Regulación de la degradación de compuestos hidroaromáticos por bacterias

Peer reviewe

Voyage of RepA protein from plasmid DNA replication through amyloid aggregation towards synthetic biology

DNA replication of plasmids in Gram-negative bacteria has been an object of study at CIB-CSIC for nearly 30 years. We have been focused on the enterobacterial antibiotic resistance factor R1 (1981-1992) and the pPS10 replicon from the phytopathogen Pseudomonas savastanoi (since 1984). Our group has used multidisciplinary (genetic, biochemical and biophysical-structural) approaches to unravel the molecular mechanism for the activation of RepA. Rep-type plasmidic proteins are either transcriptional repressors or replication initiators/inhibitors, depending on their association state (dimers vs. monomers) and targeting of alternative (operator or iteron) DNA sites. We discovered that allosteric DNA-binding remodels the structure of RepA N-terminal domain (WH1), transforming α-helical portions into β-strands. This precisely tunes the distances between the DNA reading heads in WH1 and the C-terminal domain (WH2), to match the target operator or iteron sequences. We have recently moved into engineering such structural transformation in RepA-WH1 to build-up synthetic protein devices that allow for customized ligand (DNA)-promoted amyloidogenesis. Our basic studies on plasmid DNA replication are relevant for settling the bases of a minimalist bacterial model to tackle transmissible amyloid proteinopathies and are a valuable tool for bottom-up synthetic biology.Our research is currently supported by grants from the Spanish Ministry of Science and Innovation (MICINN: BIO2009-06952 and CSD2009-00088) and the Autonomous Government of Madrid (CAM: P-BIO-0214-2006)

Twenty years of the pPS10 replicon: Insights on the molecular mechanism for the activation of DNA replication in iteron-containing bacterial plasmids

This review focuses on the contributions of the Pseudomonas replicon pPS10 to understanding the initiation of DNA replication in iteron-containing plasmids from Gram-negative bacteria. Dimers of the pPS10 initiator protein (RepA) repress repA transcription by binding to the two halves of an inverted repeat operator. RepA monomers are the active initiator species that bind to four directly repeated sequences (iterons). pPS10 initiator was the first Rep protein whose domains were defined (two “winged-helix,” WH modules) and their binding sites were identified at each half of the iteron repeat. This was confirmed by the crystal structure of the monomer of a homologous initiator (RepE from F plasmid) bound to iteron DNA. The recently solved structure of the dimeric N-terminal domain (WH1) of pPS10 RepA, when compared to the RepE monomer, shows that upon dimer dissociation an α-helix at WH1 C-terminus becomes part of an interdomain β-sheet. In solution, the iteron sequence, by itself, can induce the same kind of structural transformation in RepA. This seems to alter the package of both WH domains to adapt their DNA reading heads (HTH motifs) to the distinct spacing between half repeats in iterons and operator. Based on biochemical and spectroscopic work, structural and functional similarities were proposed between RepA and archaeal/eukaryal initiators. This was independently confirmed by the crystal structure of the archaeal initiator Cdc6. Characterization of mutants, either in pPS10 or in the Escherichia coli chromosome, has provided some evidence on a WH1-mediated interaction between RepA and the chromosomal initiator DnaA that results in a broadened-host range.Work on pPS10 has been possible thanks to the continuous support from Spanish CICyT/MCyT, currently under Grant BMC2003-00088. Finally, we are indebted to the enlightening work of many plasmid labs overseas, notably those focused in P1, F, R6K, RK2, and pSC101 replicons

Replicación , rango de huésped y mantenimiento de plásmidos en bacterias Gram-negativas: Estudios con pPS10, un plásmido de pseudomonas con R1, un factor de resistencia a antibióticos de enterobacterias

Peer reviewe

Bacterial zipper

An in-phase repetition of a leucine every seven residues in an a-helicoidal structure is a motif associated with the dimerization of proteins and, together with an adjacent basic region, is responsible for the interaction of eukaryotic regulators and specific DNA sequences. This structural and functional motif has been termed the 'leucine zipper'l.2. It is also present in membrane proteins that do not bind to DNA3-5

Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for pseudomonas isolated from Pseudomonas syringae patovar savastanoi

The sequence of a 1823 base-pair region containing the replication functions of pPS10, a narrow host-range plasmid isolated from a strain of Pseudomonas savastanoi, is reported. The origin of replication, oriV, or pPS10 is contained in a 535 base-pair fragment of this sequence that can replicate in the presence of trans-acting function(s) of the plasmid. oriV contains four iterons of 22 base-pairs that are preceded by G + C-rich and A + T-rich regions. A dnaA box located adjacent to the repeats of the origin is dispensable but required for efficient replication of pPS10; A and T are equivalent bases at the 5′ end of the box. repA, the gene of a trans-acting replication protein of 26,700 Mr has been identified by genetic and functional analysis, repA is adjacent to the origin of replication and is preceded by the consensus sequences of a typical σ70 promoter of Escherichia coli. The RepA protein has been identified, using the minicell system of E. coli, as a polypeptide with an apparent molecular mass of 26,000. A minimal pPS10 replicon has been defined to a continuous 1267 base-pair region of pPS10 that includes the oriV and repA sequences.This work was supported by grants BI088-0294 and BT-42-84 from the Spanish CICYT and CSIC. C.N. and R.G. were supported by fellowships from the Spanish MEC

Mutations within the minimal replicon of plasmid pPS10 increase its host range

Mutations in the basic replicon of the Pseudomonas plasmid pPS10 permit its establishment in E.coli, Some of the mutations affect the synthesis and/or activity of RepA, the replication protein of the plasmid, and one of the mutants, pMM141, is further analysed. Replication of pMM141 in E.coli requires functional oriV and repA sequences, and depends on the DnaA protein of the host. pMM141 can replicate in Pseudomonas.Preliminary analysis of the replication of this mutant in extracts of E.coli, shows that the plasmid replication protein RepA as well as the host proteins DnaA, DnaB, DnaG, HU, SSB, DNA gyrase and DnaK are required. In vitro replication of pMM141 can occur in the absence of transcription by host RNA polymerase. Reconstitution experiments locate the mutation in a 0.3 kb fragment of the basic replicon of pMM141 that includes the 5′ end of the repA gene and that contains the coding region for the leucine zipper motif of RepA (Giraldo et al, 1989). It is proposed that the broad host-range character of the mutant is due to the adjustment of RepA interactions to the ones required for initiation of replication in E.coli

Cloning vectors, derived from a naturally occurring plasmid of Pseudomonas savastanoi, specifically tailored for genetic manipulations in Pseudomonas

A minimal replicon of 1.8 kb isolated from a 10-kb plasmid of Pseudomonas savastanoi, pPS10, has been used to obtain a collection of small vectors specific for Pseudomonas (P. savastanoi, P. aeruginosa and P. putida). In addition, shuttle vectors that can be established both in Pseudomonas and Escherichia coli have been constructed by adding a pMB9 replicon. The vectors permit cloning of DNA fragments generated by a variety of restriction enzymes using different antibiotic resistance markers for selection and offer the possibility to screen recombinants by insertional inactivation. This cloning system can be used to establish recombinant plasmids in Pseudomonas either at low or high copy number. pPS10 derivatives are compatible with other Pseudomonas vectors derived from broad-host-range replicons of the incompatibility groups P1, P4/Q and W. Intorduction and expression of the iaaMH operon in a P. savastanoi mutant deficient in the production of indoleacetic acid has been achieved using one of these vectors.This work was supported by grant BT-42-84 of the Spanish CAICYT and by the C.S.I.C

The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication

Plasmid R1 replication in vitro is inactive in extracts prepared from a dnaK756 strain but is restored to normal levels upon addition of purified DnaK protein. Replication of R1 in extracts of a dnaKwt strain can be specifically inhibited with polyclonal antibodies against DnaK. RepA-dependent replication of R1 in dnaK756 extracts supplemented with DnaKwt protein at maximum concentration is partially inhibited by rifampicin and it is severely inhibited at sub-optimal concentrations of DnaK protein. The copy number of a run-away R1 vector is reduced in a dnaK756 background at 30°C and at 42°C the amplification of the run-away R1 vector is prevented. However a runaway R1 vector containing dnaK gene allows the amplification of the plasmid at high temperature. These data indicate that DnaK is required for both in vitro and in vivo replication of plasmid R1 and show a partial compensation for the low level of DnaK by RNA polymerase. In contrast ColE1 replication is not affected by DnaK as indicated by the fact that ColE1 replicates with the same efficiency in extracts from dnaKwt and dnaK756 strains.This work was supported by grants B1089-0497, B1088-0294 and BIO91-1055 of the Spanish 'Comision Asesora de Investigacin Cientfica y Tecnica (C.A.I.C.Y.T)'

A leucine zipper motif determines different functions in a DNA replication protein

RepA is the replication initiator protein of the Pseudomonas plasmid pPS10 and is also able to autoregulate its own synthesis. Here we report a genetic and functional analysis of a leucine zipper-like (LZ) motif located at the N-terminus of RepA. It is shown that the LZ motif modulates the equilibrium between monomeric and dimeric forms of the protein and that monomers of RepA interact with sequences at the origin of replication, oriV, while dimers are required for interactions of RepA at the repA promoter. Further, different residues of the LZ motif are seen to have different functional roles. Leucines at the d positions of the putative alpha-helix are relevant in the formation of RepA dimers required for transcriptional autoregulation. They also modulate other RepA-RepA interactions that result in cooperative binding of protein monomers to the origin of replication. The residues at the b/f positions of the putative helix play no relevant role in RepA-RepA interactions. These residues do not affect RepA autoregulation but do influence replication, as demonstrated by mutants that, without affecting binding to oriV, either increase the host range of the plasmid or are inactive in replication. It is proposed that residues in b/f positions play a relevant role in interactions between RepA and host replication factors.This work was supported by grants B1088-0294. B1091-1055, B1092-1018-CO202 and PB92007 from the Spanish D.G.I.C.Y.T. and by the European Net CHRX-CT92-0010. D.G.de V. was supported by a fellowship from the 'Comunidad de Madrid' and by contract reference BI02CT920084 of the BIOTECH Program of the EC