Isolation, characterization, and genetic manipulation of cold-tolerant, manganese-oxidizing Pseudomonas sp. strains

Abstract

Manganese-oxidizing bacteria (MnOB) produce Mn oxide minerals that can be used by humans for bioremediation, but the purpose for the bacterium is less clear. This study describes the isolation and characterization of cold-tolerant MnOB strains isolated from a compost pile in Morris, Minnesota, USA: Pseudomonas sp. MS-1 and DSV-1. The strains were preliminarily identified as members of species Pseudomonas psychrophila by 16S rRNA analysis and a multi-locus phylogenetic study using a database of 88 genomes from the Pseudomonas genus. However, the average nucleotide identity between these strains and the P. psychrophila sp. CF149 type strain was less than 93%. Thus, the two strains are members of a novel species that diverged from P. psychrophila. DSV-1 and MS-1 are cold tolerant; both grow at 4°C but faster at 24°C. Unlike the mesophilic MnOB P. putida GB-1, both strains are capable of robustly oxidizing Mn at low temperatures. Both DSV-1 and MS-1 genomes contain homologs of several Mn oxidation genes found in P. putida GB-1 (mnxG, mcoA, mnxS1, mnxS2, and mnxR). Random mutagenesis by transposon insertion was successfully performed in both strains and identified genes involved in Mn oxidation that were similar to those found in P. putida GB-1. Our results show that MnOB can be isolated from compost, supporting a role for Mn oxidation in plant waste degradation. The novel isolates Pseudomonas spp. DSV-1 and MS-1 both can oxidize Mn at low temperature and likely employ similar mechanisms and regulation as P. putida GB-1.IMPORTANCEBiogenic Mn oxides have high sorptive capacity and are strong oxidants. These two characteristics make these oxides and the microbes that make them attractive tools for the bioremediation of wastewater and contaminated environments. Identifying MnOB that can be used for bioremediation is an active area of research. As cold-tolerant MnOB, Pseudomonas sp. DSV-1 and MS-1 have the potential to expand the environmental conditions in which biogenic Mn oxide bioremediation can be performed. The similarity of these organisms to the well-characterized MnOB P. putida GB-1 and the ability to manipulate their genomes raise the possibility of modifying them to improve their bioremediation ability.

Keywords: Pseudomonas; compost; manganese oxidation; psychrotolerant.

Fig 1

Mn oxidation phenotype of new isolates compared to Pseudomonas putida GB-1. Strains were streaked onto Lept media and incubated at 24°C for 3 days. GB-1, Pseudomonas putida GB-1; MS-1, Pseudomonas sp. MS-1; DSV-1, Pseudomonas sp. DSV-1.

Fig 2

Phylogenetic tree of genus Pseudomonas. A total of 88 species of the genus Pseudomonas are represented by this tree, which uses a concatenated sequence of the 16S rRNA gene, rpoB, rpoD, and gyrB to construct proposed evolutionary relationships. Known MnOB are highlighted in red.

Fig 3

Conservation of putative Mn oxidation operons. Arrows represent predicted genes. Numbers below the arrows represent the number of base pairs (bp) between predicted genes; numbers above the arrows are the length of the predicted protein product in amino acids (aa). Written within the arrows are the gene names and/or IMG gene ID# for the gene. Genes in the putative mnxG operon are red, genes in the mcoA operon are green, and those in the mnx two-component regulatory pathway are blue. GB-1, Pseudomonas putida GB-1; MS-1, Pseudomonas sp. MS-1; DSV-1, Pseudomonas sp. DSV-1.

Fig 4

Growth of P. putida GB-1, and P. psychrophila strains DSV-1 and MS-1 at various temperatures. Datapoints represent the average of three replicates; error bars are the standard deviation. (A) 24°C, (B) 14°C, and (C) 4°C. After 80 h of growth at 4°C, cellular aggregation in the MS-1 culture made it difficult to measure OD₆₀₀.

Fig 5

Effect of temperature on Mn(II) oxidation. Strains were incubated on Lept for 5 days at (A) 24°C, (B) 14°C, and (C) 4°C. (D) Plate C after 10 months at 4°C.

Fig 6

Generation of strains with altered Mn(II) oxidation. (A) Wild-type and Tn5 mutant strains, 2 days at 24°C on Lept. (B) KG278 with empty vector (EV) and with plasmid-carrying rpoN (prpoN), 2 days at RT on Lept.