RESUMEN
The ability of bacteria to adapt to external osmotic changes is fundamental for their survival. Halotolerant microorganisms, such as Tistlia consotensis, have to cope with continuous fluctuations in the salinity of their natural environments which require effective adaptation strategies against salt stress. Changes of extracellular protein profiles from Tistlia consotensis in conditions of low and high salinities were monitored by proteogenomics using a bacterial draft genome. At low salinity, we detected greater amounts of the HpnM protein which is involved in the biosynthesis of hopanoids. This may represent a novel, and previously unreported, strategy by halotolerant microorganisms to prevent the entry of water into the cell under conditions of low salinity. At high salinity, proteins associated with osmosensing, exclusion of Na+ and transport of compatible solutes, such as glycine betaine or proline are abundant. We also found that, probably in response to the high salt concentration, T. consotensis activated the synthesis of flagella and triggered a chemotactic response neither of which were observed at the salt concentration which is optimal for growth. Our study demonstrates that the exoproteome is an appropriate indicator of adaptive response of T. consotensis to changes in salinity because it allowed the identification of key proteins within its osmoadaptive mechanism that had not previously been detected in its cell proteome.
Asunto(s)
Adaptación Fisiológica/fisiología , Presión Osmótica/fisiología , Rhodospirillaceae/fisiología , Tolerancia a la Sal/fisiología , Cloruro de Sodio/metabolismo , Adaptación Fisiológica/genética , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico/genética , Flagelos/metabolismo , Genoma Bacteriano/genética , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/genética , Proteoma/genética , Rhodospirillaceae/genética , Salinidad , Tolerancia a la Sal/genética , Análisis de Secuencia de ADNRESUMEN
Given the ease of whole genome sequencing with next-generation sequencers, structural and functional gene annotation is now purely based on automated prediction. However, errors in gene structure are frequent, the correct determination of start codons being one of the main concerns. Here, we combine protein N termini derivatization using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP Ac-OSu) as a labeling reagent with the COmbined FRActional DIagonal Chromatography (COFRADIC) sorting method to enrich labeled N-terminal peptides for mass spectrometry detection. Protein digestion was performed in parallel with three proteases to obtain a reliable automatic validation of protein N termini. The analysis of these N-terminal enriched fractions by high-resolution tandem mass spectrometry allowed the annotation refinement of 534 proteins of the model marine bacterium Roseobacter denitrificans OCh114. This study is especially efficient regarding mass spectrometry analytical time. From the 534 validated N termini, 480 confirmed existing gene annotations, 41 highlighted erroneous start codon annotations, five revealed totally new mis-annotated genes; the mass spectrometry data also suggested the existence of multiple start sites for eight different genes, a result that challenges the current view of protein translation initiation. Finally, we identified several proteins for which classical genome homology-driven annotation was inconsistent, questioning the validity of automatic annotation pipelines and emphasizing the need for complementary proteomic data. All data have been deposited to the ProteomeXchange with identifier PXD000337.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Péptidos/química , Roseobacter/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Cromatografía , Genoma Bacteriano , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Compuestos Organofosforados/química , Compuestos Organofosforados/metabolismo , Péptidos/genética , Proteómica , Roseobacter/clasificación , Roseobacter/metabolismo , Espectrometría de Masas en TándemRESUMEN
Tistlia consotensis is a halotolerant Rhodospirillaceae that was isolated from a saline spring located in the Colombian Andes with a salt concentration close to seawater (4.5%w/vol). We cultivated this microorganism in three NaCl concentrations, i.e. optimal (0.5%), without (0.0%) and high (4.0%) salt concentration, and analyzed its cellular proteome. For assigning tandem mass spectrometry data, we first sequenced its genome and constructed a six reading frame ORF database from the draft sequence. We annotated only the genes whose products (872) were detected. We compared the quantitative proteome data sets recorded for the three different growth conditions. At low salinity general stress proteins (chaperons, proteases and proteins associated with oxidative stress protection), were detected in higher amounts, probably linked to difficulties for proper protein folding and metabolism. Proteogenomics and comparative genomics pointed at the CrgA transcriptional regulator as a key-factor for the proteome remodeling upon low osmolarity. In hyper-osmotic condition, T. consotensis produced in larger amounts proteins involved in the sensing of changes in salt concentration, as well as a wide panel of transport systems for the transport of organic compatible solutes such as glutamate. We have described here a straightforward procedure in making a new environmental isolate quickly amenable to proteomics. BIOLOGICAL SIGNIFICANCE: The bacterium Tistlia consotensis was isolated from a saline spring in the Colombian Andes and represents an interesting environmental model to be compared with extremophiles or other moderate organisms. To explore the halotolerance molecular mechanisms of the bacterium T. consotensis, we developed an innovative proteogenomic strategy consisting of i) genome sequencing, ii) quick annotation of the genes whose products were detected by mass spectrometry, and iii) comparative proteomics of cells grown in three salt conditions. We highlighted in this manuscript how efficient such an approach can be compared to time-consuming genome annotation when pointing at the key proteins of a given biological question. We documented a large number of proteins found produced in greater amounts when cells are cultivated in either hypo-osmotic or hyper-osmotic conditions. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.
Asunto(s)
Adaptación Fisiológica/fisiología , Alphaproteobacteria , Proteínas Bacterianas , Manantiales Naturales/microbiología , Proteoma , Cloruro de Sodio , Microbiología del Agua , Alphaproteobacteria/clasificación , Alphaproteobacteria/genética , Alphaproteobacteria/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Lectura Abierta/fisiología , Proteoma/genética , Proteoma/metabolismoRESUMEN
Proteogenomics is the alliance of proteomics and genomics with the aim of better annotating structural genes based on experimental, protein-based data items established by tandem mass spectrometry. While, on average, more than one-tenth of protein N-termini are incorrectly annotated, there is a crucial need for methodological approaches to systematically establish the translational starts of polypeptides, and their maturations, such as N-terminal methionine processing and peptide signal excision. Refinement of genome annotation through correction of wrongly annotation initiation start site and detection of unannotated genes can be achieved after enrichment and detection of protein N-termini by mass spectrometry. Here we describe a straightforward strategy to specifically label protein N-termini with a positively charged TMPP label to selectively capture these entities with in-house-developed anti-TMPP antibodies coupled to magnetic beads and to analyze them by nanoLC-MS/MS. While most N-terminomics-oriented approaches are based on the depletion of internal peptides to retrieve N-terminal peptides, this enrichment approach is fast and the results are highly specific for improved, ionizable, TMPP-labeled peptides. The whole proteome of the model marine bacterium, Roseobacter denitrificans, was analyzed, leading to the identification of more than twice the number of N-terminal peptides compared with the nonenriched fraction. A total of 269 proteins were characterized in terms of their N-termini. In addition, three unannotated genes were identified based on multiple, redundant N-terminal peptides. Our strategy greatly simplifies the systematic and automatic proteogenomic annotation of genomes as well as degradomics-oriented approaches, focusing the mass spectrometric efforts on the most crucial enriched fractions.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/química , Separación Inmunomagnética , Péptidos/análisis , Espectrometría de Masas en Tándem/métodos , Animales , Anticuerpos Monoclonales/inmunología , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Péptidos/inmunología , Péptidos/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Microbiología Ambiental , Proteómica/métodos , Bacterias/genética , Bacterias/metabolismo , Genoma Bacteriano , Anotación de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
The use of non-aqueous solvents in desorption electrospray ionization mass spectrometry (DESI-MS) is explored by analyzing a set of 43 compounds using binary mixtures of chloroform, tetrahydrofuran, and acetonitrile as the spray solvent. Comparisons of data obtained from chloroform/tetrahydrofuran (1:1) and chloroform/acetonitrile (1:1) spray solvents with the standard aqueous-based spray solvent (methanol/water, 1:1) shows that the non-aqueous systems have practical value for DESI, especially in the analysis of hydrophobic compounds. Non-aqueous spray solvents were used to ionize thermometer molecules (benzyl pyridinium salts) and showed lower internal energies (softer DESI ionization compared with methanol/water, 1:1), a result that has parallels in known solvent effects in electrospray ionization and is explained by solvent effects on surface tension. Consideration of octanol/water partition coefficients (K(ow)) of the 43 analytes in the light of their DESI results reveals the importance of the solubility of analyte in the spray solvent in producing high quality mass spectra. This finding provides additional support for the droplet pick-up description of the DESI mechanism, which is based on analyte dissolution in the spray solvent, followed by splashing of subsequently arriving droplets in the liquid film to form microdroplets of dissolved analyte. DESI solvent optimization can be improved by the use of K(ow) of the analyte as an indication of the polarity of the most appropriate solvent system.