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Article

Comparative Genomic Analysis of the Biotechnological Potential of the Novel Species Pseudomonas wadenswilerensis CCOS 864T and Pseudomonas reidholzensis CCOS 865T

by
Dominik Rutz
1,2,†,
David Frasson
2,
Martin Sievers
2,
Jochen Blom
3,
Fabio Rezzonico
1,
Joël F. Pothier
1 and
Theo H. M. Smits
1,*
1
Environmental Genomics and Systems Biology Research Group, Institute of Natural Resource Sciences, Zurich University of Applied Sciences (ZHAW), CH-8820 Wädenswil, Switzerland
2
Microbiology and Molecular Biology Research Group, Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences (ZHAW), CH-8820 Wädenswil, Switzerland
3
Bioinformatics and Systems Biology, Justus-Liebig-Universität, D-35392 Giessen, Germany
*
Author to whom correspondence should be addressed.
†
Present address: Roche Diagnostics International AG, CH-6343 Rotkreuz, Switzerland.
Diversity 2019, 11(11), 204; https://doi.org/10.3390/d11110204
Submission received: 20 September 2019 / Revised: 18 October 2019 / Accepted: 26 October 2019 / Published: 28 October 2019
(This article belongs to the Special Issue Pseudomonas Biology and Biodiversity)
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Abstract

:
In recent years, the use of whole-cell biocatalysts and biocatalytic enzymes in biotechnological applications originating from the genus Pseudomonas has greatly increased. In 2014, two new species within the Pseudomonas putida group were isolated from Swiss forest soil. In this study, the high quality draft genome sequences of Pseudomonas wadenswilerensis CCOS 864T and Pseudomonas reidholzensis CCOS 865T were used in a comparative genomics approach to identify genomic features that either differed between these two new species or to selected members of the P. putida group. The genomes of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T were found to share genomic features for the degradation of aromatic compounds or the synthesis of secondary metabolites. In particular, genes encoding for biocatalytic relevant enzymes belonging to the class of oxidoreductases, proteases and isomerases were found, that could yield potential applications in biotechnology. Ecologically relevant features revealed that both species are probably playing an important role in the degradation of soil organic material, the accumulation of phosphate and biocontrol against plant pathogens.

1. Introduction

The genus Pseudomonas taxonomically belongs to the gamma subclass of Proteobacteria [1]. Members of Pseudomonas have a saprophytic lifestyle and are ubiquitously found in the environment surviving at most temperatures [2]. They can be isolated from samples of soil, water, air, plants and animal or human related sources [3]. Pseudomonads are highly versatile in relation to genetics, physiology and metabolism [2]. They grow rapidly under simple nutritional conditions and some species are able to use more than 100 different sources of carbon and energy. Members of the genus Pseudomonas may catabolize aromatic and aliphatic hydrocarbons (phenols, toluene, n-alkanes, cyclohexane) by using specific pathways [4,5,6].
For industrial biotechnology application, Pseudomonas putida and related species have particularly been scrutinized following the discovery of degradation pathways for natural and xenobiotic compounds [5,7]. Members of the genus Pseudomonas are being used as whole-cell biocatalyst or for the production of specific enzymes for bulk- and fine-chemicals manufactory [8]. Another advantage is that they are resistant against environmental stress, making them robust against the presence of toxins or inhibiting solvents and under extreme pH and temperature conditions [7,9]. The use of solvent-tolerant pseudomonads as biocatalysts thus provides a solution in the production of toxic products [10]. Furthermore, numerous extracellular enzymes are being produced by pseudomonads, such as for example lipases and proteases, which find important application in biotechnology [11,12]. Examples for the application are the synthesis of bio-based polymers, small molecular weight chiral compounds, biosurfactants and heterologous proteins [7,12]. In agriculture, pseudomonads are known for their potential as biocontrol agents of soilborne plant pathogens or insects [13,14,15]. Nevertheless, the biocatalytic potential of some newly found isolates is still unknown. Research in this direction would be of great interest for the biotechnology industry.
In 2014, two isolates belonging to the P. putida group were obtained from soil samples of the Reidholz forest in Richterswil, Switzerland, during the search for novel biocatalysts [16]. Genotypic and phenotypic data clarified that the isolates differed from other species of the P. putida group. Because of the clear separation of the strains, Pseudomonas wadenswilerensis CCOS 864T and Pseudomonas reidholzensis CCOS 865T were proposed as novel species [16]. Recently, both genomes were published [17,18]. In this study, we used comparative genomics to elucidate the potential of these two species as a source for novel biocatalytic features while investigating also their ecological role.

2. Materials and Methods

2.1. Bacterial Strains

P. wadenswilerensis CCOS 864T (LMG 29327T) and P. reidholzensis CCOS 865T (LMG 29328T) were both isolated from soil from the Reidholz forest (47°13′ N and 8°41′ E), located between the city of Wädenswil and the village of Richterswil, Switzerland in 2014, during research for novel biocatalysts for biotechnological application [16].

2.2. Comparative Genomics

The core genome phylogenetic relationships were obtained using EDGAR v.2.2 [19]. Briefly, the core genome was defined by iterative pairwise comparison of the gene content of each of the selected genomes using the bidirectional best hits (BBH) strategy as orthology criterion. For all calculations, protein BLAST (BLASTp) was used with BLOSUM62 as similarity matrix [20]. Genes were considered orthologous when a reciprocal best BLAST hit was found between two genes, and when both BLAST hits were based on alignments with a score ratio value [21] exceeding 30%. Multiple alignments of each orthologous gene set of the core genome were calculated using the MUSCLE software [22] and non-matching parts of the alignments were removed using GBLOCKS [23]. The resulting alignments were concatenated and used to construct an approximately-maximum-likelihood phylogenetic tree using the FastTree software [24], which computes local support values with the Shimodaira-Hasegawa test. The resulting tree was edited for display with the MEGA7 software [25].
Species differentiation was additionally checked by calculation of ANI, Tetra and GGDC values between the closest related strain in the tree and the draft genome sequences of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T. For ANI and Tetra, the online service of JSpeciesWS version 3.0.20 with default parameters was used [26], while for GGDC, the online service version 2.1 [27] was used, reporting values obtained with Formula 2 for draft genomes. Pseudomonas species names not fitting with the type strains of the designated species were not maintained and renamed as Pseudomonas sp. (Table 1).
For investigation of genomic features, both genomes were annotated in GenDB [28]. Based on the obtained annotation data, a comparative pan-genome analysis was performed. Genomic features for potential biotechnological applications or features of ecological interests were selected using EDGAR [19] and examined for their presence in other strains of the P. putida group. Information on transport proteins was obtained from the transporter classification database (TCDB) by using the BLAST search on the website [29], while for aromatic degradation the Kyoto Encyclopaedia of Genes and Genomes (KEGG) [30] and the Biocatalysis / Biodegradation Database from Eawag (EAWAG-BBD) [31] were used. Maps of individual features were created using different subroutines of the LASERGENE package version 11.0 (DNASTAR, Madison, WI, USA).

3. Results

3.1. The Genomes of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T

The genome sizes of 5′966′942 bp for P. wadenswilerensis CCOS 864T [17] and 6′163′129 bp for P. reidholzensis CCOS 865T [18] were well within the range of the minimum (4′655′082 bp) and maximum (6′663′130 bp) of other members of the P. putida group. A total number of 5′437 genes for P. wadenswilerensis CCOS 864T and 5′441 genes for P. reidholzensis CCOS 865T were annotated. When comparing the number of genes to genomes of the P. putida group, the values of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T were within the average range of 5′382.8 ± 579.9 genes (average ± standard deviation of genome metrics; Table 1) as determined for members of the P. putida group. A comparison of the G+C content values revealed that P. wadenswilerensis CCOS 864T was, with a value of 62.39%, within the average range of 62.45% ± 0.96% of the P. putida group. P. reidholzensis CCOS 865T, however, was with a value of 64.09% closer to the highest observed G+C content of 64.4%. Based on the comparison of genome characteristics, we conclude that P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T share similar genome characteristics as other members of the P. putida group.
The draft genome sequences were screened for antibiotic resistance genes using ResFinder [32] and CRISPR sequences using CRISPRfinder [33]. Analysis of the draft genome of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T revealed no similarities to known antibiotic resistance genes. Screening for CRISPR repeat sequences resulted in three questionable CRISPR repeat regions in P. wadenswilerensis CCOS 864T and eight in P. reidholzensis CCOS 865T, but none of them could be confirmed.

3.2. Selection of Genomes and Phylogeny

In order to perform a pan-genome analysis to identify genomic features that differ between the genomes of either P. wadenswilerensis CCOS 864T [17] or P. reidholzensis CCOS 865T [18], a total of 123 genome sequences were selected from GenBank. The selection criteria were: a) allocation to a species known to belong to the P. putida group; and b) covering the species with as many as possible genomes to allow statements on species-specific traits. Several reference strains and species, for which the complete 16S rRNA gene was indicating that the species could be closely related to the P. putida group, such as Pseudomonas fuscovaginae, Pseudomonas asplenii or Pseudomonas oryzihabitans, were also included in the core phylogeny to understand its phylogenetic position within the genus [3,34]. Strains indicated as Pseudomonas sp. in GenBank were not analyzed for their membership of the P. putida group. The final selection had a total of 123 genome sequences and included the genomes of 17 type strains belonging to the P. putida group (Figure 1).
The core genome tree and comparisons based on average nucleotide identities using BLASTn as performed in EDGAR using the same BLAST parameters as JSpecies [19], identified a further 14 operational taxonomic units (OTUs) each potentially representing a novel species. From this analysis, we selected 43 genomes to cover the phylogenetic groups of the P. putida group with at least one representative (Figure 2; Table 1). The table includes 20 of 41 isolates that were renamed to Pseudomonas sp. as they were in different taxonomic groups as the type strains of the species to which the sequence was assigned in GenBank [34]. Based on this selection of strains, the comparative genomics platform EDGAR [19] was used to identify some of the most relevant differential genomic features of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T in order to reveal their yet unknown potential as biocatalyst for biotechnological applications and in ecological processes.

3.3. Degradation of Aromatic Compounds

Aromatic compounds are widely distributed in nature in form of aromatic amino acids in organisms, plant cell material such as lignin or derivates of gasoline (benzene, toluene, xylene, ethylbenzene) [35]. Pseudomonas spp. are reported to be able to degrade aromatic compounds in two steps, beginning with a ring modification to yield a catechol (upper pathway) followed by a ring fission by either ortho- or meta-cleavage (lower pathway) [4].
The aromatic compound p-hydroxyphenylacetic acid (p-HPA) is a product of lignin degradation [36]. Investigation of the pan-genome revealed that P. wadenswilerensis CCOS 864T has a complete gene set for p-HPA degradation (Figure 3A), which was absent from the genome of P. reidholzensis CCOS 865T. Positive growth of P. wadenswilerensis CCOS 864T with this compound was obtained before [16], supporting our findings from the genomes. The hpaBC genes for degradation of p-HPA to homoprotocatechuic acid (HPC) and the hpaAG1G2EDF-nicT-hpaHIR gene cluster for the degradation from HPC to succinyl-CoA and acetyl-CoA by meta-cleavage [35,37] were found in the genome of P. wadenswilerensis CCOS 864T. While most genes were located in one cluster (CCOS864_03099–03108), the hpaBC genes were located at a different location in the genome (CCOS864_02954–02955). The presence of both p-HPA degradation gene clusters was confirmed for 15 of 43 members of the P. putida group (Figure 2). Comparative genomic results of this feature matched with the phenotypical results obtained before for strains that were tested in their studies [16,38]. In comparison to the cluster in P. wadenswilerensis CCOS 864T, the cluster in P. entomophila L48T differed by the absence of the putative metabolite transport protein nicT, while in Pseudomonas sp. 13.1.2 (OTU03), different genes were found in the flanking region (Figure 3B).
Another gene cluster identified in the genome of P. reidholzensis CCOS 865T may allow this organism to degrade mandelic acid. Mandelic acid occurs in its free form in plant tissues of wheat leaves and grapes [39]. Furthermore, glycosides of mandelonitrile can be found in almonds, peach and apricot pits [39]. Apart from this, the repellent mixtures of various arthropods and urine from animals are a potential source of mandelate or mandelonitrile, which can then find its way into the soil [39]. Four genes are known to encode enzymes involved in mandelic acid degradation (mdlABCD) [40]. Later studies revealed that also mandelamide dehydrolase (MdlY) is involved in the degradation pathway from mandelamide [41]. The mentioned genes were conserved in P. reidholzensis CCOS 865T (CCOS865_04122–04126) and only occurred as well in P. putida NBRC 14164T (Figure 2).
Additionally, in P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T, a putative gene cluster encoding for enzymes involved in the ortho-cleavage of benzaldehyde was found (CCOS864_00141–00148; CCOS865_04272–04279) (Figure 3A). Both species should therefore be able to degrade benzaldehyde via benzoate to catechol. Catechol will then be degraded to muconolactone and in a last step to 3-oxoadipate [30]. Furthermore, 3-oxoadipate enol-lactonases, involved in the last degradation reaction, were found at multiple locations in both genomes. Genes for the oxidation from benzene, toluene ethylbenzene or xylene (BTEX upper pathways) were not identified [42]. However, this pathway could be involved in the further degradation of mandelic acid in P. reidholzensis CCOS 865T, whereas upper pathways in P. wadenswilerensis CCOS 864T could not be identified. This ortho-cleavage pathway was also found in 32 other members of the P. putida group (Figure 2), while the cluster in Pseudomonas sp. MO2 (OTU06) was incomplete due to assembly gaps.
In the P. reidholzensis CCOS 865T genome, we identified a putative gene cluster for the degradation of gallic acid (CCOS865_04015–04020) (Figure 3A). In nature, gallic acid and structurally related compounds have been discovered in many different fruits, plants and in oak wood [43,44]. Furthermore, this compound occurs mainly in black and green tea as phenolic component together with its catechol derivatives [44]. Degradation of gallate, a derivative of gallic acid, was observed in Pseudomonas sp. (formerly P. putida) KT2440, Klebsiella pneumoniae and other Alpha-, Beta- and Gammaproteobacteria. Genes involved in the degradation process of gallic acid are commonly in a single gene cluster (galABCDPRT) [45]. However, the cluster in the genome of P. reidholzensis CCOS 865T lacked galP, encoding an outer membrane porin, responsible with GalT for the transport of gallic acid into the cell. This gene was located at another location in the genome (CCOS865_00376). The absence of galP in some gal-clusters was already reported before [45]. The putative gallic acid degradation cluster was also found in the genome of seven other P. putida group members and was not present in P. wadenswilerensis CCOS 864T (Figure 2).
Additionally, the P. reidholzensis CCOS 865T genome also contains genes for vanillin degradation (Figure 3A). Vanillin is a popular aromatic flavor compound and a natural degradation product of lignin or ferulic acid [46]. The vdh gene (CCOS865_02494) encodes the enzyme vanillin dehydrogenase, which catalyzes the oxidation of vanillin to vanillic acid [47]. The vdh gene occurred in 17 other members of the P. putida group (Figure 2). In the next step, the two subunits VanA (CCOS865_03327) and VanB (CCOS865_03328) are needed for the demethylation reaction of vanillic acid to protocatechuic acid [47]. Degradation thereof will occur via the intermediate 4-oxalomesaconate in the gallic acid degradation pathway.

3.4. Secondary Metabolites

In the genomes of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T, a complete gene cluster for hydrogen cyanide synthesis (hcnABC) was found (CCOS864_00134–00136, CCOS865_00148–00150). Hydrogen cyanide (HCN) is a secondary metabolite and functions as cytochrome c oxidase and other metalloenzymes inhibitor [48]. The synthesis of HCN was observed in several pseudomonads, like Pseudomonas aeruginosa, Pseudomonas protegens, and Pseudomonas soli [15,48,49]. In Proteobacteria, hydrogen cyanide synthase is a protein involved in the oxidation of glycine to HCN and carbon dioxide on membranes [48]. HCN synthesis is of ecological interest as it is involved in biological control of plant root diseases and as broad-spectrum antimicrobial compound [50]. Additionally, HCN increases the nutrients availability in the soil through sequestration of metals, resulting in higher phosphate concentrations necessary for plant growth [51]. As an example for biological control, HCN-producing fluorescent pseudomonads can suppress Thielaviopsis basicola, a plant pathogenic fungus involved in black root rot of tobacco [15,52]. Nevertheless, cyanide production of bacteria can have different effects on plants depending on the plant species and cyanide tolerance of the plant [53]. The hcnABC gene cluster also occurred in 13 other members of the P. putida group (Figure 2). Although the clusters in the different pseudomonads were highly similar, the flanking regions differed in most of the strains (Figure 4), which may be an indication that this cluster was introduced by horizontal gene transfer.
Furthermore, a putative carotenoid gene cluster for zeaxanthin biosynthesis in P. reidholzensis CCOS 865T (CCOS865_02931–02936) was identified. Synthesis of carotenoids is widely present in Proteobacteria and has been demonstrated among others in Erwinia, Pantoea, or Pseudomonas sp. strain Akiakane [54,55]. Zeaxanthin, phenotypically seen as a yellow color of the colony, plays likely a role in photo-oxidative damage protection [54]. In P. reidholzensis CCOS 865T, the cluster consisted of crtE-fni-crtXIYBZ, which corresponds to the cluster setup as in Cronobacter or Erwinia spp. [55,56]. When comparing with other members of the P. putida group (Figure 2), only four other strains had orthologous genes forming a complete cluster, while Pseudomonas sp. CRS01-1 (OTU12) displayed an incomplete cluster. However, closer inspection of the region downstream of crtI showed the presence of alternative crtBZ orthologs with lower sequence identity at this location (Figure 4). Although the presence of a complete gene cluster, a yellow color, indicating the synthesis of this carotenoid, was not observed phenotypically for P. reidholzensis CCOS 865T [16]. Therefore, it might be possible that it is only weak or not at all expressed in P. reidholzensis CCOS 865T under the tested conditions.
Bacteria from the genus Pseudomonas are known for possessing several protein secretion mechanisms, which are important for commensal, mutualistic or pathogenic interactions between them and their environment [57]. We identified one type I secretion (T1SS) cluster in P. wadenswilerensis CCOS 864T, while three and two type II secretion system (T2SS) gene clusters were present in P. reidholzensis CCOS 865T and P. wadenswilerensis CCOS 864T, respectively. Further observations showed also the presence of two different type VI secretion system (T6SS) gene clusters.

3.5. Secretion Systems

3.5.1. Type I Secretion Systems

The alkaline protease AprA is secreted by a type I secretion (T1SS) [58,59]. The T1SS complex is built by three proteins; a specific outer membrane protein (OMP), an ATP-binding cassette (ABC) and an adapter or membrane fusion protein (MFP) [60]. In P. aeruginosa-associated infections, alkaline protease inhibits the connection between natural killer cells and target cells and plays therefore an important role in pathogenesis [61]. It was also proven that it occurs in other bacterial species such as Dickeya dadantii, Escherichia coli and several Bacillus or fungal species [61,62]. A T1SS gene cluster for alkaline protease was found in the genome of P. wadenswilerensis CCOS 864T (CCOS864_01636–01640), but was not present in P. reidholzensis CCOS 865T. The apr gene cluster in P. wadenswilerensis CCOS 864T consists of five genes (aprDEFAI) as already observed before in other strains [60,63]. Orthologous apr gene clusters were only found in eight further strains within the P. putida group (Figure 2) although it was only partially identified in Pseudomonas donghuensis HYST (Figure 5). The sequence of the region pointed towards an assembly problem within the aprD gene due to the presence of unidentified bases (Ns).

3.5.2. Type II Secretion Systems

Analysis of the pan-genome revealed several type II secretion system gene clusters (T2SS) in numerous strains. Two T2SS clusters were identified in P. wadenswilerensis CCOS 864T, while three were present in P. reidholzensis CCOS 865T. The main function of the T2SS being used by Gram-negative bacteria is the secretion of proteins (lipases, pectinases, phospholipases, cellulases and toxins), which are associated with tissue destruction and leading to cell damage and diseases [64]. At least 12 genes (xcp genes) are encoding for the T2SS and in some species it is known that they are regulated by quorum-sensing signals [64]. The two gene clusters present in P. wadenswilerensis CCOS 864T were identified in a broad range in the P. putida group but are not omnipresent, while the third cluster in P. reidholzensis CCOS 865T only was detected in Pseudomonas sp. KCJK7865 (OTU05) and Pseudomonas sp. W619 (OTU09) (Figure 2). Differences were only observed in the flanking regions of the genes, not within the clusters, indicating a foreign origin of these systems.

3.5.3. Type VI Secretion Systems

Type VI secretion systems (T6SSs) gained a lot of attention because of their potential involvement in pathogenic bacterial-host interactions [65]. Nevertheless, this is rarely the case and T6SSs are more often used to mediate in cooperative and competitive bacterial interactions, or to encourage commensal or mutualistic relationships between bacteria and eukaryotes [65,66,67]. In co-infections with a T6SS-positive P. putida strain and a plant pathogen (Xanthomonas campestris), a reduced plant leaf necrosis was seen [68]. Based on these investigations, the T6SS may also play an important role in plant protection and biocontrol of plant pathogens [66,68,69]. Gram-negative bacteria are able to possess up to five structurally different T6SS clusters in a genome [70]. In our study, we observed a single T6SS in P. reidholzensis CCOS 865T, which was only present in two other members of the P. putida group (Figure 2). This T6SS cluster shares all conserved genes with Pseudomonas sp. W619 (OTU09) and P. putida W15Oct28 and therefore belongs to the phylogenetic group 2 as described before [68]. However, a group of hypothetical proteins, located in the center of the gene cluster, is only shared but not absolutely conserved between P. reidholzensis CCOS 865T and P. putida W15Oct28 (Figure 6A). The T6SS group 2 is related to the T6SS-3 of Erwinia or Pantoea spp. [66,68,69].
Additionally, we discovered a different T6SS in the genome of P. wadenswilerensis CCOS 864T (Figure 2), which was also existent in nine other members of the P. putida group, but was absent in P. reidholzensis CCOS 865T. The T6SS cluster from P. wadenswilerensis CCOS 864T showed structural differences due to insertions of genes encoding hypothetical proteins in the other strains (Figure 6B). However, this T6SS could not be categorized into a phylogenetic group as defined by Barnal et al. because similar clusters only occurred in species not investigated in the previous study [68]. This T6SS cluster thus represents an additional phylogenetic group of T6SS.

4. Discussion

4.1. Potential Ecological Role of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T

The aim of our study was to highlight some of the most important genomic features in the genome of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T. As both species were found in forest soil, we can now, after studying their genome sequences, use the identified features to hypothesize about their potential ecological role. In this study, we found some gene clusters encoding enzymes for the degradation of aromatic compounds such as p-HPA, mandelic acid, gallic acid and accompanying ortho- or meta-cleavage pathways (Figure 3A). We thus assume that P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T may play a role as degraders of organic material from forest litter. The presence of the T1SS in P. wadenswilerensis CCOS 864T, several T6SSs and the HCN biosynthesis gene cluster in both species indicate that P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T are most probably able to competitively act in their environment. Additionally, they could be involved in biocontrol of plant pathogens and therefore in plant protection [50,66,68,69]. Furthermore, their ability for HCN biosynthesis may lead to a better phosphate availability in the soil, which allows for better plant-growth performance [51]. In conclusion, we assume that P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T represent ecologically relevant microorganisms based on their potential for degradation of organic material, nutrient accumulation for plants and protection against plant pathogens.

4.2. Potential Role of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T as a Biocatalyst

Screening for biocatalytic enzymes has gained great importance due to the rapid increase of their usage in industrial applications [71,72]. At the same time, sequencing of genomes has become simpler, cheaper and faster [73], which has made genomics highly valuable in the search for biocatalysts and in the understanding of the whole systems biology [74,75,76]. In this study, we used the large amount of genomic information available for the P. putida group to determine the potential features of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T that could qualify them as potential biocatalysts in biotechnological applications.
The discovery of several aromatic compound degradative pathways can yield novel biocatalysts with alternative substrate spectrum or catalytic specifications. However, as we only identified an ortho-cleavage pathway as required for BTEX compounds, it would be possible the genomes still contain upper pathways, including several mono- and dioxygenases that would act on aromatic compounds [42]. A first inspection of the genomes of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T revealed several enzymes of this type, but it was impossible to assign a clear substrate from the annotation of the genes. The low sequence identity to known systems could thus yield enzymes with different catalytic properties.
The mandelic acid pathway as discovered in the genome of P. reidholzensis CCOS 865T and P. putida NBRC 14164T are highly similar to that of other species [39]. The mandelate racemase from P. putida ATCC 12633 is commonly used in biocatalysis for a broad range of conversions in which stereochemistry is relevant [77]. Here, we have shown that related enzymes are present in specific Pseudomonas strains that can be used to test stereo- and regiospecificity [77,78].
The potential for production of carotenoids by P. reidholzensis CCOS 865T might be of interest for further studies. In particular, zeaxanthin showed promising effects against human cancer cells [79,80]. However, due to the missing phenotypical manifestation [16], the activity of the carotenoid biosynthesis genes for potential biocatalysis must be studied in more details.

5. Conclusions

This study has examined the potential role of P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T as biocatalysts. This revealed many interesting gene clusters encoding for enzymes for potential use in biotechnological applications, such as proteases, isomerases, oxidoreductases and enzymes of the carotenoid biosynthesis pathway. Apart from biotechnological applications, we can assume that P. wadenswilerensis CCOS 864T and P. reidholzensis CCOS 865T are playing a role in the degradation of aromatic compounds commonly found in organic material in nature. Furthermore, we discovered many ecological relevant genomic features and we expect that, based on the presence of several secretion systems (T2SS, T6SS) and the ability to synthesize HCN, both species may be involved in biocontrol against plant pathogens and solubilization of phosphate in the soil, which may improve plant growth. Nevertheless, additional research will be necessary to confirm the expression of these enzyme systems in vivo and determine their activity. Altogether, we can conclude that both species have the potential as an alternative source for novel biocatalysts.

Author Contributions

Conceptualization, D.R. and T.H.M.S.; methodology, D.R., D.F., M.S., F.R. and J.F.P.; software, J.B. and J.F.P.; data analysis, D.R., D.F., M.S., F.R. and T.H.M.S.; data curation, D.R., J.B. and T.H.M.S.; writing—original draft preparation, D.R. and T.H.M.S.; writing—review and editing, D.R., D.F., M.S., J.B., F.R., J.F.P. and T.H.M.S.; visualization, D.R., J.F.P. and T.H.M.S.; supervision, M.S. and T.H.M.S.

Funding

This study was financially supported by a grant of the Department of Life Science and Facility Management of the ZHAW in Wädenswil, Switzerland.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. Core genome phylogeny with 123 Pseudomonas genomes, build out of a core of 471 genes per genome (131′448 amino acid residues per genome) as calculated by EDGAR version 2.2 [19]. Names in the graphic are directly derived from the NCBI database (with accession numbers). Type strains are indicated in bold. The taxonomic speciation in the P. putida group based on ANIb is indicated by the use of different backgrounds.
Figure 1. Core genome phylogeny with 123 Pseudomonas genomes, build out of a core of 471 genes per genome (131′448 amino acid residues per genome) as calculated by EDGAR version 2.2 [19]. Names in the graphic are directly derived from the NCBI database (with accession numbers). Type strains are indicated in bold. The taxonomic speciation in the P. putida group based on ANIb is indicated by the use of different backgrounds.
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Figure 2. Core genome phylogenetic tree of the Pseudomonas putida group genomes analyzed generated using EDGAR [19] based on a total of 1′558 orthologs (573′549 amino acid residues per genome) and presence of shared features to Pseudomonas wadenswilerensis CCOS 864T or Pseudomonas reidholzensis CCOS 865T. Red dot: absence; green dot: presence, yellow dot: partial presence. Species taxonomy is used as given in Table 1. Abbreviations: T1SS apr: type 1 secretion system with apr genes; T2SS: type 2 secretion system; T6SS: type 6 secretion system; p-HPA: gene cluster for degradation of p-hydroxyphenylacetic acid; gal: gene cluster for gallic acid degradation; mdl: gene cluster for mandelate degradation; xyl: lower degradation pathway of benzoate degradation; vdh: vanillate dehydrogenase; crt: carotenoid biosynthesis gene cluster; hcn: cyanide biosynthesis cluster.
Figure 2. Core genome phylogenetic tree of the Pseudomonas putida group genomes analyzed generated using EDGAR [19] based on a total of 1′558 orthologs (573′549 amino acid residues per genome) and presence of shared features to Pseudomonas wadenswilerensis CCOS 864T or Pseudomonas reidholzensis CCOS 865T. Red dot: absence; green dot: presence, yellow dot: partial presence. Species taxonomy is used as given in Table 1. Abbreviations: T1SS apr: type 1 secretion system with apr genes; T2SS: type 2 secretion system; T6SS: type 6 secretion system; p-HPA: gene cluster for degradation of p-hydroxyphenylacetic acid; gal: gene cluster for gallic acid degradation; mdl: gene cluster for mandelate degradation; xyl: lower degradation pathway of benzoate degradation; vdh: vanillate dehydrogenase; crt: carotenoid biosynthesis gene cluster; hcn: cyanide biosynthesis cluster.
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Figure 3. (A) Schematic degradation pathways for aromatic compounds in Pseudomonas wadenswilerensis CCOS 864T and Pseudomonas reidholzensis CCOS 865T. Colors represent the organisms in which they are found: blue: only P. wadenswilerensis CCOS 864T; green: only P. reidholzensis CCOS 865T, black: both species. (B) Gene cluster for p-hydroxyphenylic acid (p-HPA) degradation. Orthologous genes are marked in the same color. The grey shading shows identical gene arrangements within the genomes.
Figure 3. (A) Schematic degradation pathways for aromatic compounds in Pseudomonas wadenswilerensis CCOS 864T and Pseudomonas reidholzensis CCOS 865T. Colors represent the organisms in which they are found: blue: only P. wadenswilerensis CCOS 864T; green: only P. reidholzensis CCOS 865T, black: both species. (B) Gene cluster for p-hydroxyphenylic acid (p-HPA) degradation. Orthologous genes are marked in the same color. The grey shading shows identical gene arrangements within the genomes.
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Figure 4. Secondary metabolites. (A): Gene cluster for hydrogen cyanide (HCN) biosynthesis. Orthologous genes are marked in the same color. Identical gene arrangements within the genomes are shaded in grey. (B): Carotenoid gene cluster for the synthesis of zeaxanthin. The grey shading shows identical arrangements within the genomes, while orthologous genes are marked in the same color. Dashed color indicates weak levels of sequence identity.
Figure 4. Secondary metabolites. (A): Gene cluster for hydrogen cyanide (HCN) biosynthesis. Orthologous genes are marked in the same color. Identical gene arrangements within the genomes are shaded in grey. (B): Carotenoid gene cluster for the synthesis of zeaxanthin. The grey shading shows identical arrangements within the genomes, while orthologous genes are marked in the same color. Dashed color indicates weak levels of sequence identity.
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Figure 5. Type I secretion cluster (T1SS) for the secretion of alkaline protease. The same gene arrangement of the genomes is shaded in grey. Orthologous genes are marked in the same color. Dashed color indicates weak levels of sequence identity.
Figure 5. Type I secretion cluster (T1SS) for the secretion of alkaline protease. The same gene arrangement of the genomes is shaded in grey. Orthologous genes are marked in the same color. Dashed color indicates weak levels of sequence identity.
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Figure 6. Type VI secretion systems. (A): The type VI secretion cluster (T6SS) of Pseudomonas reidholzensis CCOS 865T and related sequences in other strains. Genes aligning to each other are marked in the same color, while the grey background marks identical alignments of the genes within the cluster. (B): The type VI secretion cluster (T6SS) of Pseudomonas wadenswilerensis CCOS 864T and related systems in other Pseudomonas strains. Identical genes are marked in the same color, while the grey shading indicates the same gene arrangement.
Figure 6. Type VI secretion systems. (A): The type VI secretion cluster (T6SS) of Pseudomonas reidholzensis CCOS 865T and related sequences in other strains. Genes aligning to each other are marked in the same color, while the grey background marks identical alignments of the genes within the cluster. (B): The type VI secretion cluster (T6SS) of Pseudomonas wadenswilerensis CCOS 864T and related systems in other Pseudomonas strains. Identical genes are marked in the same color, while the grey shading indicates the same gene arrangement.
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Table
Table 1. List of bacterial strains of the Pseudomonas putida group used for comparative genomics and features of the individual genomes. Strain names labeled with “T” represent the type strains of this species.
Table 1. List of bacterial strains of the Pseudomonas putida group used for comparative genomics and features of the individual genomes. Strain names labeled with “T” represent the type strains of this species.
Strain Name (NCBI)Taxonomic GroupName Used in this StudyAssembly LevelContigs/ScaffoldsGenome Size [bp]GenesG+C [%]GenBank Accession No.
P. alkylphenolica KL28TP. alkylphenolicaP. alkylphenolica KL28TComplete15′764′6225′45460.63CP009048
P. cremoricolorata DSM 17059TP. cremoricolorataP. cremoricolorata DSM 17059TDraft264′655′0824′07263.35AUEA00000000
P. donghuensis P482P. donghuensisP. donghuensis P482Draft695′623′9975′25962.40JHTS00000000
P. donghuensis HYSTP. donghuensisP. donghuensis HYSTDraft645′646′0285′23962.40AJJP01000000
P. donghuensis SVBP6P. donghuensisP. donghuensis SVBP6Draft715′701′3425′35562.40NWCB01000000
P. entomophila L48TP. entomophilaP. entomophila L48TComplete15′888′7805′22364.20NC_008027
P. fulva NBRC 16637T = DSM 17717TP. fulvaP. fulva NBRC 16637 T = DSM 17717TDraft464′768′2294′33161.80BBIQ00000000
P. guariconensis LMG 27394TP. guariconensisP. guariconensis LMG 27394TDraft295′079′0344′70362.20FMYX00000000
P. hunanensis P11P. hunanensisP. hunanensis P11Draft1716′644′4246′46961.20PISL00000000
P. japonica NBRC 103040T = DSM 22348 TP. japonicaP. japonica NBRC 103040T = DSM 22348 TDraft1626′663′1305′84564.20BBIR00000000
P. monteilii MO2OTU06Pseudomonas sp. MO2Draft1′5896′240′6085′94762.00JFBC00000000
P. monteilii SB3078OTU06Pseudomonas sp. SB3078Complete16′000′0875′62062.50CP006978
P. monteilii SB3101OTU06Pseudomonas sp. SB3101Complete15′945′1205′54662.50CP006979
P. monteilii GTC 10897OTU08Pseudomonas sp. GTC 10897Draft1495′547′2825′31360.40BCAO00000000
P. monteilii NBRC 103158 = DSM 14164TP. monteiliiP. monteilii NBRC 103158 = DSM 14164TDraft1326′299′9856′00561.50BBIS00000000
P. monteilii USDA-ARS-USMARC-56711OTU13Pseudomonas sp. USDA-ARS-USMARC-56711Complete14′714′3594′10064.40CP013997
P. mosselii DSM 17497TP. mosseliiP. mosselii DSM 17497TDraft556′260′8445′91664.00JHYW00000000
P. parafulva DSM 17004TP. parafulvaP. parafulva DSM 17004TDraft324′956′6224′53662.50AUEB00000000
P. parafulva PRS09-11288 P. fulvaP. fulva PRS09-11288 Complete14′690′7834′24661.70CP019952
P. parafulva CRS01-1 OTU12Pseudomonas sp. CRS01-1 Complete15′087′6194′45763.50CP009747
P. plecoglossicida KCJK7865 OTU05Pseudomonas sp. KCJK7865 Draft2055′806′3095′60663.00QANO00000000
P. plecoglossicida NyZ12OTU06Pseudomonas sp. NyZ12Complete16′233′2545′84362.40CP010359
P. plecoglossicida NBRC 103162TP. plecoglossicidaP. plecoglossicida NBRC 103162TDraft975′341′7964′94663.00BBIV00000000
P. putida DOT-T1EP. hunanensisPseudomonas sp. DOT-T1E Complete16′260′7025′80361.40CP003734
P. putida KT2440P. hunanensisPseudomonas sp. KT2440 Complete16′181′8735′42062.30AE015451
P. putida GB-1 OTU01Pseudomonas sp. GB-1 Complete16′078′4305′58661.90AAXR01000000
P. putida H8234OTU02Pseudomonas sp. H8234Complete15′956′1106′51261.60NC_021491
P. putida S13.1.2OTU03Pseudomonas sp. S13.1.2Complete16′621′8485′99362.30CP010979
P. putida NBRC 14164TP. putidaP. putida NBRC 14164TComplete16′156′7015′61062.30NC_021505
P. putida W15Oct28 P. putidaP. putida W15Oct28 Draft1196′320′5105′70362.80JENB00000000
P. putida S16 OTU06Pseudomonas sp. S16 Complete15′984′7905′58562.30NC_015733
P. putida IEC33019OTU11Pseudomonas sp. IEC33019Complete15′847′1205′44562.27CP016634
P. putida CSV86OTU14Pseudomonas sp.CSV86Draft2096′469′7805′90663.10AMWJ01000000
P. putida W619 OTU09Pseudomonas sp. W619Complete15′774′3305′37861.40NC_010501
P. reidholzensis CCOS 865TP. reidholzensisP. reidholzensis CCOS 865TDraft456′163′1295′44164.09UIDD01000000
P. soli CCOS 191P. soliP. soli CCOS 191Complete16′012′9475′30164.19LN847264
P. soli LMG 27941TP. soliP. soli LMG 27941TDraft345′644′9105′18864.00FOEQ00000000
Pseudomonas sp. TJI-51OTU04Pseudomonas sp. TJI-51Draft2085′805′0965′50862.10AEWE00000000
Pseudomonas sp. GM84 OTU10Pseudomonas sp. GM84 Draft3845′818′7725′29963.20AKJC00000000
P. taiwanensis DSM 21245TP. taiwanensisP. taiwanensis DSM 21245TDraft675′415′1345′05661.90AUEC00000000
P. taiwanensis SJ9OTU06Pseudomonas sp. SJ9Draft7366′253′0556′07561.80AXUP00000000
P. vranovensis DSM 16006TP. vranovensisP. vranovensis DSM 16006TDraft365′697′8075′29561.50AUED00000000
P. wadenswilerensis CCOS 864TP. wadenswilerensisP. wadenswilerensis CCOS 864TDraft185′966′9425′43762.39UNOZ01000000

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MDPI and ACS Style

Rutz, D.; Frasson, D.; Sievers, M.; Blom, J.; Rezzonico, F.; Pothier, J.F.; Smits, T.H.M. Comparative Genomic Analysis of the Biotechnological Potential of the Novel Species Pseudomonas wadenswilerensis CCOS 864T and Pseudomonas reidholzensis CCOS 865T. Diversity 2019, 11, 204. https://doi.org/10.3390/d11110204

AMA Style

Rutz D, Frasson D, Sievers M, Blom J, Rezzonico F, Pothier JF, Smits THM. Comparative Genomic Analysis of the Biotechnological Potential of the Novel Species Pseudomonas wadenswilerensis CCOS 864T and Pseudomonas reidholzensis CCOS 865T. Diversity. 2019; 11(11):204. https://doi.org/10.3390/d11110204

Chicago/Turabian Style

Rutz, Dominik, David Frasson, Martin Sievers, Jochen Blom, Fabio Rezzonico, Joël F. Pothier, and Theo H. M. Smits. 2019. "Comparative Genomic Analysis of the Biotechnological Potential of the Novel Species Pseudomonas wadenswilerensis CCOS 864T and Pseudomonas reidholzensis CCOS 865T" Diversity 11, no. 11: 204. https://doi.org/10.3390/d11110204

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