Genomic Insight of Alicyclobacillus mali FL18 Isolated From an Arsenic-Rich Hot Spring

Abstract

Extreme environments are excellent places to find microorganisms capable of tolerating extreme temperature, pH, salinity pressure, and elevated concentration of heavy metals and other toxic compounds. In the last decades, extremophilic microorganisms have been extensively studied since they can be applied in several fields of biotechnology along with their enzymes. In this context, the characterization of heavy metal resistance determinants in thermophilic microorganisms is the starting point for the development of new biosystems and bioprocesses for environmental monitoring and remediation. This work focuses on the isolation and the genomic exploration of a new arsenic-tolerant microorganism, classified as Alicyclobacillus mali FL18. The bacterium was isolated from a hot mud pool of the solfataric terrains in Pisciarelli, a well-known hydrothermally active zone of the Campi Flegrei volcano near Naples in Italy. A. mali FL18 showed a good tolerance to arsenite (MIC value of 41 mM), as well as to other metals such as nickel (MIC 30 mM), cobalt, and mercury (MIC 3 mM and 17 μM, respectively). Signatures of arsenic resistance genes (one arsenate reductase, one arsenite methyltransferase, and several arsenite exporters) were found interspersed in the genome as well as several multidrug resistance efflux transporters that could be involved in the export of drugs and heavy metal ions. Moreover, the strain showed a high resistance to bacitracin and ciprofloxacin, suggesting that the extreme environment has positively selected multiple resistances to different toxic compounds. This work provides, for the first time, insights into the heavy metal tolerance and antibiotic susceptibility of an Alicyclobacillus strain and highlights its putative molecular determinants.

Keywords: arsenic resistance system; bioremediation; genomic sequencing and annotation; geothermal environment; thermophilic microorganism; toxic metals.

FIGURE 1

The Pisciarelli site, the sample mud pool, and the sample area (white circle). (A) The hydrothermal setting with the fumaroles, the main boiling pool, the outflow water drainage zone, and the surrounding diffuse degassing area. (B) Southern view of the bubbling pool with the boiling points. (C) The sampled muddy area at the periphery of the mud pool.

FIGURE 2

Circular map of Alicyclobacillus mali FL18 genome performed using CGview Server. From the center to outside: genome size, with a ring showing the GC skews (positive values in green and the negatives in purple); the contigs, represented as dark blue arrows in a double ring to indicate the strands plus and minus; the CDSs are reported in light blue, together with tRNA (red), rRNA (green), tmRNA (dark red), and CRISPR (blue).

FIGURE 3

Functional annotation of Alicyclobacillus isolate using RAST server.

FIGURE 4

Phylogenetic tree based on the genome comparison of A. mali FL18 with reference genomes publicly available at NCBI.

FIGURE 5

Genome comparison between A. mali FL18 and NBRC 102425. (A) COG comparison of A. mali FL18 (blue) and A. mali NBRC 102425 (red). (B) Mauve progressive alignment of FL18 and NBRC 102425. (C) Venn diagram showing the number of shared and unique proteins.

FIGURE 6

Organization of arsenic resistance genes. Comparison of arsenic resistance genes in A. mali FL18 (Up) and A. mali NBRC 102425 (Down). In purple, arsR genes coding for arsenic responsive transcriptional regulator; in blue, arsC genes coding for arsenate reductase; in green, arsB genes coding for arsenite membrane transporter; in yellow, arsA genes coding for anion stimulated ATPase; in red, arsM genes coding for arsenite methyltransferase; in orange, arsP genes coding for organoarsenical permease; in black, genes that are not involved in arsenic resistance.

FIGURE 7

Organization of multi-drug resistance genes. Comparison of antibiotic resistance genes in A. mali FL18 (Up) and A. mali NBRC 102425 (Down). In purple, MarR genes coding for antibiotic responsive transcriptional regulator; in blue, MerR gene coding for mercury resistance transcriptional regulator; in green, multidrug transporter genes; in red, multidrug permease genes; in orange, small multidrug resistance protein; in black, genes that are not involved in antibiotic resistance.

FIGURE 8

Growth curves of A. mali FL18 in the presence of 20 mM As(III) (A) and 6 mM As(V) (B). The red curve is the As-free control growth.

FIGURE 9

Qualitative evaluation of arsenic biotransformation performed by A. mali FL18. The experiment was performed by reaction of AgNO₃ with (A) supernatants and (B) pellets of cells grown for 16 h in BAT medium (CN well) and in BAT medium supplemented with 20 mM As(III) or 5 mM As(V). (C) Color scales of different ratios of As(V)/As(III) in solutions with final arsenic concentrations of 5 and 20 mM.