A Periplasmic Lanthanide Mediator, Lanmodulin, in Methylobacterium aquaticum Strain 22A

Methylobacterium and Methylorubrum species oxidize methanol via pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca2+-dependent MDH (MxaF) and lanthanide (Ln(3+))-dependent MDH (XoxF), whose expression is regulated by the availability of Ln(3+). A set of a siderophore, TonB-dependent receptor, and an ABC transporter that resembles the machinery for iron uptake is involved in the solubilization and transport of Ln(3+). The transport of Ln(3+) into the cytosol enhances XoxF expression. A unique protein named lanmodulin from Methylorubrum extorquens strain AM1 was identified as a specific Ln(3+)-binding protein, and its biological function was implicated to be an Ln(3+) shuttle in the periplasm. In contrast, it remains unclear how Ln(3+) levels in the cells are maintained, because Ln(3+) is potentially deleterious to cellular systems due to its strong affinity to phosphate ions. In this study, we investigated the function of a lanmodulin homolog in Methylobacterium aquaticum strain 22A. The expression of a gene encoding lanmodulin (lanM) was induced in response to the presence of La3+. A recombinant LanM underwent conformational change upon La3+ binding. Phenotypic analyses on lanM deletion mutant and overexpressing strains showed that LanM is not necessary for the wild-type and XoxF-dependent mutant's methylotrophic growth. We found that lanM expression was regulated by MxcQE (a two-component regulator for MxaF) and TonB_Ln (a TonB-dependent receptor for Ln(3+)). The expression level of mxcQE was altered to be negatively dependent on Ln(3+) concentration in increment lanM, whereas it was constant in the wild type. Furthermore, when exposed to La3+, increment lanM showed an aggregating phenotype, cell membrane impairment, La deposition in the periplasm evidenced by electron microscopy, differential expression of proteins involved in membrane integrity and phosphate starvation, and possibly lower La content in the membrane vesicle (MV) fractions. Taken together, we concluded that lanmodulin is involved in the complex regulation mechanism of MDHs and homeostasis of cellular Ln levels by facilitating transport and MV-mediated excretion

Evaluation of rhizosphere, rhizoplane and phyllosphere bacteria and fungi isolated from rice in Kenya for plant growth promoters

Rice (Oryza sativa L.) is the most important staple food crop in many developing countries, and is ranked third in Kenya after maize and wheat. Continuous cropping without replenishing soil nutrients is a major problem in Kenya resulting to declining soil fertility. The use of chemical fertilizers to avert the problem of low soil fertility is currently limited due to rising costs and environmental concerns. Many soil micro-organisms are able to solubilize the unavailable phosphorus, increase uptake of nitrogen and also synthesize growth promoting hormones including auxin. The aim of this study was to isolate and characterize phyllosphere, rhizoplane and rhizosphere micro-organisms from Kenyan rice with growth promoting habits. In this study whole plant rice samples were collected from different rice growing regions of Kenya. 76.2%, over 80% and 38.5% of the bacterial isolates were positive for phosphate solubilization, nitrogenase activity and IAA production whereas 17.5% and 5% of the fungal isolates were positive for phosphate solubilization and IAA production respectively. Hence these micro-organisms have potential for utilization as bio-fertilizers in rice production

A capsidless ssRNA virus hosted by an unrelated dsRNA virus

Viruses typically encode the capsid that encases their genome, while satellite viruses do not encode a replicase and depend on a helper virus for their replication1. Here, we report interplay between two RNA viruses, yado-nushi virus 1 (YnV1) and yado-kari virus 1 (YkV1), in a phytopathogenic fungus, Rosellinia necatrix2. YkV1 has a close phylogenetic affinity to positive-sense, single-stranded (+)ssRNA viruses such as animal caliciviruses3, while YnV1 has an undivided double-stranded (ds) RNA genome with a resemblance to fungal totiviruses4. Virion transfection and infectious full-length cDNA transformation has shown that YkV1 depends on YnV1 for viability, although it probably encodes functional RNA-dependent RNA polymerase (RdRp). Immunological and molecular analyses have revealed trans-encapsidation of not only YkV1 RNA but also RdRp by the capsid protein of the other virus (YnV1), and enhancement of YnV1 accumulation by YkV1. This study demonstrates interplay in which the capsidless (+)ssRNA virus (YkV1), hijacks the capsid protein of the dsRNA virus (YnV1), and replicates as if it were a dsRNA virus

Sphingosine 1-phosphate (S1P) inhibits monocyte–endothelial cell interaction by regulating of RhoA activity

AbstractRecent studies suggest that sphingosine 1-phosphate (S1P) protects against atherosclerosis. We assessed the effects of S1P on monocyte–endothelial interaction in the presence of inflammatory mediators. Pretreatment of THP-1 cells with S1P abolished Phorbol 12 myristate 13-acetate (PMA)-induced THP-1 cell adhesion to human umbilical vein endothelial cells (HUVECs). S1P inhibited PMA-induced activation of RhoA, but not PKCs. S1P activated p190Rho GTPase activation protein (GAP) only in the presence of PMA, suggesting an inhibitory effect of S1P and PMA to suppress RhoA. In conclusion, S1P inhibited monocyte–endothelial interactions by inhibiting RhoA activity which may explain its anti-atherogenic effects

Comprehensive Comparative Genomics and Phenotyping of Methylobacterium Species

The pink-pigmented facultative methylotrophs (PPFMs), a major bacterial group found in the plant phyllosphere, comprise two genera: Methylobacterium and Methylorubrum. They have been separated into three major clades: A, B (Methylorubrum), and C. Within these genera, however, some species lack either pigmentation or methylotrophy, which raises the question of what actually defines the PPFMs. The present study employed a comprehensive comparative genomics approach to reveal the phylogenetic relationship among the PPFMs and to explain the genotypic differences that confer their different phenotypes. We newly sequenced the genomes of 29 relevant-type strains to complete a dataset for almost all validly published species in the genera. Through comparative analysis, we revealed that methylotrophy, nitrate utilization, and anoxygenic photosynthesis are hallmarks differentiating the PPFMs from the other Methylobacteriaceae. The Methylobacterium species in clade A, including the type species Methylobacterium organophilum, were phylogenetically classified into six subclades, each possessing relatively high genomic homology and shared phenotypic characteristics. One of these subclades is phylogenetically close to Methylorubrum species; this finding led us to reunite the two genera into a single genus Methylobacterium. Clade C, meanwhile, is composed of phylogenetically distinct species that share relatively higher percent G+C content and larger genome sizes, including larger numbers of secondary metabolite clusters. Most species of clade C and some of clade A have the glutathione-dependent pathway for formaldehyde oxidation in addition to the H4MPT pathway. Some species cannot utilize methanol due to their lack of MxaF-type methanol dehydrogenase (MDH), but most harbor an XoxF-type MDH that enables growth on methanol in the presence of lanthanum. The genomes of PPFMs encode between two and seven (average 3.7) genes for pyrroloquinoline quinone-dependent alcohol dehydrogenases, and their phylogeny is distinctly correlated with their genomic phylogeny. All PPFMs were capable of synthesizing auxin and did not induce any immune response in rice cells. Other phenotypes including sugar utilization, antibiotic resistance, and antifungal activity correlated with their phylogenetic relationship. This study provides the first inclusive genotypic insight into the phylogeny and phenotypes of PPFMs

High-Throughput Identification and Screening of Novel Methylobacterium Species Using Whole-Cell MALDI-TOF/MS Analysis

Methylobacterium species are ubiquitous α-proteobacteria that reside in the phyllosphere and are fed by methanol that is emitted from plants. In this study, we applied whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (WC-MS) to evaluate the diversity of Methylobacterium species collected from a variety of plants. The WC-MS spectrum was reproducible through two weeks of cultivation on different media. WC-MS spectrum peaks of M. extorquens strain AM1 cells were attributed to ribosomal proteins, but those were not were also found. We developed a simple method for rapid identification based on spectra similarity. Using all available type strains of Methylobacterium species, the method provided a certain threshold similarity value for species-level discrimination, although the genus contains some type strains that could not be easily discriminated solely by 16S rRNA gene sequence similarity. Next, we evaluated the WC-MS data of approximately 200 methylotrophs isolated from various plants with MALDI Biotyper software (Bruker Daltonics). Isolates representing each cluster were further identified by 16S rRNA gene sequencing. In most cases, the identification by WC-MS matched that by sequencing, and isolates with unique spectra represented possible novel species. The strains belonging to M. extorquens, M. adhaesivum, M. marchantiae, M. komagatae, M. brachiatum, M. radiotolerans, and novel lineages close to M. adhaesivum, many of which were isolated from bryophytes, were found to be the most frequent phyllospheric colonizers. The WC-MS technique provides emerging high-throughputness in the identification of known/novel species of bacteria, enabling the selection of novel species in a library and identification without 16S rRNA gene sequencing

A novel quadripartite dsRNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix

AbstractHere we report the biological and molecular attributes of a novel dsRNA virus isolated from Rosellinia necatrix, a filamentous phytopathogenic fungus. The virus, termed Rosellinia necatrix quadrivirus 1 (RnQV1), forms rigid spherical particles approximately 45nm in diameter in infected mycelia. The particles contain 4 dsRNA segments, dsRNA1 to dsRNA4, with a size range of 4.9 to 3.7kbp, each possessing a single large ORF. A comparison of the virus-infected and -cured isogenic fungal strains suggested that RnQV1 infection has no appreciable phenotypic effects. Phylogenetic analysis using the dsRNA3-encoded RdRp sequence revealed that RnQV1 is more distantly related to quadripartite chrysoviruses than to monopartite totiviruses, and is placed in a distinct group from other mycoviruses. No significant sequence similarities were evident between known proteins and RnQV1 structural proteins shown to be encoded by dsRNA2 or dsRNA4. These suggest that RnQV1 is a novel latent virus, belonging to a new family

Siderophore for Lanthanide and Iron Uptake for Methylotrophy and Plant Growth Promotion in Methylobacterium aquaticum Strain 22A

Methylobacterium and Methylorubrum species are facultative methylotrophic bacteria that are abundant in the plant phyllosphere. They have two methanol dehydrogenases, MxaF and XoxF, which are dependent on either calcium or lanthanides (Lns), respectively. Lns exist as insoluble minerals in nature, and their solubilization and uptake require a siderophore-like substance (lanthanophore). Methylobacterium species have also been identified as plant growth-promoting bacteria although the actual mechanism has not been well-investigated. This study aimed to reveal the roles of siderophore in Methylobacterium aquaticum strain 22A in Ln uptake, bacterial physiology, and plant growth promotion. The strain 22A genome contains an eight-gene cluster encoding the staphyloferrin B-like (sbn) siderophore. We demonstrate that the sbn siderophore gene cluster is necessary for growth under low iron conditions and was complemented by supplementation with citrate or spent medium of the wild type or other strains of the genera. The siderophore exhibited adaptive features, including tolerance to oxidative and nitrosative stress, biofilm formation, and heavy metal sequestration. The contribution of the siderophore to plant growth was shown by the repressive growth of duckweed treated with siderophore mutant under iron-limited conditions; however, the siderophore was dispensable for strain 22A to colonize the phyllosphere. Importantly, the siderophore mutant could not grow on methanol, but the siderophore could solubilize insoluble Ln oxide, suggesting its critical role in methylotrophy. We also identified TonB-dependent receptors (TBDRs) for the siderophore-iron complex, iron citrate, and Ln, among 12 TBDRs in strain 22A. Analysis of the siderophore synthesis gene clusters and TBDR genes in Methylobacterium genomes revealed the existence of diverse types of siderophores and TBDRs. Methylorubrum species have an exclusive TBDR for Ln uptake that has been identified as LutH. Collectively, the results of this study provide insight into the importance of the sbn siderophore in Ln chelation, bacterial physiology, and the diversity of siderophore and TBDRs in Methylobacterium species

A Catalytic Role of XoxF1 as La3+-Dependent Methanol Dehydrogenase in Methylobacterium extorquens Strain AM1

In the methylotrophic bacterium Methylobacterium extorquens strain AM1, MxaF, a Ca2+-dependent methanol dehydrogenase (MDH), is the main enzyme catalyzing methanol oxidation during growth on methanol. The genome of strain AM1 contains another MDH gene homologue, xoxF1, whose function in methanol metabolism has remained unclear. In this work, we show that XoxF1 also functions as an MDH and is La3+-dependent. Despite the absence of Ca2+ in the medium strain AM1 was able to grow on methanol in the presence of La3+. Addition of La3+ increased MDH activity but the addition had no effect on mxaF or xoxF1 expression level. We purified MDH from strain AM1 grown on methanol in the presence of La3+, and its N-terminal amino acid sequence corresponded to that of XoxF1. The enzyme contained La3+ as a cofactor. The ΔmxaF mutant strain could not grow on methanol in the presence of Ca2+, but was able to grow after supplementation with La3+. Taken together, these results show that XoxF1 participates in methanol metabolism as a La3+-dependent MDH in strain AM1

Honeydew-associated microbes elicit defense responses against brown planthopper in rice

Feeding of sucking insects, such as the rice brown planthopper (Nilaparvata lugens; BPH), causes only limited mechanical damage on plants that is otherwise essential for injury-triggered defense responses against herbivores. In pursuit of complementary BPH elicitors perceived by plants, we examined the potential effects of BPH honeydew secretions on the BPH monocot host, rice (Oryza sativa). We found that BPH honeydew strongly elicits direct and putative indirect defenses in rice, namely accumulation of phytoalexins in the leaves, and release of volatile organic compounds from the leaves that serve to attract natural enemies of herbivores, respectively. We then examined the elicitor active components in the honeydew and found that bacteria in the secretions are responsible for the activation of plant defense. Corroborating the importance of honeydew-associated microbiota for induced plant resistance, BPHs partially devoid of their microbiota via prolonged antibiotics ingestion induced significantly less defense in rice relative to antibiotic-free insects applied to similar groups of plants. Our data suggest that rice plants may additionally perceive herbivores via their honeydew-associated microbes, allowing them to discriminate between incompatible herbivores—that do not produce honeydew—and those that are compatible and therefore dangerous