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Selection-free markerless genome manipulations in the polyploid bacterium Thermus thermophilus.

ABSTRACT: A genome manipulation approach based on double-crossover homologous recombination was developed in the polyploid model organism Thermus thermophilus HB27 without the use of any selectable marker. The method was established and optimized by targeting the megaplasmid-encoded ?-glucosidase gene bgl. When linear and supercoiled forms of marker-free suicide vector were used for transformations, the frequencies of obtaining apparent Bgl^- mutant were 10^-?5 and 10^-?3, respectively; while the frequency could reach 10^-?2 when transformation with concatemer form of the same vector. All randomly selected Bgl^- colonies from the transformations were found to be true bgl knockout mutants. Thus, markerless gene deletion mutants could be constructed in T. thermophilus by the direct selection-free method. The functionality of this approach was further demonstrated by deletion of one chromosomal locus (TTC_0340-0341) as well as by generation of a reporter strain for the phytoene synthase promoter (PcrtB), homozygous mutants of the both targets could also be detected with a frequency of approximately 10^-?2. During the genome modification process, heterozygous cells carrying two different alleles at a same locus (e.g., bgl and pyrE) could also be generated. However, in the absence of selection pressure, these strains could rapidly convert to homozygous strains containing only one of the two alleles. This indicated that allele segregation could occur in the heterozygous T. thermophilus cells, which probably explained the ease of obtaining homozygous gene deletion mutants with high frequency (10^-?2) in the polyploid genomic background, as after the mutant allele had been introduced to the target region, allele segregation would lead to homozygous mutant cells. This marker-free genome manipulation approach does not require phenotype-based screens, and is applicable in gene deletion and tagging applications.

SUBMITTER: Li H

PROVIDER: S-EPMC6431304 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Selection-free markerless genome manipulations in the polyploid bacterium Thermus thermophilus.

Li Haijuan H

3 Biotech 20190323 4

A genome manipulation approach based on double-crossover homologous recombination was developed in the polyploid model organism Thermus thermophilus HB27 without the use of any selectable marker. The method was established and optimized by targeting the megaplasmid-encoded β-glucosidase gene bgl. When linear and supercoiled forms of marker-free suicide vector were used for transformations, the frequencies of obtaining apparent Bgl- mutant were 10- 5 and 10<sup ...[more]

PMID: 30944795

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Project description:BackgroundThermus thermophilus, an extremely thermophilic bacterium, has been widely recognized as a model organism for studying how microbes can survive and adapt under high temperature environment. However, the thermotolerant mechanisms and cellular metabolism still remains mostly unravelled. Thus, it is highly required to consider systems biological approaches where T. thermophilus metabolic network model can be employed together with high throughput experimental data for elucidating its physiological characteristics under such harsh conditions.ResultsWe reconstructed a genome-scale metabolic model of T. thermophilus, iTT548, the first ever large-scale network of a thermophilic bacterium, accounting for 548 unique genes, 796 reactions and 635 unique metabolites. Our initial comparative analysis of the model with Escherichia coli has revealed several distinctive metabolic reactions, mainly in amino acid metabolism and carotenoid biosynthesis, producing relevant compounds to retain the cellular membrane for withstanding high temperature. Constraints-based flux analysis was, then, applied to simulate the metabolic state in glucose minimal and amino acid rich media. Remarkably, resulting growth predictions were highly consistent with the experimental observations. The subsequent comparative flux analysis under different environmental conditions highlighted that the cells consumed branched chain amino acids preferably and utilized them directly in the relevant anabolic pathways for the fatty acid synthesis. Finally, gene essentiality study was also conducted via single gene deletion analysis, to identify the conditional essential genes in glucose minimal and complex media.ConclusionsThe reconstructed genome-scale metabolic model elucidates the phenotypes of T. thermophilus, thus allowing us to gain valuable insights into its cellular metabolism through in silico simulations. The information obtained from such analysis would not only shed light on the understanding of physiology of thermophiles but also helps us to devise metabolic engineering strategies to develop T. thermophilus as a thermostable microbial cell factory.

Project description:Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

Project description:UnlabelledThermus thermophilus HB8 expresses silica-induced protein (Sip) when cultured in medium containing supersaturated silicic acids. Using genomic information, Sip was identified as a Fe(3+)-binding ABC transporter. Detection of a 1-kb hybridized band in Northern analysis revealed that sip transcription is monocistronic and that sip has its own terminator and promoter. The sequence of the sip promoter showed homology with that of the σ(A)-dependent promoter, which is known as a housekeeping promoter in HB8. Considering that sip is transcribed when supersaturated silicic acids are added, the existence of a repressor is presumed. DNA microarray analysis suggested that supersaturated silicic acids and iron deficiency affect Thermus cells similarly, and enhanced sip transcription was detected under both conditions. This suggested that sip transcription was initiated by iron deficiency and that the ferric uptake regulator (Fur) controlled the transcription. Three Fur gene homologues (TTHA0255, TTHA0344, and TTHA1292) have been annotated in the HB8 genome, and electrophoretic mobility shift assays revealed that the TTHA0344 product interacts with the sip promoter region. In medium containing supersaturated silicic acids, free Fe(3+) levels were decreased due to Fe(3+) immobilization on colloidal silica. This suggests that, because Fe(3+) ions are captured by colloidal silica in geothermal water, Thermus cells are continuously exposed to the risk of iron deficiency. Considering that Sip is involved in iron acquisition, Sip production may be a strategy to survive under conditions of low iron availability in geothermal water.ImportanceThe thermophilic bacterium Thermus thermophilus HB8 produces silica-induced protein (Sip) in the presence of supersaturated silicic acids. Sip has homology with iron-binding ABC transporter; however, the mechanism by which Sip expression is induced by silicic acids remains unexplained. We demonstrate that Sip captures iron and its transcription is regulated by the repressor ferric uptake regulator (Fur). This implies that Sip is expressed with iron deficiency. In addition, it is suggested that negatively charged colloidal silica in supersaturated solution absorbs Fe(3+) ions and decreases iron availability. Considering that geothermal water contains ample silicic acids, it is suggested that thermophilic bacteria are always facing iron starvation. Sip production may be a strategy for surviving under conditions of low iron availability in geothermal water.

Dataset Information

Selection-free markerless genome manipulations in the polyploid bacterium Thermus thermophilus.

Publications

Selection-free markerless genome manipulations in the polyploid bacterium <i>Thermus thermophilus</i>.

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