Biodegradation of selected aminophosphonates by the bacterial isolate Ochrobactrum sp. BTU1.

Aminophosphonates, like glyphosate (GS) or metal chelators such as ethylenediaminetetra(methylenephosphonic acid) (EDTMP), are released on a large scale worldwide. Here, we have characterized a bacterial strain capable of degrading synthetic aminophosphonates. The strain was isolated from LC/MS standard solution. Genome sequencing indicated that the strain belongs to the genus Ochrobactrum. Whole-genome classification using pyANI software to compute a pairwise ANI and other metrics between Brucella assemblies and Ochrobactrum contigs revealed that the bacterial strain is designated as Ochrobactrum sp. BTU1. Degradation batch tests with Ochrobactrum sp. BTU1 and the selected aminophosphonates GS, EDTMP, aminomethylphosphonic acid (AMPA), iminodi(methylene-phosphonic) (IDMP) and ethylaminobis(methylenephosphonic) acid (EABMP) showed that the strain can use all phosphonates as sole phosphorus source during phosphorus starvation. The highest growth rate was achieved with AMPA, while EDTMP and GS were least supportive for growth. Proteome analysis revealed that GS degradation is promoted by C-P lyase via the sarcosine pathway, i.e., initial cleavage at the C-P bond. We also identified C-P lyase to be responsible for degradation of EDTMP, EABMP, IDMP and AMPA. However, the identification of the metabolite ethylenediaminetri(methylenephosphonic acid) via LC/MS analysis in the test medium during EDTMP degradation indicates a different initial cleavage step as compared to GS. For EDTMP, it is evident that the initial cleavage occurs at the C-N bond. The detection of different key enzymes at regulated levels, form the bacterial proteoms during EDTMP exposure, further supports this finding. This study illustrates that widely used and structurally more complex aminophosphonates can be degraded by Ochrobactrum sp. BTU1 via the well-known degradation pathways but with different initial cleavage strategy compared to GS.

Treatment with atypical rhizobia, Pararhizobium giardinii and Ochrobactrum sp. modulate the rhizospheric bacterial community, and enhances Lens culinaris growth in fallow-soil.

Diazotrophic nodule isolates are acknowledged promoters of plant growth and rhizospheric community. Consequently, in the lentil agroecosystem, inoculation of atypical rhizobial isolates could be a viable alternative to chemical fertilizers for fallow land usage optimization. The aim of this study is to evaluate and select the rhizobial isolates of lentil nodules with plant-growth-promoting (PGP) attributes and to elucidate their application in rice-fallow soil for determining the growth of lentils and its impact on the rhizospheric bacterial community. Lentil's nodule isolates were identified and screened for their PGP attributes, biofilm, exopolysaccharide (EPS) formation, and early plant growth promotion. The pot experiment with the selected atypical rhizobial isolates Pararhizobium giardinii (P1) and Ochrobactrum sp. (42S) significantly enhanced germination, vigour index, nodule formation (P1 60%, 42S 42% increase), nodule fresh weight, shoot length (65% P1 & 35% 42S), and chlorophyll content as compared to the uninoculated control treatment. The genes for nitrogen fixation nifH and nifK were detected in both isolates. Scanning Electron Microscopy (SEM) revealed successful root and nodule colonization by both isolates, while Transmission Electron Microscopy (TEM) displayed nitrogen-fixing zones within root nodules. Proteobacteria predominated in the lentil rhizosphere of all the treatments. Whereas, application of either P1 or 42S increased Rhizobium, Mesorhizobium, and Bradyrhizobium genra, thus positively modulating rhizospheric community structure. The correlation network analysis revealed an abundance of some interdependent bacterial genera with a possible role in overall plant growth. Functional genes for siderophore biosynthesis and ABC transporter were positively modulated by application of either P1 or 42S. This study showed the significant effect of P. giardinii P1 and Ochrobactrum sp. 42S of L. culinaris on lentil growth, improving fallowsoil health for optimum usage, and modulated rhizospheric community structure which strongly manifest prospects of low-cost, eco-friendly and sustainable biofertilizers.

The wheat growth-promoting traits of Ochrobactrum and Pantoea species, responsible for solubilization of different P sources, are ensured by genes encoding enzymes of multiple P-releasing pathways.

Production and release of organic acids and phosphatase enzymes by microbes are important for inorganic and organic phosphorus cycling in soil. The presence of microorganisms with corresponding traits in the plant rhizosphere lead to improved plant P uptake and ultimately growth promotion. We studied the potential of two rhizosphere-competent strains, Pantoea sp. MR1 and Ochrobactrum sp. SSR, for solubilization of different organic and inorganic P sources in vitro. In a pot experiment we further revealed the impact of the two strains on wheat seedling performance in soil amended with either phytate, rock phosphate or K2HPO4 as solely P source. To directly link P-solubilizing activity to the strain-specific genetic potential, we designed novel primers for glucose dehydrogenase (gcd), phosphatase (pho) and phytase (phy) genes, which are related to the organic and inorganic P solubilization potential. Quantitative tracing of these functional genes in the inoculated soils of the conducted pot experiment further allowed to compare strain abundances in the soil in dependency on the present P source. We observed strain- and P source-dependent patterns of the P solubilization in vitro as well as in the pot experiment, whereby P release, particularly from phytate, was linked to the strain abundance. We further revealed that the activity of microbial phosphatases is determined by the interplay between functional gene abundance, available soil P, and substrate availability. Moreover, positive impacts of microbial seed inoculation on wheat root architecture and aboveground growth parameters were observed. Our results suggest that screening for rhizosphere-competent strains with gcd, pho and phy genes may help to identify new microbial taxa that are able to solubilize and mineralize inorganic as well as organic bound P. Subsequently, the targeted use of corresponding strains may improve P availability in agricultural soils and consequently reduce fertilizer application.

Ochrobactrum cytisi IPA7.2 promotes growth of potato microplants and is resistant to abiotic stress.

Bacteria in natural associations with agricultural crops are promising for use in the improvement of clonal micropropagation of plants. We clarified the taxonomic position of Ochrobactrum cytisi strain IPA7.2 and investigated its tolerance for salinity, high temperature, and glyphosate pollution. We also tested the strain's potential to promote the growth of potato (Solanum tuberosum L.) microplants. Using the IPA7.2 draft genome (no. NZ_MOEC00000000), we searched for housekeeping genes and also for the target genes encoding glyphosate tolerance and plant-growth-promoting ability. A multilocus sequence analysis of the gap, rpoB, dnaK, trpE, aroC, and recA housekeeping genes led us to identify isolate IPA7.2 as O. cytisi. The strain tolerated temperatures up to 50 °C and NaCl concentrations up to 3-4%, and it produced 8 µg ml-1 of indole-3-acetic acid. It also tolerated 6 mM glyphosate owing to the presence of type II 5-enolpyruvylshikimate-3-phosphate synthase. Finally, it was able to colonize the roots and tissues of potato microplants, an ability preserved by several generations after subculturing. We identified the development phase of potato microplants that was optimal for inoculation with O. cytisi IPA7.2. Inoculation of in vitro-grown 15-day-old microplants increased the mitotic index of root meristem cells (by 50%), the length of shoots (by 34%), the number of leaves (by 7%), and the number of roots (by 16%). Under ex vitro conditions, the inoculated plants had a greater leaf area (by 77%) and greater shoot and root dry weight (by 84 and 61%, respectively) than did the control plants. We recommend O. cytisi IPA 7.2 for use in the growing of potato microplants to improve the production of elite seed material.

Ochrobactrum quorumnocens sp. nov., a quorum quenching bacterium from the potato rhizosphere, and comparative genome analysis with related type strains.

Ochrobactrum spp. are ubiquitous bacteria attracting growing attention as important members of microbiomes of plants and nematodes and as a source of enzymes for biotechnology. Strain Ochrobactrum sp. A44T was isolated from the rhizosphere of a field-grown potato in Gelderland, the Netherlands. The strain can interfere with quorum sensing (QS) of Gram-negative bacteria through inactivation of N-acyl homoserine lactones (AHLs) and protect plant tissue against soft rot pathogens, the virulence of which is governed by QS. Phylogenetic analysis based on 16S rRNA gene alone and concatenation of 16S rRNA gene and MLSA genes (groEL and gyrB) revealed that the closest relatives of A44T are O. grignonense OgA9aT, O. thiophenivorans DSM 7216T, O. pseudogrignonense CCUG 30717T, O. pituitosum CCUG 50899T, and O. rhizosphaerae PR17T. Genomes of all six type strains were sequenced, significantly expanding the possibility of genome-based analyses in Ochrobactrum spp. Average nucleotide identity (ANIb) and genome-to-genome distance (GGDC) values for A44T and the related strains were below the single species thresholds (95% and 70%, respectively), with the highest scores obtained for O. pituitosum CCUG 50899T (87.31%; 35.6%), O. rhizosphaerae PR17T (86.80%; 34.3%), and O. grignonense OgA9aT (86.30%; 33.6%). Distinction of A44T from the related type strains was supported by chemotaxonomic and biochemical analyses. Comparative genomics revealed that the core genome for the newly sequenced strains comprises 2731 genes, constituting 50-66% of each individual genome. Through phenotype-to-genotype study, we found that the non-motile strain O. thiophenivorans DSM 7216T lacks a cluster of genes related to flagella formation. Moreover, we explored the genetic background of distinct urease activity among the strains. Here, we propose to establish a novel species Ochrobactrum quorumnocens, with A44T as the type strain (= LMG 30544T = PCM 2957T).

Isolation and characterization of a crude oil degrading bacteria from formation water: comparative genomic analysis of environmental Ochrobactrum intermedium isolate versus clinical strains.

In this study, we isolated an environmental clone of Ochrobactrum intermedium, strain 2745-2, from the formation water of Changqing oilfield in Shanxi, China, which can degrade crude oil. Strain 2745-2 is aerobic and rod-shaped with optimum growth at 42 °C and pH 5.5. We sequenced the genome and found a single chromosome of 4 800 175 bp, with a G+C content of 57.63%. Sixty RNAs and 4737 protein-coding genes were identified: many of the genes are responsible for the degradation, emulsification, and metabolizing of crude oil. A comparative genomic analysis with related clinical strains (M86, 229E, and LMG3301(T)) showed that genes involved in virulence, disease, defense, phages, prophages, transposable elements, plasmids, and antibiotic resistance are also present in strain 2745-2.

Acquired genetic mechanisms of a multiresistant bacterium isolated from a treatment plant receiving wastewater from antibiotic production.

The external environment, particularly wastewater treatment plants (WWTPs), where environmental bacteria meet human commensals and pathogens in large numbers, has been highlighted as a potential breeding ground for antibiotic resistance. We have isolated the extensively drug-resistant Ochrobactrum intermedium CCUG 57381 from an Indian WWTP receiving industrial wastewater from pharmaceutical production contaminated with high levels of quinolones. Antibiotic susceptibility testing against 47 antibiotics showed that the strain was 4 to >500 times more resistant to sulfonamides, quinolones, tetracyclines, macrolides, and the aminoglycoside streptomycin than the type strain O. intermedium LMG 3301T. Whole-genome sequencing identified mutations in the Indian strain causing amino acid substitutions in the target enzymes of quinolones. We also characterized three acquired regions containing resistance genes to sulfonamides (sul1), tetracyclines [tet(G) and tetR], and chloramphenicol/florfenicol (floR). Furthermore, the Indian strain harbored acquired mechanisms for horizontal gene transfer, including a type I mating pair-forming system (MPFI), a MOBP relaxase, and insertion sequence transposons. Our results highlight that WWTPs serving antibiotic manufacturing may provide nearly ideal conditions for the recruitment of resistance genes into human commensal and pathogenic bacteria.

Ochrobactrum rhizosphaerae sp. nov. and Ochrobactrum thiophenivorans sp. nov., isolated from the environment.

Two Gram-negative, rod-shaped, non-spore-forming bacteria, PR17(T) and DSM 7216(T), isolated from the potato rhizosphere and an industrial environment, respectively, were studied for their taxonomic allocation. By rrs (16S rRNA) gene sequencing, these strains were shown to belong to the Alphaproteobacteria, most closely related to Ochrobactrum pseudogrignonense (98.4 and 99.3 % similarity to the type strain, respectively). Chemotaxonomic data (major ubiquinone Q-10; major polyamines spermidine, sym-homospermidine and putrescine; major polar lipids phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol and phosphatidylcholine and the Ochrobactrum-specific unidentified aminolipid AL2; major fatty acids C(18 : 1)omega7c and C(19 : 0) cyclo omega8c) supported the genus affiliation. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the isolates from all hitherto-described Ochrobactrum species. Hence, both isolates represent novel species of the genus Ochrobactrum, for which the names Ochrobactrum rhizosphaerae sp. nov. (type strain PR17(T) =CCUG 55411(T) =CCM 7493(T) =DSM 19824(T)) and Ochrobactrum thiophenivorans sp. nov. (type strain DSM 7216(T) =CCUG 55412(T) =CCM 7492(T)) are proposed.

Bacterial community dynamics during in-situ bioremediation of petroleum waste sludge in landfarming sites.

In-situ bioremediation of petroleum waste sludge in landfarming sites of Motor Oil Hellas (petroleum refinery) was studied by monitoring the changes of the petroleum composition of the waste sludge, as well as the changes in the structure of the microbial community, for a time period of 14 months. The analyses indicated an enhanced degradation of the petroleum hydrocarbons in the landfarming areas. A depletion of n-alkanes of approximately 75-100% was obtained. Marked changes of the microbial communities of the landfarms occurred concomitantly with the degradation of the petroleum hydrocarbons. The results obtained from terminal restriction fragment length polymorphism (T-RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rRNA genes demonstrated that bacteria originating from the refinery waste sludge and newly selected bacteria dominated the soil bacterial community during the period of the highest degradation activity. However, the diversity of the microbial community was decreased with increased degradation of the petroleum hydrocarbons contained in the landfarms. T-RFLP fingerprints of bacteria of the genera Enterobacter and Ochrobactrum were detected in the landfarmed soil over the entire treatment period of 14 months. In contrast, the genus Alcaligenes appeared in significant numbers only within the 10 month old landfarmed soil. Genes encoding catechol 2,3-dioxygenase (subfamily I.2.A) were detected only in DNA of the untreated refinery waste sludge. However, none of the genes known to encode the enzymes alkane hydroxylase AlkB, catechol 2,3-dioxygenase (subfamily I.2.A) and naphthalene dioxygenase nahAc could be detected in DNA of the landfarmed soils.