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Quantitative assessment of microbiomes using ultra-fast MS/MS-free metaproteomics: model 2

ABSTRACT: This study presents an adaptation of the novel ultrafast MS1-based method called DirectMS1 to characterization of the single microorganisms and microbiomes. Using DirectMS1 we demonstrate the blind identification (with no prior knowledge of the bacterias in the sample) of a single bacterial species and microbial compositions using two-step database search against the microbial part of SwissProt and TrEMBL (62Gb database size), the evaluation of fold changes in biomass of microorganisms between the metaproteomic samples, and the assessment of metabolic activity of Rhodococcus species responding to a change in growth conditions and cold shock.

INSTRUMENT(S):

ORGANISM(S): Rhodococcus Qingshengii Rhodococcus Opacus Gordonia Polyisoprenivorans Gordonia Alkanivorans Rhodococcus Erythropolis

SUBMITTER: Tomiris Kusainova

LAB HEAD: Dr Irina A. Tarasova

PROVIDER: PXD050887 | Pride | 2026-08-07

REPOSITORIES: Pride

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Project description:We compared the expression profile of TTHA1939 deletion mutant strain of Thermus thermophils HB8 with that of wild-type. The mutant strain grown in minimum essential medium (CS medium) exhibited growth arrest and cellular aggregation during the logarithm growth phase. In this phase, 99 genes were suppressed and 63 genes were activated for the mutant strain. The suppressed genes included genes of ribosomal proteins, TCA cycle, amino acid biosynthesis, fatty acid biosynthesis, cobalamin biosynthesis, transporters, and cell division including dnaB and ftsZ. The activated genes included genes of nitrogen metabolism, stress proteins, and proteases. We suggested that these transcriptional alterations in the mutant cell were induced by the ppGpp accumulation which was prevented by ppGpp degradation activity of TTHA1939 in WT cell. Keywords: gene deletion, minimum essential medium, logarithm growth phase Wild-type and the mutant strains were precultured in rich medium (TR medium) for two times at 70 oC and then subcultured to minimum essential medium (CS medium). These cells were harvested during the logarithm growth phase (800 minutes from the start of culture). Total RNA were extracted from each strain and used for the cDNA synthesis. For the biological replication, above experiments were performed four times independently. The cDNA fragments were labeled with biotin-dideoxy UTP, using terminal transferase according to the manufacturerâs instructions (ENZO Biochem. Inc., Farmingdale, NY). The 3â-terminal-labeled cDNA (2 micro g) was hybridized to a TTHB8401 GeneChip (Affymetrix, Santa Clara, CA). The Probe Array was scanned with a GeneArray Scanner (Affymetrix), and then, the image data was scaled to the target intensity by one-step Tukeyâs biweight algorithm using GeneChip Operating software, version 1.0 (Affymetrix, Santa Clara, CA). The data analysis was performed by using GeneSpring GX (Agilent Tech.). The genes which had detection call of âpresenceâ more than 4 times from 8 samples were used for following analysis. The normalized intensities for the mutant strains were calculated by using the intensities for wild-type strains of each data set, and then, the average of these intensities were used. The genes which produced P value < 0.1 in the Studentâs t-test were extracted. Among them, the genes whose expression level was different more than 2 fold between two strains were considered as significant.

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Quantitative assessment of microbiomes using ultra-fast MS/MS-free metaproteomics: model 2

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