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Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography
Published on: June 4, 2019
A Corynebacterium glutamicum mutant with a defined deletion within the rplK gene is impaired in (p)ppGpp accumulation
Lutz Wehmeier1, Olaf Brockmann-Gretza1, Agustin Pisabarro2
1Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, D-33501 Bielefeld, Germany1.
Abstract:
The rplK gene of Corynebacterium glutamicum ATCC13032 comprises 438 nucleotides and encodes a protein of 145 amino acids with a molecular mass of 15.3 kDa. The amino acid sequence revealed extensive similarities to the large ribosomal subunit protein L11 from several Gram-positive and Gram-negative bacteria. The C. glutamicum rplK gene is located downstream of secE, representing part of the protein export apparatus, and of nusG, encoding a transcription antiterminator protein. The rplK gene is followed by an ORF homologous to rplA encoding the 50S ribosomal protein L1. Northern analysis revealed that transcription of the rplK-rplA cluster resulted in two different transcripts of 1.5 and 0.6 kb. The 1.5 kb transcript corresponds to the entire rplK-rplA cluster and the short transcript originates from the rplK gene. A C. glutamicum rplK mutant strain carrying a 12 bp in-frame deletion within rplK, which resulted in the loss of the tetrapeptide Pro-Ala-Leu-Gly in the L11 protein, was constructed. The mutant failed to accumulate (p)ppGpp in response to amino acid starvation and exhibited an increased tolerance to the antibiotic thiostrepton. Evidently, the C. glutamicum rplK gene is required for (p)ppGpp accumulation upon nutritional starvation.
Insights
The Corynebacterium glutamicum rplK gene is essential for accumulating guanosine tetraphosphate (ppGpp) during nutrient scarcity. A mutant lacking functional rplK protein showed increased thiostrepton antibiotic tolerance.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The rplK gene in Corynebacterium glutamicum encodes the large ribosomal subunit protein L11.
- rplK is located near genes involved in protein export and transcription regulation.
- The rplK-rplA gene cluster produces distinct messenger RNA transcripts.
Purpose of the Study:
- To investigate the function of the rplK gene in Corynebacterium glutamicum.
- To determine the role of the L11 ribosomal protein in cellular stress responses.
Main Methods:
- Gene sequencing and analysis of the rplK gene.
- Construction of a rplK mutant strain with an in-frame deletion.
- Northern blot analysis to study gene transcription.
- Assessing ppGpp accumulation and antibiotic tolerance in wild-type and mutant strains.
Main Results:
- The rplK gene encodes a protein similar to bacterial ribosomal protein L11.
- Transcription analysis revealed two transcripts from the rplK-rplA cluster.
- A mutant with a deletion in rplK failed to accumulate (p)ppGpp under amino acid starvation.
- The rplK mutant displayed enhanced tolerance to the antibiotic thiostrepton.
Conclusions:
- The rplK gene is crucial for the accumulation of (p)ppGpp in response to nutritional stress in C. glutamicum.
- The L11 protein plays a role in the stringent response pathway.
- rplK function impacts antibiotic sensitivity, suggesting a link between ribosomal protein function and drug resistance.
