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.

Expression and characterization of 1,4-α-glucan branching enzyme from Microvirga sp. MC18 and its application in the preparation of slowly digestible starch.

Nutraceuticals containing modified starch with increased content of slowly-digestible starch (SDS) may reduce the prevalence of obesity, diabetes and cardiovascular diseases due to its slow digestion rate. Enzymatic methods for the preparation of modified starch have attracted increasing attention because of their low environmental impact, safety and specificity. In this study, the efficient glucan branching enzyme McGBE from Microvirga sp. MC18 was identified, and its relevant properties as well as its potential for industrial starch modification were evaluated. The purified McGBE exhibited the highest specificity for potato starch, with a maximal specific activity of 791.21 U/mg. A time-dependent increase in the content of α-1,6 linkages from 3.0 to 6.0% was observed in McGBE-modified potato starch. The proportion of shorter chains (degree of polymerization, DP < 13) increased from 29.2 to 63.29% after McGBE treatment, accompanied by a reduction of the medium length chains (DP 13-24) from 52.30 to 35.99% and longer chains (DP > 25) from 18.51 to 0.72%. The reduction of the storage modulus (G') and retrogradation enthalpy (ΔHr) of potato starch with increasing treatment time demonstrated that McGBE could inhibit the short- and long-term retrogradation of starch. Under the optimal conditions, the SDS content of McGBE-modified potato starch increased by 65.8% compared to native potato starch. These results suggest that McGBE has great application potential for the preparation of modified starch with higher SDS content that is resistant to retrogradation.

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 tunisiensis sp. nov., a root nodule symbiotic bacterium isolated from Lupinus micranthus and L. luteus grown in Northern Tunisia.

Three bacterial strains, LmiM8T, LmiE10 and LluTb3, isolated from nitrogen-fixing nodules of Lupinus micranthus (Lmi strains) and L. luteus (Llu strain) growing in Northern Tunisia were analysed using genetic, phenotypic and symbiotic approaches. Phylogenetic analyses based on rrs and concatenated gyrB and dnaK genes suggested that these Lupinus strains constitute a new Microvirga species with identities ranging from 95 to 83% to its closest relatives Microvirga makkahensis, M. vignae, M. zambiensis, M. ossetica, and M. lotononidis. The genome sequences of strains LmiM8T and LmiE10 exhibited pairwise Average Nucleotide Identities (ANIb) above 99.5%, significantly distant (73-89% pairwise ANIb) from other Microvirga species sequenced (M. zambiensis and M. ossetica). A phylogenetic analysis based on the symbiosis-related gene nodA placed the sequences of the new species in a divergent clade close to Mesorhizobium, Microvirga and Bradyrhizobium strains, suggesting that the M. tunisiensis strains represent a new symbiovar different from the Bradyrhizobium symbiovars defined to date. In contrast, the phylogeny derived from another symbiosis-related gene, nifH, reproduced the housekeeping genes phylogenies. The study of morphological, phenotypical and physiological features, including cellular fatty acid composition of the novel isolates demonstrated their unique profile regarding close reference Microvirga strains. Strains LmiM8T, LmiE10 and LluTb3 were able to nodulate several Lupinus spp. Based on genetic, genomic and phenotypic data presented in this study, these strains should be grouped within a new species for which the name Microvirga tunisiensis sp. nov. is proposed (type strain LmiM8T=CECT 9163T, LMG 29689T).

Microvirga calopogonii sp. nov., a novel alphaproteobacterium isolated from a root nodule of Calopogonium mucunoides in Southwest China.

In this study, a Gram-negative, rod-shaped, and non-spore-forming bacterium, which was designated as strain CCBUA 65841T, was isolated from a root nodule of Calopogonium mucunoides grown in Yunan Province of China. The sequence alignment results of 16S rRNA and four housekeeping genes (including gyrB, recA, dnaK and rpoB) indicated the isolated strain is a member of the genus Microvirga, closely related to Microvirga lotononidis WSM3557T. In addition, results of genome average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) had revealed the lower values (ANI ≤ 88.72%, dDDH ≤ 39.5%) between strain CCABU 65841T and other related Microvirga species. The genome of the novel strain exhibits a G + C content of 64.48% and contains 7296 protein-coding genes and 93 RNA genes. The major polar lipids were found to be phosphatidylcholine and phosphatidylethanolamine. The predominant cellar fatty acids were identified to be C16:0, C18:0, C19:0 cyclo ω8c, summed feature 2, summed feature 3 and summed feature 8. Moreover, menaquinone 8 (MK-8) was detected to be the predominant quinone. Based on the phylogenetic and phenotypic dissimilarity, a novel species Microvirga calopogonii sp. nov. is proposed with the type strain CCABU 65841T (= LMG 25488 T = HAMBI 3033T).

Roseomonas aestuarii sp. nov., a bacteriochlorophyll-a containing alphaproteobacterium isolated from an estuarine habitat of India.

Two strains (JC17(T) and JC19a) of orange pigmented bacteria were isolated from an estuarine sample. Cells of both the strains were Gram-negative coccobacilli, non-motile, non-spore forming and strictly aerobic. Chemo-organoheterotrophy was the growth mode for both strains and was possible on a wide range of organic compounds. Strains were non-hemolytic and contained low levels of BChl-a and carotenoids. The fatty acids (>1.0%) comprised C(18:1)omega7c, C(16:1)omega7c/iso-C(15:0)2OH, C(16:0), C(16:0) 3-OH, C(18:1)2OH, C(16:1)omega5c, and C(19:0) cycloomega8c. The genomic DNA G+C content of strain JC17(T) was 66.2mol%. A phylogenetic tree based on 16S rRNA gene sequence analysis showed that strains JC17(T) and JC19a had the highest similarity to members of the genus Roseomonas and were closely related to Roseomonas cervicalis CIP104027(T) (96.4%) and Roseomonas ludipueritiae CIP107418(T) (96.3%) of the family Acetobacteraceae within the class Alphaproteobacteria. Strains JC17(T) and JC19a shared 100% 16S rRNA gene sequence similarity, were phenotypically (morphological, physiological, biochemical characters) identical and had closely related genomes (85% DDH). Based on polyphasic taxonomic data, strain JC17(T) is classified as a novel species of the genus Roseomonas for which the name Roseomonas aestuarii sp. nov. is proposed. The type strain is JC17(T) (=CCUG 57456(T) =KCTC 22692(T) =NBRC105654(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).