bluuebunny/crossref_metadata_2025 huggingface dataset:
The sodium‐driven polar flagellar motor of marine Vibrio as the mechanosensor that regulates lateral flagellar expression
Authors: Kawagishi Ikuro , Imagawa Miho , Imae Yasuo , McCarter Linda , Homma Michio
Published: October 2006

Certain marine Vibrio species swim in sea water, propelled by a polar flagellum, and swarm over surfaces using numerous lateral flagella. The polar and the lateral flagellar motors are powered by sodium‐ and proton‐motive forces, respectively. The lateral flagella are produced in media of high viscosity, and the relevant viscosity sensor is the polar flagellum. The cell might monitor either the rotation rate of the flagellar motor or the mechanical force applied against the flagellum. To test these possibilities, we examined the effects of amiloride and its derivatives, which inhibit the rotation of the sodium‐driven motor, on lateral flagellar gene (Iaf) expression in Vibrio parahaemolyticus. Phenamil, an amiloride analogue that inhibits swimming at micromolar concentrations, induced Iaf transcription in media devoid of viscous agents in a dose‐dependent manner. The relationship between the average swimming speed and Iaf induction in the presence of various concentrations of phenamil was very similar to that observed when viscosity was changed. These results indicate that marine Vibrio sense a decrease in the rotation rate of (or the sodium influx through) the polar flagellar motor as a trigger for Iaf induction. Alternative mechanisms for Iaf induction are also discussed.
Participation of the molecular chaperone DnaK in intracellular growth of Brucella suis within U937‐derived phagocytes
Authors: Köhler Stephan , Teyssier Jacques , Cloeckaert Axel , Rouot Bruno , Liautard Jean‐Pierre
Published: October 2006

In the intracellular bacterium Brucella suis, the molecular chaperone DnaK was induced under heat‐shock conditions and at low pH. Insertional inactivation of dnaK and dnaJ within the dnaK/J locus led to the conclusion that DnaK, but not DnaJ, was required for growth at 37°C in vitro. Viability of the dnaK null mutant was also greatly affected at low pH. Under conditions allowing intracellular multiplication, the infection of U937‐derived phagocytes resulted in long‐lasting DnaK induction in the wild‐type bacteria. In infection experiments performed with both mutants at the reduced temperature of 30°C, the dnaK mutant of B. suis survived but failed to multiply within U937 cells, whereas the wild‐type strain and the dnaJ mutant multiplied normally. Complementation of the dnaK mutant with the cloned dnaK gene restored growth at 37°C, increased resistance to acid pH, and increased intracellular multiplication. This is the first report of the effects of dnaK inactivation in a pathogenic species, and of the temperature‐independent contribution of DnaK to intracellular multiplication of the pathogen B. suis.
Alternate promoters direct stress‐induced transcription of the Bacillus subtilis clpC operon
Authors: Krüger Elke , Msadek Tarek , Hecker Michael
Published: October 2006

clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co‐transcribed and encode stress‐inducible proteins. Two promoters (PA and PB) were mapped upstream of the first gene. PA resembles promoters recognized by the vegetative RNA polymerase EσA. The other promoter (PB) was shown to be dependent on σB, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σB‐dependent manner. This is the first evidence that σB in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ32 in Escherichia coli, indicating a new function of σB‐dependent general stress proteins. PB deviated from the consensus sequence of σB promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σB‐dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σA‐like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σA‐like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress‐induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.
Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli
Authors: Palmer Tracy , Santini Claire‐Lise , Iobbi‐Nivol Chantal , Eaves Deborah J. , Boxer David H. , Giordano Gerard
Published: October 2006

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl2, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over‐expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.
An environmentally regulated pilus‐like appendage involved inCampylobacterpathogenesis
Authors: Doig Peter , Yao Ruijin , Burr Donald H. , Guerry Patricia , Trust Trevor J.
Published: October 2006

Examination of strains ofCampylobacter jejuni, Campylobacter coli, andCampylobacter fetusby electron microscopy revealed that they produced peritrichous pilus‐like appendages when the bacteria were grown in the presence of bile salts. Various bile‐salt supplements were used and it was found that deoxycholate and chenodeoxycholic acid caused a significant enhancement of pilus production and resulted in a highly aggregative phenotype. Morphologically, the pili were between 4 and 7 nm in width and were greater than 1 μm in length. A gene, termedpspA, which encodes a predicted protein resembling protease IV ofEscherichia coli, was identified inC. jejunistrain 81–176. A site‐specific insertional mutation within this gene resulted in the loss of pilus synthesis as determined by electron microscopy. Insertions upstream and downstream of the gene had no effect on pilus production. The non‐piliated mutant of strain 81–176 showed no reduction in adherence to or invasion of INT 407 cellsin vitro. However, this mutant, while still possessing the ability to colonize ferrets, caused significantly reduced disease symptoms in this animal model.
Control of bacterial chemotaxis
Authors: Eisenbach Michael
Published: October 2006

Bacterial chemotaxis, which has been extensively studied for three decades, is the most prominent model system for signal transduction in bacteria. Chemotaxis is achieved by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein, CheY. This protein is activated by a stimulus‐dependent phosphorylation mediated by an autophosphorylatable kinase (CheA) whose activity is controlled by chemoreceptors. Upon phosphorylation, CheY dissociates from its kinase, binds to the switch at the base of the flagellar motor, and changes the motor rotation from the default direction (counter‐clockwise) to clockwise. Phosphorylation may also be involved in terminating the response. Phosphorylated CheY binds to the phosphatase CheZ and modulates its oligomeric state and thereby its dephosphorylating activity. Thus CheY phosphorylation appears to be involved in controlling both the excitation and adaptation mechanisms of bacterial chemotaxis. Additional control sites might be involved in bacterial chemotaxis, e.g. lateral control at the receptor level, control at the motor level, or control by metabolites that link central metabolism with chemotaxis.
Sequence specificity for DNA binding by Escherichia coli SoxS and Rob proteins
Authors: Li Ziyi , Demple Bruce
Published: October 2006

SoxS is a transcriptional activator of oxidative stress genes in Escherichia coli. SoxS in vitro binds the promoters of soxRS‐regulated genes such as micF, zwf, nfo and sodA, forms multiple protein‐DNA complexes, and recruits RNA polymerase to the promoters. E. coli Rob protein, with an N‐terminus 55% identical to SoxS, was initially identified by its binding to the oriC replication origin, but Rob in vitro binds some of the same promoters as SoxS and in vivo activates some SoxS‐regulated genes. In this work we show that the multiple complexes with SoxS arise from the presence at least two independent binding sites in each of the micF and zwf promoters. SoxS and Rob each form only a single complex with a 20 bp DNA oligonucleotide corresponding to the region immediately upstream of the ‐35 element of the micF promoter. Methylation interference identified several conserved purine residues required for binding to micF and five other SoxS‐binding sites. Together with binding studies using mutated oligonucleotides and published DNase I footprinting data, this information was used to form a consensus for SoxS sequence specificity: AN2GCAYN7CWA (where N is any base, Y is a pyrimi‐dine, and W is A or T). The sequence requirements for Rob binding differed somewhat from those of SoxS. Using the SoxS‐binding consensus, several genes potentially regulated by soxRS were identified in an E. coli genomic database; some of these genes have functions that might contribute to cellular resistance to oxidative stress.
The leucine‐responsive regulatory protein (Lrp) acts as a specific repressor for σs‐dependent transcription of the Escherichia coli aidB gene
Authors: Landini Paolo , Hajec Laurel I. , Nguyen Lam H. , Burgess Richard R. , Volkert Michael R.
Published: October 2006

The product of the Escherichia coli aidB gene is homologous to human isovaleryl‐coenzyme A dehydrogenase (IVD), an enzyme involved in the breakdown of the amino acid leucine. The aidB gene is not expressed constitutively, but its transcription is induced via distinct mechanisms in response to: (i) exposure to alkylating agents; (ii) acetate at a slightly acidic pH; and (iii) anoxia. Induction by alkylating agents is mediated by the transcriptional activator Ada, in its methylated form (meAda); the other forms of induction are Ada independent and require σs, the alternative σs factor mainly expressed during the stationary phase of bacterial growth. In this report we show that, in the absence of any transcriptional factor, aidB is efficiently transcribed in vitro by the σs, but not by the σ70, form of RNA polymerase holoenzyme. In the presence of meAda, levels of transcription by both forms of RNA polymerase are significantly increased. However, σs ‐dependent transcription of aidB is inhibited both in vitro and in vivo by binding of the transcriptional regulator Lrp (leucine responsive protein) to the aidB promoter region (PaidB)‐ Lrp acts as a specific repressor for σs ‐dependent transcription of aidB. Leucine counteracts Lrp binding to PaidB as does binding to PaidB of meAda, which causes Lrp to dissociate from the promoter. The physiological significance of aidB transcription regulation is discussed.
The Ustilago maydis virally encoded KP1 killer toxin
Authors: Park Chung‐Mo , Banerjee Nanditta , Koltin Yigal , Bruenn Jeremy A.
Published: October 2006

Some strains of the plant‐pathogenic fungus Ustilago maydis secrete toxins (killer toxins) that are lethal to susceptible strains of the same fungus. There are three well‐characterized killer toxins in U. maydis–KP1, KP4, and KP6–which are secreted by the P1, P4, and P6 subtypes, respectively. These killer toxins are small polypeptides encoded by segments of an endogenous, persistent double‐stranded RNA (dsRNA) virus in each U. maydis subtype. In P4 and P6, the M2 dsRNA segment encodes the toxin. In this work, the KP1 killer toxin was purified for internal amino acid sequence analysis, and P1M2 was identified as the KP1 toxin‐encoding segment by sequence analysis of cDNA clones. The KP1 toxin is a monomer with a predicted molecular weight of 13.4kDa and does not have extensive sequence similarity with other viral anti‐fungal toxins. The P1M2 segment is different from the P4 and P6 toxin‐encoding dsRNA segments in that the 3’non‐coding region of its plus strand has no sequence homology to the 3’ends of the plus strands of P1M1, P4M2, or P6M2.
Role of nodl and nodj in lipo‐chitooligosaccharide secretion in Azorhizobium caulinodans and Escherichia coli
Authors: Fernández‐López Manuel , D'Haeze Wim , Mergaert Peter , Verplancke Christa , Prome Jean‐Claude , Van Montagu Marc , Holsters Marcelle
Published: October 2006

Lipo‐chitooligosaccharide (LCO) Nod factors are produced and secreted by rhizobia and trigger nodule development in leguminous host plants. The products of the bacterial nodlJ genes are related to transporters of capsular polysaccharides and were proposed to be involved in LCO transport. We have studied nodlJ of Azorhizobium caulinodans ORS571 by analysis of cell‐associated and secreted radioactively labelled Nod factors in wild‐type ORS571, a nodJ mutant and a complemented strain. Secretion was strongly reduced in the nodJ mutant, and restored to wild‐type levels after complementation. Constructs were made for expression of combinations of different nod genes in Escherichia coli DH5a. The strain DH5α(pUCNABCSU) synthesized LCOs, but they were only secreted when a plasmid containing both nodl and nodJ was supplied in trans, nodi or nodJ alone was not sufficient. In E. coli as well as in Azorhizobium, the nod/J‐encoded transporter showed a specificity for more hydrophilic LCOs.
A glutamate/glutamine/aspartate/asparagine transport operon in Rhodobacter capsulatus
Authors: Zheng Su , Haselkorn Robert
Published: October 2006

A mutant of Rhodobacter capsulatus was identified in which an operon encoding a binding‐protein‐dependent transporter was interrupted by Tn5 transposition. Cloning and sequence analysis of the wild‐type operon revealed a four‐gene cluster with similarities to genes encoding periplasmic binding proteins (BztA), integral membrane proteins (BztB and BztC), and ATP‐binding proteins (BztD). To assess the function of this putative binding‐protein‐dependent transport system, a mutant was constructed in which most of the bztABCD operon was deleted and replaced by an antibiotic‐resistance marker. The deletion mutant grew more slowly than the wild type in NH‐free medium supplemented by glutamate, glutamine, aspartate or asparagine; it was resistant to toxic analogues of Glu, Asp, and Asn at concentrations that inhibited growth of the wild type; and it was defective in the uptake of Glu, Gin, and Asp. A complementing plasmid containing the wild‐type copy of bztABCD was able to rescue all the mutant phenotypes. Taken together, these results indicate that the proteins encoded by bztABCD are active in the uptake of Glu, Gin, Asp, and Asn. In addition, competition experiments, in which the ability of each of the four amino acids to compete for the transport of one another was examined, demonstrated that all four substrates share at least one component of this transport system.
Random initiation of replication of plasmids P1 and F (oriS) when integrated into the Escherichia coli chromosome
Authors: Ellasson Åsa , Bernander Rolf , Nordström Kurt
Published: October 2006

We have constructed intP1 and intFs strains of Escherichia coli in which the basic replicons of either plasmid P1 or plasmid F (oriS) were integrated into an inactivated oriC, such that chromosome replication is controlled by the integrated plasmid replicon. In this study, we have further analysed these strains, and density‐shift experiments revealed that chromosome replication occurred randomly during the cell cycle. Flow‐cytometry analyses of exponentially growing populations supported this conclusion, and also showed that the DNA/mass ratio of the strains decreased with increasing growth rate. Flow cytometry of exponentially growing cultures treated with rifampicin demonstrated that initiation of replication was uncoordinated in cells containing multiple replication origins.
PNPase modulates RNase II expression in Escherichia coli: implications for mRNA decay and cell metabolism
Authors: Zilhão Rita , Cairrão Fátima , Régnier Philippe , Arraiano Cecília M.
Published: October 2006

PNPase and RNase II are the key regulatory exo‐nucleases controlling mRNA decay in Escherichia coli The rnb transcripts were found to proceed through the terminator and PNPase was found to be involved in the 3’to 5’degradation of rnb mRNA. Analysis of these longer 3’termini revealed that they are located in UA‐rich regions. Comparison of single and double mutants suggested that PNPase and RNase II could have different roles in the degradation of these unstructured regions. We have shown that RNase II levels can vary over a fivefold range in haploid cells and that its expression depends on PNPase levels. PNPase‐deficient strains were found to have a 2–2.5‐fold increase in RNase M activity, while PNPase‐overproducing strains reduced the rnb message and RNase II levels. Conversely, the amount of PNPase in the rnb deletion strain was approximately twofold higher than that in the wild‐type strain. These observations suggest that the two main exonu‐cleases are inter‐regulated through a fine tuning mechanism. We discuss the implications of these results with regard to mRNA degradation and cell metabolism.
YopH of Yersinia pseudotuberculosis interrupts early phosphotyrosine signalling associated with phagocytosis
Authors: Andersson Kerstin , Carballeira Nivia , Magnusson Karl‐Eric , Persson Cathrine , Stendahl Olle , Wolf‐Watz Hans , Fällman Maria
Published: October 2006

The PTPase YopH of Yersinia is essential to the ability of these bacteria to block phagocytosis. Wild‐type Yersinia pseudotuberculosis, but not the yopH mutant strain, resisted phagocytosis by J774 cells. Ingestion of a yopH mutant was dependent on tyrosine kinase activity. Transcomplementation with wild‐type yopH restored the anti‐phagocytic effect, whereas introduction of the gene encoding the catalytically inactive yopHC403A was without effect. The PTPase inhibitor orthovanadate impaired the anti‐phagocytic effect of the wild‐type strain, further demonstrating the importance of bacteria‐derived PTPase activity for this event. The ability to resist phagocytosis indicates that the effect of the bacterium is immediately exerted when it becomes associated with the phagocyte. Within 30 s after the onset of infection, wild‐type Y. pseudotuberculosis caused a YopH‐dependent dephosphorylation of phosphotyrosine proteins in J774 cells. Furthermore, interaction of the cells with phagocytosable strains led to a rapid and transient increase in tyrosine phosphorylation of paxillin and some other proteins, an event dependent on the presence of the bacterial surface‐located protein invasin. Co‐infection with the phagocytosable strain and the wild‐type strain abolished the induction of tyrosine phosphorylation. Taken together, the present findings demonstrate an immediate YopH‐mediated dephosphorylation of macrophage phosphotyrosine proteins, suggesting that this PTPase acts by preventing early phagocytosis‐linked signalling in the phagocyte.
Countertranscript‐driven attenuation system of the pAMβ1 repE gene
Authors: Le Chatelier E. , Ehrlich S. D. , Jannière L.
Published: October 2006

The plasmid‐encoded RepE protein is absolutely essential and rate‐limiting for replication of the promiscuous plasmid pAMβ1 originating from Enterococcus faecalis. We previously showed that the rep gene is transcribed from a promoter that is negatively regulated (10‐fold reduction) by the CopF repressor. In this report, we show that this transcription is decreased a further 10‐times by a countertranscript‐driven transcriptional attenuation system. Extensive mutagenesis revealed that this system operates by a mechanism similar to that previously described for the unrelated repC gene of plasmid pT181.
Questions about gonococcal pilus phase‐ and antigenic variation
Authors: Seifert H. Steven
Published: October 2006

Pathogenic organisms inhabit one of several defined locations within a host where temperature, pH, and nutrients are relatively constant. While the microorganism must adapt to different environments within the host, the host immune system is the most formidable predator that can limit the growth of a pathogen. Neisseria gonorrhoeae (the gonococcus, Gc) is the causative agent of gonorrhoea, and has evolved several systems for varying the antigenicity of different surface antigens, presumably to help evade the effects of the human immune system. The On/Off/On phase variation of surface structure expression also alters the antigenic characteristics of the bacterial cell surface. Antigenic variation of the major subunit of the pilus, pilin, occurs by unidirectional, homologous recombination between a silent locus and the expression locus. The silent loci lie from 1 to 900 kb from the expression locus in the chromosome yet all can donate their sequences to the expression locus. The genetic composition of the pilin loci of two Gc strains has been elucidated, and the types of changes that lead to altered forms of the pilus have been extensively characterized. However, little is known about the precise molecular mechanisms used to allow high‐frequency, non‐reciprocal, chromosomal recombination between pilin loci or about what regulates the process of maintaining chromosome fidelity.
Genetic analysis of the transcriptional arrangement of Azotobacter vinelandii alginate biosynthetic genes: identification of two independent promoters
Authors: Lloret Lourdes , Barreto Rita , León Renato , Moreno Soledad , MartÍnez‐Salazar Jaime , Espín Guadalupe , Soberón‐Chávez Gloria
Published: October 2006

The study of alginate biosynthesis, the exopolysac charide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, might lead to different bio‐technological applications. Here we report the cloning of A. vinelandii algA, the gene coding for the bifunctional enzyme phosphomannose isomerase‐guano‐sine diphospho‐D‐mannose pyrophosphorylase (PMI‐GMP). This gene was selected by the complementation for xanthan gum production of Xanthomonas campestris pv. campestris xanB mutants, which lack this enzymatic activity. The complementing cosmid clones selected, besides containing algA, presented a gene coding for an alginate lyase activity (algL), and some of them also contained algD which codes for GDP‐mannose dehydrogenase. We present here the characterization of the A. vinelandii chromosomal region comprising algD and its promoter region, algA and algL, showing that, as previously reported for P. aeruginosa, A. vinelandii has a cluster of the biosynthetic alginate genes. We provide evidence for the presence of an algD‐independent promoter in this region which transcribes at least algL and algA, and which is regulated in a manner that differs from that of the algD promoter.
In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus
Authors: Strauss Andreas , Götz Friedrich
Published: October 2006

Many surface proteins of Gram‐positive bacteria are covalently anchored to the cell wall by a ubiquitous mechanism, involving a specific, C‐terminal sorting signal. To achieve cell‐wall immobilization of a normally secreted enzyme in vivo, we constructed a hybrid protein consisting of Staphylococcus hyicus lipase and the C‐terminal region of Staphylococcus aureus fibronectin binding protein B (FnBPB). This region comprised the authentic cell‐wall‐spanning region and cell‐wall sorting signal of FnBPB. Expression of the hybrid protein in Staphylococcus carnosus resulted in efficient cell‐wall anchoring of enzymatically active lipase. The cell‐wall‐immobilized lipase (approximately 10000 molecules per cell) retained more than 80% of the specific activity, compared to the C‐terminally unmodified S. hyicus lipase secreted by S. carnosus cells. After releasing the hybrid protein from the cell wall by lysostaphin treatment, its specific activity was indistinguishable from that of the unmodified lipase. Thus, the C‐terminal region of FnBPB per se was fully compatible with folding of the lipase to an active conformation. To study the influence of the distance between the cell‐wall sorting signal and the C‐terminus of the lipase on the activity of the immobilized lipase, the length of this spacer region was varied. Reduction of the spacer length gradually reduced the activity of the surface‐immobilized lipase. On the other hand, elongation of this spacer did not stimulate the activity of the immobilized lipase, indicating that the spacer must exceed a critical length of approx. 90 amino acids to allow efficient folding of the enzyme, which probably can only be achieved outside the pep‐tidoglycan web of the cell wall. When the lipase was replaced by another enzyme, the Escherichia coliβ‐lactamase, the resulting hybrid was also efficiently anchored in an active conformation to the cell wall of S, carnosus. These results demonstrate that it is possible to immobilize normally soluble enzymes on the cell wall of S. carnosus ‐ without radically altering their catalytic activity ‐ by fusing them to a cell‐wall‐immobilization unit, consisting of a suitable cellwall‐spanning region and a standard cell‐wall sorting signal.
The role of the Saccharomyces cerevisiae CCC1 gene in the homeostasis of manganese ions
Authors: Lapinskas Paula Jean , Lin Su‐Ju , Culotta Valeria Cizewski
Published: October 2006

We previously reported that oxidative damage in yeast lacking copper/zinc superoxide dismutase (SOD1) can be alleviated through mutations in PMR1, encoding a calcium P‐type ATPase homologue that also functions in manganese homeostasis. In an attempt to further understand the relationship between manganese ions, PMR1 and SOD1, we conducted a search for manganese homeostasis genes that interact with PMR1. A genomic library was screened for genes that, when overexpressed, suppress the manganese hypersensitivity associated with pmr1 mutations. A single clone was isolated that reduced manganese toxicity in both the pmr1 mutant and PMR1 wild‐type yeast. This gene was identified as CCC1, previously shown to function in calcium metabolism. Our studies indicate that, like PMR1, CCC1 functions in the homeostasis of both calcium and manganese ions. The Ccc1p polypeptide was found to localize to a Golgi‐like organelle in yeast cells. Ccc1 p co‐fractionated with a Golgi marker in subcellular fractionation studies and, with immunofluorescence microscopy, Ccc1 p exhibited a punctate pattern of staining typical of yeast Golgi. Our studies suggest that Ccc1 p may act to sequester manganese ions in this organelle and limit the intracellular availability of the metal. First, overexpression of CCC1 reduced manganese cytotoxicity without lowering total accumulation of the metal. Second, overexpression of CCC1 appeared to limit the intracellular availability of the manganese ions needed to support aerobic growth of SOD1 mutants. We provide a model in which Ccc1p and Pmr1p work together to control the intracellular partitioning of manganese ions.
The use of differential display‐PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages
Authors: Kwaik Yousef Abu , Pederson Lisa L.
Published: October 2006

The differential display (DD)‐PCR technique has been modified to identify prokaryotic cDNA fragments that are differentially induced by facultative intracellular bacteria in response to the intracellular environment of eukaryotic cells. Several DD‐PCR fragments identified from the intracellular bacterium Legionella pneumophila were induced at 4 h post‐infection of the U937 macrophage‐like cells. From these, a 700 bp fragment was cloned and sequenced. Neither the DNA sequence nor the predicted protein sequence from the open reading frame has similarity to other sequences in genetic databases. Transcription of the chromosomal locus containing the 700 bp fragment (eml, for early stage macrophage‐induced locus) was induced by intracellular bacteria during the first few hours post‐infection of macrophages but the expression was downregulated by 12 h post‐infection. Transcription of eml was not growth phase‐related in vitro, and was not affected by in vitro stress stimuli. A 3.7 kb EcoRI genomic fragment containing the 700 bp DD‐PCR product was cloned. Six mini‐Tn 10 insertions in the 3.7 kb EcoRI fragment were recombined into the L. pneumophila chromosome. Compared to the wild‐type strain, five of the eml isogenic mutants had a similar phenotype of reduced cytopathicity to the U937 cells, showed a 100‐fold increase in killing by macrophages during the first 5h of the intracellular infection, and showed a 100‐fold increase in killing during the first 24 h of infection of the amoeba Hartmanella vermiformis. The 6th mutant had a phenotype indistinguishable from the wild‐type strain. The cytopathicity defect of the mutants to the U937 cells was restored to wild‐type levels by complementation of the mutants with a plasmid containing the 3.7 kb EcoRI fragment. These data showed that the 3.7 kb fragment containing eml is a novel L. pneumophila locus whose expression is uniquely induced by non‐stress stimuli during early stages of the intracellular infection of phagocytic cells. Expression of this locus is required for survival of L. pneumophila within macrophages and within amoebae during early stages of the infection.
Integration host factor alleviates the H‐NS‐mediated repression of the early promoter of bacteriophage Mu
Authors: van Ulsen Peter , Hillebrand Marcel , Zulianello Laurence , van de Putte Pieter , Goosen Nora
Published: October 2006

Integration host factor (IHF), which is a histone‐like protein, has been shown to positively regulate transcription in two different ways. It can either help the formation of a complex between a transcription factor and RNA polymerase or it can itself activate RNA polymerase without the involvement of other transcription factors. In this study, we present a third mechanism for IHF‐stimulated gene expression, by counteracting the repression by another histone‐like protein, H‐NS. The early (Pe) promoter of bacteriophage Mu is specifically inhibited by H‐NS, both in vivo and in vitro. For this inhibition, H‐NS binds to a large DNA region overlapping the Pe promoter. Binding of IHF to a binding site just upstream of Pe alleviates the H‐NS‐mediated repression of transcription. This same ihf site is also involved in the direct activation of Pe by IHF. In contrast to the direct activation by IHF, however, the alleviating effect of IHF appears not to be dependent on the relevant position of the ihf site on the DNA helix, and it also does not require the presence of the C‐terminal domain of the alpha subunit of RNA polymerase. Footprint analysis shows that binding of IHF to the ihf site destabilizes the interaction of H‐NS with the DNA, not only in the IHF‐binding region but also in the DNA regions flanking the ihf site. These results suggest that IHF disrupts a higher‐order nucleoprotein complex that is formed by H‐NS and the DNA.
Examination of AsmA and its effect on the assembly of Escherichia coli outer membrane proteins
Authors: Deng Ming , Misra Rajeev
Published: October 2006

asmA mutations were isolated as extragenic suppressors of an OmpF assembly mutant, OmpF315. This suppressor locus produced a protein that was present in extremely low levels and could only be visualized by Western blotting in cells where AsmA expression was induced from a plasmid. Detailed fractionation analyses showed that AsmA localized with the inner membrane. Curiously, however, the mutant OmpF assembly step influenced by AsmA occurred in the outer membrane, perhaps indicating an indirect involvement of AsmA in the assembly of outer membrane proteins. Biochemical examination of the outer membrane showed that asmA null mutations reduce lipo‐polysaccharide (LPS) levels, thereby lowering the ratios of glycerolphospholipids to LPS and envelope proteins to LPS in the outer membrane. Despite these quantitative alterations, no apparent structural changes in LPS or major phospholipids were noted. Reduced LPS levels in asmA mutants indicate a possible role of AsmA in LPS biogenesis. Data presented in this study suggest that asmA‐mediated OmpF assembly suppression may have been achieved by altering the outer membrane fluidity, thus making it more amenable for the assembly of mutant proteins.
Differential regulation of multiple overlapping promoters in flagellar class II operons in Escherichia coli
Authors: Liu Xiaoying , Matsumura Philip
Published: October 2006

The Escherichia coli flagellar operons are divided into three categories: classes I, II and III. Expression of class II depends on expression of class I. One of the class II gene products, the FliA protein, is an alternative ß factor (ß28) required for transcription of the class III operons. In this study, we have characterized, in vitro, a role of ß28 in the regulation of the class II operons. Among the three class II operons examined, the fliA and fliL operons, but not the flhB operon, could be transcribed by both ß70 RNA polymerase holoenzyme with FlhD/C (Eß70‐FlhD/C) and ß28 RNA polymerase holoenzyme (Eß28). The flhB operon could only be transcribed by Eß70‐FlhD/C under the conditions used. Both the fliA and fliL operons contained two overlapping promoters oriented in tandem. The transcription of fliA directed by Eß28 could outcompete that by Eß70‐FlhD/C, indicating a positive autoregulation. However, Eß28 could not displace Eß70‐FlhD/C bound to the fliL promoter. The c28‐mediated positive regulation of the class II operons involved a mechanism in which ß28 competed with ß70 for core RNA polymerase. In addition, recruitment of core RNA polymerase from the ß70 ‐10 site to the ß28 ‐10 was facilitated by formation of Eß70‐FlhD/C pre‐initiation complex. Taken together, the three class II promoters investigated are different in terms of their regulation by ß28. We propose that class II operons may be further divided into different subcategories.
A secreted protein tyrosine phosphatase with modular effector domains in the bacterial pathogen Salmonella typhimurlum
Authors: Kaniga Koné , Uralil Jaimol , Bliska James B. , Galán Jorge E.
Published: October 2006

A number of bacterial pathogens have evolved sophisticated strategies to subvert host‐cell signal‐transduction pathways for their own benefit. These bacteria produce and export proteins capable of specific interactions with key mammalian cell regulatory molecules in order to derail the normal functions of the cells. In this study, we describe the identification of a modular effector protein secreted by the bacterial pathogen Salmonella typhimurium that is required for its full display of virulence. Sequence analysis revealed that a carboxy‐terminal region of this protein, which we have termed SptP, is homologous to the catalytic domains of protein tyrosine phosphatases. Purified SptP protein efficiently dephosphorylated peptide substrates phosphorylated on tyrosine. An engineered mutant of SptP in which a critical Cys residue in the catalytic domain was changed to Ser was devoid of phosphatase activity, indicating a catalytic mechanism similar to that of other tyrosine phosphatases. In addition, an amino‐terminal region of SptP exhibited sequence similarity to the ribosyltransferase exo‐enzyme S from Pseudomonas aeruginosa and the cytotoxin YopE from Yersinia spp. The modular nature of this effector protein may allow multiple interactions with host‐cell signalling functions.
Cis preference of the IS 903 transposase is mediated by a combination of transposase instability and inefficient translation
Authors: Derbyshire Keith M. , Grindley Nigel D. F.
Published: October 2006

The transposase protein encoded by the insertion element IS 903 belongs to an unusual class of DNA‐binding proteins, termed cis‐acting proteins, that act preferentially at their site of synthesis. Previous work had led us to propose that instability of the IS 903 transposase was a major determinant of its cis preference. Here we describe the isolation of two classes of mutations within the transposase gene that increased action in trans. One class specifically increased trans action without increasing the level of transposition when the mutant gene was located in cis to the transposon. In particular, a threonine‐to‐proline substitution at amino acid 25 (T25P) reduced cis preference about 60‐fold. The half‐life of this mutant transposase was significantly longer than that of the wild‐type transposase, confirming the critical role of protein instability. The second, larger, class of mutations increased the level of transposition both in trans and in cis. The behaviour and location of these mutations were consistent with an increase in gene expression by improving translational initiation. Several of these mutations exerted a disproportionate effect on the action of transposase in trans, implying that translation efficiency may affect more than just the amount of transposase made. Our results indicate that cis preference of the IS 903 transposase is mediated by a combination of transposase instability and inefficient translation initiation.
Mycoplasma pneumoniae cytadherence: unravelling the tie that binds
Authors: Krause Duncan C.
Published: October 2006

Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults. Mycoplasma adherence to the respiratory epithelium (cytadherence) is required for colonization and pathogenesis. Although considered to be among the smallest and simplest known prokaryotes, this cell‐wall‐less bacterium possesses a highly differentiated terminal structure that is thought to be functional in mycoplasma cell division, gliding motility, and cytadherence. Mutant analysis has identified mycoplasma proteins associated with cytadherence, and revealed novel regulatory features. Ultrastructural and biochemical studies have established the subcellular location and interaction of key components, several of which are phosphorylated by ATP‐dependent kinase(s) in a manner that is responsive to changing nutritional conditions. This review summarizes recent progress in defining the composition, organization and regulation of the attachment organelle. What emerges is a picture of M. pneumoniae cytadherence as a multifactorial process that extends well beyond adhesin‐receptor recognition.
Molecular genetic bases of Salmonella entry into host cells
Authors: Galán Jorge E.
Published: October 2006

Salmonella spp. can enter into non‐phagocytic cells, a property that is essential for their pathogenicity. Recently, considerable progress has been made in the understanding of the molecular genetic bases of this process. It is now evident that Salmonella entry functions are largely encoded on a 35–40 kb region of the Salmonella chromosome located at centisome 63. The majority of the loci in this region encode components of a type III or contact‐dependent secretion system homologous to those described in a variety of animal and plant‐pathogenic bacteria as well as a number of proteins that require this system for their export to the extracellular environment. A somewhat unexpected finding has been the remarkable homology between the Salmonella and Shigella proteins that mediate the entry of these organisms into cultured epithelial cells.
Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis
Authors: Salazar Leirla , Fsihi Hafida , de Rossi Edda , Riccardi Giovanna , Rios Carmen , Cole Stewart T. , Takiff Howard E.
Published: October 2006

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA‐rpmH‐dnaA‐dnaN‐recF‐orf‐gyrB‐gyrA, is the same as in other Gram‐positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA‐dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.
DNA supercoiling depends on the phosphorylation potential in Escherichia coli
Authors: van Workum Marielle , van Dooren Silvy J. M. , Oldenburg Nienke , Molenaar Douwe , Jensen Peter R. , Snoep Jacky L. , Westerhoff' Hans V.
Published: October 2006

ATP/ADP ratios were varied in different ways and the degree of negative supercoiling was determined in Escherichia coli. Independent of whether the ATP/ADP ratio was reduced by a shift to anaerobic conditions, by addition of a protonophore (dinitrophenol) or by potassium cyanide addition, DNA supercoiling decreased similarly with the ATP/ADP ratio. The experiments were performed under well‐defined conditions, where oxidative phosphorylation was the dominant route for ATP synthesis, i.e. using a minimal salts medium with succinate as the sole free‐energy and carbon source, and in the presence or absence of ammonia as the nitrogen source. The results of the different experiments were consistent with a single linear relationship between the log(ATP/ADP) and the change in linking number. The dependence of DNA supercoiling on the ATP/ADP ratio was not influenced by inhibitors of transcription or translation. Because the ATP/ADP ratio was modulated in different ways, the unique relationship suggests coupling between the phosphorylation potential and DNA supercoiling. This was most probably mediated by the DNA gyrase, independent of topoisomerase I or transcription.
Co‐operative binding of two Trp repressor dimers to α‐ or β‐centred trp operators
Authors: Günes Çagatay , Staacke Detlev , von Wilcken‐Bergmann Brigitte , Müller‐Hill Benno
Published: October 2006

The α‐centred trp operator binds one dimer of the Trp repressor, whereas the β‐centred trp operator binds two dimers of the Trp repressor (Carey et al., 1991; Haran et al., 1992). The Trp repressor with a Tyr‐Gly‐7 substitution binds almost as well as the wild‐type Trp repressor to the α‐centred trp operator, but it does not bind to the β‐centred trp operator. This confirms that Tyr‐7 is involved in the interaction between Trp repressor dimers, as seen in the crystal structure (Lawson and Carey, 1993). Further experiments with a‐centred trp operator variants showed that positions 1 of the a‐centred trp operators play a crucial role in tetramerisation. The two innermost base pairs of the α‐centred trp operator are not involved in contacts with the dimer of the Trp repressor binding to it. However, substitutions in these positions (T‐A to G‐T) effectively transform the α‐centred trp operator into a β‐centred trp operator, and thus encourage the binding of two Trp repressor dimers to this operator. Finally, we demonstrate, with suitable heterodimers, that one subunit of each dimer suffices to bind to a β‐centred trp operator.
A 38 kDa precursor protein of aqualysin I (a thermophilic subtilisin‐type protease) with a C‐terminal extended sequence: its purification and in vitro processing
Authors: Kurosaka Keisuke , Ohta Takahisa , Matsuzawa Hiroshi
Published: October 2006

The precursor of aqualysin I, an extracellular subtilisin‐type protease produced by Thermus aquaticus, consists of four domains: an N‐terminal signal peptide, an N‐terminal pro‐sequence, a protease domain, and a C‐terminal extended sequence. In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C‐terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65°C. The 38 kDa precursor protein is separated as a soluble form from denatured E. coli proteins after heat treatment. Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography. The purified precursor protein gave a single band on SDS‐polyacrylamide gels. The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The purified precursor protein was processed to the mature enzyme upon heat treatment. The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C‐terminal extended sequence occurs through an intermolecular self‐processing mechanism.
Autoregulation of luxR: the Vibrio harveyi lux‐operon activator functions as a repressor
Authors: Chatterjee Jaidip , Miyamoto Carol M. , Meighen Edward A.
Published: October 2006

Mobility‐shift assays have been used to demonstrate that the activator of the Vibrio harveyi lux operon, LuxR, binds independently, and with similar affinity, to two sites upstream of its own open reading frame. One site was located between 52 and 107 bp upstream of, and the other site in a region 25 bp downstream of, the transcriptional start site. The luxR promoter, in a transcriptional fusion with the chloramphenicol acetyl transferase (cat) gene, could readily be expressed in Escherichia coli as well as V. harveyi in the absence of LuxR. In both species, the presence of the luxR gene product resulted in repression of luxR promotion. These results show that LuxR directly regulates its own expression by functioning as an autorepressor. A mechanism for this repression is suggested by evidence showing that LuxR has a negative effect on RNA polymerase binding to the luxR promoter. In light of the fact that LuxR is also part of a regulatory family of repressors, the mechanism by which LuxR functions as a transcriptional activator of the lux operon has been re‐examined.