RESUMO
Even though several in vitro studies have focused on bacterial biology, the extent of such knowledge is not complete when considering an actual infection. As culture-independent microbiology methods such as high-throughput sequencing became available, important aspects of host-bacterium interactions will be elucidated. Based on microbiological relevance, we considered Bacteroides fragilis in a murine experimental infection as a model system to evaluate the in vivo bacterial transcriptome in host exudates. A disproportionate number of reads belonging to the host genome were retrieved in the first round of pyrosequencing, even after depletion of ribosomal RNA; the average number of reads related to the eukaryotic genome was 71.924-67.7%, whereas prokaryotic reads represented 34.338-32.3% in host exudates. Thus, different treatments were used to improve the prokaryotic RNA yield: i) centrifugation; ii) ultrasonic treatment; and iii) ultrasonic treatment followed by centrifugation. The latter treatment was found to be the most efficient in generating bacterial yields, as it resulted in a higher number of Bacteroides cells. However, the RNA extracted after this treatment was not of sufficient quality to be used in cDNA synthesis. Our results suggest that the methodology routinely used for RNA extraction in transcriptional analysis is not appropriate for in vivo studies in complex samples. Furthermore, the most efficient treatment for generating good bacterial cell yields was not suitable to retrieve high-quality RNA. Therefore, as an alternative methodological approach to enable in vivo studies on host-bacterium interactions, we advise increasing the sequencing depth despite the high costs.
Assuntos
Bacteroides fragilis/genética , Perfilação da Expressão Gênica/métodos , RNA Mensageiro/genética , Transcriptoma/genética , Animais , Bacteroides fragilis/patogenicidade , Regulação Bacteriana da Expressão Gênica/genética , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , RNA Mensageiro/isolamento & purificação , Análise de Sequência de RNARESUMO
AIMS: To isolate and to characterize the diversity of Chromobacterium violaceum from the Brazilian Amazon region. METHODS AND RESULTS: Twenty-two isolates were obtained from the waters and banks of the river Negro, in the Brazilian Amazon. All isolates were able to grow in vitro at 44 degrees C and pH 4.0, but were adversely affected by temperatures below 15 degrees C, and unable to survive at 4 degrees C, properties that may be related to the adaptation to the ecosystem. The isolates were joined at a final level of similarity of only 13% in the rep-PCR analysis. The analysis of 16S rRNA genes resulted in three main groups clustered at a final level of similarity of 97% and only three isolates were clustered with the type strain. Similar data were obtained for the 23S rRNA gene. CONCLUSIONS: A high level of genetic diversity was verified with indications that the Brazilian isolates would fit into at least two new clusters besides C. violaceum species. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show remarkable bacterial adaptability and genetic diversity of C. violaceum in the Amazon region.
Assuntos
Chromobacterium/isolamento & purificação , Rios/microbiologia , Microbiologia da Água , Brasil , Chromobacterium/genética , Chromobacterium/patogenicidade , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase , Polimorfismo Genético , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genéticaRESUMO
Chromobacterium violaceum is a Gram-negative bacterium, abundant in a variety of ecosystems in tropical and subtropical regions, including the water and borders of the Negro River, a major component of the Amazon Basin. As a free-living microorganism, C. violaceum is exposed to a series of variable conditions, such as different sources and abundance of nutrients, changes in temperature and pH, toxic compounds and UV rays. These variations, and the wide range of environments, require great adaptability and strong protective systems. The complete genome sequencing of this bacterium has revealed an enormous number and variety of ORFs associated with alternative pathways for energy generation, transport-related proteins, signal transduction, cell motility, secretion, and secondary metabolism. Additionally, the limited availability of iron in most environments can be overcome by iron-chelating compounds, iron-storage proteins, and by several proteins related to iron metabolism in the C. violaceum genome. Osmotically inducible proteins, transmembrane water-channel, and other membrane porins may be regulating the movement of water and maintaining the cell turgor, activities which play an important role in the adaptation to variations in osmotic pressure. Several proteins related to tolerance against antimicrobial compounds, heavy metals, temperature, acid and UV light stresses, others that promote survival under starvation conditions, and enzymes capable of detoxifying reactive oxygen species were also detected in C. violaceum. All these features together help explain its remarkable competitiveness and ability to survive under different types of environmental stress