Gene Expansion and Positive Selection as Bacterial Adaptations to Oligotrophic Conditions.

We examined the genomic adaptations of prevalent bacterial taxa in a highly nutrient- and ion-depleted freshwater environment located in the secondary cooling water system of a nuclear research reactor. Using genome-centric metagenomics, we found that none of the prevalent bacterial taxa were related to typical freshwater bacterial lineages. We also did not identify strong signatures of genome streamlining, which has been shown to be one of the ecoevolutionary forces shaping the genome characteristics of bacterial taxa in nutrient-depleted environments. Instead, focusing on the dominant taxon, a novel Ramlibacter sp. which we propose to name Ramlibacter aquaticus, we detected extensive positive selection on genes involved in phosphorus and carbon scavenging pathways. These genes were involved in the high-affinity phosphate uptake and storage into polyphosphate granules, metabolism of nitrogen-rich organic matter, and carbon/energy storage into polyhydroxyalkanoate. In parallel, comparative genomics revealed a high number of paralogs and an accessory genome significantly enriched in environmental sensing pathways (i.e., chemotaxis and motility), suggesting extensive gene expansions in R. aquaticus The type strain of R. aquaticus (LMG 30558T) displayed optimal growth kinetics and productivity at low nutrient concentrations, as well as substantial cell size plasticity. Our findings with R. aquaticus LMG 30558T demonstrate that positive selection and gene expansions may represent successful adaptive strategies to oligotrophic environments that preserve high growth rates and cellular productivity.IMPORTANCE By combining a genome-centric metagenomic approach with a culture-based approach, we investigated the genomic adaptations of prevalent populations in an engineered oligotrophic freshwater system. We found evidence for widespread positive selection on genes involved in phosphorus and carbon scavenging pathways and for gene expansions in motility and environmental sensing to be important genomic adaptations of the abundant taxon in this system. In addition, microscopic and flow cytometric analysis of the first freshwater representative of this population (Ramlibacter aquaticus LMG 30558T) demonstrated phenotypic plasticity, possibly due to the metabolic versatility granted by its larger genome, to be a strategy to cope with nutrient limitation. Our study clearly demonstrates the need for the use of a broad set of genomic tools combined with culture-based physiological characterization assays to investigate and validate genomic adaptations.

Isolation and characterization of soil bacteria able to rapidly degrade the organophosphorus nematicide fosthiazate.

Foshtiazate is an organophosphorus nematicide commonly used in protected crops and potato plantations. It is toxic to mammals, birds and honeybees, it is persistent in certain soils and can be transported to water resources. Recent studies by our group demonstrated, for the first time, the development of enhanced biodegradation of fosthiazate in agricultural soils. However, the micro-organisms driving this process are still unknown. We aimed to isolate soil bacteria responsible for the enhanced biodegradation of fosthiazate and assess their degradation potential against high concentrations of the nematicide. Enrichment cultures led to the isolation of two bacterial cultures actively degrading fosthiazate. Denaturating Gradient Gel Electrophoresis analysis revealed that they were composed of a single phylotype, identified via 16S rRNA cloning and phylogenetic analysis as Variovorax boronicumulans. This strain showed high degradation potential against fosthiazate. It degraded up to 100 mg l-1 in liquid cultures (DT50  = 11·2 days), whereas its degrading capacity was reduced at higher concentration levels (500 mg l-1 , DT50  = 20 days). This is the first report for the isolation of a fosthiazate-degrading bacterium, which showed high potential for use in future biodepuration and bioremediation applications. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reported for the first time the isolation and molecular identification of bacteria able to rapidly degrade the organophosphorus nematicide fosthiazate; one of the few synthetic nematicides still available on the global market. Further tests demonstrated the high capacity of the isolated strain to degrade high concentrations of fosthiazate suggesting its high potential for future bioremediation applications in contaminated environmental sites, considering high acute toxicity and high persistence and mobility of fosthiazate in acidic and low in organic matter content soils.

Polaromonas ginsengisoli sp. nov., isolated from ginseng field soil.

A Gram-reaction-negative, strictly aerobic, milky-white and rod-shaped bacterium (designated Gsoil 115T) isolated from ginseng field soil was characterized by a polyphasic approach to clarify its taxonomic position. Strain Gsoil 115T grew optimally at 30 °C and at pH 7.0 on Reasoner's 2A agar medium. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Gsoil 115T belongs to the genus Polaromonas and was most closely related to Polaromonaseurypsychrophila B717-2T (98.6 %), Polaromonasvacuolata 34-PT (98.3 %), Polaromonasjejuensis NBRC 106434T (98.1 %), Polaromonas aquatic CCUG 39402T (97.7 %) and Polaromonascryoconiti Cr4-35T (97.5 %). The DNA G+C content was 60.9 mol%. The DNA-DNA hybridization relatedness between strain Gsoil 115T and P. eurypsychrophila B717-2T, P. vacuolata 34-PT, P. jejuensis NBRC 106434T, P. aquatic CCUG 39402T and P. cryoconiti Cr4-35T were 31.2, 21.6, 16.9, 8.7 and 10.1 %, respectively. The major polar lipids were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE). The sole respiratory quinone was Q-8. The major fatty acids were C16 : 0 and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), which supported the affiliation of strain Gsoil 115T to the genus Polaromonas. Moreover, the physiological, biochemical and low level of DNA-DNA relatedness value allowed the phenotypic and genotypic differentiation of strain Gsoil 115T from the recognized species of the genus Polaromonas. Therefore, strain Gsoil 115T represents a novel species of the genus Polaromonas, for which the name Polaromonas ginsengisoli sp. nov. is proposed, with the type strain Gsoil 115T (LMG 23393T=KCTC 12577T).

Ramlibacter alkalitolerans sp. nov., alkali-tolerant bacterium isolated from soil of ginseng.

A novel bacterial strain, designated CJ661T, was isolated from soil of ginseng in Anseong, South Korea. Cells of strain CJ661T were white-coloured, Gram-staining-negative, non-motile, aerobic and rod-shaped. Strain CJ661T grew optimally at 30 °C and pH 7.0. The analysis of 16S rRNA gene sequence of strain CJ661T showed that it belongs to the genus Ramlibacter within the family Comamonadaceae and was most closely related to Ramlibacter ginsenosidimutans KCTC 22276T (98.1 %), followed by Ramlibacter henchirensis DSM 14656T (97.1 %). DNA-DNA relatedness levels of strain CJ661T were 40.6 % to R. ginsenosidimutans KCTC 22276T and 25.0 % to R. henchirensis DSM 14656T. The major isoprenoid quinone was ubiquinone (Q-8). The predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The major cellular fatty acids of strain CJ661T were summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c), C16 : 0 and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The G+C content of the genomic DNA was 65.4 mol%. On the basis polyphasic taxonomic data, strain CJ661T represents a novel species in the genus Ramlibacter, for which name Ramlibacter alkalitolerans sp. nov. is proposed; the type strain is CJ661T (=KACC 19305T=JCM 32081T).

Investigation of catechol 2,3-dioxygenase and 16S rRNA gene diversity in hypoxic, petroleum hydrocarbon contaminated groundwater.

Detection of catechol 2,3-dioxygenase genes in aromatic hydrocarbon contaminated environments gives the opportunity to measure the diversity of bacteria involved in the degradation of the contaminants under aerobic conditions. In this study, we investigated the diversity and distribution of Comamonadaceae family (Betaproteobacteria) related catechol 2,3-dioxygenase genes, which belong to the I.2.C subfamily of extradiol dioxygenase genes. These catabolic genes encode enzymes supposed to function under hypoxic conditions as well, and may play a notable role in BTEX degradation in oxygen limited environments. Therefore, their diversity was analyzed in oxygen limited, petroleum hydrocarbon contaminated groundwater by terminal restriction fragment length polymorphism and cloning. Subfamily I.2.C related catechol 2,3-dioxygenase genes were detected in every investigated groundwater sample and a dynamic change was observed in the case of the structure of C23O gene possessing bacterial communities. To link the metabolic capability to the microbial structure, 16S rRNA gene-based clone libraries were generated and it was concluded that Betaproteobacteria were abundant in the bacterial communities of the contaminated samples. These results support the opinion that Betaproteobacteria may play a significant role in BTEX degradation under hypoxic conditions.

Variovorax ginsengisoli sp. nov., a denitrifying bacterium isolated from soil of a ginseng field.

A Gram-negative, aerobic or facultatively anaerobic, non-spore-forming, motile, rod-shaped bacterium (strain Gsoil 3165(T)) was isolated from soil of a ginseng field in Pocheon, South Korea. Its taxonomic position was determined by using a polyphasic approach. On the basis of 16S rRNA gene sequence analysis, strain Gsoil 3165(T) was shown to belong to the family Comamonadaceae, class Betaproteobacteria, and was related most closely to the type strains of Variovorax boronicumulans (98.9 % similarity), Variovorax paradoxus (98.3 %), Variovorax soli (98.2 %) and Variovorax dokdonensis (96.6 %). Levels of 16S rRNA gene sequence similarity between strain Gsoil 3165(T) and the type strains of other species in the family Comamonadaceae were less than 97.0 %. The G+C content of the genomic DNA of strain Gsoil 3165(T) was 66 mol%. Phenotypic and chemotaxonomic data (Q-8 as the major ubiquinone; C(16 : 0) and C(17 : 0) cyclo as major fatty acids) supported the affiliation of strain Gsoil 3165(T) to the genus Variovorax. The results of physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain Gsoil 3165(T) from recognized Variovorax species. Gsoil 3165(T) is therefore considered to represent a novel species of the genus Variovorax, for which the name Variovorax ginsengisoli sp. nov. is proposed. The type strain is Gsoil 3165(T) (=KCTC 12583(T) =LMG 23392(T)).

DNA-based stable isotope probing enables the identification of active bacterial endophytes in potatoes.

A (13)CO2 (99 atom-%, 350 ppm) incubation experiment was performed to identify active bacterial endophytes in two cultivars of Solanum tuberosum, cultivars Desirée and Merkur. We showed that after the assimilation and photosynthetic transformation of (13)CO2 into (13)C-labeled metabolites by the plant, the most directly active, cultivar specific heterotrophic endophytic bacteria that consume these labeled metabolite scan be identified by DNA stable isotope probing (DNA-SIP).Density-resolved DNA fractions obtained from SIP were subjected to 16S rRNA gene-based community analysis using terminal restriction fragment length polymorphism analysis and sequencing of generated gene libraries.Community profiling revealed community compositions that were dominated by plant chloroplast and mitochondrial 16S rRNA genes for the 'light' fractions of (13)CO2-incubated potato cultivars and of potato cultivars not incubated with (13)CO2. In the 'heavy' fractions of the (13)CO2-incubated endophyte DNA, a bacterial 492-bp terminal restriction fragment became abundant, which could be clearly identified as Acinetobacter and Acidovorax spp. in cultivars Merkur and Desirée,respectively, indicating cultivar-dependent distinctions in (13)C-label flow. These two species represent two common potato endophytes with known plant-beneficial activities.The approach demonstrated the successful detection of active bacterial endophytes in potato. DNA-SIP therefore offers new opportunities for exploring the complex nature of plant-microbe interactions and plant-dependent microbial metabolisms within the endosphere.

Characterization of phosphobacteria isolated from eutrophic aquatic ecosystems.

Phosphobacteria are able to enhance phosphorus availability in soil and improve crop yields. To develop such biofertilizers, 14 predominant phosphobacteria were isolated from eutrophic aquatic ecosystems. Molecular identification and phylogenetic analysis revealed three groups among the nine isolates of inorganic phosphate-solubilizing bacteria (IPSB): IPSB1 and IPSB2 belonged to the actinobacteria and flavobacteria, respectively, and the other seven belonged to the gamma-proteobacteria. Among five isolates of organic phosphorus-mineralizing bacteria (OPMB), two groups were present: OPMB1 and OPMB3 belonged to the beta-proteobacteria, while the other three belonged to the gamma-proteobacteria. The IPSB isolates released 62.8-66.7 mg P 1(-1) from tricalcium phosphate under shaking conditions, and 26.8 to 43.7 mg P 1(-1) under static conditions; the OPMB strains released 23.5-30.2 mg P 1(-1) from lecithin under shaking conditions, and 16.7-27.6 mg P 1(-1) under static conditions. To the best of our knowledge, this is the first report indicating that IPSBI (designated Aureobacterium resistents) as a tricalcium phosphate-solubilizing bacterium and OPMB1 and OPMB3 (designated Acidovorax temperans and Achromobacter xylosoxidans, respectively) are lecithin-mineralizing bacteria. This investigation demonstrated that a eutrophic aquatic ecosystem is a selective source of phosphobacteria and the screened phosphobacteria are a potential alternative to the development of biofertilizers.

Caenimonas koreensis gen. nov., sp. nov., isolated from activated sludge.

A Gram-negative, rod-shaped bacterium, designated strain EMB320T, was isolated from activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor. The isolate was strictly aerobic and non-motile. Growth was observed between 10 and 35 degrees C (optimum 30 degrees C) and between pH 6.0 and 9.0 (optimum pH 7.0-8.0). The predominant cellular fatty acids of strain EMB320T were C16 : 0, C18 : 1omega7c and summed feature 3 (C16 : 1omega7c and/or iso-C15 : 0 2-OH). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Strain EMB320T contained ubiquinone-8 (Q-8) as the major respiratory quinone system and 2-hydroxyputrescine and putrescine as the major polyamines, which suggests that it belongs to the Betaproteobacteria. The G+C content of the genomic DNA was 62.7 mol%. Comparative 16S rRNA gene sequence analysis showed that strain EMB320T formed a phyletic lineage distinct from other genera within the family Comamonadaceae. On the basis of chemotaxonomic data and molecular properties, strain EMB320T represents a novel genus and species within the family Comamonadaceae, for which the name Caenimonas koreensis sp. nov. is proposed. The type strain of Caenimonas koreensis is EMB320T (=KCTC 12616T =DSM 17982T).