Louis S. Tisa

Melanin Production and Use as a Soluble Electron Shuttle for Fe(III) Oxide Reduction and as a Terminal Electron Acceptor by Shewanella algae BrY

ABSTRACT Dissimilatory metal-reducing bacteria (DMRB) utilize numerous compounds as terminal electron acceptors, including insoluble iron oxides. The mechanism(s) of insoluble-mineral reduction by DMRB is not well understood. Here we report that extracellular melanin is produced by Shewanella algae BrY. The extracted melanin served as the sole terminal electron acceptor. Upon reduction the reduced, soluble melanin reduced insoluble hydrous ferric oxide in the absence of bacteria, thus demonstrating that melanin produced by S. algae BrY is a soluble Fe(III)-reducing compound. In the presence of bacteria, melanin acted as an electron conduit to Fe(III) minerals and increased Fe(III) mineral reduction rates. Growth of S. algae BrY occurred in anaerobic minimal medium supplemented with melanin extracted from previously grown aerobic cultures of S. algae BrY. Melanin produced by S. algae BrY imparts increased versatility to this organism as a soluble Fe(III) reductant, an electron conduit for iron mineral reduction, and a sole terminal electron acceptor that supports growth.

Heavy Metal Resistance Patterns of Frankia Strains

ABSTRACT The sensitivity of 12 Frankia strains to heavy metals was determined by a growth inhibition assay. In general, all of the strains were sensitive to low concentrations (<0.5 mM) of Ag1+, AsO21−, Cd2+, SbO21−, and Ni2+, but most of the strains were less sensitive to Pb2+ (6 to 8 mM), CrO42− (1.0 to 1.75 mM), AsO43− (>50 mM), and SeO22− (1.5 to 3.5 mM). While most strains were sensitive to 0.1 mM Cu2+, four strains were resistant to elevated levels of Cu2+ (2 to 5 mM and concentrations as high as 20 mM). The mechanism of SeO22− resistance seems to involve reduction of the selenite oxyanion to insoluble elemental selenium, whereas Pb2+ resistance and Cu2+ resistance may involve sequestration or binding mechanisms. Indications of the resistance mechanisms for the other heavy metals were not as clear.

Auxin Carriers Localization Drives Auxin Accumulation in Plant Cells Infected by Frankia in Casuarina glauca Actinorhizal Nodules

Actinorhizal symbioses are mutualistic interactions between plants and the soil bacteria Frankia that lead to the formation of nitrogen-fixing root nodules. Little is known about the signaling mechanisms controlling the different steps of the establishment of the symbiosis. The plant hormone auxin has been suggested to play a role. Here we report that auxin accumulates within Frankia-infected cells in actinorhizal nodules of Casuarina glauca. Using a combination of computational modeling and experimental approaches, we establish that this localized auxin accumulation is driven by the cell-specific expression of auxin transporters and by Frankia auxin biosynthesis in planta. Our results indicate that the plant actively restricts auxin accumulation to Frankia-infected cells during the symbiotic interaction.

Significant Natural Product Biosynthetic Potential of Actinorhizal Symbionts of the Genus Frankia, as Revealed by Comparative Genomic and Proteomic Analyses

ABSTRACT Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus.

Phylogeny of members of the Frankia genus based on gyrB, nifH and glnII sequences

To construct an evolutionary hypothesis for the genus Frankia, gyrB (encoding gyrase B), nifH (encoding nitrogenase reductase) and glnII (encoding glutamine synthetase II) gene sequences were considered for 38 strains. The overall clustering pattern among Frankia strains based on the three analyzed sequences varied among themselves and with the previously established 16S rRNA gene phylogeny and they did not reliably reflect clear evolution of the four discerned Frankia clusters (1, 2, 3 and 4). Based on concatenated gyrB, nifH and glnII, robust phylogenetic trees were observed with the three treeing methods (Maximum Likelihood, Parsimony and Neighbor-Joining) and supported by strong bootstrap and posterior probability values (>75%) for overall branching. Cluster 4 (non-infective and/or non-nitrogen-fixing Frankia) was positioned at a deeper branch followed by cluster 3 (Rhamnaceae and Elaeagnaceae infective Frankia), while cluster 2 represents uncultured Frankia microsymbionts of the Coriariaceae, Datiscaceae, Rosaceae and of Ceanothus sp. (Rhamnaceae); Cluster 1 (Betulaceae, Myricaceae and Casuarinaceae infective Frankia) appears to have diverged more recently. The present study demonstrates the utility of phylogenetic analyses based upon concatenated gyrB, nifH and glnII sequences to help resolve previously unresolved or poorly resolved nodes and will aid in describing species among the genus Frankia.

Electron transfer from Shewanella algae BrY to hydrous ferric oxide is mediated by cell‐associated melanin

Shewanella algae BrY uses insoluble mineral oxides as terminal electron acceptors, but the mechanism of electron transfer from cell surface to mineral surface is not well understood. We tested the hypothesis that cell-associated melanin produced by S. algae BrY serves as an electron conduit for bacterial-mineral reduction. Results from Fourier transform infrared spectroscopy and cell surface hydrophobicity assays indicated that extracellular melanin was associated with the cell surface. With H(2) as electron donor, washed cell suspensions of melanin-coated S. algae BrY reduced hydrous ferric oxide (HFO) 10 times faster than cells without melanin. The addition of melanin (20 microg ml(-1)) to these melanin-free cells increased their HFO reduction rate two-fold. These results suggest that cell-associated melanin acts as an electron conduit for iron mineral reduction by S. algae BrY.

Draft Genome Sequence of Frankia sp. Strain CN3, an Atypical, Noninfective (Nod - ) Ineffective (Fix - ) Isolate from Coriaria nepalensis

ABSTRACT We report here the genome sequence of Frankia sp. strain CN3, which was isolated from Coriaria nepalensis. This genome sequence is the first from the fourth lineage of Frankia, strains of which are unable to reinfect actinorhizal plants. At 10 Mb, it represents the largest Frankia genome sequenced to date.

Cultivating the uncultured: growing the recalcitrant cluster-2 Frankia strains

The repeated failures reported in cultivating some microbial lineages are a major challenge in microbial ecology and probably linked, in the case of Frankia microsymbionts to atypical patterns of auxotrophy. Comparative genomics of the so far uncultured cluster-2 Candidatus Frankia datiscae Dg1, with cultivated Frankiae has revealed genome reduction, but no obvious physiological impairments. A direct physiological assay on nodule tissues from Coriaria myrtifolia infected with a closely-related strain permitted the identification of a requirement for alkaline conditions. A high pH growth medium permitted the recovery of a slow-growing actinobacterium. The strain obtained, called BMG5.1, has short hyphae, produced diazovesicles in nitrogen-free media, and fulfilled Koch’s postulates by inducing effective nodules on axenically grown Coriaria spp. and Datisca glomerata. Analysis of the draft genome confirmed its close proximity to the Candidatus Frankia datiscae Dg1 genome with the absence of 38 genes (trehalose synthase, fumarylacetoacetase, etc) in BMG5.1 and the presence of 77 other genes (CRISPR, lanthionine synthase, glutathione synthetase, catalase, Na+/H+ antiporter, etc) not found in Dg1. A multi-gene phylogeny placed the two cluster-2 strains together at the root of the Frankia radiation.

Insertion sequence content reflects genome plasticity in strains of the root nodule actinobacterium Frankia

BackgroundGenome analysis of three Frankia sp. strains has revealed a high number of transposable elements in two of the strains. Twelve out of the 20 major families of bacterial Insertion Sequence (IS) elements are represented in the 148 annotated transposases of Frankia strain HFPCcI3 (CcI3) comprising 3% of its total coding sequences (CDS). EAN1pec (EAN) has 183 transposase ORFs from 13 IS families comprising 2.2% of its CDS. Strain ACN14a (ACN) differs significantly from the other strains with only 33 transposase ORFs (0.5% of the total CDS) from 9 IS families.ResultsInsertion sequences in the Frankia genomes were analyzed using BLAST searches, PHYML phylogenies and the IRF (Inverted Repeat Finder) algorithms. To identify putative or decaying IS elements, a PSI-TBLASTN search was performed on all three genomes, identifying 36%, 39% and 12% additional putative transposase ORFs than originally annotated in strains CcI3, EAN and ACN, respectively. The distribution of transposase ORFs in each strain was then analysed using a sliding window, revealing significant clustering of elements in regions of the EAN and CcI3 genomes. Lastly the three genomes were aligned with the MAUVE multiple genome alignment tool, revealing several Large Chromosome Rearrangement (LCR) events; many of which correlate to transposase clusters.ConclusionAnalysis of transposase ORFs in Frankia sp. revealed low inter-strain diversity of transposases, suggesting that the majority of transposase proliferation occurred without recent horizontal transfer of novel mobile elements from outside the genus. Exceptions to this include representatives from the IS3 family in strain EAN and seven IS4 transposases in all three strains that have a lower G+C content, suggesting recent horizontal transfer. The clustering of transposase ORFs near LCRs revealed a tendency for IS elements to be associated with regions of chromosome instability in the three strains. The results of this study suggest that IS elements may help drive chromosome differences in different Frankia sp. strains as they have adapted to a variety of hosts and environments.

Draft Genome sequence of Frankia sp. Strain QA3, a nitrogen-fixing actinobacterium isolated from the root nodule of Alnus nitida

ABSTRACT Members of the actinomycete genus Frankia form a nitrogen-fixing symbiosis with 8 different families of actinorhizal plants. We report a high-quality draft genome sequence for Frankia sp. strain QA3, a nitrogen-fixing actinobacterium isolated from root nodules of Alnus nitida.