Motility in Periweissella Species: Genomic and Phenotypic Characterization and Update on Motility in Lactobacillaceae

The genus Weissella and the recently described genus Periweissella, to which some previously named Weissella species have been reclassified as a result of a taxogenomic assessment, includes lactic acid bacteria species with high biotechnological and probiotic potential. Only one species, namely, Periweissella (P.) beninensis, whose type strain has been shown to possess probiotic features, has so far been described to be motile. However, the availability of numerous genome sequences of Weissella and Periweissella species prompted the possibility to screen for the presence of the genetic determinants encoding motility in Weissella and Periweissellas spp. other than P. beninensis. Herein, we performed a comprehensive genomic analysis to identify motility-related proteins in all Weissella and Periweissella species described so far, and extended the analysis to the recently sequenced Lactobacillaceae spp. Furthermore, we performed motility assays and transmission electron microscopy (TEM) on Periweissella type strains to confirm the genomic prediction. The homology-based analysis revealed genes coding for motility proteins only in the type strains of P. beninensis, P. fabalis, P. fabaria and P. ghanensis genomes. However, only the P. beninensis type strain was positive in the motility assay and displayed run-and-tumble behavior. Many peritrichous and long flagella on bacterial cells were visualized via TEM, as well. As for the Lactobacillaceae, in addition to the species previously described to harbor motility proteins, the genetic determinants of motility were also found in the genomes of the type strains of Lactobacillus rogosae and Ligilactobacillus salitolerans. This study, which is one of the first to analyze the genomes of Weissella, Periweissella and the recently sequenced Lactobacillaceae spp. for the presence of genes coding for motility proteins and which assesses the associated motility phenotypes, provides novel results that expand knowledge on these genera and are useful in the further characterization of lactic acid bacteria.

Species of the genera Weissella and Periweissella are widespread in nature and have been isolated from several food matrices, including meat, fish, vegetables and fermented foods, as well as from soil, sewage and the gastrointestinal tracts of humans and animals [8,9].Despite their probiotic potential, which has recently been demonstrated by several studies [10,11], to date, none of the Weissella and Periweissella strains are recognized as GRAS (Generally Recognized as Safe) by the Food and Drug Administration (FDA) in the United States of America, nor have they been included in the Qualified Presumption of Safety (QPS) list by the European Food Safety Authority (EFSA).
Within Weissella and Periweissella spp., only P. beninensis was previously described as being motile [3].Recently, during the publication process of this manuscript, Qiao et al. [12] reported on the motility of Periweissella strains.Bacterial motility, combined with chemotaxis, provides the ability for bacterial dissemination and surface exploration, enabling movement towards favorable conditions to reach resources and allow for surface colonization.These features are also relevant for the probiotic properties of bacterial strains, as motility is important for the initial steps of adhesion to intestinal epithelial cells and biofilm formation [13].It, furthermore, provides certain advantages regarding survivability within this specific ecological niche and persistence of the microorganisms in the gut mucosa, and is crucial for enabling them to exert their protective effects and for interaction with the host [14].
The recent availability of genome sequences of all Periweissella and Weissella strains allows for investigations into the presence of genes coding for motility proteins in other species in addition to P. beninensis, and if found, these could point towards a potential motile phenotype.In addition, the probiotic potential of some Weissella and Periweissella strains, recently confirmed by Fanelli et al. [11], also encourages further investigation of functional traits of these strains.This study aimed to perform a comprehensive genomic analysis to identify motility proteins in all Weissella and Periweissella species described so far, and extended the analysis to the recently sequenced Lactobacilliaceae spp.Furthermore, motility assays and transmission electron microscopy (TEM) were carried out on Periweissella type strains to confirm our genomic predictions.

Strain Info and Culture Conditions
P. beninensis LMG 25373 T , P. fabalis LMG 26217 T and P. fabaria LMG 24289 T were purchased from the Belgian Coordinated Collection of Microorganisms (BCCM/LMG, Ghent, Belgium), while P. ghanensis DSM 19935 T and W. diestrammenae DSM 27940 T were purchased from the German Collection of Microorganisms and Cell Cultures (© DSMZ, Braunschweig, Germany).The purity of each strain was confirmed as described in Fanelli et al. [11] via streaking on de Man, Rogosa and Sharpe (MRS; Oxoid, Milan, Italy) agar plates and through microscopic observation.Strains were maintained at −80 • C as pure stock cultures in MRS broth (Oxoid) supplemented with 30% glycerol (v/v).Strains were routinely grown in MRS broth at 30 • under aerobic and static conditions, unless specified otherwise.

Motility Test
Motility was evaluated according to the method described by Shield and Cathcart [15].Periweissella strains were cultured on MRS agar medium (Oxoid).Subsequently, different media, including MRS, PCA (Oxoid) and motility-indole-lysine (MIL) medium [15], were tested to find the one that could best show the strain motility.By using a sterile needle, a colony of each strain was picked and stabbed into the relevant sterile agar medium in tubes to within 1 cm of the bottom.Tubes were incubated at 30 • C until growth became evident.The motility test was considered positive if a diffuse cloud of growth was visibly extending away from the line of inoculation.The test was considered negative if the growth was limited along the inoculation line.

Transmission Electron Microscopy
For transmission electron microscopy, all samples were essentially prepared as described by Sørensen et al. [16], with the modification that 2% (v/v) glutaraldehyde was used for fixation and 1% (w/v) uranyl acetate for negative staining.Electron micrographs were taken using a Talos L120C transmission electron microscope (Thermo Fisher Scientific, Eindhoven, The Netherlands) at an accelerating voltage of 80 kV.Digital micrographs were taken using a 4 k × 4 k Ceta camera (Thermo Fisher Scientific).The whole-genome sequencing and the evaluation of the quality of the assemblies were described in Fanelli et al. [17].The whole-genome shotgun project and GenBank accession number used for the identification of the motility proteins are listed in Supplementary Table S1.Proteins were predicted as described in Fanelli et al. [17] by using the Prokaryotic Genome Annotation Pipeline [18] and PROKKA pipeline [19] implemented in the Galaxy platform (Galaxy Version 1.14.6 + galaxy0; [20].Functional classification was performed using the RAST server [21].Genes related to motility and flagellar systems were retrieved through a keyword search within the UniProtID entry list obtained via functional annotation (by using as a query the terms 'flagella' and 'motility', and by using the RAST server [21]), and then, manually curated.

In Silico Identification of Motile Species in the Lactobacillaceae Family
The identification of motile species in the family Lactobacillaceae was performed according to the strategy used by Cousin et al. [22], with some modification.Sequences of the predicted filament protein flagellin FliC, the flagellar hook-basal body complex protein FliE, the flagellar motor switch protein FliG, the flagellar biosynthesis protein FlhA and the chemotaxis/stator protein MotA were identified in the proteomes of Periweissella spp.Consensus sequences of the selected proteins, generated using EMBOSS Cons [23], were used as query for a TBLASTN search performed against all publicly available Weissella species genomes, and then, extended to all recently sequenced and publicly available Lactobacillaceae species genomes described after the analysis performed by Cousin et al. [22], according to the taxonomic description amended by Zheng et al. [7], with a cut off E-value of 1 × 10 −10 and % identity > 30%.Where positive hits were retrieved, motility protein sequences were extracted from the respective proteomes.The homology-based relationship among proteins was determined and confirmed using the BLASTP algorithm on the NCBI site (http://blast.ncbi.nlm.nih.gov/Blast.cgiaccessed on 1 October 2023).

Phylogenetic Analysis
The DNA sequences of the 16S rRNA gene of the type strain of each Lactobacillaceae species positive for the presence of motility proteins were downloaded from the NCBI GenBank database.Phylogenetic analysis was performed by using (i) the 16S rRNA genes, (ii) a concatenated dataset of all the motility proteins identified in each species assumed as being motile by the in silico analysis, and (iii) concatenated datasets of clusters of motility proteins grouped according to their function or position in the flagellum: chemotaxis proteins (concatenated dataset of CheA, CheB, CheC, CheD, CheW and CheY); basal body-hook proteins (concatenated dataset of FlhA, FlhB, FliE, FliF, FliG, FliH, FliI, FliJ, FliM, FliO, FliP, FliQ, FliR and FliK); filament junction proteins (concatenated dataset of FlgL, FlgK, FliC, FliD and FliS); rod, ring and hook/filament junction proteins (concatenated dataset of FlgB, FlgC, FlgD, FlgE, FlgF and FlgG).Phylogeny was inferred by using an "A la Carte" Mode workflow implemented in the NGphylogeny.frplatform [24] (https://ngphylogeny.fr/workflows/alacarte accessed on 1 October 2023) integrated with MAFFT (version 7.407), BMGE (version 1.12) and FastMe (version 2.1.6.1) with default parameters.The phylogenetic reconstruction was performed using the BIONJ method; the phylogenetic robustness was inferred via a bootstrapping procedure with 1000 replications to obtain the confidence value for the aligned sequence dataset.The trees were graphically generated using iTOL version 6.6 [25].Bacillus subtilis subsp.subtilis strain 168 was used as an outgroup.

Brightfield Microscopy
Bacteria grown for 24 h in MRS broth under anaerobic conditions were diluted 1:10 in sterile MRS broth.A total of 200 µL was pipetted into 8-well ibiTreat chambered coverslips (Ibidi, Gräfelfing, Germany) and used for microscopy at 1000× magnification with oil immersion using a Nikon Eclipse TS100 (Nikon, Tokyo, Japan) inverted microscope.The bacteria were allowed to settle for at least 30 min, and videos were recorded using the NIS-Elements D software (version 4.13.03,Nikon).The videos were processed using ImageJ to remove artefacts, and the final videos were compressed to 2× speed and combined.

Motility Behavior
As reported in the Material and Methods section, different media were tested for the motility assay.P. beninensis grew in MRS and PCA, but without the possibility to take a photo and underline its motility, whereas it very clearly showed its motility in MIL.Therefore, MIL was used as a medium for the assay of the motility of all the target strains.Among the four Periweissella type strains tested, only the P. beninensis type strain was able to grow and spread diffusely within the MIL medium (Figure 1).Negative results were obtained for the P. fabalis LMG 26217 T , P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T type strains (Figure 1).P. beninensis LMG 25373 T was also the only type strain demonstrating overt motility when observed using brightfield microscopy, showing run-and-tumble movements (Supplementary Movie S1).
phylogenetic robustness was inferred via a bootstrapping procedure with 1000 replications to obtain the confidence value for the aligned sequence dataset.The trees were graphically generated using iTOL version 6.6 [25].Bacillus subtilis subsp.subtilis strain 168 was used as an outgroup.

Brightfield Microscopy
Bacteria grown for 24 h in MRS broth under anaerobic conditions were diluted 1:10 in sterile MRS broth.A total of 200 µL was pipetted into 8-well ibiTreat chambered coverslips (Ibidi, Gräfelfing, Germany) and used for microscopy at 1000× magnification with oil immersion using a Nikon Eclipse TS100 (Nikon, Tokyo, Japan) inverted microscope.The bacteria were allowed to settle for at least 30 min, and videos were recorded using the NIS-Elements D software (version 4.13.03,Nikon).The videos were processed using Im-ageJ to remove artefacts, and the final videos were compressed to 2× speed and combined.

Motility Behavior
As reported in the Material and Methods section, different media were tested for the motility assay.P. beninensis grew in MRS and PCA, but without the possibility to take a photo and underline its motility, whereas it very clearly showed its motility in MIL.Therefore, MIL was used as a medium for the assay of the motility of all the target strains.Among the four Periweissella type strains tested, only the P. beninensis type strain was able to grow and spread diffusely within the MIL medium (Figure 1).Negative results were obtained for the P. fabalis LMG 26217 T , P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T type strains (Figure 1).P. beninensis LMG 25373 T was also the only type strain demonstrating overt motility when observed using brightfield microscopy, showing run-and-tumble movements (Supplementary Movie S1).The other four strains, P. fabalis LMG 26217 T , P. fabaria LMG 24289 T , P. ghanensis DSM 19935 T and W. diestrammenae DSM 27940 T , did not demonstrate motility beyond vibrations, which did not appear directional in the observed time span (Supplementary Movie S1).

Transmission Electron Microscopy
Figure 1 also shows the electron micrographs of Periweissella type strains analyzed in this study.As indicated by the red arrows, only in the P. beninensis type strain was it possible to visualize numerous peritrichous and long flagella.In P. fabaria LMG 24289 T , only a rudimental piece of flagellum was visible (red arrow), and such short pieces were visible only in a very minor portion of cells.W. diestrammenae DSM 27940 T was used as a negative control since its genome was found not to harbor any motility protein genes.

Presence of Motility Proteins in Lactobacillaceae
The homology-based analysis of the P. beninensis LMG 25373 T , P. fabalis LMG 26217 T , P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T genomes revealed the presence of motility protein-encoding genes in all four strains.The chemotaxis-and flagella-related proteins identified in Periweissella species are listed in Table 1.FlgN was predicted to be encoded in the genomes of all Periweissella putative motile species as a hypothetical protein (indicated by hp gene near to flgM gene in Figure 2).This protein is the flagellum synthesis protein required for the efficient initiation of filament assembly.The FlgN putative homolog proteins identified in P. beninensis LMG 25373 T (WP_205144167.1) and P. ghanensis DSM 19935 T (WP_230098022) were classified by Inter-ProScan as having a FlgN-like domain (IPR036679), while no Geno Ontology (GO) term was associated with the putative FlgN homolog of P. fabalis LMG 26217 T (WP_168721789.1) and P. fabaria LMG 24289 T (WP_230097033.1),indicating no functional prediction, although the MotifFinder analysis retrieved, for all four hypothetical proteins, the PAFM domain PF05130 associated with the FlgN protein.
Some main differences between the Periweissella species positive for the presence of motility proteins could be determined.For instance, the flaG gene coding for the FlaG protein (flagella-associated protein) was identified only in P. beninensis LMG 25373 T (WP_205144163.1); the flaG gene was located in the same operon as the fliD and fliS genes reported in other species (e.g., Camphylobacter jejuni), or in the vicinity of the fliD operon on the chromosome of many different species of bacteria [26].

Comparative Analysis of Motility Locus in Periweissella Species
The organization of the flagellar locus of the Periweissella type strains analyzed in this study is illustrated in Figure 2, while motility protein IDs are listed in Table 1.
In P. fabaria LMG 24289 T , P. fabalis LMG 26217 T and P. ghanensis DSM 19935 T , the motility gene locus was annotated on a unique contig, while in P. beninensis LMG 25373 T the fliD operon (fliD operon; following nomenclature of corresponding operons in Bacillus subtilis and Salmonella enterica Typhimurium, containing the genes fliD, fliS and fliT) [27,28] was annotated in a separated contig (Figure 2).The organization of the flagellar operon was conserved in the Periweissella species, while the gene content and arrangement within the fliD operon showed some differences: (i) Downstream of the FliS coding gene in P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T were two genes (gtfA) annotated to code for a UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase GtfA subunit; (ii) in P. fabaria LMG 24289 T and P. fabalis LMG 26217 T , there were two genes coding for two hypothetical proteins, both harboring a NEAT domain, which was found in a group of iron-regulated surface determinant proteins.In addition, one of these was predicted to have an internaline surface protein domain with a leucine-rich repeat (LRR) that allowed it to provide a structural framework for the formation of protein-protein interactions.(iii) The fliD operon of P. fabalis LMG 26217 T also harbored two hag genes coding for the two flagellins FliC1 and FliC2, as previously described, while only one hag gene was present in the other Periweissella spp.(iv) In the fliD operon in P. beninensis LMG 25373 T one gene was annotated that codes for FlaG and one for a glycosyltransferase located between the flgL and hag gene, respectively, and three methyl-accepting chemotaxis protein (Mcp)-encoding genes were located upstream of the flgM operon With respect to the Weissella genus, only in the draft genome sequences of Weissella confusa L3 and L9 strains, isolated from human feces, were partial sequences of flagellin proteins detected, and they have been deposited under the accession nos.MBS7551942.1, MBS7552003.1, MCQ8173872.1 and MCQ8173814.1.No homolog of the motility proteins listed in Table 1 was identified in the other Weissella species, nor in the P. cryptocerci type strain, the only remaining species within the Periweissella genus so far described.In Leuconostoc spp., Oenococcus spp., Convivina spp.and Fructobacillus spp., no motility proteins could be determined.
In addition to the Lactobacillaceae species described by Cousin et al. [22] to harbor motility proteins, they were also identified in the genomes of the recently sequenced Lactobacillus (L.) rogosae ATCC 27753 T (and identical to those retrieved from the genome of the L. rogosae isolate avicel METABAT 177 assembled from the human gut microbiome) and Ligilactobacillus salitolerans DSM 103433 T (Figure 2).

Comparative Analysis of Motility Locus in Periweissella Species
The organization of the flagellar locus of the Periweissella type strains analyzed in this study is illustrated in Figure 2, while motility protein IDs are listed in Table 1.
In P. fabaria LMG 24289 T , P. fabalis LMG 26217 T and P. ghanensis DSM 19935 T , the motility gene locus was annotated on a unique contig, while in P. beninensis LMG 25373 T the fliD operon (fliD operon; following nomenclature of corresponding operons in Bacillus subtilis and Salmonella enterica Typhimurium, containing the genes fliD, fliS and fliT) [27,28] was annotated in a separated contig (Figure 2).The organization of the flagellar operon was conserved in the Periweissella species, while the gene content and arrangement within the fliD operon showed some differences: (i) Downstream of the FliS coding gene in P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T were two genes (gtfA) annotated to code for a UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase GtfA subunit; (ii) in P. fabaria LMG 24289 T and P. fabalis LMG 26217 T , there were two genes coding for two hypothetical proteins, both harboring a NEAT domain, which was found in a group of iron-regulated surface determinant proteins.In addition, one of these was predicted to have an internaline surface protein domain with a leucine-rich repeat (LRR) that allowed it to provide a structural framework for the formation of protein-protein interactions.(iii) The fliD operon of P. fabalis LMG 26217 T also harbored two hag genes coding for the two flagellins FliC1 and FliC2, as previously described, while only one hag gene was present in the other Periweissella spp.(iv) In the fliD operon in P. beninensis LMG 25373 T one gene was annotated that codes for FlaG and one for a glycosyltransferase located between the flgL and hag gene, respectively, and three methyl-accepting chemotaxis protein (Mcp)-encoding genes were located upstream of the flgM operon (Figure 2), which exceeded the count observed in the loci of the other Periweissella species by one.
Another difference in the motility genes of the different Periweissella species was that in P. beninensis LMG 25373 T , there were the putative homologs of proteins, annotated as FliH and FliJ (WP_205144100.1 and WP_205144102.1,respectively; Table 1 and boxed parts in Figure 2).These were two cytoplasmic ATPase complex proteins that are part of the flagellum export apparatus [29].Meanwhile, in P. fabalis LMG 26217 T , P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T , they were annotated as hypothetical proteins.The similarity among these hypothetical proteins and the respective homolog in P. beninensis LMG 25373 T was, on average, ca.35%, while between P. fabaria LMG 24289 T and the respective homolog in P. ghanensis DSM 19935 T , it reached 99%, and between P. fabalis LMG 26217 T and the respective homolog in P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T , it was around 75-80%.The MotifFinder pipeline predicted, in the three hypothetical proteins homologous to FliH of P. fabalis LMG 26217 T , P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T , the PFAM domain PF02108 associated with the flagellar assembly protein FliH, and in all three hypothetical protein homologs of FliJ, the PFAM domain PF02050 associated with the flagellar FliJ protein.
As mentioned before, a difference in the motility genes among the Periweissella species is that only in P. fabalis LMG 26217 T was a second hag (fliC2) gene coding for the flagellin FliC2 annotated in the flagellar operon (WP_168721785.1): the predicted FliC2 shares 75% similarity with FliC1 (WP_168721786.1), and 78% and 65% with the homologs in P. fabaria LMG 24289 T (WP_230097036.1) and Ligilactobacillus ruminis AM12-58A (WP_270413121.1),respectively.In Ligilactobacillus ruminis AM12-58A, the fliC2 flagellin gene was 98% identical at nucleotide level to the fliC1 gene encoded in the same strain; however, gene expression studies showed that only fliC2 was expressed at a high level in the motile strains [30].Multiple flagellin proteins were also described to occur in several species, such as Sinorhizobium meliloti, Rhizobium leguminosarum, Agrobacterium tumefaciens and Caulobacter crescentus, the latter of which has a single polar flagellum and six flagellin proteins [31][32][33][34].
The main differences between the flagellar loci of the non-motile Periweissella strains P. fabalis LMG 26217 T , P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T with respect to the motile P. beninensis LMG 25373 T thus appears to be the absence of the flaG gene and the presence of hypothetical genes showing low homology to the corresponding (fliH, fliJ, flgN) genes.The identity of the hypothetical genes was validated using HHpred, which allows for the detection of remote homologs by comparing protein profiles based on Hidden Markov Models rather than only relying on sequence homology.The hypothetical protein sequences were screened against the RCSB Protein Data Bank database (https://www.rcsb.org/accessed on 1 October 2023) and yielded hits for FliH, FliJ and FlgN, respectively.
The ATPase complex promoting the flagellar protein export machinery is composed of FliH, FliJ and FliI, where the latter exhibits enzymatic ATPase activity.The characteristic Walker A (GxxxxGK(S/T)-where x is any amino acid) and Walker B (hhhhDE-where h is any hydrophobic amino acid) motifs found in ATPases were detected in all Periweissella homologs (Supplementary Figure S1), suggesting that all homologs are catalytically active.Surprisingly, there are major differences in the conservation of the three proteins forming the ATPase complex between the analyzed Periweissella species.While FliI is highly conserved between the here determined non-motile species P. fabaria, P. fabalis and P. ghanensis (protein similarity 98.2-98.5%)and rather well conserved in P. beninensis (protein similarity 72.4-72.9%), the other two proteins forming the ATPase complex (FliH and FliJ) display a decreased homology between P. beninensis and the three herein determined as non-motile Periweissella species.Among only the latter species, FliH displays an amino acid sequence similarity of 71.5% and FliJ of 79.2-81.9%.Compared to P. beninensis, protein similarity decreases to 25.9-33.2%for FliH and 31.7-33.7%for FliJ, thus demonstrating unequal conservation of the ATPase complex.Interestingly, these genes are all present and share higher homology in all the other motile lactic acid bacteria examined in this study (L.acidipisces DSM 15836 T , L. salitotolerans DSM 103433 T and L. rogosae ATCC 27753 T (Figure 2)), the only exception being that the flaG gene is not present in the motility locus of L. salitotolerans DSM 103433 T .On the other hand, the fliD gene encoding the filament cap protein, which is a protein for chaperoning and sorting flagellin (FliC) proteins after they traverse the hollow filament and exit the growing flagellum tip [35], was only found in the flagellar loci of the P. fabalis LMG 26217 T , P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T strains, but not in the corresponding locus of the P. beninensis strain LMG 25373 T (Figure 2).As shown in Figure 2, the content and organization of the flagellar locus in Periweissella species compared to other Lactobacillaceae, such as the L. acidipiscis type strain and L. salitolerans type strain, is quite different.In L. salitolerans DSM 103433 T , motA and motB genes were located within the fliD operon, which comprised two flagellin genes and was annotated in a separate contig with respect to the flagellar operon.Also, in Lactobacillus rogosae ATCC 27753 T , the fliD operon was annotated on a separate contig with respect to the flagellar operon, which misses flbD, fliK and fliL and harbors the fapA gene coding for the flagellar assembly protein FapA, and ycgR coding for PliZ, a cyclic-di-GMP (c-di-GMP) associated with flagellar and pili-based motility.In the L. acidipiscis type strain fliD operon, there are four genes annotated as transposase, motB and motA genes, the fliS and fliD gene interspaced by a gene coding for a hypothetical protein, the flaG gene, one RNA polymerase-coding gene, two flagellin hag genes, a gene coding for the peptidoglycan-binding protein LysM and one integrase.

Phylogeny of the Motility Proteins in the Lactobacillaceae Species
Figures 3 and 4 show the concordant phylogeny of 16S rRNA genes and motility proteins in the Lactobacillaceae family.P. beninensis LMG 25373 T , P. fabalis LMG 26217 T , P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T were always clustered in one separate clade, with P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T being on the same node, and slightly divergent from P. fabalis LMG 26217 T and more divergent from P. beninensis LMG 25373 T (Figures 3 and 4).P. beninensis LMG 25373 T was clustered together with the other Periweissella species in the phylogenetic tree of the concatenated dataset of all the motility proteins, and of the groups of chemotaxis proteins, filament junction proteins, and rod-ring-hook proteins, although, interestingly, it was also grouped apart from these, showing a clear difference in the motility gene content outlined above.Furthermore, in a few cases, the phylogeny of individual proteins, such as CheB, CheR, CheC, CheD, FlgB, FlgC, etc., as well as of those occurring in the cluster of the basal body and hook proteins (shown in Supplementary Figure S2B), underlines a higher proximity of P. beninensis LMG 25373 T with species outside of the Periweissella genus.
L. rogosae ATCC 27753 T (as well as L. rogosae isolate avicel METABAT 177) was phylogenetically apart from the other Lactilobacillaceae species in each motility protein analyzed, as well as in relation to the 16S rRNA gene sequence phylogeny (Figures 3 and 4).

Discussion
The bacterial flagellum is organized into three basic components [36,37]: (1) The basal body, anchored in the cell membrane and comprising a rotary motor, connected to a rod, which powers the flagellum, the export apparatus machinery, and bearing structures.
(2) The hook [38], which functions like a universal joint and is a tubular helical structure made via the polymerization of multiple copies of a protein, FlgE; its length is controlled by FliK.The hook transmits the torque produced by the motor to the (3) filament, which is the propeller of the flagellum, with FliC as the core unit and FliD acting as a cap.The junction between the hook and the filament is the charger of FlgL and FlgK.
The flagellar apparatus in Salmonella enterica subsp.enterica serovar Typhimurium is one of the best characterized thanks to the work by Minamino and coworkers [29], and the crystal structures of most of the protein complexes that comprise the flagellar apparatus have been resolved and deposited in the RCSB Protein Data Bank.Both FliH and FliJ, which are also present in the genetic locus of motile P. beninensis LMG 25373 T , have been described to be part of the FliH( 12)-FliI(6)-FliJ(1) cytoplasmic ATPase export ring complex, which acts as an active protein transporter.Its structure resembles the cytoplasmic part of F O F 1 ATP synthase, a rotary motor that couples proton (H + ) flow through F O with ATP synthesis by F 1 [39].We found that the conservation of the subunits of the ATPase involved in protein export is dissimilar between the Periweissella strains.While FliI is rather highly conserved, FliH and FliJ display considerably lower sequence homology between P. beninensis and the three non-motile species.This could indicate that the complex might not form properly in the non-motile species due to the high evolutionary divergence, resulting in an impaired flagellar export mechanism, and thus, could explain the difference in motility.
In addition to that, FlgN was also poorly conserved.FlgN is one of the flagellar chaperones, together with FliS and FliT, specific to FlgK and FlgL, which, in Bacillus subtilis, is required for flagellum-based motility [40].The crystal structure of FlgN in Salmonella was solved in 2016 by Kinoshita et al. [41] and consists of three helices.The authors showed that a conformational change in FlgN with rearrangements of α-helices is critical for the association and dissociation of FlgK [41].Minamino et al. [42] demonstrated that FlgN promotes the docking of FlgK and FlgL to the FlhA platform of the export gate complex to facilitate the rapid and efficient export of these proteins [43].The impairing of this activity reduces the secretion levels of FlgK and FlgL, resulting in a considerable reduction in the probability of filament formation at the tip of the hook-basal body [44].In addition to its chaperone activity, FlgN interacts with FlhA, which is the export engine of the apparatus, generating a conformational change in FlhA that allows it to function as a Na + channel.
The flaG gene was identified only in P. beninensis LMG 25373 T .FlaG is one of the flagellar proteins that are actually absent in well-studied model organisms such as Escherichia coli and Salmonella enterica [45,46], but has been detected and characterized in other motile species such as Aeromonas caviae, Campylobacter jejuni, Pseudomonas aeruginosa and Pseudomonas fluorescens [47][48][49].Divergence of the protein sequences and mutational behaviors in different species suggested that FlaG had a possible role in regulating the export of flagellin proteins, filament length and the number or the expression of other flagellin genes [50][51][52].
Our study shows that hypothetical proteins with putative homology to FliH, FliJ and FlgN in the three non-motile Periweissella species have only low similarity (35-41%) when compared to those comprising the flagellar locus of P. beninensis LMG 25373 T .As FliH and FliJ are essential for the export of flagellar proteins, it is possible that a low homology of counterpart proteins in the non-motile strains implies that the flagellar components cannot be sufficiently exported for flagellar biosynthesis.Similarly, FlgN is a flagellum synthesis protein and is required for the export of the hook/filament junction proteins FlgK and FlgL.If this is absent or nonfunctional, again, this could prevent the assembly of a functional flagellum, although we have evidence that in P. fabaria LMG 24289 T , a partial flagellum could possibly be assembled, as seen in the results of electron microscopy (Figure 1).Despite the fact that P. fabalis LMG 26217 T , P. fabaria LMG 24289 T and P. ghanensis DSM 19935 T possess a complete flagellar operon, motility could be demonstrated only for P. beninensis LMG 25373 T .It is very clear from live cell video microscopy that P. beninensis LMG 25373 T bacteria are remarkably motile compared to the other Periweisella species (Supplementary Movie S1).
Our data are in agreement with previous papers describing Periweissella spp.In particular P. fabalis [4] and P. fabaria [5] were described as not motile, and for P. ghanensis, gliding motility was not observed [6], while, conversely, P. beninensis [3] was defined as motile with peritrichous flagella that can occur singly, in pairs or in short chains.
A recent paper published by Qiao et al. [12] confirmed the presence of the flagellar operon in P. beninensis, P. fabalis and P. fabaria, and the authors described all these species as motile.However, the only phenotypic assessment to prove their assumption was the stab test in semisolid MRS agar medium, which, for us, was not as adequately informative for assessing motility compared to using MIL medium.Furthermore, surprisingly, P. beninensis was not able to grow under the condition tested by Qiao et al. [12].In our work, the TEM experiments showed that with the exception of the P. beninensis type strain, the other Periweissella species were not able to assemble any visible flagellar apparatus under the tested condition.Only in P. fabaria LMG 24289 T was a rudimentary piece of flagellum visible, and very few pieces of flagella could be found only in a very minor portion of cells.This was supported by microscopic observations, which showed that only P. beninensis had high motility.Hypothetically the genomic differences reported here in the flagellar operons of the other Periweissela when compared to P. beninensis LMG 25373 T might lead to a lower expression of flagellar proteins under the condition that we tested in this study.This should, however, be investigated for media and test conditions in further studies.
It is interesting to note that, in addition to other Weissella species, in the P. cryptocerci type strain, no motility proteins could be determined.Previous work by our group [11,17] demonstrated that P. beninensis LMG 25373 T displayed the best probiotic behavior, with the highest adhesion capacity, as well as a varied carbohydrate utilization profile, and was the only one capable of using D-galactose.Although motility is not essential for gut colonization, it might provide certain advantages in survivability and persistence for these organisms in the gut mucosa, and favor colonization in the gastrointestinal tract, as occurred for motile lactobacilli compared to not-motile ones [53].
Flagella are composed, together with pili, of surface layer proteins (SLPs), capsular polysaccharide (CPS), lipoteichoic acid and lipopolysaccharide, the surface components of probiotics that constitute microbe-associated molecular patterns (MAMPs) [54], which can specifically bind to pattern recognition receptors and regulate nuclear factor kappa B, mitogen-activated protein kinases, peroxisome proliferator-activated receptor gamma, and other signaling pathways in intestinal epithelial cells [55], in addition to activating the cellular protease-dependent signaling cascade to produce a variety of cytokines and chemokines that alleviate inflammation and enhance intestinal epithelial function [56].In the probiotic E. coli Nissle 1917, the flagellum is not just responsible for motility and capable of inducing the production of human antimicrobial peptide β-defensin 2, but is the major adhesin mediating binding to human mucus [57], which enables this probiotic strain to compete efficiently for binding sites on host tissue with several pathogenic bacteria.The ability of P. beninensis LMG 25373 T to assemble a functional flagellum thus confirms the distinctiveness of this strain with respect to the other Periweissella type strains analyzed, and suggests a correlation with optimal probiotic activity, in particular related to its adhesion ability, that was recently demonstrated in vitro by Fanelli and coworkers [17].

Conclusions
The comprehensive genomic analysis herein carried out to detect the genetic determinants of motility in Weissella, Periweissella and the recently sequenced Lactobacillaceae spp.allowed us to detect genes coding for motility proteins only in the type strains of P. beninensis, P. fabalis, P. fabaria and P. ghanensis genomes, as well as in the recently genome-sequenced type strains of Lactobacillus rogosae and Ligilactobacillus salitolerans.Among the Weissella and Periweissella type strains analyzed in this study, only the P. beninensis type was positive in the motility assay, and its many peritrichous and long flagella were visualized via TEM, while, despite the type strains of P. fabalis, P. fabaria and P. ghanensis possessing a complete flagellar operon, no motility was observed in these strains under the conditions used in this study, and only the P. fabaria type strain showed a rudimentary piece of flagellum on few cells.The type strain of P. beninesis showed the highest in vitro adhesion capacity and resistance to simulated gastrointestinal digestion [17] (Fanelli et al., 2023), indicating a possible correlation between the motile behavior and the probiotic potential of this strain.
This study provides novel results that are useful in the further characterization of these genera.

2. 4 .
Comparative Analysis of Flagellar Locus and Motility Proteins 2.4.1.Identification of Motility Protein Genes in Some Periweissella Type Strains

Figure 1 .
Figure 1.Motility test tubes and TEM of Periweissella spp.W. diestrammenae DSM 27940 T was used as negative control.Flagella are indicated by red arrows.

a
in the columns, protein ID and locus tag are indicated; b annotated as hypothetical protein; na = not annotated.

Figure 2 .
Figure 2. Organization of flagellar locus in Lactobacillaceae species.Schematic representation of the flagellar locus in P. fabalis LMG 26217 T , P. fabaria LMG 24289 T , P. ghanensis DSM 19935 T , P. beninensis LMG 25373 T , L. acidipiscis DSM 15836 T , L. salitolerans DSM 103433 T , L. rogosae ATCC 27753 T and B. subtilis subsp.subtilis str.168.Genes are not drawn to scale.Rectangles highlight the fliJ, fliH and flgM locus, where the main differences among P. beninensis LMG 25373 T and the other Periweissella species were identified (see text for details).

Figure 2 .
Figure 2. Organization of flagellar locus in Lactobacillaceae species.Schematic representation of the flagellar locus in P. fabalis LMG 26217 T , P. fabaria LMG 24289 T , P. ghanensis DSM 19935 T , P. beninensis LMG 25373 T , L. acidipiscis DSM 15836 T , L. salitolerans DSM 103433 T , L. rogosae ATCC 27753 T and B. subtilis subsp.subtilis str.168.Genes are not drawn to scale.Rectangles highlight the fliJ, fliH and flgM locus, where the main differences among P. beninensis LMG 25373 T and the other Periweissella species were identified (see text for details).

Figure 3 .
Figure 3. 16S rRNA gene-based phylogeny of Lactobacillaceae species.Phylogeny was performed using the neighbor-joining method; phylogenetic robustness was inferred via a bootstrapping procedure from 1000 replications to obtain the confidence value for the aligned sequence dataset.Bacillus subtilis subsp.subtilis str.168 was used as an outgroup.The tree was drawn to scale, with branch lengths measured as the number of substitutions per site.Scaled circles are representative of bootstrap values.

Figure 4 .
Figure 4. Phylogenetic analysis of motility proteins in Lactobacillaceae species.The tree is based on the concatenated dataset of motility proteins (see text for details).Phylogenetic robustness was inferred via a bootstrapping procedure from 1000 replications to obtain the confidence value for the

Figure 3 .
Figure 3. 16S rRNA gene-based phylogeny of Lactobacillaceae species.Phylogeny was performed using the neighbor-joining method; phylogenetic robustness was inferred via a bootstrapping procedure from 1000 replications to obtain the confidence value for the aligned sequence dataset.Bacillus subtilis subsp.subtilis str.168 was used as an outgroup.The tree was drawn to scale, with branch lengths measured as the number of substitutions per site.Scaled circles are representative of bootstrap values.

Figure 3 .
Figure 3. 16S rRNA gene-based phylogeny of Lactobacillaceae species.Phylogeny was performed using the neighbor-joining method; phylogenetic robustness was inferred via a bootstrapping procedure from 1000 replications to obtain the confidence value for the aligned sequence dataset.Bacillus subtilis subsp.subtilis str.168 was used as an outgroup.The tree was drawn to scale, with branch lengths measured as the number of substitutions per site.Scaled circles are representative of bootstrap values.

Figure 4 .
Figure 4. Phylogenetic analysis of motility proteins in Lactobacillaceae species.The tree is based on the concatenated dataset of motility proteins (see text for details).Phylogenetic robustness was inferred via a bootstrapping procedure from 1000 replications to obtain the confidence value for the

Figure 4 .
Figure 4. Phylogenetic analysis of motility proteins in Lactobacillaceae species.The tree is based on the concatenated dataset of motility proteins (see text for details).Phylogenetic robustness was inferred via a bootstrapping procedure from 1000 replications to obtain the confidence value for the aligned sequence dataset.Bacillus subtilis subsp.subtilis str.168 was used as an outgroup.The tree was drawn to scale, with branch lengths measured as the number of substitutions per site.Scaled circles are representative of bootstrap values.

Table 1 .
Flagellar and chemotaxis proteins predicted in Periweissella strains a .
a in the columns, protein ID and locus tag are indicated; b annotated as hypothetical protein; na = not annotated.