The Genome of Bifidobacterium longum subsp. infantis YLGB-1496 Provides Insights into Its Carbohydrate Utilization and Genetic Stability

Bifidobacterium longum subsp. infantis YLGB-1496 (YLGB-1496) is a probiotic strain isolated from human breast milk. The application of YLGB-1496 is influenced by carbohydrate utilization and genetic stability. This study used genome sequencing and morphology during continuous subculture to determine the carbohydrate utilization characteristics and genetic stability of YLGB-1496. The complete genome sequence of YLGB-1496 consists of 2,758,242 base pairs, 2442 coding sequences, and a GC content of 59.87%. A comparison of carbohydrate transport and metabolism genes of Bifidobacterium longum subsp. infantis (B. infantis) showed that YLGB-1496 was rich in glycosyl hydrolase 13, 20, 25, and 109 gene families. During continuous subculture, the growth characteristics and fermentation activity of the strain were highly stable. The bacterial cell surface and edges of the 1000th-generation strains were progressively smoother and well-defined, with no perforations or breaks in the cell wall. There were 20 SNP loci at the 1000th generation, fulfilling the requirement of belonging to the same strain. The presence of genes associated with cell adhesion and the absence of resistance genes supported the probiotic characteristics of the strain. The data obtained in this study provide insights into broad-spectrum carbohydrate utilization, genomic stability, and probiotic properties of YLGB-1496, which provide theoretical support to promote the use of YLGB-1496.


Introduction
Probiotic development and consumption have increased globally in recent decades because of awareness of the beneficial effects their use has on promoting gut and overall health [1].Probiotics are living bacteria that, when consumed in sufficient quantities, provide health benefits to the host [2].The Lactobacillus group and Bifidobacterium genus are probiotic microorganisms that are most often utilized [3].The Bifidobacteria genus is Gram-positive, nonmotile, non-sporulating, heterofermentative, obligate anaerobes that are commonly found in the gastrointestinal tract, breast milk, and vagina [4,5].The genus contains approximately 50 species, of which only 10 are found in humans.Some strains of the Bifidobacterium genus are thought to benefit the host by stimulating and modulating both innate and adaptive host immune responses, reducing the inflammatory response, and degrading diet-derived carbohydrates [6].
As an important member of the bifidobacteria, Bifidobacterium longum (B.longum)species are classified into four subspecies: B. longum subsp.longum, B. longum subsp.suis, B. longum subsp.suillum, and B. longum subsp.infantis (B.infantis) [7].The fact that members of B. infantis have been found in breast milk, infant guts, and breastfed newborn feces suggests that they have a good impact on human health [4,5].It has been shown that five gene clusters in B. infantis may efficiently employ certain human milk oligosaccharides (HMOs) for intracellular metabolism and transport [8,9].In addition, B. infantis has a large number of glycosyl hydrolases (GH), which are hydrolases that metabolize oligosaccharides originating from plants or milk, including indigestible ones like galactooligosaccharides and fructooligosaccharides [10].B. infantis can effectively collect preferred carbon sources in the competitive ecosystem of infants' guts thanks to these transporter-dependent intracellular consumption methods, demonstrating the irreplaceable probiotic role of this strain in the host-microbe co-evolution of infants [11,12].
B. infantis R0033 [13], B. infantis ATCC15697 [9], B. infantis EVC001 [14], B. infantis CECT7210 [15], B. infantis 35624 [16], and B. infantis BI-G201 [17] are mainly used in infant-related foods, such as infant supplemented formula, solid beverages, and probiotic dietary supplements.It is crucial to employ B. infantis in milk-borne molecules that lack a nutritive value to the neonate to utilize HMOs, and it is clear that some Bifidobacterium animalis (B.animalis) strains lack this capacity [13].For a commercial strain, the ability to maintain probiotic properties and genetic stability is the primary condition and criterion for industrialization.The sequencing of B. infantis is essential since it is an attractive candidate for use in infant probiotics [18,19].Whether the strain can be transferred from laboratory to industrial production depends greatly on the strain's continuous-subculture stability [20].
This study aimed to clarify the carbohydrate utilization and genetic stability of Bifidobacterium longum subsp.infantis YLGB-1496 (YLGB-1496) by analyzing its genome and consecutive subcultures; we isolated YLGB-1496 with prospective uses for its cellfree culture supernatant, which exhibited strong oxidation resistance and enhanced skin barrier function [2].We sequenced the whole genome of YLGB-1496 (China Center for Type Culture Collection: No. M2011122) and compared it with the genomes of other B. infantis strains to understand its carbohydrate utilization capacity and probiotic characteristics.Using next-generation technologies, we performed deep sequencing on wild YLGB-1496 and consecutive subcultures to look for low-frequency variants and to evaluate genome stability.

Bacterial Growth Conditions
YLGB-1496 was initially isolated from human breast milk by our team, and the isolate was stored in cryoprotectant (12% (w/v) skim milk containing 10% glycerol) at −80 • C. YLGB-1496 was propagated twice in de Man, Rogosa, and Sharpe (MRS) broth (Oxoid Ltd., Beijing, China) at 37 • C overnight in an anaerobic incubator.

Carb Trials
The fermentation substrate concerning the mono/oligosaccharide range was determined with the API 20A assay (Biomérieux, Marcy l'Etoile, France) [24].Active strain cultures were washed twice with sterile physiological saline (0.9% w/v, NaCl), and pellets were resuspended in API 20A medium (BioMérieux, SA, Marcy l'Etoile, France).Homogenized cell suspensions were transferred into 50 wells of the API 50 CH strips using sterile Pasteur pipettes.All wells were overlaid with sterile mineral oil (Sigma, St. Louis, MO, USA) to effect anaerobiosis.Strips were moistened and incubated at 37 • C for 24-48 h.
Further validation of specific sugar sources.The strain was inoculated at 1 × 10 7 CFU/mL in an anaerobic atmosphere in a chamber in MRS containing 0.5% of a carbohydrate carbon source supplemented with 0.05% (w/v) L-cysteine hydrochloride.All incubations were carried out at 37 • C and in quadruplicate.We considered that the strain grows on the tested sugar when the turbidity at 600 nm reaches more than 0.5.The blank is represented by MRS without bacteria.

Continuous Subculture of YLGB-1496
The strain was injected in MRS broth at 2% (v/v) and continuously incubated for up to 1000th generation.Twelve hours was defined as one continuous cycle of culture.According to the strain's growth curve, the number of bacteria increases approximately 50-fold at 2% inoculum, with log 2 50 ≈ 5.64 generations of growth (i.e., the number of times the bacteria divided) in each cycle [25].The absorbance of the culture solution at 600 nm was measured every 200 generations using a UV-2800A spectrophotometer (Unico Instrument Co., Ltd., Beijing, China).At the same time, colonies were observed for morphology, size, and color.All experiments were performed in three biological replicates.

Viable Microbial Counts
The count of strain was determined by counting colonies on MRS agar supplemented with 0.05% (w/v) L-cysteine hydrochloride in an anaerobic atmosphere in a chamber at 37 • C, as described previously [21,23].The number of viable cells per gram was counted and expressed as lg CFU/mL.

Scanning Electron Microscopy (SEM) Analysis of YLGB-1496
The strain-normal culture or continuous subculture was harvested via centrifugation at 1000× g for 10 min.After washing twice with 10 mmol/L phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4), cell pellets were resuspended to 1 × 10 5 CFU.Bacterial pellets were then fixed overnight in 500 mL of 2.5% (v/v) glutaraldehyde in PBS at 4 • C. Following that, the bacteria were rinsed twice with PBS and dehydrated for 15 min in a graded ethanol series (50%, 70%, 90%, and 100%).The samples were then transferred for 20 min to a mixture (1.1, v/v) of ethanol, tertiary butanol, and pure tertiary butanol.After lyophilization and gold coating, the specimens were observed using SEM (Hitachi SU70, Tokyo, Japan).

Determination of the Fermentation Viability
The milk fermentation medium was reconstituted skim milk powder (10% (w/v); Fonterra TM , Auckland, New Zealand) with glucose (2% (w/v)).The medium was sterilized by heating for 15 min at 115 • C and inoculated with 1 × 10 7 CFU/mL of strain, followed by incubation at 37 • C for 24 h.The milk fermentation viability of the strain was determined according to the China National Standard GB 5009.239-2016, as described previously [26].One viability unit (U) was calculated as 1 µmol of lactic acid per 1 × 10 7 CFU of bacterial cells fermenting 1 mL of milk under the above conditions.

Statistical Analyses
The data were log2 transformed before analysis.Cluster analysis of YLGB-1496 was performed using IBM SPSS statistics.Each experiment was performed in triplicate.Statistical analyses were performed using SPSS13.0software (SPSS Inc, Chicago, IL, USA).All results are expressed as a mean ± standard deviation (SD).Significant differences between the sample means were determined at p < 0.05 using a Tukey's test.

Genome Sequences of YLGB-1496
According to the findings of this study, the genome of YLGB-1496 consists of single circular chromosomes of 2,758,242 bp and has no plasmid (Figure 1).The GC content was 59.87%, which was comparable to that of other strains of B. infantis [4,9].The genome contains 2442 predicted coding sequences (CDSs) and 64 structural RNAs (6 rRNA and 58 tRNA).No active phages were found by PHAge in the YLGB-1496 genomic sequences (Table 1).

Genome Sequences of YLGB-1496
According to the findings of this study, the genome of YLGB-1496 consists of single circular chromosomes of 2,758,242 bp and has no plasmid (Figure 1).The GC content was 59.87%, which was comparable to that of other strains of B. infantis [4,9].The genome contains 2442 predicted coding sequences (CDSs) and 64 structural RNAs (6 rRNA and 58 tRNA).No active phages were found by PHAge in the YLGB-1496 genomic sequences (Table 1).From outside to inside, the outermost circle of the circle diagram was the identification of genome size.The second and third circles were the CDS on the positive and negative strands, with various colors indicating different COG functional classes.The fourth circle was the rRNAs and tRNAs.The fifth circle was the GC content, and the outward red portion indicates that the GC content of the region was higher than the average GC content of the whole genome, while the inward blue part indicates that the GC content of the region was lower than the genome-wide average GC content, and higher peaks indicate a larger difference from the average GC content.The innermost circle was the GC-Skew value, and the specific algorithm is G-C/G+C, which could help to determine the leading and trailing strand; generally, the leading strand GC skew > 0 and the lagging strand GC skew < 0, and also assist in determining the replication start point (cumulative offset minimum) and the end point (cumulative offset maximum), which was especially important for the circular genome.The fourth circle was the rRNAs and tRNAs.The fifth circle was the GC content, and the outward red portion indicates that the GC content of the region was higher than the average GC content of the whole genome, while the inward blue part indicates that the GC content of the region was lower than the genome-wide average GC content, and higher peaks indicate a larger difference from the average GC content.The innermost circle was the GC-Skew value, and the specific algorithm is G-C/G+C, which could help to determine the leading and trailing strand; generally, the leading strand GC skew > 0 and the lagging strand GC skew < 0, and also assist in determining the replication start point (cumulative offset minimum) and the end point (cumulative offset maximum), which was especially important for the circular genome.

Phylogenetic Analysis of YLGB-1496
The genome-wide comparative analysis of selected strains from different Bifidobacterium species strains represents phylogenomic data (Figure 2A).The phylogenomic analysis shows that within the Bifidobacterium genus, YLGB-1496 is most closely related to B. infantis ATCC 15697, which was isolated from an infant's feces [40].It indicated that YLGB-1496 had some similarities to strains reported in the literature.To detect strain boundaries, pairwise genome comparisons were performed using average nucleotide identity (ANI) metrics (Figure 2B); the ANI values of all strains were between 83.56% and 99.90%, and it evidenced the presence of three distinct clades: one represented by B. animalis A6, another represented by B. infantis KCTC5934 and two B. longum JCM1217 and KCTC5934, and the third clades comprising the remaining nine B. infantis genomes, including YLGB-1496 (Figure 2B).However, a more in-depth study of the genomes of such isolates is required to better discover the genetic differences that lead to carbohydrate utilization characteristics.

Genetic and Phenotypic Data for Carbohydrate Metabolism
The protein sequences of the YLGB-1496 genome were subjected to an orthology prediction using OrthoMCL, with thresholds of E-Value: 1e-5, percent identity cutoff: 0, and Markov inflation index: 1.5.Compared to the other seven B. infantis strains, YLGB-1496 had the highest number of genes clustered to COG (Table 1).The 1896 genes in the YLGB-1496 genome were mapped to the bacterial COG and KEGG databases [41,42] to assess the main functional COG categories associated with the genome (Table S1).The major fraction of ortho groups (23.02%) were grouped under the category of "unknown func-

Genetic and Phenotypic Data for Carbohydrate Metabolism
The protein sequences of the YLGB-1496 genome were subjected to an orthology prediction using OrthoMCL, with thresholds of E-Value: 1e-5, percent identity cutoff: 0, and Markov inflation index: 1.5.Compared to the other seven B. infantis strains, YLGB-1496 had the highest number of genes clustered to COG (Table 1).The 1896 genes in the YLGB-1496 genome were mapped to the bacterial COG and KEGG databases [41,42] to assess the main functional COG categories associated with the genome (Table S1).The major fraction of ortho groups (23.02%) were grouped under the category of "unknown function" (S) (Figure 3A), this assignment indicating the need for additional advances in functional gene prediction.However, 13.08% of the OGs were classified as "carbohydrate transport and metabolism" (G), which showed that the ability to utilize a wide range of carbohydrates is an important attribute of the organism (Figure 3A).Further analysis of the G category genes (209 genes) through the Cazy database revealed that of the 74 genes mapped to the Cazy database, GHs (56.76%) were the most abundant, followed by glycosyl transferases (22.97%) and carbohydrate esterases (16.22%) (Figure 3B).The GH13 family was the most abundant glycoside hydrolase in YLGB-1496, followed by G11, G20, and G25.Furthermore, the GH36, GH42, GH51, and GH77 families were discovered to be specifically present in Bifidobacterium (Figure 3C).
We compared the glycoside hydrolase genes in YLGB-1496 and seven B. infantis strains (Figure 4, Table S2).The largest number of genes from the GH13 family were found in YLGB-1496, of which α-amylase, α-glucosidase, and α-1,4-glucan:maltose -1-phosphate maltosyltransferase are typical representatives; within the GH20 family, none of the six B. infantis strains has a β-hexosaminidase; LysM peptidoglycan-binding-domain-containing protein in the GH25 family involved in the degradation of bovine lactose macropeptides [43] was also found in B. infantis GRPT and B. infantis BB02.Nine strains included the GH36 and GH42 families of genes, but none of the seven B. infantis strains included inositol 2-dehydrogenase (Figure 4, Table S2).
We further analyzed carbohydrate metabolism pathways and found that eight B. infantis strains shared the same Leloir and fermentative pathway genes necessary to break down glucose and galactose through the bifid shunt (Table S3).It is also worth noting that a gene cluster was identified in YLGB-1496 containing a fucP (gene2316), lacZ (gene2344), fucA (gene2346), fucU (gene2347), and fucD gene (gene2350) (Figure 5A).Fucose-utilizing genes are conserved in all tested B. infantis genomes, all having the necessary genes to produce 1,2-propanediol (1,2-PD) (Table S4).The main products of both 2 ′ -fucosyllactose (2 ′ -FL) and lactose fermentation can be predicted as acetate, formate, pyruvate, lactate, and 1,2-PD in 2 ′ -FL fermentation.We examined the ability of YLGB-1496 to metabolize 20 carbohydrate substrates verified by a single sugar source.In addition to the basic hexoses and pentoses, the diversity of glycoside hydrolases allows them to utilize a wide range of oligosaccharides, such as breast milk, oligolactose, or oligogalactose (Figure 5B).

Morphology during YLGB-1496 Continuous Subculture
In consecutive subcultures of the 0th, 200th, 400th, 600th, 800th, and 1000th generations, the colonies of the strain had consistent morphology and were white or yellowish and circular.The colonies were 1-3 mm in diameter, with smooth, raised surfaces and tidy borders (Figure S1A).The morphology was curved or rod-shaped structures that were irregularly arranged (Figure S1B).The colony and morphology of the strain showed the characteristic morphology of Bifidobacterium and did not vary significantly across 1000 generations.The microscopic morphology of the strain during successive subcultures was observed through the use of SEM (Figure 6).The results showed that the wild strain had a typical rod-like shape with regular arrangement, as well as partial cell wall perforation and fracture.There was no flagellum or pod membrane on the surface of the bacterium (Figure 6C-0).As the continuous subculture progressed to 1000 generations, the surface and edges of the bacterium gradually became smooth and clear, and the number of perforated and fractured cell walls gradually decreased (Figure 6C-200, 400, 600, and 800), indicating that the morphology of the strain gradually entered a stable state during the continuous subculture up to 1000th generation.
Genes 2024, 15, x FOR PEER REVIEW 8 of 18 genes are conserved in all tested B. infantis genomes, all having the necessary genes to produce 1,2-propanediol (1,2-PD) (Table S4).The main products of both 2′-fucosyllactose (2′-FL) and lactose fermentation can be predicted as acetate, formate, pyruvate, lactate, and 1,2-PD in 2′-FL fermentation.We examined the ability of YLGB-1496 to metabolize 20 carbohydrate substrates verified by a single sugar source.In addition to the basic hexoses and pentoses, the diversity of glycoside hydrolases allows them to utilize a wide range of oligosaccharides, such as breast milk, oligolactose, or oligogalactose (Figure 5B).

Growth Characteristics during YLGB-1496 Continuous Subculture
The growth characteristics of the strain in continuous subculture for 1000 generations are shown in Table 2.The OD600, pH value, and culturable bacteria number of the strain did not change, and the fermentative activity remained largely unchanged at around 60.17 U (p > 0.05) in the continuous subculture, indicating that the strain's overall stability was good and that continuous subculture did not affect the strain's growth or fermentation.

Growth Characteristics during YLGB-1496 Continuous Subculture
The growth characteristics of the strain in continuous subculture for 1000 generations are shown in Table 2.The OD 600 , pH value, and culturable bacteria number of the strain did not change, and the fermentative activity remained largely unchanged at around 60.17 U (p > 0.05) in the continuous subculture, indicating that the strain's overall stability was good and that continuous subculture did not affect the strain's growth or fermentation.
Table 2.The table shows the growth characteristics during YLGB-1496 continuous subculture.One viability unit (U) was calculated as 1 µmol of lactic acid per 1 × 10 7 CFU of bacterial cells fermenting 1 mL of milk under the above conditions.Tests were performed in triplicate, and the results are expressed as mean ± standard deviation (SD).Analysis of variance (ANOVA) was performed using Tukey's range test for pairwise comparison.

Stability of Genetic Information
Consecutive subcultures of strain were carried out to compare gene single-nucleotide polymorphism (SNP) analysis via deep sequencing (approx.1000-fold data coverage).YLGB-1496 had 14 SNP loci at 200 and 400 generations, 18 at 600 and 800 generations, and 20 at 1000 generations, along with two insertional mutations and one deletion (Table 3).The findings demonstrated that the genetic makeup of the strain remained constant throughout 1000 successive generations and satisfied the requirements for belonging to the same strain [44].

Genetic Data for Extracellular Proteins Involved in Adhesion
YLGB-1496 still had its unique adhesion properties.Compared to the nearest related B. infantis ATCC 15697, twelve genes, namely, the six flp pilus and four sortase genes, as well as a gene for one tight-adherence protein and the outer-membrane-specific lipoprotein, were identified in YLGB-1496 as being responsible for the probiotic properties of cell adhesion (Table S5).The predicted and functionally annotated secretory (Sec) pathway secreted lipoproteins were found in YLGB-1496, and the results are shown in Table 4.The analysis showed that there were seven Sec pathway-secreted proteins, seven of which contained transmembrane helix structures.Seven of the seventeen genes are involved in amino acid transport and metabolism, seven in carbohydrate transport and metabolism, and three in intracellular trafficking, secretion, and vesicular transport (Table 4).We analyzed the whole genome of YLGB-1496, and we found no evidence of any genes for drug resistance to any of the antibiotics tested, including aztreonam, cycloserin, polymyxin B, mupirocin, spectinomycin, kanamycin, gentamicin, streptomycin, doxorubicin B, nalidixic acid, barongomycin, neomycin, tetracycline, and erythromycin.The result demonstrates that YLGB-1496 does not pose a risk for antimicrobial resistance or heteroresistance.Because of the possible impact on human health, Bifidobacterium spp.bacteria should not be allowed to have genes for antimicrobial resistance [45,46].The ability to pose a risk for antimicrobial resistance or heteroresistance must be considered an important parameter for selecting probiotic strains.

Discussion
ANI values are based on the pairwise alignment of genomic segments; ANI provides more robust results when the pair of genomes compared share a high degree of similarity (ANI > 90%), an ANI of 95% to 96%, which corresponds to 98.65% 16S rRNA gene sequence similarity, is widely accepted as a threshold for species demarcation [47].According to the ANI values of 98.40-99.90%for YLGB-1496 and B. infantis BB02, ATCC15697, JRPT, NLS, PI007, JCM7009, and BT1, YLGB-1496 is a member of the B. infantis species, and there is little diversity within it.
Glycoside hydrolases, widely distributed enzymes that catalyze the cleavage of glycosidic bonds in sugar or sugar conjugates, are considered to be catabolic enzymes that play key roles in carbohydrate metabolism.The GH13 family consists of glycoside hydrolases present in humans, in which the main substrate is an amylopectin-like polysaccharide.The widespread presence of this enzyme in the intestinal flora is indicative of the importance of amylopectins to the intestinal flora [48].The most common function of GH20 family glycoside hydrolases is lactose-N-biosidase activity, which acts mainly on the conversion of lactose tetrasaccharides to lactose-N-disaccharides and lactose in HMOs [9,49].According to research on B. infantis, GH36 and GH42 family glycoside hydrolases typically have α-galactosidase or β-galactosidase activities, working mostly on the decreasing terminal galactose of polysaccharides and oligosaccharides [50].Inositol 2-dehydrogenase encourages the hydrolysis of food-derived inositol in the gut and breast milk, which accelerates preterm babies' bronchopulmonary growth and organ maturation [51].YLGB-1496 possesses a large number of glycoside hydrolase genes, suggesting that it can utilize carbohydrates and may improve infant intestinal health if added to infant foods.
Human milk oligosaccharides (HMOs) are the third most abundant solid component in human milk.The core structures of HMOs consist of Gal, Glc, and GlcNAc, with the core frequently decorated with Fuc and N-acetylneuraminic acid (Neu5Ac) [52].In addition to providing direct nutrition to the infant, HMOs primarily act as specific substrates for sustaining the growth of selected beneficial bacteria that aid in the development and protection of the newborn [53].The gene clusters involved in the import and processing of HMOs were identified in B. infantis; the main enzymes encoded by the gene clusters are sialidase, fucosidase, N-acetyl-β-hexosaminidase, and β-galactosidase.Sialidases (EC 3.2.1.18)from B. infantis, which belong to GH33, act on both α-2,3 and α-2,6 linkages HMOs; it has been shown to liberate Neu5Ac from mucin glycoproteins [54,55].Growing on HMO induces fucosidase activity; the enzyme acts on Fuc residue bound to GlcNAc [53].Therefore, the novel gene cluster from YLGB-1496 may have been dedicated to co-regulation and participation in HMOs metabolic.
The fermentation activity of the strain is an important indicator for evaluating its fermentation performance, and its stability is essential for developing direct-vat-starter (DVS) starters [17,56].The fermentative activity of strains is rapidly decreased by unfavorable environmental conditions, which impacts the fermentate's stability and shelf life [57].The periodic detection of changes in the acid-producing viability of strains during continuous subculture is an important task in strain stability research and has practical applications [31,58].The fermentation activity of YLGB-1496 in continuous subculture was 59.3-60.70U, indicating that the strain's overall stability was good and that continuous subculture did not affect the strain's ability in terms of fermentation.
SNP analysis is a crucial technique for evaluating differences in the genetic information of strains [59,60].In comparison to more established methods like the 16S rRNA gene [61], pheS/atpD functional gene [62], ANI [47], and random amplified polymorphic DNA (RAPD) [63], SNP analysis has become an indispensable tool for studying genetic variation by detecting gene sequence and structural variation with its low-cost, time-consuming, and high-throughput characteristics [64,65].The established standard for homogenous strains is a strain having less than 21 SNP loci during subsequent subcultures [44].In continuous culturing for up to 1000 generations, YLGB-1496 had 20 SNP loci; this result is consistent with previous reports of LAB.There is a similarity in genomic comparisons of Bifidobacterium bifidum BGN4, B. longum JDM301, and Lactobacillus brevis KB 290, with the number of SNPs in consecutive cultures being less than 21 [10,31,66].
Type II secretion system represents the major pathway for exoprotein transport from the periplasm across the outer membrane in a wide variety of bacteria.Type IV pili are hair-like appendages of the proteinaceous mass that are primarily involved in adhesion, DNA uptake, motility, biofilm formation, and protein secretion and are essential for adhesion to host cells and molecules [67,68].Typically, the Sec pathway or the twin-arginine translocation (TAT) pathway is used to deliver proteins to the cell exterior [69].The Sec pathway recognizes unfolded protein targets and contains an N-terminal leader peptide, a hydrophobic core, and a C-terminal sequence that facilitates the binding of the Sec machinery.These proteins are either exported from the cell, or their N terminus anchors them to the membrane, depending on the peptide sequence in the C-terminal region [1,70].LPXTGanchored proteins are covalently bound to cell wall peptidoglycan after Sec targeting by the housekeeping sorting enzyme, sortase A (SrtA) enzyme.Sortase-dependent proteins are divided into four classes, of which class C sortases are transpeptidases and play a critical role in pilus assembly [71,72].The genome of YLGB-1496 contains 17 Sec pathway-secreted proteins; these lipoproteins were located on the surface of the bacterial cells and could interact directly with the external environment, acting as adhesion agents.
Bifidobacteria is naturally resistant to mupirocin by expressing a specific isoleucyl-tRNA synthetase gene (ileS).This leads to losses in the competitive inhibition of mupirocin, which reverts the bacterium's ability to synthesize proteins [46].Tetracycline-and macrolideresistant genes predominate among the resistance genes carried by bifidobacteria, e.g., erm (X), a gene encoding a ribosomal protection protein located on transposon Tn5432 in B. animalis, mediates its resistance to macrolides [73]; Tetracycline resistance genes such as tet (W), tet (M) and tet (O) were detected in B. longum, Bifidobacterium brevis and B. animalis, with tet (W) being the most prevalent.Mutations in the rpsL gene encoding the ribosomal subunit protein S12 in bifidobacteria can lead to high levels of resistance to streptomycin [74].Most bifidobacteria are insensitive to aminoglycosides, which is attributed to the lack of cytochrome-mediated drug transport.No antibiotic resistance genes, including mupirocin and tetracycline, were detected in the whole genome of YLGB-1496, suggesting that it does not pose a risk of antimicrobial resistance or heteroresistance.

Conclusions
In this work, we showed that YLGB-1496 exhibits extensive carbohydrate utilization capacity and genetic stability through whole-genome sequencing based on Illumina and PacBio sequencing.An omics-based comparison of B. infantis revealed the presence of a wide range of glycoside hydrolase genes, with the GH13, 20, 25 family, and inositol 2-dehydrogenase being typical representatives.The 1000th-generation strain exhibited a stable fermentation activity of 60.17 U; the morphology was curved or rod-shaped, regularly arranged and well-defined, with intact cell walls that were not perforated or broken.At the 1000th generation, there were 20 SNP loci meeting the criteria of belonging to the same strain.The data obtained in this study provide insights into broad-spectrum carbohydrate utilization, genomic stability, and probiotic properties, which may enhance the industrial applications of this species.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes15040466/s1, Figure S1: Clones and morphology of YLGB-1496 in the continuous subculture; Table S1: Genes and primers used in this work; Table S2: Gnens of carbohydrate transport and metabolism of seven B. infantis strains; Table S3: Glycoside hydrolase specific genes of YLGB-1496; Table S4: Annotated genes related to 2 ′ -FL utilization in eight B. infantis strains.Used in conjunction with Figure 5A to describe genes needed to make quantified metabolites.Included are gene name, KEGG enzyme class and pathway the gene is located in.presence in other B. infantis strains is noted; Table S5: Genes predicted to be involved in adhesion in YLGB-1496.
Author Contributions: Project administration, supervision and writing-review and editing, R.W.; investigation and writing-original draft preparation, X.L.; formal analysis, J.Y.; visualization and date curation, S.S.; methodology, H.L.; conceptualization, W.Z.; software, resources & Project administration, W.H. and J.H.All authors have read and agreed to the published version of the manuscript.

Figure 1 .
Figure 1.The figure shows a circular map of Bifidobacterium longum subsp.infantis YLGB-1496 (YLGB-1496).From outside to inside, the outermost circle of the circle diagram was the identification of genome size.The second and third circles were the CDS on the positive and negative strands, with various colors indicating different COG functional classes.The fourth circle was the rRNAs and tRNAs.The fifth circle was the GC content, and the outward red portion indicates that the GC content of the region was higher than the average GC content of the whole genome, while the inward blue part indicates that the GC content of the region was lower than the genome-wide average GC content, and higher peaks indicate a larger difference from the average GC content.The innermost circle was the GC-Skew value, and the specific algorithm is G-C/G+C, which could help to determine the leading and trailing strand; generally, the leading strand GC skew > 0 and the lagging strand GC skew < 0, and also assist in determining the replication start point (cumulative offset minimum) and the end point (cumulative offset maximum), which was especially important for the circular genome.

Figure 1 .
Figure 1.The figure shows a circular map of Bifidobacterium longum subsp.infantis YLGB-1496 (YLGB-1496).From outside to inside, the outermost circle of the circle diagram was the identification of genome size.The second and third circles were the CDS on the positive and negative strands, with various colors indicating different COG functional classes.The fourth circle was the rRNAs and tRNAs.The fifth circle was the GC content, and the outward red portion indicates that the GC content of the region was higher than the average GC content of the whole genome, while the inward blue part indicates that the GC content of the region was lower than the genome-wide average GC content, and higher peaks indicate a larger difference from the average GC content.The innermost circle was the GC-Skew value, and the specific algorithm is G-C/G+C, which could help to determine the leading and trailing strand; generally, the leading strand GC skew > 0 and the lagging strand GC skew < 0, and also assist in determining the replication start point (cumulative offset minimum) and the end point (cumulative offset maximum), which was especially important for the circular genome.

Figure 3 .
Figure 3.The figure shows the genetic data for carbohydrate metabolism.(A) COG function classification of genes for YLGB-1496.The genome was mapped to the bacterial COGs and KEGG database to evaluate the main functional COG categories related to the genome.(B) Carbohydrateactive enzymes classification of eight B. infantis strains.The genes of the eight B. infantis strains were comparatively analyzed using Diamond and hmmscan software after annotation through the CAZy Database (http://www.cazy.org/,accessed on 1 January 2024) (E-value ≤ 10 −5 ).PL: Polysaccharide Lyases; GT: Glycosyl Transferases; GH: Glycoside Hydrolases; CE: Carbohydrate Esterases; CBM: Carbohydrate-Binding Modules; AA: Auxiliary Activities.(C) Glycoside hydrolases family of eight B. infantis strains.

Figure 3 .
Figure 3.The figure shows the genetic data for carbohydrate metabolism.(A) COG function classification of genes for YLGB-1496.The genome was mapped to the bacterial COGs and KEGG database to evaluate the main functional COG categories related to the genome.(B) Carbohydrate-active enzymes classification of eight B. infantis strains.The genes of the eight B. infantis strains were comparatively analyzed using Diamond and hmmscan software after annotation through the CAZy Database (http://www.cazy.org/)(E-value ≤ 10 −5 ).PL: Polysaccharide Lyases; GT: Glycosyl Transferases; GH: Glycoside Hydrolases; CE: Carbohydrate Esterases; CBM: Carbohydrate-Binding Modules; AA: Auxiliary Activities.(C) Glycoside hydrolases family of eight B. infantis strains.

Figure 6 .
Figure 6.The figure shows the morphology of YLGB-1496 in the continuous subculture.The samples were prepared using ethanol gradient drying, and the surface morphology of the strain was observed using SEM at a magnification of 5000× in the 2 µm range.Red arrows indicate cell wall perforation or fracture.

Figure 6 .
Figure 6.The figure shows the morphology of YLGB-1496 in the continuous subculture.The samples were prepared using ethanol gradient drying, and the surface morphology of the strain was observed using SEM at a magnification of 5000× in the 2 µm range.Red arrows indicate cell wall perforation or fracture.

Funding:
This work was supported by National Center of Technology Innovation for Dairy (No. 2020-Ke Ji Xing Meng-National Innovation Center-2 project).

Table 1 .
The table shows the general genome feature comparison of seven Bifidobacterium longum subsp.infantis (B.infantis) strains.Genomic assembly statistics, along with subsystems (annotated from COG and KEGG), are displayed.The genome sequences of seven B. infantis strains were downloaded from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/datasets/taxonomy/1682/, accessed on 1 January 2024).A comparison of genes involved in metabolism in the genomic data of the eight strains was performed using Diamond software (E-value ≤ 10 −5 ).

Table 3 .
The table shows the genomic mutation information of YLGB-1496 with different generations.

Table 4 .
The table shows predictive analysis of lipoprotein secretion by the YLGB-1496 secretory (Sec) pathway.