Biotechnological potential of psychrotolerant methylobacteria isolated from biotopes of Antarctic oases.

Ten strains of psychrotolerant methylotrophic bacteria were isolated from the samples collected in Larsemann and Bunger Hills (Antarctica). Most of the isolates are assigned to the genus Pseudomonas, representatives of the genera Janthinobacterium, Massilia, Methylotenera and Flavobacterium were also found. Majority of isolates were able to grow on a wide range of sugars, methylamines and other substrates. Optimal growth temperatures for the isolated strains varied from 6 °C to 28 °C. The optimal concentration of NaCl was 0.5-2.0%. The optimal pH values of the medium were 6-7. It was found that three strains synthesized indole-3-acetic acid on a medium with L-tryptophan reaching 11-12 µg/ml. The values of intracellular carbohydrates in several strains exceeded 50 µg/ml. Presence of calcium-dependent and lanthanum-dependent methanol dehydrogenase have been shown for some isolates. Strains xBan7, xBan20, xBan37, xBan49, xPrg27, xPrg48, xPrg51 showed the presence of free amino acids. Bioprospection of Earth cryosphere for such microorganisms has a potential in biotechnology.

Roseateles caseinilyticus sp. nov. and Roseateles cellulosilyticus sp. nov., isolated from rice paddy field soil.

Two novel Gram-stain-negative strains designated P7T and P8T, were isolated from the soil of a paddy field in Goyang, Republic of Korea, and identified as new species within the genus Roseateles through a polyphasic taxonomic approach. These aerobic, rod-shaped, non-sporulating strains demonstrated optimal growth at 30 °C, pH 7, and in the absence of NaCl (0% w/v). Phylogenetic analysis based on 16S rRNA gene sequences indicated close relationships with Roseateles saccharophilus DSM654T (98.7%) and Roseateles puraquae CCUG 52769T (98.96%), respectively. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the isolates with the most closely related strains with publicly available whole genomes were 82.0-85.5% and 25.0-30.2%, respectively. The predominant fatty acids identified were C16:0 and summed feature 3 (composed of C16:1 ω6c and/or C16:1 ω7c), with minor amounts of C12:0, C10:0 3-OH and summed feature 8 (composed of C18:1 ω7c and/or C18:1 ω6c; 26.4%). Ubiquinone 8 was the main quinone, and the polar lipid profile included phosphatidylethanolamine, phosphatidylglycerol, two unidentified phosphoaminolipids, one unidentified phosphoglycolipid, three unidentified phospholipids, and one unidentified aminolipid. The draft genome sequences revealed genomic DNA G + C contents of 70.1% for P7T and 68.2% for P8T. Comprehensive physiological, biochemical, and 16S rRNA sequence analyses confirm these isolates as novel species of the genus Roseateles, proposed to be named Roseateles caseinilyticus sp. nov for strain P7T (= KACC 22504T = TBRC 15694T) and Roseateles cellulosilyticus sp. nov. for strain P8T (= KACC 22505T = TBRC 15695T).

Microvirga splendida sp. nov., bacteria isolated from soil.

Two bacterial strains, BT325T and BT690, were isolated from soil samples collected in Korea. Both strains were Gram stain-negative, short rod-shaped, and formed light-pink colored colonies. The 16S rRNA sequence similarity of strains BT325T and BT690 shared a sequence similarity of 99.7%. Both strains shared the highest 16S rRNA gene similarity of 98.6% with Microvirga arabica SV2184PT, followed by Microvirga ossetica V5/3 M T (98.5% and 98.2%, respectively), Microvirga soli R491T (98.3% and 98.2%, respectively), Microvirga aerilata (98.2% and 98.08%, respectively), Microvirga makkahensis (98.08% and 97.8%, respectively). Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain BT325T and BT690 were positioned in a distinct lineage within the family Methylobacteriaceae (order Rhizobiales, class Alphaproteobacteria). The genome size of strain BT325T was 5,200,315 bp and the genomic DNA G + C content was 64.3 mol%. The sole respiratory quinone of strain BT325T was Q-10 and the predominant cellular fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c) and summed feature 8 (C18:1 ω7c/C18:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. Polyphasic taxonomic analysis of biochemical, chemotaxonomic, and phylogenetic analyses suggested that strains BT325T represents a novel bacterial species within the genus Microvirga, for which the name Microvirga splendida is proposed. The type strain of Microvirga splendida is BT325T (= KCTC 72406 T = NBRC 114847 T).

Isolation and characterization of tick-borne Roseomonas haemaphysalidis sp. nov. and rodent-borne Roseomonas marmotae sp. nov.

Four novel Gram-negative, mesophilic, aerobic, motile, and cocci-shaped strains were isolated from tick samples (strains 546T and 573) and respiratory tracts of marmots (strains 1318T and 1311). The 16S rRNA gene sequencing revealed that strains 546T and 573 were 97.8% identical to Roseomonas wenyumeiae Z23T, whereas strains 1311 and 1318T were 98.3% identical to Roseomonas ludipueritiae DSM 14915T. In addition, a 98.0% identity was observed between strains 546T and 1318T. Phylogenetic and phylogenomic analyses revealed that strains 546T and 573 clustered with R. wenyumeiae Z23T, whereas strains 1311 and 1318T grouped with R. ludipueritiae DSM 14915T. The average nucleotide identity between our isolates and members of the genus Roseomonas was below 95%. The genomic G+C content of strains 546T and 1318T was 70.9% and 69.3%, respectively. Diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE) were the major polar lipids, with Q-10 as the predominant respiratory quinone. According to all genotypic, phenotypic, phylogenetic, and phylogenomic analyses, the four strains represent two novel species of the genus Roseomonas, for which the names Roseomonas haemaphysalidis sp. nov. and Roseomonas marmotae sp. nov. are proposed, with 546T (= GDMCC 1.1780T = JCM 34187T) and 1318T (= GDMCC 1.1781T = JCM 34188T) as type strains, respectively.

Microvirga pudoricolor sp. nov., and Microvirga alba sp. nov., isolated from soil in South Korea.

Two novel Gram-stain-negative, aerobic, rod-shaped, circular, convex, light-pink and white-colored bacterial strains BT291T and BT350T were isolated from soil collected in Uijeongbu city (37° 44' 55″ N, 127° 2' 20″ E) and Jeju island (33° 22' 48″ N, 126° 31' 48″ E), respectively, South Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that each of the strains BT291T and BT350T belong to a distinct lineages within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alpha Proteobacteria, phylum Proteobacteria, kingdom Bacteria). The 16S rRNA gene sequence similarity between the two strains BT291T and BT350T was 97.4%. The two strains were found to have the same quinone system, with Q-10 as the major respiratory quinone. The major polar lipids of strains BT291T and BT350T were phosphatidylethanolamine (PE), diphosphatydilglycerol (DPG), phosphatidylcholine (PC) and phosphatidylglycerol (PG). The major cellular fatty acids of strain BT291T were C18:1 ω7c (58.2%) and cyclo-C19:0 ω8c (25.7%). The major cellular fatty acids of strain BT350T were C18:1 ω7c (38.5%) and cyclo-C19:0 ω8c (27.7%). Based on the polyphasic analysis (phylogenetic, chemotaxonomic and biochemical), strains BT291T and BT350T can be suggested as two novel bacterial species within the genus Microvirga and the proposed names are Microvirga pudoricolor and Microvirga alba, respectively. The type strain of Microvirga pudoricolor is BT291T (= KCTC 72368T = NBRC 114845T) and the type strain of Microvirga alba is BT350T (= KCTC 72385T = NBRC 114848T).

A Case of Roseomonas gilardii Peritonitis Associated with a Flooded Peritoneal Dialysis Treatment Space.

Bacterial peritonitis is a key complication of Peritoneal Dialysis (PD) and a preventable cause of withdrawal from PD treatment. Infection generally arises from contamination with skin commensals during handling of the dialysis delivery system or from translocation of gastrointestinal organisms and more rarely from an environmental organism. Herein, we report the case of a 73-year-old admitted for PD-related peritonitis due to Roseomonas gilardii with an associated environmental exposure from a domestic plumbing issue. We describe the presentation, case, and antibiotic regimen progression from empiric therapy of ceftazidime and vancomycin IP to ciprofloxacin. We acknowledge the importance of performing laboratory sensitivities given the high antibiotic resistance of the Roseomonas genus. We offer that nephrologists should consider Roseomonas as a potential causative organism of peritonitis, especially when initial or further history reveals exposure to potentially contaminated water.

Expression and characterization of a novel trehalase from Microvirga sp. strain MC18.

Trehalase catalyzes the hydrolysis of trehalose into two glucose molecules and is present in nearly all tissues in various forms. In this study, a putative bacterial trehalase gene, encoding a glycoside hydrolase family 15 (GH15) protein was identified in Microvirga sp. strain MC18 and heterologously expressed in E. coli. The specific activity of the purified recombinant trehalase MtreH was 24 U/mg, with Km and Vmax values of 23.45 mg/mL and 184.23 µmol/mg/min, respectively. The enzyme exhibited optimal activity at 40 °C and pH 7.0, whereby Ca2+ had a considerable positive effects on the catalytic activity and thermostability. The optimized enzymatic reaction conditions for the bioconversion of trehalose using rMtreH were determined as 40 °C, pH 7.0, 10 h and 1% trehalose concentration. The characterization of this bacterial trehalase improves our understanding of the metabolism and biological role of trehalose in prokaryotic organism.

Metabolic engineering of Methylorubrum extorquens AM1 for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production using formate.

Formate is a promising environmentally friendly and sustainable feedstock synthesized from syngas or carbon dioxide. Methylorubrum extorquens is a type II methylotroph that can use formate as a carbon source. It accumulates polyhydroxyalkanoates (PHAs) inside the cell, mainly producing poly-3-hydroxybutyrate (PHB), a degradable biopolymer. Owing to its high melting point and stiff nature, however, mechanical property improvement is warranted in the form of copolymerization. To produce the PHA copolymer, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the endogenous gene phaC was deleted and the pathway genes bktB, phaJ1, and phaC2, with broader substrate specificities, were heterologously expressed. To improve the incorporation of 3-hydroxyvalerate (3HV), the expression level of bktB was improved by untranslated region (UTR) engineering, and the endogenous gene phaA was deleted. The engineered M. extorquens produced PHBV with 8.9% 3HV using formate as the sole carbon source. In addition, when propionate and butyrate were supplemented, PHBVs with 3HV portions of up to 70.6% were produced. This study shows that a PHBV copolymer with a high proportion of 3HV can be synthesized using formate, a C1 carbon source, through metabolic engineering and supplementation with short-chain fatty acids.

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.

The Genome Analysis of Methylobacterium populi YC-XJ1 with Diverse Xenobiotics Biodegrading Capacity and Degradation Characteristics of Related Hydrolase.

Methylobacterium populi YC-XJ1 isolated from desert soil exhibited a diverse degrading ability towards aromatic oxyphenoxypropionic acid esters (AOPPs) herbicide, phthalate esters (PAEs), organophosphorus flame retardants (OPFRs), chlorpyrifos and phoxim. The genome of YC-XJ1 was sequenced and analyzed systematically. YC-XJ1 contained a large number of exogenous compounds degradation pathways and hydrolase resources. The quizalofop-p-ethyl (QPE) degrading gene qpeh2 and diethyl phthalate (DEP) degrading gene deph1 were cloned and expressed. The characteristics of corresponding hydrolases were investigated. The specific activity of recombinant QPEH2 was 0.1 ± 0.02 U mg-1 for QPE with kcat/Km values of 1.8 ± 0.016 (mM-1·s-1). The specific activity of recombinant DEPH1 was 0.1 ± 0.02 U mg-1 for DEP with kcat/Km values of 0.8 ± 0.02 (mM-1·s-1). This work systematically illuminated the metabolic versatility of strain YC-XJ1 via the combination of genomics analysis and laboratory experiments. These results suggested that strain YC-XJ1 with diverse xenobiotics biodegrading capacity was a promising candidate for the bioremediation of polluted sites.

Microvirga arsenatis sp. nov., an arsenate reduction bacterium isolated from Tibet hot spring sediments.

Two novel Gram-stain negative, moderately thermophilic, aerobic, rod-shaped strains, designated 3D203T and 3D207, were isolated from hot spring sediment samples collected from Tibet, western China. Phylogenetic analyses based on 16S rRNA gene sequence similarities showed that two isolates belonged to the genus Microvirga and were most closely related to Microvirga makkahensis SV1470T (98.5% and 98.4%, respectively) and two strains had 99.8% similarity to each other. The average nucleotide identity (ANI) based on whole genome sequences of two strains and M. makkahensis SV1470T was 80.8% and 80.78%, respectively. Optimum growth was observed at 45 °C, pH 7.0 and 0.5% NaCl. They both could tolerate to high concentration arsenic. Ubiquinone 10 (Q10) was their predominant quinone. The differences of strains 3D203T and 3D207 were phosphatidyl dimethyl ethanolamine, phosphatidyl-N-methylethanolamine, phosphatidylglycerol, unidentified glycolipids and unidentified lipids. The major fatty acids (> 5%) were identified C18:1ω7c and/or C18:1ω6c, C18:0 and C16:0. The genomic DNA G + C contents of strain 3D203T and 3D207 based on whole genome sequences were 64.8% and 64.7%, respectively. Phenotypic, chemotaxonomic, phylogenetic and genomic analyses suggested that two strains represent a novel species of the genus Microvirga, for which the name Microvirga arsenatis sp. nov. is proposed. The type strain is 3D203T (= CGMCC 1.17691T = KCTC 72653T).

A nosocomial cluster of Roseomonas mucosa bacteremia possibly linked to contaminated hospital environment.

Roseomonas, a genus of pink-pigmented glucose non-fermentative bacteria, has been associated with various primary and hospital-acquired human infections; however, to our knowledge, its nosocomial transmission has never been reported. Clinical and epidemiological investigations were carried out after two cases of R. mucosa bacteremia occurred in our hospital in 2018. Environmental samples were taken of environmental surfaces prone to water contamination in the wards and cultured. The two clinical isolates and all environmental isolates that showed growth of pink colonies were identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry and 16S rRNA gene sequencing. Pulse-field gel electrophoresis (PFGE) was performed and fingerprinting software was used to analyze the DNA restriction patterns and determine their similarity. Two patients who developed R. mucosa bacteremia had received care from the same treatment team. Of 126 environmental samples, five showed growth of R. mucosa. Using 80% similarity as the cut-off, PFGE analysis revealed that the isolates from the two patients' blood cultures and three environmental isolates belonged to the same clone. The hospital water environment was contaminated with the same clone of R. mucosa that caused bacteremia in the two patients, suggesting nosocomial transmission linked to contaminated environment. Increased vigilance is needed to monitor the emergence of Roseomonas in healthcare settings.

Different roles of two groEL homologues in methylotrophic utiliser of dichloromethane Methylorubrum extorquens DM4.

The genome of methylotrophic bacteria Methylorubrum extorquens DM4 contains two homologous groESL operons encoding the 60-kDa and 10-kDa subunits of GroE heat shock chaperones with highly similar amino acid sequences. To test a possible functional redundancy of corresponding GroEL proteins we attempted to disrupt the groEL1 and groEL2 genes. Despite the large number of recombinants analysed and the gentle culture conditions the groEL1-lacking mutant was not constructed suggesting that the loss of GroEL1 was lethal for cells. At the same time the ∆groEL2 strain was viable and varied from the wild-type by increased sensitivity to acid, salt and desiccation stresses as well as by the impaired growth with a toxic halogenated compound-dichloromethane (DCM). The evaluation of activity of putative PgroE1 and PgroE2 promoters using the reporter gene of green fluorescent protein (GFP) showed that the expression of groESL1 operon greatly prevails (about two orders of magnitude) over those of groESL2 under all tested conditions. However the above promoters demonstrated differential regulation in response to stresses. The expression from PgroE1 was heat-inducible, while the activity of PgroE2 was upregulated upon acid shock and cultivation with DCM. Based on these results we conclude that the highly conservative groESL1 operon (old locus tags METDI5839-5840) encodes the housekeeping chaperone essential for fundamental cellular processes. On the contrary the second pair of paralogues (METDI4129-4130) is dispensable, but corresponding GroE2 chaperone promotes the tolerance to acid and salt stresses, in particular, during the growth with DCM.

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.

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).

Growth Stage-dependent Bacterial Communities in Soybean Plant Tissues: Methylorubrum Transiently Dominated in the Flowering Stage of the Soybean Shoot.

Plant-associated bacteria are critical for plant growth and health. However, the effects of plant growth stages on the bacterial community remain unclear. Analyses of the microbiome associated with field-grown soybean revealed a marked shift in the bacterial community during the growth stages. The relative abundance of Methylorubrum in the leaf and stem increased from 0.2% to more than 45%, but decreased to approximately 15%, with a peak at the flowering stage at which nitrogen metabolism changed in the soybean plant. These results suggest the significance of a time-series analysis for understanding the relationship between the microbial community and host plant physiology.

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.

Comparison of VITEK 2, MALDI-TOF MS, 16S rRNA gene sequencing, and whole-genome sequencing for identification of Roseomonas mucosa.

The bacterial species Roseomonas mucosa is pathogenic in humans, and although it is rarely detected during routine diagnostics, it is becoming increasingly important clinically. For a long time, R. mucosa was regarded as a classic environmental bacterium. Recent studies, however, revealed that it is part of the physiological human skin flora and mainly affects immunocompromised patients. Furthermore, the use of catheter systems may increase the risk of contracting R. mucosa infections. The bacterium has been linked to severe infections, such as bacteraemia, osteomyelitis and cellulitis. Therefore, it is important to discern the best method of identifying R. mucosa in routine laboratory testing. To facilitate this testing, we compared three suitable methods for routine bacterial identification in the laboratory: VITEK 2, MALDI-TOF MS and 16S rRNA gene sequencing. Additionally, we conducted whole-genome sequencing (WGS) and calculated the average nucleotide identity (ANI). ANI is seen as the gold standard of strain identification; therefore, we decided to use it as a reference method. Both MALDI-TOF MS and 16S rRNA gene sequencing confidently identified the species. However, when using the VITEK 2 technique, isolates were misidentified as Roseomonas gilardii, Rhizobium radiobacter, or Sphingomonas paucimobilis. When conducting WGS and determining the ANI, it became obvious that one isolate belonged to the species R. gilardii rather than R. mucosa. Therefore (although not yet applicable in routine diagnostics), we suggest that WGS is presently the most appropriate technique to reliably identify Roseomonas mucosa. However, after expanding the Biotyper database, MALDI-TOF MS could also be an applicable method.

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).

Proteogenomic Refinement of the Neomegalonema perideroedesT Genome Annotation.

Neomegalonema perideroedes (formerly Meganema perideroedes) str. G1 is the type strain and only described isolate of the genus Neomegalonema (formerly Meganema) which belongs to the Alphaproteobacteria. N. perideroedes is distinguished by the ability to accumulate high amounts of polyhydroxyalkanoates and has been associated with bulking problems in wastewater treatment plants due to its filamentous morphology. In 2013, its genome was sequenced as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA), which aims to improve the sequencing coverage of the poorly represented regions of the bacterial and archaeal branches of the tree of life. As N. perideroedes str. G1 is relatively distantly related to well described species-being the only sequenced member of its proposed family-the in silico prediction of genes by nucleotide homology to reference genes might be less reliable. Here, a proteomic dataset for the refinement of the N. perideroedes genome annotations is generated which clearly indicates the shortcomings of high-throughput in silico genome annotation.