RESULTS:

A rod-shaped Gram-stain-negative strictly anaerobic catalase-negative and endospore-forming bacterial strain CSC2T was isolated from corn silage preserved in Tochigi Japan. The strain CSC2T grew at 15–40 °C at pH 5.0–7.7 and with up to 0.5 % (w/v) NaCl. The main cellular fatty acids were C14 : 0 C16 : 0 and C16 : 0 dimethyl acetal. The cellular polar lipids detected were diphosphatidylglycerol phosphatidylglycerol phosphatidylethanolamine phosphatidic acid lysophosphatidylethanolamine phosphatidylserine lysophosphatidylcholine and two unidentified polar lipids. Phylogenetic analysis of the 16S rRNA gene showed that strain CSC2T was a member of the genus Clostridium and closely related to Clostridium polyendosporum DSM 57272T (95.6 % gene sequence similarity) and Clostridium fallax ATCC 19400T (95.3 %). The genomic DNA G+C content of strain CSC2T was 31.1 mol% (whole genome analysis). The average nucleotide identity based on blast and digital DNA–DNA hybridization values between strain CSC2T and the type strains of phylogenetically related species were below 71 and 24 % respectively. On the basis of the genotypic phenotypic and chemotaxonomic characteristics it is proposed to designate strain CSC2T as representing Clostridium zeae sp. nov. The type strain is CSC2T (=MAFF212476T=JCM 33766T=DSM 111242T).

The taxonomic status of the species Clostridium methoxybenzovorans was assessed. The 16S rRNA gene sequence whole-genome sequence and phenotypic characterizations suggested that the type strain deposited in the American Type Culture Collection ( C. methoxybenzovorans ATCC 700855T) is a member of the species Eubacterium callanderi . Hence C. methoxybenzovorans ATCC 700855T cannot be used as a reference for taxonomic study. The type strain deposited in the German Collection of Microorganism and Cell Cultures GmbH (DSM 12182T) is no longer listed in its online catalogue. Also both the 16S rRNA gene and the whole-genome sequences of the original strain SR3T showed high sequence identity with those of Lacrimispora indolis (recently reclassified from Clostridium indolis ) as the most closely related species. Analysis of the two genomes showed average nucleotide identity based on blast and digital DNA–DNA hybridization values of 98.3 and 87.9 % respectively. Based on these results C. methoxybenzovorans SR3T was considered to be a member of L. indolis .

In this study we isolated a novel strain of lactic acid bacteria AF129T from alfalfa silage prepared locally in Morioka Iwate Japan. Polyphasic taxonomy was used to characterize the bacterial strain. The bacterium was rod-shaped Gram-stain-positive non-spore-forming and catalase-negative. The strain grew at various temperatures (15–40°C) and pH levels (4.0–8.0). The optimum growth conditions were a temperature of 30°C and a pH of 6.0. AF129T exhibited growth at salt (NaCl) concentrations of up to 6.5 % (w/v). The G+C content of the strain’s genomic DNA was 41.5 %. The major fatty acids were C16 : 0 C18 : 1ω9c C19 : 0cyclo ω8c and summed feature 8. 16S rRNA gene sequencing revealed that AF129T represents a member of the genus Ligilactobacillus and it has higher sequence similarities with Ligilactobacillus pobuzihii (98.4 %) Ligilactobacillus acidipiscis (97.5 %) and Ligilactobacillus salitolerans (97.4 %). The digital DNA–DNA hybridization values for AF129T and phylogenetically related species of the genus Ligilactobacillus ranged from 19.8% to 24.1%. The average nucleotide identity of the strain with its closely related taxa was lower than the threshold (95 %–96 %) used for species differentiation. In the light of the above-mentioned physiological genotypic chemotaxonomic and phylogenetic evidence we confirm that AF129T represents a member of the genus Ligilactobacillus and constitutes a novel species; we propose the name Ligilactobacillus pabuli sp. nov. for this species. The type strain is AF129T =MAFF 518002T =JCM 34518T=BCRC 81335T.

A corrigendum of this article has been published full details can be found at 10.1099/ijsem.0.006564

Seven novel lactic acid bacterial strains (BF125T BF186 TKL145 YK3 YK6 YK10 and NSK) were isolated from the fresh faeces of Japanese black beef cattle and weanling piglets spent mushroom substrates or steeping water of a corn starch production plant. These strains are rod-shaped Gram-stain-positive non-motile non-spore-forming catalase-negative cytochrome oxidase-negative facultatively anaerobic and homofermentative. Strain BF125T did not produce any gas from glucose; both d- and l-lactate were produced as end-products of glucose (D/L 40 : 60). Growth occurred at 30–45 °C (optimum 37 °C) pH 5.0–8.0 (optimum pH 6.0) and with NaCl concentration of 1.0–3.0% (w/v). The G+C content of genomic DNA of strain BF125T was 37.8 mol% (whole-genome analysis). The major fatty acids were C16 : 0 C18 : 1 ω9c C19 cyclopropane 9 10 and summed feature 10. The 16S rRNA gene in strain BF125T showed high similarity to that of the type strain of Lactobacillus amylovorus (99.93%) and the other isolates were also identified as L. amylovorus based on these similarities. A phylogenetic tree based on the core genomes of L. amylovorus strains (n=54) including the seven isolates showed that they could be divided into two clusters. Strains YK3 YK6 YK10 and NSK were in the first cluster along with the type strain DSM 20531T while the second cluster included isolates BF125T BF186 TKL145 and other strains isolated from various animal origins. Phenotypic differences in fermentability were observed for lactose salicin and gentiobiose between these two groups. The intergroup digital DNA–DNA hybridization values (72.9–78.6%) and intergroup average nucleotide identity values (95.64–96.92%) were comparable to values calculated using datasets of other valid subspecies of the genus (ex-) Lactobacillus. In light of the physiological genotypic and phylogenetic evidence we propose a novel subspecies of L. amylovorus named Lactobacillus amylovorus subsp. animalis subsp. nov. (type strain BF125T=MAFF 212522T=DSM 115528T). Our findings also led to the automatic creation of Lactobacillus amylovorus subsp. amylovorus subsp. nov. and an emended description of the species L. amylovorus.

Two Gram-stain-positive rod-shaped non-motile non-spore-forming catalase-negative bacteria designated strains SG162T and NK01 were isolated from Japanese rice grain silage and total mixed ration silage respectively. They were initially identified as Lactobacillus buchneri based on the 16S rRNA gene sequence similarities. However the two strains were separated into a distinct clade from L. buchneri DSM 20057T (=JCM 1115T) through whole-genome sequence-based characterization forming an infraspecific subgroup together with strains CD034 and S42 whose genomic sequences were available in the public sequence database. Strains within the subgroup shared 99.4–99.7 % average nucleotide identity (ANI) and 97.5–99.0 % digital DNA–DNA hybridization (dDDH) with each other albeit 96.9–97.0 % ANI and 76.0–76.6 % dDDH against DSM 20057T. Strains SG162T and NK01 could utilize more substrates as sole carbon sources than DSM 20057T potentially owing to the abundance of genes involved in carbon metabolism especially the Entner–Doudoroff pathway. The inability of γ-aminobutyric acid (GABA) production was evidenced by the lack of glutamate decarboxylase and glutamate/GABA antiporter genes in the new subgroup strains. Strain SG162T grew at 10–45 °C (optimum 30 °C) pH 3.5–8.0 and 0–8 % (w/v) NaCl. Its genomic DNA G+C content was 44.1 mol%. The predominant fatty acids were C16 : 0 C19 : 0 cyclo ω8c and summed feature 8. On the basis of the polyphasic characterization findings strains SG162T and NK01 represent a novel subspecies of L. buchneri for which the name Lactobacillus buchneri subsp. silagei subsp. nov. is proposed. The type strain is SG162T (=JCM 32599T=DSM 107969T) and strains CD034 and S42 are also transferred to L. buchneri subsp. silagei.