bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA sequence variation in BpMADS2 gene in two populations of Betula pendula
Authors: Järvinen Pia , Lemmetyinen Juha , Savolainen Outi , Sopanen Tuomas
Published: March 2003

The PISTILLATA (PI) homologue, BpMADS2, was isolated from silver birch (Betula pendula Roth) and used to study nucleotide polymorphism. Two regions (together about 2450 bp) comprising mainly untranslated sequences were sequenced from 10 individuals from each of two populations in Finland. The nucleotide polymorphism was low in the BpMADS2 locus, especially in the coding region. The synonymous site overall nucleotide diversity (πs) was 0.0043 and the nonsynonymous nucleotide diversity (πa) was only 0.000052. For the whole region, the π values for the two populations were 0.0039 and 0.0045, and for the coding regions, the π values were only 0 and 0.00066 (for the corresponding coding regions of Arabidopsis thaliana PI world‐wide π was 0.0021). Estimates of π or θ did not differ significantly between the two populations, and the two populations were not diverged from each other. Two classes of BpMADS2 alleles were present in both populations, suggesting that this gene exhibits allelic dimorphism. In addition to the nucleotide site variation, two microsatellites were also associated within the haplotypes. This allelic dimorphism might be the result of postglacial re‐colonization partly from northwestern, partly from southeastern/eastern refugia. The sequence comparison detected five recombination events in the regions studied. The large number of microsatellites in all of the three introns studied suggests that BpMADS2 is a hotspot for microsatellite formation.
Degradation of the Cry1Ab protein within transgenic Bacillus thuringiensis corn tissue in the field
Authors: Zwahlen C. , Hilbeck A. , Gugerli P. , Nentwig W.
Published: March 2003

Large quantities of Bacillus thuringiensis (Bt) corn plant residue are left in the field after harvest, which may have implications for the soil ecosystem. Potential impacts on soil organisms will also depend on the persistence of the Bt toxin in plant residues. Therefore, it is important to know how long the toxin persists in plant residues. In two field studies in the temperate corn‐growing region of Switzerland we investigated degradation of the Cry1Ab toxin in transgenic Bt corn leaves during autumn, winter and spring using an enzyme‐linked immunosorbent assay (ELISA). In the first field trial, representing a tillage system, no degradation of the Cry1Ab toxin was observed during the first month. During the second month, Cry1Ab toxin concentrations decreased to ≈ 20% of their initial values. During winter, there was no further degradation. When temperatures again increased in spring, the toxin continued to degrade slowly, but could still be detected in June. In the second field trial, representing a no‐tillage system, Cry1Ab toxin concentrations decreased without initial delay as for soil‐incorporated Bt plants, to 38% of the initial concentration during the first 40 days. They then continued to decrease until the end of the trial after 200 days in June, when 0.3% of the initial amount of Cry1Ab toxin was detected. Our results suggest that extended pre‐ and post‐commercial monitoring are necessary to assess the long‐term impact of Bt toxin in transgenic plant residues on soil organisms.
Pmt1 mannosyl transferase is involved in cell wall incorporation of several proteins in Saccharomyces cerevisiae
Authors: Bourdineaud Jean‐Paul , Van Der Vaart J. Marcel , Donzeau Mariel , De Sampaïo Guillaume , Verrips C. Theo , Lauquin Guy J.‐M.
Published: March 2003

We constructed hybrid proteins containing a plant α‐galactosidase fused to various C‐terminal moieties of the hypoxic Srp1p; this allowed us to identify a cell wall‐bound form of Srp1p. We showed that the last 30 amino acids of Srp1p, but not the last 16, contain sufficient information to signal glycosyl‐phosphatidylinositol anchor attachment and subsequent cell wall anchorage. The cell wall‐bound form was shown to be linked by means of a β1,6‐glucose‐containing side‐chain. Pmt1p enzyme is known as a protein‐O‐mannosyltransferase that initiates the O‐glycosidic chains on proteins. We found that a pmt1 deletion mutant was highly sensitive to zymolyase and that in this strain the α‐galactosidase–Srp1 fusion proteins, an α‐galactosidase–Sed1 hybrid protein and an α‐galactosidase–α‐agglutinin hybrid protein were absent from both the membrane and the cell wall fractions. However, the plasma membrane protein Gas1p still receives its glycosyl‐phosphatidylinositol anchor in pmt1 cells, and in this mutant strain an α‐galactosidase–Cwp2 fusion protein was found linked to the cell wall but devoid of β1,6‐glucan side‐chain, indicating an alternative mechanism of cell wall anchorage.
Type II protein secretion by Pseudomonas aeruginosa: genetic suppression of a conditional mutation in the pilin‐like component XcpT by the cytoplasmic component XcpR
Authors: Kagami Yoshihiro , Ratliff Melanie , Surber Mark , Martinez Alejandro , Nunn David N.
Published: March 2003

Pseudomonas aeruginosa exports a number of hydrolytic enzymes and toxins using the type II or general secretion pathway, found in a variety of Gram‐negative bacteria and requiring the functions of at least 12 gene products (XcpP–Z and PilD/XcpA in P. aeruginosa). A number of these gene products are homologues of components of the type IV pilus biogenesis system, including four proteins, XcpT–W, which are highly similar to the pilin subunit in their size, localization and post‐translational modifications. These proteins, in addition to the pilin subunit, are cleaved and methylated by the PilD/XcpA prepilin peptidase, but their interactions with other components of the export apparatus are unclear. Using a medium developed for the selection of export‐proficient P. aeruginosa strains, we have isolated temperature‐sensitive mutations in the xcpT gene and extragenic suppressors for one of the mutants. These suppressors fall into two classes, one that maps outside of the xcpP–Z gene cluster and may define additional cellular functions that are required for export, and a second that maps to the xcpR gene product and indicates a potential protein–protein interaction connecting two different cellular compartments and required for the assembly or function of the export apparatus.
Specificity of the BvgAS and EvgAS phosphorelay is mediated by the C‐terminal HPt domains of the sensor proteins
Authors: Perraud Anne‐Laure , Kimmel Brigitte , Weiss Verena , Gross Roy
Published: March 2003

Despite the presence of highly conserved signalling modules, significant cross‐communication between different two‐component systems has only rarely been observed. Domain swapping and the characterization of liberated signalling modules enabled us to characterize in vitro the protein domains that mediate specificity and are responsible for the high fidelity in the phosphorelay of the unorthodox Bvg and Evg two‐component systems. Under equimolar conditions, significant in vitro phosphorylation of purified BvgA and EvgA proteins was only obtained by their histidine kinases, BvgS and EvgS respectively. One hybrid histidine kinase consisting of the BvgS transmitter and HPt domains and of the EvgS receiver domain (BvgS‐TO‐EvgS‐R) was able to phosphorylate BvgA but not EvgA. In contrast, the hybrid protein consisting of the BvgS transmitter and the EvgS receiver and HPt domains (BvgS‐T‐EvgS‐RO) was unable to phosphorylate BvgA but efficiently phosphorylated EvgA. These results demonstrate that the C‐terminal HPt domains of the sensor proteins endow the unorthodox two‐component systems with a high specificity for the corresponding regulator protein. In the case of the response regulators, the receiver but not the output domains contribute to the specific interaction with the histidine kinases, because a hybrid protein consisting of the EvgA receiver and the BvgA output domain could only be phosphorylated by the EvgS protein.
The σD‐dependent transcription of the ywcG gene from Bacillus subtilis is dependent on an excess of glucose and glutamate
Authors: Moch Christine , Schrögel Oliver , Allmansberger Rudolf
Published: March 2003

We investigated the function and transcriptional regulation of ywcG. The protein is essential for Bacillus subtilis. Biochemical characterization of the protein revealed that it is an FMN‐containing NADPH oxidase. ywcG is transcribed throughout the whole life cycle of B. subtilis. The start point of transcription is preceded by potential promoter sequences for σA, σB and σD. A boost in transcription occurs at the beginning of stationary phase in complex media containing glutamate and glucose. The induction of transcription at the beginning of stationary phase needs the activity of a different alternative σ‐factor σD. ywcG is, therefore, the first gene with a putative role in energy metabolism from B. subtilis that is transcribed in a σD‐dependent fashion, but its regulation is unique and the reverse of that described for all other σD‐dependent genes.
Non‐genetic population heterogeneity studied by in situ polymerase chain reaction
Authors: Tolker‐Nielsen Tim , Holmstrøm Kim , Boe Lars , Molin Søren
Published: March 2003

Expression of a lac operon in Salmonella typhimurium single cells was monitored using lac mRNA targeting in situ reverse transcription–polymerase chain reaction (RT–PCR). It is demonstrated that suboptimal induction of the lac operon in a culture of S. typhimurium/F′lac+ cells generates a subpopulation in which transcription of the lac operon occurs and another subpopulation in which transcription of the lac operon is repressed, whereas suboptimal induction of the lac operon in a culture of S. typhimurium/F′lacY cells generates a population with uniform levels of lac mRNA. The outcome of the single‐cell lac mRNA detection assay was compared with the outcome of a single‐cell β‐galactosidase assay. In cultures grown under different suboptimal lac induction conditions, the fraction of cells in which transcription of the lac operon occurred was concurrent with the fraction of cells showing β‐galactosidase activity. Besides supporting the hypothesis that the lactose permease has a role in generating non‐genetic heterogeneity in suboptimally induced cultures of Lac+ cells, these results demonstrate the usefulness of in situ RT–PCR for the study of non‐genetic population heterogeneities.
Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli
Authors: Suzuki Emi , Kondo Taemi , Makise Masaki , Mima Shinji , Sakamoto Kenji , Tsuchiya Tomofusa , Mizushima Tohru
Published: March 2003

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature‐sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42°C (non‐permissive temperature) and at 37°C (semi‐permissive temperature), but not at 28°C (permissive temperature), compared with the wild‐type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature‐sensitive dnaA mutants showed similar results. On the other hand, a cold‐sensitive dnaAcos mutant, in which overinitiation of DNA replication occurs at low temperature (28°C), showed a higher level of unsaturation of fatty acids at 28°C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.
Immunity proteins and their specificity for endonuclease colicins: telling right from wrong in protein–protein recognition
Authors: Kleanthous Colin , Hemmings Andrew M. , Moore Geoffrey R. , James Richard
Published: March 2003

Immunity proteins inhibit colicins, protein toxins released by bacteria during times of environmental stress, by binding and inactivating their cytotoxic domains. This protects the producing organism as it attempts to kill off competing bacteria. The cytotoxic domains of related colicins share a high degree of sequence identity, as do their corresponding immunity proteins, yet specificity and affinity are also high, with little non‐cognate biological cross‐protection evident under physiological conditions. We review recent work on DNase‐specific immunity proteins, which shows that, although both cognate and non‐cognate proteins can bind a single toxin, their affinities can differ by as much as 12 orders of magnitude. We have termed this mode of binding dual recognition, because the DNase‐binding surface of an immunity protein is made up of two components, one conserved and the other variable. The strength of the binding interaction is dominated by the conserved residues, while neighbouring variable residues control specificity. Similar dual recognition systems may exist in other biological contexts, particularly where a protein must discriminate the right binding partner from numerous, structurally homologous alternatives.
TonB‐dependent iron acquisition: mechanisms of siderophore‐mediated active transport†
Authors: Moeck Gregory S. , Coulton James W.
Published: March 2003

Cells growing in aerobic environments have developed intricate strategies to overcome the scarcity of iron, an essential nutrient. In Gram‐negative bacteria, high‐affinity iron acquisition requires outer membrane‐localized proteins that bind iron chelates at the cell surface and promote their uptake. Transport of bound chelates across the outer membrane depends upon TonB–ExbB–ExbD, a cytoplasmic membrane‐localized complex that transduces energy from the proton motive force to high‐affinity receptors in the outer membrane. Upon ligand binding to iron chelate receptors, conformational changes are induced, some of which are detected in the periplasm. These structural alterations signal the ligand‐loaded status of the receptor and, therefore, the requirement for TonB‐dependent energy transduction. Thus, TonB interacts preferentially and directly with ligand‐loaded receptors. Such a mechanism ensures the productive use of cellular energy to drive active transport at the outer membrane.
In search of higher energy: metabolism‐dependent behaviour in bacteria
Authors: Taylor Barry L. , Zhulin Igor B.
Published: March 2003

Bacteria use different strategies to navigate to niches where environmental factors are favourable for growth. Chemotaxis is a behavioural response mediated by specific receptors that sense the concentration of chemicals in the environment. Recently, a new type of sensor has been described in Escherichia coli that responds to changes in cellular energy (redox) levels. This sensor, Aer, guides the bacteria to environments that support maximal energy levels in the cells. A variety of stimuli, such as oxygen, alternative electron acceptors, light, redox carriers that interact with the electron transport system and metabolized carbon sources, effect changes in the cellular energy (redox) levels. These changes are detected by Aer and by the serine chemotaxis receptor Tsr and are transduced into signals that elicit appropriate behavioural responses. Diverse environmental signals from Aer and chemotaxis receptors converge and integrate at the level of the CheA histidine kinase. Energy sensing is widespread in bacteria, and it is now evident that a variety of signal transduction strategies are used for the metabolism‐dependent behaviours. The occurrence of putative energy‐sensing domains in proteins from cells ranging from Archaea to humans indicates the importance of this function for all living systems.
PRD — a protein domain involved in PTS‐dependent induction and carbon catabolite repression of catabolic operons in bacteria
Authors: Stülke Jörg , Arnaud Maryvonne , Rapoport Georges , Martin‐Verstraete Isabelle
Published: March 2003

Several operon‐specific transcriptional regulators, including antiterminators and activators, contain a duplicated conserved domain, the PTS regulation domain (PRD). These duplicated domains modify the activity of the transcriptional regulators both positively and negatively. PRD‐containing regulators are very common in Gram‐positive bacteria. In contrast, antiterminators controlling β‐glucoside utilization are the only functionally characterized members of this family from Gram‐negative bacteria. PRD‐containing regulators are controlled by PTS‐dependent phosphorylation with different consequences: (i) In the absence of inducer, the phosphorylated EIIB component of the sugar permease donates its phosphate to a PRD, thereby inactivating the regulator. In the presence of the substrate, the regulator is dephosphorylated, and the phosphate is transferred to the sugar, resulting in induction of the operon. (ii) In Gram‐positive bacteria, a novel mechanism of carbon catabolite repression mediated by PRD‐containing regulators has been demonstrated. In the absence of PTS substrates, the HPr protein is phosphorylated by enzyme I at His‐15. This form of HPr can, in turn, phosphorylate PRD‐containing regulators and stimulate their activity. In the presence of rapidly metabolizable carbon sources, ATP‐dependent phosphorylation of HPr at Ser‐46 by HPr kinase inhibits phosphorylation by enzyme I, and PRD‐containing regulators cannot, therefore, be stimulated and are inactive. All regulators of this family contain two copies of PRD, which are functionally specialized in either induction or catabolite repression.
The A and the extended V N‐terminal regions of streptococcal protein I/IIf mediate the production of tumour necrosis factor α in the monocyte cell line THP‐1
Authors: Chatenay‐Rivauday Christian , Yamodo Innocent , Sciotti Michel‐Angelo , Ogier Joelle A. , Klein Jean‐Paul
Published: March 2003

The induction of tumour necrosis factor (TNF)‐α from the monocytic cell line THP‐1 by the streptococcal antigen I/II from Streptococcus mutans serotype f (protein I/IIf) was studied by use of recombinant polypeptides containing discrete domains of the protein. The derivatives carrying the N‐terminal alanine‐rich region (A region) and the adjacent variable region (extended V region) of the protein bound to THP‐1 cell extracts in a saturable fashion, and one derivative lacking both the A and the extended V regions was not able to bind monocyte cell extracts, suggesting that the domains responsible for the binding of protein I/IIf to monocytes were the A and the extended V regions. Sodium metaperiodate pretreatment of THP‐1 cell extracts, tunicamycin pretreatment of monocyte cells or competition with N‐acetyl neuraminic acid (NANA) and fucose resulted in a 45–70% reduction in binding activity of the derivatives carrying the extended V region, demonstrating the lectin‐like mode of recognition of the monocytic receptor by the extended V region and the role of NANA and fucose in this recognition process. Besides, the stimulation of monocytes to release TNF‐α by the derivatives containing the A region and the extended V region was effective and was not affected by the addition of polymyxin B or vitamin D3, suggesting that CD14 does not play the role of receptor in stimulation of monocytes by protein I/IIf to release TNF‐α.
The ZnuABC high‐affinity zinc uptake system and its regulator Zur in Escherichia coli
Authors: Patzer Silke I. , Hantke Klaus
Published: March 2003

In Escherichia coli, lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein‐dependent transport system. The growth defect of these mutants on TY medium containing 5 mM EGTA was compensated for by the addition of Zn2+. In the presence of 0.5 mM EGTA, only the parental strain was able to take up 65Zn2+. This high‐affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI ) and localized at 42 min on the genetic map of E. coli. At high Zn2+ concentrations, the znu mutants took up more 65Zn2+ than the parental strain. The high‐affinity 65Zn2+ uptake was repressed by growth in the presence of 10 μM Zn2+. A znuA–lacZ operon fusion was repressed by 5 μM Zn2+ and showed a more than 20‐fold increase in β‐galactosidase activity when Zn2+ was bound to 1.5 μM TPEN [tetrakis‐(2‐pyridylmethyl) ethylenediamine]. To identify the Zn2+‐dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli. A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High‐affinity 65Zn2+ transport of the constitutive zur mutant was 10‐fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB.
De novo fatty acid synthesis is required for establishment of cell type‐specific gene transcription during sporulation in Bacillus subtilis
Authors: Schujman Gustavo E. , Grau Roberto , Gramajo Hugo C. , Ornella Leonardo , De Mendoza Diego
Published: March 2003

A hallmark of sporulation of Bacillus subtilis is the formation of two distinct cells by an asymmetric septum. The developmental programme of these two cells involves the compartmentalized activities of σE in the larger mother cell and of σF in the smaller prespore. A potential role of de novo lipid synthesis on development was investigated by treating B. subtilis cells with cerulenin, a specific inhibitor of fatty acid biosynthesis. These experiments demonstrated that spore formation requires de novo fatty acid synthesis at the onset of sporulation. The transcription of the sporulation genes that are induced before the formation of two cell types or that are under the exclusive control of σF occurred in the absence of fatty acid synthesis, as monitored by spo–lacZ fusions. However, expression of lacZ fusions to genes that required activation of σE for transcription was inhibited in the absence of fatty acid synthesis. The block in σE‐directed gene expression in cerulenin‐treated cells was caused by an inability to process pro‐σE to its active form. Electron microscopy revealed that these fatty acid‐starved cells initiate abnormal polar septation, suggesting that de novo fatty acid synthesis may be essential to couple the activation of the mother cell transcription factors with the formation of the differentiating cells.
Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation
Authors: Marsh Jane W. , Taylor Ronald K.
Published: March 2003

Cholera toxin secretion is dependent upon the extracellular protein secretion apparatus encoded by the eps gene locus of Vibrio cholerae. Although the eps gene locus encodes several type four prepilin‐like proteins, the peptidase responsible for processing these proteins has not been identified. This report describes the identification of a prepilin peptidase from the V. cholerae genomic database by virtue of its homology with the PilD prepilin peptidase of Pseudomonas aeruginosa. Plasmid disruption or deletion of this peptidase gene in either El Tor or classical V. cholerae O1 biotype strains results in a dramatic decrease in cholera toxin secretion. In the case of the El Tor biotype mutants, surface expression of the type 4 pilus responsible for mannose‐sensitive haemagglutination is abolished. The cloned V. cholerae peptidase processes either EpsI or MshA preproteins when co‐expressed in E. coli. Mutation of the V. cholerae peptidase gene also results in a defect in virulence and decreased levels of OmpU. The V. cholerae peptidase gene sequence shows 80% homology with the Vibrio vulnificus VvpD type 4 prepilin peptidase required for pilus assembly and cytolysin secretion in V. vulnificus. Accordingly, the V. cholerae type 4 prepilin peptidase required for pilus assembly and cholera toxin secretion has been designated VcpD.
Salmonella InvG forms a ring‐like multimer that requires the InvH lipoprotein for outer membrane localization
Authors: Crago Aimee M. , Koronakis Vassilis
Published: March 2003

Salmonella species translocate virulence effector proteins from the bacterial cytoplasm into mammalian host cells by means of a type III secretion apparatus, encoded by the pathogenicity island‐1 (SPI‐1). Little is known about the assembly and structure of this secretion apparatus, but the InvG protein is essential and could be an outer membrane secretion channel for the effector proteins. We observed that in recombinant Escherichia coli, the yield of InvG was enhanced by co‐expression of InvH, and showed that mutation of invH decreased the level of InvG in wild‐type Salmonella typhimurium. In E. coli, InvG alone was able to form an SDS‐resistant multimer, but InvG localization to the outer membrane was dependent upon InvH, a lipoprotein itself located in the outer membrane, and no other SPI‐1 specific protein. InvG targeted to the outer membrane by InvH became accessible to extracellular protease. InvG and InvH did not, however, appear to form a stable complex. Electron microscopy of InvG membrane protein purified from E. coli revealed that it forms an oligomeric ring‐like structure with inner and outer diameters, 7 nm and 15 nm respectively.
Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development
Authors: O'Toole George A. , Kolter Roberto
Published: March 2003

The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. We report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective (sad ). Two classes of sad mutants were analysed: (i) mutants defective in flagellar‐mediated motility and (ii) mutants defective in biogenesis of the polar‐localized type IV pili. We followed the development of the biofilm formed by the wild type over 8 h using phase‐contrast microscopy. The wild‐type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time‐lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As observed with the wild type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild‐type strain, the type IV pili mutants did not develop microcolonies over the course of the experiments, suggesting that these structures play an important role in microcolony formation. Very few cells of a non‐motile strain (carrying a mutation in flgK ) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell‐to‐surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.
Fatal attraction of mammalian cells to Legionella pneumophila
Authors: Abu Kwaik Yousef
Published: March 2003

Legionella pneumophila is a protozoan parasite that causes Legionnaires' disease. Its ability to do so is dependent on its capacity to replicate intracellularly within a phagosome that is not trafficked through the endosomal–lysosomal pathway and is surrounded by the rough endoplasmic reticulum. Within this unique niche, the bacterium undergoes alterations in gene expression. In addition, many virulence‐related phenotypes that are induced in vitro by starvation are expressed intracellularly as the bacteria exit the logarithmic growth phase. (p)ppGpp appears to signal expression of the virulence‐related genes in L. pneumophila upon starvation. This growth phase‐dependent phenotypical transition is concomitant with lysis of the host cell, in which both necrosis and apoptosis seem to play roles. Many genetic loci that are required for intracellular replication within mammalian and protozoan cells have been identified, and the majority of them are novel. Two secretion systems have been identified, one of which may be distantly related to type IV secretion systems. The other is a type II secretion system similar to the PilBCD piliation system of Pseudomonas aeruginosa.
Protein‐mediated DNA transfer into liposomes
Authors: Lambert Olivier , Plançon Laure , Rigaud Jean Louis , Letellier Lucienne
Published: March 2003

The transfer of a foreign genome into a bacterium by means of phage infection is a very efficient but poorly understood process. To analyse the mechanism of phage DNA transfer at a molecular level, we have reconstituted FhuA, the receptor for phage T5 in the outer membrane of Escherichia coli, into unilamellar vesicles made of natural phospholipids. Cryoelectron microscopy studies showed that the binding of the phage to FhuA triggered the transfer of its double‐stranded DNA (121 000 bp) into the proteoliposomes. DNA was entrapped within vesicles with a diameter ranging from 70 to 150 nm. The DNA appeared to be densely packed, but its presence did not alter the morphology of the liposomes, suggesting no DNA–lipid interactions. These liposomes represent an attractive model system for studying the mechanisms of DNA transport and condensation. They may also serve as alternative vehicles for the transfer of foreign genes into eukaryotic cells.
Cloning of the mating type loci from Pyrenopeziza brassicae reveals the presence of a novel mating type gene within a discomycete MAT 1‐2 locus encoding a putative metallothionein‐like protein
Authors: Singh Gurjeet , Ashby Alison M.
Published: March 2003

The mating type loci were cloned from Pyrenopeziza brassicae by chromosome walking from a mating type‐linked polymerase chain reaction (PCR) fragment and shown to be idiomorphic by sequence analysis. The MAT 1‐1 locus is approximately 3.2 kb and contains a single gene encoding a putative high‐mobility group (HMG) domain protein. The MAT 1‐2 locus is approximately 3.9 kb with three open reading frames (ORFs) encoding a putative HMG domain, an α‐1 domain and metallothionein‐like proteins. The putative α‐1 domain ORF on MAT 1‐2 is transcribed in the opposite orientation to the other two transcripts and extends into non‐idiomorphic sequence. This is the first report of sequence analysis of the mating type loci from a discomycete fungus, which has revealed an interesting mating type infrastructure within the MAT 1‐2 locus. Although metallothionein‐like proteins have been implicated in a number of processes in animals and plants, they have not to date been implicated in the mating process of filamentous fungi. Possible roles for metallothionein‐like proteins in the mating process are discussed.
The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase
Authors: Kravanja Melanie , Engelmann Roswitha , Dossonnet Valérie , Blüggel Martin , Meyer Helmut E. , Frank Rainer , Galinier Anne , Deutscher Josef , Schnell Norbert , Hengstenberg Wolfgang
Published: March 2003

The HPr kinase of Gram‐positive bacteria is an ATP‐dependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision‐induced dissociation of tryptic peptides derived from the HPr kinase of E. faecalis. The gene was overexpressed in Escherichia coli, which does not contain any ATP‐dependent HPr kinase or phosphatase activity. The homogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P‐Ser‐HPr phosphatase activity, which was assumed to be a separate enzyme activity. The bifunctional HPr kinase/phosphatase acts preferentially as a kinase at high ATP levels of 2 mM occurring in glucose‐metabolizing Streptococci. At low ATP levels, the enzyme hydrolyses P‐Ser‐HPr. In addition, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme may be to adjust the ratio of HPr and P‐Ser‐HPr according to the metabolic state of the cell; P‐Ser‐HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestigation of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P‐Ser‐HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in activity of the B. subtilis HPr kinase was only observed when fructose‐1,6‐bisphosphate was also present.
CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in Gram‐positive bacteria
Authors: Derré Isabelle , Rapoport Georges , Msadek Tarek
Published: March 2003

clpP and clpC of Bacillus subtilis encode subunits of the Clp ATP‐dependent protease and are required for stress survival, including growth at high temperature. They play essential roles in stationary phase adaptive responses such as the competence and sporulation developmental pathways, and belong to the so‐called class III group of heat shock genes, whose mode of regulation is unknown and whose expression is induced by heat shock or general stress conditions. The product of ctsR, the first gene of the clpC operon, has now been shown to act as a repressor of both clpP and clpC, as well as clpE, which encodes a novel member of the Hsp100 Clp ATPase family. The CtsR protein was purified and shown to bind specifically to the promoter regions of all three clp genes. Random mutagenesis, DNaseI footprinting and DNA sequence deletions and comparisons were used to define a consensus CtsR recognition sequence as a directly repeated heptad upstream from the three clp genes. This target sequence was also found upstream from clp and other heat shock genes of several Gram‐positive bacteria, including Listeria monocytogenes, Streptococcus salivarius, S. pneumoniae, S. pyogenes, S. thermophilus, Enterococcus faecalis, Staphylococcus aureus, Leuconostoc oenos, Lactobacillus sake, Lactococcus lactis and Clostridium acetobutylicum. CtsR homologues were also identified in several of these bacteria, indicating that heat shock regulation by CtsR is highly conserved in Gram‐positive bacteria.
Molecular genetic analysis of enoyl‐acyl carrier protein reductase inhibition by diazaborine
Authors: De Boer Gert‐Jan , Pielage Gerlof J. A. , Nijkamp H. John J. , Slabas Antoni R. , Rafferty John B. , Baldock Clair , Rice David W. , Stuitje Antoine R.
Published: March 2003

Diazaborine and isoniazid are, at first sight, unrelated anti‐bacterial agents that inhibit the enoyl‐ACP reductase (ENR) of Escherichia coli and Mycobacterium tuberculosis respectively. The crystal structures of these enzymes including that of the diazaborine‐inhibited E. coli ENR have been obtained at high resolution. Site‐directed mutagenesis was used to study the importance of amino acid residues in diazaborine susceptibility and enzyme function. The results show that drug binding and inhibition require the presence of a glycine residue at position 93 of E. coli ENR or at the structurally equivalent position in the plant homologue, which is naturally resistant to the drug. The data confirm the hypothesis that any amino acid side‐chain other than hydrogen at this position within the three‐dimensional structure of these enzymes will affect diazaborine resistance by encroaching into the drug binding site. Substitutions of Gly‐93 by amino acids with small side‐chains, such as serine, alanine, cysteine and valine, hardly affected the catalytic parameters and rendered the bacterial host resistant to the drug. Larger amino acid side‐chains, such as that of arginine, histidine, lysine and glutamine, completely inactivated the activity of the enzyme.
Localization and environmental regulation of MCP‐like proteins in Rhodobacter sphaeroides
Authors: Harrison David M. , Skidmore Jennifer , Armitage Judith P. , Maddock Janine R.
Published: March 2003

Chemotaxis to many compounds by Rhodobacter sphaeroides requires transport and at least partial metabolism of the chemoeffector. Previous investigations using phototrophically grown cells have failed to find any homologues of the MCP chemoreceptors identified in Escherichia coli. However, using an antibody raised against the highly conserved domain of E. coli Tsr, MCP‐like proteins were identified in R. sphaeroides WS8N. Analysis using Western blotting and immunogold electron microscopy showed that expression of these MCP‐like proteins is environmentally regulated and that receptors are targeted to two different cellular locations: the poles of the cells and the cytoplasm. In aerobically grown cells, these proteins were shown by immunoelectron microscopy to localize predominantly to the cell poles and to an electron‐dense body in the cytoplasm. Western blot analysis indicated a 17‐fold reduction in protein concentration when cells were grown in the light. The number of immunogold particles was also dramatically reduced in anaerobically light‐grown cells and their cellular distribution was altered. Fewer receptors localized to the cell poles and more particles randomly distributed within the cell, but the cytoplasmic cluster remained. These trends were more pronounced in cells grown anaerobically under dim light than in those grown anaerobically under bright light, suggesting that expression is controlled by redox state and either light intensity or the extent of photosynthetic membrane synthesis. Recent work on E. coli chemosensing suggests that oligomerization of receptors and chemosensory proteins is important for sensory signalling. The data presented here suggest that this oligomerization can occur with cytoplasmic receptors and also provides an explanation for the multiple copies of chemosensory proteins in R. sphaeroides.
ZntR is a Zn(II)‐responsive MerR‐like transcriptional regulator of zntA in Escherichia coli
Authors: Brocklehurst Kathryn R. , Hobman Jon L. , Lawley Blair , Blank Lindsay , Marshall Samantha J. , Brown Nigel L. , Morby Andrew P.
Published: March 2003

We have identified the promoter/operator region of the zntA gene of Escherichia coli and shown that Zn(II) is the primary inducer of expression of this Zn(II)/Cd(II) export gene. The promoter PzntA shows sequence similarities to the promoters of mercury resistance (mer ) operons, including a long spacer region containing an inverted repeat sequence. The gene encoding the transcriptional regulator of PzntA, designated zntR, has been identified from genome sequence data, by expression of the gene product and by insertional inactivation/complementation. The ZntR product is a member of the MerR family of transcriptional regulators and appears to act as a hypersensitive transcriptional switch. A hybrid MerR/ZntR protein has been constructed and indicates that the C‐terminal region of ZntR recognizes Zn(II).
IS903 transposase mutants that suppress defective inverted repeats
Authors: Tavakoli Norma P. , Derbyshire Keith M.
Published: March 2003

The inverted repeats (IRs) of the insertion element IS903 are composed of two functional regions. An inner region, consisting of basepairs 6–18, is the transposase binding site. The outer region (positions 1–3) is not contacted during initial transposase binding, but is essential for efficient transposition. We have examined the interaction of the IR with the transposase by isolating transposase suppressors of IR mutations. These suppressors define two patches within the N‐terminus of the protein. One class of suppressors, which rescued the majority of outer IR mutants tested, contained mutations in close proximity to an aspartate residue (D121) believed to form part of the catalytic DDE motif, suggesting that their suppressive effect is in the positioning of the catalytic site at the terminus of the transposon. The hypertransposition phenotype of mutant VA119 is also consistent with this hypothesis. The second class was more allele specific and preferentially suppressed a mutation at position 3 of the IR. Finally, we showed that mutations at the termini of the IR elevate the frequency of cointegrate formation by IS903. Other outer IR mutations did not have this effect. These data are consistent with the terminal bases of the transposon playing multiple and distinct roles in transposition.
Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP‐dependent, enzyme I‐ and HPr‐catalysed phosphorylation
Authors: Lindner Cordula , Galinier Anne , Hecker Michael , Deutscher Josef
Published: March 2003

The transcriptional antiterminator LicT regulates the induction and carbon catabolite repression of the Bacillus subtilis bglPH operon. LicT is inactive in mutants affected in one of the two general components of the phosphoenolpyruvate (PEP):glycose phosphotransferase system, enzyme I or histidine‐containing protein (HPr). We demonstrate that LicT becomes phosphorylated in the presence of PEP, enzyme I and HPr. The phosphoryl group transfer between HPr and LicT is reversible. Phosphorylation of LicT with PEP, enzyme I and HPr led to the appearance of three additional LicT bands on polyacrylamide–urea gels. These bands probably correspond to one‐, two‐ and threefold phosphorylated LicT. After phosphorylation of LicT with [32P]‐PEP, enzyme I and HPr, proteolytic digestion of [32P]‐P‐LicT, separation of the peptides by reverse‐phase chromatography, mass spectrometry and N‐terminal sequencing of radiolabelled peptides, three histidyl residues were found to be phosphorylated in LicT. These three histidyl residues (His‐159, His‐207 and His‐269) are conserved in most members of the BglG/SacY family of transcriptional antiterminators. Phosphorylation of LicT in the presence of seryl‐phosphorylated HPr (P‐Ser‐HPr) was much slower compared with its phosphorylation in the presence of HPr. The slower phosphorylation in the presence of P‐Ser‐HPr leading to reduced LicT activity is presumed to play a role in a recently described LicT‐mediated CcpA‐independent carbon catabolite repression mechanism operative for the bglPH operon.
Mycobacterium tuberculosis phagosome
Authors: Deretic V. , Fratti R. A.
Published: March 2003

The arrest of Mycobacterium tuberculosis phagosome maturation in infected macrophages is a phenomenon of dual significance both for the pathogenesis of tuberculosis and as a model system to study interference of microbes with membrane trafficking and organelle biogenesis in host cells. Among other factors, compartment‐specialized regulators of vesicular trafficking and other parts of membrane fusion machinery are likely to play a role in these processes. Here we summarize the emerging view of mycobacterial phagosome maturation arrest in the context of the dynamic processes of intracellular membrane trafficking.
IHF protein inhibits cleavage but not assembly of plasmid R388 relaxosomes
Authors: Moncalián Gabriel , Valle Mikel , Valpuesta José María , De La Cruz Fernando
Published: March 2003

Relaxosomes are specific nucleoprotein structures involved in DNA‐processing reactions during bacterial conjugation. In this work, we present evidence indicating that plasmid R388 relaxosomes are composed of origin of transfer (oriT) DNA plus three proteins TrwC relaxase, TrwA nic‐cleavage accessory protein and integration host factor (IHF), which acts as a regulatory protein. Protein IHF bound to two sites (ihfA and ihfB) in R388 oriT, as shown by gel retardation and DNase I footprinting analysis. IHF binding in vitro was found to inhibit nic‐cleavage, but not TrwC binding to supercoiled DNA. However, no differences in the frequency of R388 conjugation were found between IHF− and IHF+ donor strains. In contrast, examination of plasmid DNA obtained from IHF− strains revealed that R388 was obtained mostly in relaxed form from these strains, whereas it was mostly supercoiled in IHF+ strains. Thus, IHF could have an inhibitory role in the nic‐cleavage reaction in vivo. It can be speculated that triggering of conjugative DNA processing during R388 conjugation can be mediated by IHF release from oriT.
Physical and genetic map of the genome of Vibrio parahaemolyticus: presence of two chromosomes in Vibrio species
Authors: Yamaichi Yoshiharu , Iida Tetsuya , Park Kwon‐Sam , Yamamoto Koichiro , Honda Takeshi
Published: March 2003

We constructed a physical map of the genomic DNA (5.1 Mb) for Vibrio parahaemolyticus strain AQ4673 by combining 17 adjacent NotI fragments. This map shows two circular replicons of 3.2 and 1.9 Mb. Pulsed‐field gel electrophoresis (PFGE) of undigested genomic DNA revealed two bands of corresponding sizes. Analysis both by NotI digestion and by Southern blot of the two isolated bands confirmed the existence of two replicons. The presence of genes for 16S rRNA on both the replicons indicates that the replicons are chromosomes rather than megaplasmids. The two bands were also seen after PFGE of undigested genomic DNA of V. parahaemolyticus strains other than AQ4673, and of strains belonging to other Vibrio species, such as V. vulnificus, V. fluvialis and various serovars and biovars of V. cholerae. It is noteworthy that V. cholerae O1 strain 569B, a classical biovar, was also shown to have two replicons of 2.9 and 1.2 Mb, which does not agree with a physical map proposed in a previous study. Our results suggest that a two‐replicon structure is common throughout Vibrio species.
New insights into the ATP‐dependent Clp protease: Escherichia coli and beyond
Authors: Porankiewicz Joanna , Wang Jimin , Clarke Adrian K.
Published: March 2003

Proteolysis functions as a precise regulatory mechanism for a broad spectrum of cellular processes. Such control impacts not only on the stability of key metabolic enzymes but also on the effective removal of terminally damaged polypeptides. Much of this directed protein turnover is performed by proteases that require ATP and, of those in bacteria, the Clp protease from Escherichia coli is one of the best characterized to date. The Clp holoenzyme consists of two adjacent heptameric rings of the proteolytic subunit known as ClpP, which are flanked by a hexameric ring of a regulatory subunit from the Clp/Hsp100 chaperone family at one or both ends. The recently resolved three‐dimensional structure of the E. coli ClpP protein has provided new insights into its interaction with the regulatory/chaperone subunits. In addition, an increasing number of studies over the last few years have recognized the added complexity and functional importance of ClpP proteins in other eubacteria and, in particular, in photosynthetic organisms ranging from cyanobacteria to higher plants. The goal of this review is to summarize these recent findings and to highlight those areas that remain unresolved.