bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA replication‐coupled inactivation of DnaA protein in vitro: a role for DnaA arginine‐334 of the AAA+ Box VIII motif in ATP hydrolysis
Authors: Su'Etsugu Masayuki , Kawakami Hironori , Kurokawa Kenji , Kubota Toshio , Takata Makoto , Katayama Tsutomu
Published: March 2003

The DnaA protein, which initiates chromosomal replication in Escherichia coli, is negatively regulated by both the sliding clamp of DNA polymerase III holoenzyme and the IdaB protein. We have found that, when the amount of minichromosome is limited in an in vitro replication system, minichromosomal replication‐stimulated hydrolysis of DnaA‐bound ATP yields the ADP‐bound inactive form. The number of sliding clamps formed during replication was at least five per minichromosome, which is 2.7‐fold higher than the number formed during incubation without replication. These results support the notion that coupling of DnaA‐ATP hydrolysis to DNA replication is the outcome of enhanced clamp formation. We have also found that the amino acid substitution R334H in DnaA severely inhibits the hydrolysis of bound ATP in vitro. Whereas ATP bound to wild‐type DnaA is hydrolysed in a DNA‐dependent intrinsic manner or in a sliding clamp‐dependent manner, ATP bound to DnaA R334H protein was resistant to hydrolysis under the same conditions. This arginine residue may be located in the vicinity where ATP binds, and therefore may play an essential role in ATP hydrolysis. This residue is highly conserved among DnaA homologues and also in the Box VIII motif of the AAA+ protein family.
A homologue of the transcriptional repressor Ssn6p antagonizes cAMP signalling in Ustilago maydis
Authors: Loubradou Gabriel , Brachmann Andreas , Feldbrügge Michael , Kahmann Regine
Published: March 2003

In Ustilago maydis, cAMP signalling is crucial for successful infection of maize plants. Strains are non‐pathogenic if mutated in any of the currently identified components of this signalling pathway, such as the α‐subunit of a heterotrimeric G protein Gpa3, the adenylate cyclase Uac1 and the regulatory and catalytic subunit of protein kinase A Ubc1 and Adr1 respectively. Deletion of gpa3, uac1 or adr1 triggers filamentous growth, and certain point mutations in gpa3 and ubc1 that mimic a high cAMP level display a glossy colony phenotype. Screening an autonomously replicating U. maydis library in such a background (gpa3Q206L), we identified sql1 as a suppressor of the glossy colony phenotype. Interestingly, only alleles carrying C‐terminal truncations of Sql1 were able to complement the mutant phenotype, suggesting a gain‐of‐function by these variants. Sql1 is a functional homologue of the yeast transcriptional repressor Ssn6p and contains 10 tetratricopeptide repeats (TPRs), of which the first six are important for suppressor function. Truncated sql1 alleles that suppress the glossy colony phenotype of gpa3Q206L strains induce filamentous growth when introduced in wild type. Filamentation of these strains is reversed in the presence of cAMP. We present a model in which Sql1 is part of an evolutionary conserved Sql1–Tup1 transcriptional repressor complex that antagonizes cAMP signalling by repressing cAMP‐regulated genes.
The CbrA–CbrB two‐component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa
Authors: Nishijyo Takayuki , Haas Dieter , Itoh Yoshifumi
Published: March 2003

A novel two‐component system, CbrA–CbrB, was discovered in Pseudomonas aeruginosa;cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7 kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108 379, 983 residues) and a cognate response regulator (Mr 52 254, 478 residues) respectively. The amino‐terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy‐terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot–argR operon was not induced by arginine, indicating an essential role for this two‐component system in the expression of the ArgR‐dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild‐type strain. When succinate or other C4‐dicarboxylates were added to proline medium at 1 mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate‐dependent Pru+ property was almost abolished by 20 mM ammonia. In conclusion, the CbrA–CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB–NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.
The role of HetN in maintenance of the heterocyst pattern in Anabaena sp. PCC 7120
Authors: Callahan Sean M. , Buikema William J.
Published: March 2003

The gene hetN encodes a putative oxidoreductase that is known to suppress heterocyst differentiation when present on a multicopy plasmid in Anabaena sp. PCC 7120. To mimic the hetN null phenotype and to examine where HetN acts in the regulatory cascade that controls heterocyst differentiation, we replaced the native chromosomal hetN promoter with the copper‐inducible petE promoter. In the presence of copper, heterocyst formation was suppressed in undifferentiated filaments. When hetN expression was turned off by transferring cells to media lacking copper, the filaments initially displayed the wild‐type pattern of single heterocysts but, 48 h after the induction of heterocyst formation, a pattern of multiple contiguous heterocysts predominated. Suppression of heterocyst formation by HetN appears to occur both upstream and downstream of the positive regulator HetR: overexpression of hetN in undifferentiated filaments prevents the wild‐type pattern of hetR expression as well as the multiheterocyst phenotype normally observed when hetR is expressed from an inducible promoter. Green fluorescent protein fusions show that the expression of hetN in wild‐type filaments normally occurs primarily in heterocysts. We propose that HetN is normally involved in the maintenance of heterocyst spacing after the initial heterocyst pattern has been established, but ectopic expression of hetN can also block the initial establishment of the pattern.
Pseudomonas aeruginosa GacA, a factor in multihost virulence, is also essential for biofilm formation
Authors: Parkins Michael D. , Ceri Howard , Storey Douglas G.
Published: March 2003

We have investigated a potential role for GacA, the response regulator of the GacA/GacS two‐component regulatory system, in Pseudomonas aeruginosa biofilm formation. When gacA was disrupted in strain PA14, a 10‐fold reduction in biofilm formation capacity resulted relative to wild‐type PA14. However, no significant difference was observed in the planktonic growth rate of PA14 gacA−. Providing gacA in trans on the multicopy vector pUCP‐gacA abrogated the biofilm formation defect. Scanning electron microscopy of biofilms formed by PA14 gacA− revealed diffuse clusters of cells that failed to aggregate into microcolonies, implying a deficit in biofilm development or surface translocation. Motility assays revealed no decrease in PA14 gacA− twitching or swimming abilities, indicating that the defect in biofilm formation is independent of flagellar‐mediated attachment and solid surface translocation by pili. Autoinducer and alginate bioassays were performed similarly, and no difference in production levels was observed, indicating that this is not merely an upstream effect on either quorum sensing or alginate production. Antibiotic susceptibility profiling demonstrated that PA14 gacA− biofilms have moderately decreased resistance to a range of antibiotics relative to PA14 wild type. This study establishes GacA as a new and independent regulatory element in P. aeruginosa biofilm formation.
Mediator – a universal complex in transcriptional regulation
Authors: Gustafsson Claes M. , Samuelsson Tore
Published: March 2003

The Mediator complex is essential for basal and regulated expression of nearly all RNA polymerase II‐dependent genes in the Saccharomyces cerevisiae genome. Mediator acts as a bridge, conveying regulatory information from enhancers and other control elements to the promoter. It is now clear that Mediator‐like complexes also exist in higher eukaryotic cells and that they have an important role in metazoan transcriptional regulation. However, the exact mechanism of Mediator‐dependent transcriptional regulation remains unclear. We review here some recent advances in our understanding of Mediator structure and function. We also discuss a model to account for the functional and evolutionary relationship between yeast and metazoan Mediators. As an appendix to this review, we have created a database, MEDB, in which we have compiled information about all the S. cerevisiae Mediator subunits and their homologues in other eukaryotic cells (http://bio.lundberg.gu.se/medb/).
The promoter of the operon encoding the F0F1 ATPase of Streptococcus pneumoniae is inducible by pH
Authors: Martín‐Galiano Antonio Javier , Ferrándiz María José , De La Campa Adela G.
Published: March 2003

The genes encoding the subunits of the FoF1 membrane ATPase of Streptococcus pneumoniae were cloned and sequenced. The eight genes, transcribed to one mRNA, are organized in an operon encoding the c, a, b, delta, alpha, gamma, beta and epsilon subunits of 66, 238, 165, 178, 501, 292, 471 and 139 amino acid residues, respectively, that were expressed in an Escherichia coli system. To investigate the role of the ATPase in the regulation of the intracellular pH, the expression of the operon between pH 5.7 and 7.5 was studied. An increase in both the ATPase activity and the amount of the alpha and beta F1 subunits as shown by Western blot analysis was observed as the pH decreased. These increases were accompanied by an increase in the atp‐specific mRNA, as shown by Northern blot and slot‐blot analysis. Primer extension experiments and transcriptional fusions between the atp promoter and the reporter cat gene demonstrated that this pH‐dependent increase in the mRNA was regulated at the level of initiation of transcription. Transcription of the operon occurs from a promoter with a consensus −35 box (TTGACA) and a −10 box (TACACT) that differs from the consensus (TATAAT). A point mutation at the −10 box of the promoter (change to TGCACT) avoided this increase, suggesting a role for this sequence in the pH‐inducible regulation.
Antisense RNA regulation of the par post‐segregational killing system: structural analysis and mechanism of binding of the antisense RNA, RNAII and its target, RNAI
Authors: Greenfield Tony J. , Franch Thomas , Gerdes Kenn , Weaver Keith E.
Published: March 2003

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA regulated post‐segregational killing system (PSK) identified in a Gram‐positive organism. Par encodes two small, convergently transcribed RNAs, designated RNAI and RNAII, which are the toxin and antitoxin of the par PSK system respectively. RNAI encodes an open reading frame for a 33 amino acid toxin called Fst. Expression of fst is regulated post‐transcriptionally by RNAII. RNAII interacts with RNAI by a unique antisense RNA mechanism involving binding at the 5′ and 3′ ends of both RNAs. Par RNA interaction requires a complementary transcriptional terminator stem‐loop and a set of direct repeat sequences, DRa and DRb, located at the 5′ end of both RNAs. The secondary structures of RNAI, RNAII and the RNAI–RNAII complex were analysed by partial digestion with Pb(II) and ribonucleases. Probing data for RNAI and RNAII are consistent with previously reported computer generated models, and also confirm that complementary direct repeat and terminator sequences are involved in the formation of the RNAI–RNAII complex. Mutant par RNAs were used to show that the binding reaction occurs in at least two steps. The first step is the formation of an initial kissing interaction between the transcriptional terminator stem‐loops of both RNAs. The subsequent step(s) involves an initial pairing of the complementary direct repeat sequences followed by complete hybridization of the 5′ nucleotides to stabilize the RNAI–RNAII complex.
Replication arrests during a single round of replication of the Escherichia coli chromosome in the absence of DnaC activity
Authors: Maisnier‐Patin Sophie , Nordström Kurt , Dasgupta Santanu
Published: March 2003

We used a flow cytometric assay to determine the frequency of replication fork arrests during a round of chromosome replication in Escherichia coli. After synchronized initiation from oriC in a dnaC(Ts) strain, non‐permissive conditions were imposed, such that active DnaC was not available during elongation. Under these conditions, about 18% of the cells failed to complete chromosome replication. The sites of replication arrests were random and occurred on either arm of the bidirectionally replicating chromosome, as stalled forks accumulated at the terminus from both directions. The forks at the terminal Ter sites disappeared in the absence of Tus protein, as the active forks could then pass through the terminus to reach the arrest site, and the unfinished rounds of replication would be completed without DnaC. In a dnaC2(Ts)rep double mutant, almost all cells failed to complete chromosome replication in the absence of DnaC activity. As inactivation of Rep helicase (the rep gene product) has been shown to cause frequent replication arrests inducing double‐strand breaks (DSBs) in a replicating chromosome, DnaC activity appears to be essential for replication restart from DSBs during elongation.
The yeast transcriptome in aerobic and hypoxic conditions: effects of hap1, rox1, rox3 and srb10 deletions
Authors: Becerra Manuel , Lombardía‐Ferreira Luis J. , Hauser Nicole C. , Hoheisel Jörg D. , Tizon Belén , Cerdán M. Esperanza
Published: March 2003

The transcriptome of Saccharomyces cerevisiae was screened using the high‐density membrane hybridization method, under aerobic and hypoxic conditions, in wild‐type and mutant backgrounds obtained by the disruption of the genes encoding the regulatory proteins Hap1, Rox1 and the Srb10 and Rox3 subunits of RNA polymerase II holoenzyme. None of the mutations studied was able to fully overcome the wild‐type hypoxic response. Deletion of the hap1 gene changed the expression profiles of individual open reading frames (ORFs) under both aerobic and hypoxic conditions. Major changes associated with rox3 deletion were related to the hypoxic activation. Rox3 also caused a repressor effect (oxygen‐independent) on a subset of genes related to subtelomeric proteins. With regard to the effect brought about by the deletion of rox1 and srb10, correspondence cluster analysis revealed that the transcriptome profile in aerobic conditions is very similar in the wild‐type and both deletion strains. In contrast, however, differences were found during hypoxia between the subgroup formed by wild‐type and the Δrox1 deletant compared with the Δsrb10 deletant. An analysis of selected ORFs responding to hypoxia, in association with a dependence on the regulatory factors studied, made it possible to identify the clusters that are related to different regulatory circuits.
Lateral flagella of Aeromonas species are essential for epithelial cell adherence and biofilm formation
Authors: Gavín Rosalina , Rabaan Ali A. , Merino Susana , Tomás Juan M. , Gryllos Ioannis , Shaw Jonathan G.
Published: March 2003

Mesophilic Aeromonas strains express a single polar flagellum in all culture conditions and produce lateral flagella on solid media. Such hyperflagellated cells demonstrate increased adherence. Nine lateral flagella genes, lafA–U for Aeromonas hydrophila, and four Aeromonas caviae genes, lafA1, lafA2, lafB and fliU, were isolated. Mutant characterization, nucleotide and N‐terminal sequencing demonstrated that the A. hydrophila and A. caviae lateral flagellins were almost identical, but were distinct from their polar flagellum counterparts. The aeromonad lateral flagellins exhibited higher molecular masses on SDS–PAGE, and this aberrant migration was thought to result from post‐translational modification through glycosylation. Mutation of the Aeromonas lafB, lafS or both A. caviae lateral flagellins caused the loss of lateral flagella and a reduction in adherence and biofilm formation. Mutations in lafA1, lafA2, fliU or lafT resulted in strains that expressed lateral flagella, but had reduced adherence levels. Mutation of the lateral flagella loci did not affect polar flagellum synthesis, but the polarity of the transposon insertions on the A. hydrophila lafT/U genes resulted in non‐motility. However, mutations that abolished polar flagellum production also inhibited lateral flagella expression. We conclude that Aeromonas lateral flagella: (i) play a role in adherence and biofilm formation; (ii) are distinct from the polar flagellum; (iii) synthesis is dependent upon the presence of a polar flagellum filament; and (iv) that the motor proteins of the polar and lateral flagella systems appear to be shared.
Plasticity of a transcriptional regulation network among alpha‐proteobacteria is supported by the identification of CtrA targets in Brucella abortus
Authors: Bellefontaine Anne‐Flore , Pierreux Christophe E. , Mertens Pascal , Vandenhaute Jean , Letesson Jean‐Jacques , Bolle Xavier De
Published: March 2003

CtrA is a master response regulator found in many alpha‐proteobacteria. In Caulobacter crescentus and Sinorhizobium meliloti, this regulator is essential for viability and is transcriptionally autoregulated. In C. crescentus, it is required for the regulation of multiple cell cycle events, such as DNA methylation, DNA replication, flagella and pili biogenesis and septation. Here, we report the characterization of the ctrA gene homologue in the α2‐proteobacteria Brucella abortus, a facultative intracellular pathogen responsible for brucellosis. We detected CtrA expression in the main Brucella species, and its overproduction led to a phenotype typical of cell division defect, consistent with its expected role. A purified B. abortus CtrA recombinant protein (His6–CtrA) was shown to protect the B. abortus ctrA promoter from DNase I digestion, suggesting transcriptional autoregulation, and this protection was enhanced under CtrA phosphorylation on a conserved Asp residue. Despite the similarities shared by B. abortus and C. crescentus ctrA, the pathway downstream from CtrA may be distinct, at least partially, in both bacteria. Indeed, beside ctrA itself, only one (the ccrM gene) out of four B. abortus homologues of known C. crescentus CtrA targets is bound in vitro by phosphorylated B. abortus CtrA. Moreover, further footprinting experiments support the hypothesis that, in B. abortus, CtrA might directly regulate the expression of the rpoD, pleC, minC and ftsE homologues. Taken together, these results suggest that, in B. abortus and C. crescentus, similar cellular processes are regulated by CtrA through the control of distinct target genes. The plasticity of the regulation network involving CtrA in these two bacteria may be related to their distinct lifestyles.
Control of 26S proteasome expression by transcription factors regulating multidrug resistance in Saccharomyces cerevisiae
Authors: Owsianik Grzegorz , Balzi Lisabetta , Ghislain Michel
Published: March 2003

In eukaryotic cells, intracellular proteolysis occurs mainly via the ubiquitin–proteasome system. Expression of the yeast proteasome is under the control of the transcription factor, Rpn4p (also known as Son1p/Ufd5p). We show here that the RPN4 gene
promoter contains regulatory sequences that bind Pdr1p and Pdr3p, two homologous
zinc finger‐containing transcription factors, which mediate multiple drug resistance
through the expression of membrane transporter proteins. Mutations in the RPN4
Pdr1p/Pdr3p binding sites lead to decreased expression of the proteasome RPT6
gene and to defective ubiquitin‐mediated proteolysis. Pdr3p, but not Pdr1p, is required
for normal levels of intracellular proteolysis, indicating that the two transcription
factors have distinct functions in the control of RPN4 expression. The RPN4
promoter contains an additional sequence that binds Yap1p, a bZIP‐type transcription
factor that plays an important role in the oxidative stress response and multidrug
resistance. We also show that the Yap1p response element is important in the transactivation
of RPN4 by Yap1p. In yeast cells lacking Pdr1p, ubiquitin‐Pro‐β‐galactosidase,
a short‐lived protein used to assay proteasome activity, is stabilized by the loss
of Yap1p. These data demonstrate that the ubiquitin–proteasome system is controlled
by transcriptional regulators of multidrug resistance via RPN4 expression.
The proper folding of a long C‐terminal segment of the yeast Lys14p regulator is required for activation of LYS genes in response to the metabolic effector
Authors: Alami Mohamed El , Feller André , Piérard André , Dubois Evelyne
Published: March 2003

Transcription of lysine genes in Saccharomyces cerevisiae is dependent on

Lys14p and on α‐aminoadipate semialdehyde (αAASA), an intermediate

of the pathway. The two‐thirds C‐terminal end of Lys14p is sufficient to ensure the

activation function of the protein and its modulation by αAASA. Here,

we show that no single discrete domain of Lys14p is able to activate transcription

and that most of the deleted LexA–Lys14p proteins are inactive even in the

presence of a high αAASA concentration. The point mutations abolishing

the activation capacity of Lys14p are distributed all over the entire C‐terminal

segment. Although the deletion of 20 residues rich in leucine and located downstream

of the DNA‐binding domain converts Lys14p to a constitutive transcriptional activator,

our analysis provides evidence that the modulation process of Lys14p activity does

not involve an effector‐dependent masking/unmasking mechanism. Furthermore, we show

that the protein chaperone Hsp82p is required for full activation of LYS genes

by the αAASA‐activated Lys14p as well as by the constitutive Lys14p. Our

results suggest that the proper folding of the two‐thirds C‐terminal portion of Lys14p

is essential not only to activate transcription but also to modulate it according

to αAASA concentration.
C‐terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high‐affinity binding to bacterial cell wall carbohydrates
Authors: Loessner Martin J. , Kramer Karl , Ebel Frank , Scherer Siegfried
Published: March 2003

Listeria monocytogenes phage endolysins Ply118 and Ply500 share a unique enzymatic activity and specifically hydrolyse Listeria cells at the completion of virus multiplication in order to release progeny phage. With the aim of determining the molecular basis for the lytic specificity of these enzymes, we have elucidated their domain structure and examined the function of their unrelated and unique C‐terminal cell wall binding domains (CBDs). Analysis of deletion mutants showed that both domains are needed for lytic activity. Fusions of CBDs with green fluorescent protein (GFP) demonstrated that the C‐terminal 140 amino acids of Ply500 and the C‐terminal 182 residues of Ply118 are necessary and sufficient to direct the murein hydrolases to the bacterial cell wall. CBD500 was able to target GFP to the surface of Listeria cells belonging to serovar groups 4, 5 and 6, resulting in an even staining of the entire cell surface. In contrast, the CBD118 hybrid bound to a ligand predominantly present at septal regions and cell poles, but only on cells of serovars 1/2, 3 and 7. Non‐covalent binding to surface carbohydrate ligands occurred in a rapid, saturation‐dependent manner. We measured 4 × 104 and 8 × 104 binding sites for CBD118 and CBD500 respectively. Surface plasmon resonance analysis revealed unexpected high molecular affinity constants for the CBD–ligand interactions, corresponding to nanomolar affinities. In conclusion, we show that the CBDs are responsible for targeting the phage endolysins to their substrates and function to confer recognition specificity on the proteins. As the CBD sequences contain no repeats and lack all known sequence motifs for anchoring of proteins to the bacterial cell, we conclude that they use unique structural motifs for specific association with the surface of Gram‐positive bacteria.
Generation of Campylobacter jejuni genetic diversity in vivo
Authors: Boer Paulo de , Wagenaar Jaap A. , Achterberg René P. , Putten Jos P. M. van , Schouls Leo M. , Duim Birgitta
Published: March 2003

Molecular epidemiology studies suggest that horizontal genetic exchange is a major cause of pathogen biodiversity. We tested this concept for the bacterial enteropathogen Campylobacter jejuni by seeking direct in vivo evidence for the exchange of genetic material among Campylobacter strains. For this purpose, two antibiotic resistance markers were inserted into the hipO or htrA gene of genetically distinct and naturally transformable C. jejuni strains. Genetic exchange of the resistance markers was analysed after co‐cultivation of homologous and heterologous strains in vitro and in vivo during experimental infection of chickens. Double‐resistant recombinants were obtained both in vitro and from the chicken intestine for all combinations of strains tested. Bidirectional genetic exchange of DNA between homologous and heterologous strains was confirmed by Southern blotting in combination with flaA polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP), amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE). Extensive PFGE analyses of isolated recombinants indicated the frequent occurrence of genetic rearrangements during the experimental infection, in addition to the homologous recombination of the antibiotic resistance genes. Together, the data indicate unequivocally that interstrain genetic exchange as well as intragenomic alterations do occur in vivo during C. jejuni infection. These events probably explain the genome plasticity observed for this pathogen.
Characterization of Burkholderia pseudomallei infection and identification of novel virulence factors using a Caenorhabditis elegans host system
Authors: Gan Yunn‐Hwen , Chua Kim Lee , Chua Hui Hoon , Liu Boping , Hii Chung Shii , Chong Hwee Ling , Tan Patrick
Published: March 2003

The environmental saphrophyte Burkholderia pseudomallei is the causative
agent of melioidosis, a systemic, potentially life‐threatening condition endemic
to many parts of south‐east Asia and northern Australia. We have used the soil nematode
Caenorhabditis elegans as a model host to characterize the mechanisms by which
this bacterium mounts a successful infection. We find that C. elegans is susceptible
to a broad range of Burkholderia species, and that the virulence mechanisms
used by this pathogen to kill nematodes may be similar to those used to infect mammals.
We also find that the specific dynamics of the C. elegans–B. pseudomallei
host–pathogen interaction can be highly influenced by environmental factors,
and that nematode killing results at least in part from the presence of a diffusible
toxin. Finally, by screening for bacterial mutants attenuated in their ability to
kill C. elegans, we genetically identify several new potential virulence factors
in B. pseudomallei. The use of C. elegans as a model host should greatly
facilitate future investigations into how B. pseudomallei can interact with host organisms.
The datA locus predominantly contributes to the initiator titration mechanism in the control of replication initiation in Escherichia coli
Authors: Ogawa Tohru , Yamada Yoshitaka , Kuroda Takao , Kishi Tetsuya , Moriya Shigeki
Published: March 2003

Replication of the Escherichia coli chromosome is initiated synchronously
from all origins (oriC) present in a cell at a fixed time in the cell cycle
under given steady state culture conditions. A mechanism to ensure the cyclic initiation
events operates through the chromosomal site, datA, which titrates exceptionally
large amounts of the bacterial initiator protein, DnaA, to prevent overinitiation.
Deletion of the datA locus results in extra initiations and altered temporal
control of replication. There are many other sites on the E. coli chromosome
that can bind DnaA protein, but the contribution of these sites to the control of
replication initiation has not been investigated. In the present study, seven major
DnaA binding sites other than datA have been examined for their influence
on the timing of replication initiation. Disruption of these seven major binding
sites, either individually or together, had no effect on the timing of initiation
of replication. Thus, datA seems to be a unique site that adjusts the balance
between free and bound DnaA to ensure that there is only a single initiation event
in each bacterial cell cycle. Mutation either in the second or the third DnaA box
(a 9 basepair DnaA‐binding sequence) in datA was enough to induce asynchronous
and extra initiations of replication to a similar extent as that observed with the
datA‐deleted strain. These DnaA boxes may act as cores for the cooperative
binding of DnaA to the entire datA region.
LitR, a new transcriptional activator in Vibrio fischeri, regulates luminescence and symbiotic light organ colonization
Authors: Fidopiastis Pat M. , Miyamoto Carol M. , Jobling Michael G. , Meighen Edward A. , Ruby Edward G.
Published: March 2003

Vibrio fischeri
is the bacterial symbiont within the light‐emitting organ of the sepiolid squid
Euprymna scolopes
. Upon colonizing juvenile squids, bacterial symbionts grow on host‐supplied nutrients, while providing a bioluminescence that the host uses during its nocturnal activities. Mutant bacterial strains that are unable to emit light have been shown to be defective in normal colonization. A 606 bp open reading frame was cloned from
V. fischeri
that encoded a protein, which we named LitR, that had about 60% identity to four related regulator proteins:
Vibrio cholerae
HapR,
Vibrio harveyi
LuxR,
Vibrio parahaemolyticus
OpaR and
Vibrio vulnificus
SmcR. When grown in culture, cells of
V. fischeri
strain PMF8, in which
litR
was insertionally inactivated, were delayed in the onset of luminescence induction and emitted only about 20% as much light per cell as its parent. Protein‐binding studies suggested that LitR enhances quorum sensing by regulating the transcription of the
luxR
gene. Interestingly, when competed against its parent in mixed inocula, PMF8 became the predominant symbiont present in 83% of light organs. Thus, the
litR mutation appears to represent a novel class of mutations in which the loss of a regulatory gene function enhances the bacterium's competence in initiating a benign infection.
The role of heterologous receptors in McpB‐mediated signalling in Bacillus subtilis chemotaxis
Authors: Zimmer Michael A. , Szurmant Hendrik , Saulmon Michael M. , Collins Marissa A. , Bant Jason S. , Ordal George W.
Published: March 2003

Asparagine chemotaxis in Bacillus subtilis appears to involve two partially redundant adaptation mechanisms: a receptor methylation‐independent process that operates at low attractant concentrations and a receptor methylation‐dependent process that is required for optimal responses to high concentrations. In order to elucidate these processes, chemotactic responses were assessed for strains expressing methylation‐defective mutations in the asparagine receptor, McpB, in which all 10 putative receptors (10del), five receptors (5del) or only the native copy of mcpB were deleted. This was done in both the presence and the absence of the methylesterase CheB. We found that: (i) only responses to high concentrations of asparagine were impaired; (ii) the presence of all heterologous receptors fully compensated for this defect, whereas responses progressively worsened as more receptors were taken away; (iii) methyl‐group turnover occurred on heterologous receptors after the addition of asparagine, and these methylation changes were required for the restoration of normal swimming behaviour; (iv) in the absence of the methyleste‐rase, the presence of heterologous receptors in some cases caused impaired chemotaxis; and (v) either a certain threshold number of receptors must be present to promote basal CheA activity, or one or more of the receptors missing in the 10del background (but present in the 5del background) is required for establishing basal CheA activity. Taken together, these findings suggest that many or all chemoreceptors work as an ensemble that constitutes a robust chemotaxis system. We propose that the ability of non‐McpB receptors to compensate for the methylation‐defective McpB mutations involves lateral transmission of the adapted conformational change across the ensemble.
Proteoglycans mediate malaria sporozoite targeting to the liver
Authors: Pradel Gabriele , Garapaty Shivani , Frevert Ute
Published: March 2003

Malaria sporozoites are rapidly targeted to the liver where they pass through Kupffer cells and infect hepatocytes, their initial site of replication in the mammalian host. We show that sporozoites, as well as their major surface proteins, the CS protein and TRAP, recognize distinct cell type‐specific surface proteoglycans from primary Kupffer cells, hepatocytes and stellate cells, but not from sinusoidal endothelia. RecombinantPlasmodium falciparumCS protein and TRAP bind to heparan sulphate on hepatocytes and both heparan and chondroitin sulphate proteoglycans on stellate cells. On Kupffer cells, CS protein predominantly recognizes chondroitin sulphate, whereas TRAP binding is glycosaminoglycan independent.Plasmodium bergheisporozoites attach to heparan sulphate on hepatocytes and stellate cells, whereas Kupffer cell recognition involves both chondroitin sulphate and heparan sulphate proteoglycans. CS protein also interacts with secreted proteoglycans from stellate cells, the major producers of extracellular matrix in the liver.In situbinding studies using frozen liver sections indicate that the majority of the CS protein binding sites are associated with these matrix proteoglycans. Our data sug‐gest that sporozoites are first arrested in the sinusoid by binding to extracellular matrix proteoglycans and then recognize proteoglycans on the surface of Kupffer cells, which they use to traverse the sinusoidal cell barrier.
The cellular level of the recognition factor RssB is rate‐limiting for σS proteolysis: implications for RssB regulation and signal transduction in σS turnover in Escherichia coli
Authors: Pruteanu Mihaela , Hengge‐Aronis Regine
Published: March 2003

Degradation of the general stress sigma factor σS of Escherichia coli is a prime example of regulated proteolysis in prokaryotes. Whereas exponentially growing cells rapidly degrade σS, various stress conditions result in stabilization and, therefore, rapid accumulation of σS. Proteolysis of σS requires the response regulator RssB, a direct recognition factor with phosphorylation‐dependent affinity for σS, which targets σS to the ClpXP protease. Here, we demonstrate that a sudden increase in σS synthesis results in σS stabilization, indicating titration of an essential proteolytic component. Evidence is provided that RssB is the overall rate‐limiting factor for σS proteolysis. As a consequence, the cell has to continuously adjust the expression of RssB to σS in order to maintain σS proteolysis in growing cells, despite variations in the rate of σS synthesis. Such homeostatic feedback‐coupling is provided by rssB transcription being dependent on the σS‐controlled rssAB operon promoter. However, strong and rapid increases in σS synthesis, in re‐sponse to acute stress, exceed the compensatory potential of this feedback loop with the result that σS is stabilized because of RssB titration. We propose that RssB control of σS proteolysis functions as a genetic switch, in which (i) the ‘off’ state (low σS levels caused by proteolysis) is stabilized by a homeostatic negative feedback, and (ii) the threshold for switching to the ‘on’ state (high levels of stable σS) is dependent on the cellular level of active, i.e. phosphorylated RssB.
Thinking and decision making, bacterial style: Bacterial Neural Networks, Obernai, France, 7th–12th June 2002
Authors: Armitage Judith P. , Dorman Charles J. , Hellingwerf Klaas , Schmitt Rudiger , Summers David , Holland Barry
Published: March 2003

Bacteria exhibit a bewildering range of behavioural responses and permutations of metabolic pathways for maximum exploitation of their environment. These are based on sensory perception of external and internal signals through batteries of surface and cytoplasmic receptors, evaluation of complex information flows and rapid decision making. Appreciation of the diversity of bacterial behaviour and adaptation capacities requires the study of a broad range of organisms and at this meeting we sampled more than 30 species with new findings which included the nature of gaseous receptors, advances in chemotaxis, subversion of host defences by pathogens, adaptation to high salt, community life and its obvious benefits, cell to cell communications and even the nature of bacterial circadian rhythms. With around 80 bacterial genomes now completed, and many more almost there, it was appropriate to complete the meeting with an introduction to Systems Biology and prospects for simulating the virtual cell. The versatility and seemingly ‘intelligent’ behaviour of bacteria will continue to fascinate, and this meeting on Bacterial Neural Networks fully reflected the excitement of this field.
A screen in Saccharomyces cerevisiae identified CaMCM1, an essential gene in Candida albicans crucial for morphogenesis
Authors: Rottmann M. , Dieter S. , Brunner H. , Rupp S.
Published: March 2003

Morphogenesis in Saccharomyces cerevisiae and the pathogenic yeast Candida albicans is governed in part by the same molecular circuits. In S. cerevisiae, FLO11/MUC1 expression has been shown to be modulated by multiple signalling pathways required for pseudohyphal development. We have established a screen in S. cerevisiae to identify regulators of fungal development in C. albicans based on FLO11::lacZ expression as a reporter. This screen identified both known components of the mitogen‐activated protein kinase (MAPK) cascade and the cAMP cascade that are important for hyphal development in C. albicans, as well as genes not yet known to be involved in morphogenesis. The Candida homologue of MCM1 is one of the novel factors identified in this screen as being important for morphogenesis. CaMcm1p levels do not vary significantly in different cell types and respond to an autoregulatory feedback mechanism, arguing that CaMcm1p activity is regulated by post‐translational modifications. Both overexpression and repression of this essential gene led to the induction of hyphae. Moreover, we found that the expression of HWP1, a hyphae‐specific gene, was induced by repression of CaMCM1. The changes in morphology and HWP1 expression were not the result of a change in expression levels of NRG1 or TUP1, known repressors of hyphal development. Thus, CaMcm1p is a component of a hitherto unknown regulatory mechanism of hyphal growth.
Nucleotide sequence, functional characterization and evolution of pFKN, a virulence plasmid in Pseudomonas syringae pathovar maculicola
Authors: Rohmer Laurence , Kjemtrup Susanne , Marchesini Patrizia , Dangl Jeffery L.
Published: March 2003

Pseudomonas syringae pv. maculicola strain M6 (Psm M6) carries the avrRpm1 gene, encoding a type III effector, on a 40 kb plasmid, pFKN. We hypothesized that this plasmid might carry additional genes required for pathogenesis on plants. We report the sequence and features of pFKN. In addition to avrRpm1, pFKN carries an allele of another type III effector, termed avrPphE, and a gene of unknown function (ORF8), expression of which is induced in planta, suggesting a role in the plant–pathogen interaction. The region of pFKN carrying avrRpm1, avrPphE and ORF8 exhibits several features of pathogenicity islands (PAIs). Curing of pFKN (creating Psm M6C) caused a significant reduction in virulence on Arabidopsis leaves. However, complementation studies using Psm M6C demonstrated an obvious virulence function only for avrRpm1. pFKN can integrate and excise from the chromosome of Psm M6 at low frequency via homologous recombination between identical sequence segments located on the chromosome and on pFKN. These segments are part of two nearly identical transposons carrying avrPphE. The avrPphE transposon was also detected in other strains of P. s. pv. maculicola and in P. s. tomato strain DC3000. The avrPphE transposon was found inserted at different loci in different strains. The analysis of sequences surrounding the avrPphE transposon insertion site in the chromosome of Psm M6 indicates that pFKN integrates into a PAI that encodes type III effectors. The integration of pFKN into this chromosomal region may therefore be seen as an evolutionary process determining the formation of a new PAI in the chromosome of Psm M6.
The slow wave does not propagate across the gastroduodenal junction in the isolated feline preparation
Authors: LAMMERS  , STEPHEN  , ADEGHATE  , PONERY  , POZZAN
Published: March 2003

Detailed spatial analysis of the propagation of individual slow waves was performed in the isolated gastroduodenal preparation of the cat. Use was made of a system that allowed the simultaneous recordings from 240 extracellular electrodes, which were positioned across the gastroduodenal region. Reconstructions of the spread of propagation (n = 31) revealed that (a) the antral slow wave never propagated into the duodenum but was blocked at the pyloric ring, (b) the duodenal slow wave did not activate the antral tissue, and (c) a quiescent zone in which no slow waves could be recorded was always present at the most proximal part of the duodenum immediately distal to the pyloric ring. Furthermore, phase density distributions of duodenal cycles revealed that antral activity had no influence on the rate of discharge of duodenal pacemakers. Light microscopic study of sections of the duodenum close to the pyloric ring and further away did not show any structural differences between the quiescent zone and the active areas. In conclusion, slow waves do not propagate across the gastroduodenal junction in the isolated feline preparation and therefore do not seem to play a role in the electro‐mechanical integration between the stomach and the duodenum.
Motor activity of vascularly perfused rat duodenum. 1. Characteristics of spontaneous movement
Authors: YAMAMOTO  , KUWAHARA  , YAMAMOTO  , FUJIMURA  , MAEDA  , FUJIMIYA
Published: March 2003

We developed an ex vivo model of arterially perfused rat duodenum to examine the motor activity of intestine. In this preparation, spontaneously occurring pressure waves with regular rhythm were observed. The oxygen consumption and motor activity of the intestine were compared at different arterial perfusion rates to determine the degree of oxygenation required to elicit spontaneous motility. Pressure waves with regular rhythm occurred at a frequency of 1 min–1 when the arterial perfusion was 3–5 mL min–1, and stopped when the perfusion rate fell below 2 mL min–1. Atropine and hexamethonium reduced the percentage motor index/10 min of pressure waves in a dose‐dependent manner, and tetrodotoxin completely blocked motor activity. Acetylcholine stimulated motor activity, and this effect was not antagonized by TTX. These findings suggest that spontaneous contraction in the ex vivo perfused rat duodenum might be mediated by a cholinergic mechanism via muscarinic receptors on smooth muscle, but that noncholinergic mechanisms may also participate in this response.
Interaction between blood flow and motility in normal and inflamed ileum
Authors: Cowles  , Ryan  , Sarna
Published: March 2003

The alterations in local and superior mesenteric blood flow during ileal inflammation and their correlations with motility in the normal and the inflamed ileum were investigated in the conscious state. Ileal inflammation decreased the local mesenteric blood flow but had no significant effect on the superior mesenteric blood flow. A significant reduction or an increase in local mesenteric blood flow in the normal or the inflamed ileum had no effect on local contractile activity. The vascular reactivity to vaso‐dilators and vaso‐constrictors was significantly reduced during inflammation. Local mesenteric blood flow increased significantly in the descending segment ahead of a caudal propagating giant migrating contraction. The local mesenteric blood flow oscillated during a migrating motor complex (MMC) cycle. We conclude that a several‐fold increase or decrease in local mesenteric blood flow lasting for several minutes does not affect contractility. Ileal inflammation decreases local mesenteric blood flow but does not affect the total blood flow to the small intestine.
Inhibition by CCK of ascending contraction elicited by mucosal stimulation in the duodenum of the rat
Authors: Giralt  , Vergara
Published: March 2003

CCK released by intraluminal stimuli modifies duodenal activity contributing to a decrease in gastric emptying. However, the neural mechanisms by which CCK controls motility are not well known. The aim of this study was to investigate the interaction between CCK and the enteric nervous system through the study of the effects of CCK‐8 on ascending excitation. Anaesthetized Sprague‐Dawley rats were prepared with a strain‐gauge sutured to the duodenum wall. An electrode holder was placed in the duodenum lumen to elicit ascending contraction. Electrical field stimulation of the duodenal mucosa (4 Hz, 0.6 ms, 30 V) induced an ascending excitation which was blocked by hexamethonium (10 mg kg–1; n=5) and atropine (0.3 mg kg–1; n=5), but enlarged by
L‐NNA (10–5 mol kg–1; n=5). CCK‐8 (3 × 10–9 mol kg–1 10 min–1) blocked ascending excitation and an inhibition of the induced phasic activity was observed instead (n=18). Individually, none of the CCK receptor antagonists (L‐364 718 and L‐365 260) (3 × 10–7 mol kg–1; n=6 each) blocked the inhibition of ascending excitation induced by CCK‐8. However, simultaneous infusion of both antagonists abolished CCK‐8 effect on electrical stimulation (n=5). Similarly, none of the CCK‐8 agonists (A‐71623, A‐71378, gastrin) modified the ascending excitation. In contrast, the simultaneous infusion of A‐71623 and CCK‐4 (n=4) induced an effect similar to CCK‐8. In conclusion, CCK‐8 blocked ascending contraction elicited by electrical field stimulation of duodenal mucosa by means of simultaneous activation of CCK‐A and CCK‐B receptors.