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.

Telomere Length Variation in Model Bryophytes.

The ends of linear chromosomes of most eukaryotes consist of protein-bound DNA arrays called telomeres, which play essential roles in protecting genome integrity. Despite general evolutionary conservation in function, telomeric DNA is known to drastically vary in length and sequence between different eukaryotic lineages. Bryophytes are a group of early diverging land plants that include mosses, liverworts, and hornworts. This group of ancient land plants recently emerged as a new model for important discoveries in genomics and evolutionary biology, as well as for understanding plant adaptations to a terrestrial lifestyle. We measured telomere length in different ecotypes of model bryophyte species, including Physcomitrium patens, Marchantia polymorpha, Ceratodon purpureus, and in Sphagnum isolates. Our data indicate that all analyzed moss and liverwort genotypes have relatively short telomeres. Furthermore, all analyzed ecotypes and isolates of model mosses and liverworts display evidence of substantial natural variation in telomere length. Interestingly, telomere length also differs between male and female strains of the dioecious liverwort M. polymorpha and dioecious moss C. purpureus. Given that bryophytes are extraordinarily well adapted to different ecological niches from polar to tropical environments, our data will contribute to understanding the impact of natural telomere length variation on evolutionary adaptations in this ancient land plant lineage.

Comparative Application of Terminal Restriction Fragment Analysis Tools to Large-Scale Genomic Assays.

The analysis of telomere length is an important component of many studies aiming to characterize the role of telomere maintenance mechanisms in cellular lifespan, disease, or in general chromosome protection and DNA replication pathways. Several powerful methods to accurately measure the telomere length from Southern blots have been developed, but their utility for large-scale genomic studies has not been previously evaluated. Here, we performed a comparative analysis of two recently developed programs, TeloTool and WALTER, for the extraction of mean telomere length values from Southern blots. Using both software packages, we measured the telomere length in two extensive experimental datasets for the model plant Arabidopsis thaliana, consisting of 537 natural accessions and 65 T-DNA (transfer DNA for insertion mutagenesis) mutant lines in the reference Columbia (Col-0) genotype background. We report that TeloTool substantially overestimates the telomere length in comparison to WALTER, especially for values over 4500 bp. Importantly, the TeloTool- and WALTER-calculated telomere length values correlate the most in the 2100-3500 bp range, suggesting that telomeres in this size interval can be estimated by both programs equally well. We further show that genome-wide association studies using datasets from both telomere length analysis tools can detect the most significant SNP candidates equally well. However, GWAS analysis with the WALTER dataset consistently detects fewer significant SNPs than analysis with the TeloTool dataset, regardless of the GWAS method used. These results imply that the telomere length data generated by WALTER may represent a more stringent approach to GWAS and SNP selection for the downstream molecular screening of candidate genes. Overall, our work reveals the unanticipated impact of the telomere length analysis method on the outcomes of large-scale genomic screens.

Arabidopsis AN3 and OLIGOCELLULA genes link telomere maintenance mechanisms with cell division and expansion control.

Telomeres are conserved chromosomal structures necessary for continued cell division and proliferation. In addition to the classical telomerase pathway, multiple other genes including those involved in ribosome metabolism and chromatin modification contribute to telomere length maintenance. We previously reported that Arabidopsis thaliana ribosome biogenesis genes OLI2/NOP2A, OLI5/RPL5A and OLI7/RPL5B have critical roles in telomere length regulation. These three OLIGOCELLULA genes were also shown to function in cell proliferation and expansion control and to genetically interact with the transcriptional co-activator ANGUSTIFOLIA3 (AN3). Here we show that AN3-deficient plants progressively lose telomeric DNA in early homozygous mutant generations, but ultimately establish a new shorter telomere length setpoint by the fifth mutant generation with a telomere length similar to oli2/nop2a - deficient plants. Analysis of double an3 oli2 mutants indicates that the two genes are epistatic for telomere length control. Telomere shortening in an3 and oli mutants is not caused by telomerase inhibition; wild type levels of telomerase activity are detected in all analyzed mutants in vitro. Late generations of an3 and oli mutants are prone to stem cell damage in the root apical meristem, implying that genes regulating telomere length may have conserved functional roles in stem cell maintenance mechanisms. Multiple instances of anaphase fusions in late generations of oli5 and oli7 mutants were observed, highlighting an unexpected effect of ribosome biogenesis factors on chromosome integrity. Overall, our data implicate AN3 transcription coactivator and OLIGOCELLULA proteins in the establishment of telomere length set point in plants and further suggest that multiple regulators with pleiotropic functions can connect telomere biology with cell proliferation and cell expansion pathways.

Characterization of Bacillus pumilus Strains with Targeted Gene Editing for Antimicrobial Peptides and Sporulation Factor.

Due to their capacity to produce antimicrobial peptides that can prevent the growth of diseases, many Bacillus spp. are beneficial to plants. In this study, we looked into the antagonistic activity of the B. pumilus 3-19 strain and its derivatives following targeted genome editing. Two peptide genes with antibacterial action, bacilysin (bac) and bacteriocin (bact), and the sigF gene, which encodes the sigma factor of sporulation, were specifically inactivated using the CRISPR-Cas9 system in the genome of B. pumilus 3-19. Antibacterial activity against B. cereus and Pantoea brenneri decreased as a result of the inactivation of target genes in the B. pumilus 3-19 genome, with a noticeable effect against bacilysin. The growth dynamics of the culture changed when the bac, bact, and sigF genes were inactivated, and the altered strains had less proteolytic activity. An asporogenic mutant of B. pumilus 3-19 was obtained by inactivating the sigF gene. It has been proven that bacilysin plays a unique part in the development of B. pumilus 3-19's antagonistic action against soil microorganisms.

Identification and Analysis of Antimicrobial Activities from a Model Moss Ceratodon purpureus.

The emergence of bacterial drug resistance is often viewed as the next great health crisis of our time. While more antimicrobial agents are urgently needed, very few new antibiotics are currently in the production pipeline. Here, we aim to identify and characterize novel antimicrobial natural products from a model dioicous moss, Ceratodon purpureus. We collected secreted moss exudate fractions from two C. purpureus strains, male R40 and female GG1. Exudates from the female C. purpureus strain GG1 did not exhibit inhibitory activity against any tested bacteria. However, exudates from the male moss strain R40 exhibited strong inhibitory properties against several species of Gram-positive bacteria, including Staphylococcus aureus and Enterococcus faecium, though they did not inhibit the growth of Gram-negative bacteria. Antibacterial activity levels in C. purpureus R40 exudates significantly increased over four weeks of moss cultivation in liquid culture. Size fractionation experiments indicated that the secreted bioactive compounds have a relatively low molecular weight of less than 1 kDa. Additionally, the R40 exudate compounds are thermostable and not sensitive to proteinase K treatment. Overall, our results suggest that the bioactive compounds present in C. purpureus R40 exudates can potentially add new options for treating infections caused by antibiotic-resistant Gram-positive bacteria.

Regulating Pathways of Bacillus pumilus Adamalysin-like Metalloendopeptidase Expression.

The minor secreted proteinase of B. pumilus 3-19 MprBp classified as the unique bacillary adamalysin-like enzyme of the metzincin clan. The functional role of this metalloproteinase in the bacilli cells is not clear. Analysis of the regulatory region of the mprBp gene showed the presence of potential binding sites to the transcription regulatory factors Spo0A (sporulation) and DegU (biodegradation). The study of mprBp activity in mutant strains of B. subtilis defective in regulatory proteins of the Spo- and Deg-systems showed that the mprBp gene is partially controlled by the Deg-system of signal transduction and independent from the Spo-system.

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.

Antimicrobial Activities of Secondary Metabolites from Model Mosses.

Plants synthetize a large spectrum of secondary metabolites with substantial structural and functional diversity, making them a rich reservoir of new biologically active compounds. Among different plant lineages, the evolutionarily ancient branch of non-vascular plants (Bryophytes) is of particular interest as these organisms produce many unique biologically active compounds with highly promising antibacterial properties. Here, we characterized antibacterial activity of metabolites produced by different ecotypes (strains) of the model mosses Physcomitrium patens and Sphagnum fallax. Ethanol and hexane moss extracts harbor moderate but unstable antibacterial activity, representing polar and non-polar intracellular moss metabolites, respectively. In contrast, high antibacterial activity that was relatively stable was detected in soluble exudate fractions of P. patens moss. Antibacterial activity levels in P. patens exudates significantly increased over four weeks of moss cultivation in liquid culture. Interestingly, secreted moss metabolites are only active against a number of Gram-positive, but not Gram-negative, bacteria. Size fractionation, thermostability and sensitivity to proteinase K assays indicated that the secreted bioactive compounds are relatively small (less than <10 kDa). Further analysis and molecular identification of antibacterial exudate components, combined with bioinformatic analysis of model moss genomes, will be instrumental in the identification of specific genes involved in the bioactive metabolite biosynthesis.

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.

Comparative Genome Analysis of Two Bacillus pumilus Strains Producing High Level of Extracellular Hydrolases.

Whole-genome sequencing of a soil isolate Bacillus pumilus, strain 7P, and its streptomycin-resistant derivative, B. pumilus 3-19, showed genome sizes of 3,609,117 bp and 3,609,444 bp, respectively. Annotation of the genome showed 3794 CDS (3204 with predicted function) and 3746 CDS (3173 with predicted function) in the genome of strains 7P and 3-19, respectively. In the genomes of both strains, the prophage regions Bp1 and Bp2 were identified. These include 52 ORF of prophage proteins in the Bp1 region and 38 prophages ORF in the Bp2 region. Interestingly, more than 50% of Bp1 prophage proteins are similar to the proteins of the phi105 in B. subtilis. The DNA region of Bp2 has 15% similarity to the DNA of the Brevibacillus Jimmer phage. Degradome analysis of the genome of both strains revealed 148 proteases of various classes. These include 60 serine proteases, 48 metalloproteases, 26 cysteine proteases, 4 aspartate proteases, 2 asparagine proteases, 3 threonine proteases, and 2 unclassified proteases. Likewise, three inhibitors of proteolytic enzymes were found. Comparative analysis of variants in the genomes of strains 7P and 3-19 showed the presence of 81 nucleotide variants in the genome 3-19. Among them, the missense mutations in the rpsL, comA, spo0F genes and in the upstream region of the srlR gene were revealed. These nucleotide polymorphisms may have affected the streptomycin resistance and overproduction of extracellular hydrolases of the 3-19 strain. Finally, a plasmid DNA was found in strain 7P, which is lost in its derivative, strain 3-19. This plasmid contains five coding DNA sequencing (CDS), two regulatory proteins and three hypothetical proteins.

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.

Label-Free Multiple Reaction Monitoring, a Promising Method for Quantification Analyses of Specific Proteins in Bacteria.

Bacillus subtilis produces eight industrially important exo-proteases. For the detection of proteases, the activity- and antibody-based assays are normally used. Current activity-based assays require expensive multiplex chemical substrates which allow specificity determination of each enzyme. In this study, we provide evidences pertaining to the usefulness of the label-free multiple reaction monitoring (MRM) assay for a rapid identification and quantitation of specific proteins in bacteria. We used wild-type B. pumilus cells producing at least two serine proteases, subtilisin-like protease (AprBp) and glutamyl endopeptidase (GseBp), as well as optimized recombinant B. subtilis cells containing the same protease genes under control of the LIKE expression system. The Skyline software was used for the selection of three specific proteotypic peptides and their fragment ions for quantification and confirmation of AprBp and GseBp in complex mixtures. MRM indicated that the production of AprBp and GseBp exo-enzymes were respectively 0.9- and 26.6-fold higher in the culture medium of B. pumilus strain in comparison to the recombinant B. subtilis strains carrying optimized LIKE expression systems under identical conditions. The developed procedure in this study is fast, easy to perform and dependable. Additionally, it achieves accurate proteins identification and quantification in complex mixture.

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.

Components of the ribosome biogenesis pathway underlie establishment of telomere length set point in Arabidopsis.

Telomeres cap the physical ends of eukaryotic chromosomes to ensure complete DNA replication and genome stability. Heritable natural variation in telomere length exists in yeast, mice, plants and humans at birth; however, major effect loci underlying such polymorphism remain elusive. Here, we employ quantitative trait locus (QTL) mapping and transgenic manipulations to identify genes controlling telomere length set point in a multi-parent Arabidopsis thaliana mapping population. We detect several QTL explaining 63.7% of the total telomere length variation in the Arabidopsis MAGIC population. Loss-of-function mutants of the NOP2A candidate gene located inside the largest effect QTL and of two other ribosomal genes RPL5A and RPL5B establish a shorter telomere length set point than wild type. These findings indicate that evolutionarily conserved components of ribosome biogenesis and cell proliferation pathways promote telomere elongation.

Draft genome sequence and analysis of Klebsiella oxytoca strain NK-1 isolated from ureteral stent.

Klebsiella oxytoca is a facultative aerobic, gram-negative, rod-shaped bacterium capable of causing nosocomial infections, in particular catheter-associated urinary tract infections (CAUTIs). Data on the possible roles of uncommon pathogens such as K. oxytoca in the pathogenesis of biofilm-associated infections such as CAUTIs have been already reported. Herein, we describe the draft genome sequence of K. oxytoca strain NK-1 isolated from the surface of ureteral stent retrieved from a Russian female. The genome comprises 6,232,464 bp, with a G + C content of 55.60% and an L 50 of 7. A total of 6246 putative protein-encoding genes were predicted, including considerable number of genes responsible for adhesion, invasion, drug resistance, iron acquisition and other genes relevant for virulence. The NK-1 strain was ascribed a sequence type (ST) as ST 216 (4, 6, 19, 10, 46, 24, 31). Data comparison of the recA gene sequences confirmed that the strain belongs to the species K. oxytoca. Minimal inhibitory concentration of different antibiotics have been determined. This whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number QPKC00000000.1.

Heterologous Expression of Secreted Bacterial BPP and HAP Phytases in Plants Stimulates Arabidopsis thaliana Growth on Phytate.

Phytases are specialized phosphatases capable of releasing inorganic phosphate from myo-inositol hexakisphosphate (phytate), which is highly abundant in many soils. As inorganic phosphorus reserves decrease over time in many agricultural soils, genetic manipulation of plants to enable secretion of potent phytases into the rhizosphere has been proposed as a promising approach to improve plant phosphorus nutrition. Several families of biotechnologically important phytases have been discovered and characterized, but little data are available on which phytase families can offer the most benefits toward improving plant phosphorus intake. We have developed transgenic Arabidopsis thaliana plants expressing bacterial phytases PaPhyC (HAP family of phytases) and 168phyA (BPP family) under the control of root-specific inducible promoter Pht1;2. The effects of each phytase expression on growth, morphology and inorganic phosphorus accumulation in plants grown on phytate hydroponically or in perlite as the only source of phosphorus were investigated. The most enzymatic activity for both phytases was detected in cell wall-bound fractions of roots, indicating that these enzymes were efficiently secreted. Expression of both bacterial phytases in roots improved plant growth on phytate and resulted in larger rosette leaf area and diameter, higher phosphorus content and increased shoot dry weight, implying that these plants were indeed capable of utilizing phytate as the source of phosphorus for growth and development. When grown on phytate the HAP-type phytase outperformed its BPP-type counterpart for plant biomass production, though this effect was only observed in hydroponic conditions and not in perlite. Furthermore, we found no evidence of adverse side effects of microbial phytase expression in A. thaliana on plant physiology and seed germination. Our data highlight important functional differences between these members of bacterial phytase families and indicate that future crop biotechnologies involving such enzymes will require a very careful evaluation of phytase source and activity. Overall, our data suggest feasibility of using bacterial phytases to improve plant growth in conditions of phosphorus deficiency and demonstrate that inducible expression of recombinant enzymes should be investigated further as a viable approach to plant biotechnology.

Draft genome sequence data of Lysinibacillus fusiformis strain GM, isolated from potato phyllosphere as a potential probiotic.

Here we present the morphological and physiological properties of isolated Lysinibacillus fusiformis strain GM, its draft genome sequence as well as annotation and analysis of its genome. Initial analysis of MALDI-TOF mass spectrometry, 16S rRNA gene analysis and in silico DNA-DNA hybridization revealed that the strain belongs to the species Lysinibacillus fusiformis. The 4,678,122 bp draft genome consist of 17 scaffolds encoding 4588 proteins and 137 RNAs. Annotation of the genome sequence revealed cellulase and protease encoding genes, genes of adhesion proteins and putative genes responsible for the biosynthesis of antimicrobial metabolites. The Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number NTMQ00000000.1 (https://www.ncbi.nlm.nih.gov/nuccore/NZ_NTMQ00000000.1).

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.