Genomic insights into the plant-associated lifestyle of Kosakonia radicincitans MUSA4, a diazotrophic plant-growth-promoting bacterium.

The process of nitrogen (N) fixation by plant-associated bacteria plays an indispensable role in the development of novel agricultural solutions worldwide. In this sense, it is of extreme importance to identify and understand the properties of efficient plant-growth-promoting bacteria (PGPB) that are able to fix N. In this study, the characterization and detailed genomic analysis of the diazotrophic bacterium Kosakonia radicincitans MUSA4, isolated from the internal leaf tissues of a banana tree in Brazil, were undertaken. K. radicincitans MUSA4 presented several plant-growth-promoting traits, including indoleacetic acid, siderophore, acetoin and polyamine biosynthesis, phosphate solubilization, and nitrogen fixation. The strain was able to increase cucumber plant growth significantly, demonstrating its potential in beneficial interactions with plant hosts. Detailed genomic analysis of strain MUSA4 revealed the abundant presence of genes involved in plant colonization, stress resistance and plant-growth-promoting abilities. Moreover, the genome harbored the nif and anf gene clusters, encoding the Fe-Mo nitrogenase and Fe-Fe nitrogenase systems, respectively. Comparative genomic analysis also showed that strain MUSA4 possessed several strain-specific genes, which could be related to its evolutionary history in Brazilian mangrove environments. The results obtained in the present study revealed the plant beneficial role and biotechnological potential of K. radicincitans MUSA4, and provided new insights into plant colonization and plant growth promoting mechanisms employed by diazotrophic Kosakonia.

Genomic Analysis of the 1-Aminocyclopropane-1-Carboxylate Deaminase-Producing Pseudomonas thivervalensis SC5 Reveals Its Multifaceted Roles in Soil and in Beneficial Interactions With Plants.

Beneficial 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing bacteria promote plant growth and stress resistance, constituting a sustainable alternative to the excessive use of chemicals in agriculture. In this work, the increased plant growth promotion activity of the ACC deaminase-producing Pseudomonas thivervalensis SC5, its ability to limit the growth of phytopathogens, and the genomics behind these important properties are described in detail. P. thivervalensis SC5 displayed several active plant growth promotion traits and significantly increased cucumber plant growth and resistance against salt stress (100mmol/L NaCl) under greenhouse conditions. Strain SC5 also limited the in vitro growth of the pathogens Botrytis cinerea and Pseudomonas syringae DC3000 indicating active biological control activities. Comprehensive analysis revealed that P. thivervalensis SC5 genome is rich in genetic elements involved in nutrient acquisition (N, P, S, and Fe); osmotic stress tolerance (e.g., glycine-betaine, trehalose, and ectoine biosynthesis); motility, chemotaxis and attachment to plant tissues; root exudate metabolism including the modulation of plant phenolics (e.g., hydroxycinnamic acids), lignin, and flavonoids (e.g., quercetin); resistance against plant defenses (e.g., reactive oxygens species-ROS); plant hormone modulation (e.g., ethylene, auxins, cytokinins, and salicylic acid), and bacterial and fungal phytopathogen antagonistic traits (e.g., 2,4-diacetylphloroglucinol, HCN, a fragin-like non ribosomal peptide, bacteriocins, a lantipeptide, and quorum-quenching activities), bringing detailed insights into the action of this versatile plant-growth-promoting bacterium. Ultimately, the combination of both increased plant growth promotion/protection and biological control abilities makes P. thivervalensis SC5 a prime candidate for its development as a biofertilizer/biostimulant/biocontrol product. The genomic analysis of this bacterium brings new insights into the functioning of Pseudomonas and their role in beneficial plant-microbe interactions.

Multiple plant hormone catabolism activities: an adaptation to a plant-associated lifestyle by Achromobacter spp.

Elaborating the plant hormone catabolic activities of bacteria is important for developing a detailed understanding of plant-microbe interactions. In this work, the plant hormone catabolic and plant growth promotion activities of Achromobacter xylosoxidans SOLR10 and A. insolitus AB2 are described. The genome sequences of these strains were obtained and analysed in detail, revealing the genetic mechanisms behind its multiple plant hormone catabolism abilities. Achromobacter strains catabolized indoleacetic acid (IAA) and phenylacetic acid (PAA) (auxins); salicylic acid (SA) and its precursor, benzoic acid (BA); and the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). The inoculation of cucumber plants resulted in increased plant growth and development, indicating the beneficial properties of SOLR10 and AB2 strains. Genomic analysis demonstrated the presence of IAA, PAA and BA degradation gene clusters, as well as the nag gene cluster (SA catabolism) and the acdS gene (ACC deaminase), in the genomes of strains SOLR10 and AB2. Additionally, detailed analysis revealed that plant hormone catabolism genes were commonly detected in the Achromobacter genus but were mostly absent in the Bordetella genus, consistent with the notion that Achromobacter evolved in soils in close association with its plant hosts.

The extreme plant-growth-promoting properties of Pantoea phytobeneficialis MSR2 revealed by functional and genomic analysis.

Numerous Pantoea strains are important because of the benefit they provide in the facilitation of plant growth. However, Pantoea have a high level of genotypic diversity and not much is understood regarding their ability to function in a plant beneficial manner. In the work reported here, the plant growth promotion activities and the genomic properties of the unusual Pantoea phytobeneficialis MSR2 are elaborated, emphasizing the genetic mechanisms involved in plant colonization and growth promotion. Detailed analysis revealed that strain MSR2 belongs to a rare group of Pantoea strains possessing an astonishing number of plant growth promotion genes, including those involved in nitrogen fixation, phosphate solubilization, 1-aminocyclopropane-1-carboxylic acid deaminase activity, indoleacetic acid and cytokinin biosynthesis, and jasmonic acid metabolism. Moreover, the genome of this bacterium also contains genes involved in the metabolism of lignin and other plant cell wall compounds, quorum-sensing mechanisms, metabolism of plant root exudates, bacterial attachment to plant surfaces and resistance to plant defences. Importantly, the analysis revealed that most of these genes are present on accessory plasmids that are found within a small subset of Pantoea genomes, reinforcing the idea that Pantoea evolution is largely mediated by plasmids, providing new insights into the evolution of beneficial plant-associated Pantoea.

Plant growth-promoting activities and genomic analysis of the stress-resistant Bacillus megaterium STB1, a bacterium of agricultural and biotechnological interest.

In this work, the stress-resistant Bacillus megaterium STB1 is characterized and its ability to promote plant growth under normal and stress conditions is demonstrated. The genomic sequence of this bacterium, and a detailed analysis of the genes involved in facilitating its stress resistance and plant growth-promoting activities is also reported. The B. megaterium STB1 genome is rich in genetic elements involved in multiple stress resistance, xenobiotic degradation, pathogen antagonistic activities, and other traits related to soil and rhizosphere colonization. Moreover, genes participating in the biosynthesis of auxins and cytokinins, the modulation of polyamines, GABA, brassinosteroids and ethylene levels were also found. Ultimately, this study brings new insights into the role of B. megaterium as a plant growth-promoting bacterium and opens new opportunities for the development of novel strategies for agriculture and biotechnology.

ACC deaminase plays a major role in Pseudomonas fluorescens YsS6 ability to promote the nodulation of Alpha- and Betaproteobacteria rhizobial strains.

Ethylene acts as a major regulator of the nodulation process of leguminous plants. Several rhizobial strains possess the ability to modulate plant ethylene levels through the expression of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase; however, rhizobia present low enzymatic activities. One possible alternative to this problem resides on the use of free-living bacteria, such as Pseudomonas, presenting high levels of ACC deaminase activity that may be used as adjuvants in the nodulation process by decreasing inhibitory ethylene levels. Nevertheless, not much is understood about the specific role of ACC deaminase in the possible role of free-living bacteria as nodulation adjuvants. Therefore, this work aims to study the effect of ACC deaminase in the plant growth-promoting bacterium, Pseudomonas fluorescens YsS6, ability to facilitate alpha- and beta-rhizobia nodulation. The ACC deaminase-producing P. fluorescens YsS6 and its ACC deaminase mutant were used in co-inoculation assays to evaluate their impact in the nodulation process of alpha- (Rhizobium tropici CIAT899) and beta-rhizobia (Cupriavidus taiwanensis STM894) representatives, in Phaseolus vulgaris and Mimosa pudica plants, respectively. The results obtained indicate that the wild-type P. fluorescens YsS6, but not its mutant defective in ACC deaminase production, increase the nodulation abilities of both alpha- and beta-rhizobia, resulting in an increased leguminous plant growth. Moreover, this is the first report of the positive effect of free-living bacteria in the nodulation process of beta-rhizobia. The modulation of inhibitory ethylene levels by free-living ACC deaminase-producing bacteria plays an important role in facilitating the nodulation process of alpha- and beta-rhizobia.

Isolation and characterization of novel soil- and plant-associated bacteria with multiple phytohormone-degrading activities using a targeted methodology.

Ethylene (ET), salicylic acid (SA) and indole-3-acetic acid (IAA) are important phytohormones regulating plant growth and development, as well as plant-microbe interactions. Plant growth-promoting bacteria (PGPB) naturally associate with plants and facilitate plant growth through a variety of mechanisms, including the ability to modulate the concentrations of these phytohormones in planta. Importantly, the wide presence of phytohormone degradation mechanisms amongst symbiotic and other soil- and plant-associated bacteria indicates that the ability to modulate phytohormone concentrations plays an important role in bacterial colonization and plant-growth promotion abilities. Obtaining phytohormone-degrading bacteria is therefore key for the development of novel solutions aiming to increase plant growth and protection. In this paper, we report an optimized targeted methodology and the consequent isolation of novel soil- and plant-associated bacteria, including rhizospheric, endophytic and phyllospheric strains, with the ability to degrade the phytohormones, SA and IAA, as well as the ET precursor, 1-aminocyclopropane-1-carboxylic acid (ACC). By using an optimized targeted methodology, we rapidly isolated diverse soil- and plant-associated bacteria presenting phytohormone-degrading abilities from several plants, plant tissues and environments, without the need for prior extensive and laborious isolation and maintenance of large numbers of isolates. The developed methodology facilitates PGPB research, especially in developing countries. Here, we also report, for the first time, the isolation of bacterial strains able to concomitantly catabolize three phytohormones (SA, IAA and ACC). Ultimately, the described targeted methodology and the novel phytohormone-degrading bacteria obtained in this work may be useful tools for future plant-microbe interaction studies, and in the development of new inoculant formulations for agriculture and biotechnology.

Microbially-enriched poultry litter-derived biochar for the treatment of acid mine drainage.

Sulfate-reducing bacteria (SRB) and a biochar array were used to reduce sulfate concentrations and the levels of metals in acid mine drainage (AMD) waters. Cow manure SRB-enriched biochar promoted sulfate reductions of 41% compared to original AMD, and 39% compared to other treatments (control, AMD sediment, sludge). Treatments reduced levels of all analyzed metals below Brazilian official standards. DGGE showed a significant relation between SRB-source and SRB-structural community, where cow manure and sludge presented the more cohesive community structure throughout the monitoring (180 days). The study showed that AMD treatment alternatives can be applied and are effective in reducing the contamination of wastewaters.

Near-Complete Genome Sequence of Pseudomonas palleroniana MAB3, a Beneficial 1-Aminocyclopropane-1-Carboxylate Deaminase-Producing Bacterium Able To Promote the Growth of Mushrooms and Plants.

The near-complete genome sequence of Pseudomonas palleroniana MAB3, a 1-aminocyclopropane-1-carboxylate deaminase-producing bacterium isolated from an environmental soil Amanita mushroom, is presented here. The genome of P. palleroniana MAB3 contains a single circular chromosome of 6.29 Mb and an average GC content of 60.5%.

Improvement of Cupriavidus taiwanensis Nodulation and Plant Growth Promoting Abilities by the Expression of an Exogenous ACC Deaminase Gene.

Several rhizobial strains possess the ability to modulate leguminous plants ethylene levels by producing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase. While the effect of ACC deaminase has been studied in several rhizobia belonging to the Alphaproteobacteria class, not much is understood about its impact in the nodulation abilities of rhizobia belonging to the Betaproteobacteria class, which are common symbionts of Mimosa species. In this work, we report the impact of ACC deaminase production by the Betaproteobacterium, Cupriavidus taiwanensis STM894, and its role in the nodulation of Mimosa pudica. C. taiwanensis STM894 was studied following its transformation with the plasmid pRKACC, containing an ACC deaminase gene. The expression of the exogenous ACC deaminase led to increased nodulation and M. pudica growth promotion by C. taiwanensis STM894. These results indicate that ACC deaminase plays an important role in modulating ethylene levels that inhibit the nodulation process induced by both rhizobia belonging to the Alpha and Betaproteobacteria class.

Ethylene and 1-Aminocyclopropane-1-carboxylate (ACC) in Plant-Bacterial Interactions.

Ethylene and its precursor 1-aminocyclopropane-1-carboxylate (ACC) actively participate in plant developmental, defense and symbiotic programs. In this sense, ethylene and ACC play a central role in the regulation of bacterial colonization (rhizospheric, endophytic, and phyllospheric) by the modulation of plant immune responses and symbiotic programs, as well as by modulating several developmental processes, such as root elongation. Plant-associated bacterial communities impact plant growth and development, both negatively (pathogens) and positively (plant-growth promoting and symbiotic bacteria). Some members of the plant-associated bacterial community possess the ability to modulate plant ACC and ethylene levels and, subsequently, modify plant defense responses, symbiotic programs and overall plant development. In this work, we review and discuss the role of ethylene and ACC in several aspects of plant-bacterial interactions. Understanding the impact of ethylene and ACC in both the plant host and its associated bacterial community is key to the development of new strategies aimed at increased plant growth and protection.

The modulation of leguminous plant ethylene levels by symbiotic rhizobia played a role in the evolution of the nodulation process.

Ethylene plays an important role in regulating the rhizobial nodulation process. Consequently, numerous strains of rhizobia possess the ability to decrease plant ethylene levels by the expression of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase or via the production of rhizobitoxine, thus, leading to an increased ability to nodulate leguminous plants. Nevertheless, not much is understood about the prevalence of these ethylene modulation genes in different rhizobial groups nor their role in the evolution of the symbiotic process. In this work, we analyze the prevalence and evolution of the enzymes ACC deaminase (AcdS) and dihydrorhizobitoxine desaturase (RtxC) in 395 NodC+ genomes from different rhizobial strains isolated from a wide range of locations and plant hosts, and discuss their importance in the evolution of the symbiotic process. The obtained results show that AcdS and RtxC are differentially prevalent in rhizobial groups, indicating the existence of several selection mechanisms governed by the rhizobial strain itself and its evolutionary origin, the environment, and, importantly, the leguminous plant host (co-evolution). Moreover, it was found that the prevalence of AcdS and RtxC is increased in Bradyrhizobium and Paraburkholderia, and lower in other groups. Data obtained from phylogenetic, evolutionary as well as gene localization analysis support the previous hypotheses regarding the ancient origin of the nodulation abilities in Bradyrhizobium and Paraburkholderia, and brings a new perspective for the importance of ethylene modulation genes in the development of the symbiotic process. The acquisition of AcdS by horizontal gene transfer and a positive selection in other rhizobial groups indicates that this enzyme plays an important role in the nodulation process of many rhizobia. On the other hand, RtxC is negatively selected in most symbioses. Understanding the evolution of ethylene modulation genes in rhizobia may be the key to the development of new strategies aiming for an increased nodulation and nitrogen fixation process.

Morphological and ultrastructural characterization of the acidophilic and lipid-producer strain Chlamydomonas acidophila LAFIC-004 (Chlorophyta) under different culture conditions.

Chlamydomonas acidophila LAFIC-004 is an acidophilic strain of green microalgae isolated from coal mining drainage. In the present work, this strain was cultivated in acidic medium (pH 3.6) under phototrophic, mixotrophic, and heterotrophic regimes to determine the best condition for growth and lipid production, simultaneously assessing possible morphological and ultrastructural alterations in the cells. For heterotrophic and mixotrophic treatments, two organic carbon sources were tested: 1 % glucose and 1 % sodium acetate. Lipid content and fatty acid profiles were only determined in phototrophic condition. The higher growth rates were achieved in phototrophic conditions, varying from 0.18 to 0.82 day-1. Glucose did not result in significant growth increase in either mixotrophic or heterotrophic conditions, and acetate proved to be toxic to the strain in both conditions. Oil content under phototrophic condition was 15.9 % at exponential growth phase and increased to 54.63 % at stationary phase. Based on cell morphology (flow cytometry and light microscopy) and ultrastructure (transmission electron microscopy), similar characteristics were observed between phototrophic and mixotrophic conditions with glucose evidencing many lipid bodies, starch granules, and intense fluorescence. Under the tested conditions, mixotrophic and heterotrophic modes did not result in increased neutral lipid fluorescence. It can be concluded that the strain is a promising lipid producer when grown until stationary phase in acidic medium and under a phototrophic regime, presenting a fatty acid profile suitable for biodiesel production. The ability to grow this strain in acidic mining residues suggests a potential for bioremediation with production of useful biomass.

Non-specific transient mutualism between the plant parasitic nematode, Bursaphelenchus xylophilus, and the opportunistic bacterium Serratia quinivorans BXF1, a plant-growth promoting pine endophyte with antagonistic effects.

The aim of this study is to understand the biological role of Serratia quinivorans BXF1, a bacterium commonly found associated with Bursaphelenchus xylophilus, the plant parasitic nematode responsible for pine wilt disease. Therefore, we studied strain BXF1 effect in pine wilt disease. We found that strain BXF1 promoted in vitro nematode reproduction. Moreover, the presence of bacteria led to the absence of nematode chitinase gene (Bxcht-1) expression, suggesting an effect for bacterial chitinase in nematode reproduction. Nevertheless, strain BXF1 was unable to colonize the nematode interior, bind to its cuticle with high affinity or protect the nematode from xenobiotic stress. Interestingly, strain BXF1 was able to promote tomato and pine plant-growth, as well as to colonize its interior, thus, acting like a plant-growth promoting endophyte. Consequently, strain BXF1 failed to induce wilting symptoms when inoculated in pine shoot artificial incisions. This bacterium also presented strong antagonistic activities against fungi and bacteria isolated from Pinus pinaster. Our results suggest that B. xylophilus does not possess a strict symbiotic community capable of inducing pine wilt disease symptoms as previously hypothesized. We show that bacteria like BXF1, which possess plant-growth promoting and antagonistic effects, may be opportunistically associated with B. xylophilus, possibly acquired from the bacterial endophytic community of the host pine.

New insights into 1-aminocyclopropane-1-carboxylate (ACC) deaminase phylogeny, evolution and ecological significance.

The main objective of this work is the study of the phylogeny, evolution and ecological importance of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, the activity of which represents one of the most important and studied mechanisms used by plant growth-promoting microorganisms. The ACC deaminase gene and its regulatory elements presence in completely sequenced organisms was verified by multiple searches in diverse databases, and based on the data obtained a comprehensive analysis was conducted. Strain habitat, origin and ACC deaminase activity were taken into account when analyzing the results. In order to unveil ACC deaminase origin, evolution and relationships with other closely related pyridoxal phosphate (PLP) dependent enzymes a phylogenetic analysis was also performed. The data obtained show that ACC deaminase is mostly prevalent in some Bacteria, Fungi and members of Stramenopiles. Contrary to previous reports, we show that ACC deaminase genes are predominantly vertically inherited in various bacterial and fungal classes. Still, results suggest a considerable degree of horizontal gene transfer events, including interkingdom transfer events. A model for ACC deaminase origin and evolution is also proposed. This study also confirms the previous reports suggesting that the Lrp-like regulatory protein AcdR is a common mechanism regulating ACC deaminase expression in Proteobacteria, however, we also show that other regulatory mechanisms may be present in some Proteobacteria and other bacterial phyla. In this study we provide a more complete view of the role for ACC deaminase than was previously available. The results show that ACC deaminase may not only be related to plant growth promotion abilities, but may also play multiple roles in microorganism's developmental processes. Hence, exploring the origin and functioning of this enzyme may be the key in a variety of important agricultural and biotechnological applications.