Luis Kameyama

High Diversity and Novel Species of Pseudomonas aeruginosa Bacteriophages

ABSTRACT The diversity of Pseudomonas aeruginosa bacteriophages was investigated using a collection of 68 phages isolated from Central Mexico. Most of the phages carried double-stranded DNA (dsDNA) genomes and were classified into 12 species. Comparison of the genomes of selected archetypal phages with extant sequences in GenBank resulted in the identification of six novel species. This finding increased the group diversity by ∼30%. The great diversity of phage species could be related to the ubiquitous nature of P. aeruginosa.

Narrow-Host-Range Bacteriophages That Infect Rhizobium etli Associate with Distinct Genomic Types

ABSTRACT In this work, we isolated and characterized 14 bacteriophages that infect Rhizobium etli. They were obtained from rhizosphere soil of bean plants from agricultural lands in Mexico using an enrichment method. The host range of these phages was narrow but variable within a collection of 48 R. etli strains. We obtained the complete genome sequence of nine phages. Four phages were resistant to several restriction enzymes and in vivo cloning, probably due to nucleotide modifications. The genome size of the sequenced phages varied from 43 kb to 115 kb, with a median size of ∼45 to 50 kb. A large proportion of open reading frames of these phage genomes (65 to 70%) consisted of hypothetical and orphan genes. The remainder encoded proteins needed for phage morphogenesis and DNA synthesis and processing, among other functions, and a minor percentage represented genes of bacterial origin. We classified these phages into four genomic types on the basis of their genomic similarity, gene content, and host range. Since there are no reports of similar sequences, we propose that these bacteriophages correspond to novel species.

Analysis of some phenotypic traits of feces-borne temperate lambdoid bacteriophages from different immunity groups: a high incidence of cor+, FhuA-dependent phages

A group of previously isolated heterogeneous mEp lambdoid phages (43) from 19 different immunity groups for phage infection was further characterized to gain insight into some phenotypic traits and to assess their relationship with phage λ. Interestingly, the FhuA host receptor was required by the majority of mEp phages (37 out of 43; ~85%). The cor gene, which has been reported to be involved in FhuA-dependent exclusion of lambdoid phages, was also found in most of the FhuA-dependent phages. Accordingly, no cor amplification by PCR was obtained among the six FhuA-independent mEp lambdoid phages. In contrast, it was found that around 25% of the population (10 out of 43 phages) required the specific and essential λ N antitermination function, and the λ site-specific DNA recombination function was observed only in two members (4.6%). Thus, a larger proportion of phages require the FhuA receptor for infection, and this is frequently correlated with the cor gene.

Evidence of bar minigene expression and tRNA2Ile sequestration as peptidyl-tRNA2Ile during lambda bacteriophage development.

Lambda bacteriophage development is impaired in Escherichia coli cells defective for peptidyl (pep)-tRNA hydrolase (Pth). Single-base-pair mutations (bar(-)) that affect translatable two-codon open reading frames named bar minigenes (barI or barII) in the lambda phage genome promote the development of this phage in Pth-defective cells (rap cells). When the barI minigene is cloned and overexpressed from a plasmid, it inhibits protein synthesis and cell growth in rap cells by sequestering tRNA(2)(Ile) as pep-tRNA(2)(Ile). Either tRNA(2)(Ile) or Pth may reverse these effects. In this paper we present evidence that both barI and barII minigenes are translatable elements that sequester tRNA(2)(Ile) as pep-tRNA(2)(Ile). In addition, overexpression of the barI minigene impairs the development even of bar(-) phages in rap cells. Interestingly, tRNA or Pth may reestablish lambda phage development. These results suggest that lambda bar minigenes are expressed and tRNA(2)(Ile) is sequestered as pep-tRNA(2)(Ile) during lambda phage development.

Regulation Of λ N-Gene Expression

The N gene product of phage λ is a positive regulatory factor for the transcription of other λ genes during phage development. It acts by modifying the RNA polymerase transcription complex. In the absence of N function, RNA polymerase initiates at the early λ promoters, p L and p R,but terminates transcription shortly after initiation at terminators, t L1 and t R1 respectively. The action of N prevents polymerase from terminating at t L1 and t R1 as well as at other more distant terminators in the p L and p R operon (see review by Friedman, 1988). In each of these operons, between the promoter and the first terminator, there is a target sequence which is also required for the N- antitermination process (Friedman et al., 1973; Rosenberg et al, 1978). These targets, nutL and nutR, have been defined genetically by mutations to block antitermination in the respective operons (Salstrom and Szybalski, 1978; Olson, et al., 1984). These mutations have defined two regions of nut, boxA and boxB (Friedman & Gottesman, 1983). The boxA site is conserved in sequence among other phages and E. coli whereas the box B site is unique for each phage type and is thought to be the N recognition site (Lazinski, et al., 1989) (See Figure 1).

Comparative genomic analysis of Pseudomonas aeruginosa phage PaMx25 reveals a novel siphovirus group related to phages infecting hosts of different taxonomic classes

Bacteriophages (phages) are estimated to be the most abundant and diverse entities in the biosphere harboring vast amounts of novel genetic information. Despite the genetic diversity observed, many phages share common features, such as virion morphology, genome size and organization, and can readily be associated with clearly defined phage groups. However, other phages display unique genomes or, alternatively, mosaic genomes composed of regions that share homology with those of phages of diverse origins; thus, their relationships cannot be easily assessed. In this work, we present a functional and comparative genomic analysis of Pseudomonas aeruginosa phage PaMx25, a virulent member of the Siphoviridae family. The genomes of PaMx25 and a highly homologous phage NP1, bore sequence homology and synteny with the genomes of phages that infect hosts different than Pseudomonas. In order to understand the relationship of the PaMx25 genome with that of other phages, we employed several computational approaches. We found that PaMx25 and NP1 effectively bridged several phage groups. It is expected that as more phage genomes become available, more gaps will be filled, blurring the boundaries that currently separate phage groups.

BRAF 1799T>A Mutation Frequency in Mexican Mestizo Patients with Papillary Thyroid Cancer

Thyroid cancer is the most frequent endocrine malignancy, and its incidence and prevalence are increasing worldwide. Despite its generally good prognosis, the observed mortality rates are higher in the less-developed regions. This indicates that timely diagnosis and appropriate initial management of this disease are important to achieve a positive outcome. We performed an observational study in order to describe the frequency of the BRAF 1799T>A mutation in Mexican mestizo patients with thyroid nodules, a scarcely studied ethnic group with large populations. Competitive allele-specific Taqman PCR was performed in 147 samples of thyroid tissue DNA obtained from patients histologically diagnosed with papillary thyroid cancer (PTC), colloid goiters, and follicular adenomas. The BRAF 1799T>A mutation frequency was 61.1% in PTC samples (p = 4.99 × 10−11). Potential diagnostic values were as follows: sensitivity, 61.1%; specificity, 96%; PPV, 94.2%; NPV, 69.5%; accuracy, 77.9%. Taking into account the fact that this mutation is not frequently found in cytologically indeterminate nodules, we suggest that the BRAF mutational analysis should be implemented in the clinical setting along with other diagnostic criteria such as USG, in order to contribute to diagnosis and to surgical decision-making during the initial management of thyroid nodules in Mexican public hospitals.

Some Reflections on the Origin of Lambdoid Bacteriophages

Luis Kameyama1, Eva Martinez-Penafiel1, Omar Sepulveda-Robles1, Zaira Y. Flores-Lopez1, Leonor Fernandez2, Francisco Martinez-Perez3 and Rosa Ma. Bermudez1 1Departamento de Genetica y Biologia Molecular, Centro de Investigacion y de Estudios Avanzados del IPN, 2Facultad de Quimica, Universidad Nacional Autonoma de Mexico D. F. 3Laboratorio de Microbiologia y Mutagenesis Ambiental, Escuela de Biologia, Universidad Industrial de Santander, Bucaramanga, 1,2Mexico 3Colombia