Comparative Genomics of Microbacterium Species to Reveal Diversity, Potential for Secondary Metabolites and Heavy Metal Resistance

Abstract

Microbacterium species have been isolated from a wide range of hosts and environments, including heavy metal-contaminated sites. Here, we present a comprehensive analysis on the phylogenetic distribution and the genetic potential of 70 Microbacterium belonging to 20 different species isolated from heavy metal-contaminated and non-contaminated sites with particular attention to secondary metabolites gene clusters. The analyzed Microbacterium species are divided in three main functional clades. They share a small core genome (331 gene families covering basic functions) pointing to high genetic diversity. The most common secondary metabolite gene clusters encode pathways for the production of terpenoids, type III polyketide synthases and non-ribosomal peptide synthetases, potentially responsible of the synthesis of siderophore-like compounds. In vitro tests showed that many Microbacterium strains produce siderophores, ACC deaminase, auxins (IAA) and are able to solubilize phosphate. Microbacterium isolates from heavy metal contaminated sites are on average more resistant to heavy metals and harbor more genes related to metal homeostasis (e.g., metalloregulators). On the other hand, the ability to increase the metal mobility in a contaminated soil through the secretion of specific molecules seems to be widespread among all. Despite the widespread capacity of strains to mobilize several metals, plants inoculated with selected Microbacterium isolates showed only slightly increased iron concentrations, whereas concentrations of zinc, cadmium and lead were decreased.

Keywords: comparative genomics; heavy metals; plant associated bacteria; polyketide synthases; secondary metabolites; siderophore; terpenoids.

FIGURE 1

Maximum likelihood phylogenetic tree based on 38 concatenated unique marker genes identified using Phylosift (Darling et al., 2014). Kocuria palustris MU14/1 (CP012507) was used as outgroup. Bootstrap values > 50% are shown at branch points. Strains belonging to phylogenetic groups I, II and III are highlighted in green, blue, and red, respectively. Isolates in bold were tested in this study. Isolates marked with a black circle were isolated from heavy metal contaminated sites. The first column on the right side of the tree shows the different types of PKS gene clusters; whereas the second and third columns show the different types of NRPS gene clusters. The bars indicate the other predicted secondary metabolite gene clusters identified with antiSMASH 5.0.

FIGURE 2

Non-metric multidimensional scaling (NMDS) ordination based on Bray–Curtis dissimilarity calculated on a contingency table where functionally annotated orthologous groups (eggNOG) were counted across all Microbacterium spp. genomes. Samples are displayed in different shapes according to the isolation origin, whereas the colors represent the functional group affiliation (green, blue, and red) as described in Figure 1.

FIGURE 3

(A) Heavy metal resistance test: zinc sulfate (1–14 mM), lead nitrate (1–10 mM), and cadmium nitrate (0.5–6 mM). (B) Total number of genes related to heavy metal resistance and homeostasis (HM genes), of metallo-sensing regulators (regulators) and ArsR regulator family. Error bars show standard deviation. Asterisks indicate significantly different values of +HM compared to –HM (p < 0.05). (C) Odds ratio (OR) of genes belonging to single isolates compared to all sequenced bacteria (red and blue panels) and isolates from heavy metal contaminated sites compared from non-contaminated sites (green panel). Asterisks indicate significant deviation from the null hypothesis [ln(OR) = 0] at the 95% confidence level by one-tailed Fisher exact test. Gene categories: HM genes (blue), genes related to heavy metal resistance and homeostasis; ArsR (red), regulators belonging to the ArsR family; Regulators (green), metallo-sensing regulators; COG P (purple), genes assigned to the COG P category (Inorganic ion transport and metabolism). +HM, bacteria isolated from heavy metal contaminated sites (red); –HM, bacteria isolated from heavy metal non-contaminated sites (blue).

FIGURE 4

Mobilization of zinc (A), cadmium (B), lead (C), and iron (D) from contaminated soil by bacterial exudates. The different colors of the diagram bars represent different isolation sources: shoots in light green; roots in dark green; rhizosphere in orange; soil in brown; nodules in dark red. NC, negative control (gray). +HM, bacteria isolated from heavy metal contaminated sites; –HM, bacteria isolated from non-contaminated sited. Error bars show standard errors of the mean (n = 6, except for NC n = 3 and for bacteria producing a polysaccharide matrix difficult to filtrate: n = 5 for EX104 and 280; n = 3 for 228 and K02; n = 2 for K01; n = 1 for DSM 8608). Samples with extraction values differing significantly from the control (NC) are labeled with ^∗(p < 0.05) and ^∗∗(p < 0.01). The order of magnitude of CFU/mL prior to filtration is indicated above the diagram bars. The graphs representing the mobilization of copper and manganese can be found in the Supplementary Material, Supplementary Figure S9.