Biocontrol potential of Pseudomonas protegens ML15 against Botrytis cinerea causing gray mold on postharvest tomato (Solanum lycopersicum var. cerasiforme).

Gray mold, caused by Botrytis cinerea is a major cause of post-harvest rot of fresh fruits and vegetables. The utilization of selected microorganisms as biocontrol agents is a promising alternative to effectively control gray mold on tomatoes. The current study was conducted to explore potential biocontrol mechanisms of the Pseudomonas strain to control infections on post-harvest tomatoes. Among the 8 tested bacterial isolates, Pseudomonas protegens ML15 demonstrated antagonistic activity to Botrytis cinerea. Moreover, P. protegens ML15 exhibited the production of siderophores, hydrogen cyanide, ammonia, exopolysaccharides, lipase, biosurfactant, 2,4-diacetylphloroglucinol, and several other antifungal compounds, such as 1-tetradecanol, cyclododecane, 2,4-di-tert-butylphenol, and 2-methyl-1-hexadecanol. A comprehensive genomic analysis of P. protegens ML15 unravels 18 distinct genetic regions with the potential for biosynthesizing secondary metabolites, known for their pivotal role in biocontrol responses against plant pathogens. In vivo, experiments showed that both culture suspension and cell-free supernatant of P. protegens ML15 significantly reduced fungal growth (53.0 ± 0.63%) and mitigated disease development (52.8 ± 1.5%) in cherry tomatoes at four days post-B. cinerea inoculation. During the infection, the application of P. protegens ML15 resulted in the augmentation of total antioxidant, phenolic content, and ascorbic acids content. Thus, our results suggested that P. protegens ML15's role as a biocontrol agent against B. cinerea-induced postharvest tomato decay achieved through the secretion of antifungal substances, induction of tomato defense responses, and inhibition of mycelial growth of B. cinerea. These findings provide a significant contribution to the ongoing search for alternative, eco-friendly methods of controlling gray mold in fresh products. The utilization of P. protegens ML15 as a biocontrol agent could help to reduce the reliance on chemical fungicides and promote sustainable agriculture practices.

Structure and functions of a multireplicon genome of Antarctic Psychrobacter sp. ANT_H3: characterization of the genetic modules suitable for the construction of the plasmid-vectors for cold-active bacteria.

Among Psychrobacter spp., there are several multireplicon strains, carrying more than two plasmids. Psychrobacter sp. ANT_H3 carries as many as 11 extrachromosomal replicons, which is the highest number in Psychrobacter spp. Plasmids of this strain were subjected to detailed genomic analysis, which enables an insight into the structure and functioning of this multireplicon genome. The replication and conjugal transfer modules of ANT_H3 plasmids were analyzed functionally to discover their potential for being used as building blocks for the construction of novel plasmid-vectors for cold-active bacteria. It was shown that two plasmids have a narrow host range as they were not able to replicate in species other than Psychrobacter, while remaining plasmids had a wider host range and were functional in various Alpha- and Gammaproteobacteria. Moreover, it was confirmed that mobilization modules of seven plasmids were functional, i.e., could be mobilized for conjugal transfer by the RK2 conjugation system. Auxiliary genes were also distinguished in ANT_H3 plasmids, including these encoding putative DNA-protecting protein DprA, multidrug efflux SMR transporter of EmrE family, glycine cleavage system T protein, MscS small-conductance mechanosensitive channel protein, and two type II restriction-modification systems. Finally, all genome-retrieved plasmids of Psychrobacter spp. were subjected to complex genome- and proteome-based comparative analyses showing that Antarctic replicons are significantly different from plasmids from other locations.

Biopriming of seed with plant growth-promoting bacteria for improved germination and seedling growth.

Several seed priming methods can be used to improve seed germination, seedling vigor, and to overcome abiotic stress. In addition to these benefits, only the biopriming method provides the additional benefit of biotic stress management, earning it special attention. Seed biopriming is useful in almost all crops around the world and is an environmentally friendly alternative to chemical fungicides. Biopriming usually refers to use of beneficial microorganisms, in particular plant growth-promoting bacteria (PGPB) able to survive under various harsh environmental conditions. In this study, various bacterial strains were isolated from samples of different origins, i.e., rhizospheric soil, desert sand, and sea mud. Preliminary screening of 156 bacterial isolates was conducted on the basis of their potassium (K), phosphorus (P) solubilization ability, and production of plant growth hormone, i.e., indole acetic acid (IAA). The most efficient bacteria were identified by 16S rRNA gene nucleotide sequences and further examined for their ACC deaminase activity, ammonia production, and biocontrol activity (defined via chitinolytic activity, HCN, and siderophores production). Finally, carrot seed germination assay was conducted with 10 shortlisted most potent isolates. 68.6, 58.3, and 66.7% of tested bacterial isolates were capable of P, K, and Zn solubilization, respectively. Klebsiella aerogenes AF3II1 showed the highest P and K solubilization, while isolate AF4II5, AF7II3, and PC3 showed the highest IAA synthesis ability. Serratia plymuthica EDC15 and Pseudomonas putida AF1I1 showed the strongest chitinolytic and siderophore production activity, respectively. Seven isolates demonstrated strong HCN production ability. Five isolates improved carrot seed germination. Only selected isolates with plant growth-promoting properties can improve carrot germination. The results of this study demonstrate that mainly auxins are involved in seed germination. Furthermore, the data suggest that phosphate solubilization ability may play an additional role in seed germination.

Revealing the diversity of bacteria and fungi in the active layer of permafrost at Spitsbergen island (Arctic) - Combining classical microbiology and metabarcoding for ecological and bioprospecting exploration.

Arctic soils are constantly subjected to extreme environmental conditions such as low humidity, strong winds, high salinity, freeze-thaw cycles, UV exposition, and low nutrient availability, therefore, they have developed unique microbial ecosystems. These environments provide excellent opportunities to study microbial ecology and evolution within pristine (i.e. with limited anthropogenic influence) regions since the High Arctic is still considered one of the wildest and least explored environments on the planet. This environment is also of interest for the screening and recovery of unique microbial strains suitable for various biotechnological applications. In this study, a combination of culture-depended and culture-independent approaches was used to determine the cultivation bias in studies of the diversity of cold-active microorganisms. Cultivation bias is a reduction in recovered diversity, introduced when applying a classical culturing technique. Six different soil types, collected in the vicinity of the Polish Polar Station Hornsund (Spitsbergen, Norway), were tested. It was revealed that the used media allowed recovery of only 6.37 % of bacterial and 20 % of fungal genera when compared with a culture-independent approach. Moreover, it was shown that a combination of R2A and Marine Broth media recovered as much as 93.6 % of all cultivable bacterial genera detected in this study. Based on these results, a novel protocol for genome-guided bioprospecting, combining a culture-dependent approach, metabarcoding, next-generation sequencing, and genomic data reuse was developed. With this methodology, 14 psychrotolerant, multi-metal-resistant strains, including the highly promising Rhodococcus spp., were obtained. These strains, besides increased metal tolerance, have a petroleum hydrocarbon utilization capacity, and thus may be good candidates for future bioremediation technologies, also suited to permanently cold regions.

Diversity of antibiotic resistance gene variants at subsequent stages of the wastewater treatment process revealed by a metagenomic analysis of PCR amplicons.

Wastewater treatment plants have been recognised as point sources of various antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARG) which are considered recently emerging biological contaminants. So far, culture-based and molecular-based methods have been successfully applied to monitor antimicrobial resistance (AMR) in WWTPs. However, the methods applied do not permit the comprehensive identification of the true diversity of ARGs. In this study we applied next-generation sequencing for a metagenomic analysis of PCR amplicons of ARGs from the subsequent stages of the analysed WWTP. The presence of 14 genes conferring resistance to different antibiotic families was screened by PCR. In the next step, three genes were selected for detailed analysis of changes of the profile of ARG variants along the process. A relative abundance of 79 variants was analysed. The highest diversity was revealed in the ermF gene, with 52 variants. The relative abundance of some variants changed along the purification process, and some ARG variants might be present in novel hosts for which they were currently unassigned. Additionally, we identified a pool of novel ARG variants present in the studied WWTP. Overall, the results obtained indicated that the applied method is sufficient for analysing ARG variant diversity.

Heterologous production and characterization of a pyomelanin of Antarctic Pseudomonas sp. ANT_H4: a metabolite protecting against UV and free radicals, interacting with iron from minerals and exhibiting priming properties toward plant hairy roots.

BACKGROUND: Antarctica has one of the most extreme environments in the world. This region is inhabited by specifically adapted microorganisms that produce various unique secondary metabolites (e.g. pigments) enabling their survival under the harsh environmental conditions. It was already shown that these natural, biologically active molecules may find application in various fields of biotechnology. RESULTS: In this study, a cold-active brown-pigment-producing Pseudomonas sp. ANT_H4 strain was characterized. In-depth genomic analysis combined with the application of a fosmid expression system revealed two different pathways of melanin-like compounds biosynthesis by the ANT_H4 strain. The chromatographic behavior and Fourier-transform infrared spectroscopic analyses allowed for the identification of the extracted melanin-like compound as a pyomelanin. Furthermore, optimization of the production and thorough functional analyses of the pyomelanin were performed to test its usability in biotechnology. It was confirmed that ANT_H4-derived pyomelanin increases the sun protection factor, enables scavenging of free radicals, and interacts with the iron from minerals. Moreover, it was shown for the first time that pyomelanin exhibits priming properties toward Calendula officinalis hairy roots in in vitro cultures. CONCLUSIONS: Results of the study indicate the significant biotechnological potential of ANT_H4-derived pyomelanin and open opportunities for future applications. Taking into account protective features of analyzed pyomelanin it may be potentially used in medical biotechnology and cosmetology. Especially interesting was showing that pyomelanin exhibits priming properties toward hairy roots, which creates a perspective for its usage for the development of novel and sustainable agrotechnical solutions.

Fresh Versus Frozen Stool for Fecal Microbiota Transplantation-Assessment by Multimethod Approach Combining Culturing, Flow Cytometry, and Next-Generation Sequencing.

The objective of this work was to compare the quality of FMT preparations made from fresh feces with those made from feces frozen at -30°C without any pre-processing or cryopreservation additives. The research hypothesis was that such preservation protocol (frozen whole stool, then thawed and processed) is equipotent to classical fresh FMT preparation. For that, three complementary methods were applied, including: (i) culturing in aerobic and anaerobic conditions, (ii) measuring viability by flow cytometry, and (iii) next-generation sequencing. Flow cytometry with cell staining showed that the applied freezing protocol causes significant changes in all of the observed bacterial fractions. Alive cell counts dropped four times, from around 70% to 15%, while the other two fractions, dead and unknown cell counts quadrupled and doubled, with the unknown fraction becoming the dominant one, with an average contribution of 57.47% per sample. It will be very interesting to uncover what this unknown fraction is (e.g., bacterial spores), as this may change our conclusions (if these are spores, the viability could be even higher after freezing). Freezing had a huge impact on the structure of cultivable bacterial communities. The biggest drop after freezing in the number of cultivable species was observed for Actinobacteria and Bacilli. In most cases, selected biodiversity indices were slightly lower for frozen samples. PCoA visualization built using weighted UniFrac index showed no donor-wise clusters, but a clear split between fresh and frozen samples. This split can be in part attributed to the changes in the relative abundance of Bacteroidales and Clostridiales orders. Our results clearly show that whole stool freezing without any cryoprotectants has a great impact on the cultivability and biodiversity of the bacterial community, and possibly also on the viability of bacterial cells.

Draft Genome Sequence of Arctic, Heavy Metal-Resistant Agrococcus sp. Strain ARC_14 Isolated from Active Layer of Permafrost from Spitsbergen (Norway).

Extreme environmental conditions observed in polar regions create a unique ecological niche for microbial life. Bacteria living under these harsh, environmental conditions exhibit specific metabolic capabilities. In this report, we present multimetal-resistant Agrococcus sp. strain ARC_14, isolated from soil samples collected in Spitsbergen, Svalbard, Norway.

Marginal lands and fungi - linking the type of soil contamination with fungal community composition.

Fungi can be found in almost all ecosystems. Some of them can even survive in harsh, anthropogenically transformed environments, such as post-industrial soils. In order to verify how the soil fungal diversity may be changed by pollution, two soil samples from each of the 28 post-industrial sites were collected. Each soil sample was characterized in terms of concentration of heavy metals and petroleum derivatives. To identify soil fungal communities, fungal internal transcribed spacer 2 (ITS2) amplicon was sequenced for each sample using Illumina MiSeq platform. There were significant differences in the community structure and taxonomic diversity among the analysed samples. The highest taxon richness and evenness were observed in the non-polluted sites, and lower numbers of taxa were identified in multi-polluted soils. The presence of monocyclic aromatic hydrocarbons, gasoline and mineral oil was determined as the factors driving the differences in the mycobiome. Furthermore, in the culture-based selection experiment, two main groups of fungi growing on polluted media were identified - generalists able to live in the presence of pollution, and specialists adapted to the usage of BTEX as a sole source of energy. Our selection experiment proved that it is long-term soil contamination that shapes the community, rather than temporary addition of pollutant.

Application of Psychrotolerant Antarctic Bacteria and Their Metabolites as Efficient Plant Growth Promoting Agents.

Iron is the fourth most abundant element on earth. However, its low bioavailability is a key plant-growth limiting factor. Bacteria play an important role in plant growth promotion since they produce specific secondary metabolites that may increase macro- and micronutrient accessibility in soil. Therefore, bacterial-derived iron chelators, as well as surface-active compounds, are recognised as essential to plant welfare. In this study, three cold-active Antarctic bacterial strains, i.e. Pseudomonas sp. ANT_H12B, Psychrobacter sp. ANT_H59 and Bacillus sp. ANT_WA51, were analysed. The physiological and genomic characterisation of these strains revealed their potential for plant growth promotion, reflected in the production of various biomolecules, including biosurfactants (that may lower the medium surface tension of even up to 53%) and siderophores (including ANT_H12B-produced mixed-type siderophore that demonstrated the highest production, reaching the concentration of up to 1.065 mM), increasing the availability of nutrients in the environment and neutralising fungal pathogens. Tested bacteria demonstrated an ability to promote the growth of a model plant, alfalfa, increasing shoots' length and fresh biomass even up to 26 and 46% respectively; while their metabolites increased the bioavailability of iron in soil up to 40%. It was also revealed that the introduced strains did not disrupt physicochemical conditions and indigenous soil microbial composition, which suggests that they are promising amendments preserving the natural biodiversity of soil and increasing its fertility.

Development and validation of novel PCR primers for identification of plasmid-mediated colistin resistance (mcr) genes in various environmental settings.

Antibiotic resistance is considered one of the biggest threats to public health and has become a major concern for governments and international organizations. Combating this problem starts with improving global surveillance of antibiotic resistance genes (ARGs) and applying standardized protocols, both in a clinical and environmental context, in agreement with the One Health approach. Exceptional efforts should be directed to controlling ARGs conferring resistance to Critically Important Antimicrobials (CIA). In this study, a systematic literature review to synthesize data on the identification of mcr genes using a PCR technique was performed. Additionally, a novel set of PCR primers for mcr-1 - mcr-9 genes detection was proposed. The developed primers were in silico and experimentally validated by comparison with mcr-specific PCR primers reported in the literature. This validation, besides being a proof-of-concept for primers' usefulness, provided insight into the distribution of mcr genes in municipal wastewater, clay and river sediments, glacier moraine, manure, seagulls and auks feces and daphnids from four countries. This analysis proved that commonly used primers may deliver false results, and some mcr genes may be overlooked in tested samples. Newly-developed PCR primers turned out to be relevant for the screening of mcr genes in various environments.

Monitoring antibiotic resistance genes in wastewater environments: The challenges of filling a gap in the One-Health cycle.

Antibiotic resistance (AR) is a global problem requiring international cooperation and coordinated action. Global monitoring must rely on methods available and comparable across nations to quantify AR occurrence and identify sources and reservoirs, as well as paths of AR dissemination. Numerous analytical tools that are gaining relevance in microbiology, have the potential to be applied to AR research. This review summarizes the state of the art of AR monitoring methods, considering distinct needs, objectives and available resources. Based on the overview of distinct approaches that are used or can be adapted to monitor AR, it is discussed the potential to establish reliable and useful monitoring schemes that can be implemented in distinct contexts. This discussion places the environmental monitoring within the One-Health approach, where two types of risk, dissemination across distinct environmental compartments, and transmission to humans, must be considered. The plethora of methodological approaches to monitor AR and the variable features of the monitored sites challenge the capacity of the scientific community and policy makers to reach a common understanding. However, the dialogue between different methods and the production of action-oriented data is a priority. The review aims to warm up this discussion.

Insight Into Ecology, Metabolic Potential, and the Taxonomic Composition of Bacterial Communities in the Periodic Water Pond on King George Island (Antarctica).

Polar regions contain a wide variety of lentic ecosystems. These include periodic ponds that have a significant impact on carbon and nitrogen cycling in polar environments. This study was conducted to assess the taxonomic and metabolic diversity of bacteria found in Antarctic pond affected by penguins and sea elephants and to define their role in ongoing processes. Metabolic assays showed that of the 168 tested heterotrophic bacteria present in the Antarctic periodic pond, 96% are able to degrade lipids, 30% cellulose, 26% proteins, and 26% starch. The taxonomic classification of the obtained isolates differs from that based on the composition of the 16S rRNA relative abundances in the studied pond. The dominant Actinobacteria constituting 45% of isolates represents a low proportion of the community, around 4%. With the addition of run-off, the proportions of inhabiting bacteria changed, including a significant decrease in the abundance of Cyanobacteria, from 2.38 to 0.33%, increase of Firmicutes from 9.32 to 19.18%, and a decreasing richness (Chao1 index from 1299 to 889) and diversity (Shannon index from 4.73 to 4.20). Comparative studies of communities found in different Antarctic environments indicate a great role for penguins in shaping bacterial populations.

Metaplasmidome-encoded functions of Siberian low-centered polygonal tundra soils.

Plasmids have the potential to transfer genetic traits within bacterial communities and thereby serve as a crucial tool for the rapid adaptation of bacteria in response to changing environmental conditions. Our knowledge of the environmental pool of plasmids (the metaplasmidome) and encoded functions is still limited due to a lack of sufficient extraction methods and tools for identifying and assembling plasmids from metagenomic datasets. Here, we present the first insights into the functional potential of the metaplasmidome of permafrost-affected active-layer soil-an environment with a relatively low biomass and seasonal freeze-thaw cycles that is strongly affected by global warming. The obtained results were compared with plasmid-derived sequences extracted from polar metagenomes. Metaplasmidomes from the Siberian active layer were enriched via cultivation, which resulted in a longer contig length as compared with plasmids that had been directly retrieved from the metagenomes of polar environments. The predicted hosts of plasmids belonged to Moraxellaceae, Pseudomonadaceae, Enterobacteriaceae, Pectobacteriaceae, Burkholderiaceae, and Firmicutes. Analysis of their genetic content revealed the presence of stress-response genes, including antibiotic and metal resistance determinants, as well as genes encoding protectants against the cold.

Simple, Reliable, and Time-Efficient Manual Annotation of Bacterial Genomes with MAISEN.

Over the last 15 years, the costs of DNA sequencing have sharply fallen, effectively shifting the costs of DNA analysis from sequencing to bioinformatic curation and storage. A huge number of available DNA sequences (including genomes and metagenomes) resulted in the development of various tools for sequence annotation. While much effort has been invested into the development of automatic annotation pipelines, manual curation of their results is still necessary in order to obtain a reliable and strictly validated data. Unfortunately, due to its time-consuming nature, manual annotation is now rarely used.In this chapter, a protocol for efficient manual annotation of prokaryotic DNA sequences using a novel bioinformatic tool-MAISEN ( http://maisen.ddlemb.com ), is presented. MAISEN is a free, web-based tool designed to accelerate manual annotation, by providing the user with simple interface and precomputed alignments for each predicted feature. It was designed to be available for every scientist, regardless of their bioinformatic proficiency.

The Bad and the Good-Microorganisms in Cultural Heritage Environments-An Update on Biodeterioration and Biotreatment Approaches.

Cultural heritage objects constitute a very diverse environment, inhabited by various bacteria and fungi. The impact of these microorganisms on the degradation of artworks is undeniable, but at the same time, some of them may be applied for the efficient biotreatment of cultural heritage assets. Interventions with microorganisms have been proven to be useful in restoration of artworks, when classical chemical and mechanical methods fail or produce poor or short-term effects. The path to understanding the impact of microbes on historical objects relies mostly on multidisciplinary approaches, combining novel meta-omic technologies with classical cultivation experiments, and physico-chemical characterization of artworks. In particular, the development of metabolomic- and metatranscriptomic-based analyses associated with metagenomic studies may significantly increase our understanding of the microbial processes occurring on different materials and under various environmental conditions. Moreover, the progress in environmental microbiology and biotechnology may enable more effective application of microorganisms in the biotreatment of historical objects, creating an alternative to highly invasive chemical and mechanical methods.

Genome-Based Insights into the Production of Carotenoids by Antarctic Bacteria, Planococcus sp. ANT_H30 and Rhodococcus sp. ANT_H53B.

Antarctic regions are characterized by low temperatures and strong UV radiation. This harsh environment is inhabited by psychrophilic and psychrotolerant organisms, which have developed several adaptive features. In this study, we analyzed two Antarctic bacterial strains, Planococcus sp. ANT_H30 and Rhodococcus sp. ANT_H53B. The physiological analysis of these strains revealed their potential to produce various biotechnologically valuable secondary metabolites, including surfactants, siderophores, and orange pigments. The genomic characterization of ANT_H30 and ANT_H53B allowed the identification of genes responsible for the production of carotenoids and the in silico reconstruction of the pigment biosynthesis pathways. The complex manual annotation of the bacterial genomes revealed the metabolic potential to degrade a wide variety of compounds, including xenobiotics and waste materials. Carotenoids produced by these bacteria were analyzed chromatographically, and we proved their activity as scavengers of free radicals. The quantity of crude carotenoid extracts produced at two temperatures using various media was also determined. This was a step toward the optimization of carotenoid production by Antarctic bacteria on a larger scale.

Effect of Clinoptilolite and Halloysite Addition on Biogas Production and Microbial Community Structure during Anaerobic Digestion.

The study presents a comparison of the influence of a clinoptilolite-rich rock-zeolite (commonly used for improving anaerobic digestion processes)-and a highly porous clay mineral, halloysite (mainly used for gas purification), on the biogas production process. Batch experiments showed that the addition of each mineral increased the efficiency of mesophilic anaerobic digestion of both sewage sludge and maize silage. However, halloysite generated 15% higher biogas production during maize silage transformation. Halloysite also contributed to a much higher reduction of chemical oxygen demand for both substrates (by ~8% for maize silage and ~14% for sewage sludge) and a higher reduction of volatile solids and total ammonia for maize silage (by ~8% and ~4%, respectively). Metagenomic analysis of the microbial community structure showed that the addition of both mineral sorbents influenced the presence of key members of archaea and bacteria occurring in a well-operated biogas reactor. The significant difference between zeolite and halloysite is that the latter promoted the immobilization of key methanogenic archaea Methanolinea (belong to Methanomicrobia class). Based on this result, we postulate that halloysite could be useful not only as a sorbent for (bio)gas treatment methodologies but also as an agent for improving biogas production.

Molecular Characterization and Comparative Genomics of IncQ-3 Plasmids Conferring Resistance to Various Antibiotics Isolated from a Wastewater Treatment Plant in Warsaw (Poland).

As small, mobilizable replicons with a broad host range, IncQ plasmids are widely distributed among clinical and environmental bacteria. They carry antibiotic resistance genes, and it has been shown that they confer resistance to ß-lactams, fluoroquinolones, aminoglycosides, trimethoprim, sulphonamides, and tetracycline. The previously proposed classification system divides the plasmid group into four subgroups, i.e., IncQ-1, IncQ-2, IncQ-3, and IncQ-4. The last two subgroups have been poorly described so far. The aim of this study was to analyze five newly identified IncQ-3 plasmids isolated from a wastewater treatment plant in Poland and to compare them with all known plasmids belonging to the IncQ-3 subgroup whose sequences were retrieved from the NCBI database. The complete nucleotide sequences of the novel plasmids were annotated and bioinformatic analyses were performed, including identification of core genes and auxiliary genetic load. Furthermore, functional experiments testing plasmid mobility were carried out. Phylogenetic analysis based on three core genes (repA, mobA/repB, and mobC) revealed the presence of three main clusters of IncQ-3 replicons. Apart from having a highly conserved core, the analyzed IncQ-3 plasmids were vectors of antibiotic resistance genes, including (I) the qnrS2 gene that encodes fluoroquinolone resistance and (II) ß-lactam, trimethoprim, and aminoglycoside resistance genes within integron cassettes.

In vivo creation of plasmid pCRT01 and its use for the construction of carotenoid-producing Paracoccus spp. strains that grow efficiently on industrial wastes.

BACKGROUND: Carotenoids are natural tetraterpene pigments widely utilized in the food, pharmaceutical and cosmetic industries. Currently, chemical synthesis of these compounds outperforms their production in Escherichia coli or yeast due to the limited efficiency of the latter. The use of natural microbial carotenoid producers, such as bacteria of the genus Paracoccus (Alphaproteobacteria), may help to optimize this process. In order to couple the ability to synthesize these pigments with the metabolic versatility of this genus, we explored the possibility of introducing carotenoid synthesis genes into strains capable of efficient growth on simple low-cost media. RESULTS: We constructed two carotenoid-producing strains of Paracoccus carrying a new plasmid, pCRT01, which contains the carotenoid synthesis gene locus crt from Paracoccus marcusii OS22. The plasmid was created in vivo via illegitimate recombination between crt-carrying vector pABW1 and a natural "paracoccal" plasmid pAMI2. Consequently, the obtained fusion replicon is stably maintained in the bacterial population without the need for antibiotic selection. The introduction of pCRT01 into fast-growing "colorless" strains of Paracoccus aminophilus and Paracoccus kondratievae converted them into efficient producers of a range of both carotenes and xanthophylls. The exact profile of the produced pigments was dependent on the strain genetic background. To reduce the cost of carotenoid production in this system, we tested the growth and pigment synthesis efficiency of the two strains on various simple media, including raw industrial effluent (coal-fired power plant flue gas desulfurization wastewater) supplemented with molasses, an industrial by-product rich in sucrose. CONCLUSIONS: We demonstrated a new approach for the construction of carotenoid-producing bacterial strains which relies on a single plasmid-mediated transfer of a pigment synthesis gene locus between Paracoccus strains. This strategy facilitates screening for producer strains in terms of synthesis efficiency, pigment profile and ability to grow on low-cost industrial waste-based media, which should increase the cost-effectiveness of microbial production of carotenoids.