biostudies-arrayexpressMichał ŚmigaPorphyromonas gingivalishttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16130Porphyromonas gingivalis, the keystone pathogen responsible for dysbiosis in the oral microbiome, the development of periodontal diseases, and the contribution to systemic comorbidities, is a heme auxotroph. It encodes only four enzymes in the heme biosynthesis pathway, namely HemD, HemN, HemG, and HemH. HemH protein is a ferrochelatase involved in the final step of heme biosynthesis by inserting iron ions (Fe2+) into protoporphyrin IX. This study aimed to analyze the effect of hemH gene deletion on P. gingivalis gene expression, thereby verifying its importance for P. gingivalis.biostudies-arrayexpressSample Collection - 1 ml of bacterial culture was centrifuged (8,000×g, 10 min, 4°C), and the bacterial pellet was resuspended in 800 μl of Fenozol (A&A Biotechnology, Gdańsk, Poland) and stored in -80°CSequencing - The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms, according to effective library concentration and data amount required.Growth Protocol - P. gingivalis strains were cultured at 37℃ in anaerobic conditions (80% N2, 10% CO2, 10% H2) using in basal medium (BM) composed of 3% trypticase soy broth (Becton Dickinson), 0.5% yeast extract (Biomaxima) 0.05 mg/L vitamin K3 (Fluka), and 0.05% L-cysteine (Sigma-Aldrich). P. gingivalis was first cultured on BM agar plates supplemented with 7.7 µM heme for 4 days. Subsequently, the bacteria were transferred to liquid BM containing 2 µM heme, inoculated at an initial OD₆₀₀ of 0.2, and cultured for 24 h. Then, two 24-h passages in BM lacking heme, each initiated at an OD₆₀₀ equal to 0.2, were carried out. In the final step, a culture was established in heme-free BM at OD₆₀₀ 0.2 and incubated for 20 h, reaching an OD₆₀₀ of approximately 1–1.5.Library Construction - Firstly, ribosomal RNA was removed from total RNA, followed by ethanol precipitation. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. During the second strand cDNA synthesis, dUTPs were replaced with dTTPs in the reaction buffer. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification.Nucleic Acid Extraction - Total RNA was extracted from ~ 10^9 bacteria using the Total RNA Mini Kit (A&A Biotechnology, Gdańsk, Poland), followed by treatment with the Clean-up RNA Concentrator Kit (A&A Biotechnology) to eliminate residual genomic DNA contamination. RNA purity and integrity were evaluated using spectrophotometry and agarose gel electrophoresis, respectively. RNA samples were stored at –80 °C or in dry ice until further processing.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Raw reads were mapped to the Porphyromonas gingivalis reference genome using Bowtie2, followed by sorting and indexing. Gene expression levels were quantified based on the number of reads aligned to genomic features. To account for differences in gene length and sequencing depth, read counts were normalized using the FPKM method (Fragments Per Kilobase of transcript per Million mapped reads), enabling accurate comparison of transcript abundances across genes and samples.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XRNA-seq of coding RNAPorphyromonas gingivalisMichał ŚmigafalseAnalysis of gene expression in Porphyromonas gingivalis hemH deletion mutant strain grown under heme-depleted conditions (RNA-seq analysis)Porphyromonas gingivalis, the keystone pathogen responsible for dysbiosis in the oral microbiome, the development of periodontal diseases, and the contribution to systemic comorbidities, is a heme auxotroph. It encodes only four enzymes in the heme biosynthesis pathway, namely HemD, HemN, HemG, and HemH. HemH protein is a ferrochelatase involved in the final step of heme biosynthesis by inserting iron ions (Fe2+) into protoporphyrin IX. This study aimed to analyze the effect of hemH gene deletion on P. gingivalis gene expression, thereby verifying its importance for P. gingivalis.2026-03-03T00:00:00Z2026-03-03T02:03:24.481Z2025-11-14T13:34:04.854ZE-MTAB-16130ERP184451EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184