Manel Camps

University of California, Santa Cruz | UCSC · Department of Microbiology & Environmental Toxicology

Dengue fever is a mosquito-transmitted disease of great public health importance. Dengue lacks adequate vaccine protection and insecticide-based methods of mosquito control are proving increasingly ineffective. Here we review the emerging use of mosquitoes transinfected with the obligate intracellular bacterium Wolbachia pipientis for vector contro...

Food contamination with pathogenic Escherichia coli can cause severe disease. Here, we report the isolation of a multidrug resistant strain (A23EC) from fresh spinach. A23EC belongs to subclade C2 of ST131, a virulent clone of Extraintestinal Pathogenic E. coli (ExPEC). Most A23EC virulence factors are concentrated in three pathogenicity islands. T...

Multiple mutations often have non-additive (epistatic) phenotypic effects. Epistasis is of fundamental biological relevance but is not well understood mechanistically. Adaptive evolution, i.e., the evolution of new biochemical activities, is rich in epistatic interactions. To better understand the principles underlying epistasis during genetic adap...

We analyze 14,651 HIV1 reverse transcriptase (HIV RT) sequences from the Stanford HIV Drug Resistance Database labeled with treatment regimen in order to study the evolution this enzyme under drug selection in the clinic. Our goal is to identify distinct sectors of HIV RT's sequence space that are undergoing evolution as a way to identify individua...

The link between E. coli strains contaminating foods and human disease is unclear, with some reports supporting a direct transmission of pathogenic strains via food and others highlighting their role as reservoirs for resistance and virulence genes. Here we take a genomics approach, analyzing a large set of fully-assembled genomic sequences from E....

Fluoroquinolones are one of the most widely used class of antibiotics. They target two type II topoisomerase enzymes: gyrase and topoisomerase IV. Resistance to these drugs, which is largely caused by mutations in their target enzymes, is on the rise and becoming a serious public health risk. In this work, we analyze the sequences of 352 extraintes...

Mechanisms of Theta Plasmid Replication in Enterobacteria and Implications for Adaptation to Its Host, Page 1 of 2 Abstract Plasmids are autonomously replicating sequences that help cells adapt to diverse stresses. Theta plasmids are the most frequent plasmid class in enterobacteria. They co-opt two host replication mechanisms: replication at ori...

Antibiotic resistance is a powerful model for studying evolutionary biology and population genetics. For the purpose of these evolutionary studies, fitness data have been approximated through susceptibility testing methods designed for clinical use in providing appropriate antibiotic therapies. An alternative approach for measuring fitness of micro...

ColE1-like plasmid vectors are widely used for expression of recombinant genes in E. coli. For these vectors, segregation of individual plasmids into daughter cells during cell division appears to be random, making them susceptible to loss over time when no mechanisms ensuring their maintenance are present. Here we use the plasmid pGFPuv in a recA...

We evaluate the distribution of genetic markers for antibiotic resistance in 276 genomic sequences of Extraintestinal Pathogenic E. coli from two hospitals on the U.S. West coast. Plasmid-borne genes encoding drug-inactivating enzymes dominate the distribution of aminoglycoside and β-lactam resistance markers. These genes can be assigned based on t...

The evolution of new biochemical activities frequently involves complex dependencies between mutations and rapid evolutionary radiation. Mutation co-occurrence and co-variation have previously been used to identify compensating mutations that are the result of physical contacts and preserve protein function and fold. Here, we model pairwise functio...

Mutagenesis in model organisms following exposure to chemicals is used as an indicator of genotoxicity. Mutagenesis assays are also used to study mechanisms of DNA homeostasis. This chapter focuses on detection of mutagenesis in prokaryotes, which boils down to two approaches: reporter inactivation (forward mutation assay) and reversion of an inact...

Mechanisms of Theta Plasmid Replication, Page 1 of 2 Abstract Plasmids have been used as convenient models for the study of molecular mechanisms of replication and DNA repair due to their small size, dispensability to the host, and easy manipulation. In addition, plasmids are key facilitators for the evolution and dissemination of drug resistance...

Directed evolution is an approach that mimics natural evolution in the laboratory with the goal of modifying existing enzymatic activities or of generating new ones. The identification of mutants with desired properties involves the generation of genetic diversity coupled with a functional selection or screen. Genetic diversity can be generated usi...

A central problem of evolutionary biology is the genetic basis of protein adaptation, i.e. the evolution of new functions. In a widely accepted paradigm, each protein mutation represents a discrete step sequence space along adaptive trajectories toward new functions. Predicting the functional effect of multiple mutations is, however, a very challen...

ColE1 plasmid replication is unidirectional and requires two DNA polymerases: DNA polymerase I (Pol I) and DNA polymerase III (Pol III). Pol I initiates leading-strand synthesis by extending an RNA primer, allowing the Pol III holoenzyme to assemble and finish replication of both strands. The goal of the present work is to study the interplay betwe...

Our laboratory specializes in directed protein evolution, i.e., evolution of proteins under defined selective pressures in the laboratory. Our target genes are encoded in ColE1 plasmids to facilitate the generation of libraries in vivo. We have observed that when random mutations are not restricted to the coding sequence of the target genes, direct...

In this issue of Molecular Cell, Dango and Mosammaparast discover that the human oxidative demethylase ALKBH3 functions in complex with a DNA helicase to eliminate N3-methylcytosine lesions from ssDNA and that specific cancer cell lines are dependent on this activity for proliferation (Dango et al., 2011).

Structural impact of extended-spectrum antibiotic resistance mutations. (A) Mutations at residue 164. An arginine to serine (or arginine to histidine) substitution at position 164 (blue spheres) has been hypothesized to collapse the critical Ω-loop (green) in the active site, thus opening the active site to β-lactams with larger side chains [9], [7...

Calculation of conversion factor used to extrapolate growth to the gradient concentration used for wild-type. A conversion factor was used to compare results from gradients containing different drug concentrations. To that end, we measured the growth distance for a number individual reference clones at two contiguous concentrations and averaged the...

Supporting Methods and Results sections. The Methods section outlines important processing steps for TEM mutations before their inclusion in the network. Key node network centrality metrics are also presented. The Results section highlights the observation that the evolutionary trajectories we predicted by our betweenness centrality ranking are enr...

Locations of amino acid residues in the TEM coevolution network and the TEM extended-spectrum community network, mapped onto the TEM tertiary structure (PDB 1ero). (A) Residues in the TEM coevolution network and their three major communities (Figure 1). Residues are colored by community membership: gray (broad-spectrum resistance), blue (extended-s...

Presence of triple mutants predicted to be of special significance for adaptation within cefotaxime-driven evolutionary trajectories. Cefotaxime-driven evolutionary trajectories reported by Salverda et al. [36] are listed in column 2. This work investigated the role of contingency of the first mutation to be fixed under a given selective pressure o...

FASTA formatted protein sequences used in the construction of the TEM coevolution network. Sequences are collected from clinical isolates that have been demonstrated to confer resistance to β-lactamase inhibitors. (FA)

Lists of all naturally occurring TEM-1 mutants in our database. Each mutant is listed in column 1 and amino acid substitution mutations are indicated by residue position according to the Ambler system [65]. The mutants are listed in three worksheets by Bush-Jacoby phenotype class [27]: broad-spectrum antibiotic resistance (2b), extended-spectrum an...

Primers used for site-directed mutagenesis of TEM β–lactamase. The primers used for site-directed mutagenesis by the megaprimer protocol [70] are listed, grouped by orientation. For each primer, the name, sequence, and amino acid substitution are shown. (XLSX)

Calculation of average effect of mutations at individual positions tested in a variety of sequence contexts. 14 out of 16 positions identified in high betweenness centrality trajectories were tested experimentally in a variety of sequence contexts. For each position, the most frequent mutation found in our database was used (see Table 6). The posit...

FASTA formatted protein sequences used in the construction of the TEM coevolution network. Sequences are collected from published laboratory evolution experiments selecting for resistance to extended-spectrum β-lactam antibiotics. (FA)

FASTA formatted protein sequences used in the construction of the TEM coevolution network. Sequences are collected from clinical isolates that have been demonstrated to confer resistance to broad-spectrum β-lactam antibiotics. (FA)

The weighted degree distribution of the TEM coevolution network (Figure 1). The distribution of nodes by aggregate weight of links per node (weighted degree centrality, Equation S1) is shown. Many nodes (residue positions) with high weighted degree are functionally important (Table 1). The distribution reveals that the network contains very few hig...

Lists of all laboratory-evolved TEM-1 mutants in our database. Each mutant is listed in column 1, and amino acid substitution mutations are indicated by residue position according to the Ambler system [65]. The mutants are listed in three worksheets by Bush-Jacoby phenotype class [27]: broad-spectrum antibiotic resistance (2b), extended-spectrum an...

Codon-based analysis of positive selection. We performed a PAML (codeml) [67] analysis for the naturally occurring sequences. Residue position number according to the Ambler system [65] (column 1); wild-type amino acid residue in TEM-1 (column 2); ω value (ratio of non-synonymous to synonymous nucleotide substitutions at a codon position) (column 3...

Cefotaxime gradient measurements. All mutants and controls tested experimentally for cefotaxime resistance are listed in column A. The concentration empirically found to produce adequate resolution (i.e. intermediate level of growth in the gradient) is listed in column B. Columns C through AF list measurements (in centimeters) of continuous growth...

Network link weight normalization is predictive of epistasis. Links between nodes were weighted according to the frequency in which the relevant pair (i.e. the mutant positions joined by the link) occurred in our database. This metric was further modified using a normalization factor comparing frequency of co-occurrence to that of the corresponding...

FASTA formatted protein sequences used in the construction of the TEM coevolution network. Sequences are collected from published laboratory evolution experiments selecting for resistance to broad-spectrum β-lactam antibiotics. (FA)

References for the laboratory-evolved TEM sequences in Datasets S1 through S3. (XLSX)

FASTA formatted protein sequences used in the construction of the TEM coevolution network. Sequences are collected from published laboratory evolution experiments selecting for resistance to β-lactamase inhibitors. (FA)

FASTA formatted protein sequences used in the construction of the TEM coevolution network. Sequences are collected from clinical isolates that have been demonstrated to confer resistance to extended-spectrum β-lactam antibiotics. (FA)

FASTA formatted coding DNA sequences used in the TEM phylogenetic analysis. Sequences are collected from clinical isolates that have been demonstrated to confer resistance to extended- or broad-spectrum β-lactam antibiotics, or to β-lactamase inhibitors. (FA)

Understanding how novel functions evolve (genetic adaptation) is a critical goal of evolutionary biology. Among asexual organisms, genetic adaptation involves multiple mutations that frequently interact in a non-linear fashion (epistasis). Non-linear interactions pose a formidable challenge for the computational prediction of mutation effects. Here...

DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations gen...

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>10(11)) mutant libraries for a given target sequence. This method is based on...

ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression becaus...

ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression becaus...

Evolution requires the generation and optimization of new traits ("adaptation") and involves the selection of mutations that improve cellular function. These mutations were assumed to arise by selection of neutral mutations present at all times in the population. Here we review recent evidence that indicates that deleterious mutations are more freq...

The protozoan parasite, Toxoplasma gondii, interconverts between fast-growing tachyzoites and slow-growing bradyzoites within intermediate hosts. The surface of T. gondii is covered by the SAG1-related sequence (SRS) superfamily of glycosyl phosphatidyl inositol-anchored proteins, many of which are stage-specific. Previous transient transfection of...

Blocks in replication result from impediments to the advancing replication machinery and are lethal if not resolved. The replication fork must be reassembled for DNA synthesis to proceed. Fork assembly outside the chromosomal origin of replication (oriC) is mediated by recombination or via a helicase-dependent pathway. ColE1 plasmid origins of repl...

Pol I is the most abundant polymerase in E. coli and plays an important role in short patch repair. In accord with this role in the cell, the purified polymerase exhibits low processivity and high fidelity in vitro. Pol I is also the polymerase responsible for leader strand synthesis during ColE1 plasmid replication. In a previous publication, we d...

Extract: Mutagenesis is widely used in the fields of genetics, enzyme catalysis, ligand-binding recognition, metabolic regulation, control of gene expression, and to study mechanisms of DNA repair. Mutations are also introduced to optimize enzyme performance. This area has attracted renewed interest with the increased use of biocatalysts in chemica...

We present a system for random mutagenesis in Escherichia coli for the evolution of targeted genes. To increase error rates of DNA polymerase I (Pol I) replication, we introduced point mutations in three structural domains that govern Pol I fidelity. Expression of error-prone Pol I in vivo results in strong mutagenesis of a target sequence encoded...

ColE1 plasmid replication is unidirectional and requires two DNA polymerases: DNA polymerase I (Pol I) and DNA polymerase III (Pol III). Pol I initiates leading-strand synthesis by extending an RNA primer, allowing the Pol III holoenzyme to assemble and finish replication of both strands. The goal of the present work is to study the interplay betwe...

Toxoplasma gondii is a protozoan sensitive to several inhibitors of prokaryotic translation (e.g. clindamycin, macrolides and tetracyclines). A priori, two prokaryotic-like organelles, the 'apicoplast' (a non-photosynthetic plastid) and the mitochondrion, are likely targets for these drugs. Without using overt mutagenesis, we selected two independe...

Toxoplasma gondii is an obligate intracellular protozoan parasite with an exceptionally broad host range. Recently, it has become apparent that the number of surface antigens (SAGs) it expresses may rival the number of genera it can infect. Most of these antigens belong to the developmentally regulated and distantly related SAG1 or SAG2 families. T...

Extracellular Toxoplasma parasites are sensitive to pyrrolidine dithiocarbamate (PDTC) at low micromolar concentrations. Loss of parasite viability following PDTC treatment is shown to be mediated by oxidation, which is reminiscent of PDTC killing in mammalian cells. Intracellular parasites, by contrast, are resistant to PDTC killing, although trea...

Drug resistance generated in vitro in the protozoan parasite Toxoplasma gondii is described. We focus on drugs that are in use in patients, that show some promise for such use, or that represent lead compounds for further development. No instance has yet been reported where resistance to any of these drugs has arisen in a patient or in the field al...

The plant Arabidopsis thaliana contains five isoforms of the catalytic subunit of protein phosphatase 2A (PP2A) that can be grouped into two families, one composed by isoforms PP2A-1, -2 and -5 and the other composed by isoforms PP2A-3 and PP2A-4. An Arabidopsis genomic library was screened and several clones corresponding to genes PP2A-3 and PP2A-...

Protein phosphatases PPZ1 and PPZ2 represent a novel form of Ser/Thr phosphatases structurally related to type 1 phosphatases and characterized by an unusual amino-terminal region. We have found that the deletion of PPZ1 gene results in increased tolerance to Na+ and Li+ cations. Simultaneous deletion of PPZ2 gene results in an additional increase...

Two DNA fragments, AP-1 and AP-2, encoding amino acid sequences closely related to Ser/Thr protein phosphatases were amplified from Arabidopsis thaliana genomic DNA. Fragment AP-1 was used to screen A. thaliana cDNA libraries and several positive clones were isolated. Clones EP8a and EP14a were sequenced and found to encode almost identical protein...