Pangenomic and functional investigations for dormancy and biodegradation features of an organic pollutant-degrading bacterium Rhodococcus biphenylivorans TG9.

Environmental bacteria contain a wealth of untapped potential in the form of biodegradative genes. Leveraging this potential can often be confounded by a lack of understanding of fundamental survival strategies, like dormancy, for environmental stress. Investigating bacterial dormancy-to-degradation relationships enables improvement of bioremediation. Here, we couple genomic and functional assessment to provide context for key attributes of the organic pollutant-degrading strain Rhodococcus biphenylivorans TG9. Whole genome sequencing, pangenome analysis and functional characterization were performed to elucidate important genes and gene products, including antimicrobial resistance, dormancy, and degradation. Rhodococcus as a genus has strong potential for degradation and dormancy, which we demonstrate using R. biphenylivorans TG9 as a model. We identified four Resuscitation-promoting factor (Rpf) encoding genes in TG9 involved in dormancy and resuscitation. We demonstrate that R. biphenylivorans TG9 grows on fourteen typical organic pollutants, and exhibits a robust ability to degrade biphenyl and several congeners of polychlorinated biphenyls. We further induced TG9 into a dormant state and demonstrated pronounced differences in morphology and activity. Together, these results expand our understanding of the genus Rhodococcus and the relationship between dormancy and biodegradation in the presence of environmental stressors.

Antibiotic tolerance and degradation capacity of the organic pollutant-degrading bacterium Rhodococcus biphenylivorans TG9T.

Antibiotics are ubiquitous in soil due to natural ecological competition, as well as emerging contaminants due to anthropogenic inputs. Under environmental factors like antibiotic stress, some bacteria, including those that degrade environmental pollutants, can enter a dormant state as a survival strategy, thereby limiting their metabolic activity and function. Dormancy has a critical influence on the degradative activity of bacteria, dramatically decreasing the rate at which they transform organic pollutants. To better understand this phenomenon in environmental pollutant-degrading bacteria, we investigated dormancy transitions induced with norfloxacin in Rhodococcus biphenylivorans TG9T using next-generation proteomics, proteogenomics, and additional experiments. Our results suggest that exposure to norfloxacin inhibited DNA replication, which led to damage to the cell. Dormant cells then likely triggered DNA repair, particularly homologous recombination, for continued survival. The results also indicated that substrate transport (ATP-binding cassette transporter), ATP production, and the tricarboxylic acid (TCA) cycle were repressed during dormancy, and degradation of organic pollutants was down-regulated. Given the widespread phenomenon of dormancy among bacteria involved in pollutant removal systems, this study improves our understanding of possible implications of antibiotic survival strategies on biotransformation of mixtures containing antibiotics as well as other organics.

Taking the shortcut for high-throughput shotgun proteomic analysis of bacteria.

Currently, proteomic tools are able to establish a complete list of the most abundant proteins present in a sample, providing the opportunity to study at high resolution the physiology of any bacteria for which the genome sequence is available. For a comprehensive list, proteins should be first resolved into fractions that are then proteolyzed by trypsin. The resulting peptide mixtures are analyzed by a high-throughput tandem mass spectrometer that records thousands of MS/MS spectra for each fraction. These spectra are then assigned to peptides, which are used as evidence of the existence of proteins. In addition to generating a list of protein identifications, this shortcut to proteomics uses the number of spectra recorded for each protein to quantify the observations. Here, we describe one of the most simple sample preparation methods for high-throughput proteomics of bacteria, as well as the subsequent data processing to extract quantitative information based on the spectral count approach.

Proteogenomics for environmental microbiology.

Proteogenomics sensu stricto refers to the use of proteomic data to refine the annotation of genomes from model organisms. Because of the limitations of automatic annotation pipelines, a relatively high number of errors occur during the structural annotation of genes coding for proteins. Whether putative orphan sequences or short genes encoding low-molecular-weight proteins really exist is still frequently a mystery. Whether start codons are well defined is also an open debate. These problems are exacerbated for genomes of microorganisms belonging to poorly documented genera, as related sequences are not always available for homology-guided annotation. The functional annotation of a significant proportion of genes is also another well-known issue when annotating environmental microorganisms. High-throughput shotgun proteomics has recently greatly evolved, allowing the exploration of the proteome from any microorganism at an unprecedented depth. The structural and functional annotation process may be usefully complemented with experimental data. Indeed, proteogenomic mapping has been successfully performed for a wide variety of organisms. Specific approaches devoted to systematically establishing the N-termini of a large set of proteins are being developed. N-terminomics is giving rise to datasets of experimentally proven translational start codons as well as validated peptide signals for secreted proteins. By extension, combining genomic and proteomic data is becoming routine in many research projects. The proteomic analysis of organisms with unfinished genome sequences, the so-called composite proteomics, and the search for microbial biomarkers by bottom-up and top-down combined approaches are some examples of proteogenomic-flavored studies. They illustrate the advent of a new era of environmental microbiology where proteomics and genomics are intimately integrated to answer key biological questions.