Analysis of heme and iron influence on Porphyromonas gingivalis A7436 and ATCC 33277 strains genes expression (microarray results)

The aim of this study was to analyze phenotypic differences between P. gingivalis more virulent A7436 and less virulent ATCC 33277 (33277) strains. The analysis comprised the influence of heme and iron on P. gingivalis gene expression. 
P. gingivalis A7436 and 33277 strains were cultured in basal medium (3% trypticase soy broth and 0.5% yeast extract), supplemented with 3.6 mM L-cysteine hydrochloride, and 0.5 mg/l menadione, in anaerobic conditions (80% N2, 10% H2 and 10% CO2). To generate heme and iron-limited conditions, the medium was supplemented with 0.16 mM of the iron chelator 2,2-dipyridyl (DIP conditions). To generate heme and iron-rich conditions, the medium was supplemented with 0.0077mM hemin chloride (Hm conditions). Three sample replicates of A7436 and 33277 strains were grown in Hm or DIP conditions for 20 hours. RNA isolation and microarray analysis were performed in IMGM laboratories (Martinsried, Germany), as described by Śmiga et al. (2023).
The online tool eArray (http://earray.chem.agilent.com/; Agilent Technologies, Santa Clara, CA, USA) was used to design an Agilent Custom Porphyromonas gingivalis A7436 Gene Expression Microarray (8×15K format). Probes were prepared based on P. gingivalis transcriptome information derived from the NCBI reference sequence NZ_CP011995.1. Total RNA isolation, RNA quantity, and quality were determined as described by Curaszkiewicz et al. 2014. For internal labeling control, the total RNA was spiked with in vitro synthesized polyadenylated transcripts (One-Color RNA Spike-In Mix; Agilent Technologies). Subsequently, samples were reverse transcribed into cDNA and then converted into cyanine-3-labeled complementary RNA (cRNA) with Low Input Quick-Amp Labeling Kit One-Color (Agilent Technologies). For microarray hybridization, a Gene Expression Hybridization Kit (Agilent Technologies) was used. Labeled cRNA was hybridized for 17 hours at 65℃ on Agilent Custom GE 8×15K Microarrays, washed according to the manufacturer's protocol, and dried with acetonitrile (Sigma-Aldrich). The fluorescence of samples was detected with Scan Control A.8.4.1 software (Agilent Technologies) on the Agilent DNA Microarray Scanner (Agilent Technologies) and extracted from the images using Feature Extraction 10.7.3.1 software (Agilent Technologies). For data analysis, Feature Extraction 10.7.3.1 (Agilent Technologies), GeneSpring GX 13.1.1 (Agilent Technologies), and Excel 2010 (Microsoft, Redmond, WA, USA) were used. For statistical analysis, Welch's approximate t-test was used. Differences in gene expression are shown as fold change values (FC). The average was calculated from the normalized signal values and they were transformed from the log2 to the linear scale. Increases and decreases in gene expression are shown as positive and negative numbers, respectively. The fold change in gene expression was considered significant for FC ≥ 2 or FC ≤ -2 and P-value ≤ 0.05

Ciuraszkiewicz J, Śmiga M, Mackiewicz P, Gmiterek A, Bielecki M, Olczak M, Olczak T. 2014. Fur homolog regulates Porphyromonas gingivalis virulence under low-iron/heme conditions through a complex regulatory network. Mol Oral Microbiol 29:333-353. doi: 10.1111/omi.12077.
Śmiga M, Ślęzak P, Olczak T. 2023. Comparative analysis of Porphyromonas gingivalis A7436 and ATCC 33277 strains reveals differences in the expression of heme acquisition systems. Microbiol Spectr (revised manuscript under revision).