Methylotroph Quorum Sensing Signal Identification by Inverse Stable Isotopic Labeling.

Natural products are an essential source of bioactive compounds. Isotopic labeling is an effective way to identify natural products that incorporate a specific precursor; however, this approach is limited by the availability of isotopically enriched precursors. We used an inverse stable isotopic labeling approach to identify natural products by growing bacteria on a 13C-carbon source and then identifying 12C-precursor incorporation by mass spectrometry. We applied this approach to methylotrophs, ecologically important bacteria predicted to have significant yet underexplored biosynthetic potential. We demonstrate that this method identifies N-acyl homoserine lactone quorum sensing signals produced by diverse methylotrophs grown on three different one-carbon compounds. We then apply this approach to simultaneously detect five previously unidentified signals produced by a methylotroph and link these compounds to their synthases. We envision that this method can be used to identify other natural product classes synthesized by methylotrophs and other organisms that grow on relatively inexpensive 13C-carbon sources.

Bioinformatics of a Novel Nitrile Hydratase Gene Cluster of the N2-Fixing Bacterium Microvirga flocculans CGMCC 1.16731 and Characterization of the Enzyme.

Microvirga flocculans CGMCC 1.16731 can degrade many cyano group-containing neonicotinoid insecticides. Here, its genome was sequenced, and a novel nitrile hydratase gene cluster was discovered in a plasmid. The NHase gene cluster (pnhF) has gene structure ß-subunit 1, α-subunit, and ß-subunit 2, which is different from previously reported NHase gene structures. Phylogenetic analysis of α-subunits indicated that NHases containing the three subunit (ß1αß2) structure are independent from NHases containing two subunits (αß). pnhF was successfully expressed in Escherichia coli, and the purified PnhF could convert the nitrile-containing insecticide flonicamid to N-(4-trifluoromethylnicotinoyl)glycinamide. The enzymatic properties of PnhF were investigated using flonicamid as a substrate. Homology models revealed that amino acid residue ß1-Glu56 may strongly affect the catalytic activity of PnhF. This study expands our understanding of the structures and functions of NHases and the enzymatic mechanism of the environmental fate of flonicamid.

Microvirga sp. symbiovar mediterranense nodulates Lupinus cosentinii grown wild in Morocco.

AIM: To analyse the diversity of nodule-forming bacteria isolated from Lupinus cosentinii naturally grown in the Maamora cork oak forest (Rabat, Morocco). METHODS AND RESULTS: Of the 31 bacterial strains, four were selected based on their REP-PCR fingerprinting that were studied by sequencing and phylogenetic analysis of their 16S rRNA, gyrB, dnaK, recA and rpoB housekeeping genes as well as the nodC symbiotic gene. The nearly complete 16S rRNA gene sequence of the four representative strains showed that they are related to Tunisian strains of genus Microvirga isolated from L. micranthus with nucleotide identity values ranging from 98·67 to 97·13%. The single and concatenated sequences of the 16S rRNA, gyrB, dnaK, recA and rpoB housekeeping genes indicated that the L. cosentinii-isolated strains had 99·2-99·9% similarities with the Tunisian L. micranthus microsymbionts. The nodC gene phylogeny revealed that the Moroccan strains clustered in the newly described mediterranense symbiovar, and nodulation tests showed that they nodulated not only L. cosentinii but also L. angustifolius, L. luteus and L. albus. CONCLUSIONS: To the best of our knowledge, this is the first report concerning the isolation, molecular identification and phylogenetic diversity of L. cosentinii nodule-forming endosymbionts and of their description as members of the Microvirga genus. SIGNIFICANCE AND IMPACT OF THE STUDY: In this work, we show that Microvirga sp. can be isolated from root nodules of wild-grown L. cosentinii in Northeast Africa, that selected strains also nodulate L. angustifolius, L. luteus and L. albus, and that they belong to symbiovar mediterranense. In addition, our data support that the ability of Microvirga to nodulate lupines could be related to the soil pH, its geographical distribution being more widespread than expected.

New chromosomal lineages within Microvirga and Bradyrhizobium genera nodulate Lupinus angustifolius growing on different Tunisian soils.

Thirty-one rhizobial isolates nodulating native Lupinus angustifolius (blue lupine) plants growing in Northern Tunisian soils were isolated and analysed using different chromosomal and symbiotic gene markers. Phylogenetic analyses based on recA partial sequences grouped them into at least five groups: four of them within the genus Bradyrhizobium (26 isolates) and one into the genus Microvirga (5 isolates). Representative strains were analysed by multilocus sequence analysis of three housekeeping genes rrs-recA-glnII and rrs-gyrB-dnaK for Bradyrhizobium and Microvirga isolates, respectively. Based on this analysis, eight isolates clustered with the previously described strains Bradyrhizobium lupini USDA3051 and Bradyrhizobium canariense BTA-1. However, five of the isolates clustered separately and may constitute a new species within the Bradyrhizobium genus. The remaining five isolates were closely related to the strain Microvirga sp. LmiM8 and may constitute a new Microvirga species. The analysis of the nodC gene showed that all Bradyrhizobium strains nodulating blue lupine belong to the symbiovar genistearum, whereas the Microvirga isolates are associated with the symbiovar mediterranense. The results of this study support that the L. angustifolius root nodule symbionts isolated in Northern Tunisia belong mostly to the B. canariense/B. lupini lineages. However, new clades of Bradyrhizobium and Microvirga have been identified as L. angustifolius endosymbionts.

Roseomonas alkaliterrae sp. nov., isolated from an alkali geothermal soil sample in Tengchong, Yunnan, South-West China.

An alkalitolerant, thermotolerant and Gram-stain negative bacterium, designated strain YIM 78007(T), was isolated from an alkaline geothermal soil sample from Hehua hot spring, Tengchong, Yunnan province, south-west China. Cells of strain YIM 78007(T) were observed to be aerobic and short rod-shaped. The colonies were observed to be orange-red, convex and circular. 16S rRNA gene sequence-based phylogenetic analysis showed that strain YIM 78007(T) clustered with members of the genus Roseomonas (with similarities from 97.2 to 92.2 %). Optimal growth of strain YIM 78007 occurs at 40-50 °C and pH 8.0-10.0. The predominant ubiquinone was identified as Q-10 and the major fatty acids were identified as C18:1 ω7c and C16:0. The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified aminolipids and one unknown phospholipid. The G + C content of the genomic DNA was determined to be 63 mol %. The levels of DNA-DNA hybridization relatedness between strain YIM 78007(T) and its closet neighbours (Roseomonas lacus JCM 13283(T) and Roseomonas terrae JCM 14592(T)) were well below the threshold required for the proposal of a novel species. The results of physiological and biochemical characteristics, the phylogenetic analysis, as well as low DNA-DNA hybridization values, allowed the phenotypic and genotypic differentiation of strain YIM 78007(T) from its closest phylogenetic neighbours. Therefore, strain YIM 78007(T) is considered to represent a novel species of the genus Roseomonas, for which the name Roseomonas alkaliterrae sp. nov. is proposed. The type strain is YIM 78007(T) (=BCRC 80644(T) = JCM 19656(T)).

Roseomonas tokyonensis sp. nov. isolated from a biofilm sample obtained from a cooling tower in Tokyo, Japan.

Strain K-20(T), a Gram-negative, nonmotile, nonspore-forming and strictly aerobic coccobacillus, which produces a pale pink pigment (R2A agar medium, 30℃, seven days) was isolated from a sample of biofilm obtained from a cooling tower in Tokyo, Japan. A phylogenetic analysis of the 16S rRNA partial gene sequences (1,439 bp) showed that the strain (accession number: AB297501) was related to Roseomonas frigidaquae CW67(T) and Roseomonas stagni HS-69(T) with 97.4% and 96.9% sequence similarity, respectively. Strain K-20(T) formed a distinct cluster with Roseomonas frigidaquae CW67(T) in the phylogenetic tree at a high bootstrap value (93%); however, distance was recognized between the strains. In addition, the DNA-DNA hybridization level between strain K-20(T) and Roseomonas frigidaquae JCM 15073(T) was 33%. The taxonomic data indicate that K-20(T) (=JCM 14634(T) =KCTC 32152(T)) should be classified in the genus Roseomonas as the type strain of a novel species, Roseomonas tokyonensis sp. nov.

Nodule morphology, symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the Southern African crotalarioid clade Lotononis s.l.

BACKGROUND AND AIMS: The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules. METHODS: Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N2 fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy. KEY RESULTS: Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen. CONCLUSIONS: Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The seasonally waterlogged habitat of Listia species may favour the development of symbiotic specificity.

Roseomonas musae sp. nov., a new bacterium isolated from a banana phyllosphere.

A Gram-negative, coccobacilli, non-spore forming and non-motile bacterium, designated PN1(T), was isolated from a banana leaf collected in Mattra island, Thailand. This isolate was observed to grow optimally at 30 °C and pH 7.0, and to grow with 0-3 % NaCl. Comparative 16S rRNA gene sequence analysis showed that strain PN1(T) is closely related to members of the genus Roseomonas, exhibiting the highest 16S rRNA gene sequence similarity to Roseomonas aestuarii JC17(T) (96.5 %). The DNA G + C content of strain PN1(T) was determined to be 69.7 mol %. Based on physiological and biochemical tests, and genotypic differences between strain PN1(T) and the validly named species of the genus Roseomonas, it is proposed that the strain be classified as a new species of Roseomonas for which the name Roseomonas musae sp. nov. is proposed. The type strain is PN1(T) (= BCC 44863(T) = NBRC 107870(T)).

[Methanotrophic bacteria in cold seeps of the floodplains of northern rivers].

Small mud volcanoes (cold seeps), which are common in the floodplains of northern rivers, are a potentially important, although poorly studied sources of atmospheric methane. Field research on the cold seeps of the Mukhrina River (Khanty-Mansiysk Autonomous okrug, Russia) revealed methane fluxes from these structures to be orders of magnitude higher than from equivalent areas of the mid-taiga bogs. Microbial communities developing around the seeps were formed under conditions of high methane concentrations, low temperatures (3-5 degrees C), and near-neutral pH. Molecular identification of methane-oxidizing bacteria from this community by analysis of the pmoA gene encoding particulate methane monooxygenase revealed both type I and type II methanotrophs (classes Gammaproteobacteria and Alphaproteobacteria, respectively), with predomination of type I methanotrophs. Among the latter, microorganisms related to Methylobacterpsychrophilus and Methylobacter tundripaludum, Crenothrix polyspora (a stagnant water dweller), and a number of methanotrophs belonging to unknown taxa were detected. Growth characteristics of two isolates were determined. Methylobactersp. CMS7 exhibited active growth at 4-10 degrees C, while Methylocystis sp. SB12 grew better at 20 degrees C. Experimental results confirmed the major role ofmethanotrophic gammaproteobacteria in controlling the methane emission from cold river seeps.

Genome sequence of Roseomonas sp. strain B5, a quorum-quenching N-acylhomoserine lactone-degrading bacterium isolated from Malaysian tropical soil.

Roseomonas sp. strain B5 was isolated from Malaysian tropical soil that showed N-acylhomoserine lactone degradation. This is the first genome announcement of a member from the genus of Roseomonas and the first report on the quorum-quenching activity of Roseomonas spp.

Roseomonas pecuniae sp. nov., isolated from the surface of a copper-alloy coin.

Strain N75(T) was isolated from the surface of a copper-alloy 50 Euro cent coin collected from general circulation. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain N75(T) formed a distinct branch within the genus Roseomonas and placed it in the Alphaproteobacteria. Strain N75(T) showed 16S rRNA gene sequence similarities of 92.4-97.1  % to type strains of species of the genus Roseomonas. Strain N75(T) was a Gram-negative, non-spore-forming, non-motile coccoid, with an optimum growth temperature of about 30 °C; the strain did not grow at 5 or 37 °C. Strain N75(T) did not grow in medium containing NaCl. The major respiratory quinone was ubiquinone 10 (Q-10). The major fatty acids were unsaturated C16:1ω7c/C16:1ω6c and C18:1ω7c (around 70 % of the total fatty acids); the third most abundant fatty acid was the hydroxylated C18:1 2-OH. The major polar lipids were phosphatidylcholine, phosphatidylethanolamine and an unknown aminolipid. The DNA G+C content was 72.8 mol%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics, we conclude that strain N75(T) represents a novel species of the genus Roseomonas, for which we propose the name Roseomonas pecuniae sp. nov. (type strain N75(T) =LMG 25481(T) =CIP 110074(T)).

Microvirga guangxiensis sp. nov., a novel alphaproteobacterium from soil, and emended description of the genus Microvirga.

A Gram-negative-staining bacterium, designated strain 25BT, was isolated from a soil sample from a rice field in Guangxi Province, China, and its taxonomic position was investigated by using a polyphasic approach. Cells were rod-shaped, non-spore-forming, non-motile and strictly aerobic. Strain 25BT grew optimally at 37 degrees C and pH 7.0. The predominant fatty acids of this soil isolate were C18:1omega7c, C19:0 cyclo omega8c and C16:0. Phylogenetic analysis based on the almost-complete 16S rRNA gene sequence showed that strain 25BT formed a monophyletic clade with the type strain of Microvirga subterranea; the two organisms shared 97.2% 16S rRNA gene sequence similarity. However, the two strains shared low DNA-DNA relatedness. Strain 25BT was also readily distinguishable from Microvirga subterranea DSM 14364T by various phenotypic characteristics. The combination of genotypic and phenotypic data suggests that the isolate represents a novel species of the genus Microvirga, for which the name Microvirga guangxiensis sp. nov. is proposed. The type strain is 25BT (=CGMCC 1.7666T=JCM 15710T).

Transfer of Teichococcus ludipueritiae and Muricoccus roseus to the genus Roseomonas, as Roseomonas ludipueritiae comb. nov. and Roseomonas rosea comb. nov., respectively, and emended description of the genus Roseomonas.

Phylogenetic analyses based on 16S rRNA gene sequences revealed that Teichococcus ludipueritiae and Muricoccus roseus are closely related to the species of the genus Roseomonas. The type strain of Teichococcus ludipueritiae, 170/96(T), exhibited 16S rRNA gene sequence similarity levels of 96.4 % to Roseomonas cervicalis ATCC 49957(T), 95.0 % to Roseomonas aquatica TR53(T), 94.5 % to Muricoccus roseus 173/96(T), 93.4 % to Roseomonas mucosa ATCC BAA-692(T) and 93.5 % to Roseomonas gilardii subsp. gilardii ATCC 49956(T), while Muricoccus roseus 173/96(T) showed 16S rRNA gene sequence similarity values of 95.7 % to R. mucosa ATCC BAA-692(T), 95.7 % to R. aquatica TR53(T) and 95.3 % to R. gilardii subsp. gilardii ATCC 49956(T) and R. gilardii subsp. rosea ATCC BAA-691(T). Different phylogenetic analysis methods (neighbour-joining, maximum-likelihood and maximum-parsimony) confirmed that both species are within the Roseomonas branch. Neither polyamine patterns (spermidine predominant) nor major characteristics in the polar lipid profiles distinguished the two species from representatives of the genus Roseomonas. The fatty acid composition of the two species exhibited alphaproteobacterial characteristics but, like Roseomonas species, they also showed considerable amounts of the rarely encountered C(18 : 1) 2-OH. On the other hand, they showed some phenotypic differences, but their features are compatible with the transfer of these two species to the genus Roseomonas. We propose the reclassification of Teichococcus ludipueritiae and Muricoccus roseus as Roseomonas ludipueritiae comb. nov. (type strain 170/96(T) =CIP 107418(T) =DSM 14915(T)) and Roseomonas rosea comb. nov. (type strain 173/96(T) =CIP 107419(T) =DSM 14916(T)), respectively. Emended descriptions of the genus Roseomonas and the species Roseomonas gilardii (and its subspecies Roseomonas gilardii subsp. gilardii and Roseomonas gilardii subsp. rosea), Roseomonas aquatica, Roseomonas cervicalis, Roseomonas mucosa and Roseomonas lacus are provided.

Roseomonas aerilata sp. nov., isolated from an air sample.

A polyphasic study was carried out to clarify the taxonomic position of a pink-coloured, Gram-negative bacterium isolated from air in the Suwon region of Korea. The novel strain, 5420S-30(T), grew in the temperature range of 5-35 degrees C (optimum 30 degrees C) and pH range of 5-9 (optimum pH 6-7). Growth did not occur in the presence of as little as 1 % (w/v) NaCl. It contained Q-10 as the predominant ubiquinone. The major fatty acids were C(18 : 1)omega7c, summed feature 3 (C(16 : 1)omega7c/iso-C(15 : 0) 2-OH) and C(16 : 0) (together representing 77.7 % of the total). The DNA G+C content was 72.1 mol%. The 16S rRNA gene sequence similarities between strain 5420S-30(T) and members of the genus Roseomonas were in the range 91.6-95.1 %, with the highest sequence similarities (95.1 and 94.5 %, respectively) to Roseomonas aquatica TR53(T) and Roseomonas gilardii ATCC 49956(T). On the basis of its phenotypic and genotypic characteristics, strain 5420S-30(T) represents a novel species of the genus Roseomonas, for which the name Roseomonas aerilata sp. nov. is proposed, with 5420S-30(T) (=KACC 12521(T) =DSM 19363(T)) as the type strain.

Roseomonas terrae sp. nov.

A Gram-negative, non-motile, coccobacilli-shaped bacterium, DS-48T, was isolated from soil from Dokdo, Korea, and its taxonomic position was investigated by means of a polyphasic study. Strain DS-48T grew optimally at 25 degrees C and pH 7.0-8.0 in the presence of 0.5% (w/v) NaCl. It contained Q-10 as the predominant ubiquinone and C18:1omega7c and C18:1 2-OH as the major fatty acids. The DNA G+C content was 69.3 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain DS-48T fell within the genus Roseomonas, clustering with Roseomonas lacus TH-G33T (at a bootstrap confidence level of 100%). The levels of similarity between the 16S rRNA gene sequence of strain DS-48T and those of the type strains of recognized Roseomonas species were in the range 93.2-98.0%. DNA-DNA relatedness data and differential phenotypic properties, together with the phylogenetic distinctiveness of DS-48T, revealed that this strain differs from recognized Roseomonas species. On the basis of phenotypic, phylogenetic and genetic data, therefore, strain DS-48T represents a novel species within the genus Roseomonas, for which the name Roseomonas terrae sp. nov. is proposed. The type strain is DS-48T (=KCTC 12874T=JCM 14592T).

[Moderately haloalkaliphilic aerobic methylobacteria].

Aerobic methylobacteria utilizing oxidized and substituted methane derivatives as carbon and energy sources are widespread in nature and involved in the global carbon cycle, being a unique biofilter on the path of these C1 compounds from different ecosystems to the atmosphere. New data on the biological features of moderately halophilic, neutrophilic, and alkaliphilic methylobacteria isolated from biotopes with higher osmolarity (seas, saline and soda lakes, saline soils, and deteriorating marble) are reviewed. Particular attention is paid to the latest advances in the study of the mechanisms of osmoadaptation of aerobic moderately haloalkaliphilic methylobacteria: formation of osmolytes, in particular, molecular and genetic aspects of biosynthesis of the universal bioprotectant ectoine. The prospects for further studies of the physiological and biochemical principles of haloalkalophily and for the application of haloalkaliphilic aerobic methylobacteria in biosynthesis and biodegradation are discussed.

Roseomonas aquatica sp. nov., isolated from drinking water.

Strain TR53(T), a Gram-negative, non-motile, non-spore-forming and strictly aerobic coccobacillus, isolated from the drinking water distribution system of Seville, Spain, was subjected to polyphasic taxonomic analysis using a combination of phenotypic, genotypic and phylogenetic features. Phylogenetic analysis of 16S rRNA gene sequences showed that strain TR53(T) had highest similarity to members of the genus Roseomonas, with sequence similarity values between 95.7 % (to Roseomonas genomospecies 5 strain ATCC 49960) and 94.0 % (to Roseomonas gilardii subsp. rosea ATCC 49956(T)). On the basis of its phenotypic characteristics, 16S rRNA gene sequence data and DNA G+C content (68.6 mol%), strain TR53(T) represents a novel species of the genus Roseomonas, for which the name Roseomonas aquatica sp. nov. is proposed. The type strain of Roseomonas aquatica is TR53(T) (=CECT 7131(T)=JCM 13556(T)).

Roseomonas lacus sp. nov., isolated from freshwater lake sediment.

An aerobic, Gram-negative bacterial strain, TH-G33T, was isolated from freshwater sediment of Taihu Lake in China. The taxonomy of strain TH-G33T was studied by using phenotypic and phylogenetic methods. Cells of strain TH-G33T were coccoid rods or rods and formed colourless to pale-pink colonies on nutrient agar. Phylogenetic analysis based on nearly complete 16S rRNA gene sequences showed that strain TH-G33T was related to Roseomonas mucosa (94.4 %), Roseomonas gilardii subsp. gilardii (94.1 %), Roseomonas gilardii subsp. rosea (94.8 %) and Roseomonas cervicalis (93.9 %). Cells contained ubiquinone 10 (Q-10) as the major quinone and the G+C content was 71.9 mol%. Thus, strain TH-G33T represents a novel species of the genus Roseomonas, for which the name Roseomonas lacus sp. nov. is proposed. The type strain is TH-G33T (= CGMCC 1.3617T = JCM 13283T).

Aerobic methanotrophic bacteria of cold ecosystems.

This review summarizes the recent advances in understanding the ecophysiological role and structure-function features of methanotrophic bacteria living in various cold ecosystems. The occurrence of methanotrophs in a majority of psychrosphere sites was verified by direct measurement of their methane-utilizing activity, by electron microscopy and immunofluorescent observations, and analyses of specific signatures in cellular phospholipids and total DNAs extracted from environmental samples. Surprisingly, the phenotypic and genotypic markers of virtually all extant methanotrophs were detected in various cold habitats, such as underground waters, Northern taiga and tundra soils, polar lakes and permafrost sediments. Also, recent findings indicated that even after long-term storage in permafrost, some methanotrophs can oxidize and assimilate methane not only at positive but also at subzero temperatures. Pure cultures of psychrophilic and psychrotolerant methanotrophs were isolated and characterized as new genera and species: Methylobacter psychrophilus, Methylosphaera hansonii, Methylocella palustris, Methylocella silvestris, Methylocella tundrae, Methylocapsa acidiphila and Methylomonas scandinavica. However, our knowledge about their adaptive mechanisms and survival in cold ecosystems remains limited and needs to be established using both traditional and molecular microbiological methods.