Karakterisasi Genom Bacillus cereus DCN1 dan Identifikasi Klaster Gen Terkait Toleransi Timbal (Pb) dan Besi (Fe)

Abstract

Pencemaran logam berat seperti timbal (Pb) dan besi (Fe) menjadi masalah lingkungan yang signifikan karena bersifat toksik terhadap berbagai organisme, Paparan Pb diketahui dapat menyebabkan gangguan sistem saraf, fungsi ginjal, dan reproduksi, serta bersifat karsinogenik. Sementara itu, Fe merupakan logam esensial yang penting dalam berbagai proses biologis, namun keberadaan dalam kadar berlebih dapat memicu stres oksidatif, kerusakan jaringan, dan gangguan metabolisme. Tantangan ini mendorong upaya pengembangan pendekatan berkelanjutan berbasis mikroorganisme toleran logam yang dikenal memiliki adaptasi ekologis yang luas dan potensi tinggi dalam remediasi lingkungan. Pemahaman toleransi logam pada mikroorganisme memerlukan integrasi antara analisis fenotipik dan pendekatan genomik. Whole Genome Sequencing (WGS) berperan dalam mengidentifikasi klaster gen dan mekanisme molekuler yang mendasari respons terhadap cekaman logam. Kombinasi data genomik dan fenotipik memungkinkan evaluasi komprehensif terhadap strategi adaptasi dan potensi fungsional suatu mikroorganisme. Penelitian ini bertujuan mengeksplorasi potensi Bacillus cereus DCN1, yang diisolasi dari tanaman cengkeh (Syzygium aromaticum L.), dalam menghadapi cekaman logam Pb dan Fe melalui pendekatan genomik dan fenotipik. Analisis genomik diawali dengan proses Whole Genome Sequencing (WGS) menggunakan platform MGI DNBSEQ-G400. Hasil sekuensing menunjukkan kualitas data yang tinggi dengan total panjang genom sebesar 5.846.109 bp dan jumlah pembacaan fragmen sebesar 11.411.106, dengan 59,66% fragmen berhasil dipetakan (6.808.421 fragmen). Sebanyak 4.169 gen berhasil diidentifikasi dengan depth of coverage sebesar 30x. Hasil perakitan genom menunjukkan jumlah total contig sebanyak 774 dan kandungan GC sebesar 35,38%. Selain itu, nilai Q20 dan Q30 masing-masing mencapai 97,55% dan 92,75%, menandakan akurasi pembacaan basa yang baik dan validitas data yang kuat untuk dianalisis lanjut. Analisis komparatif genom menggunakan OrthoVenn3 mengungkapkan bahwa DCN1 berbagi 1.911 klaster ortolog dengan tujuh spesies Bacillus lainnya, dengan kekerabatan terdekat terhadap B. cereus ATCC 14579 dan B. thuringiensis serovar berliner ATCC 10792. Kedekatan ini didukung oleh analisis filogenetik dan visualisasi BRIG yang menunjukkan tingkat kesamaan genomik yang tinggi, mengindikasikan potensi kesamaan adaptasi fisiologis, termasuk terhadap logam berat. Identifikasi klaster biosintesis metabolit sekunder melalui metode antiSMASH mendeteksi sembilan region, terdiri dari tipe NRPS-like, azole-containing RiPP, NI-siderophore, NRP-metallophore/NRPS, betalactone, dua RiPP-like, terpene, dan terpene-precursor. Di antara region tersebut, region 1.3 dan 1.4 menunjukkan kemiripan signifikan dengan klaster biosintesis petrobactin dan bacillibactin, dua jenis siderofor yang diketahui berperan dalam pengikatan dan transportasi besi serta mekanisme homeostasis terhadap logam berat. Anotasi lanjut secara fungsional menggunakan BlastKOALA berhasil mengidentifikasi 2.322 entri protein (47,9%) dengan keterlibatan dalam berbagai lintasan biologis, termasuk sistem homeostasis besi, respons terhadap stres oksidatif, detoksifikasi logam berat, dan perbaikan kerusakan molekuler. Temuan dari ketiga pendekatan ini secara sinergis mendukung hipotesis bahwa toleransi logam berat pada DCN1 dapat dimediasi melalui biosintesis siderofor dan eksopolisakarida, yang dikodekan oleh gen-gen seperti dhbB, dhbE, dhbF, besA, epsA, dan epsB. Evaluasi fenotipik dilakukan untuk menilai kesesuaian antara potensi genomik dan kemampuan adaptif nyata dari DCN1 terhadap paparan logam berat. Uji pertumbuhan awal pada empat jenis logam (Fe, Pb, Cd, dan Hg) menunjukkan bahwa hanya Fe dan Pb yang mampu mendukung pertumbuhan stabil, menunjukkan spesifisitas toleransi terhadap kedua logam tersebut. Nilai Minimum Inhibitory Concentration (MIC) tercatat sebesar 2.500 ppm untuk FeCl3 dan 5.000 ppm untuk Pb(NO3)2, sementara itu nilai Minimum Bactericidal Concentration (MBC) dari keduanya melebihi 10.000 ppm, mengindikasikan tingkat toleransi yang tinggi. Temuan bioinformatika terkait keberadaan klaster gen siderofor dan Extracellular Polymeric Substances (EPS) mendorong pengujian pembentukan biofilm oleh DCN1. Hasil uji menunjukkan produksi biofilm yang tinggi, terutama pada konsentrasi FeCl3 sebesar 2.500 ppm dan Pb(NO3)2 sebesar 5.000 ppm. Uji biosorpsi menggunakan Atomic Absorption Spectrophotometry (AAS) menunjukkan penurunan konsentrasi Fe dan Pb terlarut dalam medium setelah inkubasi. Pengamatan Scanning Electron Microscopy (SEM) memperlihatkan sel bakteri berukuran lebih besar dibandingkan kontrol, dengan permukaan sel tertutup oleh struktur menyerupai matriks biofilm. Hasil penelitian ini menunjukkan bahwa DCN1 memiliki potensi genetik dan fisiologis dalam menoleransi logam berat Pb dan Fe. Kombinasi pendekatan genomik dan fenotipik memberikan pemahaman yang komprehensif terhadap strategi adaptasi bakteri, serta memperkuat potensi DCN1 sebagai kandidat agen bioremediasi di lingkungan yang tercemar logam berat.

Heavy metal pollution, such as lead (Pb) and iron (Fe), is a significant environmental issue due to its toxicity to various organisms. Exposure to Pb is known to cause nervous system disorders, kidney dysfunction, and reproductive issues, as well as being carcinogenic. Meanwhile, Fe is an essential metal important in various biological processes, but its presence in excessive amounts can trigger oxidative stress, tissue damage, and metabolic disorders. This challenge has driven efforts to develop sustainable approaches based on metal-tolerant microorganisms, which are known for their broad ecological adaptations and high potential in environmental remediation. Understanding metal tolerance in microorganisms requires the integration of phenotypic analysis and genomic approaches. Whole Genome Sequencing (WGS) plays a role in identifying gene clusters and molecular mechanisms underlying responses to metal stress. The combination of genomic and phenotypic data enables a comprehensive evaluation of an organism's adaptive strategies and functional potential. This study aims to explore the potential of Bacillus cereus DCN1, isolated from clove plants (Syzygium aromaticum L.), in coping with Pb and Fe metal stress through genomic and phenotypic approaches. Genomic analysis began with Whole Genome Sequencing (WGS) using the MGI DNBSEQ-G400 platform. The sequencing results showed high-quality data with a total reference genome length of 5,846,109 bp and a total of 11,411,106 reads, where 59.66% of the reads were successfully mapped (6,808,421 reads). A total of 4,169 genes were identified with a depth of coverage of 30x. The assembly results showed a total of 774 contigs with an N50 value of 24 kb and a GC content of 35.38%. Additionally, the Q20 and Q30 values reached 97.55% and 92.75%, respectively, indicating excellent base reading accuracy and data validity for further analysis. Comparative genome analysis using OrthoVenn3 revealed that DCN1 shares 1,911 orthologous clusters with seven other Bacillus species, with the closest relationship to B. cereus ATCC 14579 and B. thuringiensis serovar berliner ATCC 10792. This proximity is supported by phylogenetic analysis and BRIG visualization, which show a high level of genomic similarity, indicating potential physiological adaptation similarities, including toward heavy metals. Identification of secondary metabolite biosynthesis clusters via antiSMASH detected nine regions, comprising NRPS-like, azole-containing RiPP, NI-siderophore, NRP-metallophore/NRPS, betalactone, two RiPP-like, terpene, and terpene-precursor types. Among these regions, regions 1.3 and 1.4 show significant similarity to the petrobactin and bacillibactin biosynthesis clusters, two types of siderophores known to play a role in iron binding and transport as well as heavy metal homeostasis mechanisms. Functional annotation using BlastKOALA successfully identified 2,322 protein entries (47.9%) involved in various biological pathways, including iron homeostasis systems, oxidative stress responses, heavy metal detoxification, and molecular damage repair. Findings from these three approaches synergistically support the hypothesis that heavy metal tolerance in DCN1 may be mediated through siderophore and exopolysaccharide biosynthesis, encoded by genes such as dhbB, dhbE, dhbF, besA, epsA, and epsB. Phenotypic evaluation was conducted to assess the alignment between genomic potential and the actual adaptive capacity of DCN1 toward heavy metal exposure. Initial growth tests on four types of metals (Fe, Pb, Cd, and Hg) showed that only Fe and Pb could support stable growth, indicating specificity in tolerance toward these two metals. The Minimum Inhibitory Concentration (MIC) values were recorded at 2,500 ppm for FeCl3 and 5,000 ppm for Pb(NO3)2, while the Minimum Bactericidal Concentration (MBC) of both exceeded 10,000 ppm, indicating a high level of tolerance. Bioinformatics findings related to the presence of siderophore and Extracellular Polymeric Substances (EPS) gene clusters prompted testing of biofilm formation by DCN1. The results showed high biofilm production, especially at FeCl3 concentrations of 2,500 ppm and Pb(NO3)2 concentrations of 5,000 ppm. Biosorption tests using Atomic Absorption Spectrophotometry (AAS) showed a decrease in the concentration of dissolved Fe and Pb in the medium after incubation. Scanning Electron Microscopy (SEM) observations revealed that the bacterial cells were larger than the control, with the cell surface covered by a structure resembling a biofilm matrix. The results of this study indicate that DCN1 has genetic and physiological potential to tolerate heavy metals Pb and Fe. The combination of genomic and phenotypic approaches provides a comprehensive understanding of bacterial adaptation strategies and reinforces the potential of DCN1 as a candidate bioremediation agent in heavy metal-contaminated environments.