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The petroleum hydrocarbons in seawater have been worldwide concern contaminants. Biological method, with the advantages of low cost, minimal environmental impact, and no secondary pollution, is a promising method for petroleum hydrocarbon treatment. In this study, two strains, identified as Stenotrophomonas acidaminiphila and Ochrobactrum, were demonstrated to possess the ability to degrade petroleum hydrocarbons. The mixed culture composed of Stenotrophomonas acidaminiphila and Ochrobactrum at a 2:1 ratio was able to achieve 79.41% degradation of the total petroleum hydrocarbons after 5 days. Besides, the average removal efficiencies of C10-C30 components in petroleum hydrocarbons by Stenotrophomonas acidaminiphila, Ochrobactrum, and mixed culture were 62.98%, 59.14% and 73.30%, respectively. The possible degradation pathways of petroleum hydrocarbons had been speculated through gas chromatography-mass spectrometry (GC-MS) and differential gene expression metabolomics analyses. The toxicity of products from the biodegradation of petroleum hydrocarbons was greatly reduced., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Yin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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)., Competing Interests: The authors have declared that no competing interests exist.

The genus Brucella comprises various species of both veterinary and human medical importance. All species are genetically highly related to each other, sharing intra-species average nucleotide identities (ANI) of > 99%. Infections occur among various warm-blooded animal species, marine mammals, and humans. Until recently, amphibians had not been recognized as a host for Brucella. In this study, however, we show that novel Brucella species are distributed among exotic frogs worldwide. Comparative recA gene analysis of 36 frog isolates from various continents and different frog species revealed an unexpected high genetic diversity, not observed among classical Brucella species. In phylogenetic reconstructions the isolates consequently formed various clusters and grouped together with atypical more distantly related brucellae, like B. inopinata, strain BO2, and Australian isolates from rodents, some of which were isolated as human pathogens. Of one frog isolate (10RB9215) the genome sequence was determined. Comparative genome analysis of this isolate and the classical Brucella species revealed additional genetic material, absent from classical Brucella species but present in Ochrobactrum, the closest genetic neighbor of Brucella, and in other soil associated genera of the Alphaproteobacteria. The presence of gene clusters encoding for additional metabolic functions, flanked by tRNAs and mobile genetic elements, as well as by bacteriophages is suggestive for a different ecology compared to classical Brucella species. Furthermore it suggests that amphibian isolates may represent a link between free living soil saprophytes and the pathogenic Brucella with a preferred intracellular habitat. We therefore assume that brucellae from frogs have a reservoir in soil and, in contrast to classical brucellae, undergo extensive horizontal gene transfer., Competing Interests: The authors have declared that no competing interests exist.

Ochrobactrum tritici SCII24T is a highly As-resistant bacterium, with two previously described arsenic resistance operons, ars1 and ars2. Among a large number of genes, these operons contain the arsB and Acr3 genes that encode the arsenite efflux pumps responsible for arsenic resistance. Exploring the genome of O. tritici SCII24T, an additional putative operon (ars3) was identified and revealed the presence of the Acr3_2 gene that encodes for an arsenite efflux protein but which came to prove to not be required for full As resistance. The genes encoding for arsenite efflux pumps, identified in this strain, were inactivated to develop microbial accumulators of arsenic as new tools for bioremediation. Six different mutants were produced, studied and three were more useful as biotools. O. tritici wild type and the Acr3-mutants showed the highest resistance to As(III), being able to grow up to 50 mM of arsenite. On the other hand, arsB-mutants were not able to grow at concentrations higher than 1 mM As(III), and were the most As(III) sensitive mutants. In the presence of 1 mM As(III), the strain with arsB and Acr3_1 mutated showed the highest intracellular arsenic concentration (up to 17 ng(As)/mg protein), while in assays with 5 mM As(III), the single arsB-mutant was able to accumulate the highest concentration of arsenic (up to 10 ng(As)/mg protein). Therefore, arsB is the main gene responsible for arsenite resistance in O. tritici. However, both genes arsB and Acr3_1 play a crucial role in the resistance mechanism, depending on the arsenite concentration in the medium. In conclusion, at moderate arsenite concentrations, the double arsB- and Acr3_1-mutant exhibited a great ability to accumulate arsenite and can be seen as a promising bioremediation tool for environmental arsenic detoxification.

Ochrobactrum intermedium is considered as an emerging human environmental opportunistic pathogen with mild virulence. The distribution of isolates and sequences described in literature and databases showed frequent association with human beings and polluted environments. As population structures are related to bacterial lifestyles, we investigated by multi-locus approach the genetic structure of a population of 65 isolates representative of the known natural distribution of O. intermedium. The population was further surveyed for genome dynamics using pulsed-field gel electrophoresis and genomics. The population displayed a clonal epidemic structure with events of recombination that occurred mainly in clonal complexes. Concerning biogeography, clones were shared by human and environments and were both cosmopolitan and local. The main cosmopolitan clone was genetically and genomically stable, and grouped isolates that all harbored an atypical insertion in the rrs. Ubiquitism and stability of this major clone suggested a clonal succes in a particular niche. Events of genomic reduction were detected in the population and the deleted genomic content was described for one isolate. O. intermedium displayed allopatric characters associated to a tendancy of genome reduction suggesting a specialization process. Considering its relatedness with Brucella, this specialization might be a commitment toward pathogenic life-style that could be driven by technological selective pressure related medical and industrial technologies.

Neonicotinoid insecticides are one of the most important commercial insecticides used worldwide. The potential toxicity of the residues present in environment to humans has received considerable attention. In this study, a novel Ochrobactrum sp. strain D-12 capable of using acetamiprid as the sole carbon source as well as energy, nitrogen source for growth was isolated and identified from polluted agricultural soil. Strain D-12 was able to completely degrade acetamiprid with initial concentrations of 0-3000 mg · L(-1) within 48 h. Haldane inhibition model was used to fit the special degradation rate at different initial concentrations, and the parameters q max, K s and K i were determined to be 0.6394 (6 h)(-1), 50.96 mg · L(-1) and 1879 mg · L(-1), respectively. The strain was found highly effective in degrading acetamiprid over a wide range of temperatures (25-35 °C) and pH (6-8). The effects of co-substrates on the degradation efficiency of acetamiprid were investigated. The results indicated that exogenously supplied glucose and ammonium chloride could slightly enhance the biodegradation efficiency, but even more addition of glucose or ammonium chloride delayed the biodegradation. In addition, one metabolic intermediate identified as N-methyl-(6-chloro-3-pyridyl)methylamine formed during the degradation of acetamiprid mediated by strain D-12 was captured by LC-MS, allowing a degradation pathway for acetamiprid to be proposed. This study suggests the bacterium could be a promising candidate for remediation of environments affected by acetamiprid.

Ochrobactrum tritici 5bvl1 is able to resist to high concentrations of chromate through the expression of an inducible chromate-resistant determinant, found in a mobile element (TnOtChr), which carries the genes, chrB, chrA, chrC and chrF. The regulation of chr operon present in TnOtChr, which is controlled by a transcriptional regulator, ChrB, was characterized in the current work. Fusions of chr promoter, or chr promoter and chrB gene, upstream of a gfp reporter gene, identified the most probable promoter sequence within the tnpR-chrB intergenic region. This region contains an AT-rich imperfect inverted repeat sequence, which overlaps a part of the -10 sequence. The results of the in vitro DNA-binding assays with purified ChrB (His- or no-tagged) showed that the protein binds directly to the chr promoter region. In order to identify the ChrB functional domain for sensing chromate stress and for DNA-binding, site-directed mutagenesis of ChrB was performed. Among several single amino acid mutants, three mutants (R180; R187 and H229) prevented chromate induction without any modification to the protein's stability. Interestingly, two ChrB mutants (R18 and R23) were constitutively active, regardless of chromate stress conditions, indicating that the residues most probably belong to the protein-DNA binding site. As such, the ChrB was classified as a transcriptional regulator that recognizes a specific DNA sequence, regulating the expression of a chromate resistance determinant.

Microbial bioreporters offer excellent potentialities for the detection of the bioavailable portion of pollutants in contaminated environments, which currently cannot be easily measured. This paper describes the construction and evaluation of two microbial bioreporters designed to detect the bioavailable chromate in contaminated water samples. The developed bioreporters are based on the expression of gfp under the control of the chr promoter and the chrB regulator gene of TnOtChr determinant from Ochrobactrum tritici 5bvl1. pCHRGFP1 Escherichia coli reporter proved to be specific and sensitive, with minimum detectable concentration of 100 nM chromate and did not react with other heavy metals or chemical compounds analysed. In order to have a bioreporter able to be used under different environmental toxics, O. tritici type strain was also engineered to fluoresce in the presence of micromolar levels of chromate and showed to be as specific as the first reporter. Their applicability on environmental samples (spiked Portuguese river water) was also demonstrated using either freshly grown or cryo-preserved cells, a treatment which constitutes an operational advantage. These reporter strains can provide on-demand usability in the field and in a near future may become a powerful tool in identification of chromate-contaminated sites.

Background: During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some alpha-Proteobacteria are stealthy intracellular pathogens not readily detected by this system. Brucella members follow this strategy and are highly virulent, but other Brucellaceae like Ochrobactrum are rhizosphere inhabitants and only opportunistic pathogens. To gain insight into the emergence of the stealthy strategy, we compared these two phylogenetically close but biologically divergent bacteria., Methodology/principal Findings: In contrast to Brucella abortus, Ochrobactrum anthropi did not replicate within professional and non-professional phagocytes and, whereas neutrophils had a limited action on B. abortus, they were essential to control O. anthropi infections. O. anthropi triggered proinflammatory responses markedly lower than Salmonella enterica but higher than B. abortus. In macrophages and dendritic cells, the corresponding lipopolysaccharides reproduced these grades of activation, and binding of O. anthropi lipopolysaccharide to the TLR4 co-receptor MD-2 and NF-kappaB induction laid between those of B. abortus and enteric bacteria lipopolysaccharides. These differences correlate with reported variations in lipopolysaccharide core sugars, sensitivity to bactericidal peptides and outer membrane permeability., Conclusions/significance: The results suggest that Brucellaceae ancestors carried molecules not readily recognized by innate immunity, so that non-drastic variations led to the emergence of stealthy intracellular parasites. They also suggest that some critical envelope properties, like selective permeability, are profoundly altered upon modification of pathogen-associated molecular patterns, and that this represents a further adaptation to the host. It is proposed that this adaptive trend is relevant in other intracellular alpha-Proteobacteria like Bartonella, Rickettsia, Anaplasma, Ehrlichia and Wolbachia.