E. G. Ivanova

Aerobic Methylobacteria Are Capable of Synthesizing Auxins

Obligately and facultatively methylotrophic bacteria with different pathways of C1 metabolism were found to be able to produce auxins, particularly indole-3-acetic acid (IAA), in amounts of 3–100 μg/ml. Indole-3-pyruvic acid and indole-3-acetamide were detected only in methylobacteria with the serine pathway of C1 metabolism (Methylobacterium mesophilicumand Aminobacter aminovorans).The production of auxins by methylobacteria was stimulated by the addition of L-tryptophan to the growth medium and was inhibited by ammonium ions. The methylobacteria under study lacked tryptophan decarboxylase and tryptophan side-chain oxidase. At the same time, they were found to contain several aminotransferases. IAA is presumably synthesized by methylobacteria through indole-3-pyruvic acid.

Aerobic methylotrophic bacteria as phytosymbionts

This paper deals with the physiological, biochemical, and molecular genetic aspects of the interaction of aerobic methylotrophic bacteria with plants by means of phytohormones (such as cytokinins and auxins) and other physiologically active substances (vitamins, exopolysaccharides, bioprotectants, and others). An overview of the field and the prospects of research in the field of bacteria–plant interactions and the application of aerobic methylotrophs in plant biotechnology is discussed.

Facultative and obligate aerobic methylobacteria synthesize cytokinins

The presence and expression of genes controlling the synthesis and secretion of cytokinins by the pink-pigmented facultative methylotroph Methylobacterium mesophilicum VKM B-2143 with the serine pathway and nonpigmented obligate methylotroph Methylovorus mays VKM B-2221 with the ribulose monophosphate pathway of C1 metabolism were shown using the polymerase chain reaction (PCR) and reverse transcription–PCR methods. The presence of the corresponding mRNA in M. mesophilicum cells grown on methanol or succinate suggests that the expression of these genes is constitutive. The cytokinin activity of culture liquid and its fractions was determined by a biotest with Amarantus caudatus L. seedlings. Using enzyme-linked immunosorbent analysis, we detected zeatin (riboside) in the culture liquid of both bacteria studied. The data obtained show that the aerobic methylobacteria are phytosymbionts that are able to utilize the single- and polycarbon compounds secreted by symbiotic plants and to synthesize cytokinins.

New Evidence for the Ability of Methylobacteria and Methanotrophs to Synthesize Auxins

Indole-3-acetic acid (IAA) is one of the most widespread auxins, a group of plant hormones that are derivatives of indole. The formation of indole and its derivatives from L-tryptophan is a taxonomically valuable characteristic of aerobic gram-negative bacteria [1]. According to the identification criteria of Bergey’s Manual, methylobacteria are unable to produce indoles [2]. However, our previous study [3] showed that four species of methylobacteria, Methylobacterium mesophilicum, Aminobacter aminovorans, Methylovorus mays , and Paracoccus kondratievae , are able to produce IAA. This ability was established using the Salkowski and van Urk reagents, which allow an indole concentration as low as 2 μ g/ml to be detected. At the same time, the Ehrlich and Kovacs reagents, which are used in routine microbiological tests, failed to detect indoles in the methylobacterial cultures because of the low sensitivity of these reagents (40 μ g/ml). IAA synthesis in the methylobacteria studied was found to be inhibited by ammonium ions present in the growth medium. This finding prompted us to investigate the ability of a wider range of methylotrophic bacteria to synthesize indole derivatives from L-tryptophan.

Methanotrophs and methylobacteria are found in woody plant tissues within the winter period

Samples of tree seeds, buds, and needles collected within the winter period at ambient temperatures from −11 to −17°C were analyzed for the presence of methylotrophic microflora. Thin sections of blue spruce needles were found to contain bacteria morphologically close to pink-pigmented methylobacteria. The methylobacteria that were isolated in pure cultures from samples of linden seeds and buds and pine and blue spruce needles, as well as of lilac, maple, and apple buds, were classified into the genera Methylobacterium and Paracoccus based on the data of morphological studies, enzyme assay, and DNA-DNA hybridization analysis. The methanotrophs that were isolated in pure cultures from samples of linden buds and blue spruce needles were referred to the genus Methylocystis based on the data of morphological studies, enzyme assay, DNA-DNA hybridization, and the phylogenetic analysis of the particulate methane monooxygenase gene pmoA sequences. The inference is made that aerobic methylotrophic bacteria are permanently associated with plants. At the beginning of the vegetative period in spring, the phyllosphere of coniferous and deciduous trees is colonized by methylotrophic bacteria that have wintered inside plant tissues.

[Methylophaga murata sp. nov.: a haloalkaliphilic aerobic methylotroph from deteriorating marble].

The haloalkaliphilic methylotrophic bacterium (strain Kr3) isolated from material scraped off the deteriorating marble of the Moscow Kremlin masonry has been found to be able to utilize methanol, methylamine, trimethylamine, and fructose as carbon and energy sources. Its cells are gram-negative motile rods multiplying by binary fission. Spores are not produced. The isolate is strictly aerobic and requires vitamin B12 and Na+ ions for growth. It is oxidase- and catalase-positive and reduces nitrates to nitrites. Growth occurs at temperatures between 0 and 40°C (with the optimum temperatures being 20–32°C), pH values between 6 and 11 (with the optimum at 8–9), and NaCl concentrations between 0.05 and 3 M (with the optimum at 0.5–1.5 M). The dominant cellular phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. The major cellular fatty acids are palmitic (C16:0), palmitoleic (C16:1), and octadecenoic (C18:1) acids. The major ubiquinone is Q8. It accumulates ectoine and glutamate, as well as a certain amount of sucrose, to function as osmoprotectants and synthesizes an exopolysaccharide composed of carbohydrate and protein components. It is resistant to heating at 70°C, freezing, and drying; utilizes methanol, with the resulting production of formic acid, which is responsible for the marble-degrading activity of the isolate; and implements the 2-keto-3-deoxy-6-phosphogluconate variant of the ribulose monophosphate pathway. The G+C content of its DNA is 44.6 mol %. Based on 16S rRNA gene sequencing and DNA-DNA homology levels (23–41%) with neutrophilic and alkaliphilic methylobacteria from the genus Methylophaga, the isolate has been identified as a new species, Methylophaga murata (VKM B-2303T = NCIMB 13993T).

A new system of degenerate oligonucleotide primers for detection and amplification of nifHD genes

247 Biological nitrogen fixation is a process of dinitrogen reduction to assimilable ammonium [1, 2]. Many prokaryotes fix nitrogen by means of nitrogenase, a highly conservative enzyme complex consisting of dinitrogenase (MoFe-protein) and dinitrogenase reductase (Fe-protein) encoded by the nifDK and nifH genes, respectively [1]. The most convenient method of assessing the nitrogen-fixing ability in different prokaryotes is amplification of nitrogenase structural genes ( nif genes) using degenerate oligonucleotide primers in polymerase chain reaction (PCR). Nitrogen fixation can be assessed by other methods, such as acetylene reduction, hybridization of genome DNA with labeled probes, or methods using 15 N. However, these methods require expensive equipment and reagents, and their results are not always reliable.

The Effect of Aerobic Methylotrophic Bacteria on the in vitro Morphogenesis of Soft Wheat (Triticum aestivum)

The effects of four aerobic methylotrophic bacteria on the morphogenesis of soft wheat (Triticum aestivum) were studied in vitro using immature embryos as explants. The inoculation of the embryos with methylotrophic bacteria led to their stable colonization with the bacteria. The colonization of the explants with the strains of Methylobacterium sp. D10 and Methylophilus glucoseoxidans stimulated the formation of morphogenic calli and shoots and also promoted development of the regenerated plants. These regenerated plants manifested bright green leaves and a well-developed root system. The colonization of immature wheat embryos with methylotrophic bacteria can be employed as a tool for raising the efficiency of genetic transformation of various wheat cultivars.

Vitamin B12-independent strains of Methylophaga marina isolated from Red Sea algae

Two strains (KM3 and KM5) of halophilic methylobacteria isolated from Red Sea algae do not require vitamin B12 for growth and can use methanol, methylamine, dimethylamine, trimethylamine, dimethyl sulfide, and fructose as sources of carbon and energy. The cells of these strains are gram-negative motile monotrichous (strain KM3) or peritrichous (strain KM5) rods. The strains are strictly aerobic and require Na+ ions but not growth factors. They are oxidase-and catalase-positive and reduce nitrates to nitrites. Both strains can grow in a temperature range of 4 to 37°C (with optimal growth at 29–34°C), at pH between 5.5 and 8.5 (with optimal growth at pH 7.5–8.0), and in a range of salt concentrations between 0.5 and 15% NaCl (with optimal growth at 5–9% NaCl). The phospholipids of these strains are dominated by phosphatidylethanolamine and phosphatidylglycerol and also include phosphatidylcholine, phosphatidylserine, and cardiolipin. The dominant fatty acids are C16:1ω7c and C16:0. The major ubiquinone is Q8. The cells accumulate ectoin, glutamate, and sucrose as intracellular osmoprotectants. The strains implement the 2-keto-3-deoxy-6-phosphogluconate-dependent variant of the ribulose monophosphate pathway. The G+C content of the DNA is 44.4–44.7 mol%. Analysis of the 16S rRNA genes showed that both strains belong to Gammaproteobacteria and have a high degree of homology (99.4%) to Methylophaga marina ATCC 35842T. Based on the data of polyphasic taxonomy, isolates KM3 and KM5 are identified as new strains M. marina KM3 (VKM B-2386) and M. marina KM5 (VKM B-2387). The ability of these strains to produce auxins (indole-3-acetic acid) suggests their metabolic association with marine algae.

Plant Growth and Morphogenesis in vitroIs Promoted by Associative Methylotrophic Bacteria

The effects of aerobic methylotrophic bacteria Methylovorus mayson growth and morphogenesis were studied in in vitropropagated tobacco, potato, and flax. Colonization of plant explants with the methylo-trophic bacteria led to the stable association of bacteria and plants and enhanced the growth and the capacity of the latter for regeneration and root formation. When colonized by the methylotrophic bacteria, the rootless transgenic tobacco plants carrying the agrobacterial cytokinin gene iptrestored their ability to form roots. These data indicate the possibility to employ methylotrophic bacteria as a tool in experimental biology and plant biotechnology.