Stenotrophomonas goyi sp. nov., a novel bacterium associated with the alga Chlamydomonas reinhardtii

Isolation of Stenotrophomonas goyi sp. nov.

This study took place at Campus Universitario de Rabanales, Cordoba, Spain. S. goyi sp. nov. where it was isolated from a fortuitously contaminated Chlamydomonas reinhardtii culture in the laboratory. Initially, the Chlamydomonas reinhardtii culture was simultaneously contaminated with three different bacteria (Fakhimi et al., 2023b). Individual members of this bacterial community were isolated by sequential rounds of plate streaking in Yeast Extract Mannitol (YEM) medium (handmade in our lab, described here), until three different types of bacterial colonies were visually identified. Colonies were grown separately, and the subsequent isolated DNA was used for PCR-amplification of their partial RNA 16S sequences. After sequencing, the three independently isolated bacteria were identified as members of the genus Microbacterium, Stenotrophomonas, and Bacillus (Fakhimi et al., 2023b).

Genome sequencing and assembling of S. goyi sp. nov.

DNA extraction and whole genome sequencing using PacBio (Pacific Biosciences) RS II Sequencing System (RRID:SCR_017988) were performed by SNPsaurus LLC (https://www.snpsaurus.com/). Whole genome sequencing generated 102,238 reads yielding 832,209,774 bases for 166 read depth over the genome (Table 1). Genome was assembled with Canu (RRID:SCR_015880) 1.7 (Koren et al., 2017) generating a 4,487,389 pb circular genome. The genome completeness was checked by BUSCO 3.0.2 (RRID:SCR_015008) (Manni et al., 2021) and was 94.6% complete, with 94.6% of the genome single copy and 0.0% duplicated. Any other prokaryotic contamination was discarded using ContEst16S 1.0 (RRID:SCR_000595) (Lee et al., 2017).

Phylogenetic analysis

Phylogenetic analyses were performed using the Type (Strain) Genome Server (TYGS) at Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures GmbH) (Camacho et al., 2009; Farris, 1972; Kreft et al., 2017; Lagesen et al., 2007; Lefort et al., 2015; Meier-Kolthoff et al., 2022, 2013; Meier-Kolthoff and Göker, 2019; Ondov et al., 2016). Information on nomenclature, synonymy and associated taxonomic literature was provided by TYGS's sister database, the List of Prokaryotic names with Standing in Nomenclature (LPSN). Trees were inferred with FastME 2.1.6.1. Phylogenetic trees were drawn with iTOL (RRID:SCR_018174) (Letunic and Bork, 2021).

Annotation

Tentative annotation of the S. goyi sp. nov. genome was performed using the RAST tool kit, RASTtk (RRID:SCR_014606) at The Genome Annotation Service (Brettin et al., 2015; Overbeek et al., 2014). BlastKOALA service was used to automatically assign K numbers to the predicted proteins, which allowed Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology assignments, the putative characterization of individual gene functions, and the reconstruction of KEGG pathways (Kanehisa et al., 2016). The PHAge Search Tool Enhanced Release (PHASTER) (RRID:SCR_005184) was used to locate potential phage sequences within the S. goyi sp. nov. genome (Arndt et al., 2016)

Bacterium growth media and culturing conditions

Bacterial precultures were grown on Yeast Extract Mannitol (YEM) or Luria Broth (LB) media (handmade in our lab). Some growth experiments were done in Mineral Medium (MM) (Harris, 2008) supplemented with different nutrient sources (handmade in our lab). Tris-Acetate-Phosphate (TAP) medium (Harris, 2008) (handmade in our lab, described here) was also used occasionally. In some experiments, a vitamin cocktail (riboflavin, 0.5 mg⋅L^-1; p-aminobenzoic acid, 0.1 mg⋅L^-1; nicotinic acid 0.1 mg⋅L^-1; pantothenic acid, 0.1 mg⋅L^-1; pyridoxine, 0.1 mg⋅L^-1; biotin, 0.001 mg⋅L^-1; vitamin B12, 0.001 mg⋅L^-1; thiamine, 0.001 mg⋅L^-1) was added to bacterial cultures. More specific details for each experiment can be found in the corresponding figure and table legends. All the bacterium cultures were incubated at 24-28°C and under continuous agitation (130 rpm).

Coculturing algae and bacteria

Chlamydomonas cells were cultured for 3-4 days in TAP medium until mid-logarithmic growth phase, harvested by centrifugation (5.000 rpm for 5 min) and washed twice with fresh MM. Bacterial batch-cultures were incubated in TYM or LB medium until the Optical Density at 600 nm (OD₆₀₀) reached 0.8-1, then harvested by centrifugation (12.000 rpm for 5 min) and washed twice with fresh MM. Algae and bacteria were cocultured in 250 mL flasks containing 100 mL of the corresponding medium. Alga-bacterium cocultures were set to initial chlorophyll concentration of 10 μg·mL⁻¹ for the alga and an initial OD₆₀₀ of 0.1 for the bacterium. Algal and bacterial monocultures were used as controls. All cultures were incubated at 24°C with continuous agitation (120-140 rpm) and under continuous illumination (80 PPFD).

Determinations of algal and bacterial growth

The algal growth was assessed in terms of chlorophyll content. Chlorophyll measurements were done by mixing 200 μL of the cultures with 800 μL of ethanol 100%. The mix was incubated at room temperature for 2-3 min, and afterward centrifuged for 1 min at 12.000 rpm. The supernatant was used to measure chlorophyll (a + b) spectrophotometrically (DU 800, Beckman Coulter) at 665 and 649 nm (Wintermans and de Mots, 1965).

Bacterial growth in monocultures was estimated spectrophotometrically in terms of OD₆₀₀ evolution (DU 800, Beckman Coulter). However, estimation of the bacterial growth in cocultures required bacterium cells separation from the alga cells. To do this, a customized Selective Centrifugal Sedimentation (SCS) approach was used. This approach consisted in finding the centrifugation parameters that led to maximize algal cell sedimentation while minimizing bacterial cell sedimentation (Torres et al., 2022). Thus, measuring the OD of the supernatant after centrifugation can provide an estimation of the bacterial growth in the cocultures. To do this, the percentages of precipitated cells of each monoculture were calculated at different forces (from 100 to 500 x g) and times (1 and 2 min) using the measured OD before (A_BC) and after (A_AC) the centrifugation. Centrifugation at 200 x g for 1 min led to 87.9% of Chlamydomonas sedimentation, while only 2.1% of the bacterial cells dropped (meaning that 97.9% of the S. goyi cells remained in the supernatant). These parameters were chosen as a good compromise for SCS and used to evaluate the contribution of the bacteria to the OD in cocultures (^SCSOD₆₀₀).

Identification of Stenotrophomonas goyi sp. nov.

A fortuitous contaminated Chlamydomonas reinhardtii culture (strain 704; CC-3597; https://www.chlamycollection.org/) was studied due to its enhanced hydrogen production capability. This alga culture turned out to be contaminated with three different bacterial strains (Fakhimi et al., 2023b), one of them consisting in a white-pigmented bacterium (Figure 1). This bacterium was isolated after several rounds of plate streaking in TYM medium. First, partial PCR amplification and sequencing of the ribosomal 16S gene allowed the identification of this bacterium as a member of the Stenotrophomonas genus. Afterwards, the whole genome sequence was obtained. Genome assembling provided one single circular contig of 4,487,389 pb (Table 1). No plasmids or extrachromosomal elements were identified.

Figure 1. Plate with Chlamydomonas reinhardtii and Stenotrophomonas goyi sp. nov.

The RAST server identified 4,147 genes (4,066 CDS + 81 rRNAs and tRNAs) (Table 2). Out of these 4,066 CDS identified by RAST, 1,096 of them were in subsystems. Tentative genome annotation derived from the RAST server is available in Supplemental Table 1 as Extended data (González-Ballester et al., 2023).

Phylogenetic analyses were performed with both, the whole genome (Figure 2) and the inferred 16S rDNAs (Figure 3). Pairwise comparisons with the closest type strains genomes are shown in Table 3. These phylogenetic analyses revealed that the sequenced genome belonged to a new Stenotrophomonas sp.; all dDDH values (d0, d4 and d6) were below 70% (Meier-Kolthoff et al., 2013) (Table 3). This new bacterial species was named as Stenotrophomonas goyi sp. nov. The closest related bacteria in terms of whole genome and 16S rDNA similarities were Stenotrophomonas rhizophila DSM 14405 and Stenotrophomonas nematodicola W5, respectively (Figures 2 and 3). S. goyi sp. nov. genome was deposited in the NCBI as SUB12103906.

Figure 2. Phylogenetic tree for Stenotrophomonas goyi genome and related closest bacteria.

Tree inferred with FastME 2.1.6.1 using the Genome BLAST Distance Phylogeny (GBDP) distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above the branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 91.6%. The tree was rooted at the midpoint. Branch lengths (black) and bootstraps (red) values are indicated. Genome sizes: 3,906,271–5,177,426 pb. Average δ statistics: 0.078 (Holland et al., 2002). Phylogenetic tree drawn with iTOL (Letunic and Bork, 2021).

Figure 3. Phylogenetic tree for Stenotrophomonas goyi 16S rDNA and related closest bacteria.

Tree inferred with FastME 2.1.6.1 using the Genome BLAST Distance Phylogeny (GBDP) distances calculated from 16S rDNA gene sequences. The branch lengths are scaled in terms of GBDP distance formula d5. The numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with an average branch support of 78.6%. The tree was rooted at the midpoint. Branch lengths (black) and bootstraps (red) values are indicated. RNA16S lengths: 1,385–1,535 pb. Average δ statistics: 0.236 (Holland et al., 2002). Phylogenetic tree drawn with iTOL (Letunic and Bork, 2021).

BlastKOALA (Kanehisa et al., 2016) service allowed KEGG orthology assignments to characterize individual gene functions and reconstruct KEGG pathways of S. goyi genome (Supplemental Table 2; Extended data (González-Ballester et al., 2023)). Some important pathways were either absent or incomplete in S. goyi sp. nov. including assimilation of nitrate (the whole assimilatory pathway is missing including nitrate transporters) and sulfate (only sulfite reductase is present). On the other hand, putative complete pathways for the glyoxylate cycle and biosynthesis of biotin, coenzyme A, pantothenate, riboflavin, tetrahydrofolate, glutathione, pyridoxal-P, lipoic acid, dTDP-L-rhamnose, UDP-N-acetyl-D-glucosamine, C5 isoprenoids, bacterial lipopolysaccharides, and antimicrobial proteins, among others, were present. Incomplete pathways for the degradation of aromatic compounds (including phenol, toluene, xylene, methylnaphthalene, 3-hydroxytoluene, and terephthalate) and myo-inositol biosynthesis, were also present.

Search with PHASTER (Arndt et al., 2016) revealed one intact prophage (PHAGE_Erwini_phiEt88_NC_015295) located at position 753112-799783 of the S. goyi sp. nov. genome.

Nutrient requirements of S. goyi sp. nov.

S. goyi sp. nov. showed no growth on MM, or in MM supplemented with different C sources (sucrose, glucose, lactose, mannitol, or glycerol) (Table 4). The addition of vitamins to the MM supplemented with glucose or lactose did not support the bacterium growth either (Table 4). However, the bacterium showed an excellent growth when cultivated in MM supplemented with yeast extract, tryptone, peptone or even Bovine Serum Albumin (BSA) (Table 4), suggesting that this bacterium has a great capacity to use peptides/amino acids as C source, and probably also as N source. Moreover, the peptides/amino acids could also provide, in addition to C and N sources, other essential nutrients or even palliate potential amino acids auxotrophies. Note that MM medium has sulfate as only S source. As commented before, the genome of S. goyi sp. nov. is lacking a functional sulfate assimilation pathway. Thereby S-containing amino acids, such as cysteine and methionine, could support the growth in medium rich in peptides/amino acids.

To confirm this hypothesis, S. goyi sp. nov. was inoculated in plates of MM + glucose supplemented with different combinations of cysteine, methionine, biotin, and thiamine. Only plates containing cysteine and methionine supported the bacterial growth for several culturing rounds (Figure 4). This result confirms the cysteine and methionine growth dependence of S. goyi. sp. nov. Cysteine and methionine could provide either a reduced S source or complement an auxotrophy for these two amino acids. Since S. goyi sp. nov. genome has complete pathways for all the essential amino acids, is more likely that cysteine and methionine are being used as reduced S sources. Similar results were found for M. forte, where cysteine and methionine are required as S sources (Fakhimi et al., 2023a).

Figure 4. Cysteine and methionine requirements of S. goyi to grow.

S. goyi sp. nov. was inoculated in: A) plates of Mineral Medium (MM) + glucose (5 g·L^-1); B) MM + glucose + biotin (0.001 mg·L^-1) + thiamine (0.001 mg·L^-1); C) MM + glucose + cysteine (4 mM) + methionine (4 mM); and D) MM + glucose + cysteine + methionine + biotin + thiamine.

S. goyi sp. nov. showed optimal growth between 24 and 32°C and pH 5-9 (Table 5). Despite the presence of the complete multidrug resistance efflux pump MexJK-OprM in the genome (Chuanchuen et al., 2005), no resistance to tetracycline, rifampicin, chloramphenicol and polymyxin (50 μg/mL each) was observed.

Growth of S. goyi sp. nov -C. reinhardtii consortium

Torres et al., (2022) reported that cocultures of S. goyi sp. nov. (published as Stenotrophomonas sp.) and C. reinhardtii promoted the growth of the microalga (nearly doubled) when incubated in MM supplemented with glucose and mannitol, but not when supplemented with acetic acid (Torres et al., 2022).

Here, it was also checked if the bacterium also benefited when co-cultivated with C. reinhardtii in glucose- and mannitol-containing media. First, we observed that the chlorophyll content in the cocultures was 2.4 times higher than in the C. reinhardtii monocultures after 13 days (Figure 5A), which is in accordance with previous results (Torres et al., 2022). Additionally, the dry biomass resulting from the consortia was 2.2 times higher than the sum of the respective monocultures (Figure 5B). Finally, the growth of the bacterium in cocultures was very efficient, unlike S. goyi sp. nov. monocultures (Figure 5C).

Figure 5. S. goyi sp. nov. and C. reinhardtii growth in consortium.

S. goyi-C. reinhardtii consortium, and respective control monocultures, were incubated in Mineral Medium (MM) supplemented with glucose (5 g·L^-1) and mannitol (5 g·L^-1). A) Chlorophyll content; B) dry weight biomass after 13 days; C) bacterial growth in terms of OD^SCS; D) actual picture of the cultures after 13 days.

These results indicate that S. goyi sp. nov. and C. reinhardtii can establish a mutualistic relationship when incubated in sugars-containing media. On the one hand, S. goyi sp. nov. can greatly support the growth of the C. reinhardtii in media supplemented with glucose or mannitol, which are two carbon sources that the alga cannot utilize. Likely, this growth promotion is due to the release of acetate and/or CO₂ from the bacteria after the sugar fermentation. Acetate is the sole organic carbon source that C. reinhardtii can utilize during heterotrophic/mixotrophic growth (Chaiboonchoe et al., 2014). On the other hand, S. goyi, sp. nov. can grow in media without amino acids/peptides supplementation when cocultured with C. reinhardtii, suggesting that the alga must provide some essential nutrients for the bacterium. Reduced S forms excreted by the alga (e.g., cysteine or methionine) could potentially explain the bacterium growth in the consortium.