Importance of RNA stabilization: evaluation of ansB, ggt, and rpoA transcripts in microaerophilic Campylobacter jejuni 81-176

Abstract

A subset of food-borne Campylobacter jejuni strains utilizes amino acids asparagine and glutamine as carbon sources that may enhance the ability of this microaerophilic pathogen to colonize specific tissues. In this study, we analyzed the transcript sizes of the ansB and ggt genes encoding the periplasmic asparaginase and γ-glutamyltranspeptidase in C. jejuni 81-176, respectively, and compared the expression level of mRNAs at different time points during the growth in vitro. In addition, we included the housekeeping rpoA gene, encoding the α-subunit of DNA-directed RNA polymerase, to monitor sample processing as it has been described as a stable reference gene in gene expression studies in C. jejuni. Our results revealed that both the ansB and ggt genes were expressed in the end of the logarithmic growth phase and their corresponding monocistronic mRNAs were not affected by sample processing steps. In contrast, the mRNAs of the polycistronic operon containing rpoA gene were highly induced at earlier stage of the logarithmic growth and were clearly differentially responding to external factors during cell harvesting step.

Morphology heterogeneity within a Campylobacter jejuni helical population: the use of calcofluor white to generate rod‐shaped C. jejuni 81‐176 clones and the genetic determinants responsible for differences in morphology within 11168 strains

“…Relative quantification was carried out by normalizing against unit mass (10 ng cDNA) and relative expression of pgp1 was calculated by the ΔC T method. A reference gene was not used for relative quantification as the amplification efficiency calculated from a standard curve with various template preparations and different sets of primers to the commonly used C. jejuni reference genes rrs (16S rRNA; Hyytiäinen et al ., ) or rpoA (α‐subunit of DNA‐directed RNA polymerase; (Ritz et al ., ) was too low (<90%).…”

Comparison of Proteomics Profiles of Campylobacter jejuni Strain Bf under Microaerobic and Aerobic Conditions

“…Samples of RNA were submitted to reverse transcription using RevertAid H Minus First-Strand cDNA synthesis kit (Euromedex) and random hexamer primers (Eurobio) according to the manufacturer's instructions and Bièche et al ( 2012 ) with the following modifications: DNA removal was checked by PCR using 341F/758R primers and the gene expression of kat A, sod B, ahp C, tpx, trxB , and rrs was assessed using specific primers (Table 1 ). The rrs gene was used as the endogenous control as described previously (Li and Schellhorn, 2007 ; Hyytiäinen et al, 2012 ). The composition of the PCR mix was as follows: 5.0 μL of sample, reverse primer (1 μM), forward primer (1 μM), and 12.5 μL of SYBR Green I Master Mix.…”

Description of Campylobacter jejuni Bf, an atypical aero-tolerant strain

“…The quantitative real-time PCR assay was performed with SYBR Green I (Applied Biosystems, USA) using an MJ Research PTC-200 Thermal Cycler and mreB and rrs primers (Table 1 ). The rrs gene was used as the internal control [ 52 ]. The composition of the PCR mix was as follows: 5.0 μL of sample, reverse primer (1 μM), forward primer (1 μM), and 12.5 μL of SYBR Green I Master Mix.…”