A novel pink-pigmented facultative methylotroph, Methylobacterium thiocyanatum sp. nov., capable of growth on thiocyanate or cyanate as sole nitrogen sources

Abstract

The isolation and properties of a novel species of pink-pigmented methylotroph, Methylobacterium thiocyanatum, are described. This organism satisfied all the morphological, biochemical, and growth-substrate criteria to be placed in the genus Methylobacterium. Sequencing of the gene encoding its 16S rRNA confirmed its position in this genus, with its closest phylogenetic relatives being M. rhodesianum, M. zatmanii and M. extorquens, from which it differed in its ability to grow on several diagnostic substrates. Methanol-grown organisms contained high activities of hydroxypyruvate reductase -3 micromol NADH oxidized min-1 (mg crude extract protein)-1], showing that the serine pathway was used for methylotrophic growth. M. thiocyanatum was able to use thiocyanate or cyanate as the sole source of nitrogen for growth, and thiocyanate as the sole source of sulfur in the absence of other sulfur compounds. It tolerated high concentrations (at least 50 mM) of thiocyanate or cyanate when these were supplied as nitrogen sources. Growing cultures degraded thiocyanate to produce thiosulfate as a major sulfur end product, apparently with the intermediate formation of volatile sulfur compounds (probably hydrogen sulfide and carbonyl sulfide). Enzymatic hydrolysis of thiocyanate by cell-free extracts was not demonstrated. Cyanate was metabolized by means of a cyanase enzyme that was expressed at approximately sevenfold greater activity during growth on thiocyanate [Vmax 634 +/- 24 nmol NH3 formed min-1 (mg protein)-1] than on cyanate [89 +/- 9 nmol NH3 min-1 (mg protein)-1]. Kinetic study of the cyanase in cell-free extracts showed the enzyme (1) to exhibit high affinity for cyanate (Km 0.07 mM), (2) to require bicarbonate for activity, (3) to be subject to substrate inhibition by cyanate and competitive inhibition by thiocyanate (Ki 0.65 mM), (4) to be unaffected by 1 mM ammonium chloride, (5) to be strongly inhibited by selenocyanate, and (6) to be slightly inhibited by 5 mM thiosulfate, but unaffected by 0.25 mM sulfide or 1 mM thiosulfate. Polypeptides that might be a cyanase subunit (mol.wt. 17.9 kDa), a cyanate (and/or thiocyanate) permease (mol.wt. 25.1 and 27.2 kDa), and a putative thiocyanate hydrolase (mol.wt. 39.3 kDa) were identified by SDS-PAGE. Correlation of the growth rate of cultures with thiocyanate concentration (both stimulatory and inhibitory) and the kinetics of cyanase activity might indicate that growth on thiocyanate involved the intermediate formation of cyanate, hence requiring cyanase activity. The very high activity of cyanase observed during growth on thiocyanate could be in compensation for the inhibitory effect of thiocyanate on cyanase. Alternatively, thiocyanate may be a nonsubstrate inducer of cyanase, while thiocyanate degradation itself proceeds by a carbonyl sulfide pathway not involving cyanate. A formal description of the new species (DSM 11490) is given.

Methylobacterium platani sp. nov., isolated from a leaf of the tree Platanus orientalis

“…The genus Methylobacterium was first described by Patt et al (1976) and at the time of writing consists of the following 24 recognized species (http://www.bacterio.cict.fr/m/methylobacterium. html): Methylobacterium adhaesivum (Gallego et al, 2006), M. aminovorans (Urakami et al, 1993), M. aquaticum (Gallego et al, 2005a), M. chloromethanicum (McDonald et al, 2001), M. dichloromethanicum (Doronina et al, 2000), M. extorquens (Bousfield & Green, 1985), M. fujisawaense (Green et al, 1988), M. hispanicum (Gallego et al, 2005a), M. isbiliense (Gallego et al, 2005c), M. jeotgali (Aslam et al, 2007), M. lusitanum (Doronina et al, 2002), M. mesophilicum , M. nodulans (Jourand et al, 2004), M. organophilum (Patt et al, 1976), M. oryzae (Madhaiyan et al, 2007), M. podarium (Anesti et al, 2004), M. populi (Van Aken et al, 2004), M. radiotolerans , M. rhodesianum (Green et al, 1988), M. rhodinum , M. suomiense (Doronina et al, 2002), M. thiocyanatum (Wood et al, 1998), M. variabile (Gallego et al, 2005b) and M. zatmanii (Green et al, 1988). Here, we provide a detailed taxonomic characterization of a Methylobacterium-like bacterial strain, PMB02 T , which was isolated from a tree leaf.…”

Methylobacterium adhaesivum sp. nov., a methylotrophic bacterium isolated from drinking water

“…It includes strictly aerobic, Gram-negative, rod-shaped, pink-pigmented, facultatively methylotrophic (PPFM) bacteria, which can grow on single carbon compounds such as formate, formaldehyde and methanol as the sole source of carbon and energy, as well as on a wide range of multi-carbon growth substrates (Green, 2001). At the time of writing, the genus comprises 20 recognized species (Patt et al, 1976;Bousfield & Green, 1985;Green et al, 1988;Urakami et al, 1993;Wood et al, 1998; Doronina et al, 2000Doronina et al, , 2002 McDonald et al, 2001;Van Aken et al, 2004;Jourand et al, 2004; Gallego et al, 2005a, b, c), with Methylobacterium organophilum as the type species (Patt et al, 1976).Members of the genus Methylobacterium are ubiquitous in terrestrial habitats (Green & Bousfield, 1981, including soil, dust, freshwater, tap water systems, lake sediments, leaf surfaces and nodules, rice grains and air, and as contaminants in various products and processes. The PPFM bacteria are strict aerobes and can be isolated from almost any freshwater environment where some dissolved oxygen exists, e.g.…”

Reclassification of Methylobacterium chloromethanicum and Methylobacterium dichloromethanicum as later subjective synonyms of Methylobacterium extorquens and of Methylobacterium lusitanum as a later subjective synonym of Methylobacterium rhodesianum

“…Methylobacterium strains produce carotenoid pigment and bacteriochlorophyll in their cells, and they belong to a group of aerobic bacteriochlorophyll-containing (ABC) bacteria (Hiraishi and Shimada, 2001). Currently, the genus Methylobacterium consists of 14 valid species, Methylobacterium extorquens (Bousfield and Green, 1985), Methylobacterium rhodesianum, Methylobacterium zatmanii and Methylobacterium fujisawaense (Green et al, 1988), Methylobacterium aminovorans (Urakami et al, 1993), Methylobacterium thiocyanatum (Wood et al, 1998), Methylobacterium dichloromethanicum (Doronina et al, 2000), Methylobacterium chloromethanicum (McDonald et al, 2001), Methylobacterium suomiense, and Methylobacterium lusitanum (Doronina et al, 2002). The taxonomic study based on phenotypic properties (Green and Bousfield, 1982) showed that Methylobacterium species were divided into two physiological groups.…”

“…1), they are likely to be the same strain. Compared to reference data of phylogenetic analysis (Doronina et al, 2002;Urakami et al, 1993;Wood et al, 1998), JCM 2833 T is thought to be the authentic type strain. Based on these facts, it is regarded that DSM 760 T had been mislabeled.…”