Identification and Classification for the Lactobacillus casei Group

Abstract

Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus are phenotypically and genotypically closely related, and together comprise the L. casei group. Although the strains of this group are commercially valuable as probiotics, the taxonomic status and nomenclature of the L. casei group have long been contentious because of the difficulties in identifying these three species by using the most frequently used genotypic methodology of 16S rRNA gene sequencing. Long used as the gold standard for species classification, DNA–DNA hybridization is laborious, requires expert skills, and is difficult to use routinely in laboratories. Currently, genome-based comparisons, including average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH), are commonly applied to bacterial taxonomy as alternatives to the gold standard method for the demarcating phylogenetic relationships. To establish quick and accurate methods for identifying strains in the L. casei group at the species and subspecies levels, we developed species- and subspecies-specific identification methods based on housekeeping gene sequences and whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral pattern analysis. By phylogenetic analysis based on concatenated housekeeping gene sequences (dnaJ, dnaK, mutL, pheS, and yycH), 53 strains were separated into four clusters corresponding to the four species: L. casei, L. paracasei and L. rhamnosus, and Lactobacillus chiayiensis sp. nov. A multiplex minisequencing assay using single nucleotide polymorphism (SNP)-specific primers based on the dnaK gene sequences and species-specific primers based on the mutL gene sequences provided high resolution that enabled the strains at the species level to be identified as L. casei, L. paracasei, and L. rhamnosus. By MALDI-TOF MS analysis coupled with an internal database and ClinProTools software, species- and subspecies-level L. casei group strains were identified based on reliable scores and species- and subspecies-specific MS peaks. The L. paracasei strains were distinguished clearly at the subspecies level based on subspecies-specific MS peaks. This article describes the rapid and accurate methods used for identification and classification of strains in the L. casei group based on housekeeping gene sequences and MALDI-TOF MS analysis as well as the novel speciation of this group including L. chiayiensis sp. nov. and ‘Lactobacillus zeae’ by genome-based methods.

Essential and Ubiquitous: The Emergence of Lanthanide Metallobiochemistry

“…[8] Thefunction of xoxF had been long unknown, in fact the first "x" in xoxF stands for its (previously long) unknown role. [9] In the same year,t he authors also reported ac erium(III)-dependent MDH in Bradyrhizobium sp.M AFF211645. [10] One year later, Nakagawa and Kawai reported that the model methylotroph strain M. extorquens AM1 also produced al anthanide-dependent MDH and that this bacterium could grow with La 3+ instead of Ca 2+ .…”

“…[2e, 22] While the affinity of Ln of LanM in in vitro experiments is very similar for all lanthanides, [20] the choice of Ln has been shown to affect the growth rate of bacteria and also the activity of alcohol dehydrogenases. [9,23] Mainly,t he earlier lanthanides (La to Sm) are known to stimulate the growth of Ln-utilizing bacteria (Figure 3). It is possible that efficient 18] In the presence of lanthanides,XoxF (Ln-MDH, golden pathway)i se xpressed and MxaF (Ca-MDH, grey pathway)i srepressed.…”

Genome-based reclassification of Lactobacillus casei: emended classification and description of the species Lactobacillus zeae

“…The amplification and sequencing of the 16S rRNA gene and housekeeping genes (dnaJ, dnaK, mutL, pheS and yycH) were carried out as previously described [19][20][21][22][23]. Multiple sequence alignments were performed with the program clustal_x version 2.1 [24].…”

Essenziell und weitverbreitet: Lanthanoid‐Metalloproteine

“…[2e,21] Während die Ln-Affinitätv on LanM bei In-vitro-Experimenten füra lle Lanthanoide ähnlich ist, [19] konnte gezeigt werden, dass die Wahl von Ln das Wachstum von Bakterien und die Aktivitätv on Alkohol-Dehydrogenasen beeinflusst. [9,22] [23] Des Weiteren haben Wachstumsstudien mit M. fumariolicum SolV gezeigt, dass La 3+ schneller aus dem Medium abgereichert wird als Eu 3+ -e ine Beobachtung,d ie mit der Wachstumsgeschwindigkeit korreliert. [26] Abbildung 3z eigt, dass besonders die früheren, häufiger vorkommenden Lanthanoide das Wachstum von Bakterien, die Ln-abhängige Enzyme exprimieren, unterstützen.…”

“…[8] Die Funktion von xoxF war lange unbekannt, das erste "x" in xoxF steht tatsächlich fürdiese lange unbekannte Rolle. [9] Im selben Jahr noch berichteten die Autoren über eine Cer(III)abhängige MDH in Bradyrhizobium sp.MAFF211645. [10] Ein Jahr später konnten Nakagawa, Kawai et al berichten, dass der Modell-Bakterienstamm Methylorubrum extorquens AM1 ebenfalls eine Lanthanoid-abhängige MDH exprimiert und dass diese Bakterien mit La 3+ anstatt von Ca 2+ wachsen kçnnen.…”