The great GATC: DNA methylation in E. coli.

In Escherichia coli the methylation of the adenine in the sequence 5'-GATC-3' is catalysed by the dam gene product, a DNA adenine methylase. We review the proposed roles for this methylation, and the sequence it modifies, in mismatch repair, DNA-protein interaction, gene expression, the initiation of chromosome replication, chromosome segregation, chromosome structure and the occurrence of mutational hotspots.

Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL.

Erwinia chrysanthemi secretes, by the type II secretory pathway, a large number of enzymes, including cellulases and pectinases. This process requires the products of the out genes, which are widely conserved in Gram-negative bacteria. The Out proteins are thought to form a membrane-associated multiprotein complex. Here, we investigated interaction between OutE, the putative ATP binding component, and OutL, an inner membrane protein. We showed, by limited proteolysis, genetic suppression and the yeast two-hybrid system, that OutE and OutL interact directly. Analysis of truncated forms of OutE demonstrated that the N terminus of OutE (residues 1-97) is important for the OutE/OutL interaction. Moreover, results from the yeast two-hybrid system suggested that OutE and OutL are each able to form homomultimers. The region required for homomultimerisation of OutE is located in its C terminus. Limited proteolysis assay indicated that OutE induces a conformational change in OutL, in both its cytoplasmic and periplasmic domains. Moreover, the secretion process requires a conformational change in OutE which depends on both the interaction with OutL and on the presence of an intact Walker A motif in OutE. Our results support the view that interaction occurring on the cytoplasmic side influences the events occurring in the outer membrane. We discuss a model in which OutE uses ATP to control the assembly of the type II secretion machinery.

Alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the Erwinia chrysanthemi Cel5 cellulase (ex-EGZ) via the type II system.

Cel5 (formerly known as endoglucanase Z) of Erwinia chrysanthemi is secreted by the Out type II pathway. Previous studies have shown that the catalytic domain (CD), linker region (LR) and cellulose-binding domain (CBD) each contain information needed for secretion. The aim of this work was to further investigate the secretion-related information present in the CBD(Cel5). Firstly(, )deleting a surface-exposed flexible loop had no effect on secretion. This indicated that some structural freedom is tolerated by the type II system. Secondly, mutation of a single tryptophan residue, previously shown to be important for binding to cellulose, i.e. Trp43, was found also to impair secretion. This indicated that the flat cellulose-binding surface of CBD(Cel5 )contains secretion-related information. Thirdly, CBD(Cel5) was substituted by the CBD(EGG) of Alteromonas haloplanctis endoglucanase G, yielding a hybrid protein CD(Cel5)-LR(Cel5)-CBD(EGG) that exhibited 90 % identity with Cel5, including the Trp43 residue. The hybrid protein was not secreted. This indicated that the Trp43 residue is necessary but not sufficient for secretion. Here we propose a model in which the secretion of Cel5 involves a transient intramolecular interaction between the cellulose-binding surface of CBD(Cel5) and a region close to the entry into the active site in CD(Cel5). Once secreted, the protein may then open out to allow the cellulose-binding surface of CBD(Cel5 )to interact with the surface of the cellulose substrate. An implication of this model is that protein molecules fold to a specific secretion-competent conformation prior to secretion that is different from the folding state of the secreted species.

Informational suppression to investigate structural functional and evolutionary aspects of the Erwinia chrysanthemi cellulase EGZ.

The cellulase EGZ produced by the plant pathogen Erwinia chrysanthemi belongs to family 5 of the beta-glycohydrolases (also referred to as cellulase family A), which contains over 40 members from Gram-negative and Gram-positive bacteria and fungi. Amber mutations were introduced into 16 codons of the celZ gene encoding EGZ. Targeted residues included: (1) two Glu, two His and one Arg residue, strictly conserved throughout family 5; (2) one Arg and one His residue conserved in sub-family 5-2; and (3) one His and six Arg residues not conserved at all. Each amber allele was introduced into 13 Escherichia coli strains each carrying a different suppressor tRNA that inserts an amino acid at the mutated position. In vivo stability of the mutated forms of EGZ and their cellulase activity were analysed as well as suppression efficiency. For some positions of particular interest, missense mutations were introduced into the celZ gene either to confirm the effect of the suppressor-mediated amino acid substitution or to broaden the spectrum of mutations available. The substitution patterns of the two Glu positions were interpretable in the light of the stereospecificity of the reaction catalysed by EGZ: Glu133 and Glu220 are proposed to act as a proton donor and as a nucleophile, respectively, forming the glycosyl-enzyme intermediate. Substitution at His-occupied positions, including two non-conserved positions, yielded proteins affected in their catalytic activity but not their in vivo stability. In particular, evidence was obtained for His at position 98 to be involved in interactions with the substrate. The view that Arg residues are important in stabilizing proteins was supported by the identification of three Arg residues, whose substitution yielded thermosensitive forms of EGZ. In addition, Pro substitutions of any of the six Arg residues altered protein stability in vivo but the substitutions scored almost neutral for activity. Five positions, predicted to be within alpha-helices, were found to be susceptible to Pro substitutions (but not to Ala) with respect to stability in vivo. Overall, the systematic alteration of all His and Arg residues coupled with the simultaneous analysis of activity and in vivo stability allowed us to demonstrate that substitution matrices vary at each position and for each biological property considered. Ideally, therefore, substitution matrices used in sequence alignment procedures should be reconsidered as position-specific and as property-specific.(ABSTRACT TRUNCATED AT 400 WORDS)

Type II protein secretion in gram-negative pathogenic bacteria: the study of the structure/secretion relationships of the cellulase Cel5 (formerly EGZ) from Erwinia chrysanthemi.

Erwinia chrysanthemi, a Gram-negative plant pathogen, secretes the cellulase Cel5 (formerly EGZ) via the type II secretion pathway (referred to as Out). Cel5 is composed of two domains, a large N-terminal catalytic domain (390 amino acid residues) and a small C-terminal cellulose-binding domain (62 amino acid residues) separated by a linker region. A combination of mutagenesis and structural analysis permitted us to investigate the structure/secretion relationships with respect to the catalytic domain of Cel5. The 3D structure of the catalytic domain was solved by molecular replacement at 2.3 A resolution. Cel5 exhibits the (beta/alpha)8 structural fold and two extra-barrel features. Our previous genetic study based upon tRNA-mediated suppression allowed us to predict positions of importance in the molecule in relation to structure and catalysis. Remarkably, all of the predictions proved to be correct when compared with the present structural information. Mutations of Arg57, which is located at the heart of the catalytic domain, allowed us to test the consequences of structural modifications on the secretion efficiency. The results revealed that secretability imposes remarkably strong constraints upon folding. In particular, an Arg-to-His mutation yielded a species that folded to a stable conformation close to, but distinct from the wild-type, which however was not secretable. We discuss the relationships between folding of a protein in the periplasm, en route to the cell exterior, and presentation of secretion information. We propose that different solutions have been selected for type II secreted exoproteins in order to meet the constraints imposed by their interaction with their respective secretion machineries. We propose that evolutionary pressure has led to the adaptation of different secretion motifs for different type II exoproteins.

Lesion-induced differential expression and cell association of Neurocan, Brevican, Versican V1 and V2 in the mouse dorsal root entry zone.

Lack of regeneration in the CNS has been attributed to many causes, including the presence of inhibitory molecules such as chondroitin sulfate proteoglycans (CSPGs). However, little is known about the contribution of CSPGs to regeneration failure in vivo, in particular at the dorsal root entry zone (DREZ), a unique CNS region that blocks regeneration of sensory fibers following dorsal root injury without glial scar formation. The goal of the present study was to evaluate the presence, regulation, and cellular identity of the proteoglycans Brevican, Neurocan, Versican V1 and Versican V2 in the DREZ using CSPG-specific antibodies and nucleic acid probes. Brevican and Versican V2 synthesized before the lesion were still present at high levels in the extracellular matrix of the DREZ several weeks after injury. In addition, Brevican was transiently expressed by reactive oligodendrocytes, and by a subset of astrocytes thereafter. Versican V2 mRNA appeared in NG2-positive cells with the morphology of oligodendrocyte precursor cells. Neurocan and Versican V1 levels were low before injury, and appeared in nestin-positive astrocytes and in NG2-positive cells, respectively, following lesion. Versican V1, but not V2, was also transiently increased in the peripheral dorsal root post-lesion. This is the first thorough description of the expression and cell association of individual proteoglycans following dorsal root lesion. It demonstrates that the proteoglycans Brevican, Neurocan, Versican V1, and Versican V2 are abundant in the DREZ at the time regenerating sensory fibers reach the PNS/CNS border and may therefore participate in growth-inhibition in this region.

[Chronic tinnitus]. / L'acouphène neurosensoriel chronique.

Chronic tinnitus is a frequent symptom in the current medical practice. Patients presenting with chronic tinnitus have to be evaluated by comprehensive examination, including ENT status, audiometry and complete neuro-otological evaluation if required, to exclude an organic cause of tinnitus, as an external--or middle ear lesion, or a retro-cochlear process. Most often, chronic tinnitus is only associated with a sensorineural hearing-loss. Overall, no drug treatment can be proposed. However, the patient needs help and two techniques can be proposed to make the symptom more tolerable: the bio-psycho-social model, with a medical and psychological sustain, and the habituation auditory therapy, using noisers to mask the tinnitus.

External pH: an environmental signal that helps to rationalize pel gene duplication in Erwinia chrysanthemi.

The phytopathogenic bacterium Erwinia chrysanthemi produces five major pectate lyases that are key virulence factors in soft-rot disease development. Using transcriptional fusions, we studied the regulation of pelA, pelD, and pelE gene expression as a function of variation of the external pH. pelA and pelD were expressed when bacteria were grown in an acidic medium while pelE was transcribed only in basic medium. Using phenol red, we observed that, in chicory leaves, pH value of infected tissue varies from acidic to basic. Taken together, these findings are discussed in the context of a model unifying both catalysis and regulation to account for pel gene evolution. In particular, we propose that the three isoenzymes are produced sequentially during the infection process.

The small proline-rich proteins constitute a multigene family of differentially regulated cornified cell envelope precursor proteins.

Loricrin, involucrin, small proline-rich protein (SPRR)1, SPRR2, and SPRR3 genes are located within a cluster of 1.5 Mbp on chromosome 1q21 and most likely evolved from a common ancestor. Monospecific polyclonal antibodies and cDNA probes were produced to investigate SPRR transcripts and proteins. SPRR expression was restricted to terminally differentiating squamous cells, preferentially located at the cell periphery, and immunoreactivity was greatly reduced in cells with a mature cornified cell envelope. Furthermore, detectable SPRR2 and SPRR3 levels were strongly increased in differentiating keratinocyte cultures after addition of LTB-2, a specific inhibitor of transglutaminases, suggesting that they are precursor proteins of the cornified cell envelope. In normal epidermis, SPRR1 was restricted to appendageal areas, SPRR2 was expressed coherently, and SPRR3 was completely absent. In the upper digestive tract, SPRR1 was expressed in sublingual and tongue epithelium, SPRR2 was mostly restricted to lingual papillae, and SPRR3 was abundant in oral and esophageal epithelium. In psoriatic epidermis, SPRR1 and SPRR2 were expressed at much higher levels than in normal epidermis. Addition of 10(-7) M retinoic acid to cultured differentiating keratinocytes significantly down-regulated the expression of SPRR2 and SPRR3 transcripts and slightly decreased that of SPRR1. Thus, SPRR1, SPRR2, and SPRR3 are differentially expressed in vivo and in vitro, suggesting that the SPRR multigene family evolved to serve as highly specialized cornified cell envelope precursor proteins in stratified epithelia.

Sequence analysis of the cellulase-encoding celY gene of Erwinia chrysanthemi: a possible case of interspecies gene transfer.

The Erwinia chrysanthemi (strain 3937) celY gene encoding the minor endoglucanase (EGY) was sequenced. The analysis of the upstream region allowed us to identify an in vivo active promoter recognized by the NtrA (sigma 54) holoenzyme. No similarity was found between the predicted amino acid (aa) sequences of EGY and either the Er. chrysanthemi major endoglucanase, EGZ, or the Er. carotovora CelS endoglucanase. In contrast, a very high level of identity, both at the nucleotide and the predicted aa levels, was found between celY and an EG-encoding gene from Cellulomonas uda, a Gram + bacterium taxonomically distant from Er. chrysanthemi. By comparing the molar G + C% of the cellulase-encoding genes and that of Er. chrysanthemi and C. uda chromosomal DNAs, we speculate that celY was transferred from Er. chrysanthemi to C. uda.

Stereochemistry of the hydrolysis reaction catalyzed by endoglucanase Z from Erwinia chrysanthemi.

Endoglucanase Z from the phytopathogenic bacterium Erwinia chrysanthemi (strain 3937) was purified by affinity chromatography on microcrystalline cellulose Avicel PH101. A kinetic characterization using p-nitrophenyl beta-D-cellobioside and p-nitrophenyl beta-D-lactosde as substrates was conducted: endoglucanase Z exhibited Km values of 3 mM and 7.5 mM and Vm values of 129 and 40 nmol.min-1.mg-1 towards p-nitrophenyl beta-D-cellobioside (kcat = 0.1 s-1) and p-nitrophenyl beta-D-lactoside (kcat = 0.03 s-1), respectively). The hydrolysis of cellotetraitol by endoglucanase Z was followed by HPLC and 1H NMR. Results show that cellobiitol and beta-cellobiose are initially formed, demonstrating that the enzyme is acting by a molecular mechanism retaining the anomeric configuration. This suggests the involvement of a glycosyl-enzyme intermediate.

Cobalt stress in Escherichia coli and Salmonella enterica: molecular bases for toxicity and resistance.

Cobalt (Co) is present in trace amounts in the environment but it can be toxic when it accumulates in cells. The question of how Co produces its toxic effects and how living organisms protect themselves from, and resist to, such a stress remains to be clarified. Studies pertaining to these issues were recently carried out in Escherichia coli and Salmonella enterica. Iron-sulfur proteins were identified as primary targets of Co ions. Perturbation of iron homeostasis, oxidative stress and possible effects on sulfur assimilation were noticed. Cells were found to respond by up-regulating genes involved in the biosynthesis of Fe-S clusters as well as genes involved in Co efflux. These data are summarized in this review article to provide a preliminary general view of Co toxicity mechanisms in these two bacterial models.

Mechanisms of iron-sulfur cluster assembly: the SUF machinery.

Biosynthesis of iron-sulfur clusters is a cellular process which depends on complex protein machineries. Escherichia coli contains two such biosynthetic systems, ISC and SUF. In this review article we specifically make a presentation of the various Suf proteins and discuss the molecular mechanisms by which these proteins work together to assemble Fe and S atoms within a scaffold and to transfer the resulting cluster to target proteins.

Influence of gyrA mutation on expression of Erwinia chrysanthemi clb genes cloned in Escherichia coli.

Erwinia chrysanthemi clb genes cloned into Nals Escherichia coli allowed growth on cellobiose, arbutin, or salicin. In contrast, Nalr isogenic strains grew only on cellobiose. It is proposed that expression of cloned E. chrysanthemi clb genes is reduced by the E. coli chromosomal gyrA (Nalr) mutation, resulting in apparent segregation of the Clb and Arb Sal characters.

Genetic analysis of the pelA-pelE cluster encoding the acidic and basic pectate lyases in Erwinia chrysanthemi EC16.

In Erwinia chrysanthemi (EC16) the clustered pelA and pelE genes encode an acidic (pI 4.2) and a basic (pI 10.0) pectate lyase (Pel), respectively. The pelA gene has been isolated on a 1.2 kb restriction fragment and the direction of transcription determined. DNA hybridization analysis showed that the pelE sequence shares DNA homology with pelA but not with pelB or pelC, two genes encoding other Pel species in EC16. Since Pel A and Pel E enzymes showed little similarity in terms of catalytic properties, it is proposed that pelA and pelE are duplicates which have highly diverged.

Arrangement of Dam methylation sites (GATC) in the Escherichia coli chromosome.

The occurrence of GATC (Dam-recognition) sites in available E. coli DNA sequences (representing about 2% of the chromosome) has been determined by a simple numerical analysis. Our approach was to analyze the nucleotide composition of nine large sequenced DNA stretches ("cantles") in order to identify patterns of GATC distribution and to rationalize such patterns in biological/structural terms. The following observations were made: (i) In addition to oriC, GATC-rich regions are present in numerous locations. (ii) There is a wide variation in GATC frequency both between and within DNA cantles which led to the identification of a void-cluster pattern of GATC arrangement. The distance between two GATCs was never greater than 2 kb. (iii) GATC sites are found more frequently in translated regions than (in decreasing order) non-coding or non-translated regions. In particular, rRNA and tRNA encoding genes exhibit the lowest GATC content.

An inner membrane platform in the type II secretion machinery of Gram-negative bacteria.

The type II secretion machinery allows most Gram-negative bacteria to deliver virulence factors into their surroundings. We report that in Erwinia chrysanthemi, GspE (the putative NTPase), GspF, GspL and GspM constitute a complex in the inner membrane that is presumably used as a platform for assembling other parts of the secretion machinery. The GspE-GspF-GspL-GspM complex was demonstrated by two methods: (i) co-immunoprecipitation of GspE-GspF-GspL with antibodies raised against either GspE or GspF; (ii) interactions in the yeast two-hybrid system between GspF and GspE, GspF and GspL, GspL and GspM. GspL was found to have an essential role in complex formation. We propose a model in which the GspE-GspF-GspL-GspM proteins constitute a building block within the secretion machinery on top of which another building block, referred to as a pseudopilus, assembles. By analogy, we predict that a similar platform is required for the biogenesis of the type IV pilus.

Functional analysis of the carbohydrate-binding domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli putative chitinase.

The Cel5 cellulase (formerly known as endoglucanase Z) from Erwinia chrysanthemi is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBD(Cel5) has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBD(Cel5) exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from Escherichia coli. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the E. coli chromosome and purified by metal chelating chromatography. While CBD(Cel5) bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.