Paloma Acebo

The structure of plasmid‐encoded transcriptional repressor CopG unliganded and bound to its operator

The structure of the 45 amino acid transcriptional repressor, CopG, has been solved unliganded and bound to its target operator DNA. The protein, encoded by the promiscuous streptococcal plasmid pMV158, is involved in the control of plasmid copy number. The structure of this protein repressor, which is the shortest reported to date and the first isolated from a plasmid, has a homodimeric ribbon–helix–helix arrangement. It is the prototype for a family of homologous plasmid repressors. CopG cooperatively associates, completely protecting several turns on one face of the double helix in both directions from a 13‐bp pseudosymmetric primary DNA recognition element. In the complex structure, one protein tetramer binds at one face of a 19‐bp oligonucleotide, containing the pseudosymmetric element, with two β‐ribbons inserted into the major groove. The DNA is bent 60° by compression of both major and minor grooves. The protein dimer displays topological similarity to Arc and MetJ repressors. Nevertheless, the functional tetramer has a unique structure with the two vicinal recognition ribbon elements at a short distance, thus inducing strong DNA bend. Further structural resemblance is found with helix–turn–helix regions of unrelated DNA‐binding proteins. In contrast to these, however, the bihelical region of CopG has a role in oligomerization instead of DNA recognition. This observation unveils an evolutionary link between ribbon–helix–helix and helix–turn–helix proteins.

Replication control of plasmid pLS1: efficient regulation of plasmid copy number is exerted by the combined action of two plasmid components, CopG and RNA II

Two elements, the products of genes copG and rnall, are involved in the copy‐number control of plasmid pLS1. RNA II is synthesized in a dosage‐dependent manner. Mutations in both components have been characterized. To determine the regulatory role of the two genes, we have cloned copG, rnall or both elements at various gene dosages into pLS1‐compatible plasmids. Assays of incompatibility towards wild‐type or mutant pLS1 plasmids showed that: (i) the rnall gene product, rather than the DNA sequence encoding it, is responsible for the incompatibility, and (ii) CopG and RNA II act in trans and are able to correct up fluctuations in pLS1 copy number. A correlation between the gene dosage at which the regulatory elements were supplied and the incompatibility effect on the resident plasmid was observed. The entire copG‐rnall circuit has a synergistic effect when compared with any of its components in the correction of pLS1 copy‐number fluctuations, indicating that, in the homoplasmid steady‐state situation, the control of pLS1 replication is exerted by the co‐ordinate action of CopG and RNA II.

Molecular characterization of the safracin biosynthetic pathway from Pseudomonas fluorescens A2‐2: designing new cytotoxic compounds

Safracin is an antibiotic with anti‐tumour activity produced by Pseudomonas fluorescens A2‐2. The entire safracin synthetic gene cluster spanning 17.5 kb has been identified, cloned and sequenced. The safracin cluster comprises 10 open reading frames (ORFs) encoding proteins for three non‐ribosomal peptide synthetases (NRPS), three safracin precursor biosynthetic enzymes, two safracin tailoring enzymes, a safracin resistance protein and a small hypothetical protein of unknown function. These genes are organized in two divergent operons of eight and two genes respectively. This pathway exhibits unusual features when compared with other NRPS systems. We have demonstrated by heterologous expression of the cluster that it is able to direct the synthesis of safracin in other strains. Cross‐feeding experiments have confirmed that 3‐hydroxy‐5‐methyl‐O‐methyltyrosine is the precursor of two amino acids of the molecule. Genetic analyses have allowed us to demonstrate that the bicistronic operon encodes the hydroxylation and N‐methylation activities of the pathway. The cloning and expression of the safracin cluster has settled the basis for the in vivo and in vitro production of a wide variety of compounds, such as the promising ecteinascidins anti‐cancer compounds.

Replication control of plasmid pLS1: the antisense RNA II and the compact rnaII region are involved in translational regulation of the initiator RepB synthesis

Replication of the streptococcal plasmid pLS1 is controlled by two plasmid‐encoded gene products: the repressor protein CopG and the antisense RNA, RNA II. Two different mutants in rnaII have been isolated. The 5′‐end and the levels of RNA II synthesized by pneumococcal cells harbouring the wild‐type pLS1 or mutant plasmids (affected in either genes copG or rnaII ) were analysed. One of the rnaII mutants exhibited a high‐copy‐number phenotype, whereas an in vitro‐constructed mutation, which affects the −10 region of the rnaII promoter, resulted in plasmids lacking copy‐number phenotype. The latter mutation had a pleiotropic effect: it abolished RNA II synthesis, but it also affected the initiation of translation signals of the gene encoding the RepB initiator protein. Transcriptional and translational fusions, together with in vitro inhibition of RepB synthesis by specific oligonucleotides, showed translational inhibition of RepB synthesis by RNA II, perhaps by directly blocking the accessibility of the ribosomes to the repB initiation of translation signals.

Quantitative detection of Streptococcus pneumoniae cells harbouring single or multiple copies of the gene encoding the green fluorescent protein

A modified gfp gene from Aequorea victoria, encoding a variant of the green fluorescent protein (GFP), was subcloned into the mobilizable plasmid pMV158. gfp was placed under the control of the inducible P(M) promoter of the Streptococcus pneumoniae gene malM, cloned in plasmid pLS70. The P(M) promoter is regulated by the product of the pneumococcal malR gene, which is inactivated by growing the cells in maltose-containing media. By homologous recombination, the P(M)-gfp construction was integrated into the host chromosome in a single copy. In both conditions (single and multiple copies), the pneumococcal cells were able to express GFP in an inducible or constitutive form, depending on whether the S. pneumoniae strain harboured a wild-type or a mutant malR gene. Quantification of the levels of GFP expressed by cultures supplemented with sucrose or maltose as carbon sources was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed pneumococcal cells expressing GFP in mixed cultures to be distinguished from those not carrying the gfp gene.

The mer operon of the acidophilic bacterium Thiobacillus T3.2 diverges from its Thiobacillus ferrooxidans counterpart

Abstract The chromosomal mercury resistance (mer) region of the acidophilic bacterium Thiobacillus T3.2 was cloned, characterized, and compared to reported homologous sequences. The Thiobacillus T3.2 mer resistance system is organized as an operon that transcribes into a polycistronic mRNA encoding the Hg2+ ion transport T and Mer P proteins and the mercuric reductase MerA. In contrast to the Thiobacillus ferrooxidans mer determinant, no merC gene was detected. Transcription of structural genes is regulated by the product of the regulatory merR gene. On the basis of sequence data and expression experiments in E. coli, both merTPA and merR transcription units could be located close to each other and in different strands, with their promoters (PTPA and PR, respectively) overlapping the putative MerR binding site in the intergenic operator/promoter (O/P) region. Amino acid sequences of mer gene products were compared to their homologs. Some sequence features, such as the number and position of cysteine residues, are unique for the Mer proteins of this bacterium. Similarities (−10 and −35 boxes are 19 bp apart in both PR and PTPA promoters) and differences (inverted repeats in the Thiobacillus T3.2 MerR-binding site are 2 bp shorter than in Thiobacillus ferrooxidans) exist between the O/P intergenic regions of both Thiobacilli. In vivo experiments showed inducible expression of mercury resistance in E. coli cells transformed with the entire Thiobacillus T3.2 mer genetic determinant (structural plus regulatory genes), and little or no expression in clones containing only the structural merT, merP, and merA genes.

Structural features of the plasmid pMV158‐encoded transcriptional repressor CopG, a protein sharing similarities with both helix‐turn‐helix and β‐sheet DNA binding proteins

The small transcriptional repressor CopG protein (45 amino acids) encoded by the streptococcal plasmid pMV158 was purified to near homogeneity. Gel filtration chromatography and analytical ultracentrifugation showed that the native protein is a spherical dimer of identical subunits. Circular dichroism measurements of CopG indicated a consensus average content of more than 50% α‐helix and 10–35% β‐strand and turns, which is compatible with the predicted secondary structure of the protein. CopG exhibited a prolonged intracellular half‐life, but deletions in regions other than the C‐terminal affected the global structure of the protein, severely reducing the half‐lives of the CopG variants. This indicates that CopG has a compact structure, perhaps constituted by a single domain. Molecular modeling of CopG showed a good fitting between the helix‐turn‐helix motifs of well‐known repressor proteins and a bihelical unit of CopG. However, modeling of CopG with ribbon‐helix‐helix class of DNA binding proteins also exhibited an excellent fit. Eleven out of the 12 replicons belonging to the pMV158 plasmid family could also encode Cop proteins, which share features with both helix‐turn‐helix and β‐sheet DNA binding proteins. Proteins 32:248–261, 1998.

Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the pMV158-encoded plasmid transcriptional repressor protein CopG.

Plasmid pMV158 encodes a 45 amino acid transcriptional repressor, CopG, which is involved in copy number control. A new procedure for overproduction and purification of the protein has been developed. The CopG protein thus obtained retained its ability to specifically bind to DNA and to repress its own promoter. Purified CopG protein has been crystallized using the sitting‐drop vapor diffusion method. The crystals, belonging to orthorhombic space group C2221 (cell constants a=67.2 Å, b=102.5 Å, c=40.2 Å), were obtained from a solution containing methylpentanediol, benzamidine and sodium chloride, buffered to pH 6.7. Complete diffraction data up to 1.6 Å resolution have been collected. Considerations about the Matthews parameter account for the most likely presence of three molecules in the asymmetric unit (2.27 Å3/Da).

Research letterIsolation and characterization of pLS 1 plasmid mutants with increased copy numbers

Streptococcus pneumoniae genetic systems designed for isolation of plasmid mutants with copy-up phenotypes have been developed. The target plasmids have the pLS1 replicon, and two different strategies have been followed: (i) selection of clones exhibiting augmented resistance to antibiotics, or (ii) obligatory co-existence of incompatible plasmids. We have isolated 23 plasmid mutants exhibiting increased number of copies. All the mutations corresponded to four different alleles of the copG gene of plasmid pLS1. These strategies could be used with other plasmids.