Comparative Genomics of Transporter Proteins in Lactic Acid Bacteria

Abstract

Although lactic acid bacteria (LABs) possess unique metabolic and physiological characteristics that have crucial effects on the transport of substances both into and out of the cell, there is still a lack of systematic research on membrane transporters in LABs and their roles in material transport. In this study, genomic data for the species Lactobacillus delbrueckii, Streptococcus thermophilus, Leuconostoc lactis, Pediococcus lactis, Lactococcus garvieae, and Bifidobacterium lactis were analyzed to identify the associated transport systems, including what kind of substances are transported. As part of a comparative genomics approach, we used the G-BLAST and AveHAS programs in the TCDB database to screen for transport proteins and clarify the distribution of these proteins in different Lactobacillus strains, allowing for further prediction of their transport substrates. Studies have shown that the distributions of these transporters differ among the selected LAB strains. Through screening and tabulation, we found that the content of transporters in the six LAB proteomes was greater than 20%, with the dominance of the large transporter group indicating complex metabolic and probiotic effects. Furthermore, it was found that the LAB strains contain a variety of homologs of drug-efflux proteins, which may make them resistant to antibiotics, as well as a large number of toxin-related transporters. This study allowed for reasonable predictions of the roles of toxin-related proteins in LABs, and further research on these proteins may be valuable for understanding the probiotic effects of LABs that arise through competition. The study of LAB transporters and the prediction of their functions might support a better understanding of the metabolic and physiological activities of these bacteria. In the future, we aim to extract DNA from laboratory strains and perform PCR amplification using suitable primers designed by us. Through comparison of the obtained gene sequences with those reported in this study, we can explore the differences among them.

1. Introduction

Lactic acid bacteria (LABs) is a collective term for a group of probiotic bacteria that can produce large amounts of lactic acid [1]. They usually use carbohydrates as their main carbon source, are Gram-positive, and are facultative anaerobes that typically exist in anaerobic or microaerobic environments. Their optimal growth temperatures range from 30 °C to 40 °C, with a relatively low required temperature, and they can be inactivated by high-temperature treatment. They present optimal growth at pH values in the range of 5.5 to 5.8 [2]. To date, more than 10 genera and 200 species of LABs have been discovered in nature. LABs are usually cocci or bacilli and are acid-tolerant. In the glycolytic pathway, LABs ferment sugars to produce pyruvic acid, which is then reduced to lactic acid via catalysis by lactate dehydrogenase [3].

LABs, as essential fermentation strains in food production, possess several significant metabolic characteristics, such as acid production, aroma hydrolysis of proteins, production of viscous exopolysaccharides, and bacteriostasis. At the same time, as probiotics, LABs have significant probiotic functions, which are mainly distributed among three categories: intestinal functions, immune functions, and metabolic functions [4].

The intestinal functions of LABs include the prevention and treatment of diarrhea. Research [5] has indicated that diarrhea in infants can be prevented by giving formula milk supplemented with Lactobacillus reuteri or Lactococcus lactis. In addition, LABs can prevent lactose intolerance. Vincent et al. [6] expressed the β-galactosidase gene of Lactococcus lactis IL403 in Escherichia coli to achieve the decomposition of lactose. The transport of β-galactosidase by LABs involves transport proteins. In addition, LABs can also prevent common diseases of the digestive system such as peptic ulcers, the main causes of which are Helicobacter pylori infection and weakened protection by the gastric mucosa. In particular, Apostolidis et al. [7] found that lactic acid and phenolic substances produced by LAB fermentation can inhibit the growth and reproduction of Helicobacter pylori. LABs with a larger number of lactic acid and phenolic-substance transport proteins can play a stronger preventive role.

The immune functions of LABs include enhancement of immunity. Research [8] has shown that the new strain Lactobacillus rhamnosus KF5, which was isolated from the feces of healthy people, can produce extracellular polysaccharides in skim milk and enhance immune function. In this process, a transport protein involved in the export of extracellular polysaccharides plays an important role. In addition, LABs also exert antibacterial effects. The Lactobacillus plantarum strain NTU102 obtained by Lin et al. [9] was shown to inhibit the activity of various pathogenic bacteria. The antibacterial substance involved in this process is an ester. Therefore, ester transport proteins can affect the antibacterial activity of LABs. Finally, Bleau et al. [10] found that the extracellular polysaccharides produced by Lactobacillus rhamnosus RW-9595M, when transported into mice by transport proteins, can promote the production of the anti-inflammatory factor interleukin-10 by the mouse peritoneal macrophages, thus reducing inflammatory responses.

The metabolic functions of LABs include the reduction of cholesterol levels. Wilck et al. [11] found that taking LABs can ameliorate the hypertension caused by high cholesterol. In this process, tryptophan plays an important regulatory role; therefore, tryptophan transporters are also considered to be involved in such functions. In addition, LABs can improve indicators of type 2 diabetes. Chen et al. [12] found that supplementing type 2 diabetic mice with the CCFM0412 strain significantly reduced levels of reactive oxygen species, while levels of glutathione peroxidase, superoxide dismutase, and glutathione were significantly increased. In this process, the transporters of these substances play roles in regulating their concentrations inside and outside the cells. Moreover, LABs can also alleviate the symptoms of fatty liver disease. Kobyliak et al. [13] found that, under the influence of LABs, indicators of fatty liver significantly decreased. Finally, LABs reduce the risk of colorectal cancer. Previous research [14] has initially confirmed that the strains BL23 and STCRL807 can effectively alleviate the symptoms of colon cancer in mice by promoting the outward transport of antioxidant enzymes.

Cell metabolism is one of the foundational processes of life, and an important part of metabolism is material transport. Through material transport, cells can complete a variety of metabolic and signal-transduction activities [15]. Metabolic regulation in cells is achieved through material transport, and the continuous transport of metabolites is one of the foundations for maintaining life. Transporters play an important role in this whole process, as they can carry external information into internal contexts and regulate metabolic reactions [16]. Therefore, a comparative analysis of the differences in transporters can reflect some of the differences in the functions of LAB strains, thus benefiting future research.

Common genera used in food fermentation [17] include Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, and Enterococcus. Therefore, in this study, six LAB strains were selected: Lactobacillus delbrueckii, Streptococcus thermophilus, Streptococcus lactis, Lactobacillus acidophilus, Lactococcus lactis, and Bifidobacterium lactis. First, these six strains are widely used in the food industry at present and are considered the most commonly used food LABs. In addition, complete genomes and proteomes for these six strains are available in the NCBI database, and the availability of these data was conducive to the development of this study. Unlike previous studies, our research involved a systematic screening and analysis of the full set of transport proteins of the selected LABs. Moreover, we conducted interspecies comparisons, which will support better utilization of LABs and the selection of appropriate strains based on their capabilities. More specifically, we studied and analyzed the distributions of transport systems and substrates in order to identify the metabolic differences among these six LAB strains. The basic characteristics of these six LAB strains are presented in

Table 1. Basic information of the six LAB strains used in this study.

Genus	Bacteria	Genome Accession Number	Genome Size (Mbp)	Total Number of Proteins	Number of Transporters	Proportion of Transporters (%)
Lactobacillus	Lactobacillus delbrueckii	GCA_001888925.1	1.9	1630	447	27.4
Streptococcus	Stretpococcus thermophilus	GCA_903886475.1	1.8	2009	446	22.2
Leuconostoc	Leuconostoc lactis	GCA_007954605.1	1.8	1703	359	21.1
Pediococcus	Pediococcus acidilactici	GCA_013127755.1	2	1901	525	27.6
Lactococcus	Lactococcus garvieae	GCA_016026695.1	2.1	1926	516	26.8
Bifidobacterium	Bifidobacterium lactis	GCA_000022965.1	1.9	1518	414	27.3

.

2. Materials and Methods

2.1. Acquisition of the LAB Proteome

In this study, six strains were selected from the NCBI database based on their high-quality and complete genome sequences, and the genomic sequences of these six strains across different genera were collected. The chosen strains included Lactobacillus, Lactobacillus delbrueckii (GCA_001888925.1); Streptococcus, Streptococcus thermophilus (GCA_903886475.1); Leuconostoc, Leuconostoc lactis (GCA_007954605.1); Pediococcus, Pediococcus acidilactici (GCA_013127755.1); Lactococcus, Lactococcus garvieae (GCA_016026695.1); and Bifidobacterium, Bifidobacterium lactis (GCA_000022965.1).

2.2. Detection of Transport-Protein Homologs

The detection of proteins was conducted using the Transporter Classification Database (TCDB) (www.tcdb.org), and transporter homologs were identified using the database’s G-BLAST [16], which is a mature and stable tool. Moreover, the G-BLAST program is highly accurate and is capable of identifying homologs with high confidence. Simultaneously, while maintaining precision, it allows for rapid searches, which is essential for handling the extensive sequence dataset used in this study. G-BLAST utilizes protein sequences in FASTA format from genomic databases to search for homologous transport proteins within the TCDB. Subsequently, it retrieves information for the identified transport proteins—including TCID number, amino acid residue count (aas), predicted transmembrane segment count (TMS), TMS overlap between the query and hit transport proteins, and e-value—to indicate the degree of similarity between the input protein sequence and the identified proteins [18]. In order to predict the quantity of TMS, G-BLAST employs a web-based program known as WHAT, which analyzes hydrophilicity, amphipathicity, and topology. This program aligns hydrophobic and amphipathic plots based on the length of the proteins [19,20]. The WHAT program uses 19 aas, an α-helix window with a viewing angle of 100° and 9 aas, and a β-chain window with a viewing angle of 180° to determine the amphiphilicity and hydrophilicity of each protein [21]. Due to the presence in many multicomponent systems of soluble components that may potentially be homologous to transport proteins, proteins lacking TMS were still taken into consideration.

The initial threshold value for the G-BLAST search was set to an e-value of 0.0001, and proteins presenting a value less than or equal to this were identified as transporters. Proteins with an e-value greater than 0.0001 were manually examined using topological data in order to determine whether the protein was a transporter homolog. As the e-values for two proteins with similar hydrophilic regions are typically very small, it is necessary to manually check the overlapping regions in order to avoid selecting proteins that score highly but are not homologous in transmembrane domains. The findings of proteins with medium e-values (i.e., between 0.0001 and e⁻⁸) indicate that there may be a range of remote homologous proteins, which were examined more closely in the following steps. The WHAT program was used to generate a hydrophilic profile in order to determine whether the transmembrane segments were missed or the transmembrane segments in the error region were predicted. Utilizing the AveHAS program, we confirmed the predictions regarding homologous substances [20]. This program was developed based on the TREEMOMENT and Hydro programs and visualizes the average hydropathy, amphipathicity, and similarity within a multiple-sequence alignment. Transport proteins with low-score hits that were recognizable via examination were included in the TCDB.

2.3. Identification of Substrate Transporters

Based on the hit results for transporter homologs, the hit transporters were used to assign transport substrates to the transporter homologs. For hit transporters with unknown functions, their transport substrates were inferred according to the genomic background of the coding gene or the scientific literature.

3. Results and Discussion

3.1. Overview of Transporter Types

According to the TCDB, there are seven types of transporters, comprising five distinct types—(1) channels; (2) secondary carriers; (3) main active transporters; (4) group transporters; and (5) transmembrane electron-flow carriers—and two less-clear types: (8) auxiliary transporters and (9) putative transporters with unknown transport function or mechanism [21,22].

To analyze the distribution of transport proteins, G-BLAST was employed to conduct TCDB screening on the proteomes of six strains of LABs.

Table 2. Analysis of LAB transporters based on TC subclass.

TC Subclass and Description	Number of Transporters						%
	LD	ST	LL	PA	LG	BL	LD	ST	LL	PA	LG	BL
1.A: α-Type channels	15	16	13	27	25	23	3.4	3.6	3.6	5.1	4.8	5.6
1.B: β-Barrel porins	3	2	2	3	2	1	0.7	0.4	0.6	0.6	0.4	0.2
1.C: Pore-forming toxins (proteins and peptides)	7	8	6	8	6	6	1.6	1.8	1.7	1.5	1.2	1.4
1.E: Holins	0	2	2	7	8	3	0	0.4	0.6	1.3	1.6	0.7
1.I: Membrane-bounded channels	1	1	2	1	3	2	0.2	0.2	0.6	0.2	0.6	0.5
1.Q: Fungal septal pores	0	0	0	0	0	3	0	0	0	0	0	0.7
1.S: Bacterial micro/nanocompartment shell protein pores	0	0	0	3	0	0	0	0	0	0.6	0	0
1.W: Phage portal protein subclass	0	0	2	1	0	1	0	0	0.6	0.2	0	0.2
2.A: Porters (uniporters, symporters, antiporters)	77	88	73	122	99	74	17.2	19.7	20.3	23.2	19.2	17.9
2.C: Ion gradient-driven energizers	1	0	0	2	2	1	0.2	0	0	0.4	0.4	0.2
3.A: P-P bond hydrolysis-driven transporters	205	222	149	172	185	181	45.9	49.8	41.5	32.8	35.9	43.7
3.B: Decarboxylation-driven transporters	3	3	3	3	3	1	0.7	0.7	0.8	0.6	0.6	0.2
3.D: Oxidoreduction-driven transporters	5	3	5	6	11	8	1.1	0.7	1.4	1.1	2.1	1.9
3.E: Light absorption-driven transporters	4	5	3	5	4	3	0.9	1.1	0.8	1	0.8	0.7
4.A: Phosphotransfer-driven group translocators (PTS)	19	9	8	49	30	0	4.3	2	2.2	9.3	5.9	0
4.B: Nicotinamide ribonucleoside uptake transporters	0	1	1	1	2	0	0	0.2	0.3	0.2	0.4	0
4.C: Acyl CoA ligase-coupled transporters	5	4	6	4	8	7	1.1	0.9	1.7	0.8	1.6	1.7
4.D: Polysaccharide synthase/exporters	4	1	2	6	4	4	0.9	0.2	0.6	1.1	0.8	1
4.F: Choline/Ethanolamine phosphotransferase 1 (CEPT1)	1	1	1	1	1	3	0.2	0.2	0.3	0.2	0.2	0.7
4.H: Lysylphosphatidylglycerol synthase/flippases	0	1	1	0	1	2	0	0.2	0.3	0	0.2	0.5
5.A: Transmembrane 2-electron transfer carriers	0	0	0	1	0	0	0	0	0	0.2	0	0
5.B: Transmembrane 1-electron transfer carriers	2	2	4	1	12	2	0.4	0.4	1.1	0.2	2.3	0.5
8.A: Auxiliary transport proteins	27	15	18	35	34	29	6	3.4	5	6.7	6.6	7
9.A: Recognized transporters of unknown biochemical mechanism	8	6	5	3	10	5	1.8	1.3	1.4	0.6	1.9	1.2
9.B: Putative transport proteins	60	56	53	64	66	55	13.4	12.6	14.8	12.2	12.8	13.3
Total	447	446	359	525	516	414	100	100	100	100	100	100

Note: LD, L. delbrueckii; ST, S. thermophilus; LL, L. lactis; PA, P. acidilactici; LG, L. garvieae; BL, B. lactis.

summarizes the results and the distribution of transport-protein subclasses.

It is evident from Table 1 that P. acidilactici exhibited the greatest number of transporters, with a total of 525, while the number of transporters of B. lactis was the lowest (with only 359). Coincidentally, in terms of the proportion of transporters to total proteins, P. acidilactici also had the greatest proportion (27.6%) while L. lactis had the lowest (21.1%).

In terms of overall distribution, the numbers of P. acidilactici and L. garvieae transporters were relatively close (525 and 516, respectively); L. delbrueckii, S. thermophilus, and B. lactis were also close (447, 446, and 414, respectively). In terms of the proportion of transporters, the overall proportion ranged from 21.1% to 27.6; L. delbrueckii, P. acidilactici, L. garvieae, and B. lactis had very similar proportions (27.4%, 27.6%, 26.8%, and 27.3%, respectively), and the proportions of S. thermophilus and L. lactis were also similar (22.2% and 21.1%, respectively).

From Table 2, the following information can be derived.

TC subclass 1.A comprises α-type channels. This type of transmembrane channel protein is widely present in the membranes of various organisms, and members of this class typically facilitate the movement of solutes through aqueous transmembrane pores or channels, relying on energy-independent processes without the need for carrier-mediated transport. Although there may be β-chains that contribute to the channels, these channel proteins are usually composed mainly of α-helices. For members of this subgroup, the distribution in the six strains ranged from 13 to 27 proteins, with L. lactis having the fewest and P. acidilactici having the most. The proportions in the six strains ranged from 3.4% to 5.6%.

TC subclass 1.B comprises to β-barrel pores. This class of proteins typically forms transmembrane pores on the outer membrane of Gram-negative bacteria, thereby facilitating the passage of solutes [23,24]. Moreover, this type of protein has been identified in mitochondria, plastids, Gram-negative bacteria, and potentially acid-resistant Gram-positive bacteria. This may also be the reason why the number in the six strains was relatively small (only 1–3 proteins), as LABs are acid-resistant Gram-positive bacteria.

TC subclass 1.C comprises pore-forming toxins (PFTs). PFTs are the most prevalent bacterial cytotoxic proteins. Unexpectedly, the six strains carried six to eight such proteins. It is surprising that LABs, as probiotics, harbor such a large number of multidrug transporters. Notably, there is no research demonstrating a significant correlation between the quantity of toxic proteins and the pathogenic potential across different species, so the results relating to this subclass do not affect the probiotic status of these strains.

TC subclass 1.E comprises holins, which serve multiple functions in prokaryotes, including biofilm formation, lysis, virulence, and toxin release. Additionally, they may function as antibacterial agents [25]. For proteins in this subclass, the distribution across the six strains was similar (ranging from two to two proteins). The exception was L. delbrueckii, which had no transporter in this subclass.

TC subclass 1.I comprises the membrane-bound channels, which allow the transit of molecules across cellular or organellar membranes. For example, the nuclear pore complex (NPC;1.I.1) allows small molecules and macromolecules to flow between the nucleus and the cytoplasm. It has been reported that Planctomycetes bacteria possess nuclear-pore-like complexes. The distribution of this subclass in the six strains was similar, ranging from one to three proteins.

TC subclass 1.Q comprises fungal septal pores. Among the species studied, only B. lactis hadhad three proteins within this subclass. Single cells are connected to each other through the diaphragm hole, which closes when the cells are damaged. Many proteins that are thought to be septum-associated proteins (SPA) have been shown to be associated with these septa. Therefore, B. lactis may possess a diaphragm hole.

TC subclass 1.S comprises bacterial micro/nano composite shell protein pores [26], which constitute a class of protein organelles that perform specific metabolic functions. Metabolic enzymes are adsorbed by shell proteins, many of which are homologous and form oligomeric structures containing substrate-selective pores. Substrates/products/intermediates can pass through the pores, which confer higher permeability. This subclass was represented only in P. acidilactici, with three proteins.

TC subclass 1.W comprises the phage portal protein subclass [27]. Of the proteins in this subclass, L. lactis had two, while P. acidilactici and B. lactis each had one.

TC subclass 2.A, the porters, represents a large transport-system group in the selected LAB strains, with 17.2–23.2% of the transport proteins belonging to this category in each strain. These transport proteins are typically characterized as single-component systems. Strain P. acidilactici had 122 such proteins—the highest number among the six strains—while the other strains carried between 73 and 99 proteins of this type, notably fewer than P. acidilactici. This discrepancy may suggest that P. acidilactici exhibits superior metabolic activity.

The TC subclass 2.C comprises an ion gradient-driven energy organelle that appears on the outer membrane of Gram-negative bacteria. Therefore, the presence of a member of this subclass in the Gram-positive bacteria L. delbrueckii, P. acidilactici, L. garvieae, and B. lactis (1, 2, 1, 1) was quite unexpected.

TC subclass 3.A comprises transporters driven by P–P bond hydrolysis, which were the largest transporter subclass in the six strains. These transporters are usually multicomponent systems. S. thermophilus had the greatest number (222); L. lactis had the lowest number (149); and other strains had between 172 and 205.

TC subclass 3.B comprises transporters that are driven by decarboxylation processes, which are currently regarded as being exclusive to prokaryotic organisms. Transport systems drive solutes (e.g., ions) either into or out of the cell via decarboxylation of cytoplasmic substrates (generally Na⁺). These systems are additionally characterized by their multicomponent nature [28]. Five of the strains carried three such proteins, while only B. lactis carried only one. These Na⁺ pumps may be involved in ion homeostasis [29].

The TC subclass 3.D comprises oxidoreduction-driven transporters, which are present in many prokaryotes and in organelles in eukaryotes, such as mitochondria and chloroplasts. In bacteria, Brooijmans et al. [30] have demonstrated that L. lactis, facultative anaerobes, and other LABs that mainly depend on fermentation have functional electron transport chains. Of the proteins in this subclass, L. garvieae had the most (eleven), while the other five strains had between three and eight proteins.

TC subclass 3.E comprises transporters driven by light absorption. For the transporter proteins in this subclass, the numbers in the six strains were relatively close, with all strains carrying three to five proteins.

TC subclass 4.A comprises phosphate transfer-driven transporters, namely, those involved in the bacterial phosphoenolpyruvate:glucose phosphate transferase system (PTS). The reaction product originates from extracellular sugars and consists of cytoplasmic sugar phosphates. Consequently, these systems function exclusively as uptake mechanisms. The PTS [31] plays a role in the regulation of various physiological processes in bacteria, for example, cytoplasmic K⁺ concentration, induction of substance exclusion, nitrogen metabolism, transcriptional regulation through various mechanisms, cyclic AMP synthesis, formation of a biological membrane, and pathogenesis. In terms of the proteins in this subclass, the six strains presented very significant differences: P. acidilactici had 49; L. garvieae had 30; B. lactis, astonishingly, had 0; and the other three strains had 8–19 proteins.

TC subclass 4.B comprises transporters responsible for the uptake of nicotinamide ribonucleoside, including several (hypothetical) vitamin transporters. Of the proteins in this subclass, L. garvieae had two; S. thermophilus, L. lactis, and P. acidilactici are had one; and L. delbrueckii and B. lactis had none.

TC subclass 4.C comprises the putative acyl-CoA ligase-coupled transporters, which are involved in the activation of fatty acids for lipid biosynthesis and may function through the mechanism of group translocation [32]. All six strains had four to eight proteins of this subclass.

The TC subclass 4.D comprises the integral membrane polysaccharide synthase/export enzyme (glycosyltransferase), which plays a role in the biosynthesis of polysaccharides and complex oligosaccharides. Furthermore, members of this subclass may be involved in diverse biological processes, such as cell signal transduction, cell–cell interactions [33], and pathogenesis. In each of the six strains, one to six members of this subclass were observed.

The TC subclass 4.F comprises the choline/ethanolamine phosphate transferase 1 (CEPT 1) family. B. lactis had three of these proteins, while all the other strains had one each.

The TC subclass 4.H comprises lysylphosphatidylglycerol synthase/turnover enzymes, which contain two domains: one for derivatization of phospholipids and the other for turnover of lipids across the membrane. The coupling of these two processes is assumed. L. delbrueckii and P. acidilactici did not carry any of these proteins, while the other strains had one to two proteins each.

TC subclass 5.A comprises transmembrane two-electron transfer proteins, through which two electrons (electron pairs) are simultaneously transferred from the electron donor on one side of the membrane to the electron acceptor on the other side. Only P. acidilactici had a protein of this type.

TC subclass 5.B comprises transmembrane single-electron transfer proteins, through which a single electron is transferred from the electron donor on one side of the membrane to the electron acceptor on the other side. L. garvieae had twelve proteins in this subclass, while the other strains had one to four proteins.

TC subclass 8.A comprises auxiliary transporters; proteins that work together with known transporters or are combined with known transporters are included in this category. The six strains had 15 to 35 such proteins each.

TC subclass 9.A comprises transporters with unknown biochemical mechanisms. The six strains had three to ten such proteins each.

TC subclass 9.B comprises putative transporters, and the six strains carried 53 to 66 of these proteins each.

3.2. α-Type Channel Proteins (TC Subclass 1.A)

A substantial quantity of α-channel proteins was observed within the six LABs. The six strains all have MIT (CorA/Mrs 2) family (TC#1.A.35) transporters, which play a role in the transport of metal cations (mainly divalent cations).

Furthermore, all strains (except S. thermophilus) had a homolog in the Large Conductance Mechanosensitive Ion Channel (MscL) Family (TC#1.A.22). These channels facilitate the efflux of cations, osmotic substances, and small proteins under conditions of hypotonic shock. At the same time, five strains (all except L. lactis) carried proteins from the Small Conductance Mechanosensitive Ion Channel (MscS) Family (TC#1.A.23), members of which helps to maintain osmotic stability when the cell is under low osmotic pressure. The proteins of these two families allow for adaptation to osmotic pressure, which is essential for maintaining stability under such conditions [34,35].

Four strains (all except S. thermophilus and L. garvieae) carried homologs of the Camphor Resistance or Fluoride Exporter (Fluc) Family (TC#1.A.43). The cytoplasmic fluoride ions of bacteria are discharged outside the cells through these export channels, thereby reducing the concentration of fluoride ions in the bacteria and avoiding or mitigating the toxic effect of F⁻ [36]. Surprisingly, four strains (all except S. thermophilus and L. lactis) carried homologs of the main K⁺ selective channel KEL or TMEM 175 [37] (TC#1.A.78) in the nucleosome and lysosome; as LABs are prokaryotes, this finding is worthy of further exploration.

All six strains carried a homolog of the Calcium Transporter A (CaTA) Family (TC#1.A.14), members of which have a C-terminal domain that forms a Ca²⁺-permeable channel; the homolog was mainly YbhL. At the same time, all six strains carried members of the Cyclin M Mg²⁺ Exporter (CNNM) Family (TC#1.A.112), members of which may be Mg²⁺ outward transporters.

All six strains carried multiple homologs of the Pore-Forming NADPH-Dependent 1-Acyldihydroxyacetone Phosphate Reductase (Ayr1) Family (TC#1.A.115). The short-chain adenylate enzyme-related protein Ayr1 forms NADPH-dependent regulatory channels in lipid bilayers and mitochondrial outer membranes [38], facilitating ion transport across these membranes [39].

L. delbrueckii, S. thermophilus, and L. garvieae carried a homolog of the Formate-Nitrite Transporter (FNT) Family (TC#1.A.16), members of which may be involved in the transport of formate, nitrite, and hydrogen sulfide-related compounds.

All six strains carried a homolog of the Cation Channel-Forming Heat Shock Protein-70 (Hsp70) Family. These were all homologs of TC#1.A.33.1.4, which can be inserted into the membrane in eukaryotes to form channels [40]; however, this protein has not been found in prokaryotes.

Four strains (all except L. delbrueckii and L. lactis) carried homologs of the Mildew-Resistance Locus O (MLO) Family (TC#1.A.130.1.3), members of which are generally plant-specific and thus are surprising to observe in LABs. L. delbrueckii, S. thermophilus, and B. lactis had a homolog of the Ammonium Channel Transporter (Amt) Family (TC#1.A.11).

All strains carried homologs of the Major Intrinsic Protein (MIP) Family (TC#1.A.8), members of which are potentially involved in the transport of glycerol, water, and dihydroxyacetone. Except for L. delbrueckii, all strains had homologs of the Mechanosensitive Calcium Channel (MCA) Family (TC#1.A.87), specifically, the alkane-desulfurizing sarcina subtype (TC#1.A.87.2.10).

L. lactis, P. acidilactici, and L. garvieae carried aMg²⁺ Transporter-E (MgtE) Family homolog (TC#1.A.26). P. acidilactici and B. lactis carried homologs of the Voltage-gated Ion Channel (VIC) superfamily transporter (TC#1.A.1). L. garvieae carried a homolog of the Mercuric Ion Pore (Mer) Superfamily (TC #1.A.72.3.2). B. lactis carried a homolog of the Homotrimeric Cation Channel (TRIC) Family (TC#1.A.62.3.2). In prokaryotes, these transporters are involved in the export of various metabolites, including amino acids and nucleotides.

3.3. β-Type Porins (TC Subclass 1.B)

Five strains (excluding B. lactis) carried a homolog of the Autotransporter-2 (AT-2) Family (TC#1.B.40). Both L. delbrueckii and P. acidilactici had a homolog of the Copper Resistance Putative Porin (CopB) Family (TC#1.B.76.1.8). Additionally, P. acidilactici carried a homolog of the Intimin/Invasin (Int/Inv) or Autotransporter-3 (AT-3) Family (TC#1.B.54.1.5), while L. garvieae carried a homolog of the Autotransporter-1 (AT-1) Family (TC#1.B.12.5.11).

3.4. Pore-Forming Toxins (TC Subclass 1.C)

Pore-forming toxins (PFTs) are bacterial cytotoxic proteins. PFTs usually cause damage to the host cell membrane and can be found in both Gram-positive and -negative bacteria [41].

From

Table 3. Distribution of PFTs in six LAB strains.

TCID	Family	LD	ST	LL	PA	LG	BL
1.C.39	The Membrane Attack Complex/Perforin (MACPF)	1	1	0	1	1	0
1.C.57	The Clostridial Cytotoxin (CCT)	0	0	1	0	0	0
1.C.82	HP2-20	1	1	1	1	1	1
1.C.95	ESAT-6	0	0	0	0	0	1
1.C.102	Cerein	0	2	0	0	0	0
1.C.105	The Bacillus thuringiensis Vegetative Insecticidal Protein-3 (Vip3)	2	2	2	4	2	0
1.C.109	TlyA	1	1	1	1	1	1
1.C.113	Hly III	1	1	1	1	1	2
1.C.126	HlyC	1	0	0	0	0	1

Note: LD, L. delbrueckii; ST, S. thermophilus; LL, L. lactis; PA, P. acidilactici; LG, L. garvieae; BL, B. lactis.

, four strains (all except L. lactis and B. lactis) carried homologs of the Membrane Attack Complex/Perforin (MACPF) Family (TC#1.C.39.4.5). Five strains (all except B. lactis) carried a homolog of the Bacillus thuringiensis Vegetative Insecticidal Protein-3 (Vip3) Family (TC#1.C.105). All strains carried homologs of the Hemolysin III (Hly III) Family (TC#1.C.113), as well as the Bacterial Hemolysin A (TlyA) Family (TC#1.C.109). The basis of the pore-forming activity of mycobacterial proteins is derived from their TlyA sequence and conserved domain, and their roles as virulence proteins have been further confirmed [42]. All strains carried homologs of the Pore-Forming Amphipathic Helical Peptide HP (2-20) (HP2-20) Family (TC#1.C.82.1.1). L. delbrueckii and B. lactis carried homologs of the HlyC Hemolysin (HlyC) Family (TC#1.C.126.1.2). S. thermophilus carried a homolog of the Cerein (Cerein) Family (TC#1.C.102.1.2). The proteins of the Cerein family can be derived from Gram-positive Firmicutes and Bacteroidetes and belong to the class II sec-independent bacteriocins [43]. L. lactis carried a homolog of the Clostridial Cytotoxin (CCT) Family (TC#1.C.57.1.2). B. lactis carried a homolog of the Pore-Forming ESAT-6 Protein (ESAT-6) Family (TC#1.C.95.1.8). The distribution of PFTs in the selected strains is detailed in Table 3.

3.5. Holins (TC Subclass 1.E)

Holins have been identified in both Gram-negative and Gram-positive bacteria, as well as in bacteriophages. They secrete murine protein hydrolases that traverse the cytoplasmic membrane to reach the cell wall, thereby initiating the hydrolysis of peptidoglycan as a precursor to cell lysis. The cells themselves may also be dissolved, as the chromosomes in the cells encode lysosomes, which break down the cell via enzymes called autolysins [44].

S. thermophilus, P. acidilactici, and L. garvieae carried homologs of the CidA/LrgA Holin (CidA/LrgA Holin) family (TC#1.E.14). Four strains (all except for L. delbrueckii and L. lactis) carried a homolog of the Putative 3-4 TMS Transglycosylase-associated Holin (T-A Hol) Family (TC#1.E.43.1.9); however, the functions of proteins in this family still need further analysis and elaboration. L. lactis, P. acidilactici, and B. lactis carried a homolog of the Mycobacterial 4 TMS Phage Holin (MP4 Holin) Family (TC#1.E.40). L. lactis carried a homolog of the Holin Hol44 (Hol44) Family (TC#1.E.29.2.2). L. lactis carried homologs of the φ11 Holin (φ11 Holin) Family (TC#1.E.11.1.11) and the Clostridium difficile TcdE Holin (TcdE Holin) Family (TC#1.E.19.1.7). L. garvieae carried homologs of the Firmicute Prophage XhlA Holin (XhlA) Family (TC # 1.E.65.1.2), the Lactococcus lactis Phage r1t Holin (r1t Holin) Family (TC#1.E.18.1.1), and the Firmicute phage φU53 Holin (φU53 Holin) Family (TC#1.E.13.1.3).

3.6. Membrane-Bound Channels (TC Subclass 1.I)

Molecules can pass through cell or organelle membranes through membrane-bound channels. All strains carried homologs of the Nuclear Pore Complex (NPC) Family (TC#1.I.1). L. garvieae carried a homolog of the Bacterial (Planctomycetes) Nuclear Pore-Like Complex (B-NPC) Family (TC#1.I.3).

3.7. Fungal Septal Pores (TC Subclass 1.Q)

Only B. lactis carried homologs of the Fungal Septal Pores (TC#1.Q.1.1.1) family.

3.8. Bacterial Micro/Nanocompartment Shell Protein Pores (TC Subclass 1.S)

Only P. acidilactici carried homologs of the Bacterial Microcompartment Shell/Pore-Forming Protein-1 (BMC-SP1) Family (TC#1.S.1.1.1) and the Bacterial Microcompartment Shell/Pore-Forming Protein-2 (BMC-SP2) Family (TC#1.S.2.1.2).

3.9. Phage Portal Protein Subclass (TC Subclass 1.W)

L. lactis and P. acidilactici carried homologs of the Phage Portal Protein 2 (PPP2) Family (TC#1.W.2). L. lactis carried a homolog of the (Bacillus Phage SPP1) Portal Protein 7 (PPP7) Family (TC#1.W.7). B. lactis carried a homolog of the (Bacillus Phage phi29) Portal Protein 6 (PPP6) Family (TC#1.W.6).

3.10. Porters (Uniporters, Symporters, Antiporters) (TC Subclass 2.A)

The MFS superfamily (TC#2.A.1) is currently recognized as the largest secondary vector family. Moreover, its members accounted for the highest number and greatest proportion of secondary vectors in the six selected strains. Homologs of the Drug:H⁺ Antiporter-1 (12 Spanner) (DHA1) Family (TC#2.A.1.2) were found in all strains, while the Drug:H⁺ Antiporter-2 (14 Spanner) (DHA2) Family (TC#2.A.1.3) was missing only from S. thermophilus. P. acidilactici had the most MFS family homologs (twenty-three), while S. thermophilus had only eight such proteins.

L. delbrueckii had no homologs in the Glycoside-Pentoside-Hexuronide (GPH) Cation Symporter Family (TC#2.A.2). Sugars and their derivatives can be transported via catalysis by monovalent cations and the proteins of this family. Studies have shown that the LacS of the GPH family of S. thermophilus may be a co-dimer with a sugar-translocation pathway for each monomer [45]. The proteins in the Amino Acid-Polyamine-Organocation (APC) Superfamily (TC#2.A.3) play critical roles in the transport of amino acids and polyamines. The homologs of this family accounted for the second largest proportion of secondary vectors in all strains. L. lactis lacked a homolog of the Cation Diffusion Facilitator (CDF) Family (TC#2.A.4) and, as the CDF proteins are related to the transport of heavy metal ions, L. lactis may exhibit weak functioning in this area. All strains carried homologs of the Resistance-Nodulation-Cell Division (RND) Superfamily (TC#2.A.6.6.11), members of which play a significant role in cholesterol uptake. The Drug/Metabolite Transporter (DMT) Superfamily (TC#2.A.7) is also a larger superfamily of secondary vectors that play roles in the context of the uptake of small metabolites and drug export. All strains carried homologs of the Glucose/Ribose Porter (GRP) Family (TC#2.A.7.5), which may function via H⁺ co-transport. Homologs of the Membrane Protein Insertase (YidC/Alb3/Oxa1) Family (TC#2.A.9) were also found in all strains. The proteins in the Tellurite-resistance/Dicarboxylate Transporter (TDT) Family (TC#2.A.16) transport dicarboxylate [46], and homologs were found in all strains. All strains carried a homolog of the Nucleobase/Ascorbate Transporter (NAT) or Nucleobase: Cation Symporter-2 (NCS2) Family (TC#2.A.40), which is the only widely distributed transporter family, although four other transporter families act on nucleobases [47].

The Multidrug/Oligosaccharidyl-lipid/Polysaccharide (MOP) Flippase Superfamily (TC#2.A.66) had homologs in all strains. Homologs of the Multi Antimicrobial Extrusion (MATE) Family (TC#2.A.66.1) and the Polysaccharide Transport (PST) Family (TC#2.A.66.2) were found in all strains; members of these families are related to the excretion of antimicrobial agents and the transport of polysaccharides, respectively. B. lactis carried a homolog of the Mouse Virulence Factor (MVF) Family (TC#2.A.66.4), which is a key virulence factor for disease in mice [48]. All strains had two homologs of the Autoinducer-2 Exporter (AI-2E) Family (TC#2.A.86). The expression of the Putative Sulfate Exporter (PSE) Family (TC#2.A.98) protein can be induced in the presence of cysteine or taurine [49], and its homologs were found in all strains.

All strains carried a homolog of the Bacterial Murein Precursor Exporter (MPE) Family (TC#2.A.103), members of which play a role in the formation of bacterial cell walls. As homologs of the Multidrug Resistance Exporter VanZ (VanZ) Family (TC#2.A.128) can enhance bacterial resistance, their discovery in all six strains may be beneficial in the control of bacterial resistance. Notably, B. lactis lacked homologs of the Novobiocin Exporter (NbcE) Family (TC#2.A.115) and the Enterobacterial Cardiolipin Transporter (CLT) Family (TC#2.A.127).

3.11. Ion Gradient-Driven Energizers (TC Subclass 2.C)

Four strains, excluding S. thermophilus and L. lactis, carried homologs of the TonB/TolA family (TC#2.C.1). These protein homologs are generally identified exclusively in Gram-negative bacteria and cyanobacteria, which makes their presence here unusual.

3.12. P-P Bond Hydrolysis-Driven Transporters (TC Subclass 3.A)

The ABC superfamily (TC#3.A.1) was the largest superfamily found in the six strains. The proteins in this family were found in all species. The transport substrates of this family are rich in variety and size and contribute to the mechanisms of uptake and efflux [50]. From this superfamily, the number of proteins in strain L. delbrueckii was 138, that for S. thermophilus was 173, that for L. lactis was 95, that for P. acidilactici was 93, that for L. garvieae was 106, and that for B. lactis was 128. In the subclass, S. thermophilus had a more prominent number (222). There are four categories of substrates involved in the external transport of ABC: 1) drugs; 2) proteins and peptides; 3) lipids and lipoproteins; and 4) secondary metabolites. P. acidilactici lacked homologs of the Carbohydrate Uptake Transporter-1 (CUT1) Family (TC#3.A.1.1) and the Carbohydrate Uptake Transporter-2 (CUT2) Family (TC#3.A.1.2), while other strains carried these families of proteins, which are involved in the transport of arabinose, arabinose, and glucose. All strains carried homologs of the Polar Amino Acid Uptake Transporter (PAAT) Family (TC#3.A.1.3), and the proteins of this family constituted the largest number and highest proportion of ABC superfamily members in all strains. P. acidilactici and L. garvieae lacked homologs of the Hydrophobic Amino Acid Uptake Transporter (HAAT) Family (TC#3.A.1.4). Meanwhile, all strains carried homologs of the Phosphate Uptake Transporter (PhoT) Family (TC#3.A.1.7), the Manganese/Zinc/Iron Chelate Uptake Transporter (MZT) Family (TC#3.A.1.15), the Methionine Uptake Transporter (MUT) Family (TC#3.A.1.24), and the Biotin Uptake Transporter (BioMNY) Family (TC#3.A.1.25). Four strains (not P. acidilactici or B. lactis) carried homologs of the Polyamine/Opine/Phosphonate Uptake Transporter (POPT) Family (TC#3.A.1.11). The proteins in this subclass are related to the transport of polyamines, typically putrescine and spermidine. As polyamines have the functions of maintaining chromosome homeostasis, regulating the synthesis of siderophores, scavenging free radicals, and promoting growth [51], the proteins in this family also play indirect roles in transport.

All strains carried homologs of the F-ATPase superfamily (TC#3.A.2), but only of those belonging to the F-type family (TC#3.A.2.1), which exhibit reversible functionality in proton translocation or ATP synthesis [52]. Homologs of the P-type ATPase (P-ATPase) Superfamily (TC#3.A.3) were present in all strains; the structure, function, and kinetics of this family of proteins have been previously reviewed [53]. Additionally, homologs of the General Secretory Pathway (Sec) Family (TC#3.A.5) were found in all strains, with the specific distribution detailed in

Table 4. The Sec family components identified in the six selected LAB strains: Y, the strain had homologs; -, no homologs were found.

Components	LD	ST	LL	PA	LG	BL
SecG	Y	Y	Y	Y	Y	Y
SecY	Y	Y	Y	Y	Y	Y
SecA	Y	Y	Y	Y	Y	Y
Ffh	Y	Y	Y	Y	Y	-
FtsY	Y	Y	Y	Y	Y	Y
SecE	Y	-	Y	Y	-	Y

Note: LD, L. delbrueckii; ST, S. thermophilus; LL, L. lactis; PA, P. acidilactici; LG, L. garvieae; BL, B. lactis.

.

The distributions of T4SS (TC#3.A.7) and T6SS (TC#3.A.11) across strains differed. The homologs of the T4SS family are involved in the transport of virulence factors and DNA–protein conjugates [54], while T6SS is related only to bacterial competent-related DNA transformation [55]. The specific distributions of the two secretion systems in the selected strains are given in

Table 5. Distributions of T4SS and T6SS homologs in the six selected LAB strains.

Family	TCID	LD	ST	LL	PA	LG	BL
T4SS	3.A.7.7	2	1	1	1	1	1
	3.A.7.11	1	0	1	0	0	2
	3.A.7.13	0	0	0	1	1	0
	3.A.7.14	6	7	5	6	17	4
	3.A.7.16	1	1	1	1	1	0
	3.A.7.18	0	0	0	0	0	1
	3.A.7.19	1	2	4	5	5	1
T6SS	3.A.23.1	0	0	0	2	0	0
	3.A.23.6	3	3	2	3	4	3

Note: LD, L. delbrueckii; ST, S. thermophilus; LL, L. lactis; PA, P. acidilactici; LG, L. garvieae; BL, B. lactis.

.

3.13. Decarboxylation-Driven Transporters (TC Subclass 3.B)

All strains carried a homolog of the Na⁺-transporting Carboxylic Acid Decarboxylase (NaT-DC) Family (TC#3.B.1), members of which catalyze the decarboxylation of substrate carboxylic acids, utilizing the energy generated from this process to extrude one or two sodium ions from the cytoplasm of the cell [29].

3.14. Oxidoreduction-Driven Transporters (TC Subclass 3.D)

All strains carried homologs of the H⁺- or Na⁺-translocating NADH Dehydrogenase (NDH) Family (TC#3.D.1). P. acidilactici, L. garvieae, and B. lactis carried homologs of the Proton-Translocating Transhydrogenase (PTH) Family (TC#3.D.2). Bacterial PTHs have a three-dimensional structure, and the structures and mechanisms of these proteins have been previously reviewed [56]. S. thermophilus and B. lactis carried homologs of the Proton-translocating Quinol:Cytochrome c Reductase (QCR) Superfamily (TC#3.D.3). P. acidilactici, L. garvieae, and B. lactis carried homologs of the Prokaryotic Succinate Dehydrogenase (SDH) Family (TC#3.D.10). L. lactis and L. garvieae carried homologs of the Proton-translocating Cytochrome Oxidase (COX) Superfamily (TC#3.D.4). L. delbrueckii carried a homolog of the Nitrogen Fixation Complex (FixABCX) Family (TC#3.D.12).

3.15. Light Absorption-Driven Transporters (TC Subclass 3.E)

All strains carried homologs of the Ion-Translocating Microbial Rhodopsin (MR) Family (TC#3.E.1.5.3), which may be a photoinhibiting guanylate cyclase [57] homolog.

3.16. Phosphotransfer-Driven Group Translocators (PTS) (TC Subclass 4.A)

B. lactis had no homolog from this subclass. All of the other five strains carried homologs of the Phosphotransfer-Driven Group Translocators (PTS) (TC#4.A.1). These five strains carried homologs of the PTS Mannose-Fructose-Sorbose (Man) Family (TC#4.A.6) and the PTS Lactose-N, N′-Diacetylchitobiose-β-glucoside (Lac) Family (TC#4.A.3), the latter of which includes several sequenced lactose (β-galactoside) transporters in Gram-positive bacteria and N, N′-diacetylchitobiose (Chb) transporters in E.coli and Borrelia burgdorferi. Four strains (all except for S. thermophilus) carried homologs of the PTS Fructose-Mannitol (Fru) Family (TC#4.A.2). L. delbrueckii and P. acidilactici carried homologs of the PTS Galactitol (Gat) Family (TC#4.A.5). L. garvieae carried a PTS Glucose-Glucoside (Glc) Family (TC#4.A.1) homolog. Similarly to other PTS protein homologs, SgaT homologs have been identified in a wide array of evolutionarily divergent bacteria; however, they are absent in eukaryotes [58].

3.17. Nicotinamide Ribonucleoside Uptake Transporters (TC Subclass 4.B)

The transporters in this subclass use ATP as a phosphoryl donor and phosphorylate external nicotinamide ribonucleotides to produce cytoplasmic nicotinamide mononucleotide (NMN), as well as ADP. A review of several predicted substrates of PnuC homologs indicates that at least some of these substrates are not phosphorylated during transport. Four strains (all except L. delbrueckii and B. lactis) carried homologs of the Nicotinamide Ribonucleoside (NR) Uptake Permease (PnuC) Family (TC#4.B.1), one representative of which, the deoxynucleoside transporter PnuN, is regulated by the NrdR repressor [59].

3.18. Acyl CoA Ligase-Coupled Transporters (TC Subclass 4.C)

All strains carried homologs of the Fatty Acid Group Translocation (FAT) Family (TC#4.C.1), as well as homologs of the Acyl-CoA Thioesterase (ACoA-T) Family (TC#4.C.3).

3.19. Polysaccharide Synthase/Exporters (TC Subclass 4.D)

All strains carried homologs of the Putative Vectorial Glycosyl Polymerization (VGP) Family (TC#4.D.1). These transporters function as catalytic carriers for glycosyl polymerization. L. delbrueckii, L. lactis, and P. acidilactici carried homologs of the glycosyltransferase 2 (GT2) family (TC#4.D.1), which possess several different functions, including drug resistance, and various enzyme activities involving glycosyl transfer; these are accompanied by transmembrane output. P. acidilactici carried a homolog of the Glycan Glucosyl Transferase (OpgH) Family (TC#4.D.3). The glucosyltransferase OpgH has been identified as a nutrient-dependent regulator of E. coli that affects the size of monomer cells.

3.20. Choline/Ethanolamine Phosphotransferase 1 (CEPT1) (TC Subclass 4.F)

Proteins in this subclass are usually involved in the process of phospholipid transport in cells with endoplasmic reticulum; they also catalyze the biosynthesis of phosphatidylcholine and phosphatidylethanolamine via CDP-choline and CDP-ethanolamine and distribute phosphatidylcholine to the lumen surface of the endoplasmic reticulum [60]; therefore, it is surprising that LABs possess homologs of this subclass. All strains carried homologs of the Choline/Ethanolamine phosphotransferase 1 (CEPT1) Family (TC#4.F.1.1.7), which were homologous to PgsA in L. mucilaginosus.

3.21. Lysyl Phosphatidylglycerol Synthase/Flippases (TC Subclass 4.H)

Four strains (all except L. delbrueckii and P. acidilactici) carried homologs of the Lysyl Phosphatidylglycerol Synthase/Flippase (MprF) Family (TC#4.H.1). The C-termini of MprF-integrated membrane proteins found in several prokaryotes are modified with lysine or alanine, which modifies phosphatidylglycerol (PG) to regulate the surface charge of the membrane, causing cells to become resistant to cationic antimicrobial agents such as daptomycin [61].

3.22. Transmembrane 2-Electron Transfer Carriers (TC Subclass 5.A)

Only P. acidilactici carried a homolog of the Disulfide Bond Oxidoreductase D (DsbD) Family (TC#5.A.1.6.1), which may be a heavy-metal-transport detoxification protein [62].

3.23. Transmembrane 1-Electron Transfer Carriers (TC Subclass 5.B)

All strains carried homologs of the Phagocyte (gp91^phox) NADPH Oxidase Family (TC#5.B.1), which are homologous to proteins from animals, myxomycetes, plants, fungi, and bacteria, with the homologs in all organisms forming six phylogenetic clusters [63].

3.24. Auxiliary Transport Proteins (TC Subclass 8.A)

These proteins are involved in one or more established transport systems and can facilitate energy transfer coupled with transport processes, serve a structural role in complex assembly, and exert stabilizing or regulatory functions.

The Voltage-Gated K⁺ Channel β-subunit (Kvβ) Family (TC#8.A.5) had homologs in all strains, particularly homologs (TC#8.A.5.1.4) of the Bacillus subtilis GSP69 protein; however, whether this protein regulates the bacterial transport system is still uncertain. In this subclass of proteins, the oxygen-sensitive vascular Kvβ protein controls myocardial blood flow [64]. All strains carried homologs of the Apoptosis Cell Death Regulator (ACDR) Family (TC#8.A.217.1.1), which is related to apoptosis and may lead to the apoptosis of LABs. All strains carried homologs of the Chlamydial Inclusion Membrane Protein MrcA (MrcA) Family (TC#8.A.229), which can regulate intracellular calcium (Ca²⁺) homeostasis [65]. Homologs of the Collapsin Response Mediator Protein 2 (CRMP2) Family were also found in all strains, and all were homologs of (TC#8.A.228.1.2) Paenibacillus putida HydA. This family of proteins is involved in neuronal growth-cone collapse, axon growth and guidance, neuronal development and polarity, and cell migration [66,67]. All strains carried a homolog of the DnaJ Homolog (DnaJ) Family (TC#8.A.192); one member of this family, DnaJC 5, is involved in the secretion of associated synaptic proteins and misfolding-related proteins [68].

Homologs of the rBAT Transport Accessory Protein (rBAT) Family (TC#8.A.9) were present in five strains (all except S. thermophilus). The extracellular domain of rBAT may specifically promote L-cystine uptake [69], and drug-resistant mutations that mediate cystineuria in human rBAT have been identified [70]. Four strains carried homologs of the Klotho Auxiliary Protein (Klotho) Family (TC#8.A.49), which affects aging and aging-related diseases.

3.25. Recognized Transporters of Unknown Biochemical Mechanisms (TC Subclass 9.A) and Putative Transport Proteins (TC Subclass 9.B)

L. delbrueckii, S. thermophilus, and L. lactis carried homologs of the Niacin/Nicotinamide Transporter (NNT) Family (TC#9.A.23), members of which are presumed to possess functions related to the transport of niacin (vitamin B3 or niacin) and/or nicotinamide [71]. Four strains (all except L. lactis and P. acidilactici) carried homologs of the Ferrous Iron Uptake (FeoB) Family (TC#9.A.8), members of which play a role in the G protein-coupled transport of Fe²⁺; in particular, they are unique in that the G protein is directly connected to the membrane domain [72]. Four strains (all except L. delbrueckii and B. lactis) carried the YggT or Fanciful K⁺ Uptake-B (FkuB; yggT) Family (TC#9.A.4) members, which may be related to K⁺ uptake.

The members of TC subclass 9.B are classified as putative transport proteins. When the transport function of one of these proteins is determined, it will be re-classified into the corresponding subclass; on the other hand, if it is determined that it does not possess a transport function, it will be deleted from the TCDB system. All strains had multiple homologs in the Xanthan Glycosyl Transferase, GumD (GumD) Family (TC#9.B.18), members of which are putatively related to the synthesis and export of glial polysaccharides [73]. All strains carried homologs of the Death Effector Domain A (DedA) Family (TC#9.B.27), members of which may play a role in membrane homeostasis [74]. With respect to the Putative Mg²⁺ Transporter-C (MgtC) Family (TC#9.B.20), L. delbrueckii, P. acidilactici, and L. garvieae carried homologs, which may mean that they have stronger Mg²⁺ transport function.

3.26. Transport Substrates of the Six Strains

The transport substrates of the six selected strains were predicted using the TCDB G-blast program, and the findings are presented in

Table 6. Predicted transport substrates of the six selected LAB strains.

Substrate Category	Transport Proteins
	LD	ST	LL	PA	LG	BL
Anions	23	23	20	18	14	23
Cations	55	70	44	91	93	74
Electrons	7	4	5	3	13	2
Water	2	1	1	1	3	2
Amines	5	16	8	11	5	3
Amino acids	67	64	47	63	45	40
Carboxylates	9	5	7	18	10	8
Non-selective	7	4	4	6	5	10
Drugs	41	46	34	49	42	33
Nucleobases, nucleosides, nucleotides	13	8	16	8	22	6
Proteins, peptides	69	73	53	68	54	53
Siderophores	8	14	2	10	18	5
Sugars	24	16	16	39	30	27
Sugar derivatives	30	15	7	24	12	7
Lipids	10	11	11	14	14	12
Sugar alcohols	0	1	2	4	2	0
Vitamins	16	10	12	15	16	10
Unknown	61	65	60	83	118	99
Total	447	446	349	525	516	414

Note: LD, L. delbrueckii; ST, S. thermophilus; LL, L. lactis; PA, P. acidilactici; LG, L. garvieae; BL, B. lactis.

.

From Table 6, we can clearly see that L. garvieae had the fewest cation transporters (14 proteins) compared with the other strains (18–23 proteins). As cation transporters can maintain ion and osmotic homeostasis and enhance heavy-metal resistance, L. garvieae may have the weakest ability in this respect among the six strains. In addition, S. thermophilus had the most amine transporters, far more than the other strains (16 vs. 5–10 proteins), based on which we can reasonably infer that S. thermophilus has better free radical-scavenging function and may be able to play an anti-aging role. All six strains had multiple carboxylate transporters (5–18 proteins), which may be associated with the transport of malic acid, succinic acid, and fumaric acid, reflecting the anaerobic respiration of LABs. The number of sugar transporters varied greatly among the strains, with that of P. acidilactici (39 proteins) being two times greater than those of S. thermophilus and L. lactis (16 proteins), which may indicate that the sugar-uptake abilities of these strains were quite different. All six strains presented a large number of drug-efflux proteins but, at the same time, there were differences among the various strains. P. acidilactici has the most such proteins, which may indicate that its drug-resistance ability is stronger.

3.27. T4SS and T6SS

From Table 5, a total of seven types of T4SS secretion pathways were identified in the six strains. The protein complexes involved in these pathways transport proteins or single-stranded DNA–protein complexes outside the cell, then enter either the medium or cytoplasm of the recipient cell [75]. In addition, the involved family of proteins also contributes to the process of DNA conjugate transfer and transport of effector proteins to recipient cells.

Surprisingly, all strains carried the T6SS secretion system, which is worth exploring in terms of the probiotic identity of LABs. Existing studies have shown that this may be due to the fact that T6SS is conducive to competition between bacteria [76], as it can allow bacteria possessing this system to seize the living space of pathogens. For LABs, this may be an embodiment of their probiotic effect.

4. Conclusions

With the assistance of the TCDB database, we systematically screened six selected LAB strains to identify their transporter homologs, allocate related transport substrates, and analyze the distributions of transporters in these LABs. As classic food probiotics, LABs possess numerous sugar-transporting proteins and their derivatives, which enable them to produce substantial amounts of energy and compounds that have beneficial effects on human health. First, we were surprised to find (from Table 3) that all six of these LABs—which are commonly used as food probiotics—carried homologs of pore-forming toxins (PFTs), which initially seemed to contradict their probiotic status. However, this is actually reasonable: PFTs in LABs help to effectively dissolve harmful bacteria, kill infected cells, and eliminate pathogens [77,78]. Moreover, PFTs also help LABs to obtain nutrients, thereby giving LABs a competitive advantage over pathogens [79].

From Table 5, we can draw the conclusion that all the strains carried T6SS components; however, the results indicated that this system is incomplete. Some possible reasons for this observation are as follows: (1) the detection method may not have been sensitive enough to identify all components; (2) differences among bacterial species might result in variations, with specific LABs potentially having a complete T6SS; and (3) evolutionary divergence could lead to disordered or missing components as evolution progresses. These findings suggest that T6SS is associated with the adaptability of LAB strains in the intestine. As previously mentioned, the presence of T6SS in LABs may reflect their probiotic function, considering that T6SS would enable these bacteria to interact with other micro-organisms by injecting toxins into recipient cells, thereby gaining a competitive advantage. At present, there is no research on the T6SS transport proteins of LABs. However, previous studies have demonstrated that certain bacteria can compete with other bacteria through the T6SS system to gain a competitive advantage [80]. Additionally, research has shown that a non-toxic probiotic can eliminate a toxic probiotic through T6SS, thereby protecting the host mice [81]. Therefore, we believe that T6SS enhances the probiotic functions of LABs.

From Table 6, we can draw the following conclusion: in an anaerobic environment, both LABs and pathogens require iron to ensure their survival. However, in this context, the iron content is limited. The presence of LAB iron carriers gives them an advantage in the competition for iron resources, thereby weakening and eliminating pathogens through competition. The competitive edge of LABs also stems from the transport of bioactive metabolites. The quantity of butyrate transporters may be positively correlated with the immunomodulatory potential of the strain [82]. Additionally, the secretion of antimicrobial peptides mediated by ABC transporters endows LABs with a competitive advantage, allowing them to inhibit pathogenic bacteria in the intestinal microenvironment.

Table 1 and Table 2 indicate that over 20% of the proteomes of these six LABs is made up of transport proteins, a large proportion of which belong to the large transport protein family. This high abundance of transporters contributes to their complex metabolic capabilities and probiotic effects. Furthermore, the identification of multiple drug-efflux proteins suggests that LABs possess excellent antibiotic resistance. These drug-efflux proteins can extrude a wide range of compounds within a certain mass range [83]; moreover, they also affect the pharmacokinetics of drugs [84]. Further research in this line may reveal how the population ecology of LABs maintains stability under the action of antibiotics.

As a result of this study, the discovery of PFTs in LABs offers the possibility of developing LAB-based health foods in the future, achieving the effect of eliminating certain pathogenic bacteria while intaking nutrients. Additionally, the competitive advantages of the T6SS system and iron carriers also contribute to this possibility. However, we should also pay more attention to the multidrug transporters in LABs, in order to prevent the spread of LAB drug-resistance genes to other bacteria, potentially facilitating the creation of superbugs. In addition, this study provides a reference for the selection of industrial lactic acid bacteria. For instance, strains with high expression of ABC transporters may exhibit stronger tolerance to adverse conditions in the host’s intestinal tract or in a fermentation environment (such as resistance to bile salts or antibiotic pressure), which has direct significance for guiding the development of probiotic formulations. Moreover, the number of monocarboxylate transporters directly affects the efficiency of lactic acid excretion. Strains with high expression of such proteins can be prioritized to enhance fermentation efficiency. In terms of laboratory research, this study provides a foundation for a series of subsequent experiments. Whether such research is conducted to verify and apply the unique discoveries reported in this study or to incorporate more LABs into this system, it is expected that notable benefits can be obtained.

In the future, we will continue to explore the findings of this study in depth. We will design primers for the target genes of interest through RT-qPCR technology and have them synthesized by a specialized company. Using the target genes as the internal reference genes, we will conduct relative quantitative analysis of the gene expression levels by the 2^−ΔΔCt method.