Skip to main content
NCBI home page
Microbial Biotechnology logoLink to Microbial Biotechnology
. 2024 May 27;17(5):e14484. doi: 10.1111/1751-7915.14484

Mutualistic interactions of lactate‐producing lactobacilli and lactate‐utilizing Veillonella dispar: Lactate and glutamate cross‐feeding for the enhanced growth and short‐chain fatty acid production

Shi‐Min Zhang 1, Jia‐He Hung 2, Tran Ngoc Yen 3, Shir‐Ly Huang 3,✉
PMCID: PMC11129673  PMID: 38801349

Abstract

The human gut hosts numerous ecological niches for microbe–microbe and host–microbe interactions. Gut lactate homeostasis in humans is crucial and relies on various bacteria. Veillonella spp., gut lactate‐utilizing bacteria, and lactate‐producing bacteria were frequently co‐isolated. A recent clinical trial has revealed that lactate‐producing bacteria in humans cross‐feed lactate to Veillonella spp.; however, their interspecies interaction mechanisms remain unclear. Veillonella dispar, an obligate anaerobe commonly found in the human gut and oral cavity, ferments lactate into acetate and propionate. In our study, we investigated the interaction between V. dispar ATCC 17748T and three representative phylogenetically distant strains of lactic acid bacteria, Lactobacillus acidophilus ATCC 4356T, Lacticaseibacillus paracasei subsp. paracasei ATCC 27216T, and Lactiplantibacillus plantarum ATCC 10241. Bacterial growth, viability, metabolism and gene level adaptations during bacterial interaction were examined. V. dispar exhibited the highest degree of mutualism with L. acidophilus. During co‐culture of V. dispar with L. acidophilus, both bacteria exhibited enhanced growth and increased viability. V. dispar demonstrated an upregulation of amino acid biosynthesis pathways and the aspartate catabolic pathway. L. acidophilus also showed a considerable number of upregulated genes related to growth and lactate fermentation. Our results support that V. dispar is able to enhance the fermentative capability of L. acidophilus by presumably consuming the produced lactate, and that L. acidophilus cross‐feed not only lactate, but also glutamate, to V. dispar during co‐culture. The cross‐fed glutamate enters the central carbon metabolism in V. dispar. These findings highlight an intricate metabolic relationship characterized by cross‐feeding of lactate and glutamate in parallel with considerable gene regulation within both L. acidophilus (lactate‐producing) and V. dispar (lactate‐utilizing). The mechanisms of mutualistic interactions between a traditional probiotic bacterium and a potential next‐generation probiotic bacterium were elucidated in the production of short‐chain fatty acids.

The study investigates the symbiotic interaction between Veillonella dispar and three lactic acid bacteria strains, revealing a robust mutualism primarily with Lactobacillus acidophilus. Through co‐culture experiments and transcriptomic analysis, the research elucidates a complex metabolic relationship involving cross‐feeding of lactate and glutamate, underscoring the potential for enhancing short‐chain fatty acid production. These findings offer insights into the mechanisms driving synergy between traditional and next‐generation probiotics.

graphic file with name MBT2-17-e14484-g001.jpg

INTRODUCTION

Microbes in natural environments usually exist as multi‐species communities (Kolenbrander et al., 2010), and such an interactive ecosystem encompasses a wide range of processes, such as metabolite cross‐feeding, coaggregation, biofilm formation, and quorum sensing (Moutinho et al., 2017; Rickard et al., 2003). A predominant form of microbial mutual interaction is metabolite cross‐feeding (Figueiredo & Kramer, 2020; Stubbendieck et al., 2016). This metabolite cross‐feeding interaction is characterized by one bacterium, the producer, releasing a by‐product that then used by another bacterium, the consumer (Rivière et al., 2015). Cross‐metabolism allows efficient nutrient utilization within their common environment and expands the metabolic niches available to both of the interacting bacteria (Oña et al., 2021).

Lactobacilli are human anaerobic commensal microbes and are known to adhere to the gut mucus (Tassell & Miller, 2011); in this niche, they interact with various pathogens and other commensal microbes (Colautti et al., 2022). Lactobacilli produce substantial amounts of lactate in the intestinal milieu. The accumulated lactate has been shown to be associated with several disease states including sepsis, arthritis, and inflammation (Ivashkiv, 2020; Pucino et al., 2019; Yang et al., 2022). The accumulation of lactate may be regulated by lactate‐utilizing bacteria, which have been proposed to play a crucial role in preventing the excessive buildup of lactate in the gut, thereby contributing to the stability of the intestinal ecosystem (Li et al., 2022). For instance, lactate produced by lactobacilli can be metabolized by Veillonella species. Veillonella are Gram‐negative, strictly anaerobic commensals that are typically found in human oral cavity and intestinal tracts (Mashima et al., 2016). They utilize lactate as primary carbon source and produce acetate and propionate during lactate fermentation (Ng & Hamilton, 1971). Veillonella are potential next generation probiotics because they produce beneficial short‐chain fatty acids (SCFAs), such as acetate and propionate (Scheiman et al., 2019; Strati et al., 2017; Tan et al., 2016). The co‐administration of L. acidophilus and Veillonella ratti has been shown to improve gut inflammation in a mice study (Li et al., 2023).

Lactobacilli and Veillonella have frequently been observed to be co‐localized, suggesting a potential relationship between these species (Bradshaw & Marsh, 1998; Gross et al., 2012; Wu et al., 2023; Xing et al., 2023; Zhou et al., 2021). Coculturing L. acidophilus and faecal microbiota significantly promoted the growth of Veillonella (Shimizu & Benno, 2015). Individuals who consumed Lacticaseibacillus paracasei have a higher abundance of Veillonella in their gut microbiota (Zhang et al., 2021). Furthermore, lactate‐producing bacteria have been shown to cross‐feed lactate to Veillonella spp. in humans (Button et al., 2023). Nonetheless, the underlying mechanisms of the microbial interaction between lactate‐producing and utilizing bacteria remain relatively unexplored.

Among Veillonella species, V. dispar has been primarily isolated from humans, particularly from the oral cavity and the intestine (Mashima et al., 2016; Rogosa & Bishop, 1964). Furthermore, V. dispar is known to be able to reprogram its lactate metabolism and SCFA production as nutrients fluctuate (Zhang & Huang, 2023), which makes V. dispar a suitable candidate bacterium for exploring the relationship of V. dispar with lactobacilli. To investigate the physiology and mechanism of the relationship between lactobacilli and Veillonella, we focused on three important and phylogenetically distinct lactobacillus strains: Lactobacillus acidophilus ATCC 4356T, Lacticaseibacillus paracasei subsp. paracasei ATCC 27216T, and Lactiplantibacillus plantarum ATCC 10241 (Zheng et al., 2020). These lactobacilli are homofermentative and predominantly produce lactate without producing significant amounts of acetate. This is important because V. dispar produce acetate and propionate during lactate fermentation. The aim was to avoid the complexities that might be introduced if acetate was produced from both bacteria. Our analysis of their growth profile and metabolic profiles revealed a pronounced interaction between V. dispar and L. acidophilus. The molecular dynamics underpinning this mutualism, focusing on metabolite cross‐feeding and gene regulation, were further investigated.

EXPERIMENTAL PROCEDURES

Bacterial strains, growth media and culture conditions

We utilized several standard bacterial strains: Lactobacillus acidophilus ATCC 4356T, Lacticaseibacillus paracasei ATCC 27216, Lactiplantibacillus plantarum ATCC 10241, and Veillonella dispar ATCC 17748T. These standard strains were acquired from the Bioresource Collection and Research Center (BCRC), Taiwan (https://www.bcrc.firdi.org.tw/en/home/). They were all routinely cultured in tryptone‐yeast‐lactate‐glucose (TYLG) medium. The TYLG medium consisted of (per litre): 5.0 g of tryptone (BD Difco, Franklin Lakes, USA), 3.0 g of yeast extract (BD Difco, Franklin Lakes, USA), 1.12 g of sodium L‐lactate (Sigma‐Aldrich, St. Louis, USA), 1.8 g of dextrose (Sigma‐Aldrich, St. Louis, USA), 1 g of Tween 80 (Sigma‐Aldrich, St. Louis, USA), 4.672 g of K2HPO4 (Sigma‐Aldrich, St. Louis, USA), and 3.154 g of KH2PO4 (Sigma‐Aldrich, St. Louis, USA). The pH of the medium was adjusted to 7.0 using a 1 M NaOH (TCI, Tokyo, Japan) solution. For the enumeration of L. acidophilus and V. dispar, TYG (tryptone‐yeast‐glucose) agar plates were utilized for enumeration of L. acidophilus, while TYL (tryptone‐yeast‐lactate) agar plates were utilized for enumeration of V. dispar. TYG agar shares the same formulation as TYLG medium except that L‐lactate is not added and the final glucose concentration is 1.2% (w/v). Similarly, TYL agar shares the same formulation as TYLG medium, but glucose, is absent and there is a final lactate concentration of 1.2% (w/v). All agar plates contain 1.5% (w/v) agar (BD Difco, Franklin Lakes, USA). All bacterial cultures were incubated in an anaerobic glove chamber (Forma Scientific model 1025, ThermoFisher Scientific, Waltham, USA) under an atmosphere of 85% N2, 10% CO2, and 5% H2. The cultures were maintained at 37°C without shaking.

Monoculture and co‐culture of lactobacilli and V. dispar

Experiments using both monoculture and co‐culture bacterial samples were conducted anaerobically using 100 mL of TYLG medium in glass flasks. For monocultures, 3 mL of inoculum was taken from an overnight culture of each species. For co‐culture equal aliquots of 1.5 mL of overnight cultures of two species were used (Milho et al., 2019). Samples were removed from each culture at various intervals (0, 1.5, 3.5, 5, 7, 9, 11, 13.5, 16, and 24 h) to assess turbidity, viable cell counts, pH, and the presence of metabolites in the supernatant; the metabolites assessed were lactate, acetate, propionate, and glucose.

Determination of bacterial numbers

Bacterial growth was evaluated using two separate methods: indirect quantification via turbidity measurement and direct quantification of viable cells by plate counting. For the turbidity measurement approach, samples were taken from the bacterial cultures and their turbidity was evaluated at 600 nm with a spectrophotometer (UV‐1800, Shimadzu, Kyoto, Japan). On the other hand, for viable cell quantification through plate counting, bacterial culture samples were taken at each time‐point and 10‐fold then these were serially diluted to an appropriate concentration. An appropriate concentration refers to the concentration where 100 μL of the suspension yielded 30–300 single colonies on one agar plate (Reynolds, 2005). Subsequently, 100 μL aliquot of the diluted bacterial suspension was evenly dispersed onto the corresponding agar plate for incubation and enumeration. For monoculture, the viable cell number of L. acidophilus was quantified using TYG agar, while that of V. dispar was quantified using TYL agar. For dual‐species co‐culture, 100 μL of diluted co‐culture suspension was dispersed onto TYG and TYL agar plates to individually enumerate L. acidophilus and V. dispar, respectively. Specifically, L. acidophilus in the co‐culture was quantified by calculating the number of single colonies formed on the TYG agar, while V. dispar in the co‐culture was quantified by calculating the number of single colonies formed on the TYL agar. The agar plates were subjected to anaerobic incubation in an anaerobic glove chamber (Forma Scientific model 1025, ThermoFisher Scientific, Waltham, USA) under an atmosphere of 85% N2, 10% CO2, and 5% H2 at a temperature of 37°C for a period of 48 h. The individual bacterial morphology was confirmed by colony morphology and cell morphology by light microscopy (ZEISS Axio Lab A1, ZEISS, Oberkochen, Germany) and comparing the results with the corresponding standard strains. In order to calculate the concentration of viable cells (expressed in CFU/mL), the number of colonies counted was multiplied by both the serial dilution factor and then by a factor of 10, the latter to account for the fact that only 100 μL of the bacterial suspension was plated.

Quantitative analysis of main carbon sources and metabolites in the medium

The glucose content in the medium was assessed using Glucose (Glu) Colorimetric Assay Kits (Elabscience, Houston, USA) by following the manufacturer's protocol. Lactate and the resulting short‐chain fatty acids (SCFAs) were extracted using a previously described liquid–liquid extraction method (De Baere et al., 2013). During the SCFA extraction, the bacterial suspension underwent centrifugation and 1000 μL of the supernatant was isolated. An internal standard, 50 μL of 0.2 M succinic acid (TCI, Tokyo, Japan), was then introduced. After thorough mixing, 100 μL of concentrated HCl (Sigma‐Aldrich, St. Louis, USA) was added. Diethyl ether (TCI, Tokyo, Japan) was subsequently added to allow extraction. The aqueous phase was discarded, and the retained organic phase was combined with 1000 μL of 1 M NaOH (TCI, Tokyo, Japan) solution for another extraction. The resulting aqueous phase was transferred to a microcentrifuge tube and mixed with additional concentrated HCl (Sigma‐Aldrich, St. Louis, USA). Following filtration through a 0.22 μm nylon membrane (Dikma, Foothill Ranch, USA), the samples were analysed using a HPLC–PDA system (Shimadzu LC‐2030, Shimadzu, Kyoto, Japan) to determine lactate, acetate, and propionate concentrations.

The preparation of the spent medium of L. acidophilus and V. dispar and analysis of their content

Lactobacillus acidophilus and V. dispar were each grown as monocultures, as well as in co‐culture, in TYLG medium until they reached the late stationary phase (24 h). Following this, the supernatant from each of the above cultures was separated by centrifugation (10,000 g for 10 min). Each supernatant was then sterilized using a 0.45 μm polyvinylidene difluoride membrane (Millipore, Burlington, USA) to generate the various spent media. For sterility verification, 50 μL of the spent media was spread onto both TYL and TYG agar plates. The spent media was then either directly used for bacterial growth or combined with TYLG medium at a ratio of 10% (v/v) to create a spent media‐supplemented TYLG medium. Subsequent bacterial growth and metabolic activities in these media were evaluated as previously described.

To analyse the constituents of the spent media derived from L. acidophilus monoculture (L‐spent), V. dispar monoculture (V‐spent), and the co‐culture (C‐spent), 50 μL of the medium was mixed with 450 μL of a methanol/water solution (8:1, v/v). This mixture was centrifuged at 15,000 g at 4°C for 15 min to separate the proteins. The resultant supernatant was then dried and redissolved in 40 μL of water. These processed samples were further analysed using LC–MS/MS. The employed LC–MS system consisted of an ACQUITY UPLC (Waters, Milford, USA) interfaced with a Xevo Q‐TOF G2‐XS mass spectrometer (Waters, Milford, USA). MS evaluations were conducted in positive ion mode, utilizing an electrospray ionization technique with full‐scan analysis spanning 50–1200 m/z. Data interpretation was facilitated by Progenesis QI software.

Dual‐RNA sequencing of V. dispar and L. acidophilus during co‐culture

Bacteria were harvested via centrifugation at 15,000 g for 1 min at 25°C. Monoculture of L. acidophilus and V. dispar were each harvested during their log phase (7 h for L. acidophilus and 6 h for V. dispar) and early‐stationary phase (13.5 h for both L. acidophilus and V. dispar). Co‐cultures of L. acidophilus and V. dispar were harvested during the log (6.5 h) and early stationary phase (13.5 h). Total RNA was extracted using the Presto mini RNA bacteria kit (Geneaid, Taiwan) by following the manufacturer's instructions. The concentration of the extracted RNA samples was determined using a Qubit fluorometer (Qubit 4, ThermoFisher Scientific, Waltham, USA), and their purity and integrity were assessed with a NanoPhotometer and an automated CE System (Agilent FA5200, Agilent, Santa Clara, USA), respectively. For RNA sequencing, cDNA libraries were prepared using an Illumina Ribo‐Zero Plus rRNA Depletion kit (Illumina, San Diego, USA), adhering to the manufacturer's protocols. RNA‐seq, using Illumina pair‐end reads (150‐bp), was conducted on a Novaseq 6000 platform (Illumina, San Diego, USA) by Genomics Co. in Taiwan. The reads were trimmed and filtered to ensure data quality, using a Phred score threshold of 30.

For the monoculture of L. acidophilus ATCC 4356T, the reads were aligned to the L. acidophilus ATCC 4356T reference genome. For the monoculture of V. dispar ATCC 17748T, reads were mapped to the V. dispar ATCC 17748T reference genome. The co‐culture samples were independently mapped in two cycles to genomes of L. acidophilus and V. dispar, respectively (Mutha et al., 2019). The statistics for aligning the transcripts to specific species are shown in Table S1. The raw read counts were then analysed using iDEP (Ge et al., 2018), an online platform that offers integrated RNA‐seq data analysis tools. Differential gene expression analysis and principal‐component analysis (PCA) were performed using this application. Differential gene expression analysis was conducted using the DEseq2 method. A Log2 fold change greater than 1 or less than −1 was established as the threshold for identifying differentially expressed genes. Lastly, KEGG enrichment pathway analysis was conducted using i‐KOBAS (Bu et al., 2021).

Statistical analysis

All data were analysed using unpaired t‐tests using GraphPad Prism 8.3. Differences were considered to be statistically significant at a p‐value of less than 0.05. The results are presented as mean ± standard error of the mean (SEM) based on three independent experiments.

RESULTS

Interaction between V. dispar and lactate‐producing lactobacilli demonstrates species preference

To determine whether a mutualistic relationship exists between the lactate utilizer V. dispar and lactate‐producing lactobacilli, V. dispar was co‐cultured with three representative lactobacillus species: L. acidophilus, L. paracasei, and L. plantarum in the TYLG medium. The TYLG medium, in terms of its carbon sources contains both L‐lactate and glucose. Initially, growth, lactate levels, and short‐chain fatty acid (SCFA) production were monitored in monocultures of L. acidophilus (Figure 1A), L. paracasei (Figure 1B), L. plantarum (Figure 1C), and V. dispar (Figure 1D). Additionally, the same parameters were monitored in co‐cultures of V. dispar with L. acidophilus (Figure 1E), L. paracasei (Figure 1F), and L. plantarum (Figure 1G). In the lactobacillus monoculture, the metabolic profiles are quite similar. All strains consume glucose and produce lactate without generating acetate. However, the lactate production activity varies among the three strains. By the late stationary phase (24 h), once all the glucose has been consumed, L. acidophilus has produced 12.4 ± 1.26 mM of lactate, while L. paracasei has produced 14.65 ± 0.64 mM, and L. plantarum has produced 14.12 ± 1.11 mM.

FIGURE 1.

FIGURE 1

Open in a new tab

Growth and metabolic pattern of monoculture and co‐culture of lactobacilli and Veillonella dispar. The growth and metabolic pattern of monoculture of (A) Lactobacillus acidophilus, (B) L. paracasei, (C) L. plantarum (D) V. dispar and the dual‐species co‐culture of (E) L. acidophilus/V. dispar, (F) L. paracasei/V. dispar and (G) L. plantarum/V. dispar. (H) Late stationary phase (24 h) optical density of monoculture of L. acidophilus, L. paracasei and L. plantarum and their co‐culture with V. dispar (I) Late stationary phase (24 h) acetate production of monoculture of V. dispar and co‐culture of L. acidophilus/V. dispar, L. paracasei/V. dispar, and L. plantarum/V. dispar. (J) Late stationary phase (24 h) propionate production of monoculture of V. dispar and co‐culture of L. acidophilus/V. dispar, L. paracasei/V. dispar, and L. plantarum/V. dispar. L. acidophilus (La), L. paracasei (Lpc), L. plantarum (Lpl), V. dispar (Vd). O.D., optical density. All the data are mean ± SEM of three biological replicates; unpaired two‐tailed t test was used for statistical analysis. ns, not significant; *p < 0.05; **p < 0.005; ***p < 0.001.

When V. dispar was co‐cultured with L. acidophilus, high lactate levels persisted throughout the early cultivation stages. However, after the 7‐h co‐culture, a notable decline in lactate was observed, and there was a concomitant rise in the production of acetate and propionate. This shift suggests that there has been an initiation of an active cross‐feeding. Remarkably, during the stationary phase, acetate levels in co‐culture were 2.50 times higher than in Veillonella monoculture, while propionate levels were 2.27 times higher compared to the V. dispar monoculture (Figure 1E).

In the case of V. dispar co‐cultured with L. paracasei, lactate levels rose steadily during initial cultivation and then began to fall around after 9 h of incubation. By the late stationary phase (24 h), acetate levels in the co‐culture were 1.99 times higher, and propionate levels were 1.76 times higher than in V. dispar monoculture. These patterns suggest a lactate consumer/provider relationship between V. dispar and L. paracasei during their co‐culture (Figure 1F).

When V. dispar was co‐cultured with L. plantarum, the onset of lactate cross‐feeding behaviour was delayed compared to the other two lactobacillus strains, with a decrease in lactate concentration occurring after 11 h. After 24 h, the level of acetate in the co‐culture was 1.45 times higher and propionate levels were 1.26 times higher compared to V. dispar monoculture. These findings point to a less pronounced metabolic cross‐feeding relationship compared to that between V. dispar and L. acidophilus (Figure 1G).

The co‐culture of V. dispar and L. acidophilus exhibited the most significant increase in growth (Figure 1H) and SCFA production (Figure 1I,J). Thus, our results suggest that the interaction between a particular lactate‐producer and a specific lactate‐utilizer is species‐specific, with V. dispar and L. acidophilus showing the strongest microbe‐microbe interaction in terms of growth promotion and metabolite cross‐feeding. In light of this, we subsequently focused on the relationship between V. dispar and L. acidophilus in order to explore in more detail the relationship between these two strains during co‐culture.

Mutualism between V. dispar and L. acidophilus enhances growth, viability, and metabolic activity

We next confirmed the mutualistic relationship between V. dispar and L. acidophilus by showing the co‐culture promoted viability, increased growth, and enhanced the metabolic activity of the two co‐cultured strains.

The growth rate, maximum cell count, and viability of V. dispar and L. acidophilus in both monoculture and co‐culture were monitored during their growth in the TYLG medium. All the following cell count refers to colony‐forming units (CFUs). V. dispar in monoculture and co‐culture had a similar growth rate during the log phase. However, V. dispar in co‐culture demonstrated a notably elevated maximum viable cell count compared to monoculture (1.1 × 109 vs. 7.1 × 108 CFU/mL). In addition, during the late stationary phase (24 h), the viable cell count of V. dispar in co‐culture was 5.0 times higher than that in monoculture (4.4 × 108 vs. 7.5 × 107 CFU/mL), suggesting the presence of L. acidophilus conferred a growth advantage on V. dispar during co‐culture (Figure 2A).

FIGURE 2.

FIGURE 2

Open in a new tab

Mutualism between Veillonella dispar and Lactobacillus acidophilus and their growth, viability, and metabolic activity. (A) Viable cell counts of V. dispar during the monoculture and co‐culture. Statistical analysis was conducted during the late stationary phase (24 h). (B) Viable cell counts of L. acidophilus during the monoculture and co‐culture. Statistical analysis was conducted during the late stationary phase (24 h). (C) Growth (optical density), (D) pH, (E) glucose concentration, (F) lactate concentration, (G) acetate concentration, and (H) propionate concentration during monoculture of V. dispar and L. acidophilus and during dual‐species co‐culture. ND, not detected. All the data are mean ± SEM of three biological replicates; an unpaired two‐tailed t‐test was used for statistical analysis. ns, not significant; *p < 0.05; **p < 0.005; ***p < 0.001.

As for L. acidophilus, the growth rates in the log phase of L. acidophilus in monoculture and co‐culture were also similar; however, the lag phase of L. acidophilus in co‐culture was shortened, indicating that the presence of V. dispar resulted in a growth‐promoting effect at the initial stages of L. acidophilus growth. Furthermore, L. acidophilus in the co‐culture exhibited higher viability during the late stationary phase (24 h) compared to monoculture (1.1 × 108 vs. 6.7 × 107 CFU/mL), suggesting that L. acidophilus also benefited from the interaction with V. dispar (Figure 2B).

The growth in monoculture and co‐culture was also assessed by measuring optical density during growth. The maximal optical density of the co‐culture was 1.99 ± 0.02, which was significantly higher than the optical densities of 1.2 ± 0.01 (monoculture of L. acidophilus) and 0.34 ± 0.001 (monoculture of V. dispar). These results show that there was growth enhancement relating to the interactions between the two bacteria (Figure 2C). The fluctuation in the pH of the medium also provided information on the metabolic activity of the lactate‐producing/utilizing bacteria. The pH of the V. dispar monoculture remained stable, while that of the L. acidophilus monoculture dropped significantly due to lactate production. By way of contrast, the pH of the co‐culture was slightly elevated compared to that of L. acidophilus monoculture (Figure 2D). During co‐culture, glucose and lactate were utilized. It should be noted that the levels of short‐chain fatty acids, such as acetate and propionate were both significantly increased during the stationary phase in co‐culture compared to V. dispar monoculture. These results show that L. acidophilus promoted the metabolic activity of V. dispar during co‐culture (Figure 2E–H).

Lactobacillus acidophilus provided not only lactate but also glutamate to V. dispar during co‐culture

Microbes modify their surrounding environments during growth and often interact with other microbial species (Figure 3A). To investigate whether the chemical environment created by one species exerts a directional effect on another species, we collected cell‐free spent media from late‐stage cultures (24 h). We then analysed the metabolic activity and growth of each species via this spent media. The samples obtained included L. acidophilus spent media (L‐spent) and V. dispar spent media (V‐spent). These spent media were either used directly for bacterial incubation (100% spent media) or combined with TYLG medium to generate a 10% (v/v) spent media‐supplemented TYLG medium. We also collected co‐cultured spent media (C‐spent) in order to carry out amnio acid content analysis.

FIGURE 3.

FIGURE 3

Open in a new tab

Directional nutrient exchanges during the co‐culture of Lactobacillus acidophilus and Veillonella dispar. (A) Schematic graph showing the proposed directional nutrient exchange. La, L. acidophilus; Vd, V. dispar. (B) The growth and metabolic pattern of monoculture of V. dispar grown in 10% L‐spent TYLG medium. The pattern of monoculture of V. dispar grown in the TYLG medium is previously shown in Figure 1D. (C) Late stationary phase (24 h) optical density of V. dispar during growth in the TYLG medium and 10% L‐spent medium. The data were compared with those in Figure 1D and Figure 3B. (D) The amino acid catabolism and biosynthesis of L. acidophilus, V. dispar, or co‐culture were analysed by comparing the increase or decrease in amino acid content in the spent media (V‐spent, L‐spent, or C‐spent) versus that of TYLG medium. *p < 0.05; **p < 0.005; ***p < 0.001. (E) Glutamate levels in TYLG medium, V‐spent medium, C‐spent medium, or L‐spent medium. (F) Aspartate levels in TYLG medium, V‐spent medium, C‐spent medium, or L‐spent medium. The detailed relative quantification of other amino acids was compared and is shown in Figure S1. a.u., arbitrary unit. All the data are mean ± SEM of three biological replicates; unpaired two‐tailed t test was used for statistical analysis; ns, not significant; *p < 0.05; **p < 0.005; ***p < 0.001.

To investigate the influence of L. acidophilus on V. dispar, we studied the growth and metabolic activity of V. dispar in 10% L‐spent medium or 100% L‐spent medium (Figure 3B). We found that L. acidophilus promoted the growth of V. dispar by acting as more than just a lactate provider. The late stationary phase (24 h) turbidity of V. dispar was 1.2 times higher in the 10% L‐spent medium (0.41 ± 0.02) than in TYLG medium (0.34 ± 0.001) even though both 10% L‐spent and TYLG media started with an initial lactate level of 10 mM (Figure 3C). Thus, our results suggest that L. acidophilus enhances V. dispar growth in way or ways other than just lactate provision.

To investigate whether the mutualistic relationship between L. acidophilus and V. dispar involves the cross‐feeding of other nutrients, such as amino acids, we studied the biosynthesis and catabolism of amino acids by both L. acidophilus and V. dispar in TYLG medium (Figure 3D; Figure S1). We found that L. acidophilus and V. dispar exhibited an amino acid production‐utilization relationship that involved glutamate (Figure 3D,E). In the L‐spent medium, the glutamate level showed an 81% increase relative to the TYLG medium, suggesting that L. acidophilus produced glutamate during growth. Conversely, for V. dispar, in the V‐spent medium, the glutamate level decreased by 56% compared to the TYLG medium. However, during co‐culture with V. dispar, the elevation in glutamate by L. acidophilus was markedly diminished (Figure 3E). This suggests that co‐cultured V. dispar consumed glutamate, highlighting another cross‐feeding relationship. We also found an augmented utilization of aspartate during the co‐culture (Figure 3F). Collectively, our findings imply that L. acidophilus supplies V. dispar not just with lactate, but also with glutamate.

The environmental changes induced by V. dispar are linked to an increase in the fermentation ability of L. acidophilus

To explore the influence of V. dispar on L. acidophilus, we evaluated the growth and metabolic activities of L. acidophilus in either 10% V‐spent medium or 100% V‐spent medium. The growth pattern of L. acidophilus grown in 10% V‐spent medium or 100% V‐spent medium were comparable to that in TYLG medium (Figure 4A,B). We further investigated the effect of V. dispar on the fermentation ability of L. acidophilus. When grown in the 100% V‐spent medium, the net lactate production by L. acidophilus surged by 37% compared to when it was cultivated in the TYLG medium (17.0 ± 0.11 mM vs 12.4 ± 0.68 mM), despite the initial glucose concentration in the 100% V‐spent being the same as TYLG medium (Figure 4B). On the other hand, when grown in the 10% V‐spent medium, the net lactate production of L. acidophilus in 10% V‐spent medium was not significantly higher than in TYLG medium (Figure 4C,D). Our findings suggest that the changes to the environment brought about by V. dispar (100% V‐spent medium versus 10% V‐spent medium) has a directional effect on L. acidophils, leading to an increase in its lactate fermentation ability.

FIGURE 4.

FIGURE 4

Open in a new tab

Effects of the culture supernatant from Veillonella dispar (V‐spent) on the lactate fermentation ability of Lactobacillus acidophilus. (A) The growth and metabolic pattern of monoculture of L. acidophilus (La) grown in 10% V‐spent TYLG medium. (B) The growth and metabolic pattern of monoculture of L. acidophilus (La) grown in 100% V‐spent TYLG medium. (C) Net production of lactate during the growth of L. acidophilus in 100% V‐spent medium, 10% V‐spent medium, and TYLG medium. The background L‐lactate concentration at zero time was non‐detectable in 100% V‐spent medium, 8.94 mM in 10% V‐spent medium, and 10 mM in TYLG medium based on the analytical data shown in Figure 1D and Figure 4A,B. (D) Bar graph of the net lactate production at 24 h (referring to the time points in Figure 4C) of L. acidophilus during growth in 100% V‐spent medium, 10% V‐spent medium, and TYLG medium. All the data are mean ± SEM of three biological replicates. ns, not significant; *p < 0.05; **p < 0.005; ***p < 0.001.

Dual‐RNA sequencing of L. acidophilus and V. dispar revealed metabolic adaptation during co‐culture

To assess the potential changes in gene expression of L. acidophilus and V. dispar during co‐culture, we employed dual‐RNA sequencing. Our results indicated significant gene expression variations in both L. acidophilus and V. dispar during log phase co‐culture. Specifically, for co‐cultured L. acidophilus, 192 genes were upregulated, and 221 genes were downregulated, relative to monoculture. Similarly, for co‐cultured V. dispar, 253 genes were upregulated, while 211 were downregulated when compared to monoculture. Furthermore, principal component analysis distinctly separated the co‐culture samples from the monoculture samples for both species (Figure S2).

A gene set enrichment analysis based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database was conducted on the identified differentially expressed genes (DEGs). For co‐cultured L. acidophilus, the identified upregulated pathways were related to ribosomal proteins, purine/pyrimidine metabolism, aspartate catabolism, glutamate catabolism, and lactate fermentative pathway (glycolysis) (Table S2). The detailed differentially expressed genes and their expression fold‐changes are listed in Table 1. The shortened lag phase and enhanced growth found during coculturing might be explained by upregulation of ribosomal function, increased glycolysis, and greater purine/pyrimidine metabolism (Figure 5). In relation to the fermentation pathway of lactate production, the genes fba, gmpA, and eno were upregulated. Furthermore, an increase in expression was observed for the ldh gene, which is responsible for the conversion of pyruvate to lactate (Figure 5). The upregulated lactate fermentative pathway in co‐cultured L. acidophilus thus may enhance its interaction with V. dispar during co‐culture via lactate cross‐feeding.

TABLE 1.

Selected genes differently expressed of co‐cultured Lactobacillus acidophilus during the log phase.

Gene Log2 fold‐change Product
Purine metabolism
gmk 1.12 Guanylate kinase
purQ 1.49 Phosphoribosylformylglycinamidine synthase
purB 1.16 Adenylosuccinate lyase
purC 1.48 Phosphoribosylaminoimidazole‐succinocarboxamide synthase
purD 1.60 Phosphoribosylamine‐glycine ligase
purE 2.33 5‐(carboxyamino)imidazole ribonucleotide mutase
purH 2.22 Phosphoribosylaminoimidazolecarboxamide formyltransferase
purK 1.17 5‐(carboxyamino)imidazole ribonucleotide synthase
purL 2.34 Phosphoribosylformylglycinamidine synthase
purM 2.90 Phosphoribosylformylglycinamidine cyclo‐ligase
purN 1.60 Phosphoribosylglycinamide formyltransferase 1
prsA 1.36 Foldase protein
Pyrimidine metabolism
pyrG 1.31 CTP synthase
pyrF 1.83 Orotidine‐5′‐phosphate decarboxylase
pyrE 1.01 Orotate phosphoribosyltransferase
pyrB 1.03 Aspartate carbamoyltransferase catalytic subunit
pyrR 1.66 Uracil phosphoribosyltransferase
Glycolysis
fba 1.02 Fructose‐bisphosphate aldolase
gpmA 1.52 2,3‐bisphosphoglycerate‐dependent phosphoglycerate mutase
adhE 1.43 Acetaldehyde dehydrogenase
eno 1.08 Enolase
ldh 1.46 L‐lactate dehydrogenase
Alanine, aspartate and glutamate catabolism
purB 1.16 Adenylosuccinate lyase
purQ 1.49 Phosphoribosylformylglycinamidine synthase
pyrB 1.03 Aspartate carbamoyltransferase catalytic subunit
purL 2.35 Phosphoribosylformylglycinamidine synthase
Cysteine and methionine metabolism
ilvE 1.27 Branched‐chain amino acid aminotransferase
metA 1.25 Homoserine O‐succinyltransferase
ldh 1.46 L‐lactate dehydrogenase
luxS 1.94 S‐ribosylhomocysteine lyase
Open in a new tab

Note: All genes listed were differentially expressed compared to their expression in monoculture.

FIGURE 5.

FIGURE 5

Open in a new tab

Mutualistic relationship between Lactobacillus acidophilus and Veillonella dispar and their gene regulation and major metabolites during co‐culture. The gene highlighted in red indicates upregulation, and black denotes no difference in expression compared to monoculture. The upregulated pathways were analysed through gene set enrichment analysis of differentially expressed genes during co‐culture in comparison to monoculture, utilizing the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. During co‐culture, both species displayed enhanced cell growth and viability. Gene regulation patterns associated with nutrient adaptation were summarized. L. acidophilus exhibited significant upregulation in growth‐related pathways, specifically involving in ribosomal proteins, purine/pyrimidine metabolism, and glycolysis. Furthermore, the lactate fermentation pathway showed heightened activity, with key upregulated genes including fba, gmpA, eno, and ldh—integral players in lactate production. Amino acids metabolic pathway such as glutamine catabolism to glutamate was upregulated. Similarly, V. dispar demonstrated upregulated growth‐related pathways related to pyrimidine metabolism. Additionally, amino acid metabolic pathways exhibited heightened activity of catabolism of aspartate and the biosynthesis of histidine, phenylalanine, tyrosine, tryptophan, and arginine. Glutamate secreted by L. acidophilus enters the central carbarn metabolism via the gene rocG to form α‐ketoglutarate. The higher production of acetate and propionate from co‐culture and beneficial effects of SCFAs are illustrated. Ala, alanine; Arg, arginine; argH, argininosuccinate lyase; Asp, aspartate; carA, carbamoyl‐phosphate synthase small subunit; carB, carbamoyl‐phosphate synthase small subunit; Cys, cysteine; eno, enolase; fba, fructose‐bisphosphate aldolase; Fum, fumarate; Gln, glutamine; gltB, glutamate synthase (NADPH) large chai; Glu, glutamate; gmpA, 2,3‐bisphosphoglycerate‐dependent phosphoglycerate mutase; His, histidine; ldh, L‐lactate dehydrogenase; Met, methionine; Phe, phenylalanine; purQ, phosphoribosylformylglycinamidine synthase; Pyu, pyruvate; rocG, NAD‐specific glutamate dehydrogenase; Trp, tryptophan; Tyr, tyrosine; α‐KG, α‐ketoglutarate.

In the case of co‐cultured V. dispar, the metabolic pathways associated with amino acid biosynthesis/catabolism and pyrimidine metabolism were upregulated (Table S3). The upregulated pathways included the biosynthesis of histidine, arginine, phenylalanine, tyrosine, and tryptophan, as well as the catabolism of aspartate and glutamine. The detailed differentially expressed genes are listed in Table 2. For V. dispar, the genes argH, carA, carB, pyrB, and glmS were significantly upregulated. The argH gene (argininosuccinate lyase) is involved in the aspartate‐to‐fumarate catabolic pathway, and the fumarate converted from aspartate can then enter central carbon metabolism via the reverse TCA cycle pathway. In our metabolomic data, V. dispar actively consumed aspartate during monoculture, (Figure 3D) and the consumed aspartate was further augmented during co‐culture (Figure 3F), indicating consistency between our transcriptomic data and metabolomic data. The expression of genes related to glutamate catabolism was not significantly different. These included rocG (glutamate dehydrogenase), which catalyses the conversion of glutamate into α‐ketoglutarate. The converted α‐ketoglutarate is then able to enter central carbon metabolism via the reverse TCA cycle pathway. Another gene related to glutamate catabolism, gltB (glutamate synthase (NADPH) large chain), also exhibited conserved gene expression. Gene gltB catalyses the interconversion of glutamate to α‐ketoglutarate and glutamine. The genes carAB (carbamoyl‐phosphate synthase) were found to be upregulated and are involved in the arginine biosynthesis pathway from glutamine. In addition to converging into the biosynthesis of arginine, we noted that the intermediate of the glutamine catabolism to arginine biosynthesis pathway, carbamoyl‐phosphate, can link pyrimidine metabolism with aspartate, as detailed in Figure S3. Our findings suggest significant gene expression reprogramming in both L. acidophilus and V. dispar during co‐culturing, indicating gene‐level interactions between the two strains.

TABLE 2.

Selected genes differently expressed of co‐cultured Veillonella dispar during the log phase.

Gene Log2 fold‐change Product
Pyrimidine metabolism
carA 1.95 Carbamoyl‐phosphate synthase small subunit
carB 1.77 Carbamoyl‐phosphate synthase large subunit
pyrC 2.19 Dihydroorotase
pyrB 2.36 Aspartate carbamoyltransferase catalytic subunit
pyrK 1.97 Dihydroorotate dehydrogenase electron transfer subunit
Alanine, aspartate and glutamate catabolism
argH 2.11 Argininosuccinate lyase
carA 1.95 Carbamoyl‐phosphate synthase small subunit
carB 1.77 Carbamoyl‐phosphate synthase large subunit
pyrB 2.36 Aspartate carbamoyltransferase catalytic subunit
glmS 1.32 Glutamine‐fructose‐6‐phosphate transaminase (isomerizing)
rocG a 0.60 Glutamate dehydrogenase (NAD(P)+)
gltB a 0.66 Glutamate synthase (NADPH) large chain
Histidine biosynthesis
hisA 2.55 Phosphoribosylformimino‐5‐aminoimidazole carboxamide ribotide isomerase
hisB 2.75 Imidazoleglycerol‐phosphate dehydratase
hisC 1.36 Histidinol‐phosphate aminotransferase
hisD 2.75 Histidinol dehydrogenase
hisE 1.02 Phosphoribosyl‐ATP pyrophosphohydrolase
Phenylalanine, tyrosine and tryptophan biosynthesis
pheA 1.27 Chorismate mutase/prephenate dehydratase
hisC 1.36 Histidinol‐phosphate aminotransferase
aroA 1.57 3‐phosphoshikimate 1‐carboxyvinyltransferase
aroB 1.79 3‐dehydroquinate synthase
aroC 1.69 Chorismate synthase
aroF 1.25 3‐deoxy‐7‐phosphoheptulonate synthase
Arginine biosynthesis
argB 1.90 Acetylglutamate kinase
argC 1.73 N‐acetyl‐gamma‐glutamyl‐phosphate reductase
argD 1.65 Acetylornithine/N‐succinyldiaminopimelate aminotransferase
argH 2.11 Argininosuccinate lyase
Open in a new tab

Note: All genes listed were differentially expressed compared to their expression in monoculture.

a

Not differentially expressed but relating to glutamate/glutamine catabolism.

Co‐cultured L. acidophilus and V. dispar alter their gene expression during the growth phase transition

To understand the temporal dynamics of gene expression for L. acidophilus and V. dispar in co‐culture, we analysed the transcriptomes of both species during the early‐stationary and log phases. Our analysis revealed significant differences in gene expression between the early stationary phase and the log phase samples in both species during co‐culture.

Specifically, in the stationary phase of co‐cultured L. acidophilus, 490 genes were upregulated, and 433 genes were downregulated, compared to the log phase. In V. dispar, 359 genes were upregulated, and 363 genes were downregulated relative to the log phase.

In L. acidophilus, genes associated with the utilization of specific disaccharides and glycogen metabolism were upregulated during the early stationary phase, relative to the log phase. In contrast, purine metabolism and aminoacyl‐tRNA biosynthesis were downregulated in the stationary phase (Table S4), implying a reduction in microbial growth. For V. dispar, we identified an upregulation of a number of metabolic pathways including gluconeogenesis (Table S5). Conversely, there was a downregulation of the biosynthesis pathways of certain amino acids (phenylalanine, tyrosine, tryptophan, and histidine) and aminoacyl‐tRNA biosynthesis, suggesting a decrease in growth requirements. Overall, both species demonstrated downregulation of growth‐related genes, which suggested dynamic gene regulation is involved in the adaptation to the changing environment.

DISCUSSION

This study sheds light on the species‐specific mutualistic relationship between V. dispar and lactobacilli, particularly focusing on L. acidophilus. Our results emphasize the importance of species‐specific mutualistic interactions among lactate‐producing and lactate‐utilizing bacteria. Collectively, all three examined lactobacilli strains underwent lactate cross‐feeding with V. dispar. However, only L. acidophilus displayed a substantial growth benefit when co‐cultured with V. dispar (Figure 1H). This was not observed for L. paracasei and L. plantarum, which further highlight the unique nature of the interactions between L. acidophilus and V. dispar. Despite their common co‐occurrence, the relationship between lactobacilli and Veillonella has been rarely addressed. Our findings indicate that the dynamics and mechanisms between lactobacilli and Veillonella are more complex than previously thought.

One prevailing viewpoint suggests that V. dispar ferments excess lactate into weaker acids, like acetate and propionate, resulting in a less acidic milieu (Rogosa, 1964). This is consistent with our results; whereby, when lactobacilli were co‐cultured with V. dispar, the medium's pH rose compared to monoculture (Figure 1A–G). Apart from pH changes, the fermentation of lactate to propionate by Veillonella not only provides an additional energy source for the host but also enhances interactions with other commensal microbes (Scheiman et al., 2019). Within the human intestine, commensal anaerobic bacteria ferment indigestible fibre and lactate, to produce SCFAs (Sun et al., 2017). These SCFAs influence the growth of neighbouring bacteria (Huang et al., 2011). Therefore, in such a complex microbial ecosystem, production of acetate and propionate from Veillonella is likely to benefit both the host and other commensal bacteria (Figure 5). In particular, both Veillonella and lactobacilli are commonly identified as part of the ileum microbiota (Liu et al., 2023).

We found that L. acidophilus provides V. dispar with a growth advantage in the TYLG medium compared to monoculture. One foundational concept in understanding the evolution of cooperation is the utilization of the waste products of one species by another species (Buck et al., 2005). Consequently, the metabolite cross‐feeding between L. acidophilus and V. dispar is likely to underpin their cooperative and mutualistic relationship. In recent years, the identification of next‐generation probiotics candidate has become an important issue (Kumar et al., 2022). Veillonella species are potential next‐generation candidate due to its ability to produce acetate and propionate (SCFAs) during lactate fermentation (Scheiman et al., 2019). SCFAs are known to have beneficial effects on human such as maintain human gut ecosystem, improving immune regulation, and helping metabolic homeostasis (Canfora et al., 2015; Furusawa et al., 2013; Morrison & Preston, 2016; Parada Venegas et al., 2019). The combined metabolism of V. dispar and L. acidophilus will most likely result in health‐promotional effects on the host (Figure 5). In one recent study, co‐administration of L. acidophilus and Veillonella ratti, a murine species, improved DSS‐induced colitis in a mouse model (Li et al., 2023), which is in agreement with our hypothesis. In addition, it has been recently shown that the lactate‐producing gut probiotics Bifidobacterium longum subspecies infantis modulate the human microbiota and cross‐feeds lactate to Veillonella spp. in human in vivo (Button et al., 2023).

We also identified that V. dispar exerts a positive effect on the growth and fermentative prowess of L. acidophilus. It is worth noting that V. dispar might indirectly bolster the fermentative ability of L. acidophilus by consuming lactate that has accumulated in the common environment, resulting in increased lactate production (Figure 4C). The mutualistic growth dynamics when L. acidophilus and V. dispar are cocultured could stem from the optimization of growth conditions during their co‐culture. One plausible rationale is that the fast consumption of the metabolic byproduct lactate by V. dispar prevents its accumulation. Drawing a parallel, Veillonella atypica releases a signalling molecule that triggers upregulation of the α‐amylase encoding gene, amyB, in Streptococcus gordonii. This then enhances carbohydrate fermentation in an S. gordonii dual‐species biofilm (Egland et al., 2004). A similar signalling interplay between L. acidophilus and V. dispar might be in play.

Microbial interactions, especially mutualistic ones, are pivotal in determining the metabolic behaviour of complex communities. Our study underscores the deep‐rooted metabolic synergy between L. acidophilus and V. dispar, suggesting that their relationship extends beyond the traditional viewpoint of lactate as the exclusive mediator. Our analysis pinpoints the production and utilization dynamics of other amino acids, specifically glutamate, confirming their significance in the mutualism between L. acidophilus and V. dispar. In essence, microbial dynamics go beyond isolated nutrient interactions, and probably manifests as intricate webs of metabolic reliance.

Understanding gene regulation within microbial interactions is essential for elucidating the mechanisms by which co‐culturing modifies the metabolic landscapes. The dual‐RNA sequencing technique has previously been leveraged to explore the interplay and gene regulation between co‐aggregated S. gordonii and V. parvula (Mutha et al., 2019). In the current study, we utilized dual‐RNA sequencing to obtain insights into the transcriptomic adaptations of V. dispar and L. acidophilus during co‐culture. The upregulated pathways of V. dispar in co‐culture, particularly those associated with amino acid biosynthesis and metabolism, underscore the metabolic adaptability of this species. On the other hand, L. acidophilus undergoes an upregulation of fermentative and growth‐associated pathways, indicating a metabolic approach tailored to rapid proliferation. The augmented pathways are linked to ribosomal proteins synthesis, glycolysis, and purine/pyrimidine metabolism and these are, in turn, associated with a reduced lag phase and enhanced growth during co‐culture (Figure 2B). Such patterns suggest higher translational activity and increased nucleotide synthesis, positioning L. acidophilus with a growth advantage. Additionally, the prominent expression of glycolytic genes, especially the ldh gene, hints at an enhanced fermentative process, which is indicative of metabolic realignment to bolster the survival and growth of the species in the co‐culture. We have elucidated the diverse roles of the amino acids including aspartate, glutamate, and glutamine in the co‐culture for downstream catabolism of V. dispar (Figure 5). Glutamate, either made from glutamine or cross‐fed from L. acidophilus, is able to enter central carbon metabolism via the rocG gene (glutamate dehydrogenase). Meanwhile, the catabolism of aspartate and glutamine converges into pyrimidine metabolism, revealing an intricate connection between these differentially expressed pathways (Figure S3). In recent years, the manipulation of the gene expression of traditional probiotics such as L. plantarum in order to gain better health‐promoting effects is also being extensively explored (Blanch‐Asensio et al., 2024).

The in‐depth analysis presented in this study elucidates the nuanced, species‐specific mutualistic interplay between V. dispar and lactobacilli, notably L. acidophilus. We spotlight the comprehensive associations between lactate‐producing and lactate‐consuming bacteria. The identified growth benefits in dual‐species co‐culture, between L. acidophilus and V. dispar, underscore the imperative of understanding these bacterial interactions. Insights garnered from both metabolomic and transcriptomic evaluations further accentuate the significance of co‐culture in terms of potentially altering metabolic landscapes. Our findings highlight the profound complexity of inter‐bacterial interactions by representative lactate‐producing and utilizing bacteria.

CONCLUSION

We have identified that lactobacilli undergo a metabolic mutualistic interaction with V. dispar, with this process exhibiting specific species preferences. V. dispar showed the highest degree of mutualism with L. acidophilus, among the three examined lactobacilli in terms of growth and SCFA production. Both V. dispar and L. acidophilus experienced increased growth and viability during co‐culture. Additionally, L. acidophilus not only cross‐fed lactate but also glutamate to V. dispar during co‐culture. Co‐culture transcriptomic analysis revealed metabolic adaptations in both bacteria during co‐culture. The health promotion effect from their interaction may be further elucidated via animal studies. Our findings highlight the metabolites cross‐feeding between the traditional probiotics and potential next‐generation probiotics that may be able to produce better health‐promoting metabolites. These potential new probiotics or the combining of interactive bacteria may serve as a future direction for novel probiotic product development.

AUTHOR CONTRIBUTIONS

Shi‐Min Zhang: Conceptualization; formal analysis; investigation; methodology; project administration; writing – original draft; writing – review and editing. Jia‐He Hung: Conceptualization; formal analysis; investigation; methodology; project administration; writing – original draft; writing – review and editing. Tran Ngoc Yen: Conceptualization. Shir‐Ly Huang: Conceptualization; funding acquisition; resources; supervision; writing – review and editing.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest in this study.

Supporting information

Appendix S1.

MBT2-17-e14484-s001.docx (531.5KB, docx)

ACKNOWLEDGEMENTS

This research was supported by the National Science and Technology Council, Taiwan (NSTC 112‐2320‐B‐A49‐042).

Zhang, S.‐M. , Hung, J.‐H. , Yen, T.N. & Huang, S.‐L. (2024) Mutualistic interactions of lactate‐producing lactobacilli and lactate‐utilizing Veillonella dispar: Lactate and glutamate cross‐feeding for the enhanced growth and short‐chain fatty acid production. Microbial Biotechnology, 17, e14484. Available from: 10.1111/1751-7915.14484

DATA AVAILABILITY STATEMENT

The RNA‐seq raw sequences data have been submitted to the Sequence Read Archive (SRA) under the BioProject number PRJNA1009256.

REFERENCES

  1. Blanch‐Asensio, M. , Dey, S. , Tadimarri, V.S. & Sankaran, S. (2024) Expanding the genetic programmability of Lactiplantibacillus plantarum . Microbial Biotechnology, 17, e14335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradshaw, D.J. & Marsh, P.D. (1998) Analysis of pH–driven disruption of Oral microbial communities in vitro. Caries Research, 32, 456–462. [DOI] [PubMed] [Google Scholar]
  3. Bu, D. , Luo, H. , Huo, P. , Wang, Z. , Zhang, S. , He, Z. et al. (2021) KOBAS‐i: intelligent prioritization and exploratory visualization of biological functions for gene enrichment analysis. Nucleic Acids Research, 49, W317–W325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buck, B.L. , Altermann, E. , Svingerud, T. & Klaenhammer, T.R. (2005) Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM. Applied and Environmental Microbiology, 71, 8344–8351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Button, J.E. , Cosetta, C.M. , Reens, A.L. , Brooker, S.L. , Rowan‐Nash, A.D. , Lavin, R.C. et al. (2023) Precision modulation of dysbiotic adult microbiomes with a human‐milk‐derived synbiotic reshapes gut microbial composition and metabolites. Cell Host & Microbe, 31, 1523–1538. e10. [DOI] [PubMed] [Google Scholar]
  6. Canfora, E.E. , Jocken, J.W. & Blaak, E.E. (2015) Short‐chain fatty acids in control of body weight and insulin sensitivity. Nature Reviews Endocrinology, 11, 577–591. [DOI] [PubMed] [Google Scholar]
  7. Colautti, A. , Orecchia, E. , Comi, G. & Iacumin, L. (2022) Lactobacilli, a weapon to counteract pathogens through the inhibition of their virulence factors. Journal of Bacteriology, 204, e0027222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. De Baere, S. , Eeckhaut, V. , Steppe, M. , De Maesschalck, C. , De Backer, P. , Van Immerseel, F. et al. (2013) Development of a HPLC–UV method for the quantitative determination of four short‐chain fatty acids and lactic acid produced by intestinal bacteria during in vitro fermentation. Journal of Pharmaceutical and Biomedical Analysis, 80, 107–115. [DOI] [PubMed] [Google Scholar]
  9. Egland, P.G. , Palmer, R.J. & Kolenbrander, P.E. (2004) Interspecies communication in Streptococcus gordonii–Veillonella atypica biofilms: signaling in flow conditions requires juxtaposition. Proceedings of the National Academy of Sciences of the United States of America, 101, 16917–16922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Figueiredo, A.R. & Kramer, J. (2020) Cooperation and conflict within the microbiota and their effects on animal hosts. Frontiers in Ecology and Evolution, 8, 132. [Google Scholar]
  11. Furusawa, Y. , Obata, Y. , Fukuda, S. , Endo, T.A. , Nakato, G. , Takahashi, D. et al. (2013) Commensal microbe‐derived butyrate induces the differentiation of colonic regulatory T cells. Nature, 504, 446–450. [DOI] [PubMed] [Google Scholar]
  12. Ge, S.X. , Son, E.W. & Yao, R. (2018) iDEP: an integrated web application for differential expression and pathway analysis of RNA‐seq data. BMC Bioinformatics, 19, 1–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gross, E.L. , Beall, C.J. , Kutsch, S.R. , Firestone, N.D. , Leys, E.J. & Griffen, A.L. (2012) Beyond Streptococcus mutans: dental caries onset linked to multiple species by 16S rRNA community analysis. PLoS One, 7, e47722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Huang, C.B. , Alimova, Y. , Myers, T.M. & Ebersole, J.L. (2011) Short‐ and medium‐chain fatty acids exhibit antimicrobial activity for oral microorganisms. Archives of Oral Biology, 56, 650–654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ivashkiv, L.B. (2020) The hypoxia–lactate axis tempers inflammation. Nature Reviews Immunology, 20, 85–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kolenbrander, P.E. , Palmer, R.J. , Periasamy, S. & Jakubovics, N.S. (2010) Oral multispecies biofilm development and the key role of cell–cell distance. Nature Reviews Microbiology, 8, 471–480. [DOI] [PubMed] [Google Scholar]
  17. Kumar, R. , Sood, U. , Kaur, J. , Anand, S. , Gupta, V. , Patil, K.S. et al. (2022) The rising dominance of microbiology: what to expect in the next 15 years? Microbial Biotechnology, 15, 110–128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li, N. , Wang, H. , Zhao, H. , Wang, M. , Cai, J. , Hao, Y. et al. (2023) Cooperative interactions between Veillonella ratti and Lactobacillus acidophilus ameliorate DSS‐induced ulcerative colitis in mice. Food & Function, 14(23), 10475–10492. [DOI] [PubMed] [Google Scholar]
  19. Li, X. , Yang, Y. , Zhang, B. , Lin, X. , Fu, X. , An, Y. et al. (2022) Lactate metabolism in human health and disease. Signal Transduction and Targeted Therapy, 7, 305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liu, Q. , He, M. , Zeng, Z. , Huang, X. , Fang, S. , Zhao, Y. et al. (2023) Extensive identification of serum metabolites related to microbes in different gut locations and evaluating their associations with porcine fatness. Microbial Biotechnology, 16, 1293–1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mashima, I. , Theodorea, C.F. , Thaweboon, B. , Thaweboon, S. & Nakazawa, F. (2016) Identification of Veillonella species in the tongue biofilm by using a novel one‐step polymerase chain reaction method. PLoS One, 11, e0157516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Milho, C. , Silva, M.D. , Alves, D. , Oliveira, H. , Sousa, C. , Pastrana, L.M. et al. (2019) Escherichia coli and Salmonella enteritidis dual‐species biofilms: interspecies interactions and antibiofilm efficacy of phages. Scientific Reports, 9, 18183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Morrison, D.J. & Preston, T. (2016) Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism. Gut Microbes, 7, 189–200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moutinho, T.J. , Panagides, J.C. , Biggs, M.B. , Medlock, G.L. , Kolling, G.L. & Papin, J.A. (2017) Novel co‐culture plate enables growth dynamic‐based assessment of contact‐independent microbial interactions. PLoS One, 12, e0182163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mutha, N.V. , Mohammed, W.K. , Krasnogor, N. , Tan, G.Y. , Wee, W.Y. , Li, Y. et al. (2019) Transcriptional profiling of coaggregation interactions between Streptococcus gordonii and Veillonella parvula by dual RNA‐seq. Scientific Reports, 9, 7664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ng, S.K. & Hamilton, I.R. (1971) Lactate metabolism by Veillonella parvula . Journal of Bacteriology, 105, 999–1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Oña, L. , Giri, S. , Avermann, N. , Kreienbaum, M. , Thormann, K.M. & Kost, C. (2021) Obligate cross‐feeding expands the metabolic niche of bacteria. Nature Ecology & Evolution, 5, 1224–1232. [DOI] [PubMed] [Google Scholar]
  28. Parada Venegas, D. , De la Fuente, M.K. , Landskron, G. , González, M.J. , Quera, R. , Dijkstra, G. et al. (2019) Short chain fatty acids (SCFAs)‐mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases. Frontiers in Immunology, 10, 277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pucino, V. , Certo, M. , Bulusu, V. , Cucchi, D. , Goldmann, K. , Pontarini, E. et al. (2019) Lactate buildup at the site of chronic inflammation promotes disease by inducing CD4+ T cell metabolic rewiring. Cell Metabolism, 30, 1055–1074. e58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rickard, A.H. , Gilbert, P. , High, N.J. , Kolenbrander, P.E. & Handley, P.S. (2003) Bacterial coaggregation: an integral process in the development of multi‐species biofilms. Trends in Microbiology, 11, 94–100. [DOI] [PubMed] [Google Scholar]
  31. Rivière, A. , Gagnon, M. , Weckx, S. , Roy, D. & De Vuyst, L. (2015) Mutual cross‐feeding interactions between Bifidobacterium longum subsp. longum NCC2705 and Eubacterium rectale ATCC 33656 explain the bifidogenic and butyrogenic effects of arabinoxylan oligosaccharides. Applied and Environmental Microbiology, 81, 7767–7781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rogosa, M. (1964) The genus Veillonella I: general cultural, ecological, and biochemical considerations. Journal of Bacteriology, 87, 162–170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rogosa, M. & Bishop, F.S. (1964) The genus Veillonella III: hydrogen sulfide production by growing cultures. Journal of Bacteriology, 88, 37–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Scheiman, J. , Luber, J.M. , Chavkin, T.A. , Macdonald, T. , Tung, A. , Pham, L.‐D. et al. (2019) Meta‐omics analysis of elite athletes identifies a performance‐enhancing microbe that functions via lactate metabolism. Nature Medicine, 25, 1104–1109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shimizu, H. & Benno, Y. (2015) Membrane filter method to study the effects of Lactobacillus acidophilus and Bifidobacterium longum on fecal microbiota. Microbiology and Immunology, 59, 643–652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Strati, F. , Cavalieri, D. , Albanese, D. , De Felice, C. , Donati, C. , Hayek, J. et al. (2017) New evidences on the altered gut microbiota in autism spectrum disorders. Microbiome, 5, 24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stubbendieck, R.M. , Vargas‐Bautista, C. & Straight, P.D. (2016) Bacterial communities: interactions to scale. Frontiers in Microbiology, 7, 1234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sun, M. , Wu, W. , Liu, Z. & Cong, Y. (2017) Microbiota metabolite short chain fatty acids, GPCR, and inflammatory bowel diseases. Journal of Gastroenterology, 52, 1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tan, J. , McKenzie, C. , Vuillermin, P.J. , Goverse, G. , Vinuesa, C.G. , Mebius, R.E. et al. (2016) Dietary fiber and bacterial SCFA enhance Oral tolerance and protect against food allergy through diverse cellular pathways. Cell Reports, 15, 2809–2824. [DOI] [PubMed] [Google Scholar]
  40. Tassell, M.L.V. & Miller, M.J. (2011) Lactobacillus adhesion to mucus. Nutrients, 3, 613–636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wu, C.M. , Wheeler, K.M. , Cárcamo‐Oyarce, G. , Aoki, K. , McShane, A. , Datta, S.S. et al. (2023) Mucin glycans drive oral microbial community composition and function. npj Biofilms and Microbiomes, 9, 11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Xing, H. , Liu, H. & Pan, J. (2023) High‐throughput sequencing of Oral microbiota in Candida carriage Sjögren's syndrome patients: a pilot cross‐sectional study. Journal of Clinical Medicine, 12, 1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yang, K. , Fan, M. , Wang, X. , Xu, J. , Wang, Y. , Tu, F. et al. (2022) Lactate promotes macrophage HMGB1 lactylation, acetylation, and exosomal release in polymicrobial sepsis. Cell Death and Differentiation, 29, 133–146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhang, S.‐M. & Huang, S.‐L. (2023) The commensal anaerobe Veillonella dispar reprograms its lactate metabolism and short‐chain fatty acid production during the stationary phase. Microbiology Spectrum, 11, e0355822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zhang, X. , Chen, S. , Zhang, M. , Ren, F. , Ren, Y. , Li, Y. et al. (2021) Effects of fermented Milk containing Lacticaseibacillus paracasei strain Shirota on constipation in patients with depression: a randomized, double‐blind, placebo‐controlled trial. Nutrients, 13, 2238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zheng, J. , Wittouck, S. , Salvetti, E. , Franz, C.M. , Harris, H.M. , Mattarelli, P. et al. (2020) A taxonomic note on the genus Lactobacillus: description of 23 novel genera, emended description of the genus lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. International Journal of Systematic and Evolutionary Microbiology, 70, 2782–2858. [DOI] [PubMed] [Google Scholar]
  47. Zhou, P. , Manoil, D. , Belibasakis, G.N. & Kotsakis, G.A. (2021) Veillonellae: beyond bridging species in oral biofilm ecology. Frontiers in Oral Health, 2, 774115. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Appendix S1.

MBT2-17-e14484-s001.docx (531.5KB, docx)

Data Availability Statement

The RNA‐seq raw sequences data have been submitted to the Sequence Read Archive (SRA) under the BioProject number PRJNA1009256.

Articles from Microbial Biotechnology are provided here courtesy of Wiley

RESOURCES