Stenotrophomonas comparative genomics reveals genes and functions that differentiate beneficial and pathogenic bacteria

Abstract

Background: In recent years, the number of human infections caused by opportunistic pathogens has increased dramatically. Plant rhizospheres are one of the most typical natural reservoirs for these pathogens but they also represent a great source for beneficial microbes with potential for biotechnological applications. However, understanding the natural variation and possible differences between pathogens and beneficials is the main challenge in furthering these possibilities. The genus Stenotrophomonas contains representatives found to be associated with human and plant host.

Results: We used comparative genomics as well as transcriptomic and physiological approaches to detect significant borders between the Stenotrophomonas strains: the multi-drug resistant pathogenic S. maltophilia and the plant-associated strains S. maltophilia R551-3 and S. rhizophila DSM14405T (both are biocontrol agents). We found an overall high degree of sequence similarity between the genomes of all three strains. Despite the notable similarity in potential factors responsible for host invasion and antibiotic resistance, other factors including several crucial virulence factors and heat shock proteins were absent in the plant-associated DSM14405T. Instead, S. rhizophila DSM14405T possessed unique genes for the synthesis and transport of the plant-protective spermidine, plant cell-wall degrading enzymes, and high salinity tolerance. Moreover, the presence or absence of bacterial growth at 37°C was identified as a very simple method in differentiating between pathogenic and non-pathogenic isolates. DSM14405T is not able to grow at this human-relevant temperature, most likely in great part due to the absence of heat shock genes and perhaps also because of the up-regulation at increased temperatures of several genes involved in a suicide mechanism.

Conclusions: While this study is important for understanding the mechanisms behind the emerging pattern of infectious diseases, it is, to our knowledge, the first of its kind to assess the risk of beneficial strains for biotechnological applications. We identified certain traits typical of pathogens such as growth at the human body temperature together with the production of heat shock proteins as opposed to a temperature-regulated suicide system that is harnessed by beneficials.

Figure 1

Genome-scale comparison for the three Stenotrophomonas strains. The plant-beneficial strains S. maltophilia R551-3 (top), S. rhizophila DSM14405^T (middle), and the human pathogenic S. maltophilia K279a (bottom). The original genomic sequence of S. rhizophila DSM14405^T was converted into its reverse complement to achieve the same direction for all three genomes. Homologous DNA segments among the strains are marked by boxes with the same color, while gaps correspond to non-homologous regions. There are vast regions of homology between the genome of S. rhizophila DSM14405^T and both S. maltophilia R551-3 and K279a. The figure was generated using nucleotide sequences of the genomes using Mauve 2.3.

Figure 2

Circular genome map of S. rhizophila DSM14405 ^T . Predicted coding sequences (CDSs) are assigned various colors with respect to cellular functions. The circles show from the outermost to the innermost: 1. DNA coordinates; 2, 3. Function-based color coded mapping of the CDSs predicted on the forward and reverse strands. Various functions are assigned different colors. 4. tRNA genes; 5. rRNA genes; 6. GC plot with regions above and below average in gray and black, respectively; 7. GC skew showing regions above and below average in dark yellow and magenta, respectively (window size: 10,000 bp). The circular genome map was constructed using DNAPlotter.

Figure 3

Gene orthology analyses between S. rhizophila DSM14405 ^T , S. maltophilia R551-3 and the clinical S. maltophilia K279a. Circles show from the outermost to the innermost: 1. DNA coordinates; 2, 3. Function-based color-coded mapping of the CDSs predicted on the forward and reverse strands of the S. rhizophila DSM14405^T genome, respectively. 4. Orthologous CDSs shared between S. rhizophila DSM14405^T and S. maltophilia R551-3. 5. S. rhizophila-specific CDSs, compared with S. maltophilia R551-3. 6. Orthologous CDSs shared between S. rhizophila and S. maltophilia K279a. 7. S. rhizophila-specific CDSs, compared with S. maltophilia K279a. 8. GC plot depicting regions above and below average in gray and black, respectively; 9. GC skew showing regions above and below average in yellow green and magenta, respectively (window size: 10,000 bp). The assessment of orthologous CDSs was carried out using the reciprocal best BLASTp hit approach with an identity threshold of 30% and evalue of 10^-6.

Figure 4

Orthology analysis and the distribution of the S. rhizophila -specific CDSs with regard to cellular functions. a: Venn diagram showing the number of CDSs shared between the three strains. S. rhizophila DSM14405^T was used as the reference genome. 3171 and 3149 CDSs are shared between S. rhizophila and S. maltophilia R551-3 and K279a, respectively. 3049 CDSs are shared between all three strains, as a trio-analysis of the three genomes revealed. 762 CDSs are absent in both S. maltophilia R551-3 and S. maltophilia K279a, and hence unique to S. rhizophila. b: Diagram showing the percentage distribution of the 762 S. rhizophila-specific CDSs with regard to the predicted cellular functions. Most of these are hypothetical genes (71.26%) or CDSs of general function (4.46%). Other S. rhizophila-unique genes showing a relative abundance are involved in carbohydrate transport and metabolism (4.07%), and cell wall, outer membrane, and cytoplasmic membrane biogenesis (3.02%).

Figure 5

Model showing the specific and shared mechanisms used by S. rhizophila and S. maltophilia K279a. The plant growth promoting and biocontrol agent S. rhizophila DSM14405^T and the human-pathogenic clinical S. maltophilia K279a each use particular mechanisms that rely on species-specific genes to best adapt and perform in their habitats. The S. rhizophila-specific genes are shown in black while those genes in gray are specific to S. maltophilia K279a. Nevertheless, other crucial mechanisms such as ensuring access to biologically available iron and resistance against antibiotics are shared between both species (middle).

Figure 6

The impact of heat shock (35°C) on the gene expression of S. rhizophila DSM14405 ^T . Genes responsible for translation and post-translational modification, the metabolism and transport of amino acids, nucleotides, lipids, and the production and conversion of energy are up-regulated while genes involved in cell motility and intracellular trafficking are strongly down-regulated. The values above each column correspond to the percentage abundance of the corresponding functional gene group relative to the total count of the up and down-regulated genes. The transcription fold change for each CDS corresponds to the ratio calculated for S. rhizophila treated with 35°C compared to 30°C. Data are presented as the mean value of two independent replicates. The error bar shown on each functional group corresponds to the mean value of errors for all genes belonging to that functional group.