Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

Abstract

Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.

Phenotypic and Transcriptomic Analyses of Seven Clinical Stenotrophomonas maltophilia Isolates Identify a Small Set of Shared and Commonly Regulated Genes Involved in the Biofilm Lifestyle

“…To identify the most relevant and strongly expressed genes in biofilm-grown cells, we decided to focus on the top 250 expressed genes (excluding ribosomal proteins) in our analyses (Table S4). This arbitrarily chosen cutoff correlated in all strains with the expression level of the rpoD gene, which is used in many studies as a reference gene ( 35 , 36 ). This initial analysis included all strongly expressed genes, independent of whether these were regulated or constitutively expressed in biofilm versus planktonic cells.…”

Validation of stable reference genes in Staphylococcus aureus to study gene expression under photodynamic treatment: a case study of SEB virulence factor analysis

“…Identical observations were demonstrated by DeLorenzo and Moon 41 . Unfortunately, according to the literature, there were some discrepancies in the results obtained by BestKeeper, geNorm and NormFinder software 36,39,40 . It should be emphasized that such calculation differences may be a result of these three programs being based on different algorithms.…”

“…Subsequently, Krzyżanowska et al . proved that the 16S rRNA-coding gene was characterized by poor expression stability in Ochrobactrum quorumnocens (Cp value = 10.26) and was one of the most unstable candidate reference genes under 10 different tested culture conditions 39 . The 16S rRNA gene has also been proven unsuitable for the analysis of iron-regulated gene expression in Pseudomonas brassicacearum (it ranked sixth of the eight genes tested) 40 .…”

Deep (Meta)genomics and (Meta)transcriptome Analyses of Fungal and Bacteria Consortia From Aircraft Tanks and Kerosene Identify Key Genes in Fuel and Tank Corrosion

“…For this, transcripts were mapped to the reference genomes of both organisms ( Figure 6 and Supplementary Table 6 ). Expression levels (RPKM) were normalized against rpoD for bacteria and TFIIB genes for fungi, where a log2 fold value above 2.0 was understood as highly expressed ( Supplementary Table 6 ) ( Knutson and Hahn, 2013 ; Gelev et al, 2014 ; McMillan and Pereg, 2014 ; Krzyżanowska et al, 2019 ). In Figure 6 , highly expressed genes are marked.…”