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.

A new OCH ß-lactamase from a Brucella pseudintermedia (Ochrobactrum pseudintermedium) strain isolated from Zophobas morio larvae.

OBJECTIVES: OCH class C ß-lactamases have been reported in several species belonging to the Brucella genus that were formerly known as Ochrobactrum. Moreover, only one complete genome of Brucella pseudintermedia has been published. In this work, we describe the genome of a B. pseudintermedia strain possessing a new blaOCH gene that was isolated from Zophobas morio larvae. METHODS: Hybrid whole-genome sequencing analysis (Illumina and Nanopore) was used to identify and characterise the strain (Ops-OCH-23). Phylogenetic analyses based on the 16S rRNA gene sequence and a core-genome alignment were performed to study the relationships among Ops-OCH-23 and deposited genomes. Moreover, all deposited blaOCH genes were compared to the one found in Ops-OCH-23. RESULTS: Ops-OCH-23 showed a susceptibility profile consistent with the production of AmpC ß-lactamase(s). Its genome consisted of two chromosomes, of which one carried the blaOCH gene. Such gene encoded a new class C OCH ß-lactamase among the fifteen so far reported. Two plasmids (120-Kb and 59-Kb) without any associated antimicrobial resistance genes were also found. Analysis of 16S rRNA revealed that Ops-OCH-23 shared 100% homology with four deposited B. pseudintermedia strains. Moreover, the core-genome analysis indicated that the closest match (279 ΔSNVs) to Ops-OCH-23 was strain CTOTU49018 isolated from an urban environment in Germany in 2013. CONCLUSION: We described the second complete genome of a B. pseudintermedia that also encoded a new OCH ß-lactamase variant. Overall, this report expands our knowledge regarding this rarely isolated Brucella species that have been reported so far only a few times in human sources.

Ochrobactrum chromiisoli sp. nov., Isolated from Chromium-Contaminated Soil.

A Gram-stain-negative, non-spore-forming, flagellated, motile, aerobic, rod-shaped bacteria strain, designated YY2XT, was isolated from chromium-contaminated soil. Phylogenetic analysis based on 16S rRNA gene, recA gene, and whole genome indicated that the strain represented a new member of the genus Ochrobactrum, family Brucellaceae, class Alphaproteobacteria. The phylogenetic trees based on 16 s rRNA gene, revealed that Falsochrobactrum ovis DSM26720T (96.7%), Ochrobactrum gallinifaecis DSM15295T (96.2%), and Pseudochrobactrum asaccharolyticum DSM25619T (96.2%) are the most closely related phylogenetic neighbors of strain YY2XT. The draft genome of YY2XT was approximately 4,650,646 bp in size with a G + C content of 53.0 mol%. Average nucleotide identity and digital DNA-DNA hybridization values among strain YY2XT and the selected Brucellaceae species were 71.4-83.1% and 13.5-42.7%, which are below the recommended cut-off values for species delineation. Growth of strain YY2XT occurred within pH 5-10 (optimum, pH 7-8), 4 â„ƒ-42 °C (optimum, 30 °C), and NaCl concentrations of 0.0-6.0% (optimum, 1.0%). Major quinone system was ubiquinone 10, the major fatty acids were C16:0, C18:1ω7c, and C16:1ω7c and the major polyamines were spermidine and putrescine. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, and four undefined lipids. On the basis of the phenotypic, genotypic and chemotaxonomic traits, strain YY2XT was considered to represent a novel species of the genus Ochrobactrum, for which the name Ochrobactrum chromiisoli sp. nov. is proposed. The type strain is YY2XT (= CCTCC AB 2023035T = JCM 36000T).

If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella.

Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.

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.

Biodegradation characteristics and mechanism of quinoline by Ochrobactrum sp. strain C2.

A quinoline-degrading strain, C2, which could completely degrade 250 mg/L of quinoline within 24 h, was isolated from coking wastewater. Strain C2 was identified as Ochrobactrum sp. on the basis of 16S rDNA sequence analysis According to 16S rDNA gene sequence analysis, Strain C2 was identified as Ochrobactrum sp. Strain C2 could utilize quinoline as the sole carbon sources and nitrogen sources to grow and degrade quinoline well under acidic conditions. The optimum inoculum concentration, temperature and shaking speed for quinoline degradation were 10%, 30 °C and 150 r/min, respectively. The degradation of quinoline at low concentration by the strain followed the first-order kinetic model. The growth process of strain C2 was more consistent with the Haldane model than the Monod model, and the kinetic parameters were: Vmax = 0.08 h-1, Ks = 131.5 mg/L, Ki = 183.1 mg/L. Compared with suspended strains, strain C2 immobilized by sodium alginate had better degradation efficiency of quinoline and COD. The metabolic pathway of quinoline by Strain C2 was tentatively proposed, quinoline was firstly converted into 2(1H) quinolone, then the benzene ring was opened with the action of catechol 1,2-dioxygenase and subsequently transformed into benzaldehyde, 2-pentanone, hydroxyphenyl propionic acid and others.

Enhancement of L-ribulose Production from L-ribose Through Modification of Ochrobactrum sp. CSL1 Ribose-5-phosphate Isomerase A.

L-ribulose, a kind of high-value rare sugar, could be utilized to manufacture L-form sugars and antiviral drugs, generally produced from L-arabinose as a substrate. However, the production of L-ribulose from L-arabinose is limited by the equilibrium ratio of the catalytic reaction, hence, it is necessary to explore a new biological enzymatic method to produce L-ribulose. Ribose-5-phosphate isomerase (Rpi) is an enzyme that can catalyze the reversible isomerization between L-ribose and L-ribulose, which is of great significance for the preparation of L-ribulose. In order to obtain highly active ribose-5-phosphate isomerase to manufacture L-ribulose, ribose-5-phosphate isomerase A (OsRpiA) from Ochrobactrum sp. CSL1 was engineered based on structural and sequence analyses. Through a rational design strategy, a triple-mutant strain A10T/T32S/G101N with 160% activity was acquired. The enzymatic properties of the mutant were systematically investigated, and the optimum conditions were characterized to achieve the maximum yield of L-ribulose. Kinetic analysis clarified that the A10T/T32S/G101N mutant had a stronger affinity for the substrate and increased catalytic efficiency. Furthermore, molecular dynamics simulations indicated that the binding of the substrate to A10T/T32S/G101N was more stable than that of wild type. The shorter distance between the catalytic residues of A10T/T32S/G101N and L-ribose illuminated the increased activity. Overall, the present study provided a solid basis for demonstrating the complex functions of crucial residues in RpiAs as well as in rare sugar preparation.

Efficient gene targeting in soybean using Ochrobactrum haywardense-mediated delivery of a marker-free donor template.

Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.

Development of an efficient marker-free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1.

We have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium-derived vir and T-DNA-mediated transformation. Oh H1 is a unique, non-phytopathogenic species, categorized as a BSL-1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1-8 strain containing a binary vector system. Oh H1-8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy-. Oh H1-8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1-8 generated high-quality, transgenic events with single-copy, plasmid backbone-free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock-inducible excision system resulting in marker-free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null-segregant lines to wild-type controls. We have demonstrated that Oh H1-8, combined with spectinomycin selection, is an efficient, rapid, marker-free and yield-neutral transformation system for elite soybean.

The first case of Ochrobactrum intermedium bacteremia in a pediatric patient with malignant tumor.

BACKGROUND: Ochrobactrum spp. are non-fermenting, Gram-negative bacilli that are regarded as emerging human pathogens of low virulence that can cause infections. The first identified case of Ochrobactrum intermedium was reported in 1998 in a liver transplantation patient with liver abcess. There are no reports of infections in pediatric patients. Here, we report the first case of O. intermedium bacteremia in a pediatric patient. CASE PRESENTATION: A two and a half years old male was admitted with fever, chills and nausea. He had been diagnosed as pineoblastoma and underwent surgical resection and chemotherapy. O. intermedium was isolated from his blood cultures and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), however, the Vitek II automated system failed to identify the organism. Then the pathogen was confirmed by 16S rDNA sequencing and average nucleotide identity result (ANI) confirmed the precise identification of O. intermedium at genomic level. In addition, the patient recovered well after antibiotic combined therapy. CONCLUSIONS: This, to our knowledge, is the first case of O. intermedium bacteremia in a pediatric patient with malignant tumor. Traditional biochemical identification methods such as API 20NE or VITEK2 system cannot differentiate O. anthropi and O. intermedium. MALDI-TOF may be a promising tool for rapid identification of microorganisms such as O. intermedium.

Genomics of Ochrobactrum pseudogrignonense (newly named Brucella pseudogrignonensis) reveals a new blaOXA subgroup.

Ochrobactrum pseudogrignonense (newly named Brucella pseudogrignonensis) is an emerging pathogen in immunodeficient and immunocompetent patients. Most documented cases associated with Ochrobactrum are frequently catheter-related and exhibit wide-spectrum ß-lactam resistance. Misidentification of this pathogen using commercial bacterial identification kits is common. We identified a case of O. pseudogrignonense infection associated with cholelithiasis. The O. pseudogrignonense genome was sequenced and reconstructed using a Nanopore and Illumina hybrid strategy. A novel blaOXA-919 divergent from existing OXA members was identified and subsequent analysis revealed its existence in all available O. pseudogrignonense genomes, which forms a new phylogenetic subgroup distinct from other OXA clusters. Further analysis demonstrated the presence of the novel blaOXA-919 in the chromosome of several other Ochrobactrum species. Our study indicated that Ochrobactrum chromosomes may be a reservoir of blaOXA-919 ß-lactamases.

Malathion biodegradation by a psychrotolerant bacteria Ochrobactrum sp. M1D and metabolic pathway analysis.

An organophosphorus pesticide malathion biodegradation was investigated by using the bacteria Ochrobactrum sp. M1D isolated from a soil sample of peach orchards in Palampur, District Kangra, Himachal Pradesh (India). The bacterium was able to utilize malathion as the sole source of carbon and energy. The isolated bacterium was found psychrotolerant and could degrade 100% of 100 mg l-1 malathion in minimal salt medium at 20°C, pH 7·0 within 12 days with no major significant metabolites left at the end of the study. Through GCMS analysis, methyl phosphate, diethyl maleate, and diethyl 2-mercaptosuccinate were detected and identified as the major pathway metabolites. Based on the GCMS profile, three probable degradation pathways were interpreted. The present study is the first report of malathion biodegradation at both the psychrophilic and mesophilic conditions by any psychrotolerant strain and also through multiple degradation pathways. In the future, the strain can be explored to bio-remediate the malathion contaminated soil in the cold climatic region and to utilize the enzymatic systems for advanced biotechnology applications.

Enzyme-catalyzed biodegradation of penicillin fermentation residues by ß-lactamase OtLac from Ochrobactrum tritici.

BACKGROUND: Biodegradation of antibiotics is a promising method for the large-scale removal of antibiotic residues in the environment. However, the enzyme that is involved in the biodegradation process is the key information to be revealed. RESULTS: In this study, the beta-lactamase from Ochrobactrum tritici that mediates the biodegradation of penicillin V was identified and characterized. When searching the proteins of Ochrobactrum tritici, the ß-lactamase (OtLac) was identified. OtLac consists of 347 amino acids, and predicted isoelectric point is 7.0. It is a class C ß-lactamase according to BLAST analysis. The coding gene of OtLac was amplified from the genomic DNA of Ochrobactrum tritici. The OtLac was overexpressed in E. coli BL21 (DE3) and purified with Ni2+ column affinity chromatography. The biodegradation ability of penicillin V by OtLac was identified in an in vitro study and analyzed by HPLC. The optimal temperature for OtLac is 32 â„ƒ and the optimal pH is 7.0. Steady-state kinetics showed that OtLac was highly active against penicillin V with a Km value of 17.86 µM and a kcat value of 25.28 s-1 respectively. CONCLUSIONS: OtLac demonstrated biodegradation activity towards penicillin V potassium, indicating that OtLac is expected to degrade penicillin V in the future.

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.

Functional analysis of the ocnE gene involved in nicotine-degradation pathways in Ochrobactrum intermedium SCUEC4 and its enzymatic properties.

The SCUEC4 strain of Ochrobactrum intermedium is a newly isolated bacterium that degrades nicotine can use nicotine as the sole carbon source via a series of enzymatic catalytic processes. The mechanisms underlying nicotine degradation in this bacterium and the corresponding functional genes remain unclear. Here, we analyzed the function and biological properties of the ocnE gene involved in the nicotine-degradation pathways in strain SCUEC4. The ocnE gene was cloned by PCR with total DNA of strain SCUEC4 and used to construct the recombinant plasmid pET28a-ocnE. The overexpression of the OcnE protein was detected by SDS-PAGE analysis, and study of the function of this protein was spectrophotometrically carried out by monitoring the changes of 2,5-dihydroxypyridine. Moreover, the effects of temperature, pH, and metal ions on the biological activities of the OcnE protein were analyzed. The optimal conditions for the biological activities of OcnE, a protein of approximately 37.6 kDa, were determined to be 25 °C, pH 7.0, and 25 µmol/L Fe2+, and the suitable storage conditions for the OcnE protein were 0 °C and pH 7.0. In conclusion, the ocnE gene is responsible for the ability of 2,5-dihydroxypyridine dioxygenase. These findings will be beneficial in clarifying the mechanisms of nicotine degradation in O. intermedium SCUEC4.

A comparative analysis of heavy metal bioaccumulation and functional gene annotation towards multiple metal resistant potential by Ochrobactrum intermedium BPS-20 and Ochrobactrum ciceri BPS-26.

The present study describes the heavy metal bioaccumulation potential of Ochrobactrum intermedium BPS-20 and Ochrobactrum ciceri BPS-26. A total of 27 isolates were retrieved from the soils of industrial areas and these two were selected based on their maximum metal tolerance. They can resist up to 2400 mg/L and 2000 mg/L of Lead and 850 mg/L and 1200 mg/L of Nickel respectively. The atomic absorption spectroscopic analysis showed considerably good bioaccumulation by O. intermedium BPS-20 (85.34% and 74.87%) and O. ciceri BPS-26 (71.20% and 88.48%) for Lead and Nickel respectively. The growth rate studies also demonstrated no inhibitory effects of heavy metals in the medium. Further the SEM analysis showed the presence of extracellular polymeric substances around bacterial cells. Moreover, the functional gene annotation confirmed the presence of ATPase, ABC, and HoxN/HupN/NixA families of transporters. Thus, both the isolates provide a better solution for the removal of metal pollutants.

Pan-genomics of Ochrobactrum species from clinical and environmental origins reveals distinct populations and possible links.

Ochrobactrum genus is comprised of soil-dwelling Gram-negative bacteria mainly reported for bioremediation of toxic compounds. Since last few years, mainly two species of this genus, O. intermedium and O. anthropi were documented for causing infections mostly in the immunocompromised patients. Despite such ubiquitous presence, study of adaptation in various niches is still lacking. Thus, to gain insights into the niche adaptation strategies, pan-genome analysis was carried out by comparing 67 genome sequences belonging to Ochrobactrum species. Pan-genome analysis revealed it is an open pan-genome indicative of the continuously evolving nature of the genus. The presence/absence of gene clusters also illustrated the unique presence of antibiotic efflux transporter genes and type IV secretion system genes in the clinical strains while the genes of solvent resistance and exporter pumps in the environmental strains. A phylogenomic investigation based on 75 core genes depicted better and robust phylogenetic resolution and topology than the 16S rRNA gene. To support the pan-genome analysis, individual genomes were also investigated for the mobile genetic elements (MGE), antibiotic resistance genes (ARG), metal resistance genes (MRG) and virulence factors (VF). The analysis revealed the presence of MGE, ARG, and MRG in all the strains which play an important role in the species evolution which is in agreement with the pan-genome analysis. The average nucleotide identity (ANI) based on the genetic relatedness between the Ochrobactrum species indicated a distinction between individual species. Interestingly, the ANI tool was able to classify the Ochrobactrum genomes to the species level which were assigned till the genus level on the NCBI database.

Identification and Characterization of the First Virulent Phages, Including a Novel Jumbo Virus, Infecting Ochrobactrum spp.

The Ochrobactrum genus consists of an extensive repertoire of biotechnologically valuable bacterial strains but also opportunistic pathogens. In our previous study, a novel strain, Ochrobactrum sp. POC9, which enhances biogas production in wastewater treatment plants (WWTPs) was identified and thoroughly characterized. Despite an insightful analysis of that bacterium, its susceptibility to bacteriophages present in WWTPs has not been evaluated. Using raw sewage sample from WWTP and applying the enrichment method, two virulent phages, vB_OspM_OC and vB_OspP_OH, which infect the POC9 strain, were isolated. These are the first virulent phages infecting Ochrobactrum spp. identified so far. Both phages were subjected to thorough functional and genomic analyses, which allowed classification of the vB_OspM_OC virus as a novel jumbo phage, with a genome size of over 227 kb. This phage encodes DNA methyltransferase, which mimics the specificity of cell cycle regulated CcrM methylase, a component of the epigenetic regulatory circuits in Alphaproteobacteria. In this study, an analysis of the overall diversity of Ochrobactrum-specific (pro)phages retrieved from databases and extracted in silico from bacterial genomes was also performed. Complex genome mining allowed us to build similarity networks to compare 281 Ochrobactrum-specific viruses. Analyses of the obtained networks revealed a high diversity of Ochrobactrum phages and their dissimilarity to the viruses infecting other bacteria.

Isolation and Characterization of Ochrobactrum tritici for Penicillin V Potassium Degradation.

Substantial concentrations of penicillin V potassium (PVK) have been found in livestock manure, soil, and wastewater effluents, which may pose potential threats to human health and contribute to the emergence of penicillin-resistant bacterial strains. In this study, bacterial strains capable of degrading PVK were isolated from sludge and characterized. Strain X-2 was selected for biodegradation of PVK. Based on morphological observations and 16S rRNA gene sequencing, strain X-2 was identified as an Ochrobactrum tritici strain. To enhance the PVK degradation ability of PVK, a whole-cell biodegradation process of Ochrobactrum tritici X-2 was established and optimized. In the whole-cell biodegradation process, the optimal temperature and pH were 30°C and 7.0, respectively. Under the optimized conditions, the degradation rate using 0.5 mg/ml PVK reached 100% within 3 h. During biodegradation, two major metabolites were detected: penicilloic acid and phenolic acid. The present study provides a novel method for the biodegradation of PVK using Ochrobactrum tritici strains, which represent promising candidates for the industrial biodegradation of PVK.IMPORTANCE Substantial concentrations of penicillin V potassium (PVK) have been found in the environment, which may pose potential threats to human health and contribute to the emergence of penicillin-resistant bacterial strains. In this study, antibiotic-degrading bacterial strains for PVK were isolated from sludge and characterized. Ochrobactrum tritici was selected for the biodegradation of PVK with high efficiency. To enhance its PVK degradation ability, a whole-cell biodegradation process was established and optimized using Ochrobactrum tritici The degradation rate with 0.5 mg/ml PVK reached 100% within 3 h. The potential biodegradation pathway was also investigated. To the best of our knowledge, the present study provides new insights into the biodegradation of PVK using an Ochrobactrum tritici strain, a promising candidate strain for the industrial biodegradation of ß-lactam antibiotics.