bluuebunny/crossref_metadata_2025 huggingface dataset:
Insertion of EspD into epithelial target cell membranes by infecting enteropathogenic Escherichia coli
Authors: Wachter Clemens , Beinke Christina , Mattes Michael , Schmidt M. Alexander
Published: March 2003

Diffusely adhering Escherichia coli (DAEC) strains have been implicated in epidemiological studies as a cause of diarrhoea in children. However, the molecular interactions of these pathogens with target cells have remained largely obscure. We found that some DAEC strains contain homologues of the locus of enterocyte effacement (LEE) pathogenicity island and secrete EspA, EspB and EspD proteins necessary for the formation of the attaching and effacing (A/E) lesions. To characterize the function of the EspD protein further, we cloned and sequenced the espD genes of two DA‐EPEC strains and compared their deduced amino‐acid sequences with known EspD sequences. A pattern of two conserved transmembrane regions and one conserved coiled‐coil region is predicted in EspD and also in the type III system secreted proteins YopB, PopB, IpaB and SipB of Yersinia, Pseudomonas, Shigella and Salmonella respectively. The EspD protein is inserted into a trypsin‐sensitive location in the HeLa cell membrane at sites of bacterial contact, but is not translocated into the cytoplasm. Secretion of EspD increases upon contact with host cells. We propose that the membrane‐located EspD protein is part of the translocation apparatus for Esp proteins into the target host cell performing functions similar to YopB in Yersinia.
Autoregulation of lactose uptake through the LacYpermease by enzyme IIAGlc of the PTS in Escherichia coli K‐12
Authors: Hogema Boris M. , Arents Jos C. , Bader Rechien , Postma Pieter W.
Published: March 2003

Bacterial growth on one or more carbon sources requires careful control of the uptake and metabolism of these carbon sources. In Escherichia coli, the phosphorylation state of enzyme IIAGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is involved in this control in two ways. The unphosphorylated form of IIAGlc causes ‘inducer exclusion’, the inhibition of uptake of a number of non‐PTS carbon sources, including lactose uptake by the lactose permease. The phosphorylated form of enzyme IIAGlc probably activates adenylate cyclase. In cells growing on lactose, enzyme IIAGlc was approximately 50% dephosphorylated, suggesting that lactose could inhibit its own uptake. This inhibition could be demonstrated by comparing lactose uptake rates in the wild‐type strain and in a mutant in which the lactose carrier was insensitive to inducer exclusion. In this deregulated mutant strain, lactose was consumed much faster, and large amounts of glucose were excreted. It was shown that enzyme IIAGlc was dephosphorylated more strongly and that the cAMP level was lower in the mutant, most probably causing the observed decrease in lac expression level. When the lac expression level in the mutant strain was increased to that of the parent strain by adding exogenous cAMP, growth on lactose was slower, suggesting that enzyme IIAGlc‐mediated inhibition of lactose uptake and downregulation of the lac expression level protected the cells against excessive lactose influx. An even stronger increase in the lac expression level in a mutant lacking enzyme IIAGlc caused complete growth arrest. We conclude that the autoregulatory mechanism that controls lactose uptake is an important mechanism for the cells in adjusting the uptake rate to their metabolic capacity.
σM, an ECF RNA polymerase sigma factor of Bacillus subtilis 168, is essential for growth and survival in high concentrations of salt
Authors: Horsburgh Malcolm J. , Moir Anne
Published: March 2003

The Bacillus subtilis 168 genome encodes seven extracytoplasmic function (ECF) RNA polymerase sigma factors of unknown physiological function. The sigM(yhdM ) gene, encoding an ECF sigma factor σM, is essential for growth and survival in nutrient broth (NB) containing 1.4 M NaCl. Strains insertionally inactivated in the sigM gene form aberrantly shaped cells, which swell and lyse spontaneously during growth in NB medium containing increased levels (0.35–0.7 M) of a wide range of different salts. The sigM gene was co‐transcribed with the yhdL and yhdK genes with transcription initiating from two promoters, PA and PM. The transcript from PM was not detected in a sigM mutant, indicating that the expression of sigM was positively autoregulated. Expression of sigM was maximal during exponential growth and was increased by 50% in NB medium containing 0.7 M NaCl. The activity of σM is negatively regulated by the proteins encoded by the yhdL and yhdK genes.
Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!
Authors: Fisher Susan H.
Published: March 2003

Nitrogen metabolism genes of Bacillus subtilis are regulated by the availability of rapidly metabolizable nitrogen sources, but not by any mechanism analogous to the two‐component Ntr regulatory system found in enteric bacteria. Instead, at least three regulatory proteins independently control the expression of gene products involved in nitrogen metabolism in response to nutrient availability. Genes expressed at high levels during nitrogen‐limited growth are controlled by two related proteins, GlnR and TnrA, which bind to similar DNA sequences under different nutritional conditions. The TnrA protein is active only during nitrogen limitation, whereas GlnR‐dependent repression occurs in cells growing with excess nitrogen. Although the nitrogen signal regulating the activity of the GlnR and TnrA proteins is not known, the wild‐type glutamine synthetase protein is required for the transduction of this signal to the GlnR and TnrA proteins. Examination of GlnR‐ and TnrA‐regulated gene expression suggests that these proteins allow the cell to adapt to growth during nitrogen‐limited conditions. A third regulatory protein, CodY, controls the expression of several genes involved in nitrogen metabolism, competence and acetate metabolism in response to growth rate. The highest levels of CodY‐dependent repression occur in cells growing rapidly in a medium rich in amino acids, and this regulation is relieved during the transition to nutrient‐limited growth. While the synthesis of amino acid degradative enzymes in B. subtilis is substrate inducible, their expression is generally not regulated in response to nitrogen availability by GlnR and TnrA. This pattern of regulation may reflect the fact that the catabolism of amino acids produced by proteolysis during sporulation and germination provides the cell with substrates for energy production and macromolecular synthesis. As a result, expression of amino acid degradative enzymes may be regulated to ensure that high levels of these enzymes are present in sporulating cells and in dormant spores.
The genetic basis of tetrathionate respiration in Salmonella typhimurium
Authors: Hensel Michael , Hinsley Andrew P. , Nikolaus Thomas , Sawers Gary , Berks Ben C.
Published: March 2003

A range of bacteria are able to use tetrathionate as a terminal respiratory electron acceptor. Here we report the identification and characterization of the ttrRSBCA locus required for tetrathionate respiration in Salmonella typhimurium LT2a. The ttr genes are located within Salmonella pathogenicity island 2 at centisome 30.5. ttrA, ttrB and ttrC are the tetrathionate reductase structural genes. Sequence analysis suggests that TtrA contains a molybdopterin guanine dinucleotide cofactor and a [4Fe–4S] cluster, that TtrB binds four [4Fe–4S] clusters, and that TtrC is an integral membrane protein containing a quinol oxidation site. TtrA and TtrB are predicted to be anchored by TtrC to the periplasmic face of the cytoplasmic membrane implying a periplasmic site for tetrathionate reduction. It is inferred that the tetrathionate reductase, together with thiosulphate and polysulphide reductases, make up a previously unrecognized class of molybdopterin‐dependent enzymes that carry out the reductive cleavage of sulphur–sulphur bonds. Cys‐256 in TtrA is proposed to be the amino acid ligand to the molybdopterin cofactor. TtrS and TtrR are the sensor and response regulator components of a two‐component regulatory system that is absolutely required for transcription of the ttrBCA operon. Expression of an active tetrathionate reduction system also requires the anoxia‐responsive global transcriptional regulator Fnr. The ttrRSBCA gene cluster confers on Escherichia coli the ability to respire with tetrathionate as electron acceptor.
Phage Mu transposition immunity reflects supercoil domain structure of the chromosome
Authors: Manna Dipankar , Higgins N. Patrick
Published: March 2003

Transposition immunity is the negative influence that the presence of one transposon sequence has on the probability of a second identical element inserting in the same site or in sites nearby. A transposition‐defective Mu derivative (MudJr1) produced transposition immunity in both directions from one insertion point in the Salmonella typhimurium chromosome. To control for the sequence preference of Mu transposition proteins, Tn10 elements were introduced as targets at various distances from an immunity‐conferring MudJr1 element. Mu transposition into a Tn10 target was not detectable when the distance of separation from MudJr1 was 5 kb, and transposition was unencumbered when the separation was 25 kb. Between 5 kb and 25 kb, immunity decayed gradually with distance. Immunity decayed more sharply in a gyrase mutant than in a wild‐type strain. We propose that Mu transposition immunity senses the domain structure of bacterial chromosomes.
Heat‐induced cell cycle arrest of Saccharomyces cerevisiae : involvement of the RAD6/UBC2 and WSC2 genes in its reversal
Authors: Raboy Bilha , Marom Anat , Dor Yuval , Kulka Richard G.
Published: March 2003

The Saccharomyces cerevisiae RAD6 (UBC2 ) gene encodes a ubiquitin‐conjugating enzyme that is involved in a wide range of cellular processes including DNA repair, sporulation and N‐end rule protein degradation. Under mild heat stress conditions (37–38°C) rad6 null and rad6–149 mutant cells are unable to grow. The molecular basis for this failure to grow is unknown. Here we show that the heat sensitivity of rad6 mutants is not due to cell death but to an inability to progress in the cell cycle. The temperature‐induced cell cycle arrest of these mutants is due to a block in a branch of the RAD6 pathway distinct from the DNA repair and the N‐end rule protein degradation pathways. Wild‐type cells heated to 38°C arrest transiently in the late G1 phase and then resume growth. At 38°C rad6 mutant cells arrest in late G1 but, unlike wild‐type cells, are unable to resume cell cycle progression. In both wild‐type and in rad6 mutant cells, CLN1 and CLN2 transcript levels fall sharply upon temperature increase. In wild‐type cells levels of these transcripts recover rapidly, whereas in the rad6 mutant they recover slowly. As rad6 cells remain arrested even after CLN1 and CLN2 mRNAs regain their preheat stress levels, factors additional to reduced G1 cyclin gene expression must cause the temperature‐induced cell cycle block of the mutant. To identify genes involved in the relief of the cell cycle arrest under heat stress, we screened a multicopy yeast genomic library for clones that restore the growth of the rad6‐149 mutant. A plasmid was isolated carrying the WSC2 gene, which is closely related to WSC1/SLG1/HCS77, a putative membrane heat sensor. Overexpression of WSC2 reverses the heat‐induced cell cycle arrest of rad6‐149 but not of rad6 null mutants. Taken together the findings point to the existence of an unidentified heat stress‐activated cell cycle checkpoint pathway, which is antagonized by Rad6p by a mechanism also involving Wsc2p.
The identification of three biologically relevant globotriaosyl ceramide receptor binding sites on the Verotoxin 1 B subunit
Authors: Bast D. J. , Banerjee L. , Clark C. , Read R. J. , Brunton J. L.
Published: March 2003

The Verotoxin 1 (VT1) B subunit binds to the glycosphingolipid receptor globotriaosylceramide (Gb3). Receptor‐binding specificity is associated with the terminally linked Galα(1–4) Galβ disaccharide sequence of the receptor. Recently, three globotriose (Galα[1–4] Galβ [1–4] Glcβ) binding sites per B‐subunit monomer were identified by crystallography. Two of these sites (sites I and II) are located adjacent to phenylalanine‐30. Site I was originally predicted as a potential Gb3 binding site on the basis of sequence conservation, and site II was additionally predicted based on computer modelling and receptor docking. The third (site III) was also identified by crystallography and is located at the N‐terminal end of the α‐helix. To determine the biological significance of sites II and III, and to support our previous findings of the significance of site I, we examined the binding properties and cytotoxicity of VT1 mutants designed to block Gb3 binding at each site selectively. The Scatchard analysis of saturation‐binding data for each mutant revealed that only the amino acid substitutions predicted to affect site I (D‐17E) or site II (G‐62T) caused reductions in the binding affinity and capacity of VT1 for Gb3. Similarly, those mutations at sites I and II also caused significant reductions in both Vero and MRC‐5 cell cytotoxicity (by seven and five logs, respectively, for G‐62T and by four and two logs, respectively, for D‐17E). In contrast, the substitution of alanine for W‐34 at site III did not reduce the high‐affinity binding of the B subunit, despite causing a fourfold reduction in the receptor‐binding capacity. The corresponding mutant W‐34A holotoxin had a two‐log reduction in cytotoxicity on Vero cells and no statistically significant reduction on MRC‐5 cells. We conclude that the high‐affinity receptor binding most relevant for cell cytotoxicity occurs at sites I and II. In contrast, site III appears to mediate the recognition of additional Gb3 receptor epitopes but with lower affinity. Our results support the significance of the indole ring of W‐34 for binding at this site.
The 5′ region of cnf1 harbours a translational regulatory mechanism for CNF1 synthesis and encodes the cell‐binding domain of the toxin
Authors: Fabbri A. , Gauthier M. , Boquet P.
Published: March 2003

The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) is organized into three functional domains: the N‐terminal part containing the cell‐binding domain, a putative central membrane‐spanning region, and a C‐terminal catalytic region. On the basis of competition assays between CNF1 and GST‐recombinant proteins containing different N‐terminal fragments, and point mutations, we restricted the binding region to the first 190 amino acids. Hydrophilic amino acids 53–75 are strictly necessary to cell receptor recognition. Using different cnf1–lacZ translational fusions, we demonstrated that the mRNA corresponding to the first 48 codons of cnf1 is involved in the translational regulation of CNF1 synthesis. This regulation consists of both a positive and a negative control. The positive control is exerted by codons 6–20, including a putative downstream box that enhances the translational expression of cnf1. The negative control depends on codons 45–48. In this region, an anti‐Shine–Dalgarno sequence, highly homologous to the core of the internal complementary sequence already reported for growth rate‐regulated metabolic genes, has been detected. To some extent, the inner structural organization of CNF1 would thus suggest the compiling of several functions in a single mRNA protein system.
Na+ translocation by the NADH:ubiquinone oxidoreductase (complex I) from Klebsiella pneumoniae
Authors: Krebs Walter , Steuber Julia , Gemperli Anja C. , Dimroth Peter
Published: March 2003

Complex I is the site for electrons entering the respiratory chain and therefore of prime importance for the conservation of cell energy. It is generally accepted that the complex I‐catalysed oxidation of NADH by ubiquinone is coupled specifically to proton translocation across the membrane. In variance to this view, we show here that complex I of Klebsiella pneumoniae operates as a primary Na+ pump. Membranes from Klebsiella pneumoniae catalysed Na+‐stimulated electron transfer from NADH or deaminoNADH to ubiquinone‐1 (0.1–0.2 μmol min−1 mg−1). Upon NADH or deaminoNADH oxidation, Na+ ions were transported into the lumen of inverted membrane vesicles. Rate and extent of Na+ transport were significantly enhanced by the uncoupler carbonylcyanide‐m‐chlorophenylhydrazone (CCCP) to values of ≈0.2 μmol min−1 mg−1 protein. This characterizes the responsible enzyme as a primary Na+ pump. The uptake of sodium ions was severely inhibited by the complex I‐specific inhibitor rotenone with deaminoNADH or NADH as substrate. N‐terminal amino acid sequence analyses of the partially purified Na+‐stimulated NADH:ubiquinone oxidoreductase from K. pneumoniae revealed that two polypeptides were highly similar to the NuoF and NuoG subunits from the H+‐translocating NADH:ubiquinone oxidoreductases from enterobacteria.
Co‐ordination of Legionella pneumophila virulence with entry into stationary phase by ppGpp
Authors: Hammer Brian K. , Swanson Michele S.
Published: March 2003

Legionella pneumophila survives in aquatic environments, but replicates within amoebae or the alveolar macrophages of immunocompromised individuals. Here, the signal transduction pathway that co‐ordinates L. pneumophila virulence expression in response to amino acid depletion was investigated. To facilitate kinetic and genetic studies, a phenotypic reporter of virulence was engineered by fusing flaA promoter sequences to a gene encoding green fluorescent protein. When subjected to amino acid depletion, L. pneumophila accumulated ppGpp and converted from a replicative to a virulent state, as judged by motility and sodium sensitivity. ppGpp appeared to initiate this response, as L. pneumophila induced to express the Escherichia coli RelA ppGpp synthetase independently of nutrient depletion accumulated ppGpp, exited the exponential growth phase and expressed flaAgfp, motility, sodium sensitivity, cytotoxicity and infectivity, five traits correlated with virulence. Although coincident with the stationary phase, L. pneumophila virulence expression appeared to require an additional factor: mutant Lp120 accumulated ppGpp and acquired two stationary phase traits but none of six virulence phenotypes analysed. We propose that, when nutrients are limiting, ppGpp acts as an alarmone, triggering the expression of multiple traits that enable L. pneumophila to escape its spent host, to survive and disperse in the environment and to re‐establish a protected intracellular replication niche.
The 213‐amino‐acid leucine‐rich repeat region of the Listeria monocytogenes InlB protein is sufficient for entry into mammalian cells, stimulation of PI 3‐kinase and membrane ruffling
Authors: Braun Laurence , Nato Farida , Payrastre Bernard , Mazié Jean‐Claude , Cossart Pascale
Published: March 2003

The Listeria monocytogenes InlB protein is a 630‐amino‐acid surface protein that mediates entry of the bacterium into a wide variety of cell types, including hepatocytes, fibroblasts and epithelial cells such as Vero, HEp‐2 and HeLa cells. Invasion stimulates host proteins tyrosine phosphorylation, PI 3‐kinase activity and rearrangements in the actin cytoskeleton. We previously showed that InlB is sufficient for entry of InlB‐coated latex beads into cells and recent results indicate that purified InlB can stimulate PI 3‐kinase activity and is thus the first bacterial agonist of this lipid kinase. In this study, we identified the region of InlB responsible for entry and stimulation of signal transduction events. Eight monoclonal antibodies directed against InlB were raised and, of those, five inhibited bacterial entry. These five antibodies recognized epitopes within the leucine‐rich repeat (LRR) region and/or the inter‐repeat (IR) region. InlB‐staphylococcal protein A (SPA) fusion proteins and recombinant InlB derivatives were generated and tested for their capacity to mediate entry into cultured mammalian cells. All the InlB derivatives that carried the amino‐terminal 213‐amino‐acid LRR region conferred invasiveness to the normally non‐invasive bacterium L. innocua or to inert latex beads and the corresponding purified polypeptides inhibited bacterial entry. In addition, the 213‐amino‐acid LRR region was able to stimulate PI 3‐kinase activity and changes in the actin cytoskeleton (membrane ruffling). These properties were not detected with purified internalin, another invasion protein of L. monocytogenes that displays LRRs similar to those of InlB. Taken together, these results show that the first 213 amino acids of InlB are critical for its specific properties.
Probing secretion and translocation of a β‐autotransporter using a reporter single‐chain Fv as a cognate passenger domain
Authors: Veiga Esteban , De Lorenzo Víctor , Fernández Luis A.
Published: March 2003

The mechanism of protein secretion mediated by the β‐domain of theNeisseria gonorrhoeaeIgA protease, a paradigm of a family of secreted polypeptides of Gram‐negative bacteria called autotransporters, has been examined using a single‐chain antibody (scFv) as a reporter passenger domain to monitor the translocation process. Fusion of a scFv to the β‐module of the IgA protease allowed us to investigate the passage of the chimeric protein through the periplasm, its insertion into the outer membrane and the movement of the N‐terminal moiety towards the cell surface. As the binding activity of the scFv to its target antigen is entirely dependent on the formation of disulphide bonds, the relationship between secretion, folding and formation of S–S bridges could be analysed in detail. In contrast to the current notion that only an unfolded N‐passenger domain can be translocated through the β‐domain, our results show that the scFv is able to pass through the outer membrane, albeit at a threefold reduced level, in an active conformation with its disulphide bonds preformed in the periplasm through the action of the DsbA product. These data call for a re‐evaluation of the prevailing model for secretion of the N‐domain of autotransporters.
A hierarchical quorum‐sensing system in Yersinia pseudotuberculosis is involved in the regulation of motility and clumping
Authors: Atkinson Steve , Throup John P. , Stewart Gordon S.A.B. , Williams Paul
Published: March 2003

In cell‐free Yersinia pseudotuberculosis culture supernatants, we have chemically characterized three N‐acyl homoserine lactone (AHL) molecules, N‐octanoyl homoserine lactone (C8‐HSL), N‐(3‐oxohexanoyl)homoserine lactone (3‐oxo‐C6‐HSL) and N‐hexanoyl homoserine lactone (C6‐HSL). We have identified, cloned and sequenced two pairs of LuxR/I homologues termed YpsR/I and YtbR/I. In Escherichia coli at 37°C, YpsI and YtbI both synthesize C6‐HSL, although YpsI is responsible for 3‐oxo‐C6‐HSL and YtbI for C8‐HSL synthesis respectively. However, in a Y. pseudotuberculosis ypsI‐negative background, YtbI appears capable of adjusting the AHL profile from all three AHLs at 37°C and 22°C to the absence of 3‐oxo‐C6‐HSL at 28°C. Insertion deletion mutagenesis of ypsR leads to the loss of C8‐HSL at 22°C, which suggests that at this temperature the YpsR protein is involved in the hierarchical regulation of the ytbR/I locus. When compared with the parent strain, the ypsR and ypsI mutants exhibit a number of phenotypes, including clumping (ypsR mutant), overexpression of a major flagellin subunit (ypsR mutant) and increased motility (both ypsR and ypsI mutants). The clumping and motility phenotypes are both temperature dependent. These data are consistent with a hierarchical quorum‐sensing cascade in Y. pseudotuberculosis that is involved in the regulation of clumping and motility.
Hierarchy in the expression of the locus of enterocyte effacement genes of enteropathogenic Escherichia coli
Authors: Friedberg Devorah , Umanski Tatiana , Fang Yuan , Rosenshine Ilan
Published: March 2003

Enteropathogenic Escherichia coli (EPEC) elicit changes in host cell morphology and cause actin rearrangement, a phenotype that has commonly been referred to as attaching/effacing (AE) lesions. The ability of EPEC to induce AE lesions is dependent upon a type III protein secretion/translocation system that is encoded by genes clustered in a 35.6 kb DNA segment, named the locus of enterocyte effacement (LEE). We used transcriptional fusions between the green fluorescent protein (gfp) reporter gene and LEE genes rorf2, orf3, orf5, escJ, escV and eae, together with immunoblot analysis with antibodies against Tir, intimin, EspB and EspF, to analyse the genetic regulation of the LEE. The expression of all these LEE genes was strictly dependent upon the presence of a functional integration host factor (IHF). IHF binds specifically upstream from the ler (orf1) promoter and appears to activate expression of ler, orf3, orf5 and rorf2 directly. The ler‐encoded Ler protein was involved in activating the expression of escJ, escV, tir, eae, espB and espF. Expression of both IHF and Ler was needed to elicit actin rearrangement associated with AE lesions. In conclusion, IHF directly activates the expression of the ler and rorf2 transcriptional units, and Ler in turn mediates the expression of the other LEE genes.
Emergency derepression: stringency allows RNA polymerase to override negative control by an active repressor
Authors: Kvint Kristian , Hosbond Carsten , Farewell Anne , Nybroe Ole , Nyström Thomas
Published: March 2003

The uspA promoter, driving production of the universal stress protein A in response to diverse stresses, is demonstrated to be under dual control. One regulatory pathway involves activation of the promoter by the alarmone guanosine 3′,5′‐bisphosphate, via the β‐subunit of RNA polymerase, whereas the other consists of negative control by the FadR repressor. In contrast to canonical dual control by activation and repression circuits, which depends on concomitant activation and derepression for induction to occur, the ppGpp‐dependent activation of the uspA promoter overrides repression by an active FadR under conditions of severe cellular stress (starvation). The ability of RNA polymerase to overcome repression during stringency depends, in part, on the strength of the FadR operator. This emergency derepression is operative on other FadR‐regulated genes induced by starvation and is argued to be an essential regulatory mechanism operating during severe stress.
Expression cloning of the Candida albicans CSA1 gene encoding a mycelial surface antigen by sorting of Saccharomyces cerevisiae transformants with monoclonal antibody‐coated magnetic beads
Authors: Lamarre Claude , Deslauriers Noëlla , Bourbonnais Yves
Published: March 2003

The mycelial surface antigen recognized by monoclonal antibody (mAb) 4E1 has previously been shown to be present predominantly in the terminal third of the hyphal structures in Candida albicans. We report here the expression cloning of the corresponding gene (CSA1 ) by mAb 4E1‐coated magnetic beads sorting of Saccharomyces cerevisiae transformants expressing a C. albicans genomic library. The strategy is both highly selective and highly sensitive and provides an additional genetic tool for the cloning and characterization of C. albicans genes encoding surface proteins. CSA1 is an intronless gene encoding a 1203‐residue protein composed of repetitive motifs and domains. Northern analysis indicates that CSA1 is preferentially expressed during the mycelial growth phase, although a low level of CSA1 mRNA can be detected in the yeast form. As evidenced by indirect immunofluorescence microscopy with mAb 4E1, Csa1p is not randomly distributed over the surface of yeast cells, but localizes predominantly in the growing buds. This suggests that the distribution of Csa1p may be restricted to sites of cell surface elongation. Both heterozygous and homozygous C. albicans csa1Δ mutants are viable. Upon induction of mycelial growth, the number and size of hyphal structures derived from the mutants are similar to those observed in the parental wild‐type strain. The physiological role of Csa1p has yet to be determined. However, the presence in Csa1p of repeated cysteine‐rich hydrophobic domains with significant sequence similarity to motifs found in surface proteins (Ag2 and Pth11) from two distantly related fungal pathogens (Coccidioides immitis and Magnaporthe grisea respectively) suggests a common function in host interaction.
In vivo and in vitro studies of transmembrane β‐strand deletion, insertion or substitution mutants of the Escherichia coli K‐12 maltoporin
Authors: Charbit Alain , Andersen Christian , Wang Jiang , Schiffler Bettina , Michel Valérie , Benz Roland , Hofnung Maurice
Published: March 2003

LamB of Escherichia coli K12, also called maltoporin, is an outer membrane protein, which specifically facilitates the diffusion of maltose and maltodextrin through the bacterial outer membrane. Each monomer is composed of an 18‐stranded antiparallel β‐barrel. In the present work, on the basis of the known X‐ray structure of LamB, the effects of modifications of the β‐barrel domain of maltoporin were studied in vivo and in vitro. We show that: (i) the substitution of the pair of strands β13–β14 of the E. coli maltoporin with the corresponding pair of strands from the functionally related maltoporin of Salmonella typhimurium yielded a protein active in vivo and in vitro; and (ii) the removal of one pair of β‐strands (deletion β13–β14) from the E. coli maltoporin, or its replacement by a pair of strands from the general porin OmpF of E. coli, leads to recombinant proteins that lost in vivo maltoporin activities but still kept channel formation and carbohydrate binding in vitro. We also inserted into deletion β13–β14 the portion of the E. coli LamB protein comprising strands β13 to β16. This resulted in a protein expected to have 20 β‐strands and which completely lost all LamB‐specific activities in vivo and in vitro.
The development of a FACS‐based strategy for the isolation of Shigella flexneri mutants that are deficient in intercellular spread
Authors: Rathman Michelle , Jouirhi Noureddine , Allaoui Abdelmounaaïm , Sansonetti Philippe , Parsot Claude , Tran Van Nhieu Guy
Published: March 2003

In the disease course of bacillary dysentery, pathogenic Shigella flexneri invade colonic epithelial cells and spread both within and between host cells. The ability to spread intercellularly allows the organism to infect an entire epithelial layer without significant contact with the extracellular milieu. Using fluorescence activated cell sorter (FACS)‐based technology, we developed a rapid and powerful selection strategy for the isolation of S. flexneri mutants that are unable to spread from cell to cell. The majority of mutants identified using this strategy harbour mutations that affect the structure of their lipopolysaccharide or the ability of the bacteria to move intracellularly via actin‐based motility; both factors have previously been shown to be essential for cell‐to‐cell spread. However, using a modified strategy that eliminated both of these types of mutants, we identified several mutants that provide us with evidence that bacterial proteins of the type III secretion system, which are essential for bacterial entry into host cells, also play a role in cell‐to‐cell spread.
NtcA represses transcription of gifA and gifB, genes that encode inhibitors of glutamine synthetase type I from Synechocystis sp. PCC 6803
Authors: García‐Domínguez Mario , Reyes José C. , Florencio Francisco J.
Published: March 2003

Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) activity is controlled by direct interaction with two inactivating factors (IF7 and IF17). IF7 and IF17 are homologous polypeptides encoded by the gifA and gifB genes respectively. We investigated the transcriptional regulation of these genes. Expression of both genes is maximum in the presence of ammonium, when GS is inactivated. Nitrogen starvation attenuates the ammonium‐mediated induction of gifA and gifB as well as the ammonium‐mediated inactivation of GS. Putative binding sites for the transcription factor NtcA were identified at −7.5 and −30.5 bp upstream of gifB and gifA transcription start points respectively. Synechocystis NtcA protein binding to both promoters was demonstrated by gel electrophoresis mobility shift assays. Constitutive high expression levels of both genes were found in a Synechocystis NtcA non‐segregated mutant (SNC1), which showed a fourfold reduction in the ntcA expression. These experiments indicate a repressive role for NtcA on the transcription of gifA and gifB genes. Our results demonstrate that NtcA plays a central role in GS regulation in cyanobacteria, stimulating transcription of the glnA gene (GS structural gene) and suppressing transcription of the GS inactivating factor genes gifA and gifB.
Two types of cold sensitivity associated with the A184→V change in the DnaA protein
Authors: Nyborg Marlene , Atlung Tove , Skovgaard Ole , Hansen Flemming G.
Published: March 2003

Multicopy dnaA(Ts) strains carrying the dnaA5 or dnaA46 allele are high‐temperature resistant but are cold sensitive for colony formation. The DnaA5 and DnaA46 proteins both have an A184→V change in the ATP binding motif of the protein, but they also have one additional mutation. The mutations were separated, and it was found that a plasmid carrying exclusively the A184→V mutation conferred a phenotype virtually identical to that of the dnaA5 plasmid. Strains carrying plasmids with either of the additional mutations behaved like a strain carrying the dnaA+ plasmid. In temperature downshifts from 42°C to 30°C, chromosome replication was stimulated in the multicopy dnaA46 strain. The DNA per mass ratio increased threefold, and exponential growth was maintained for more than four mass doublings. Strains carrying plasmids with the dnaA(A184→V) or the dnaA5 gene behaved differently. The temperature downshift resulted in run out of DNA synthesis and the strains eventually ceased growth. The arrest of DNA synthesis was not due to the inability to initiate chromosome replication because marker frequency analysis showed high initiation activity after temperature downshift. However, the marker frequencies indicated that most, if not all, of the newly initiated replication forks were stalled soon after the onset of chromosome replication. Thus, it appears that the multicopy dnaA(A184→V) strains are cold sensitive because of an inability to elongate replication at low temperature. The multicopy dnaA46 strains, on the contrary, exhibit productive initiation and normal fork movement. In this case, the cold‐sensitive phenotype may be due to DNA overproduction.
Developmental arrest of the human malaria parasite Plasmodium falciparum within the mosquito midgut via CTRP gene disruption
Authors: Templeton Thomas J. , Kaslow David C. , Fidock David A.
Published: March 2003

Apicomplexan protozoa possess a family of micronemal and cell surface‐associated proteins, each comprised a combination of cell‐adhesive vertebrate von Willebrand factor (vWF)‐like A domains and thrombospondin (TSP) type 1‐like domains. The human malaria parasite Plasmodium falciparum has in the extracellular portion of the CS protein TRAP‐related protein (CTRP) six tandemly arrayed A domains followed by seven TSP type 1‐like domains, whereas a second member of this family, thrombospondin‐related anonymous protein (TRAP), contains a single vWF‐like A domain and a single TSP type 1‐like domain. Here we show that CTRP transcripts are present within the infected mosquito midgut and that CTRP protein is expressed with a punctate distribution and a predominance at the apical end of mosquito midgut‐stage ookinetes. This expression pattern is analogous to micronemal expression of TRAP in Plasmodium sporozoites. Disruption of the CTRP gene by homologous recombination in cultures of the human malaria parasite P. falciparum demonstrates that CTRP is essential for mosquito midgut development. Oocyst formation was never observed following membrane feeds of CTRP disruptant lines to Anopheline mosquitoes, despite the development of mature ookinetes. We propose that CTRP is involved in essential recognition or motility processes at the ookinete cell surface within the mosquito midgut.
NADP and NAD utilization in Haemophilus influenzae
Authors: Reidl Joachim , Schlör Stefan , Kraiß Anita , Schmidt‐Brauns Joachim , Kemmer Gabriele , Soleva Elizabeth
Published: March 2003

Exogenous NAD utilization or pyridine nucleotide cycle metabolism is used by many bacteria to maintain NAD turnover and to limit energy‐dependent de novo NAD synthesis. The genus Haemophilus includes several important pathogenic bacterial species that require NAD as an essential growth factor. The molecular mechanisms of NAD uptake and processing are understood only in part for Haemophilus. In this report, we present data showing that the outer membrane lipoprotein e(P4), encoded by the hel gene, and an exported 5′‐nucleotidase (HI0206), assigned as nadN, are necessary for NAD and NADP utilization. Lipoprotein e(P4) is characterized as an acid phosphatase that uses NADP as substrate. Its phosphatase activity is inhibited by compounds such as adenosine or NMN. The nadN gene product was characterized as an NAD‐nucleotidase, responsible for the hydrolysis of NAD. H. influenzae hel and nadN mutants had defined growth deficiencies. For growth, the uptake and processing of the essential cofactors NADP and NAD required e(P4) and 5′‐nucleotidase. In addition, adenosine was identified as a potent growth inhibitor of wild‐type H. influenzae strains, when NADP was used as the sole source of nicotinamide‐ribosyl.
The 5′‐proximal hairpin loop of lbi RNA is a key structural element in repression of d‐galactan II biosynthesis in Klebsiella pneumoniae serotype O1
Authors: Warnecke Jens M. , Nitschke Martin , Moolenaar Catharina E. C. , Rietschel Ernst TH. , Hartmann Roland K. , Mamat Uwe
Published: March 2003

The lbi (lipopolysaccharide biosynthesis interfering) RNA of phage Acm1, an untranslated RNA transcript of 97 nucleotides, previously shown to affect O‐polysaccharide biosynthesis in various Escherichia coli strains, was found to downregulate the synthesis of the d‐galactan II component of the O‐specific polysaccharide in Klebsiella pneumoniae serotype O1. Enzymatic and Pb2+ probing experiments revealed that lbi RNA consists of two consecutive stem–loop structures, the 5′‐proximal hairpin loop of 15 nucleotides being particularly accessible to single strand‐specific probes. Based on the assumption that the 5′‐proximal hairpin loop may be involved in an antisense interaction with cellular target RNAs, we randomly mutagenized one or two of its central nucleotides. Expression of mutated lbi RNA variants in K. pneumoniae serotype O1 relieved at least partly the repression of d‐galactan II formation. In addition, a truncated version of lbi RNA lacking the 3′‐proximal hairpin loop was almost as efficient as the wild‐type RNA in downregulating d‐galactan II synthesis. The results obtained indicate that the 5′‐proximal hairpin loop of lbi RNA functions as a key structural element in the mechanism leading to the inhibition of d‐galactan II biosynthesis in K. pneumoniae serotype O1.
Peptide deformylase as a target for new generation, broad spectrum antimicrobial agents
Authors: Giglione Carmela , Pierre Michèle , Meinnel Thierry
Published: March 2003

Peptide deformylase was discovered 30 years ago, but as a result of its unusually unstable activity it was not fully characterized until very recently. The aim of this paper is to review the many recent data concerning this enzyme and to try to assess its potential as a target for future antimicrobial drugs.
PAS domain residues involved in signal transduction by the Aer redox sensor of Escherichia coli
Authors: Repik Alexandre , Rebbapragada Anuradha , Johnson Mark S. , Haznedar Joshua Ö. , Zhulin Igor B. , Taylor Barry L.
Published: March 2003

PAS domains sense oxygen, redox potential and light, and are implicated in behaviour, circadian rhythmicity, development and metabolic regulation. Although PAS domains are widespread in archaea, bacteria and eukaryota, the mechanism of signal transduction has been elucidated only for the bacterial photo sensor PYP and oxygen sensor FixL. We investigated the signalling mechanism in the PAS domain of Aer, the redox potential sensor and aerotaxis transducer in Escherichia coli. Forty‐two residues in Aer were substituted using cysteine‐replacement mutagenesis. Eight mutations resulted in a null phenotype for aerotaxis, the behavioural response to oxygen. Four of them also led to the loss of the non‐covalently bound FAD cofactor. Three mutant Aer proteins, N34C, F66C and N85C, transmitted a constant signal‐on bias. One mutation, Y111C, inverted signalling by the transducer so that positive stimuli produced negative signals and vice versa. Residues critical for signalling were mapped onto a three‐dimensional model of the Aer PAS domain, and an FAD‐binding site and ‘active site’ for signal transduction are proposed.
The control of intracellular glycerol in Saccharomyces cerevisiae influences osmotic stress response and resistance to increased temperature
Authors: Siderius Marco , Van Wuytswinkel Olivier , Reijenga Karin A. , Kelders Marco , Mager Willem H.
Published: March 2003

Glycerol has been demonstrated to serve as the major osmolyte of Saccharomyces cerevisiae. Consistently, mutant strains gpd1gpd2 and gpp1gpp2, which are devoid of the main glycerol biosynthesis pathway, have been shown to be osmosensitive. In addition, the primary hyperosmotic stress response is affected in these strains. Hog1p phosphorylation turned out to be prolonged and osmostress‐induced gene expression is delayed compared with the kinetics observed in wild‐type cells. A hog1 deletion strain was previously found to contain lower internal glycerol and therefore displays an osmosensitive phenotype. Here, we show that the osmosensitivity of hog1 is suppressed by growth at 37°C. We reasoned that this temperature‐remedial osmoresistance might be caused by a higher intracellular glycerol level at the elevated temperature. This hypothesis was confirmed by measurement of the glycerol concentration, which was shown to be similar for wild type and hog1 cells only at elevated growth temperatures. In agreement with this finding, hog1 cells containing an fps1 allele, encoding a constitutively open glycerol channel, have lost their temperature‐remedial osmoresistance. Furthermore, gpd1gpd2 and gpp1gpp2 strains were found to be temperature sensitive. The growth defect of these strains could be suppressed by adding external glycerol. In conclusion, the ability to control glycerol levels influences proper osmostress‐induced signalling and the cellular potential to grow at elevated temperatures. These data point to an important, as yet unidentified, role of glycerol in cellular functioning.
The DNA binding and pairing preferences of the archaeal RadA protein demonstrate a universal characteristic of DNA strand exchange proteins
Authors: Seitz Erica M. , Kowalczykowski Stephen C.
Published: March 2003

The archaeal RadA protein is a homologue of the Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins and possesses the same biochemical activities. Here, using in vitro selection, we show that the Sulfolobus solfataricus RadA protein displays the same preference as its homologues for binding to DNA sequences that are rich in G residues, and under‐represented in A and C residues. The RadA protein also displays enhanced pairing activity with these in vitro‐selected sequences. These parallels between the archaeal, eukaryal and bacterial proteins further extend the universal characteristics of DNA strand exchange proteins.
A SeqA hyperstructure and its interactions direct the replication and sequestration of DNA
Authors: Norris V. , Fralick J. , Danchin A.
Published: March 2003

A level of explanation in biology intermediate between macromolecules and cells has recently been proposed. This level is that of hyperstructures. One class of hyperstructures comprises the genes, mRNA, proteins and lipids that assemble to fulfil a particular function and disassemble when no longer required. To reason in terms of hyperstructures, it is essential to understand the factors responsible for their formation. These include the local concentration of sites on DNA and their cognate DNA‐binding proteins. In Escherichia coli, the formation of a SeqA hyperstructure via the phenomenon of local concentration may explain how the binding of SeqA to hemimethylated GATC sequences leads to the sequestration of newly replicated origins of replication.
The Escherichia coli SeqA protein destabilizes mutant DnaA204 protein
Authors: Torheim Norunn K. , Boye Erik , Løbner‐Olesen Anders , Stokke Trond , Skarstad Kirsten
Published: March 2003

In wild‐type Escherichia coli cells, initiation of DNA replication is tightly coupled to cell growth. In slowly growing dnaA204 (Ts) mutant cells, the cell mass at initiation and its variability is increased two‐ to threefold relative to wild type. Here, we show that the DnaA protein concentration was two‐ to threefold lower in the dnaA204 mutant compared with the wild‐type strain. The reason for the DnaA protein deficiency was found to be a rapid degradation of the mutant protein. Absence of SeqA protein stabilized the DnaA204 protein, increased the DnaA protein concentration and normalized the initiation mass in the dnaA204 mutant cells. During rapid growth, the dnaA204 mutant displayed cell cycle parameters similar to wild‐type cells as well as a normal DnaA protein concentration, even though the DnaA204 protein was highly unstable. Apparently, the increased DnaA protein synthesis compensated for the protein degradation under these growth conditions, in which the doubling time was of the same order of magnitude as the half‐life of the protein. Our results suggest that the DnaA204 protein has essentially wild‐type activity at permissive temperature but, as a result of instability, the protein is present at lower concentration under certain growth conditions. The basis for the stabilization in the absence of SeqA is not known. We suggest that the formation of stable DnaA–DNA complexes is enhanced in the absence of SeqA, thereby protecting the DnaA protein from degradation.
Green fluorescent protein‐tagged sarco(endo)plasmic reticulum Ca2+‐ATPase overexpression in Paramecium cells: isoforms, subcellular localization, biogenesis of cortical calcium stores and functional aspects
Authors: Hauser Karin , Pavlovic Nada , Klauke Norbert , Geissinger Deisy , Plattner Helmut
Published: March 2003

We have followed the time‐dependent transfection of Paramecium cells with a vector containing the gene of green fluorescent protein (GFP) attached to the C‐terminus of the PtSERCA1 gene. The outlines of alveolar sacs (ASs) are labelled, as is the endoplasmic reticulum (ER) throughout the cell. When GFP fluorescence is compared with previous anti‐PtSERCA1 antibody labelling, the much wider distribution of GFP (ER+ASs) indicates that only a small amount of SERCA molecules is normally retained in the ER. A second isoform, PtSERCA2, also occurs and its C‐terminal GFP‐tagging results in the same distribution pattern. However, when GFP is inserted in the major cytoplasmic loop, PtSERCA1 and two fusion proteins are mostly retained in the ER, probably because of the presence of the overt C‐terminal KKXX ER‐retention signal and/or masking of a signal for transfer into ASs. On the overall cell surface, new SERCA molecules seem to be permanently delivered from the ER to ASs by vesicle transport, whereas in the fission zone of dividing cells ASs may form anew. In cells overexpressing PtSERCA1 (with C‐terminal GFP) in ASs, [Ca2+]i regulation during exocytosis is not significantly different from controls, probably because their Ca2+ pump has to mediate only slow reuptake.