Multiple-omics analysis of three novel haloalkaliphilic species of Kocuria revealed that the phenolic acid-degrading abilities are ubiquitous in the genus

Abstract

Phenolic acids (PAs), which can exert toxic effects on seed germination and plant growth, are the most common allelopathic substances found in soils. To better understand the degradation fates of PAs in the rhizosphere of halophytes, five haloalkaliphilic PA-degrading bacteria, which were identified as three novel species of Kocuria (namely, Kocuria rhizosphaerae sp. nov., Kocuria kalidii sp. nov., and Kocuria rhizosphaericola sp. nov.), were obtained from the rhizosphere and bulk soil of the halophyte Kalidium cuspidatum. All five Kocuria strains could efficiently degrade ferulic acid (FA) and cinnamic acid (CA) under saline-alkaline conditions. Genomic and transcriptomic analyses revealed that the acrylic groups of FA and CA were first converted to a carboxyl via the coenzyme A (CoA)-dependent non-β-oxidation pathway by the five Kocuria strains. However, the five Kocuria strains selected different aromatic ring-cleavage ways for the degradation of the benzoic derivatives intermediates of the two compounds. The protocatechuate result from FA was then thoroughly degraded through an aromatic ring-opening reaction catalyzed by protocatechuate 3,4-dioxygenase (PcaGH), and the β-ketoadipic acid pathway. At the same time, the yield of benzoate originated from CA was subsequently converted to catechol by the benzoate 1,2-dioxygenase system (BenABCD) or phenylacetyl-CoA epoxidase (PaaABCD) and further completed the ring-cleavage by catechol 1,2-dioxygenase or catechol 2,3-dioxygenase (two non-PcaGH dioxygenases). The comparative genomic analysis revealed that the genes for phenolic acids hydroxylation, protocatechuate 3,4-dioxygenation, and those involved in the β-ketoadipic acid pathways are universal in the Kocuria strains. It is also demonstrated that the Kocuria strains maintain their osmotic balance by accumulating potassium, rather than biosynthesizing organic osmoprotectants, under hypersaline conditions.

Keywords: Kocuria; biodegradation; comparative genomic analysis; osmotic stress; phenolic acids; polyphasic taxonomy.

Figure 1

A phylogenetic tree based on the 16S rRNA gene sequences constructed using the neighbor-joining algorithm (a), and a phylogenomic tree based on 876 core genes using OrthoFinder (b) Both bars 0.1 substitutions per nucleotide (a)/amino acids (b) position. The isolation source and clusters for strains were marked with different colors. The ANI and AAI values between the Kocuria strains (c).

Figure 2

(A,C) The five Kocuria strains degraded FA and CA in MMSM (pH 8.0 and 3.0% NaCl) as the sole carbon and energy source for the strains’ growth. The initial concentration of both PAs is 150 mg L⁻¹. (B,D) The biomass of the strains was determined by measuring the optical density at 600 nm (OD₆₀₀)_. The values in the figures are the average of the 3 times, while the bars are error bars. MMSM, which contained FA or CA but without inoculating any strains, was used as a control (CK) to evaluate the natural loss of compounds. All five strains did not obviously grow in MMSMs without any PAs (data not shown).

Figure 3

Genes related to aromatic compounds degradation in the genomes of the five strains (a), and the predicted degradation pathways according to the annotated genes (b), and the regulation of aromatic compounds-degrading genes in strains M1R5S2^T (C1), M4R5S9^T (C2) and M4R2S49^T (C3) by FA and CA. The values of the genes were first normalized using the GDPAH gene as a reference and using cells cultivated in LB as the control. The normalized values were then used for logarithmic calculation using 1.5 as the base number. The X-axis represents the expression of the genes induced by CA, while the Y-axis reflects the expression of the related genes induced by FA. The values are positive for the expression of the genes. A value >0 means gene upregulation, while a value <0 means gene downregulation (c). More detailed gene names are listed in Supplementary Table S3.

Figure 4

The phylogenetic trees based on aromatic compounds hydroxylase (a) and aromatic ring-opening dioxygenase (b), and the distribution of enzymes relating to aromatic compounds degradation, antibiotic resistance, osmotic balance maintenance, and oxidative stress defense (c).

Figure 5

Correlation of the gene distribution and the strain isolation source.

Figure 6

Phylogenomic tree of Kocuria strains (including non-type strains) based on core genome using GTDB (a), and the phylogenetic trees based on the single amino sequence of PcaG (b), CatE (c), HcaE (d), BphC (e), MobA (f), PraI (g), TfdB (h), CbdA (i), Acr (j), CzcD (k), and HcaB (l). The five clusters were subdivided according to the topological relation on the phylogenomic tree.