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. 2016 May 26:7:782.
doi: 10.3389/fmicb.2016.00782. eCollection 2016.

When Genome-Based Approach Meets the "Old but Good": Revealing Genes Involved in the Antibacterial Activity of Pseudomonas sp. P482 against Soft Rot Pathogens

Dorota M Krzyżanowska  1 , Adam Ossowicki  1 , Magdalena Rajewska  1 , Tomasz Maciąg  1 , Magdalena Jabłońska  1 , Michał Obuchowski  2 , Stephan Heeb  3 , Sylwia Jafra  1
Affiliations

When Genome-Based Approach Meets the "Old but Good": Revealing Genes Involved in the Antibacterial Activity of Pseudomonas sp. P482 against Soft Rot Pathogens

Dorota M Krzyżanowska et al. Front Microbiol. .

Abstract

Dickeya solani and Pectobacterium carotovorum subsp. brasiliense are recently established species of bacterial plant pathogens causing black leg and soft rot of many vegetables and ornamental plants. Pseudomonas sp. strain P482 inhibits the growth of these pathogens, a desired trait considering the limited measures to combat these diseases. In this study, we determined the genetic background of the antibacterial activity of P482, and established the phylogenetic position of this strain. Pseudomonas sp. P482 was classified as Pseudomonas donghuensis. Genome mining revealed that the P482 genome does not contain genes determining the synthesis of known antimicrobials. However, the ClusterFinder algorithm, designed to detect atypical or novel classes of secondary metabolite gene clusters, predicted 18 such clusters in the genome. Screening of a Tn5 mutant library yielded an antimicrobial negative transposon mutant. The transposon insertion was located in a gene encoding an HpcH/HpaI aldolase/citrate lyase family protein. This gene is located in a hypothetical cluster predicted by the ClusterFinder, together with the downstream homologs of four nfs genes, that confer production of a non-fluorescent siderophore by P. donghuensis HYS(T). Site-directed inactivation of the HpcH/HpaI aldolase gene, the adjacent short chain dehydrogenase gene, as well as a homolog of an essential nfs cluster gene, all abolished the antimicrobial activity of the P482, suggesting their involvement in a common biosynthesis pathway. However, none of the mutants showed a decreased siderophore yield, neither was the antimicrobial activity of the wild type P482 compromised by high iron bioavailability. A genomic region comprising the nfs cluster and three upstream genes is involved in the antibacterial activity of P. donghuensis P482 against D. solani and P. carotovorum subsp. brasiliense. The genes studied are unique to the two known P. donghuensis strains. This study illustrates that mining of microbial genomes is a powerful approach for predictingthe presence of novel secondary-metabolite encoding genes especially when coupled with transposon mutagenesis.

Keywords: Dickeya; Pectobacterium; antiSMASH; genome mining; nfs; secondary metabolites.

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Figures

Figure 1
Figure 1
Phylograms depicting the results of 16S rRNA analysis (A) and MLSA (B) for P482 and related Pseudomonas spp. type strains. The phylogram based on 16S rRNA gene analysis was constructed using maximum likelihood method with Kimura 2-parameter using MEGA 6 software. Bootstrap values are shown at the nodes if the value is >60%. Except for Pseudomonas sp. P482, all strains used in the analysis are type strains. Pseudomonas aeruginosa SNP 0614 was used as the outgroup. Accession numbers of all the gene sequences included are listed in Table S1. The MLSA was performed for a set of partial nucleotide sequences of three genes: gyrB, rpoB, and rpoD (8328 nucleotides). Pseudomonas sp. P482 and five other Pseudomonas spp. strains were included because of the short genetic distance between them (see panel A). The MLSA-based phylogram was constructed using maximum likelihood method with GTR + I + G model estimated by jModelTest2 software. Bootstrap values are shown at the nodes. Cellvibrio japonicas Ueda107 was used as the outgroup.
Figure 2
Figure 2
Venn diagram for the comparison of four Pseudomonas spp. genomes, including P. donghuensis P482 and three closely related strains. The calculated core genome (3226 ORFs, shown in large font) represents 60–64% of each genome. The results for P. donghuensis HYST are not shown for clarity. The number of unique ORFs found for the P482 and HYST was 222 (4.3%) and 345 (6.5%), respectively.
Figure 3
Figure 3
Antibacterial activity of Pseudomonas sp. P482 and the type strains of P482-related species against D. solani and P. carotovorum subsp. brasiliense. Four soft rot bacterial strains were tested: D. solani type strain IPO 2222T, D. solani IFB 0102, P. carotovorum subsp. brasiliense type strain LMG21371T, and P. carotovorum subsp. brasiliense JJ 56. The order of the Pseudomonas strains from left to right reflects the their degree of their relatedness to P482, as estimated from ANI calculations. The histogram shows the mean of three independent experiments, and error bars show standard deviations.
Figure 4
Figure 4
Genomic region conferring the antibacterial activity of strain Pseudomonas sp. P482 toward soft rot bacteria. Genes marked with stars were inactivated by mutagenesis and the corresponding P482 mutants were impaired in antibacterial activity. ORFs shown in gray encode proteins that have none or few homologs in other Pseudomonas spp. The locations of the promoters (green arrows) and the terminators (red pins) are not drawn to scale. Their precise locations in contig JHTS01000055.1 are provided in Supplementary Materials (Tables S9, S10). Annotations of the depicted genes: 4705—bacterial regulatory, tetR family protein; 4706—HpcH/HpaI aldolase/citrate lyase family protein; 4707—short chain dehydrogenase family protein; 4708—thioesterase superfamily protein; 4709—acyl-CoA dehydrogenase, C-terminal domain protein; 4710—phenylacetate-CoA ligase; 4711—thiamine pyrophosphate enzyme.
Figure 5
Figure 5
Antibacterial activity of Pseudomonas sp. P482 and its mutants toward D. solani and P. carotovorum subsp. brasiliense strains. The histogram shows the mean of four independent experiments (n = 4), and error bars show standard deviations. Results statistically different from those obtained for the reference strain (P482 wt) in two-tailed Student's t-test assuming equal variances (α = 0.05) are marked with stars.
Figure 6
Figure 6
Total siderophore production on CAS blue agar. The histogram shows the relative siderophore production (%) by the P482 mutants (gray bars) with respect to the wild type strain (black bar). Error bars indicate standard deviations resulting from two independent experiments.
Figure 7
Figure 7
Pyoverdine production in CAA and MKB media. The level of pyoverdine production by Pseudomonas sp. P482 mutants and the related Pseudomonas spp. strains (P. donghuensis HYST and P. vranovensis DSM 16006T), was measured in two different iron-poor media: CAA (black bars) and MKB (white bars). Results are presented in relative fluorescence units (RFU), calculated as the ratio of fluorescence level (excitation 400 nm, emission 460 nm) to the optical density of the culture at 600 nm. Error bars on the histogram show standard deviations between eight technical replicates from a single experiment.
Figure 8
Figure 8
Influence of iron availability on the antimicrobial activity of Pseudomonas sp. P482 and its mutants. The histogram shows the mean of four independent experiments, and error bars show standard deviations. Black bars represent the diameter of the respective pathogen growth inhibition zone on LB agar medium. The gray bars are the results of the assay performed on LB agar supplemented with 15 μM FeSO4.
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