Authors: Huston Wilhelmina M. , Jennings Michael P. , McEwan Alastair G.
Published: March 2003

Recently it has been observed that multicopper oxidases are present in a number of microbial genomes, raising the question of their function in prokaryotes. Here we describe the analysis of an mco mutant from the opportunistic pathogen Pseudomonas aeruginosa. Unlike wild‐type Pseudomonas aeruginosa, the mco mutant was unable to grow aerobically on minimal media with Fe(II) as sole iron source. In contrast, both the wild‐type and mutant strain were able to grow either anaerobically via denitrification with Fe(II) or aerobically with Fe(III). Analysis of iron uptake showed that the mco mutant was impaired in Fe(II) uptake but unaffected in Fe(III) uptake. Purification and analysis of the MCO protein confirmed ferroxidase activity. Taken together, these data show that the mco gene encodes a multicopper oxidase that is involved in the oxidation of Fe(II) to Fe(III) subsequent to its acquisition by the cell. In view of the widespread distribution of the mco gene in bacteria, it is suggested that an iron acquisition mechanism involving multicopper oxidases may be an important and hitherto unrecognized feature of bacterial pathogenicity.
MotX and MotY, specific components of the sodium‐driven flagellar motor, colocalize to the outer membrane in Vibrio alginolyticus
Authors: Okabe Mayuko , Yakushi Toshiharu , Kojima Masaru , Homma Michio
Published: March 2003

Rotation of the sodium‐driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY. MotX and MotY, which are unique components of the sodium‐driven motor of Vibrio, have been believed to be localized in the inner (cytoplasmic) membrane via their N‐terminal hydrophobic segments. Here we show that MotX and MotY colocalize to the outer membrane. Both proteins, when expressed together, were detected in the outer membrane fraction separated by sucrose density gradient centrifugation. As mature MotX and MotY proteins do not have N‐terminal hydrophobic segments, the N‐termini of the primary translation products must have signal sequences that are removed upon translocation across the inner membrane. Moreover, MotX and MotY require each other for efficient localization to the outer membrane. Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane. This is the first case that describes motor proteins function in the outer membrane for flagellar rotation.
A novel class of microbial phosphocholine‐specific phospholipases C
Authors: Stonehouse Martin J. , Cota‐Gomez Adela , Parker Sarah K. , Martin Wesley E. , Hankin Joseph A. , Murphy Robert C. , Chen Weibin , Lim Kheng B. , Hackett Murray , Vasil Adriana I. , Vasil Michael L.
Published: March 2003

In this report we describe the 1500‐fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17 117.2 Da (PlcR2) and 78 417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline‐containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the con‐clusion that the members of this PC‐PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.
The Salmonella enterica sv. Typhimurium smvA, yddG and ompD (porin) genes are required for the efficient efflux of methyl viologen
Authors: Santiviago Carlos A. , Fuentes Juan A. , Bueno Susan M. , Trombert A. Nicole , Hildago Alejandro A. , Socias L. Teresa , Youderian Philip , Mora Guido C.
Published: March 2003

In Gram‐negative bacteria, a subset of inner membrane proteins in the major facilitator superfamily (MFS) acts as efflux pumps to decrease the intracellular concentrations of multiple toxic substrates and confers multidrug resistance. The Salmonella enterica sv. Typhimurium smvA gene encodes a product predicted to be an MFS protein most similar to QacA of Staphylococcus aureus. Like mutations in qacA, mutations in smvA confer increased sensitivity to methyl viologen (MV). Mutations in the adjacent ompD (porin) and yddG (drug/metabolite transporter) genes also confer increased sensitivity to MV, and mutations in smvA are epistatic to mutations in ompD or yddG for this phenotype. YddG and OmpD probably comprise a second efflux pump in which the OmpD porin acts as an outer membrane channel (OMC) protein for the efflux of MV and functions independently of the SmvA pump. In support of this idea, the pump dependent on YddG and OmpD has a different substrate specificity from the pump dependent on SmvA. Mutations in tolC, which encodes an OMC protein, confer increased resistance to MV. TolC apparently facilitates the import of MV, and a subset of OMC proteins including the OmpD porin and TolC may facilitate both import and export of distinct subsets of toxic substrates.
Retrotransposition of the Ll.LtrB group II intron proceeds predominantly via reverse splicing into DNA targets
Authors: Ichiyanagi Kenji , Beauregard Arthur , Lawrence Stacey , Smith Dorie , Cousineau Benoit , Belfort Marlene
Published: March 2003

Catalytic group II introns are mobile retroelements that invade cognate intronless genes via retrohoming, where the introns reverse splice into double‐stranded DNA (dsDNA) targets. They can also retrotranspose to ectopic sites at low frequencies. Whereas our previous studies with a bacterial intron, Ll.LtrB, supported frequent use of RNA targets during retrotransposition, recent experiments with a retrotransposition indicator gene indicate that DNA, rather than RNA, is a prominent target, with both dsDNA and single‐stranded DNA (ssDNA) as possibilities. Thus retrotransposition occurs in both transcriptional sense and antisense orientations of target genes, and is largely independent of homologous DNA recombination and of the endonuclease function of the intron‐encoded protein, LtrA. Models based on both dsDNA and ssDNA targeting are presented. Interestingly, retrotransposition is biased toward the template for lagging‐strand DNA synthesis, which suggests the possibility of the replication folk as a source of ssDNA. Consistent with some use of ssDNA targets, many retrotransposition sites lack nucleotides critical for the unwinding of target duplex DNA. Moreover, in vitro the intron reverse spliced into ssDNA more efficiently than dsDNA substrates for some of the retrotransposition sites. Furthermore, many bacterial group II introns reside on the lagging‐strand template, hinting at a role for DNA replication in intron dispersal in nature.
Intracellular localization modulates targeting of ExoS, a type III cytotoxin, to eukaryotic signalling proteins
Authors: Pederson Kristin J. , Krall Rebecca , Riese Matthew J. , Barbieri Joseph T.
Published: March 2003

ExoS is a bifunctional type III cytotoxin produced by Pseudomonas aeruginosa. Residues 96–232 comprise the Rho GTPase activating protein (Rho GAP) domain, whereas residues 233–453 comprise the 14‐3‐3‐dependent ADP‐ribosyltransferase domain. Earlier studies showed that the N‐terminus targeted ExoS to intracellular membranes within eukaryotic cells. This N‐terminal targeting region is now characterized for cellular and biological contributions to intoxications by ExoS. An ExoS(1–107)–green fluorescent protein (GFP) fusion protein co‐localized with α‐mannosidase, which indicated that the fusion protein localized near the Golgi. Residues 51–72 of ExoS (termed the membrane localization domain, MLD) were necessary and sufficient for membrane localization within eukaryotic cells. Deletion of the MLD did not inhibit type III secretion of ExoS from P. aeruginosa or type III delivery of ExoS into eukaryotic cells. Type III‐delivered ExoS(ΔMLD) localized within the cytosol of eukaryotic cells, whereas type III‐delivered ExoS was membrane associated. Although type III‐delivered ExoS(ΔMLD) stimulated the reorganization of the actin cytoskeleton (a Rho GAP activity), it did not ADP‐ribosylate Ras. Type III‐delivered ExoS(ΔMLD) and ExoS showed similar capacities for eliciting a cytotoxic response in CHO cells, which uncoupled the ADP‐ribosylation of Ras from the cytotoxicity elicited by ExoS.
A role for division‐site‐selection protein MinD in regulation of internucleoid jumping of Soj (ParA) protein in Bacillus subtilis
Authors: Autret Sabine , Errington Jeffery
Published: March 2003

The Bacillus subtilis soj‐spo0J locus encodes two proteins belonging to a family of proteins (the ParAB proteins) with dual roles in plasmid or chromosome segregation and transcriptional regulation. Soj protein was previously shown to be capable of abrupt subcellular relocation. The movement was highly co‐operative and at any moment, most of the Soj in any cell formed a single large ‘patch’ covering all or part of one nucleoid. Movement, and co‐operativity, in the sense of formation of a single patch, was dependent on Spo0J. Movement, but not co‐operativity, was also shown to be dependent, directly or indirectly, on FtsZ protein. We now report that the ftsZ effect arises because jumping onto a nucleoid is promoted by proximity to a cell pole. Studies of other mutants affected in cell division suggest that the attraction to the cell pole is mediated by the division‐site‐selection protein, MinD (which localizes at the cell poles). It does not require MinC, the main effector of the division site selection system. A mutant form of Soj, putatively locked in the ATP form of the protein, interacts with the cell pole (dependent on MinD) but not with the nucleoid. These results identify a novel function for MinD and demonstrate an intriguing link between proteins involved in the cell division and chromosome segregation machineries.