Whole-genome Mining of a Lead-resistant Bacterial Strain (Bacillus cereus)

Heavy metals cause neurotoxicity/neuroinflammation in animals, including humans, and heavy metal contamination of soil/water is one of the major factors in neurodegenerative diseases. Soil bacteria that persist in metal-impacted environments often carry genomic determinants for metal detoxification alongside biosynthetic pathways for the production of structurally diverse secondary metabolites. We combined phenotypic identification, whole-genome sequencing and annotation-based genome mining to characterise a soil Bacillus cereus isolate (designated as VLR6) recovered alongside five additional bacterial isolates (VLR1-VLR5) from the same sample set.

Annotation-based mining to identify heavy-metal resistance genes involved in metal transformation, which is essential for understanding metal detoxification mechanisms and their potential implications for neurodegenerative diseases.

Isolates were identified by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (Bruker Biotyper). The VLR6 genome (5,411,809 bp, single contig) was annotated with Bakta v1.12.0 (database v6.0) and screened for acquired antimicrobial resistance determinants with the Comprehensive Antibiotic Resistance Database Resistance Gene Identifier (CARD-RGI). Biosynthetic gene clusters (BGCs) were predicted using antiSMASH v8.0.4 (default rules; Minimum Information about a Biosynthetic Gene Cluster 3.0 KnownClusterBlast comparisons) and identified through systematic keyword screening of the Bakta functional annotation.

VLR4-VLR6 were identified as B. cereus, VLR1 as Escherichia coli, VLR2 as Citrobacter braakii (within the C. freundii complex), while VLR3 yielded no interpretable spectrum. The sequenced isolate (VLR6) genome encoded 5,504 coding sequences, 108 tRNAs, 39 rRNAs and one clustered regularly interspaced short palindromic repeats array (35.3% GC). CARD-RGI identified 14 antibiotic-resistance-associated loci, dominated by glycopeptide-resistance gene cluster components (vanT/vanW/vanY homologues) and B. cereus class A/B1 beta-lactamases. antiSMASH identified 12 complete BGC regions, including an NI-siderophore region matching petrobactin (BGC0000942) independently corroborated by annotation keyword screening of the asb/fpu/fat loci, non-ribosomal peptide synthetases-metallophore region matching bacillibactin and an azole-containing RiPP region matching the thiopeptides thiocillin I/micrococcin P1; six regions returned no significant known-cluster match. Annotation-based mining additionally identified a heavy-metal-resistance gene set including a zntA-annotated Pb(II)/Cd(II)/Zn(II)-class P-type ATPase homologue, a complete ars operon (arsRBC) and two czcR-family regulators.

The VLR6 genome carries a genomically confirmed set of BGCs, most confidently a petrobactin siderophore cluster corroborated by two independent methods alongside an annotation-supported heavy-metal-resistance gene set. Phenotypic lead-MIC determination and metabolomic or transcriptomic validation are still required before any bioactivity, neuroactive or otherwise, can be attributed to this strain or its predicted metabolites.