Characterization and Molecular Insights of a Chromium-Reducing Bacterium Bacillus tropicus

Abstract

Environmental pollution from metal toxicity is a widespread concern. Certain bacteria hold promise for bioremediation via the conversion of toxic chromium compounds into less harmful forms, promoting environmental cleanup. In this study, we report the isolation and detailed characterization of a highly chromium-tolerant bacterium, Bacillus tropicus CRB14. The isolate is capable of growing on 5000 mg/L Cr (VI) in an LB (Luria Bertani) agar plate while on 900 mg/L Cr (VI) in LB broth. It shows an 86.57% reduction ability in 96 h of culture. It can also tolerate high levels of As, Cd, Co, Fe, Zn, and Pb. The isolate also shows plant growth-promoting potential as demonstrated by a significant activity of nitrogen fixation, phosphate solubilization, IAA (indole acetic acid), and siderophore production. Whole-genome sequencing revealed that the isolate lacks Cr resistance genes in their plasmids and are located on its chromosome. The presence of the chrA gene points towards Cr(VI) transport, while the absence of ycnD suggests alternative reduction pathways. The genome harbors features like genomic islands and CRISPR-Cas systems, potentially aiding adaptation and defense. Analysis suggests robust metabolic pathways, potentially involved in Cr detoxification. Notably, genes for siderophore and NRP-metallophore production were identified. Whole-genome sequencing data also provides the basis for molecular validation of various genes. Findings from this study highlight the potential application of Bacillus tropicus CRB14 for bioremediation while plant growth promotion can be utilized as an added benefit.

Keywords: Bacillus tropicus; bioremediation; chromium-reducing bacteria; heavy metal tolerant; whole-genome sequencing.

Figure 1

Graphical representation of the complete workflow for characterizing the chromium-reducing bacterium CRB14, starting with bacterial isolation, growth tolerance, and PGP analyses, and progressing through genome sequencing, functional annotation, and comparative analysis.

Figure 2

Bar plot depicting the growth of isolate CRB14 at different concentrations of Cr. The cells were cultured on LB broth supplemented with 25, 50, 100 and 200, 300, 400, 500, 600, 700, 800, 900 mg/L Cr (VI). The optical density was measured after incubation for 24 h, 48 h, 72 h, and 96 h at 35 °C.

Figure 3

Reduction in Cr (VI) by isolate CRB14. The cells were cultured in Luria Bertani broth supplemented with 0, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, and 900 mg/L Cr (VI). The Cr (VI) reduction activity was measured after incubation for 24 h, 48 h, 72 h, and 96 h at 35 °C.

Figure 4

Bar plot representing the growth of isolate CRB14 in the presence of various heavy metals.

Figure 5

Nuclear genome circle diagram of CRB14. From outside to inside, coding genes (positive-sense strand), coding genes (negative-sense strand), tRNA (blue) and rRNA (orange), tmRNA (black), CRISPR (green), Cas cluster (cyan), and GC ratio and GC-skew.

Figure 6

A maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences of Bacillus tropicus CRB14 and other closely related strains. The isolate of interest is highlighted in red.

Figure 7

COG classifications of the genome. Each bar corresponds to a specific classification, highlighting these proteins’ diverse roles in metabolism and physiological processes. The abscissa represents the various COG categories, while the ordinate shows the number of genes assigned to each category.

Figure 8

KEGG classification of the predicted coding sequences. The x-axis denotes the various pathways, and the y-axis indicates the number of genes assigned to each pathway. The bars are color coded according to the six major pathway classes, indicating that the majority of genes are involved in metabolism.

Figure 9

GO functional classification of CRB14. The x-axis shows the GO categories, and the y-axis represents the −log10 (p-value) for the top 10 terms in biological process (blue), cellular component (yellow), and molecular function (green).

Figure 10

Schematic diagram of nine secondary metabolite biosynthetic gene clusters in B. tropicus CRB14. Potential secondary metabolite biosynthetic gene clusters were predicted using antiSMASH. Color-coded blocks indicate different gene functions: dark red for core biosynthetic genes, light red for additional biosynthetic genes, blue for transport-related genes, green for regulatory genes, and gray for other genes.