biostudies-arrayexpressMichał ŚmigaPorphyromonas gingivalishttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16197Porphyromonas gingivalis is considered a keystone pathogen responsible for dysbiosis in the oral microbiome, leading to the development of periodontal disease and contributing to various systemic comorbidities. Although P. gingivalis requires heme for growth and virulence, it is unable to synthesize heme de novo and therefore relies on host hemoproteins as its source. To acquire heme, the bacterium utilizes the Hmu system, which is composed of six proteins (HmuYRSTUV). HmuY functions as a hemophore-like protein that sequesters heme from host hemoproteins; HmuR serves as an outer membrane receptor responsible for heme uptake. The remaining four proteins (HmuS, HmuT, HmuU, and HmuV) are uncharacterized. HmuS has been proposed to act as a reverse ferrochelatase, due to its homology with CobN cobaltochelatases; therefore, it could catalyze the removal of iron from heme. The released iron could then be transported into the cytoplasm via the HmuTUV proteins. However, the specific role of HmuS in P. gingivalis has never been studied or confirmed. Therefore, this study aimed to investigate the effect of hmuS gene deletion on global gene expression in P. gingivalis, thereby assessing its role in iron acquisition and overall bacterial fitness.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°CLibrary 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.Growth Protocol - P. gingivalis W83 wild-type (WT), and hmuS deltetion mutant (ΔhmuS) strain constructed in W83 strains were cultured at 37℃ in anaerobic conditions (80% N2, 10% CO2, 10% H2). on Schaedler anaerobe blood agar (ABA) plates supplemented with 5% sheep blood and vitamin K3 (Argenta, Poznań, Poland) for 3-5 days, and then in a liquid basal medium (BM) composed of 3% trypticase soy broth (Becton Dickinson, Sparks, MD, USA), 0.5% yeast extract (Biomaxima), 0.05 mg/L vitamin K3 (Fluka, Munich, Germany), and 0.05% L-cysteine (Sigma-Aldrich, St. Louis, MO, USA). To grow bacteria in iron and heme-rich conditions, BM was supplemented with 7.7 μM hemin chloride (Pol-Aura, Morąg, Poland) (BM+Hm). Bacteria were grown for 3 passages in Bm+Hm medium. In the final passage, a culture was established in BM+Hm at OD₆₀₀ 0.2 and incubated for ~20 h.Sequencing - The library was checked with Qubit and real-time PCR for quantification and a bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina platforms, according to effective library concentration and the amount of data required.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.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XRNA-seq of coding RNAPorphyromonas gingivalisMichał ŚmigafalseAnalysis of gene expression in Porphyromonas gingivalis hmuS deletion mutant strain (RNA-seq analysis)Porphyromonas gingivalis is considered a keystone pathogen responsible for dysbiosis in the oral microbiome, leading to the development of periodontal disease and contributing to various systemic comorbidities. Although P. gingivalis requires heme for growth and virulence, it is unable to synthesize heme de novo and therefore relies on host hemoproteins as its source. To acquire heme, the bacterium utilizes the Hmu system, which is composed of six proteins (HmuYRSTUV). HmuY functions as a hemophore-like protein that sequesters heme from host hemoproteins; HmuR serves as an outer membrane receptor responsible for heme uptake. The remaining four proteins (HmuS, HmuT, HmuU, and HmuV) are uncharacterized. HmuS has been proposed to act as a reverse ferrochelatase, due to its homology with CobN cobaltochelatases; therefore, it could catalyze the removal of iron from heme. The released iron could then be transported into the cytoplasm via the HmuTUV proteins. However, the specific role of HmuS in P. gingivalis has never been studied or confirmed. Therefore, this study aimed to investigate the effect of hmuS gene deletion on global gene expression in P. gingivalis, thereby assessing its role in iron acquisition and overall bacterial fitness.2026-04-24T00:00:00Z2026-04-24T09:24:31.313Z2025-11-19T11:46:43.37ZE-MTAB-16197ERP185324EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184