Phenotypic and resistome analysis of antibiotic and heavy metal resistance in the Antarctic bacterium Pseudomonas sp. AU10.

Resistance to antibiotics and heavy metals in Antarctic bacteria has been investigated due to anthropogenic impact on the continent. However, there is still much to learn about the genetic determinants of resistance in native bacteria. In this study, we investigated antibiotic, heavy metal, and metalloid resistance in Pseudomonas sp. AU10, isolated from King George Island (Antarctica), and analyzed its genome to look for all the associated genetic determinants (resistome). We found that AU10 displayed resistance to Cr(VI), Cu(II), Mn(II), Fe(II), and As(V), and produced an exopolysaccharide with high Cr(VI)-biosorption capacity. Additionaly, the strain showed resistance to aminopenicillins, cefotaxime, aztreonam, azithromycin, and intermediate resistance to chloramphenicol. Regarding the resistome, we did not find resistance genes in AU10's natural plasmid or in a prophage context. Only a copper resistance cluster indicated possible horizontal acquisition. The mechanisms of resistance found were mostly efflux systems, several sequestering proteins, and a few enzymes, such as an AmpC ß-lactamase or a chromate reductase, which would account for the observed phenotypic profile. In contrast, the presence of a few gene clusters, including the terZABCDE operon for tellurite resistance, did not correlate with the expected phenotype. Despite the observed resistance to multiple antibiotics and heavy metals, the lack of resistance genes within evident mobile genetic elements is suggestive of the preserved nature of AU10's Antarctic habitat. As Pseudomonas species are good bioindicators of human impact in Antarctic environments, we consider that our results could help refine surveillance studies based on monitoring resistances and associated resistomes in these populations.

Draft genome sequence of Bradyrhizobium sp. strain Oc8 isolated from Crotalaria ochroleuca nodule.

In this study, we report the draft genome sequence of Bradyrhizobium sp. strain Oc8, a rhizobium isolated from Crotalaria ochroleuca,efficient in C. ochroleuca, C. juncea, C. spectabilis, and Cajanus cajan. The whole genome of the strain Oc8 contains 46 scaffolds, 8,283,342 bp, and 63.27% of GC content. Bradyrhizobium sp. Oc8 is an effective nitrogen-fixing bacterium with potential use as an inoculant for legumes used as cover crops and green manures.

Fighting plant pathogens with cold-active microorganisms: biopesticide development and agriculture intensification in cold climates.

Cold-adapted (CA) microorganisms (= psychrophiles or psychrotolerants) are key players of many ecological interactions in natural ecosystems. Some of them can colonize the rhizosphere of plants and cause damage to their hosts; others, on the contrary, protect plants from their pathogens through direct and indirect mechanisms, thus promoting plant growth and development. These "protective" microbes are known as biocontrol agents (BCA). BCA either limit or inhibit the growth of plant pathogens, owing to the excretion of a panoply of secondary metabolites (including soluble and volatile antibiotics, siderophores, quorum sensing interfering agents). BCA can also control plant pathogens through indirect mechanisms, including competence for nutrients and space, or else by interfering with their chemical communication. That explains why some of these BCA have been included in the formulation of commercial biopesticides, which are environmentally friendly products containing live cells used to control plant diseases and pests. At present, the development of biopesticides from mesophilic microorganisms is an established technology. Unfortunately, these biopesticides are not active at low temperatures. On the other hand, the information concerning the potential use of CA-BCA for the same goal is at its infancy. Here, we review the current knowledge concerning the isolation, identification, and characterization of CA microbes which act as antagonists of plant pathogens, including the mechanisms they deploy to antagonize plant pathogens. We also illustrate their biotechnological potential to develop CA biopesticides and discuss their utility in the context of mountainous agriculture. KEY POINTS: • Many naturally occurring cold-active microbes antagonize plant pathogens. • The mechanisms of biocontrol exerted by these microbes are either direct or indirect. • Cold-active biocontrol agents can be used to develop biopesticides. • Cold-active biopesticides are crucial for sustainably intensifying agriculture in cold climates.

Identification of Plant Compounds Involved in the Microbe-Plant Communication During the Coinoculation of Soybean with Bradyrhizobium elkanii and Delftia sp. strain JD2.

Delftia sp. strain JD2 is a betaproteobacterium characterized as a plant growth-promoting bacterium with a 'helper' function, enhancing the performance of rhizobial inoculant strains during the coinoculation of alfalfa and clover. In this work we analyzed i) the effect of the coinoculation with Bradyrhizobium elkanii and Delftia sp. strain JD2 strains on the performance of soybean plants and ii) the production of a few secondary plant metabolites that would explain the positive effect of coinoculation on the growth and development of soybean plants. The results showed a beneficial effect of coinoculation on soybean growth, nodulation rate, and pulse yield, with the concomitant benefit for the agricultural economy. In addition, based on a metabolomics approach, we demonstrated that a different pattern of plant metabolites is being produced at different stages of plant growth. The new information suggests that the coinoculation of soybean changes the primary and secondary metabolism of the plant, including changes in the metabolic status of main and secondary nodules within the plant. The relevance of producing a different pattern of photosynthetic and photoprotective pigments, flavonoids, organic acids, and carbohydrates are discussed. Finally, we propose that JD2 could be used together with bradyrhizobia to manipulate the chemical composition of plant tissues, promoting the nutritional benefits and health of soybean.

Searching for novel photolyases in UVC-resistant Antarctic bacteria.

Ultraviolet (UV) light irradiation has serious consequences for cell survival, including DNA damage by formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6,4) pyrimidone photoproducts. In general, the Nucleotide Excision Repair pathway repairs these lesions; however, all living forms, except placental mammals and some marsupials, produce a flavoprotein known as photolyase that directly reverses these lesions. The aim of this work was the isolation and identification of Antarctic UVC-resistant bacteria, and the search for novel photolyases. Two Antarctic water samples were UVC-irradiated (254 nm; 50-200 J m- 2) and 12 UVC-resistant bacteria were isolated and identified by 16S rDNA amplification/analysis as members of the genera Pseudomonas, Janthinobacterium, Flavobacterium, Hymenobacter and Sphingomonas. The UVC 50% lethal dose and the photo-repair ability of isolates were analyzed. The occurrence of photolyase coding sequences in Pseudomonas, Hymenobacter and Sphingomonas isolates were searched by PCR or by searching in the draft DNA genome. Results suggest that Pseudomonas and Hymenobacter isolates produce CDP-photolyases, and Sphingomonas produces two CPD-photolyases and a 6,4-photolyase. Results suggest that the Antarctic environment is an important source of genetic material for the identification of novel photolyase genes with potential biotechnological applications.

The versatility of Delftia sp. isolates as tools for bioremediation and biofertilization technologies.

Two Pb(II)-resistant bacteria isolated from a soil containing 2,500 mg/kg of Pb were identified by 16S rRNA sequencing analysis as Delftia sp. and designated as 3C and 6C. Both isolates grew at a Pb(II) concentration of 62 mg/L and at the stationary phase showed a Pb(II)-sorption capability of 10 ± 1.5 (3C) and 5 ± 0.8 (6C) mg/g of biomass. Biochemical properties related to heavy metal resistance and plant growth promotion were analyzed and compared with the Cr(VI)-resistant plant growth-promoting Delftia sp. JD2, previously reported by our group. Both isolates and JD2 were resistant to Cr(VI), Pb(II) and many antibiotics, produced siderophores and the phytohormone indole-3-acetic, and showed clover growth-promoting activity in greenhouse conditions. Interestingly, the occurrence of integron class 1 was shown in all isolates. Our results add to previous reports and suggest that bacteria of the genus Delftia could be consider as good candidates for the design of technologies for cleaning up contaminated environments and/or the production of biofertilizers.

Delftia sp. JD2: a potential Cr(VI)-reducing agent with plant growth-promoting activity.

A chromium (Cr)-resistant bacterium isolated from soil containing 6,000 mg/kg of Cr was identified based on 16S rRNA gene sequence analysis as Delftia, and designated as JD2. Growth of JD2 was accompanied with reduction of Cr(VI) to Cr(III) in liquid medium initially containing 100 mg/L Cr(VI), the maximum concentration allowing growth. JD2 showed NADH/NADPH-dependent reductase activity associated with the soluble fraction of cells. The results suggest that JD2 might be a good candidate for the treatment of highly Cr(VI)-contaminated water and/or industrial effluents. The isolate produced indole-3-acetic acid in the presence and absence of Cr(VI) and showed free-living nitrogen-fixing activity possibly attributable to a V-nitrogenase. JD2 did not counteract the harmful effect of Cr(VI) during leguminous plant growth and nodulation by rhizobial strains but functioned as a "helper" bacterium to enhance the performance of rhizobial inoculant strains during inoculation of alfalfa and clover (used as model plants to study plant growth-promoting activity) in the absence of Cr(VI).

Cellular and biochemical response to Cr(VI) in Stenotrophomonas sp.

Chromium (Cr)-resistant bacteria isolated from a soil with 6 g kg(-1) of Cr were identified based on 16S rRNA gene sequence analysis as a Stenotrophomonas, and designated as JD1. Growth of JD1 was accompanied by transformation of Cr(VI) to Cr(III) in liquid medium initially containing 300 mg L(-1) Cr(VI), the maximum concentration allowing growth. JD1 produced the highest levels of a Cr(VI)-binding exopolysaccharide when grown in medium with 100 mg L(-1) Cr(VI). The relative exopolysaccharide monosaccharide composition was analysed by HPLC, which showed that rhamnose+galactose was the major component, and that its relative level increased when cells were grown with Cr(VI). JD1 grew as a biofilm on various inert surfaces. Biofilm macromolecular composition analysis indicated that the relative levels of exopolysaccharide and protein were more abundant in biofilms grown in 100 mg L(-1) Cr(VI), whereas relative uronic acid levels remained constant. Biofilm cells exposed to Cr(VI) were elongated, grouped in clusters and exopolysaccharide obtained from the biofilm extracellular matrix had an enhanced capacity to bind Cr(VI). Exopolysaccharide production and composition, and biofilm growth are discussed as a mechanism of protection that allows survival during Cr(VI) stress.