Biomedical subjects

B Hallet

Publications and source records attributed to B Hallet.

16 recordsLinked to original sources

PubMed2001

Playing Dr Jekyll and Mr Hyde: combined mechanisms of phase variation in bacteria.

Phase variation is the adaptive process by which bacteria undergo frequent and reversible phenotypic changes resulting from genetic alterations in specific loci of their genomes. This process is crucial for the survival of pathogens and commensals in hostile and ever-changing host environments. Despite important differences in the molecular mechanisms that mediate and regulate phase variation, related strategies have evolved to generate high levels of genetic diversity through complex and combinatorial reshuffling of genetic information. Recent studies, supported by the emergence of global genomic approaches, have revealed that bacterial pathogens often use a combination of different mechanisms to vary the expression of a variety of biological functions, providing new insights into bacterial adaptation and virulence mechanisms. Recent advances in the understanding of the molecular mechanisms of phase variation are reviewed, and differences in these mechanisms outlined.

Adaptation, Physiological↗

PubMed2000

Coordinated control of XerC and XerD catalytic activities during Holliday junction resolution.

Site-specific recombinases XerC and XerD function in the segregation of circular bacterial replicons. In a recombining nucleoprotein complex containing two molecules each of XerC and XerD, coordinated reciprocal switches in recombinase activity ensure that only XerC or XerD is active at any one time. Mutated recombinases that carry sub?stitutions of a catalytic arginine residue stimulate cleavage and strand exchange mediated by the partner recombinase on DNA substrates that are normally recombined poorly by the partner. This is associated with a reciprocal impairment of the recombinase's own ability to initiate catalysis. The extent of this switch in catalysis is modulated by changes in recombination site sequence and is not a direct consequence of any catalytic defect. We propose that altered interactions between the mutated proteins and their wild-type partners lead to an increased level of an alternative Holliday junction intermediate that has a conformation appropriate for resolution by the partner recombinase. The results indicate how subtle changes in protein-DNA architecture at a Holliday junction can redirect recombination outcome.

Amino Acid Substitution↗

PubMed2000

Pentapeptide scanning mutagenesis: encouraging old proteins to execute unusual tricks.

Pentapeptide scanning mutagenesis is a facile transposon-based procedure for the random insertion of a variable five amino acid cassette into a target protein. The analysis of a library of proteins harbouring pentapeptide insertions can provide invaluable information on the essential and inessential regions of a target protein, as well as revealing surprising aspects of target protein function and activity.

Amino Acid Sequence↗

PubMed2000

Changes in cspL, cspP, and cspC mRNA abundance as a function of cold shock and growth phase in Lactobacillus plantarum.

An inverse PCR strategy based on degenerate primers has been used to identify new genes of the cold shock protein family in Lactobacillus plantarum. In addition to the two previously reported cspL and cspP genes, a third gene, cspC, has been cloned and characterized. All three genes encode small 66-amino-acid proteins with between 73 and 88% identity. Comparative Northern blot analyses showed that the level of cspL mRNA increases up to 17-fold after a temperature downshift, whereas the mRNA levels of cspC and cspP remain unchanged or increase only slightly (about two- to threefold). Cold induction of cspL mRNA is transient and delayed in time as a function of the severity of the temperature downshift. The cold shock behavior of the three csp mRNAs contrasts with that observed for four unrelated non-csp genes, which all showed a sharp decrease in mRNA level, followed in one case (bglH) by a progressive recovery of the transcript during prolonged cold exposure. Abundance of the three csp mRNAs was also found to vary during growth at optimal temperature (28 degrees C). cspC and cspP mRNA levels are maximal during the lag period, whereas the abundance of the cspL transcript is highest during late-exponential-phase growth. The differential expression of the three L. plantarum csp genes can be related to sequence and structural differences in their untranslated regions. It also supports the view that the gene products fulfill separate and specific functions, under both cold shock and non-cold shock conditions.

Amino Acid Sequence↗

PubMed1999

Reciprocal control of catalysis by the tyrosine recombinases XerC and XerD: an enzymatic switch in site-specific recombination.

In Xer site-specific recombination, sequential DNA strand exchange reactions are catalyzed by a heterotetrameric complex composed of two recombinases, XerC and XerD. It is demonstrated that XerC and XerD catalytic activity is controlled by an interaction involving the C-terminal end of each protein (the donor region) and an internal region close to the active site (the acceptor region). Mutations in these regions reciprocally alter the relative activity of XerC and XerD, with their combination producing synergistic effects on catalysis. The data support a model in which C-terminal intersubunit interactions contribute to coupled protein-DNA conformational changes that lead to sequential activation and reciprocal inhibition of pairs of active sites in the recombinase tetramer during recombination.

Amino Acid Sequence↗

PubMed1997

Structure-function correlations in the XerD site-specific recombinase revealed by pentapeptide scanning mutagenesis.

Xer-mediated site-specific recombination contributes to the stability of circular chromosomes in bacteria by resolving plasmid multimers and chromosome dimers to monomers prior to cell division. Two related site-specific recombinases, XerC and XerD, each catalyse one pair of strand exchange during Xer recombination. In order to relate the recently determined structure of XerD to its function, the XerD protein was subjected to pentapeptide scanning mutagenesis, which leads to a variable five amino acid cassette being introduced randomly into the target protein. This has allowed identification of regions of XerD involved in specific DNA binding, in communicating with the partner recombinase, XerC, and in catalysis and its control. The C-terminal domain of XerD, comprising two-thirds of the protein, contains the catalytic active site and comprises ten alpha helices (alphaE to alphaN) and a beta hairpin. A flexible linker connects this domain to the N-terminal domain that comprises four alpha helices (alphaA to alphaD). Pentapeptide insertions into alphaB, alphaD, alphaG, or alphaJ interfered with DNA binding. Helices alphaG and alphaJ comprise a pseudo helix-turn-helix DNA binding motif that may provide specificity of recombinase binding. An insertion in alphaL, adjacent to an active site arginine residue, led to loss of cooperative interactions between XerC and XerD and abolished recombination activity. Other insertions close to active site residues also abolished recombination activity. Proteins with an insertion in the beta hairpin turn bound DNA, interacted cooperatively with XerC and had a phenotype that is consistent with the protein being defective in XerD catalysis. This beta hairpin appears to be highly conserved in related proteins. Insertions at a number of dispersed locations did not impair XerD catalytic activity or DNA binding, but failed to allow XerC catalysis in vivo, indicating that several sites of interaction between XerD and XerC may be important for activation of XerC catalysis by XerD.

Amino Acid Sequence↗

PubMed1997

Insertion mutagenesis as a tool in the modification of protein function. Extended substrate specificity conferred by pentapeptide insertions in the omega-loop of TEM-1 beta-lactamase.

The TEM-1 beta-lactamase enzyme efficiently hydrolyzes beta-lactam antibiotics such as ampicillin but cleaves third generation cephalosporin antibiotics poorly. Variant beta-lactamases that conferred elevated levels of resistance to the cephalosporin ceftazidime were identified in a set of beta-lactamase derivatives previously generated by pentapeptide scanning mutagenesis in which a variable 5-amino acid cassette was introduced randomly in the target protein. This mutagenesis procedure was also modified to allow the direct selection of variant beta-lactamases with pentapeptide insertions that conferred extended substrate specificities. All insertions associated with enhanced resistance to ceftazidime were targetted to the 19-amino acid Omega-loop region, which forms part of the catalytic pocket of the beta-lactamase enzyme. However, pentapeptide insertions in the C- and N-terminal halves of this region had different effects on the ability of the enzyme to hydrolyze ampicillin in vivo. Larger insertions that increased the length of the Omega-loop by up to 2-fold also retained catalytic activity toward ampicillin and/or ceftazidime in vivo. In accord with previous substitution mutation studies, these results emphasize the extreme flexibility of the Omega-loop with regards the primary structure requirements for ceftazidime hydrolysis by beta-lactamase. The potential of pentapeptide scanning mutagenesis in mimicking evolution events that result from the insertion and excision of transposons in nature is discussed.

Amino Acid Sequence↗

PubMed1997

Xer recombination in Escherichia coli. Site-specific DNA topoisomerase activity of the XerC and XerD recombinases.

Xer site-specific recombination functions in maintaining circular replicons in the monomeric state in Escherichia coli. Two recombinases of the bacteriophage lambda integrase family, XerC and XerD, are required for recombination at the chromosomal site, dif, and at a range of plasmid-borne sites. Xer recombination core sites contain the 11-base pair binding sites for each recombinase separated by a 6 to 8-base pair central region. We report that both XerC and XerD act as site-specific type I topoisomerases by relaxing supercoiled plasmids containing a dif site. Relaxation by either XerC or XerD occurs in the absence of the partner recombinase and requires only a single recombination core site. XerC or XerD relaxation activities are completely inhibited by the addition of the partner recombinase, providing that the DNA recognition sequence for the inhibiting partner is present.

Binding Sites↗

PubMed1997

Pentapeptide scanning mutagenesis: random insertion of a variable five amino acid cassette in a target protein.

A new insertion method for probing protein functional organization was developed. The method relies on the random insertion of transposon Tn 4430 and subsequent in vitro deletion of the bulk of the transposon after which a 15 bp insertion remains within the target gene. This results in pentapeptide insertions randomly distributed in the target protein. Characterization of 23 pentapeptide insertions in TEM-1beta-lactamase demonstrated the utility of the method. The phenotypes associated with the mutated beta-lactamase proteins equated both with the sorts of local peptide structures in which the pentapeptide insertions occurred and their position in the three-dimensional structure of the enzyme.

DNA Transposable Elements↗

PubMed1997

Transposition and site-specific recombination: adapting DNA cut-and-paste mechanisms to a variety of genetic rearrangements.

In bacteria, two categories of specialised recombination promote a variety of DNA rearrangements. Transposition is the process by which genetic elements move between different locations of the genome, whereas site-specific recombination is a reaction in which DNA strands are broken and exchanged at precise positions of two target DNA loci to achieve determined biological function. Both types of recombination are represented by diverse genetic systems which generally encode their own recombination enzymes. These enzymes, generically called transposases and site-specific recombinases, can be grouped into several families on the basis of amino acid sequence similarities, which, in some cases, are limited to a signature of a few residues involved in catalysis. The well characterised site-specific recombinases are found to belong to two distinct groups whereas the transposases form a large super-family of enzymes encompassing recombinases from both prokaryotes and eukaryotes. In spite of important differences in the catalytic mechanisms used by these three classes of enzymes to cut and rejoin DNA molecules, similar strategies are used to coordinate the biochemical steps of the recombination reaction and to control its outcome. This review summarises our current understanding of transposition and site-specific recombination, attempting to illustrate how relatively conserved DNA cut-and-paste mechanisms can be used to bring about a variety of complex DNA rearrangements.

Bacteria↗

PubMed1994

IS231 and other Bacillus thuringiensis transposable elements: a review.

Bacillus thuringiensis is an entomopathogenic bacterium whose toxicity is due to the presence in the sporangia of delta-endotoxin crystals active against agricultural pests and vectors of human and animal diseases. Most of the genes coding for these toxin proteins are plasmid-borne and are generally structurally associated with insertion sequences (IS231, IS232, IS240, ISBT1 and ISBT2) and transposons (Tn4430 and Tn5401). Several of these mobile elements have been shown to be active and are believed to participate in the crystal gene mobility, thereby contributing to the variation of bacterial toxicity. Structural analysis of the iso-IS231 elements indicates that they are related to IS1151 from Clostridium perfringens and distantly related to IS4 and IS186 from Escherichia coli. Like the other IS4 family members, they contain a conserved transposase-integrase motif found in other IS families and retroviruses. Moreover, functional data gathered from IS231A in Escherichia coli indicate a non-replicative mode of transposition, with a marked preference for specific targets. Similar results were also obtained in Bacillus subtilis and B. thuringiensis, and a working model for DNA-protein interactions at the target site is proposed.

Amino Acid Sequence↗

PubMed1994

IS231A insertion specificity: consensus sequence and DNA bending at the target site.

In its natural host, Bacillus thuringiensis, the insertion sequence IS231A is preferentially inserted into the terminal inverted repeats of the transposon Tn4430. Using a novel transposition assay, we demonstrate that the Tn4430 ends behave as insertion hot spots for IS231A in Escherichia coli. Sequence analysis reveals that IS231A insertion sites match the 5'-GGG(N)5CCC-3' consensus. However, this consensus is not the only determinant of IS231A insertion specificity. Although both Tn4430 ends have identical sequences, one is strongly preferred to the other and the orientation of insertion into this end is not random. We demonstrate that this preference is determined by the flanking regions of the site. These regions display a conserved periodic organization of their sequence which, by conferring anisotropic flexibility, would induce the DNA to bend in a roughly 'S'-shaped structure centered on the target consensus. DNA conformation analysis by polyacrylamide gel electrophoresis indeed shows that the preferred target site of IS231A is flanked by DNA segments curved in opposite directions. We present a model in which DNA bendability and curvature would contribute to the positioning of IS231A transposase on the target DNA.

Bacillus thuringiensis↗

PubMed1993

IS231V and W from Bacillus thuringiensis subsp. israelensis, two distant members of the IS231 family of insertion sequences.

IS231 constitutes a family of related insertion sequences (IS) from Bacillus thuringiensis. Two new IS231-related elements, IS231V and IS231W, have been isolated from the 72-MDa plasmid of B. thuringiensis subsp. israelensis. These closely related 1964-bp IS are delimited by 22-bp imperfect inverted repeats strongly similar to those of the other iso-IS231. Although the other known IS231 harbor a single long open reading frame (ORF), IS231V and W display two slightly overlapping ORF on the same DNA strand. They show about 50% identity with the transposase of the other iso-IS231. A frameshifting model is proposed for the synthesis of a fusion product which would constitute their active transposase.

Amino Acid Sequence↗

PubMed1993

The IS4 family of insertion sequences: evidence for a conserved transposase motif.

The eight IS231 variants characterized so far (IS231 A-F, V and W) display similar transposases with an overall 40% identity. Comparison with all the prokaryotic transposable elements sequenced so far revealed that the IS231 transposases share two conserved regions with those of 35 other insertion sequences of wide origins. These insertion sequences, defining the IS4 family, have a common bipartite organization of their ends and are divided into two similarity groups. Interestingly, the transposase domains conserved within this family display similarities with the well known integrase domain shared by transposases of the IS3 and IS15 families, and integrases of retroelements. This domain is also found in IS30-related elements and Tn7 TnsB protein. Amino acid residues conserved throughout all these prokaryotic and eukaryotic mobile genetic elements define a major transposase/integrase motif, likely to play an important role in the transposition process.

Amino Acid Sequence↗

PubMed1992

IS231D, E and F, three new insertion sequences in Bacillus thuringiensis: extension of the IS231 family.

IS231 constitutes a family of insertion sequences widespread among Bacillus thuringiensis subspecies. Three new IS231 variants have been isolated from B. thuringiensis subspecies finitimus (IS231 D and E) and israelensis (IS231F). Like the previously described IS231A, B and C, these 1.7 kb elements display single open reading frames encoding 477/478-amino-acid proteins which share between 72% and 88% identity with those of the other members of the family. Sequence comparisons also reveal that all the iso-IS231 terminal inverted repeats are strongly conserved 20 bp sequences. A region susceptible to forming a stable hairpin structure is found just upstream of the open reading frame. Nucleotide substitutions occurring on one strand of the hairpin stems are compensated for by complementary changes at facing positions, giving credence to the hypothesis that this secondary structure plays a role in the regulation of transposition. Examination of IS231 D, E and F flanking sequences reveals that IS231F is bordered by a 12 bp direct repeat. No direct repeats were found flanking IS231D or IS231E.

Amino Acid Sequence↗

PubMed1991

IS231A from Bacillus thuringiensis is functional in Escherichia coli: transposition and insertion specificity.

A kanamycin resistance gene was introduced within the insertion sequence IS231A from Bacillus thuringiensis, and transposition of the element was demonstrated in Escherichia coli. DNA sequencing at the target sites showed that IS231A transposition results in direct repeats of variable lengths (10, 11, and 12 bp). These target sequences resemble the terminal inverted repeats of the transposon Tn4430, which are the preferred natural insertion sites of IS231 in B. thuringiensis.

Bacillus thuringiensis↗