Pb2+ tolerance by Frankia sp. strain EAN1pec involves surface-binding.

Several Frankia strains have been shown to be lead-resistant. The mechanism of lead resistance was investigated for Frankia sp. strain EAN1pec. Analysis of the cultures by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDAX) and Fourier transforming infrared spectroscopy (FTIR) demonstrated that Frankia sp. strain EAN1pec undergoes surface modifications and binds high quantities of Pb+2. Both labelled and unlabelled shotgun proteomics approaches were used to determine changes in Frankia sp. strain EAN1pec protein expression in response to lead and zinc. Pb2+ specifically induced changes in exopolysaccharides, the stringent response, and the phosphate (pho) regulon. Two metal transporters (a Cu2+-ATPase and cation diffusion facilitator), as well as several hypothetical transporters, were also upregulated and may be involved in metal export. The exported Pb2+ may be precipitated at the cell surface by an upregulated polyphosphate kinase, undecaprenyl diphosphate synthase and inorganic diphosphatase. A variety of metal chaperones for ensuring correct cofactor placement were also upregulated with both Pb+2 and Zn+2 stress. Thus, this Pb+2 resistance mechanism is similar to other characterized systems. The cumulative interplay of these many mechanisms may explain the extraordinary resilience of Frankia sp. strain EAN1pec to Pb+2. A potential transcription factor (DUF156) binding site was identified in association with several proteins identified as upregulated with heavy metals. This site was also discovered, for the first time, in thousands of other organisms across two kingdoms.

The ins and outs of metal homeostasis by the root nodule actinobacterium Frankia.

BACKGROUND: Frankia are actinobacteria that form a symbiotic nitrogen-fixing association with actinorhizal plants, and play a significant role in actinorhizal plant colonization of metal contaminated areas. Many Frankia strains are known to be resistant to several toxic metals and metalloids including Pb(2+), Al(+3), SeO2, Cu(2+), AsO4, and Zn(2+). With the availability of eight Frankia genome databases, comparative genomics approaches employing phylogeny, amino acid composition analysis, and synteny were used to identify metal homeostasis mechanisms in eight Frankia strains. Characterized genes from the literature and a meta-analysis of 18 heavy metal gene microarray studies were used for comparison. RESULTS: Unlike most bacteria, Frankia utilize all of the essential trace elements (Ni, Co, Cu, Se, Mo, B, Zn, Fe, and Mn) and have a comparatively high percentage of metalloproteins, particularly in the more metal resistant strains. Cation diffusion facilitators, being one of the few known metal resistance mechanisms found in the Frankia genomes, were strong candidates for general divalent metal resistance in all of the Frankia strains. Gene duplication and amino acid substitutions that enhanced the metal affinity of CopA and CopCD proteins may be responsible for the copper resistance found in some Frankia strains. CopA and a new potential metal transporter, DUF347, may be involved in the particularly high lead tolerance in Frankia. Selenite resistance involved an alternate sulfur importer (CysPUWA) that prevents sulfur starvation, and reductases to produce elemental selenium. The pattern of arsenate, but not arsenite, resistance was achieved by Frankia using the novel arsenite exporter (AqpS) previously identified in the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Based on the presence of multiple tellurite resistance factors, a new metal resistance (tellurite) was identified and confirmed in Frankia. CONCLUSIONS: Each strain had a unique combination of metal import, binding, modification, and export genes that explain differences in patterns of metal resistance between strains. Frankia has achieved similar levels of metal and metalloid resistance as bacteria from highly metal-contaminated sites. From a bioremediation standpoint, it is important to understand mechanisms that allow the endosymbiont to survive and infect actinorhizal plants in metal contaminated soils.

Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport.

Several Frankia strains have been shown to be copper-tolerant. The mechanism of their copper tolerance was investigated for Frankia sp. strain EuI1c. Copper binding was shown by binding studies. Unusual globular structures were observed on the surface of the bacterium. These globular structures were composed of aggregates containing many relatively smaller "leaf-like" structures. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDAX) analysis of these structures indicated elevated copper and phosphate levels compared to the control cells. Fourier transform infrared spectroscopy (FTIR) analysis indicated an increase in extracellular phosphate on the cell surface of copper-stressed cells. Bioinformatics' analysis of the Frankia sp. strain EuI1c genome revealed five potential cop genes: copA, copZ, copC, copCD, and copD. Experiments with Frankia sp. strain EuI1c using qRT-PCR indicated an increase in messenger RNA (mRNA) levels of the five cop genes upon Cu(2+) stress. After 5 days of Cu(2+) stress, the copA, copZ, copC, copCD, and copD mRNA levels increased 25-, 8-, 18-, 18-, and 25-fold, respectively. The protein profile of Cu(2+)-stressed Frankia sp. strain EuI1c cells revealed the upregulation of a 36.7 kDa protein that was identified as FraEuI1c_1092 (sulfate-binding periplasmic transport protein). Homologues of this gene were only present in the genomes of the Cu(2+)-resistant Frankia strains (EuI1c, DC12, and CN3). These data indicate that copper tolerance by Frankia sp. strain EuI1c involved the binding of copper to the cell surface and transport proteins.

Casuarina root exudates alter the physiology, surface properties, and plant infectivity of Frankia sp. strain CcI3.

The actinomycete genus Frankia forms nitrogen-fixing symbioses with 8 different families of actinorhizal plants, representing more than 200 different species. Very little is known about the initial molecular interactions between Frankia and host plants in the rhizosphere. Root exudates are important in Rhizobium-legume symbiosis, especially for initiating Nod factor synthesis. We measured differences in Frankia physiology after exposure to host aqueous root exudates to assess their effects on actinorhizal symbioses. Casuarina cunninghamiana root exudates were collected from plants under nitrogen-sufficient and -deficient conditions and tested on Frankia sp. strain CcI3. Root exudates increased the growth yield of Frankia in the presence of a carbon source, but Frankia was unable to use the root exudates as a sole carbon or energy source. Exposure to root exudates caused hyphal "curling" in Frankia cells, suggesting a chemotrophic response or surface property change. Exposure to root exudates altered Congo red dye binding, which indicated changes in the bacterial surface properties at the fatty acid level. Fourier transform infrared spectroscopy (FTIR) confirmed fatty acid changes and revealed further carbohydrate changes. Frankia cells preexposed to C. cunninghamiana root exudates for 6 days formed nodules on the host plant significantly earlier than control cells. These data support the hypothesis of early chemical signaling between actinorhizal host plants and Frankia in the rhizosphere.

Development of a semi-high-throughput growth assay for the filamentous actinobacteria Frankia.

Filamentous bacteria pose unique challenges for testing multiple variables or growth parameters limiting the use of high-throughput methods. A semi-high-throughput growth assay system was developed to overcome these obstacles and validated for the filamentous actinobacteria Frankia. The 24-well plate assay was versatile for testing multiple growth medium parameters and provided reproducible results across wells and between plates. Under conditions of increased complexity, statistical analysis demonstrated that the variance was dependent on the experimental parameters and not the assay system. The 24-well plate assay was shown to be multipurpose for testing numerous variables on cell growth or other biological properties.

The small molecule rhodomyrtone suppresses TNF-α and IL-17A-induced keratinocyte inflammatory responses: A potential new therapeutic for psoriasis.

Psoriasis is a common skin disease pathogenically driven by TNF and IL-17A-induced epidermal hyperproliferation and inflammatory responses. The ongoing need for new therapeutic agents for psoriasis has highlighted medicinal plants as sources of phytochemicals useful for treating psoriatic disease. Rhodomyrtone, a bioactive phytochemical from Rhodomyrtus tomentosa, has well-established anti-proliferative activities. This study assessed the potential of rhodomyrtone for curtailing TNF/IL-17A-driven inflammation. Stimulating human skin organ cultures with TNF+IL-17A to model the skin inflammation in psoriasis, we found that rhodomyrtone significantly decreased inflammatory gene expression and the expression and secretion of inflammatory proteins, assessed by qRT-PCR, immunohistochemistry and ELISA assays respectively. RNA-seq analysis of monolayer primary keratinocytes treated with IL-17A/TNF showed that rhodomyrtone inhibited 724/1587 transcripts >2-fold altered by IL-17A/TNF (p<0.01), a number of which were confirmed at the mRNA and protein level. Suggesting that rhodomyrtone acts by modulating MAP kinase and NF-κB signaling pathways, rhodomyrtone inhibited TNF-induced ERK, JNK, p38, and NF-κBp65 phosphorylation. Finally, assessing the in vivo anti-inflammatory potential of rhodomyrtone, we examined its effects on imiquimod-induced skin inflammation in mice, finding rhodomyrtone reversed imiquimod-induced skin hyperplasia and epidermal thickening (p< 0.001). Taken together, these results suggest that rhodomyrtone may be useful in preventing or slowing the progression of inflammatory skin disease.

Permanent draft genome sequence of Frankia sp. NRRL B-16219 reveals the presence of canonical nod genes, which are highly homologous to those detected in Candidatus Frankia Dg1 genome.

Frankia sp. NRRL B-16219 was directly isolated from a soil sample obtained from the rhizosphere of Ceanothus jepsonii growing in the USA. Its host plant range includes members of Elaeagnaceae species. Phylogenetically, strain NRRL B-16219 is closely related to "Frankia discariae" with a 16S rRNA gene similarity of 99.78%. Because of the lack of genetic tools for Frankia, our understanding of the bacterial signals involved during the plant infection process and the development of actinorhizal root nodules is very limited. Since the first three Frankia genomes were sequenced, additional genome sequences covering more diverse strains have helped provide insight into the depth of the pangenome and attempts to identify bacterial signaling molecules like the rhizobial canonical nod genes. The genome sequence of Frankia sp. strain NRRL B-16219 was generated and assembled into 289 contigs containing 8,032,739 bp with 71.7% GC content. Annotation of the genome identified 6211 protein-coding genes, 561 pseudogenes, 1758 hypothetical proteins and 53 RNA genes including 4 rRNA genes. The NRRL B-16219 draft genome contained genes homologous to the rhizobial common nodulation genes clustered in two areas. The first cluster contains nodACIJH genes whereas the second has nodAB and nodH genes in the upstream region. Phylogenetic analysis shows that Frankia nod genes are more deeply rooted than their sister groups from rhizobia. PCR-sequencing suggested the widespread occurrence of highly homologous nodA and nodB genes in microsymbionts of field collected Ceanothus americanus.

Draft Genome Sequence of Frankia sp. Strain DC12, an Atypical, Noninfective, Ineffective Isolate from Datisca cannabina.

Frankia sp. strain DC12, isolated from root nodules of Datisca cannabina, is a member of the fourth lineage of Frankia, which is unable to reinfect actinorhizal plants. Here, we report its 6.88-Mbp high-quality draft genome sequence, with a G+C content of 71.92% and 5,858 candidate protein-coding genes.

Draft genome sequence of Frankia sp. strain CN3, an atypical, noninfective (Nod-) ineffective (Fix-) isolate from Coriaria nepalensis.

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.

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

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.

Draft Genome Sequence of Frankia sp. Strain BMG5.12, a Nitrogen-Fixing Actinobacterium Isolated from Tunisian Soils.

Members of the actinomycete genus Frankia form a nitrogen-fixing symbiosis with 8 different families of actinorhizal plants. We report a draft genome sequence for Frankia sp. strain BMG5.12, a nitrogen-fixing actinobacterium isolated from Tunisian soils with the ability to infect Elaeagnus angustifolia and Myrica gale.

Draft Genome Sequence of Frankia sp. Strain BCU110501, a Nitrogen-Fixing Actinobacterium Isolated from Nodules of Discaria trinevis.

Frankia forms a nitrogen-fixing symbiosis with actinorhizal plants. We report a draft genome sequence for Frankia sp. strain BCU110501, a nitrogen-fixing actinobacterium isolated from nodules of Discaria trinevis grown in the Patagonia region of Argentina.

Analysis of codon usage patterns and predicted highly expressed genes for six phytopathogenic Xanthomonas genomes shows a high degree of conservation.

Members of the genus Xanthomonas are significant phytopathogens, which cause diseases in several economically important crops including rice, canola, tomato, citrus, etc. We have analyzed the genomes of six recently sequenced Xanthomonas strains for their synonymous codon usage patterns for all of protein coding genes and specific genes associated with pathogenesis, and determined the predicted highly expressed (PHX) genes by the use of the codon adaptation index (CAI). Our results show considerable heterogeneity among the genes of these moderately G+C rich genomes. Most of the genes were moderate to highly biased in their codon usage. However, unlike ribosomal protein genes, which were governed by translational selection, those genes associated with pathogenesis (GAP) were affected by mutational pressure and were predicted to have moderate to low expression levels. Only two out of 339 GAP genes were in the PHX category. PHX genes present in clusters of orthologous groups of proteins (COGs) were identified. Genes in the plasmids present in two strains showed moderate to low expression level and only a couple of genes featured in the PHX list. Common genes present in the top-20 PHX gene-list were identified and their possible functions are discussed. Correspondence analysis showed that genes are highly confined to a core in the plot.