Genomic insights and anti-phytopathogenic potential of siderophore metabolome of endolithic Nocardia mangyaensis NH1.

Actinobacteria are one of the predominant groups that successfully colonize and survive in various aquatic, terrestrial and rhizhospheric ecosystems. Among actinobacteria, Nocardia is one of the most important agricultural and industrial bacteria. Screening and isolation of Nocardia related bacteria from extreme habitats such as endolithic environments are beneficial for practical applications in agricultural and environmental biotechnology. In this work, bioinformatics analysis revealed that a novel strain Nocardia mangyaensis NH1 has the capacity to produce structurally varied bioactive compounds, which encoded by non-ribosomal peptide synthases (NRPS), polyketide synthase (PKS), and post-translationally modified peptides (RiPPs). Among NRPS, five gene clusters have a sequence homology with clusters encoding for siderophore synthesis. We also show that N. mangyaensis NH1 accumulates both catechol- and hydroxamate-type siderophores simultaneously under iron-deficient conditions. Untargeted LC-MS/MS analysis revealed a variety of metabolites, including siderophores, lipopeptides, cyclic peptides, and indole-3-acetic acid (IAA) in the culture medium of N. mangyaensis NH1 grown under iron deficiency. We demonstrate that four CAS (chrome azurol S)-positive fractions display variable affinity to metals, with a high Fe3+ chelating capability. Additionally, three of these fractions exhibit antioxidant activity. A combination of iron scavenging metabolites produced by N. mangyaensis NH1 showed antifungal activity against several plant pathogenic fungi. We have shown that the pure culture of N. mangyaensis NH1 and its metabolites have no adverse impact on Arabidopsis seedlings. The ability of N. mangyaensis NH1 to produce siderophores with antifungal, metal-chelating, and antioxidant properties, when supplemented with phytohormones, has the potential to improve the release of macro- and micronutrients, increase soil fertility, promote plant growth and development, and enable the production of biofertilizers across diverse soil systems.

Serratia marcescens DUF1471-Containing Protein SrfN Is Needed for Adaptation to Acid and Oxidative Stresses.

Bacteria can quickly adapt to constantly changing environments through a number of mechanisms, including secretion of secondary metabolites, peptides, and proteins. Serratia marcescens, an emerging pathogen with growing clinical importance due to its intrinsic resistance to several classes of antibiotics, can cause an array of infections in immunocompromised individuals. To better control the spread of S. marcescens infections, it is critical to identify additional targets for bacterial growth inhibition. We found that extracellular metabolites produced by the wild-type organism in response to peroxide exposure had a protective effect on an otherwise-H2O2-sensitive ΔmacAB indicator strain. Detailed analysis of the conditioned medium demonstrated that the protective effect was associated with a low-molecular-weight heat-sensitive and proteinase K-sensitive metabolite. Furthermore, liquid chromatography-tandem mass spectrometry analysis of the low-molecular-weight proteins present in the conditioned medium led to identification of the previously uncharacterized DUF1471-containing protein TBU67220 (SrfN). We found that loss of the srfN gene did not have an impact on the production of extracellular enzymes. However, the S. marcescens mutant lacking SrfN was significantly more sensitive to growth in medium with a low pH and to exposure to oxidative stress. Both defects were fully rescued by complementation. Thus, our results indicate that SrfN, a low-molecular-weight DUF1471-containing protein, is involved in S. marcescens SM6 adaptation to adverse environmental conditions. IMPORTANCE Serratia marcescens is ubiquitous in the environment and can survive in water, soil, plants, insects, and animals, and it can also cause infections in humans. In the face of disturbances such as oxidative or low-pH stress, bacteria adapt, survive, and recover through several mechanisms, including changes in their secretome. We show that a hydrogen peroxide-exposed S. marcescens milieu contains a small previously uncharacterized DUF1471-containing protein similar to the SrfN protein in Salmonella enterica serovar Typhimurium, and we illustrate the role of this protein in bacterial survival during acid and oxidative stresses.

Genomic and metabolomic profiling of endolithic Rhodococcus fascians strain S11 isolated from an arid serpentine environment.

Genomic and metabolomic studies of endolithic bacteria are essential for understanding their adaptations to extreme conditions of the rock environment and their contributions to mineralization and weathering processes. The endoliths of arid serpentine rocks are exposed to different environmental stresses, including desiccation and re-hydration, temperature fluctuations, oligotrophy, and high concentrations of heavy metals. Bacteria of the genus Rhodococcus commonly inhabit endolithic environments. Here, we describe genomic and metabolomic analyses of the non-pathogenic wild-type Rhodococcus fascians strain S11, isolated from weathered serpentine rock at the arid Khalilovsky massif, Russia. We found that strain S11 lacks the virulence plasmid that functions in the phytopathogenecity of some R. fascians strains. Phenotypic profiling revealed a high pH tolerance, phytase activity and siderophore production. A widely untargeted metabolome analysis performed using an Orbitrap LC-MS/MS method demonstrated the presence of chrysobactin-type siderophores in the culture medium of strain S11. The natural variation of secondary metabolites produced by strain S11 might provide a practical basis for revealing antibacterial, fungicide or insecticidal activities. Finally, plant infection and plant growth stimulation studies showed no observable effect of exposure strain S11 bacteria on the aerial and root parts of Arabidopsis thaliana plants. Based on our findings, R. fascians strain S11 might be promising tool for investigations of organo-mineral interactions, heavy metal bioremediation, and mechanisms of bacterial mediated weathering of plant-free serpentine rock to soil.

The ABC-Type Efflux Pump MacAB Is Involved in Protection of Serratia marcescens against Aminoglycoside Antibiotics, Polymyxins, and Oxidative Stress.

Serratia marcescens is an emerging pathogen with increasing clinical importance due to its intrinsic resistance to several classes of antibiotics. The chromosomally encoded drug efflux pumps contribute to antibiotic resistance and represent a major challenge for the treatment of bacterial infections. The ABC-type efflux pump MacAB was previously linked to macrolide resistance in Escherichia coli and Salmonella enterica serovar Typhimurium. The role of the MacAB homolog in antibiotic resistance of S. marcescens is currently unknown. We found that an S. marcescens mutant lacking the MacAB pump did not show increased sensitivity to the macrolide antibiotic erythromycin but was significantly more sensitive to aminoglycoside antibiotics and polymyxins. We also showed that, in addition to its role in drug efflux, the MacAB efflux pump is required for swimming motility and biofilm formation. We propose that the motility defect of the ΔmacAB mutant is due, at least in part, to the loss of functional flagella on the bacterial surface. Furthermore, we found that the promoter of the MacAB efflux pump was active during the initial hours of growth in laboratory medium and that its activity was further elevated in the presence of hydrogen peroxide. Finally, we demonstrate a complete loss of ΔmacAB mutant viability in the presence of peroxide, which is fully restored by complementation. Thus, the S. marcescens MacAB efflux pump is essential for survival during oxidative stress and is involved in protection from polymyxins and aminoglycoside antibiotics.IMPORTANCE The opportunistic pathogen Serratia marcescens can cause urinary tract infections, respiratory infections, meningitis, and sepsis in immunocompromised individuals. These infections are challenging to treat due to the intrinsic resistance of S. marcescens to an extensive array of antibiotics. Efflux pumps play a crucial role in protection of bacteria from antimicrobials. The MacAB efflux pump, previously linked to efflux of macrolides in Escherichia coli and protection from oxidative stress in Salmonella enterica serovar Typhimurium, is not characterized in S. marcescens We show the role of the MacAB efflux pump in S. marcescens protection from aminoglycoside antibiotics and polymyxins, modulation of bacterial motility, and biofilm formation, and we illustrate the essential role for this pump in bacterial survival during oxidative stress. Our findings make the MacAB efflux pump an attractive target for inhibition to gain control over S. marcescens infections.

Genomic and phenotypic analysis of siderophore-producing Rhodococcus qingshengii strain S10 isolated from an arid weathered serpentine rock environment.

The success of members of the genus Rhodococcus in colonizing arid rocky environments is owed in part to desiccation tolerance and an ability to extract iron through the secretion and uptake of siderophores. Here, we report a comprehensive genomic and taxonomic analysis of Rhodococcus qingshengii strain S10 isolated from eathered serpentine rock at the arid Khalilovsky massif, Russia. Sequence comparisons of whole genomes and of selected marker genes clearly showed strain S10 to belong to the R. qingshengii species. Four prophage sequences within the R. qingshengii S10 genome were identified, one of which encodes for a putative siderophore-interacting protein. Among the ten non-ribosomal peptides synthase (NRPS) clusters identified in the strain S10 genome, two show high homology to those responsible for siderophore synthesis. Phenotypic analyses demonstrated that R. qingshengii S10 secretes siderophores and possesses adaptive features (tolerance of up to 8% NaCl and pH 9) that should enable survival in its native habitat within dry serpentine rock.

Deconstruction of plant biomass by a Cellulomonas strain isolated from an ultra-basic (lignin-stripping) spring.

Plant material falling into the ultra-basic (pH 11.5-11.9) springs within The Cedars, an actively serpentinizing site in Sonoma County, California, is subject to conditions that mimic the industrial pretreatment of lignocellulosic biomass for biofuel production. We sought to obtain hemicellulolytic/cellulolytic bacteria from The Cedars springs that are capable of withstanding the extreme alkaline conditions wherein calcium hydroxide-rich water removes lignin, making cell wall polysaccharides more accessible to microorganisms and their enzymes. We enriched for such bacteria by adding plant debris from the springs into a synthetic alkaline medium with ground tissue of the biofuel crop switchgrass (Panicum virgatum L.) as the sole source of carbon. From the enrichment culture we isolated the facultative anaerobic bacterium Cellulomonas sp. strain FA1 (NBRC 114238), which tolerates high pH and catabolizes the major plant cell wall-associated polysaccharides cellulose, pectin, and hemicellulose. Strain FA1 in monoculture colonized the plant material and degraded switchgrass at a faster rate than the community from which it was derived. Cells of strain FA1 could be acclimated through subculturing to grow at a maximal concentration of 13.4% ethanol. A strain FA1-encoded ß-1, 4-endoxylanase expressed in E. coli was active at a broad pH range, displaying near maximal activity at pH 6-9. Discovery of this bacterium illustrates the value of extreme alkaline springs in the search for microorganisms with potential for consolidated bioprocessing of plant biomass to biofuels and other valuable bio-inspired products.

Microbial diversity and mineral composition of weathered serpentine rock of the Khalilovsky massif.

Endolithic microbial communities survive nutrient and energy deficient conditions while contributing to the weathering of their mineral substrate. This study examined the mineral composition and microbial communities of fully serpentinized weathered rock from 0.1 to 6.5 m depth at a site within the Khalilovsky massif, Orenburg Region, Southern Ural Mountains, Russia. The mineral composition includes a major content of serpentinite family (mostly consisting of lizardite and chrysotile), magnesium hydrocarbonates (hydromagnesite with lesser amounts of hydrotalcite and pyroaurite) concentrated in the upper layers, and clay minerals. We found that the deep-seated weathered serpentinites are chrysotile-type minerals, while the middle and surface serpentinites mostly consist of lizardite and chrysotile types. Microbial community analysis, based on 16S rRNA gene sequencing, showed a similar diversity of phyla throughout the depth profile. The dominant bacterial phyla were the Actinobacteria (of which unclassified genera in the orders Acidimicrobiales and Actinomycetales were most numerous), Chloroflexi (dominated by an uncultured P2-11E order) and the Proteobacteria (predominantly class Betaproteobacteria). Densities of several groups of bacteria were negatively correlated with depth. Occurrence of the orders Actinomycetales, Gaiellales, Solirubrobacterales, Rhizobiales and Burkholderiales were positively correlated with depth. Our findings show that endolithic microbial communities of the Khalilovsky massif have similar diversity to those of serpentine soils and rocks, but are substantially different from those of the aqueous environments of actively serpentinizing systems.

Genome Sequence of Pigmented Siderophore-Producing Strain Serratia marcescens SM6.

Here we present a draft genome sequence of laboratory strain Serratia marcescens SM6. Using the antiSMASH 5.0 prediction tool, we identified five biosynthetic gene clusters involved in secondary metabolite production (two siderophores and a biosurfactant serratamolide, a glucosamine derivative, and a thiopeptide). Whole-genome sequencing information will be useful for the detailed study of metabolites produced by Serratia marcescens.

Comparative Metagenomic Analysis of Electrogenic Microbial Communities in Differentially Inoculated Swine Wastewater-Fed Microbial Fuel Cells.

Bioelectrochemical systems such as microbial fuel cells (MFCs) are promising new technologies for efficient removal of organic compounds from industrial wastewaters, including that generated from swine farming. We inoculated two pairs of laboratory-scale MFCs with sludge granules from a beer wastewater-treating anaerobic digester (IGBS) or from sludge taken from the bottom of a tank receiving swine wastewater (SS). The SS-inoculated MFC outperformed the IGBS-inoculated MFC with regard to COD and VFA removal and electricity production. Using a metagenomic approach, we describe the microbial diversity of the MFC planktonic and anodic communities derived from the different inocula. Proteobacteria (mostly Deltaproteobacteria) became the predominant phylum in both MFC anodic communities with amplification of the electrogenic genus Geobacter being the most pronounced. Eight dominant and three minor species of Geobacter were found in both MFC anodic communities. The anodic communities of the SS-inoculated MFCs had a higher proportion of Clostridium and Bacteroides relative to those of the IGBS-inoculated MFCs, which were enriched with Pelobacter. The archaeal populations of the SS- and IGBS-inoculated MFCs were dominated by Methanosarcina barkeri and Methanothermobacter thermautotrophicus, respectively. Our results show a long-term influence of inoculum type on the performance and microbial community composition of swine wastewater-treating MFCs.

Proteomic Analysis of 2,4,6-Trinitrotoluene Degrading Yeast Yarrowia lipolytica.

2,4,6-trinitrotoluene (TNT) is a common component of many explosives. The overproduction and extensive usage of TNT significantly contaminates the environment. TNT accumulates in soils and aquatic ecosystems and can primarily be destroyed by microorganisms. Current work is devoted to investigation of Yarrowia lipolytica proteins responsible for TNT transformation through the pathway leading to protonated Meisenheimer complexes and nitrite release. Here, we identified a unique set of upregulated membrane and cytosolic proteins of Y. lipolytica, which biosynthesis increased during TNT transformation through TNT-monohydride-Meisenheimer complexes in the first step of TNT degradation, through TNT-dihydride-Meisenheimer complexes in the second step, and the aromatic ring denitration and degradation in the last step. We established that the production of oxidoreductases, namely, NADH flavin oxidoreductases and NAD(P)+-dependent aldehyde dehydrogenases, as well as transferases was enhanced at all stages of the TNT transformation by Y. lipolytica. The up-regulation of several stress response proteins (superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase) was also detected. The involvement of intracellular nitric oxide dioxygenase in NO formation during nitrite oxidation was shown. Our results present at the first time the full proteome analysis of Y. lipolytica yeast, destructor of TNT.

Magnet-Facilitated Selection of Electrogenic Bacteria from Marine Sediment.

Some bacteria can carry out anaerobic respiration by depositing electrons on external materials, such as electrodes, thereby creating an electrical current. Into the anode chamber of microbial fuel cells (MFCs) having abiotic air-cathodes we inoculated microorganisms cultured from a magnetic particle-enriched portion of a marine tidal sediment, reasoning that since some external electron acceptors are ferromagnetic, electrogenic bacteria should be found in their vicinity. Two MFCs, one inoculated with a mixed bacterial culture and the other with an axenic culture of a helical bacterium isolated from the magnetic particle enrichment, termed strain HJ, were operated for 65 d. Both MFCs produced power, with production from the mixed culture MFC exceeding that of strain HJ. Strain HJ was identified as a Thalassospira sp. by transmission electron microscopic analysis and 16S rRNA gene comparisons. An MFC inoculated with strain HJ and operated in open circuit produced 47% and 57% of the maximal power produced from MFCs inoculated with the known electrogen Geobacter daltonii and the magnetotactic bacterium Desulfamplus magnetomortis, respectively. Further investigation will be needed to determine whether bacterial populations associated with magnetic particles within marine sediments are enriched for electrogens.

Assessment of a biogas-generating microbial community in a pilot-scale anaerobic reactor.

In this work bacteria and methanogenic archaea utilizing agricultural wastes in a pilot-scale biogas reactor were examined using sequencing and terminal restriction fragment length polymorphism analysis. Based on the analyses of 16S rRNA genes, Clostridia represented the most diverse group in the digester. Of the Clostridia, unclassified Clostridiales and the members of the genera Anaerotruncus and Tissierella were detected at high abundances. The representatives of the bacterial phyla Bacteroidetes and Proteobacteria were also defined, but in minor proportions, and were assigned to non-dominant communities. Within the phylum Euryarchaeota, the members of the orders Methanosarcinales and Methanomicrobiales were found at high levels. Methanogenic archaea were analyzed using both 16S rRNA and mcrA genes. Actually good results were received using both approaches; however, the rRNA gene method missed the non-dominant order Methanobacteriales.

Effect of ferrihydrite on 2,4,6-trinitrotoluene biotransformation by an aerobic yeast.

This study investigated the impact of ferrihydrite on the pathway and rate of 2,4,6-trinitrotoluene (TNT) transformation by Yarrowia lipolytica AN-L15. The presence of ferrihydrite in the culture medium decreased the rate of TNT biotransformation but resulted in the accumulation of the same TNT metabolites as in the absence of ferrihydrite, albeit at slightly different concentrations. Transformation products observed included aromatic ring reduction products, such as hydride-Meisenheimer complexes, and nitro group reduction products, such as hydroxylamino- and amino-dinitrotoluenes. Independently of the presence of ferrihydrite the subsequent degradation of the hydride complex(es) resulted in the release of nitrite followed by its conversion to nitrate and nitric oxide at the low pH values observed during yeast cultivation. Nitric oxide generation was ascertained by electron spin resonance spectroscopy. In addition, increased Fe(3+)-reduction was observed in the presence of TNT and Y. lipolytica. This study demonstrates that in the presence of yeast cells, TNT-hydride complexes were formed at approximately the same level as in the presence of ferrihydrite, opening up the possibility of aromatic ring cleavage, instead of promoting the production of potentially toxic nitro group reduction products in the presence of iron minerals.