Functional Genome Analysis of a Conditionally Pathogenic Rhizobacterial Strain, Pseudomonas putida AKMP7.

This manuscript reports the whole genome sequence of a conditionally pathogenic rhizobacterial strain, Pseudomonas putida AKMP7, which has been previously reported by us to be beneficial to Arabidopsis thaliana under well-watered conditions and pathogenic to the plant under water stress. As part of a study to understand this unique behavior, the whole genome sequence of this strain was analyzed. Based on the results, it was identified that the total length of the AKMP7 genome is 5,764,016 base pairs, and the total GC content of the genome is 62.93% (typical of P. putida). Using RAST annotation pipeline, it was identified that the genome has 5605 coding sequences, 80 repeat regions, 71 tRNA genes, and 22 rRNA genes. A total of 4487 functional proteins and 1118 hypothetical proteins were identified. Phylogenetic analysis has classified it as P. putida species, with a P value of 0.03. In order to identify close relatives of this strain, comparative genomics was performed with 30 other P. putida strains, taken from publicly available genome databases, using Average Nucleotide Identity (ANI) analysis. Whole genome comparison with these strains reveals that AKMP7 possesses Type-IV Secretion System (T4SS) with conjugative transfer functionality. Interestingly, the T4SS feature is absent in all the beneficial/harmless strains of P. putida that we analyzed. All the plant pathogenic bacteria that were analyzed had the T4SS feature in their genome, indicating its role in pathogenesis. This study aims to address important gaps in understanding the molecular mechanisms involved in the conditional/opportunistic pathogenesis of plant-associated, beneficial soil bacteria, using genomics approaches.

Genomic Reclassification and Phenotypic Characterization of Pseudomonas putida Strains Deposited in Japanese Culture Collections.

Pseudomonas putida is a major species belonging to the genus Pseudomonas. Although several hundred strains of P. putida have been deposited in culture collections, they potentially differ from the genetically defined "true Pseudomonas putida" because many were classified as P. putida based on their phenotypic and metabolic characteristics. A phylogenetic ana-lysis based on the concatenated sequences of the 16S rRNA and rpoD genes revealed that 46 strains of P. putida deposited in Japanese culture collections were classified into nine operational taxonomic units (OTUs) and eleven singletons. The OTU7 strain produces N-acylhomoserine lactone as a quorum-sensing signal. One of the OTU7 strains, JCM 20066, exhibited a ppuI-rsaL-ppuR quorum-sensing system that controls biofilm formation and motility. The P. putida type strain JCM 13063T and six other strains were classified as OTU4. Classification based on the calculation of whole-genome similarity revealed that three OTU4 strains, JCM 20005, 21368, and 13061, were regarded as the same species as JCM 13063T and defined as true P. putida. When orthologous genes in the whole-genome sequences of true P. putida strains were screened, PP4_28660 from P. putida NBRC 14164T (=JCM 13063T) was present in all true P. putida genome sequences. The internal region of PP4_28660 was successfully amplified from all true P. putida strains using the specific primers designed in this study.

Re-identification of strains deposited as Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas putida in GenBank based on whole genome sequences.

The taxonomic classification of Pseudomonas species has been revised and updated several times. This study utilized average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) cutoff values of 95 and 70 %, respectively, to re-identify the species of strains deposited in GenBank as P. aeruginosa, P. fluorescens and P. putida. Of the 264 deposited P. aeruginosa strains, 259 were correctly identified as P. aeruginosa, but the remaining five were not. All 28 deposited P. fluorescens strains had been incorrectly identified as P. fluorescens. Four of these strains were re-identified, including two as P. kilonensis and one each as P. aeruginosa and P. brassicacearum, but the remaining 24 could not be re-identified. Similarly, all 35 deposited P. putida strains had been incorrectly identified as P. putida. Nineteen of these strains were re-identified, including 12 as P. alloputida, four as P. asiatica and one each as P. juntendi, P. monteilii and P. mosselii. These results strongly suggest that Pseudomonas bacteria should be identified using ANI and dDDH analyses based on whole genome sequencing when Pseudomonas species are initially deposited in GenBank/DDBJ/EMBL databases.

A new type strain of Pseudomonas putida NM-CH-I15-I isolated from a nickel-contaminated soil in southwest Slovakia harbouring the czcA-NM15I resistance determinant.

A soil bacterium MR-CH-I15-I was on the base of phylogenetic analysis of almost the whole (1,492 bp) 16S rRNA sequence and sequences of selected 9 marker genes identified as Pseudomonas putida strain NM-CH-I15-I. The bacterium exhibited typical morphological features and biochemical properties for this species, the highest resistance to nickel and copper and multidrug resistance to different antibiotic groups. In addition, the whole czcA-NM15I heavy-metal resistance gene sequence (3,126 bp, 1,042 amino acids, MW 112, 138 Da) was obtained and on the base of phylogenetic analysis was assigned to CzcA protein from Pseudomonas reidholzensis with 93% similarity. This gene was significantly induced mainly by the addition of zinc, cadmium and cobalt and in a lesser extent of nickel. Furthermore, an increased expression of the CzcA-NM15I protein was confirmed by immunoblot analysis after heterologous expression of the czcA-NM15I synthetic variant gene in E. coli BL21 (DE3). Finally, the location of amino acids (R83, R673, D402, D408, D619, E415, E568) in the homology model of the CzcA-NM15I protein suggested that these amino acids may play an important role in the transport of cations such as cobalt, zinc or cadmium. This soil bacterium can represent a new type strain of P. putida NM-CH-I15-I.

Biodegradation of 3-chlorobenzoic acid with electron shuttle systems: pathways and molecular identification.

A synergy of biodegradation and electron shuttle systems is a promising strategy for eliminating pollutants including chlorinated aromatic compounds. The present work studies the degradation products of 3-chlorobenzoic acid by Pseudomonas putida in the presence of an electron shuttle system (ESS) composed of citrate and pyruvate as electron donors and the pollutant as an electron acceptor. Chromatographic results showed different pathways involved in the biodegradation process under the influence of electron shuttle systems. These routes depend on oxidation and reduction reactions for output byproducts to be easily mineralized by the bacterium under investigation. A nucleotide sequence with about 380 bp of a ton B gene was detected in P. putida and it resembles Escherichia coli Ton B. The relatedness tree of the selected gene reveals a high similarity and is comparable to P. aeruginosa (100%) and the highest variation with that of P. citronellolis (21.99%). Accordingly, in the presence of electron shuttle systems, the genes responsible for bacterial influx were activated to ease the biodegradation process. In an application model, the remediated-water samples were handled by two recycling processes using Scenedesmus obliquus and Trigonella foenum-graecum to evaluate the efficiency of this non-conventional treatment. In conclusion, this strategy succeeded in remediating the polluted water with chlorinated aromatic compounds for further applications.

Chromosomal toxin-antitoxin systems in Pseudomonas putida are rather selfish than beneficial.

Chromosomal toxin-antitoxin (TA) systems are widespread genetic elements among bacteria, yet, despite extensive studies in the last decade, their biological importance remains ambivalent. The ability of TA-encoded toxins to affect stress tolerance when overexpressed supports the hypothesis of TA systems being associated with stress adaptation. However, the deletion of TA genes has usually no effects on stress tolerance, supporting the selfish elements hypothesis. Here, we aimed to evaluate the cost and benefits of chromosomal TA systems to Pseudomonas putida. We show that multiple TA systems do not confer fitness benefits to this bacterium as deletion of 13 TA loci does not influence stress tolerance, persistence or biofilm formation. Our results instead show that TA loci are costly and decrease the competitive fitness of P. putida. Still, the cost of multiple TA systems is low and detectable in certain conditions only. Construction of antitoxin deletion strains showed that only five TA systems code for toxic proteins, while other TA loci have evolved towards reduced toxicity and encode non-toxic or moderately potent proteins. Analysis of P. putida TA systems' homologs among fully sequenced Pseudomonads suggests that the TA loci have been subjected to purifying selection and that TA systems spread among bacteria by horizontal gene transfer.

Biodegradation of endocrine disruptor Bisphenol A by Pseudomonas putida strain YC-AE1 isolated from polluted soil, Guangdong, China.

BACKGROUND: Bisphenol A is an important organic chemical as an intermediate, final and inert ingredient in manufacturing of many important products like polycarbonate plastics, epoxy resins, flame retardants, food-drink packaging coating, and other. BPA is an endocrine disruptor compound that mimics the function of estrogen causing damage to reproductive organs. Bacterial degradation has been consider as a cost effective and eco-friendly method for BPA degradation compared with physical and chemical methods. This study aimed to isolate and identify bacterial strain capable to degrade and tolerate high concentrations of this pollutant, studying the factors affecting the degradation process and study the degradation mechanism of this strain. RESULTS: YC-AE1 is a Gram negative bacterial strain isolated from soil and identified as Pseudomonas putida by 16S rRNA gene sequence and BIOLOG identification system. This strain found to have a high capacity to degrade the endocrine disruptor Bisphenol A (BPA). Response surface methodology using central composite design was used to statistically optimize the environmental factors during BPA degradation and the results obtained by significant model were 7.2, 30 °C and 2.5% for optimum initial pH, temperature and inoculum size, respectively. Prolonged incubation period with low NaCl concentration improve the biodegradation of BPA. Analysis of variance (ANOVA) showed high coefficient of determination, R2 and Adj-R2 which were 0.9979 and 0.9935, respectively. Substrate analysis found that, strain YC-AE1 could degrade a wide variety of bisphenol A-related pollutants such as bisphenol B, bisphenol F, bisphenol S, Dibutyl phthalate, Diethylhexyl phthalate and Diethyl phthalate in varying proportion. Pseudomonas putida YC-AE1 showed high ability to degrade a wide range of BPA concentrations (0.5-1000 mg l- 1) with completely degradation for 500 mg l- 1 within 72 h. Metabolic intermediates detected in this study by HPLC-MS were identified as 4,4-dihydroxy-alpha-methylstilbene, p-hydroxybenzaldeyde, p-hydroxyacetophenone, 4-hydroxyphenylacetate, 4-hydroxyphenacyl alcohol, 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl) propanoate. CONCLUSIONS: This study reports Pseudomonas putida YC-AE1 as BPA biodegrader with high performance in degradation and tolerance to high BPA concentration. It exhibited strong degradation capacity and prominent adaptability towards a wide range of environmental conditions. Moreover, it degrades BPA in a short time via two different degradation pathways.

Original sequence divergence among Pseudomonas putida CadRs drive specificity.

Bacteria, especially those living in soils, are in constant contact with metals. Transition metals like Fe or Zn, are required for proper growth. Some other metals like Cd or Hg are only toxic. Several systems exist to detoxify cells when these metals are present in concentrations harmful to biological systems. The expression of these systems is under control of specialized regulatory proteins able to detect metals and to regulate cognate detoxifying systems. In this work we report on the characterisation of the metallo-regulator CadR from Pseudomonas putida KT2440. By using gene reporter assays, we investigated the repertoire of metals detected by CadR. We show that CadR is much more responsive to Hg than to Cd, as compared to CadR from P. putida 06909. CadR from P. putida KT2440 differs in only 3 amino-acids in its metal-binding domain with respect to CadR from P. putida 06909. We show that these residues are important determinants of metal selectivity by engineering a modified CadR.

Genomic and metabolic differences between Pseudomonas putida populations inhabiting sugarcane rhizosphere or bulk soil.

Pseudomonas putida is one of 13 major groups of Pseudomonas spp. and contains numerous species occupying diverse niches and performing many functions such as plant growth promotion and bioremediation. Here we compared a set of 19 P. putida isolates obtained from sugarcane rhizosphere or bulk soil using a population genomics approach aiming to assess genomic and metabolic differences between populations from these habitats. Phylogenomics placed rhizosphere versus bulk soil strains in separate clades clustering with different type strains of the P. putida group. Multivariate analyses indicated that the rhizosphere and bulk soil isolates form distinct populations. Comparative genomics identified several genetic functions (GO-terms) significantly different between populations, including some exclusively present in the rhizosphere or bulk soil strains, such as D-galactonic acid catabolism and cellulose biosynthesis, respectively. The metabolic profiles of rhizosphere and bulk soil populations analyzed by Biolog Ecoplates also differ significantly, most notably by the higher oxidation of D-galactonic/D-galacturonic acid by the rhizosphere population. Accordingly, D-galactonate catabolism operon (dgo) was present in all rhizosphere isolates and absent in the bulk soil population. This study showed that sugarcane rhizosphere and bulk soil harbor different populations of P. putida and identified genes and functions potentially associated with their soil niches.

Pseudomonas hutmensis sp. nov., a New Fluorescent Member of Pseudomonas putida Group.

A rod-shaped, Gram-negative and aerobic bacterium, strain XWS2, was isolated from rhizosphere soil of a camphor tree in Hubei University of Chinese Medicine Huangjiahu Campus, Wuhan, China. Cells grew at 4-37 °C (optimum 28 °C), pH 5.0-9.0 (optimum 7.0) and with 0-5% NaCl (optimum 1%). Colonies growing on tryptone soybean agar are round, beige in color and approximately 2 mm in diameter after 24 h incubation at 28 °C. Pellicle formation during liquid culture and strong fluorescent pigment production on King's B medium are typical features of strain XWS2. The genome of XWS2 is 6,170,117 bp, containing 5682 predicted genes and 4770 genes are functionally annotated. Phylogenetic analysis based on 16S rRNA sequence showed that strain XWS2 formed an independent branch within the Pseudomonas putida group, with P. putida NBRC 14164T (99.86% similarity) and P. alkylphenolica KL28T (99.36% similarity) as the most closely related type strains. Genome sequence analysis based on average nucleotide identity and digital DNA-DNA hybridization are below the threshold values for species delineation. Phenotypic characteristics, physiological and biochemical tests also supported the strain represents a separate novel species within the Pseudomonas genus. The name Pseudomonas hutmensis sp. nov. is proposed, with type strain XWS2T (= CCTCC AB 2018189 T = KACC 19898T).

Genomic, phylogenetic and catabolic re-assessment of the Pseudomonas putida clade supports the delineation of Pseudomonas alloputida sp. nov., Pseudomonas inefficax sp. nov., Pseudomonas persica sp. nov., and Pseudomonas shirazica sp. nov.

Bacteria of the Pseudomonas putida group are studied for a large panel of properties ranging from plant growth promotion and bioremediation to pathogenicity. To date, most of the classification of individual pseudomonads from this group relies on 16S RNA gene analysis, which is insufficient for accurate taxonomic characterization within bacterial species complexes of the Pseudomonas putida group. Here, a collection of 20 of these bacteria, isolated from various soils, was assessed via multi-locus sequence analysis of rpoD, gyrB and rrs genes. The 20 strains clustered in 7 different clades of the P. putida group. One strain per cluster was sequenced and results were compared to complete genome sequences of type strains of the P. putida group. Phylogenetic analyses, average nucleotide identity data and digital DNA hybridizations, combined to phenotypic characteristics, resulted in the proposition and description of four new species i.e. Pseudomonas alloputida Kh7 T (= LMG 29756 T = CFBP 8484 T) sp. nov., Pseudomonas inefficax JV551A3 T (= DSM108619 T = CFBP 8493 T) sp. nov., Pseudomonas persica RUB6 T (= LMG 29757 T = CFBP 8486 T) sp. nov. and Pseudomonas shirazica VM14 T (= LMG 29953 T = CFBP 8487 T) sp. nov.

Detection of the carbapenemase gene blaVIM-5 in members of the Pseudomonas putida group isolated from polluted Nigerian wetlands.

There are increasing concerns about possible dissemination of clinically relevant antibiotic resistance genes, including genes encoding for carbapenemases in the environment. However, little is known about environmental distribution of antibiotic resistance in Africa. In this study, four polluted urban wetlands in Nigeria were investigated as potential reservoirs of carbapenem-resistant bacteria (CRB). CRB were isolated from the wetlands, characterized by Blue-Carba test, MIC determinations and whole genome sequencing (WGS). Nine of 65 bacterial isolates identified as members of the Pseudomonas putida group (P. plecoglossicida and P. guariconensis, respectively) harboured the metallo-beta-lactamase gene blaVIM-5. WGS revealed the blaVIM-5 in three novel Tn402-like class 1 integron structures containing the cassette arrays aadB|blaVIM-5|blaPSE-1, aadB|blaVIM-5|aadB|blaPSE-1, and blaVIM-5|aadB|tnpA|blaPSE-1|smr2|tnpA, respectively. Strains carrying the aadB|blaVIM-5|blaPSE-1 cassette also carried an identical integron without blaVIM-5. In addition, the strains harboured another Tn402-like class 1 integron carrying bcr2, several multidrug resistance efflux pumps, and at least one of ampC, aph(3")-lb, aph(6)-ld, tetB, tetC, tetG, floR, and macAB. This is the first report of a carbapenemase gene in bacteria from environmental sources in Nigeria and the first report of blaVIM-5 in environmental bacteria isolates. This result underscores the role of the Nigerian environment as reservoir of bacteria carrying clinically relevant antibiotic resistance genes.

Pushing the limits of de novo genome assembly for complex prokaryotic genomes harboring very long, near identical repeats.

Generating a complete, de novo genome assembly for prokaryotes is often considered a solved problem. However, we here show that Pseudomonas koreensis P19E3 harbors multiple, near identical repeat pairs up to 70 kilobase pairs in length, which contained several genes that may confer fitness advantages to the strain. Its complex genome, which also included a variable shufflon region, could not be de novo assembled with long reads produced by Pacific Biosciences' technology, but required very long reads from Oxford Nanopore Technologies. Importantly, a repeat analysis, whose results we release for over 9600 prokaryotes, indicated that very complex bacterial genomes represent a general phenomenon beyond Pseudomonas. Roughly 10% of 9331 complete bacterial and a handful of 293 complete archaeal genomes represented this 'dark matter' for de novo genome assembly of prokaryotes. Several of these 'dark matter' genome assemblies contained repeats far beyond the resolution of the sequencing technology employed and likely contain errors, other genomes were closed employing labor-intense steps like cosmid libraries, primer walking or optical mapping. Using very long sequencing reads in combination with assembly algorithms capable of resolving long, near identical repeats will bring most prokaryotic genomes within reach of fast and complete de novo genome assembly.

Simultaneous biodegradation of phenol and cyanide present in coke-oven effluent using immobilized Pseudomonas putida and Pseudomonas stutzeri

ABSTRACT Discharge of coke-oven wastewater to the environment may cause severe contamination to it and also threaten the flora and fauna, including human beings. Hence before dumping it is necessary to treat this dangerous effluent in order to minimize the damage to the environment. Conventional technologies have inherent drawbacks however, biological treatment is an advantageous alternative method. In the present study, bacteria were isolated from the soil collected from the sites contaminated by coke-oven effluent rich in phenol and cyanide. Nucleotides sequence alignment and phylogenetic analysis showed the identity of the selected phenol and cyanide degrading isolates NAUN-16 and NAUN-1B as Pseudomonas putida and Pseudomonas stutzeri, respectively. These two isolates tolerated phenol up to 1800 mg L-1 and cyanide up to 340 mg L-1 concentrations. The isolates were immobilized on activated charcoal, saw dust and fly ash. The effluent was passed through the column packed with immobilized cells with a flow rate of 5 mL min-1. The isolates showed degradation of phenol up to 80.5% and cyanide up to 80.6% and also had the ability to reduce biological oxygen demand, chemical oxygen demand and lower the pH of effluent from alkaline to near neutral. The study suggests the utilization of such potential bacterial strains in treating industrial effluent containing phenol and cyanide, before being thrown in any ecosystem.

Simultaneous biodegradation of phenol and cyanide present in coke-oven effluent using immobilized Pseudomonas putida and Pseudomonas stutzeri.

Discharge of coke-oven wastewater to the environment may cause severe contamination to it and also threaten the flora and fauna, including human beings. Hence before dumping it is necessary to treat this dangerous effluent in order to minimize the damage to the environment. Conventional technologies have inherent drawbacks however, biological treatment is an advantageous alternative method. In the present study, bacteria were isolated from the soil collected from the sites contaminated by coke-oven effluent rich in phenol and cyanide. Nucleotides sequence alignment and phylogenetic analysis showed the identity of the selected phenol and cyanide degrading isolates NAUN-16 and NAUN-1B as Pseudomonas putida and Pseudomonas stutzeri, respectively. These two isolates tolerated phenol up to 1800mgL-1 and cyanide up to 340mgL-1 concentrations. The isolates were immobilized on activated charcoal, saw dust and fly ash. The effluent was passed through the column packed with immobilized cells with a flow rate of 5mLmin-1. The isolates showed degradation of phenol up to 80.5% and cyanide up to 80.6% and also had the ability to reduce biological oxygen demand, chemical oxygen demand and lower the pH of effluent from alkaline to near neutral. The study suggests the utilization of such potential bacterial strains in treating industrial effluent containing phenol and cyanide, before being thrown in any ecosystem.

Tropical soils are a reservoir for fluorescent Pseudomonas spp. biodiversity.

Fluorescent Pseudomonas spp. are widely studied for their beneficial activities to plants. To explore the genetic diversity of Pseudomonas spp. in tropical regions, we collected 76 isolates from a Brazilian soil. Genomes were sequenced and compared to known strains, mostly collected from temperate regions. Phylogenetic analyses classified the isolates in the P. fluorescens (57) and P. putida (19) groups. Among the isolates in the P. fluorescens group, most (37) were classified in the P. koreensis subgroup and two in the P. jessenii subgroup. The remaining 18 isolates fell into two phylogenetic subclades distinct from currently recognized P. fluorescens subgroups, and probably represent new subgroups. Consistent with their phylogenetic distance from described subgroups, the genome sequences of strains in these subclades are asyntenous to the genome sequences of members of their neighbour subgroups. The tropical isolates have several functional genes also present in known fluorescent Pseudomonas spp. strains. However, members of the new subclades share exclusive genes not detected in other subgroups, pointing to the potential for novel functions. Additionally, we identified 12 potential new species among the 76 isolates from the tropical soil. The unexplored diversity found in the tropical soil is possibly related to biogeographical patterns.

Micropredator niche differentiation between bulk soil and rhizosphere of an agricultural soil depends on bacterial prey.

Predation is a fundamental mechanism of all food webs, but its drivers and organismic connectivities, especially at microbial level, are still poorly understood. Specifically, competitive carbon flows in the presence of multiple micropredators, as well as trophic links within and between microbial kingdoms have rarely been resolved. Here, using maize-planted agricultural soil as a model system, we have investigated the predation of amended bacterial prey by both prokaryotic and eukaryotic micropredators. We have queried how soil compartment (rhizosphere vs bulk soil) and nature of prey (Gram-positive vs Gram-negative) influence predation outcomes. We added 13C-labelled biomass of Pseudomonas putida and Arthrobacter globiformis to soil microcosms and found that P. putida was consumed much more rapidly. Bacteria and microeukaryotes specifically responsive to the biomass amendments were identified by RNA-stable isotope probing. Amongst the bacteria, only a few myxobacteria sequestered C from A. globiformis, whereas a considerable diversity of predatory bacteria incorporated C derived from P. putida. Diverse groups of heterotrophic protists, especially amoeba including Glaeseria, Hartmanella and Vahlkampfia spp., were observed to incorporate 13C from both amendments, but with pronounced niche differentiation between rhizosphere and bulk soil. This provides novel insights into niche partitioning between bacterial and eukaryotic micropredators in soil, driven not only by the nature of bacterial prey itself, but also by soil compartments.

Interspecies cross-talk between co-cultured Pseudomonas putida and Escherichia coli.

Pseudomonas putida and Escherichia coli are ubiquitous microorganisms that can be isolated from soil rhizosphere, the surface of vegetables, fresh waters and wastewaters - environments in which they likely co-exist. Despite this, the potential interactions between these microbes have not been studied in detail. To analyse these interactions, we carried out RNA-seq transcriptomic analysis of these microbes as monocultures and as co-cultures. Our results show that co-culture of these microbes significantly alters transcriptional profiles. The most dramatic transcriptional changes in both microorganisms were involved in central carbon metabolism, as well as adhesion to surfaces and the activation of drug efflux pumps. We also found that acetate production was one of the mechanisms used by E. coli K-12 MG1655 in response to the presence of P. putida DOT-T1E.

Phylogenetic analysis reveals the taxonomically diverse distribution of the Pseudomonas putida group.

Pseudomonas putida is well-known for degradation activities for a variety of compounds and its infections have been reported. Thus, P. putida includes both clinical and nonclinical isolates. To date, no reports have examined the phylogenetic relationship between clinical and nonclinical isolates of the P. putida group. In this study, fifty-nine strains of P. putida group containing twenty-six clinical, and thirty-three nonclinical, isolates, were subjected to phylogenetic and taxonomic analyses based on 16S rRNA gene sequences and nine housekeeping gene sequences, including argS, dnaN, dnaQ, era, gltA, gyrB, ppnK, rpoB, and rpoD, to obtain insights into the diversity of species in this group. More than 97.6% similarity was observed among the 16S rRNA gene sequences of all the strains examined, indicating that the resolution of 16S rRNA gene sequences is inadequate. Phylogenetic analysis based on the individual housekeeping genes listed above improved the resolution of the phylogenetic trees, which are different from each other. Multilocus sequence analysis (MLSA) based on the concatenated sequences of the nine genes significantly improved the resolution of the phylogenetic tree, and yielded approximately the same results as average nucleotide identity (ANI) analysis, suggesting its high reliability. ANI analysis classified the fifty-nine strains into twenty-six species containing seventeen singletons and nine strain clusters based on the 95% threshold. It also indicated the mixed distribution of clinical and nonclinical isolates in the six clusters, suggesting that the genomic difference between clinical and nonclinical isolates of the P. putida group is subtle. The P. putida type strain NBRC 14164T is a singleton that is independently located from the P. putida strains distributed among the six clusters, suggesting that the classification of these strains and the differentiation of species in the P. putida group should be re-examined. This study greatly expands insights into the phylogenetic diversity of the P. putida group.

Specific Gene Loci of Clinical Pseudomonas putida Isolates.

Pseudomonas putida are ubiquitous inhabitants of soils and clinical isolates of this species have been seldom described. Clinical isolates show significant variability in their ability to cause damage to hosts because some of them are able to modulate the host's immune response. In the current study, comparisons between the genomes of different clinical and environmental strains of P. putida were done to identify genetic clusters shared by clinical isolates that are not present in environmental isolates. We show that in clinical strains specific genes are mostly present on transposons, and that this set of genes exhibit high identity with genes found in pathogens and opportunistic pathogens. The set of genes prevalent in P. putida clinical isolates, and absent in environmental isolates, are related with survival under oxidative stress conditions, resistance against biocides, amino acid metabolism and toxin/antitoxin (TA) systems. This set of functions have influence in colonization and survival within human tissues, since they avoid host immune response or enhance stress resistance. An in depth bioinformatic analysis was also carried out to identify genetic clusters that are exclusive to each of the clinical isolates and that correlate with phenotypical differences between them, a secretion system type III-like was found in one of these clinical strains, a determinant of pathogenicity in Gram-negative bacteria.