Characterization of a Δnth mutant in Staphylococcus carnosus TM300 and a ΔPII-like mutant in Staphylococcus aureus

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Dokumentart: Dissertation
Date: 2015
Language: English
Faculty: 7 Mathematisch-Naturwissenschaftliche Fakultät
Department: Biologie
Advisor: Götz, Friedrich (Prof. Dr.)
Day of Oral Examination: 2015-05-18
DDC Classifikation: 000 - Computer science, information and general works
500 - Natural sciences and mathematics
570 - Life sciences; biology
Keywords: Staphylococcus
Other Keywords:
endonuclease III
transformation efficiency
PII-like (PstA)
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DNA nucleases catalyze the cleavage of phosphodiester bonds. These enzymes play crucial roles in various DNA repair processes, which involve DNA replication, base excision repair, nucleotide excision repair, mismatch repair, and double strand break repair. Some of the nucleases limit DNA uptake efficiency in bacteria by degrading foreign DNA. Rosenstein et al., have annotated several genes within the genome of Staphylococcus carnosus TM300 with similarity to nucleases. Among these genes, there was one homologous to the putative endonuclease III gene, nth. Endonuclease III is described to cleave phosphodiester bonds at apurinic or apyrimidinic sites in DNA and has important functions in some steps during base-excision repair of DNA. Homologous nth endonucleases are found nearly in all organisms, including humans. In this study we aimed to generate an nth (sca_1086) deletion mutant in S. carnosus TM300 and investigate its function by comparative phenotypic analysis. To delete nth, a knock-out plasmid pBT2Δnth::ermB was constructed including lox sites. Resistant colonies against erythromycin and chloramphenicol were screened and the resulting Δnth mutant was verified by DNA sequencing. Subsequently an erythromycine resistance cassette (ermB) was excised from genomic DNA by Cre recombinase which is expressed from plasmid pRAB1. Wild type (WT) and Δnth mutant strains have been characterized comparatively. Nuclease activity in cell crude extracts was assayed using undigested λ-DNA as a substrate. Indeed, in Δnth mutant, degradation of λ-DNA was significantly decreased compared to WT strain. As S. carnosus nth might also be involved in DNA repair the susceptibility to various mutagens was investigated. Two differently acting mutagens were tested: hydrogen peroxide and mitomycin-C. H2O2 is not only a strong oxidizer, it is also a mutagen, which damages DNA, and mitomycin-C is a potent DNA-crosslinker and efficiently kills bacteria. While the minimal inhibition concentration (MIC) of mitomycin-C was unchanged, the survival rate of the Δnth mutant to H2O2 or streptomycin stress was clearly impaired. The mutagenic survival assay was carried out by the classical Ames-test. It turned out that the frequency of spontaneous resistant mutants against streptomycin and mitomycin-C was approximately three-fold increased in Δnth mutant, suggesting that the DNA repair activity was impaired. Finally, the transformation efficiency of Δnth and WT strain was investigated and it was found that Δnth mutant showed a three-fold higher transformation efficiency compared to WT. These results show that the putative endonuclease III in S. carnosus TM300 has important functions in both, DNA repair and degradation of exogenic DNA. PII proteins are signal transduction proteins in bacteria, archaea and plants, which coordinate and regulate many aspects of nitrogen metabolism by interacting with enzymes, transcription factors and membrane transport proteins. The genome of Staphylococcus aureus NCTC8325 does not encode for a homologue of a canonical PII protein. However, the gene SAOUHSC_00452, annotated as a hypothetical protein, encodes a protein, which has predicted structural similarity to canonical PII and is therefore termed as PII-like protein. In this study, we generated a PII-like deletion mutant (∆PII-like) in S. aureus NCTC8325 and studied its function by comparative phenotypic analysis. Deletion of the putative PII-like gene resulted in severe impairment during the exponential growth phase in nitrogen deficient and excess media. In addition, the ΔPII-like mutant exhibited an increased biofilm formation compared to the wild type strain. These results indicated a possible role of PII-like in central metabolic pathways, which had to be elucidated. We investigated the thermodynamic binding properties of PII-like protein to various molecules such as ATP, ADP, AMP, GTP, TTP, cAMP and cyclic diadenosine monophosphate (c-di-AMP) using Isothermal Titration Calorimetry (ITC) and Biacore Surface Plasmon Resonance (SPR). The bacterial second messenger c-di-AMP was shown to bind tightly to PII-like, whereas cAMP, another second messenger molecule, just interacted loosely with PII-like. Unfortunately, we could not observed any binding interactions between ATP, ADP, AMP, GTP and TTP.

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