Membrane proteomics of Caldalkalibacillus thermarum TA2.A1
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ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Caldalkalibacillus Thermarum Ta2.a1
SUBMITTER: 
Martin Pabst
LAB HEAD: Martin Pabst
PROVIDER: PXD042369 | Pride | 2023-07-11
REPOSITORIES: Pride
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Bacterial strains The wild-type (WT) Y. pestis biovar microtus strain 201 is avirulent to humans but highly lethal to mice [1]. The 43 to 666 base pairs of ompR (720bp in total length) were replaced by the kanamycin resistance cassette using the one-step inactivation method based on the lambda Red phage recombination system, with the helper plasmid pKD46, to generate the ompR mutants of Y. pestis (designated as ΔompR) [2]. Chromosomal integration of the mutagenic cassette was confirmed by PCR and sequencing using oligonucleotides external to the integrated cassette (data not shown). The elimination of pKD46 in ΔompR was verified by PCR. Bacterial growth and RNA isolation Overnight cultures (an OD620 of about 1.0) of WT or ΔompR in the chemically defined TMH medium [3] were diluted 1:20 into the fresh TMH. Bacterial cells were grown at 26°C to the middle exponential growth phase (an OD620 of about 1.0). To trigger the high osmolarity conditions in OmpR-related experiments, a final concentration of 0.5 M sorbitol was added, after which the cell cultures were allowed to grow for another 20 min. Total RNA of bacterial cells was extracted using the TRIzol Reagent (Invitrogen) without the DNA removal step (for RT-PCR and primer extension) or by using MasterPureTM RNA Purification kit (Epicenter) with the removal of contaminated DNA (for microarray). Immediately before harvesting, bacterial cultures were mixed with RNAprotect Bacteria Reagent (Qiagen) to minimize RNA degradation. RNA quality was monitored by agarose gel electrophoresis, and RNA quantity was determined using a spectrophotometer. Microarray expression analysis Gene expression profiles were compared between WT and ΔompR using a Y. pestis whole-genome cDNA microarray as described in a previous work [4]. RNA samples were isolated from four individual bacterial cultures as biological replicates for each strain. The dual-fluorescently (Cy3 or Cy5 dye) labeled cDNA probes, for which the incorporated dye was reversed, were synthesized from the RNA samples. These were then hybridized to 4 separated microarray slides. A ratio of mRNA levels was calculated for each gene. Significant changes of gene expression were identified using the SAM software [5]. After the SAM analysis, only genes with at least two-fold changes in expression were collected for further analysis. References 1. Zhou D, Tong Z, Song Y, Han Y, Pei D, et al. (2004) Genetics of metabolic variations between Yersinia pestis biovars and the proposal of a new biovar, microtus. J Bacteriol 186: 5147-5152. 2. Zhan L, Han Y, Yang L, Geng J, Li Y, et al. (2008) The cyclic AMP receptor protein, CRP, is required for both virulence and expression of the minimal CRP regulon in Yersinia pestis biovar microtus. Infect Immun 76: 5028-5037. 3. Straley SC, Bowmer WS (1986) Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins. Infect Immun 51: 445-454. 4. Zhou D, Qin L, Han Y, Qiu J, Chen Z, et al. (2006) Global analysis of iron assimilation and fur regulation in Yersinia pestis. FEMS Microbiol Lett 258: 9-17. 5. Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98: 5116-5121.
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