RESUMEN
Thermoacidophilic archaea lack sigma factors and the large inventory of heat shock proteins (HSPs) widespread in bacterial genomes, suggesting other strategies for handling thermal stress are involved. Heat shock transcriptomes for the thermoacidophilic archaeon Saccharolobus (f. Sulfolobus) solfataricus 98/2 revealed genes that were highly responsive to thermal stress, including transcriptional regulators YtrASs (Ssol_2420) and FadRSs (Ssol_0314), as well as type II toxin-antitoxin (TA) loci VapBC6 (Ssol_2337, Ssol_2338) and VapBC22 (Ssol_0819, Ssol_0818). The role, if any, of type II TA loci during stress response in microorganisms, such as Escherichia coli, is controversial. But, when genes encoding YtrASs , FadRSs , VapC22, VapB6, and VapC6 were systematically mutated in Sa. solfataricus 98/2, significant up-regulation of the other genes within this set was observed, implicating an interconnected regulatory network during thermal stress response. VapBC6 and VapBC22 have close homologues in other Sulfolobales, as well as in other archaea (e.g. Pyrococcus furiosus and Archaeoglobus fulgidus), and their corresponding genes were also heat shock responsive. The interplay between VapBC TA loci and heat shock regulators in Sa solfataricus 98/2 not only indicates a cellular mechanism for heat shock response that differs from bacteria but one that could have common features within the thermophilic archaea.
Asunto(s)
Antitoxinas , Sulfolobus solfataricus , Toxinas Biológicas , Antitoxinas/genética , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismo , Respuesta al Choque Térmico/genética , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismo , Escherichia coli/genéticaRESUMEN
A mutant ('lab strain') of the hyperthermophilic archaeon Pyrococcus furiosus DSM3638 exhibited an extended exponential phase and atypical cell aggregation behavior. Genomic DNA from the mutant culture was sequenced and compared to wild-type (WT) DSM3638, revealing 145 genes with one or more insertions, deletions, or substitutions (12 silent, 33 amino acid substitutions, and 100 frame shifts). Approximately, half of the mutated genes were transposases or hypothetical proteins. The WT transcriptome revealed numerous changes in amino acid and pyrimidine biosynthesis pathways coincidental with growth phase transitions, unlike the mutant whose transcriptome reflected the observed prolonged exponential phase. Targeted gene deletions, based on frame-shifted ORFs in the mutant genome, in a genetically tractable strain of P. furiosus (COM1) could not generate the extended exponential phase behavior observed for the mutant. For example, a putative radical SAM family protein (PF2064) was the most highly up-regulated ORF (>25-fold) in the WT between exponential and stationary phase, although this ORF was unresponsive in the mutant; deletion of this gene in P. furiosus COM1 resulted in no apparent phenotype. On the other hand, frame-shifting mutations in the mutant genome negatively impacted transcription of a flagellar biosynthesis operon (PF0329-PF0338).Consequently, cells in the mutant culture lacked flagella and, unlike the WT, showed minimal evidence of exopolysaccharide-based cell aggregation in post-exponential phase. Electron microscopy of PF0331-PF0337 deletions in P. furiosus COM1 showed that absence of flagella impacted normal cell aggregation behavior and, furthermore, indicated that flagella play a key role, beyond motility, in the growth physiology of P. furiosus.
Asunto(s)
Flagelos/genética , Genes Bacterianos , Mutación , Pyrococcus furiosus/genética , Proliferación Celular , Flagelos/ultraestructura , Fenotipo , Pyrococcus furiosus/crecimiento & desarrollo , Pyrococcus furiosus/metabolismo , Pyrococcus furiosus/fisiología , TranscriptomaRESUMEN
The genus Thermotoga comprises extremely thermophilic (Topt > or = 70 degrees C) and hyperthermophilic (Topt > or = 80 degrees C) bacteria, which have been extensively studied for insights into the basis for life at elevated temperatures and for biotechnological opportunities (e.g. biohydrogen production, biocatalysis). Over the past decade, genome sequences have become available for a number of Thermotoga species, leading to functional genomics efforts to understand growth physiology as well as genomics-based identification and characterization of novel high-temperature biocatalysts. Discussed here are recent developments along these lines for this group of microorganisms.
Asunto(s)
Genoma Bacteriano , Thermotoga maritima , Biopelículas , Metabolismo de los Hidratos de Carbono/genética , Genómica , ARN Ribosómico 16S/genética , Thermotoga maritima/enzimología , Thermotoga maritima/genética , Thermotoga maritima/metabolismo , Thermotoga maritima/fisiologíaRESUMEN
The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms, no doubt harbors technologically important enzymes and other thermostable biomolecules that, to date, have been characterized only to a limited extent. In the first of a two-part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS(2) analysis and prediction servers suggested probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments, all including the C(18) bead technique, demonstrated that two levels of sample purification were necessary to effectively desalt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described, as enzymatic and putative uncharacterized proteins, anticipate consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion, and 11 contained transmembrane domain stretches suggested by Phobius and transmembrane hidden Markov model. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study, involved acetone precipitation followed by the C(18) bead method in which 2.4% (63 proteins) of the predicted proteome was identified, including proteins suggested to have secretion and transmembrane moieties.
Asunto(s)
Proteínas Bacterianas/metabolismo , Espacio Extracelular/metabolismo , Bacterias Grampositivas/metabolismo , Proteoma/análisis , Proteínas Bacterianas/análisis , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TemperaturaRESUMEN
Probing the intracellular proteome of Thermotoga maritima and Caldicellulosiruptor saccharolyticus in pure and co-culture affords a global investigation into the machinery and mechanisms enduring inside the bacterial thermophilic cell at the time of harvest. The second of a two part study, employing GeLC-MS(2) a variety of proteins were confidently identified with <1% false discovery rate, and spectral counts for label-free relative quantification afforded indication of the dynamic proteome as a function of environmental stimuli. Almost 25% of the T. maritima proteome and 10% of the C. saccharolyticus proteome were identified. Through comparison of growth temperatures for T. maritima, a protein associated with chemotaxis was uniquely present in the sample cultivated at the non-optimal growth temperature. It is suspected that movement was induced due to the non-optimal condition as the organism may need to migrate in the culture to locate more nutrients. The inventory of C. saccharolyticus proteins identified in these studies and attributed to spectral counting, demonstrated that two CRISPR-associated proteins had increased expression in the pure culture versus the co-culture. Further focusing on this relationship, a C. saccharolyticus phage-shock protein was identified in the co-culture expanding a scenario that the co-culture had decreased antiviral resistance and accordingly an infection-related protein was present. Alterations in growth conditions of these bacterial thermophilic microorganisms offer a glimpse into the intricacy of microbial behavior and interaction.