Lacticaseibacillus huelsenbergensis sp. nov., isolated from grass silage and corn silage

Abstract

Two rod-shaped, facultative anaerobic, Gram-stain-positive lactic acid bacteria were isolated from corn silage and grass silage. They were characterized using a polyphasic approach and designated as HO 1656T and HO 0673. Analysis of 16S rRNA gene sequence of both strains indicated that they belong to the Lacticaseibacillus group. The most closely related species, Lacticaseibacillus casei DSM 20011T and Lacticaseibacillus zeae DSM 20178T, have digital DNA–DNA hybridization (dDDH) values of 63.9 and 53.4%, respectively, with the novel strains. In contrast, the dDDH value between strains HO 1656T and HO 0673 is 99.3 %, clearly showing that these two isolated strains belong to the same species. According to analysis of the housekeeping genes (dnaK, mutL and pheS), both strains form a distinct cluster within the Lacticaseibacillus group. Strains HO 0673 and HO 1656T could produce acid from d-arabinose, adonitol, ribose, rhamnose, dulcitol, sorbitol, turanose, l-fucose and l-arabitol, unlike their nearest phylogenetic neighbour L. casei DSM 20011T. The major cellular fatty acids of both strains are C16 : 0 and C18 : 1 ω9c. The G+C content of the genomic DNA of both strains is 48.0 mol%. Thus, strains HO 1656T and HO 0673 represent a novel species based on their chemotaxonomic, phenotypic and phylogenetic characteristics. The name Lacticaseibacillus huelsenbergensis sp. nov. is proposed with the type strain HO 1656T (=DSM 115425T=NCIMB 15466T).

Lacticaseibacillus parahuelsenbergensis sp. nov., Lacticaseibacillus styriensis sp. nov. and Lacticaseibacillus zeae subsp. silagei subsp. nov., isolated from different grass and corn silage

“…DNA fingerprinting by amplification of repetitive genome sequences was often used to determine the species or subspecies of bacteria until genome sequencing became available [15, 16, 23, 24]. The method remains a rapid and effective means of distinguishing between species.…”

“…The sequence similarities of the genes compared to the reference strains were <97% for the pheS gene of LD0937 T and <98.5 % for the rpoB gene of SCR0063 T . As previously published [15, 18, 46], primers were designed to specifically amplify the housekeeping gene of the newly discovered species and subspecies after identifying gene regions of variability: Lzsil_ argS _fw1 (5′-CGGCAATGCCAAAATGGGGA-3′) and Lzsil_ argS _rev2 (5′-CAAGCGTCTTAACCACGCTGT-3′) for amplification of the argS gene of the novel subspecies, pheS _fw3 (5′-AGACCATCCGGCACGC-3′) and pheS _rev4 (5′-ATGCTGACACATGGCCAGTAAG-3′) specifically amplifying the pheS gene of LD0937 T , and rpoB _fw2 (5′-GAACTCCCCACTGAACGATGAT-3′) and rpoB _rev4 (5′-CATCAACGTCAACATGATCGC-3′) for the amplification of the rpoB gene of SCR0063 T . The PCR was performed using 25 µl in total, containing 1 U Dream Taq polymerase, 2 mM MgCl 2 , 0.2 mM dNTP and 0.4 µM of each primer.…”

“…Sequence similarity of the 16S rRNA genes within the Lacticaseibacillus group is reported to be too high to guarantee a species identification using this method [10,11,13,22]. Due to the high sequence similarity of EB0058 T , LD0937 T DNA fingerprinting by amplification of repetitive genome sequences was often used to determine the species or subspecies of bacteria until genome sequencing became available [15,16,23,24]. The method remains a rapid and effective means of distinguishing between species.…”

“…Due to the high sequence similarity of this gene in many Lacticaseibacillus species (>99.7 % for Lacticaseibacillus casei and Lacticaseibacillus zeae), this method is not always applicable [10,11]. Genome sequencing followed by reconstruction of a phylogenetic tree using >90 housekeeping genes is a standard method to classify species or subspecies [12][13][14][15].…”

“…Lacticaseibacillus absinus [16], Lacticaseibacillus kribbianus [17], Lacticaseibacillus chiayiensis [18], Lacticaseibacillus saniviri [19] and Lacticaseibacillus parakribbianus [20] were isolated from cecum or faeces of animals. Lacticaseibacillus huelsenbergensis [15] was isolated from grass and corn silage, showing a wide distribution of Lacticaseibacilli .…”