Screening of heat-killed lactic acid bacteria based on inhibitory activity against oral bacteria and effects of oral administration of heat-killed Ligilactobacillus salivarius CP3365 on periodontal health in healthy participants: a double-blinded, randomized, placebo-controlled trial.

Objectives: The aims of this study were to select heat-killed lactic acid bacteria (HKL) with antibiotic activity and investigate the efficacy of this bacteria in maintaining periodontal parameters in healthy participants. Materials and methods: An in vitro evaluation was conducted to assess the inhibitory efficacy of lactic acid bacteria against Porphyromonas gingivalis and Fusobacterium nucleatum subsp. nucleatum. The effects of HKL administration on various parameters (plaque control record, bleeding on probing, and probing pocket depth) were assessed in a randomized, placebo-controlled trial. Participants in the test and placebo groups (n = 32) consumed oral tablets containing placebo or HKL daily for 8 weeks. Oral bacteria in supra-plaque and saliva were identified using 16S rRNA gene community profiling analysis. Results: Heat-killed Ligilactobacillus salivarius CP3365 significantly (p < 0.05) decreased the viability of oral bacteria and was selected for clinical trials. Administration of HKL CP3365 significantly (p < 0.05) inhibited increases in each parameter. No changes in the relative abundance of P. gingivalis or F. nucleatum subsp. nucleatum were detected by HKL CP3365, but the relative abundance of oral bacteria (genera Porphyromonas, Fusobacterium, and Haemophilus) was significantly (p < 0.05) decreased. Conclusion: HKL CP3365 effectively inhibited oral bacteria growth and was useful for maintaining periodontal health. Clinical Trial Registration: [https://www.umin.ac.jp/ctr/index.htm], identifier [UMIN000045656].

Emergence of putative energy parasites within Clostridia revealed by genome analysis of a novel endosymbiotic clade.

The Clostridia is a dominant bacterial class in the guts of various animals and are considered to nutritionally contribute to the animal host. Here, we discovered clostridial endosymbionts of cellulolytic protists in termite guts, which have never been reported with evidence. We obtained (near-)complete genome sequences of three endosymbiotic Clostridia, each associated with a different parabasalid protist species with various infection rates: Trichonympha agilis, Pseudotrichonympha grassii, and Devescovina sp. All these protists are previously known to harbor permanently-associated, mutualistic Endomicrobia or Bacteroidales that supplement nitrogenous compounds. The genomes of the endosymbiotic Clostridia were small in size (1.0-1.3 Mbp) and exhibited signatures of an obligately-intracellular parasite, such as an extremely limited capability to synthesize amino acids, cofactors, and nucleotides and a disrupted glycolytic pathway with no known net ATP-generating system. Instead, the genomes encoded ATP/ADP translocase and, interestingly, regulatory proteins that are unique to eukaryotes in general and are possibly used to interfere with host cellular processes. These three genomes formed a clade with metagenome-assembled genomes (MAGs) derived from the guts of other animals, including human and ruminants, and the MAGs shared the characteristics of parasites. Gene flux analysis suggested that the acquisition of the ATP/ADP translocase gene in a common ancestor was probably key to the emergence of this parasitic clade. Taken together, we provide novel insights into the multilayered symbiotic system in the termite gut by adding the presence of parasitism and present an example of the emergence of putative energy parasites from a dominant gut bacterial clade.

Transconjugant range of PromA plasmids in microbial communities is predicted by sequence similarity with the bacterial host chromosome.

Nucleotide sequence similarity, including k-mer plasmid composition, has been used for prediction of plasmid evolutionary host range, representing the hosts in which a plasmid has replicated at some point during its evolutionary history. However, the relationships between the bacterial taxa of experimentally identified transconjugants and the predicted evolutionary host ranges are poorly understood. Here, four different PromA group plasmids showing different k-mer compositions were used as model plasmids. Filter mating assays were performed with a donor harbouring plasmids and recipients of bacterial communities extracted from environmental samples. A broad range of transconjugants was obtained with different bacterial taxa. A calculation of the dissimilarities in k-mer compositions as Mahalanobis distance between the plasmid and its sequenced transconjugant chromosomes revealed that each plasmid and transconjugant were significantly more similar than the plasmid and other non-transconjugant chromosomes. These results indicate that plasmids with different k-mer compositions clearly have different host ranges to which the plasmid will be transferred and replicated. The similarity of the nucleotide compositions could be used for predicting not only the plasmid evolutionary host range but also future host ranges.

Effects of Prebiotic Yeast Mannan on Gut Health and Sleep Quality in Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled Study.

Human gut health is closely related to sleep. We aimed to evaluate the efficacy of yeast mannan (YM) in improving bowel habits and sleep quality, along with metabolomics in fecal samples. A total of 40 healthy adults (age range, 22-64 years) with discomfort in defecation were enrolled and randomly allocated to receive either YM (n = 20; 1.1 g/day) or placebo (n = 20) for four weeks. Participants recorded their defecation habits throughout the test periods. Sleep electroencephalogram (EEG) recording using an EEG device and fecal sampling were performed pre- and post-treatment. The YM group significantly increased defecation frequency and stool volumes compared to the placebo group. After 4 weeks of treatment, the non-REM sleep stage 3 (N3) duration in the YM group was significantly higher than that in the placebo group. YM ingestion significantly lengthened total time in bed (TIB) and significantly shortened N3 latency compared to placebo intake during the trial. The metabolomics analysis found a total of 20 metabolite differences between the YM and placebo groups. As a result of stepwise linear regression, changes in fecal propionate and gamma-aminobutyric acid (GABA) levels were identified as the primary factors explaining changes in TIB and N3 latency, respectively. Our findings suggest that the prebiotic YM could be beneficial to gut health and sleep quality.

Hitherto-Unnoticed Self-Transmissible Plasmids Widely Distributed among Different Environments in Japan.

Various conjugative plasmids were obtained by exogenous plasmid capture, biparental mating, and/or triparental mating methods from different environmental samples in Japan. Based on phylogenetic analyses of their whole-nucleotide sequences, new IncP/P-1 plasmids that could be classified into novel subgroups were obtained. Mini-replicons of the plasmids were constructed, and each of them was incompatible with at least one of the IncP/P-1 plasmids, although they showed diverse iteron sequences in their oriV regions. There were two large clades of IncP/P-1 plasmids, clade I and II. Plasmids in clade I and II included antibiotic resistance genes. Notably, nucleotide compositions of newly found plasmids exhibited different tendencies compared with those of the previously well-studied IncP/P-1 plasmids. Indeed, the host range of plasmids of clade II was different from that of clade I. Although few PromA plasmids have been reported, the number of plasmids belonging to PromAß, and -γ subgroups detected in this study was close to that of IncP/P-1 plasmids. The host ranges of PromAγ and PromAδ plasmids were broad and transferred to different and distinct classes of Proteobacteria. Interestingly, PromA plasmids and many IncP/P-1 plasmids do not carry any accessory genes. These findings indicate the presence of "hitherto-unnoticed" conjugative plasmids, including IncP/P-1 or PromA derivative ones in nature. These plasmids would have important roles in the exchange of various genes, including antibiotic resistance genes, among different bacteria in nature. IMPORTANCE Plasmids are known to spread among different bacteria. However, which plasmids spread among environmental samples and in which environments they are present is still poorly understood. This study showed that unidentified conjugative plasmids were present in various environments. Different novel IncP/P-1 plasmids were found, whose host ranges were different from those of known plasmids, showing wide diversity of IncP/P-1 plasmids. PromA plasmids, exhibiting a broad host range, were diversified into several subgroups and widely distributed in varied environments. These findings are important for understanding how bacteria naturally exchange their genes, including antibiotic resistance genes, a growing threat to human health worldwide.

Nanobdella aerobiophila gen. nov., sp. nov., a thermoacidophilic, obligate ectosymbiotic archaeon, and proposal of Nanobdellaceae fam. nov., Nanobdellales ord. nov. and Nanobdellia class. nov.

A co-culture of a novel thermoacidophilic, obligate symbiotic archaeon, designated as strain MJ1T, with its specific host archaeon Metallosphaera sedula strain MJ1HA was obtained from a terrestrial hot spring in Japan. Strain MJ1T grew in the co-culture under aerobic conditions. Coccoid cells of strain MJ1T were 200-500 nm in diameter, and attached to the MJ1HA cells in the co-culture. The ranges and optima of the growth temperature and pH of strain MJ1T in the co-culture were 60-75 °C (optimum, 65-70 °C) and pH 1.0-4.0 (optimum, pH 2.5), respectively. Core lipids of dialkyl glycerol tetraethers (GDGT)-3 and GDGT-4 were highly abundant in MJ1T cells concentrated from the co-culture. Strain MJ1T has a small genome (0.67 Mbp) lacking genes for biosynthesis of essential biomolecules, such as nucleotides, lipids and ATP. The genomic DNA G+C content was 24.9 mol%. The 16S rRNA gene sequence of strain MJ1T was most closely related to that of the cultivated species, 'Nanopusillus acidilobi' strain N7A (85.8 % similarity). Based on phylogenetic and physiological characteristics, we propose the name Nanobdella aerobiophila gen. nov., sp. nov. to accommodate the strain MJ1T (=JCM 33616T=DSM 111728T). In addition, we propose the names Nanobdellaceae fam. nov., Nanobdellales ord. nov., and Nanobdellia class. nov. to accommodate the novel genus.

Insight into the symbiotic lifestyle of DPANN archaea revealed by cultivation and genome analyses.

Decades of culture-independent analyses have resulted in proposals of many tentative archaeal phyla with no cultivable representative. Members of DPANN (an acronym of the names of the first included phyla Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota, and Nanoarchaeota), an archaeal superphylum composed of at least 10 of these tentative phyla, are generally considered obligate symbionts dependent on other microorganisms. While many draft/complete genome sequences of DPANN archaea are available and their biological functions have been considerably predicted, only a few examples of their successful laboratory cultivation have been reported, limiting our knowledge of their symbiotic lifestyles. Here, we investigated physiology, morphology, and host specificity of an archaeon of the phylum "Candidatus Micrarchaeota" (ARM-1) belonging to the DPANN superphylum by cultivation. We constructed a stable coculture system composed of ARM-1 and its original host Metallosphaera sp. AS-7 belonging to the order Sulfolobales Further host-switching experiments confirmed that ARM-1 grew on five different archaeal species from three genera-Metallosphaera, Acidianus, and Saccharolobus-originating from geologically distinct hot, acidic environments. The results suggested the existence of DPANN archaea that can grow by relying on a range of hosts. Genomic analyses showed inferred metabolic capabilities, common/unique genetic contents of ARM-1 among cultivated micrarchaeal representatives, and the possibility of horizontal gene transfer between ARM-1 and members of the order Sulfolobales Our report sheds light on the symbiotic lifestyles of DPANN archaea and will contribute to the elucidation of their biological/ecological functions.

Comparative genomics and proposal of Streptomyces radicis sp. nov., an endophytic actinomycete from roots of plants in Thailand.

Strains DS1-2T and AZ1-7, which were isolated from roots of plants, were taxonomically characterized based on polyphasic taxonomic and taxogenomic approaches. Both strains were Gram-stain-positive and filamentous bacteria which contained LL-diaminopimelic acid in cell-wall peptidoglycan and glucose and ribose in whole-cell hydrolysates. MK-9(H6), MK-10(H6), MK-9(H8), MK-10(H8) and MK-10(H4) were major menaquinones; iso-C16:0 and iso-C16:1G were predominant cellular fatty acids; diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol mannoside presented as major phospholipids; and the DNA G+C contents of 73.2 mol%. Strains DS1-2T and AZ1-7 showed 97.6-98.0 % 16S rRNA gene sequence similarity, 81.0-82.0 % ANIb, 84.8-85.3 % ANIm and 22.0-23.1 % digital DDH to their related type strains: S. specialis GW41-1564T and S. hoynatensis S1412T. Comparative genomics results of these strains and their related type strains also revealed the differences and distributions of key genes associated with stress responses, environmental variables, plant interactions and bioactive metabolites. Based on the phenotypic, chemotaxonomic and genomic data, strains DS1-2T and AZ1-7 could be assigned to the novel species within the genus Streptomyces for which the name Streptomyces radicis sp. nov. is proposed. The type strain is DS1-2T (=JCM 32152T =KCTC 39738T =TISTR 2403T).

Actinomadura violacea sp. nov., a madurastatin A1-producing strain isolated from lichen in Thailand.

An actinomycete strain, LCR2-06T, isolated from a lichen sample on rock collected from Chiang Rai Province (Pong Phra Bat Waterfall), Thailand, was characterized using a polyphasic approach. The strain grew at 25-45 °C, pH 6-11 and on International Streptomyces Project 2 agar plate with 5 % (w/v) NaCl. It contained meso-diaminopimelic acid as the diamino acid in whole-cell hydrolysates. Rhamnose, ribose, xylose, madurose, glucose and galactose were detected as whole-cell sugar hydrolysates. Mycolic acids were absent. The N-acyl type of muramic acid was acetyl. The strain contained C16 : 0, TBSA 10-methyl C18 : 0 and 2-hydroxy C16 : 0 as the predominant fatty acids and MK-9(H6), MK-9(H4) and MK-9(H8) as the major menaquinones. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol and unidentified phospholipid. The draft genome of strain LCR2-06T was closely related to Actinomadura barringtoniae TBRC 7225T (99.2 %), Actinomadura nitritigenes NBRC 15918T (98.8 %), Actinomadura montaniterrae TISTR 2400T (98.5 %) and Actinomadura physcomitrii JCM 33455T (97.9 %). The draft genome of LCR2-06T was 11.1 Mb with 10 588 coding sequences with an average G+C content of 72.7 mol%. Results of genomic analysis revealed that the ANIb and ANIm values between strain LCR2-06T and A. montaniterrae TISTR 2400T were 90.0 and 92.0 %, respectively. The digital DNA-DNA hybridization value was 43.9 % in comparison with the draft genome of A. montaniterrae TISTR 2400T. The strain produced an antibacterial compound active against Bacillus subtilis ATCC 6633 and Kocuria rhizophila ATCC 9341. The results of taxonomic analysis suggested that strain LCR2-06T represented a novel species of the genus Actinomadura for which the name Actinomadura violacea sp. nov. is proposed. The type strain is LCR2-06T (=JCM 33065T=KCTC 49547T=NBRC 114810T=LMG 32136T=TISTR 2935T).

Adlercreutzia hattorii sp. nov., an equol non-producing bacterium isolated from human faeces.

Two obligately anaerobic, Gram-stain-positive, rod-shaped bacteria were isolated from faecal samples of healthy humans in Japan. 16S rRNA gene sequence analysis indicated that these two strains (8CFCBH1T and 9CBH6) belonged to the genus Adlercreutzia, which is known as an equol-producing bacterium. The closest neighbours of strain 8CFCBH1T were Adlercreutzia equolifaciens subsp. equolifaciens DSM 19450T (98.6%), Adlercreutzia equolifaciens subsp. celatus do03T (98.4%), Adlercreutzia muris WCA-131-CoC-2T (96.6%), Parvibacter caecicola NR06T (96.4%), Adlercreutzia caecimuris B7T (95.3%) and Adlercreutzia mucosicola Mt1B8T (95.3%). The closest relatives to strain 9CBH6 were A. equolifaciens subsp. equolifaciens DSM 19450T (99.8%), A. equolifaciens subsp. celatus do03T (99.6%) and A. muris WCA-131-CoC-2T (96.8%). Strain 8CFCBH1T showed 22.3-53.5% digital DNA-DNA hybridization (dDDH) values with its related species. In addition, the average nucleotide identity (ANI) values between strain 8CFCBH1T and its related species ranged from 75.4 to 93.3%. On the other hand, strain 9CBH6 was considered as A. equolifaciens based on the dDDH and ANI values (>70% dDDH and >95-96% ANI). Strain 9CBH6 showed daidzein-converting activity, as expected from the result of genome analysis. The genome of strain 8CFCBH1T lacked four genes involved in equol production. Growing cells of strain 8CFCBH1T were not capable of converting daidzein. Based on the collected data, strain 8CFCBH1T represents a novel species in the genus Adlercreutzia, for which the name Adlercreutzia hattorii sp. nov. is proposed. The type strain of A. hattorii is 8CFCBH1T (=JCM 34083T=DSM 112284T).

Actinoplanes lichenicola sp. nov. and Actinoplanes ovalisporus sp. nov., isolated from lichen in Thailand.

Two actinobacteria, designated as strain LDG1-01T and LDG1-06T, were isolated from lichen samples collected in Thailand. Results of morphological characterization, chemotaxonomic studies and 16S rRNA gene analysis indicated that both strains were members of the genus Actinoplanes. MK-9(H4) was found as the major menaquinone. The major fatty acids were anteiso-C15 : 0, iso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. Phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol were observed as the polar lipids, but differences in minor unidentified polar lipids were found between the strains. Results of comparative genome analysis based on average nucleotide identity and digital DNA-DNA hybridization calculations revealed that both strains showed values below 95 and 70 %, respectively, from each other and closely related Actinoplanes type strains. Based on data from this polyphasic study, strains LDG1-01T and LDG1-06T represent novel species of the genus Actinoplanes. The names proposed are Actinoplanes lichenicola sp. nov. (type strain, LDG1-01T (=JCM 33066T=TISTR 2982T) and Actinoplanes ovalisporus sp. nov. (type strain, LDG1-06T=JCM 33067T=TISTR 2983T).

Oxygen concentration affects frequency and range of transconjugants for the incompatibility (Inc) P-1 and P-7 plasmids pBP136 and pCAR1.

The frequency of transconjugants were compared for the incompatibility (Inc) P-1 and P-7 plasmids pBP136 and pCAR1 under aerobic and anaerobic conditions. Filter mating assays were performed with one donor strain and one recipient strain using different donors of Pseudomonas and recipient strains, including Pseudomonas, Pantoea, and Buttiauxella. Under anaerobic condition, frequencies of transconjugants for both plasmids were 101-103-fold lower than those under aerobic condition regardless of whether aerobically or anaerobically grown donors and recipients were used. To compare the transconjugant ranges under aerobic and anaerobic conditions, conjugation was performed between the donor of pBP136 and recipient bacteria extracted from environmental samples. Several transconjugants were uniquely obtained from each aerobic or anaerobic condition. Our findings indicate that a plasmid can differently spread among bacteria depending on the oxygen concentrations of the environment.

Actinomadura decatromicini sp. nov., isolated from mountain soil in Thailand.

A novel actinomycete strain CYP1-5T was isolated from the mountain soil sample collected from Chaiyaphum province, Thailand and its taxonomic position was clarified by using a polyphasic taxonomic approach. The chemotaxonomic properties of strain CYP1-5T were consistent within the genus Actinomadura. Cell-wall peptidoglycan of this strain contained meso-diaminopimelic acid. Galactose, madurose, and ribose were presented as the diagnostic sugars in whole-cell hydrolysates. The major menaquinone was MK-9(H6). Major cellular fatty acids were iso-C16:0 and C16:0. Phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, and phosphatidylinositol mannoside were observed as predominant phospholipids. Based on the results of phylogenetic analyses of 16S rRNA gene sequence, strain CYP1-5T was constituent with the genus Actinomadura and was closely related to Actinomadura syzygii GKU157T (99.5%) and Actinomadura chibensis IFM 10266T (= JCM 14158T) (98.2%). The draft genome size of strain CYP1-5T was 9.30 Mb with 72.2 mol% of G + C content. Strain CYP1-5T showed ANIb values of 94.9% with A. syzygii GKU157T and 93.2% with A. chibensis JCM 14158T. Phenotypic characteristics, phylogenetic analysis and genome data support that strain CYP1-5T could be discriminated from its closest relatives, representing a novel species of the genus Actinomadura, for which the name Actinomadura decatromicini sp. nov. is proposed. The type strain is CYP1-5T (= JCM 16996T = KCTC 19916T = TISTR 2901T).

Nocardia terrae sp. nov., an actinomycete isolated from soil in Thailand.

A novel actinomycete strain ET3-3T isolated from soil collected from Chachoengsao province, Thailand was taxonomic evaluated using a polyphasic approach. Chemotaxonomic analysis indicated that meso-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan. Mycolic acids were present. Ribose, arabinose, and galactose were detected in its whole-cell hydrolysates. The strain comprised C16:0 and TBSA 10-methyl C18:0 as the main fatty acids and MK-8(H4ω-cycl) as the predominant menaquinone. Major polar lipids were phosphatidylinositol, phosphatidylinositol mannoside, phosphatidylethanolamine, and diphosphatidylglycerol. The results of 16S rRNA gene sequence analysis showed that strain ET3-3T was closely related to Nocardia seriolae JCM 30082T (99.2%), Nocardia yunnanensis JCM 30082T (98.4%), and Nocardia concava JCM 12351T (98.2%). The draft genome of ET3-3T was 9.31 Mb with 8826 coding sequences with an average G + C content of 68.0%. The comparison of the draft genome of strain ET3-3T and N. seriolae NBRC 15557T showed the ANIb, ANIm and the digital DNA-DNA hybridization values of 86.3%, 88.5% and 32.9%, respectively. The results of the taxonomic analysis suggested that strain ET3-3T represented a novel species of the genus Nocardia for which the name Nocardia terrae sp. nov. is proposed. The type strain is ET3-3T (= JCM 33776T = TISTR 2837T).

Conexivisphaera calida gen. nov., sp. nov., a thermophilic sulfur- and iron-reducing archaeon, and proposal of Conexivisphaeraceae fam. nov., Conexivisphaerales ord. nov., and Conexivisphaeria class. nov. in the phylum Thaumarchaeota.

A thermoacidophilic, anaerobic, and iron- and sulfur-reducing archaeon, strain NAS-02T, was isolated from a terrestrial hot spring in Japan, as previously reported. This organism is the first non-ammonia-oxidizing isolate in the phylum Thaumarchaeota. Here, we propose Conexivisphaera calida gen. nov., sp. nov. to accommodate this strain. The type strain of the type species is NAS-02T (=JCM 31663T=DSM 105898T). The values of 16S rRNA gene similarity and average amino acid identity between NAS-02T and its closest relatives are <86 and <42 %, respectively. Based on the phylogeny and physiology, we propose the family Conexivisphaeraceae fam. nov., the order Conexivisphaerales ord. nov. and the class Conexivisphaeria class. nov. to accommodate the novel genus.

Nocardia aurantiaca sp. nov., isolated from soil in Thailand.

A novel actinomycete strain, CT2-14T, belonging to the genus Nocardia, was isolated from a soil sample collected from Phichit Province, Thailand. The taxonomic position of the strain was characterized using a polyphasic approach. The strain grew at 15-40 °C (optimum, 28-37 °C), pH 6-11 (optimum, pH 6-8) and on an International Streptomyces Project 2 with 4 % (w/v) NaCl agar plate. Meso-diaminopimelic acid was detected in the cell-wall peptidoglycan. Ribose, arabinose and galactose were detected in its whole-cell hydrolysates. Mycolic acids were present. The strain contained C16 : 0, summed feature 3, C17 : 0 10-methyl and C18 : 1 ω9c as the major fatty acids and MK-8(H4ω-cycl) as the major menaquinone. The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylinositol mannosides. Strain CT2-14T showed the highest 16S rRNA gene similarity to Nocardia veterana JCM 11307T (98.4 %), Nocardia africana JCM 11438T (98.2 %) and Nocardia kruczakiae JCM 13032T (98.0 %). The draft genome of strain CT2-14T was 7.37 Mb with 6685 coding sequences with an average G+C content of 67.9 mol %. Based on the phylogenomic tree analysis, the strain was closely related to Nocardia niigatensis NBRC 100131T. On the basis of polyphasic and genome analyses, strain CT2-14T represented a novel species of the genus Nocardia for which the name Nocardia aurantiaca sp. nov. is proposed. The type strain is CT2-14T (=JCM 33775T=TISTR 2838T).

Lactococcus insecticola sp. nov. and Lactococcus hodotermopsidis sp. nov., isolated from the gut of the wood-feeding lower termite Hodotermopsis sjostedti.

Two strains of lactic acid bacteria, designated Hs20B0-1T and Hs30E4-3T, were isolated from the gut of the damp-wood termite Hodotermopsis sjostedti. These strains were characterized genetically and phenotypically. Strain Hs20B0-1T was related to Lactococcus piscium DSM 6634T showing 96.3 and 84.2 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. Strain Hs30E4-3T was related to Lactococcus plantarum DSM 20686T showing 94.8 and 82.2 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. The 16S rRNA gene sequence similarity between strains Hs20B0-1T and Hs30E4-3T was 95.7 %. Furthermore, genomic comparisons using pairwise average nucleotide identity (ANI) and digital DNA-DNA hybridization (DDH) analyses between strain Hs20B0-1T and L. piscium DSM 6634T resulted in values of 73.5 and 20.1 %, respectively. Strain Hs30E4-3T had 72.8 % ANI similarity and 21.3 % DDH similarity to L. plantarum DSM 20686T. Strains Hs20B0-1T and Hs30E4-3T had 75.4 % ANI similarity and 21.1 % DDH similarity to each other. The cell-wall peptidoglycan types of strains Hs20B0-1T and Hs30E4-3T were A4α, Lys-Asp and A3α, Lys-Thr-Ala, respectively. The two strains, Hs20B0-1T and Hs30E4-3T, are distinguishable from each other and other established Lactococcus species phylogenetically and phenotypically. In conclusion, two novel species of the genus Lactococcus are proposed, namely Lactococcus insecticola Hs20B0-1T (=JCM 33485T=DSM 110147T) and Lactococcus hodotermopsidis Hs30E4-3T (=JCM 33486T=DSM 110148T), respectively.

Determination of Plasmid pSN1216-29 Host Range and the Similarity in Oligonucleotide Composition Between Plasmid and Host Chromosomes.

Plasmids are extrachromosomal DNA that can be horizontally transferred between different bacterial cells by conjugation. Horizontal gene transfer of plasmids can promote rapid evolution and adaptation of bacteria by imparting various traits involved in antibiotic resistance, virulence, and metabolism to their hosts. The host range of plasmids is an important feature for understanding how they spread in environmental microbial communities. Earlier bioinformatics studies have demonstrated that plasmids are likely to have similar oligonucleotide (k-mer) compositions to their host chromosomes and that evolutionary host ranges of plasmids could be predicted from this similarity. However, there are no complementary studies to assess the consistency between the predicted evolutionary host range and experimentally determined replication/transfer host range of a plasmid. In the present study, the replication/transfer host range of a model plasmid, pSN1216-29, exogenously isolated from cow manure as a newly discovered self-transmissible plasmid, was experimentally determined within microbial communities extracted from soil and cow manure. In silico prediction of evolutionary host range was performed with the pSN1216-29 using its oligonucleotide compositions independently. The results showed that oligonucleotide compositions of the plasmid pSN1216-29 had more similarities to those of hosts (transconjugants genera) than those of non-hosts (other genera). These findings can contribute to the understanding of how plasmids behave in microbial communities, and aid in the designing of appropriate plasmid vectors for different bacteria.