gyrA and rpoB as alternative markers for bacterial identification

Bakteriyalarni tur darajasida identifikatsiya qilishda 16S rRNA geni yetarli bo‘lmagan holatlar uchun muqobil gyrA va rpoB markerlarining samaradorligi in silico tahlil va molekulyar-genetik usullar yordamida o‘rganildi.

FUNDAMENTAL VA AMALIY MIKROBIOLOGIYANING BUGUNGI HOLATI VA RIVOJLANISH ISTIQBOLLARI: YOSH OLIMLAR NIGOHI Respublika ilmiy-amaliy anjumani · Toshkent, 2026-yil 30-oktabr

II BIOTEXNOLOGIYA, MOLEKULYAR BIOLOGIYA VA BIOINFORMATIKA DOI 10.66073/ReFocus-Conf/3010045

gyrA AND rpoB AS ALTERNATIVE MARKERS FOR BACTERIAL IDENTIFICATION Karimov Nurbek Rayimovich1,4, Reyimberdiyeva Marjon Zafar qizi2, Rahimova Sh.Sh.², Nazirov M.M.3, Kobilov Fazliddin Bozorovich4 ¹ National University of Uzbekistan named after Mirzo Ulugbek ² Tashkent Institute of Chemical Technology ³ Scientific Research Institute of Cotton Breeding, Seedling, and Growing Agrotechnologies, Tashkent, Uzbekistan 4 Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan karimovnmr@gmail.com Abstract: We evaluated the effectiveness of gyrA and rpoB as alternative markers for specieslevel bacterial identification when the 16S rRNA gene is insufficient, using in silico analysis and molecular genetic methods. Keywords: 16S rRNA, gyrA, rpoB, nucleotide diversity Annotatsiya: Bakteriyalarni tur darajasida identifikatsiya qilishda 16S rRNA geni yetarli bo‘lmagan holatlar uchun muqobil gyrA va rpoB markerlarining samaradorligi in silico tahlil va molekulyar-genetik usullar yordamida o‘rganildi. Kalit so‘zlar: 16S rRNA, gyrA, rpoB, nukleotid diversligi Аннотация: Эффективность альтернативных маркеров gyrA и rpoB для идентификации бактерий на видовом уровне в случаях, когда ген 16S рРНК недостаточен, оценивалась с помощью in silico анализа и молекулярно-генетических методов. Ключевые слова: 16S рРНК, gyrA, rpoB, нуклеотидное разнообразие The 16S rRNA gene is the most widely used marker for bacterial identification and phylogenetic analysis. However, because it is highly conserved, present in multiple copies per genome, and divergent among those copies, the 16S rRNA gene may be insufficient to reliably discriminate closely related species. Consequently, single-copy, protein-coding genes that evolve faster than 16S rRNA are being considered as alternative markers, notably gyrA, which encodes the DNA gyrase A subunit, and rpoB, which encodes the RNA polymerase β subunit. For the in silico analysis, complete genomes of Bacillus representatives were retrieved from the NCBI GenBank database, and the 16S rRNA, gyrA, and rpoB sequences were extracted from them. Intraspecific variability was assessed in a total of 230 genomes of B. subtilis (93), B. cereus (79), and B. safensis (58). Interspecific variability was examined in a dataset of 283 genomes representing 17 species. Sequences were aligned with MUSCLE, nucleotide diversity (π) was calculated in DnaSP using a 10-bp sliding window, and the distribution of SNPs along the genes was analyzed. In the interspecific comparison, gyrA exhibited the highest nucleotide diversity (π ≈ 0.15– 0.28), rpoB showed intermediate values (π ≈ 0.06–0.27), and the 16S rRNA gene was characterized by very low diversity (π ≈ 0.01–0.03, reaching ≈ 0.11 only in isolated variable regions). Within species, the 16S rRNA gene was nearly invariant (π ≈ 0), π in rpoB did not exceed 0.03, and π in gyrA rose to ≈ 0.09, with the highest value observed in B. cereus. Five bacterial isolates (N1, N2, N3, N4, and N5) were used in this study. Genomic DNA was extracted from the isolates by the Marmur method. The 16S rRNA gene was amplified by PCR with the universal primers 27F and 1492R under the following conditions: initial denaturation at 95 °C for

FOR CITATION Karimov N.R., Reyimberdiyeva M.Z., Rahimova Sh.Sh., Nazirov M.M. et al. gyrA and rpoB as alternative markers for bacterial identification // Proceedings of the Republican Scientific and Practical Conference “Status and Development Prospects of Fundamental and Applied Microbiology: The Viewpoint of Young Scientists”. – Tashkent, 2026. – P. 177–178. DOI: https://doi.org/10.66073/ReFocus-Conf/3010045

FUNDAMENTAL VA AMALIY MIKROBIOLOGIYANING BUGUNGI HOLATI VA RIVOJLANISH ISTIQBOLLARI: YOSH OLIMLAR NIGOHI 30.10.2026

3 min; 30 cycles of denaturation at 95 °C for 40 s, annealing at 56 °C for 40 s, and extension at 72 °C for 1.5 min; and a final extension at 72 °C for 7 min. Amplification products were verified by electrophoresis on a 0.8% agarose gel and sequenced on a Sanger sequencer. The resulting sequences were compared against the NCBI BLAST database, and the isolates were identified to the genus level. A fragment of the gyrA gene was amplified by PCR with the degenerate primers: gyrA3-F (5′-GCDGCHGCNATGCGTTAYAC-3′) gyrA3-R (5′-ACAAGMTCWGCKATTTTTTC-3′) PCR was initially performed under the following conditions: initial denaturation at 95 °C for 3 min; 30 cycles of denaturation at 95 °C for 40 s, annealing at 56 °C for 40 s, and extension at 72 °C for 40 s; and a final extension at 72 °C for 7 min. However, owing to the high degeneracy of the primers and the low Tm of the reverse primer, these conditions yielded nonspecific products. A stepwise annealing program was therefore applied, consisting of 2 cycles each at 50, 51, 52, 53, and 54 °C (10 cycles in total), followed by 10 cycles at 55 °C and 10 cycles at 56 °C (30 cycles in total). The denaturation and extension steps were left unchanged. Amplification products were verified by electrophoresis on a 0.8% agarose gel. A fragment of the rpoB gene was amplified by PCR with the degenerate primers: rpoB_F (5′-GGYTWYGAAGTNCGHGACGTDCA-3′) rpoB_R (5′-TGACGYTGCATGTTBGMRCCCATMA-3′) PCR was performed under the following conditions: initial denaturation at 95 °C for 3 min; 30 cycles of denaturation at 95 °C for 40 s, annealing at 56 °C for 40 s, and extension at 72 °C for 40 s; and a final extension at 72 °C for 7 min. Amplification products were verified by electrophoresis on a 0.8% agarose gel. The in silico analysis showed that, in the genus Bacillus, gyrA and rpoB have higher nucleotide diversity than the 16S rRNA gene (π: gyrA ≈ 0.15–0.28, rpoB ≈ 0.06–0.27, 16S rRNA ≈ 0.01–0.03). Whereas the 16S rRNA gene was nearly invariant within species, variability was detected in both gyrA and rpoB. These genes are therefore promising supplementary markers to 16S rRNA for discriminating closely related species. The conditions for amplifying gyrA and rpoB fragments were optimized. gyrA and rpoB can serve as effective supplementary markers for identifying closely related species that 16S rRNA cannot resolve.