Metallobiology of Lactobacillaceae in the gut microbiome

Abstract

Lactobacillaceae are a diverse family of lactic acid bacteria found in the gut microbiota of humans and many animals. These bacteria exhibit beneficial effects on intestinal health, including modulating the immune system and providing protection against pathogens, and many species are frequently used as probiotics. Gut bacteria acquire essential metal ions, like iron, zinc, and manganese, through the host diet and changes to the levels of these metals are often linked to alterations in microbial community composition, susceptibility to infection, and gastrointestinal diseases. Lactobacillaceae are frequently among the organisms increased or decreased in abundance due to changes in metal availability, yet many of the molecular mechanisms underlying these changes have yet to be defined. Metal requirements and metallotransporters have been studied in some species of Lactobacillaceae, but few of the mechanisms used by these bacteria to respond to metal limitation or excess have been investigated. This review provides a current overview of these mechanisms and covers how iron, zinc, and manganese impact Lactobacillaceae in the gut microbiota with an emphasis on their biochemical roles, requirements, and homeostatic mechanisms in several species.

1. Introduction

Lactobacillaceae comprise just up to ~1–2% of the total bacterial population in the distal human gut, yet these bacteria are frequently positively or negatively correlated with human disease, infection, and changes in diet or nutrient levels, including metals like zinc and iron [1–5]. The human microbiota is crucial for physiological functions including pathogen protection, immunity, and intestinal barrier regulation, and maintaining balance in this community of microbes is important for human health [6–11]. Like the hosts they inhabit, intestinal microbes require metal ions for their growth and function. Since these nutrients cannot be synthesized, bacteria must compete with host metal uptake mechanisms (Fig. 1) [5]. Conditions that change metal availability at the host-microbe interface include diet, drug treatment, and host-imposed metal withholding or overload [5,12–14]. The mechanisms by which invading pathogenic bacteria adapt to these changes have received significant attention, but resident intestinal species must also adapt and the mechanisms by which they acquire and regulate metals without harming the host is a more recent area of interest [5,14,15]. Host diet is well-recognized to impact the composition of the gut microbiota, and numerous studies have linked changes in dietary metal content with changes in the relative abundance of different groups of bacteria present in the intestinal contents [3–5,16–18]. The results of these studies correlate host dietary metal intake and gut microbiota composition but the changes in the gut microbiota are not consistent and poorly predictable, likely due to a lack of understanding of the precise molecular mechanisms by which metal ions interact with the gut microbiota. Specifically, few studies have examined how metal ions affect bacterial physiology in this complex ecosystem, including, for example, how beneficial gut bacteria acquire metal nutrients during intestinal inflammation or disease. Despite their low relative abundance in the gastrointestinal tract, Lactobacillaceae are intricately linked with human health and are often impacted by changes in iron, zinc, and manganese availability that correlate with gut pathologies and infection.

Fig. 1.

Intestinal absorption and homeostasis mechanisms for iron, zinc, and manganese. Heme iron is imported through heme carrier protein 1 (HCP1) and non-heme iron is reduced by duodenal cytochrome b (DcytB) then absorbed through the divalent metal transporter (DMT1) at the apical epithelial cell surface. Cellular heme is degraded by heme oxygenase (HO) and absorbed iron is used intracellularly, stored bound to ferritin, or exported through the basolateral membrane to the bloodstream as mediated by ferroportin (Fpn1). Zinc is primarily imported by the Zrt-/Irt-like protein (ZIP), ZIP4, and transported to intracellular organelles, bound to metallothionein (MT), or exported by ZnT1 to the bloodstream or ZnT5B back to the lumen. Zinc can also be imported at the basolateral membrane through ZIP5 or ZIP14. Manganese is absorbed through DMT1 on the apical membrane and exported by Fpn1 to the bloodstream where it can bind circulating proteins transferrin or albumin. Manganese can be imported by ZIP14 at the basolateral membrane and exported to the lumen via ZnT10.

2. Lactobacillaceae and the gut microbiota

In the complex microbial ecosystem of the gut microbiota, hundreds of species of bacteria and other microorganisms are competing for nutrients [19,20]. These microbes impact host health by modifying the nutrient supply, converting metabolites, and interacting with host cells [21,22]. Host-microbe interactions are crucial for regulating the immune system and providing protection against environmental and opportunistic resident pathogens [8,11]. Molecular approaches based on 16S rRNA analysis have identified the dominant taxa along the adult intestine [23]. The small intestine is rich in simple sugars and amino acids, supporting growth of Proteobacteria and Lactobacillales [24]. The majority of carbohydrates in the large intestine are host-indigestible or host-derived complex carbohydrates. These polysaccharides support dominant populations of Bacteroides and Clostridiales, which have numerous enzymes for digesting and using complex sugars as energy sources [25]. Lactobacillales are comparatively much less abundant, but also have enzymes that can digest polysaccharides [26,27]. Many gut bacteria metabolize indigestible dietary compounds to produce beneficial metabolites like short chain fatty acids (SCFAs), including acetate, butyrate, and propionate.

The composition of the microbiota is affected by various factors, including diet and antibiotic use [16]. It is not well defined whether the type of diet can significantly contribute to host health or disease via alteration of the gut microbiota composition, but perturbations of the balanced microbiota that lead to gut dysbiosis, including those induced by antibiotic treatment, have been associated with gastrointestinal disorders like inflammatory bowel disease and metabolic disorders like obesity [28]. Efforts have long been made to modify the gut microbial community to treat these diseases or generally improve human health. One approach is to use probiotic organisms, which are beneficial to human health through several general mechanisms involving strengthening the intestinal barrier, modulating the immune response, and protecting against pathogens by producing antimicrobial compounds or competing for mucosal binding sites [29].

Lactic acid bacteria (Fig. 2), a group which includes Lactobacillaceae, represent ~0.01–1.8% of the total intestinal bacterial community [30]. Lactobacillaceae are commonly used as probiotics and have various documented beneficial effects including protection against pathogens and immune system stimulation [31,32]. These bacteria are also found in various foods and so not all are necessarily part of the resident microbiota but may be passing through from the host environment or diet. Species considered to permanently colonize the gastrointestinal tract include Lactobacillus acidophilus, Limosilactobacillus reuteri (formerly Lactobacillus reuteri), Levilactobacillus brevis (formerly Lactobacillus brevis), Lacticaseibacillus casei (formerly Lactobacillus casei), Lactobacillus crispatus, Lactobacillus delbrueckii, Limosilactobacillus fermentum (formerly Lactobacillus fermentum), Lactobacillus gasseri, Lacticaseibacillus paracasei (formerly Lactobacillus paracasei), Lactiplantibacillus plantarum (formerly Lactobacillus plantarum), Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus), Ligilactobacillus ruminis (formerly Lactobacillus ruminis), Latilactobacillus sakei (formerly Lactobacillus sakei), Ligilactobacillus salivarius (formerly Lactobacillus salivarius), and Limosilactobacillus vaginalis (formerly Lactobacillus vaginalis) [33,34]. Recent genetic analyses and the heterogeneity within the Lactobacillus genus led to a new taxonomy assigning numerous species to different genera [34]. Here we note the new and former names and to simplify the literature discussion we will use the higher order term for the family of Lactobacillaceae, which includes all Lactobacillus species and the other newly reclassified members. Lactobacillaceae are documented in numerous studies to be significantly enriched or diminished in the distal gut during health or disease [1]. We will discuss known and predicted metal requirements and homeostatic mechanisms for several Lactobacillaceae, including some of the intestinal species listed above.

Fig. 2.

Taxonomy of lactic acid bacteria covered in this review.

3. Iron

3.1. Iron and Lactobacillaceae in the gut microbiota

Disorders that affect iron balance are among the most prevalent human diseases. Iron deficiency anemia is the most common and widespread human nutritional disorder worldwide and is generally treated by oral iron administration. Only ~5–20% of total dietary iron is ultimately absorbed by humans, and most of this iron is absorbed in the duodenum of the small intestine [35,36]. Unabsorbed excess iron may affect the microbiota and stimulate growth and virulence of bacterial pathogens in the gastrointestinal tract. It is challenging to evaluate the availability of this luminal iron to the microbiota (and host) because numerous dynamic parameters cause iron levels to fluctuate [37]. These factors include diet, pH, and microbiota activity. The presence of different chemical forms of iron bound to food-derived components, mucus, lactate, and amino acids can further affect available iron levels [37,38]. During infection or inflammation, iron levels are expected to be restricted due to the host immune response that uses chelation of iron to prevent acquisition by pathogens (nutritional immunity) [15]. The microbiota can also influence local and systemic iron regulation, further modulating the bioavailable iron levels. For example, in iron deficient conditions Lactobacillaceae species produce small molecule metabolites such as 1,3-diaminopropane and reuterin, which can repress hypoxia-inducible factor 2α (HIF2α) and reduce transcription of iron transporters (Fig. 3) [39].

Fig. 3.

Mechanisms for regulation of iron uptake during iron deficiency. Under iron deficiency duodenal hypoxia-inducible factor 2α (HIF2α) is stabilized, which transcriptionally upregulates expression of DMT1, Dcytb, and ferroportin to maximize iron uptake. Lactobacillaceae also produce small molecule metabolites that can repress HIF2α and reduce transcription of the iron transporters.

Several studies have investigated how iron affects the gut microbiota and Lactobacillaceae (Table 1). The outcomes of in vivo and in vitro trials are complex, but a relatively consistent finding is an iron-induced decrease in beneficial Lactobacillaceae and Bifidobacteria towards Enterobacteriaceae (a family of bacteria including opportunistic E. coli and Salmonella species) [37,40]. Several oral iron supplementation trials demonstrate an increased risk of diarrhea linked to higher levels of luminal iron, which shifts the gut microbiota composition toward a more pathogenic profile [40–45]. Some studies, however, have shown decreases in beneficial bacteria with increased dietary iron, but no increases in pathogenic or opportunistic Enterobacteriaceae [46–48]. In one small group of children no significant effects were observed [49]. A separate study examining the effects of iron in formula on infants found that a difference in iron content did not affect Lactobacillaceae or Bifidobacteria but led to an increase in Clostridia and decrease in Bacteroidetes [50]. A study on young women with iron deficiency anemia found that fecal levels of L. acidophilus were significantly lower in the women with low blood iron and hemoglobin [51]. Animal studies have yielded similar results, including that iron supplementation can lead to decreased Lactobacillaceae and/or Bifidobacteria [52,53]. Other varied observations include minimal effects from iron supplementation on the microbiota, increased Lactobacillaceae levels, or effects on a variety of other bacteria, likely due to the variety of animal models and study conditions [54–58]. Some mouse and rat studies and a study using in vitro fermentation models have found evidence that iron deprivation leads to an increase in Lactobacillaceae [59–62]. In addition to the direct impacts of altered luminal iron levels on microbes, excess iron may indirectly affect the microbiota and host-microbe interactions via induced inflammation [63].

Table 1.

Summary of studies on the effects of iron on Lactobacillaceae.

	Research subject	Research design	Lactobacillaceae change	Reference
Iron supplementation	Dutch infants	5 mg/L Ferric citrate in milk for 1st week of life	No change	[45]
	UK infants	Iron fortified formula for 2 weeks	No change	[84]
	UK infants	Iron and lactoferrin fortified formula up to 15 weeks	No change	[50]
	Sprague-Dawley Rats	200 mg/kg FeSO4 (males, single dose) and 50 and 150 mg/kg FeSO4 (females, over 4 weeks)	Increase	[56]
	Male Swiss-Webster Mice	121 mg or 1.59 g FeCl3/kg compared to control with 2 mg/kg Fe from base diet	Decrease	[59]
	Weaning piglets	50, 100, or 250 mg FeSO4/kg from birth to 56 days	No change	[54]
	African children	Fortified biscuits with 20 mg Fe/day 4 days/week for 6 months	Decrease	[44]
	U.S. infants (5-mo old breastfed)	Iron-fortified infant cereal for 4–5 months	Decrease	[46]
	Germ-free female Fischer 344 rats	Fe-deficient diet for 12 weeks and 35 mg/kg FeSO4 or 70 mg/kg FeSO4/ferric citrate diet for 4 weeks	No change	[57]
	South African children	50 mg FeSO4 4 days/week for 38 weeks	No change	[49]
	Kenyan infants (6-mo old breastfed)	Micronutrient powder with 2.5 mg Fe (as NaFeEDTA) or 12.5 mg Fe as ferrous fumarate daily for 4 months	Decrease	[43]
	Female C57BL/6 mice	High iron (225 mg FeSO4/kg) compared to deficient (2–6 mg/kg) and normal (45 mg FeSO4/kg) levels for 27 days	Decrease	[52]
	In vitro fermentation model	50 (medium) or 250 (high) μM FeSO4, 50 or 250 μM ferric citrate, or 50 μM hemin compared to no iron	Decrease	[85]
	Male C57BL/6 weaning mice	704 mg/kg FeSO4 compared to 202 mg/kg (basal) and 450 mg/kg for 30 days	Decrease	[53]
Iron deficiency	Wildtype and heterozygous TNFΔARE/WT mice	FeSO4 free diet (<10 mg/kg) for 11 weeks	Increase	[60]
	Male Sprague-Dawley rats	Fe-deficient diet for 24 days, repleted with FeSO4 or electrolytic Fe at 10 and 20 mg/kg for 14 days	Increase	[61]
	In vitro colonic fermentation model	Low iron (3.91 mg Fe/L media)	Increase	[62]
	Germ-free female Fischer 344 rats	Fe-deficient diet for 12 weeks	No change	[57]
	Sprague-Dawley rat pups	30 or 150 μg FeSO4/day for 56 days	Increase in growth-restricted rats	[55]

The gut microbiome composition is also affected by host iron status, further complicating studies aiming to unravel the effects of dietary iron on the gut microbiota. Genetic modification of iron metabolism genes can affect gut microbes without changes in dietary iron [64]. When iron regulatory protein 2 (IRP2) was knocked out in mice, resulting in increased iron, Lactobacillus intestinalis and Ligilactobacillus murinus (formerly Lactobacillus murinus) increased. In wildtype mice, however, another species, Lactobacillus johnsonii, was more abundant. In mice where the hereditary hemochromatosis gene was knocked out, which did not affect fecal iron levels compared to wildtype (although mutations can lead to iron overload), L. johnsonii was significantly decreased compared to wildtype and other bacteria were increased.

Host iron levels affect the microbiota, yet the microbiota can also affect host iron sensing. In germ-free and microbiota-colonized mice, microbial colonization decreases iron concentration in the cecum but not the plasma or liver [65]. In another study, germ-free mice placed on an iron-deficient diet exhibited milder iron deficiency anemia, perhaps due to more efficient iron uptake in the absence of a competing microbiota [39]. This work also showed that some bacteria (e.g. Lactobacillaceae) produce metabolites that suppress HIF2α, resulting in decreased host iron absorption (Fig. 3). Production of these metabolites, specifically 1,3-diaminopropane, was enhanced during dietary iron deficiency. Conversely, earlier work found that Limosilactobacillus fermentum (formerly Lactobacillus fermentum) excretes a molecule, p-hydroxyphenyllactic acid (HPLA), that reduces Fe³⁺ to Fe²⁺, promoting iron uptake by enterocytes in a manner that likely mimics the function of the DcytB ferric-reducing protein (Fig. 1) [66]. This study was the first to demonstrate a possible mechanism explaining several observations that some probiotics can enhance iron absorption, as discussed below [67]. These examples highlight how the microbiota can modulate luminal iron availability and local host iron homeostasis. Mouse models also suggest that the microbiota can affect hepcidin-regulated systemic iron homeostasis by modulating systemic inflammatory responses [68].

Probiotics have been linked to modulating iron absorption. Several studies and a meta-analysis showed that L. plantarum 299v enhances iron absorption in humans [69–75]. In a colonic epithelial cell line, L. acidophilus increased iron uptake while Bifidobacterium infantis decreased iron uptake [76]. Other studies also correlated both L. acidophilus and L. fermentum with increased iron absorption [77,78]. Proposed mechanisms for increased enterocyte iron uptake include metabolite formation (e.g. HPLA as for L. fermentum) [66], enhanced mucin production at the intestinal surface [79], and promotion of an anti-inflammatory immune response that suppresses hepcidin [80,81]. Lactobacillaceae could also increase iron absorption by metabolizing dietary compounds like phytic acid, which typically bind iron and zinc and restrict the bioavailability of these metals [82].

Taken together, it is apparent that Lactobacillaceae are a family of bacteria that can both be affected by dietary iron levels and play roles in modulating host iron homeostasis. As will be discussed below, Lactobacillaceae are recognized in inorganic biochemistry for their low iron requirement. This characteristic could explain their ability to grow well in milk and predominate in the gut microbiota of breast-fed infants, given that milk is a highly iron-restricted medium with high concentrations of the iron-binding protein, lactoferrin [83]. It could also explain some of the above studies where iron-deficient conditions have correlated with increased relative levels of Lactobacillaceae [59–62]. At the same time, the opposite effect is reported, and the disparity could be due to differences in iron metabolism for different species and strains and/or different experimental and environmental conditions of the studies. Studies showing that some Lactobacillaceae make metabolites that minimize host iron uptake while others make molecules that enhance it further complicate the picture [39,66]. Despite significant progress in recent years identifying possible mechanisms by which iron interacts with the gut microbiota, much of the molecular basis and biochemical mechanisms remain unclear, highlighting the importance of detailed mechanistic studies on these bacteria and their interactions with each other and the host.

3.2. Iron metabolism and transport in Lactobacillaceae

Lactobacillaceae are perhaps most famous in the bioinorganic literature for their general lack of an iron requirement. From the earliest studies on their mineral requirements, Lactobacillaceae were found to grow in the presence of little to no iron [86,87]. In 1983, Archibald reported that there was no significant effect on growth of L. plantarum in complex All purpose Tween 80 (APT)-glucose medium treated with an iron chelator [88]. Furthermore, cells grown in ⁵⁹FeCl₃-supplemented medium had minimal uptake of ⁵⁹Fe compared to E. coli grown under the same conditions. Subsequent studies showed that several species of Lactobacillaceae and related organisms could grow well in iron-chelated medium and when non-iron-specific chelators inhibited growth, it could not be restored with iron addition [89,90]. No ⁵⁹Fe uptake or siderophore production was detected among 23 lactic acid bacteria species across several genera including those within the Lactobacillaceae family [90]. These studies were all carried out in various types of complex media, making it challenging to identify individual factors that affect the bacteria. In E. coli, for example, different proteomes can be detected when grown in different media [91]. In an earlier study that contrasts the above findings, a defined medium was used to study the effects of each component on growth of 11 different organisms, including L. acidophilus ATCC 4356, and found growth inhibition of all strains in the absence of Mn²⁺ and/or Fe²⁺ [92]. Later work investigating iron and manganese requirements in the same chemically defined medium under aerobic and anaerobic conditions found that supplementation of chelated iron in the presence of manganese did not affect bacterial growth for the four strains studied, including L. acidophilus ATCC 4356 and L. plantarum LMG 6907^T [93]. L. acidophilus ATCC 4356 grown under aerobic conditions showed a slight positive effect with the addition of iron in the absence of manganese. Another study using chemically defined medium to study the nutrient requirements of several Lactobacillaceae, including L. johnsonii ATCC 11506 but not L. acidophilus ATCC 4356, found that iron sensitivity depended upon the nucleotides present in the growth medium for some species [94]. Iron was required for Lactobacillaceae when grown using medium with limited nucleotide composition, such as when only inosine and uracil are supplied as the essential purine and pyrimidine precursors. In our recent study investigating the effects of metals and varied subculture conditions on growth of L. plantarum ATCC 14917 and L. acidophilus ATCC 4356, only high iron (100–150 μM) enhanced growth of both species when subcultured in rich de Man, Rogosa and Sharpe (MRS) medium prior to chemically defined medium [95]. The underlying causes of this effect are not known, but the effect was not observed at <100 μM added iron and prior studies that saw no growth enhancements with iron supplementation all used iron concentrations below 100 μM [88,90,93,94,96]. Although most studies show that iron deprivation does not affect the growth of lactic acid bacteria, there are several species, including L. lactis and L. plantarum that require exogenous heme to activate aerobic and respiratory growth [97]. Another member of the lactic acid bacteria group, Lactococcus lactis, showed improved respiration ability with the acquisition of heme [98]. L. lactis and all known Lactobacillaceae are auxotrophic for heme [97,99].

Fewer studies have also quantified iron uptake. Archibald reported minimal uptake of ⁵⁹FeCl₃ from APT-glucose medium for L. plantarum ATCC 14917. Later, Posey and Gherardini also found little ⁵⁹Fe uptake for the same strain grown in MRS in air and in APT medium [100]. Conversely, L. bulgaricus, L. acidophilus, and L. plantarum accumulated iron in Trypticase-phytone-yeast extract (TPY) medium, but accumulation was reduced if glucose was removed [101]. Compared to the studies that found little to no iron uptake, the latter work uses ferrous rather than ferric iron, raising the question of whether the form of iron may affect quantitative iron uptake by different species or strains of Lactobacillaceae depending on which iron transport genes are encoded and activated. Some reports indicate that L. acidophilus and L. delbrueckii subsp. bulgaricus can bind ferric hydroxide at their cell surface, possibly making it unavailable to pathogens [102–104]. In Lactobacillaceae, the oxidation of Fe²⁺ to Fe³⁺ and subsequent binding of Fe(OH)₃ to the bacterial cell is increased with H₂O₂ in the medium under aerobic conditions and is sensitive to treatment with the protease trypsin. Detailed studies in Bifidobacteria on the nature of the Fe(OH)₃ bond proposed that a ferroxidase oxidizes Fe²⁺ and the resulting Fe³⁺ is stored in a particulate locus in the cell [101–106]. Several factors can affect this chemistry in both Bifidobacteria and Lactobacillaceae, including calcium and magnesium concentrations, glucose, lactate, pO₂, and pH. Our recent work used inductively coupled plasma-mass spectrometry (ICP-MS) to show that both L. plantarum and L. acidophilus accumulate iron when grown in MRS and a chemically defined minimal medium [95]. Another group measured similar iron uptake levels for two additional strains of L. plantarum grown in similar media [107].

The genome sequences of lactic acid bacteria reveal that several species have genes involved in iron transport [108]. Specifically, the genome sequence of Latilactobacillus sakei (formerly Lactobacillus sakei), which is a species well-adapted to meat, was found to have several putative iron transport systems and three iron-dependent transcriptional regulators from the Fur family [109]. Given the presence of some cytochrome genes as well, Duhutrel and coauthors investigated the effects of various iron sources (FeSO₄, FeCl₃, siderophore-complexed iron ferrichrome, ferrioxamine, ferric citrate, hematin, hemoglobin, myoglobin, h-transferrin, and lactoferrin) on strain 23K of L. sakei [110]. Neither iron deprivation or supplementation modified the growth of L. sakei. This result is consistent with most prior studies on other Lactobacillaceae, but the authors found that heminic sources (hematin, myoglobin, and hemoglobin) significantly enhanced survival during the stationary phase up to 144–168 h. Although iron is dispensable for L. sakei growth, iron sources in meat benefit this species by supporting its long-term survival. The authors used electron energy loss and secondary-ion mass spectrometry methods to show that L. sakei accumulates iron intracellularly, in a manner that depends on the identity of the iron source and which correlates with bacterial survival. Notably, a nonhomogeneous signal observed for iron in the cells was attributed to differences in the physiological status of cells within the overall population. More recently, Verplaetse and coauthors established the heme uptake function of an L. sakei energy coupling factor (ECF)-like transport system (Fig. 4) and showed that cells with this system rapidly accumulated ~3×10⁵ atoms of ⁵⁷Fe per cell in the presence of 1 μM ⁵⁷Fe-hemin and up to 260,000 atoms in the presence of 40 μM ⁵⁷Fe-hemin [111].

Fig. 4.

Energy coupling factor (ECF)-like heme uptake transporter from L. sakei. The heme transporter is comprised of the transmembrane substrate-specific binding S protein (Ecf-S), the transmembrane T protein (Ecf-T), and two nucleotide binding proteins (Ecf-A and Ecf-A’). Lsa1839 and Lsa1840 are structurally similar to the α and β subunits of the transcobalamin binding domain and are proposed to be extracellular heme-scavenging soluble proteins.

Collectively, these results show that Lactobacillaceae have low iron requirements for their metabolism, but the interplay of other factors like nucleotide synthesis and carbon source can induce higher iron requirements. Furthermore, cells might accumulate iron when present even if it is not strictly required. Few studies have systematically examined how the iron source can affect iron uptake in Lactobacillaceae. The example of L. sakei, which rapidly accumulates iron from heminic sources and not the iron salts used in earlier work also highlights that there are species- and strain-specific effects for which these studies have only just scratched the surface. In L. sakei, heme acquisition may represent a fitness trait for surviving in the heme-rich meat ecosystem. Acquisition of exogenous heme, however, allows several lactic acid bacteria, including L. lactis and L. plantarum, to activate a respiratory metabolism when grown aerobically. This implies that heme crosses the thick gram-positive cell wall and heme transporters may also be present in species and strains that are not meat-adapted [97,112,113]. Additionally, examination of the genomes of several Lactobacillaceae, including species of intestinal origin, reveals the presence of various ferric and ferrous transporters. Basic Local Alignment Search Tool (BLAST) analysis uncovers genes for iron ATP-binding cassette (ABC) transporter permeases, major facilitator superfamily (MFS) transporters, ferrous iron transport B proteins, and the ferric uptake regulator (Fur) in some Lactobacillaceae species [109,114–119]. It is worth examining the growth conditions under which these transporters are expressed and determining whether they are relevant to the survival and function of Lactobacillaceae in the gut microbiota.

4. Manganese

4.1. Manganese and Lactobacillaceae in the gut microbiota

Although there are few documented cases of manganese deficiency in humans, toxic manganese overload, especially due to environmental or occupational exposure, is more common. Manganese overload is associated with neurodegeneration, bone loss, and cardiovascular diseases [120–122]. Damage induced by manganese involves oxidative stress, alterations of ion homeostasis (e.g. iron), and inflammation [123]. Gut microbes are exposed to dietary manganese prior to host absorption in the gastrointestinal tract, but few studies have directly examined how manganese impacts the gut microbiota (Table 2) [124–127]. Manganese exposure may perturb the gut microbiota and gut bacteria could potentially influence the toxicity and physiological effects of manganese. For chickens infected with Salmonella Typhimirium, dietary manganese supplementation increased relative colonization of beneficial Lactobacillaceae and Bifidobacteria while reducing Salmonella and E. coli [126]. Manganese also significantly improved the intestinal barrier in chickens. When mice were supplemented with manganese chloride for 13 weeks, the gut microbiota was affected in a sex-specific way [125]. Mn²⁺-exposed male mice experienced a significantly increased abundance of Firmicutes and female mice underwent a decrease in bacteria from the same phylum. Furthermore, Lactobacillaceae were specifically increased in the female mice after manganese exposure. This work also supports a role for the gut microbiome in influencing the toxic effects of manganese. An increase in bacterial manganese transporter genes in female mice suggests that gut bacteria may sequester Mn²⁺, thereby reducing its toxic effects by limiting host cell manganese adsorption. Conversely, the microbiota may increase manganese toxicity by oxidizing Mn²⁺ to Mn³⁺, which is more reactive and toxic [128]. An increase in the gene for multicopper oxidase was found in the female mice. Bacteria can use multicopper oxidase to oxidize Mn²⁺ to Mn³⁺, so Mn²⁺-exposed females may experience enhanced toxic effects of manganese [129]. A separate study on a colitis mouse model concluded that manganese was important for maintaining the intestinal barrier and could provide protection against dextran sulfate sodium-induced colon injury but did not find any changes to the microbiome composition when comparing diets containing <0.01, 35, and 300 ppm manganese, although the treatment time was relatively short (2 weeks) [127]. The study comparing female and male mice used a 13-week treatment time. More trials are needed to gain a clear understanding of the interactions between dietary manganese and the gut microbiota, but it is intriguing that three of these studies found that Lactobacillaceae levels are impacted by manganese. Given that many Lactobacillaceae have long been known to sequester high concentrations of manganese, studies investigating the mechanisms by which manganese interacts with gut Lactobacillaceae are well warranted [88,96,130].

Table 2.

Summary of studies on the effects of manganese on Lactobacillaceae.

	Research subject	Research design	Lactobacillaceae change	Reference
Manganese supplementation	C57BL/6 mice	100 ppm (20 mg/kg/day) MnCl2 for 13 weeks	Increase in females	[125]
	Chickens infected with Salmonella Typhimirium	Mn control (40 mg/kg) Mn surfeit (100 mg/kg) compared to Mn deficient (none added)	Increase	[126]
	C57BL/6 mice, colitis model	Mn deficient (<0.01 ppm), Mn adequate (35 ppm), or Mn supplemented (300 ppm) for two weeks then colitis induction	No change	[127]
	Male Sprague-Dawley Rats	200 mg/L MnCl2 in water compared to water alone for 5 weeks	Decrease	[131]
Manganese deficiency	C57BL/6 mice, colitis model	Mn deficient (<0.01 ppm), Mn adequate (35 ppm), or Mn supplemented (300 ppm) for two weeks then colitis induction	No change	[127]

4.2. Biochemistry and regulation of manganese in Lactobacillaceae

Manganese is required by all known bacteria, but several lactic acid bacteria have higher requirements for manganese and can accumulate high intracellular levels of this metal [130,132,133]. For example, L. plantarum can accumulate up to 20 mM total manganese compared to micromolar levels typically acquired by bacteria like E. coli [130,134]. Manganese and iron play important roles in biological oxygen chemistry. Both metals serve as enzyme cofactors for mitigating oxidative stress by removing reactive oxygen species like superoxide $\left({\text{O}}_2^{-}\right)$ and hydrogen peroxide, yet the propensity of these metals to cause oxidative damage is distinct. Given its higher reduction potential, manganese is less prone to toxic hydroxyl radical-generating Fenton chemistry than iron. Both elements can be cofactors for superoxide dismutase (SOD) enzymes, which disproportionate superoxide into hydrogen peroxide and oxygen. Manganese bound to non-proteinaceous metabolites can also have antioxidant activity in a variety of organisms [135]. A di-manganese active site is found in some catalases, which can disproportionate toxic H₂O₂ into O₂ and water [136]. Evidence suggests that manganese can compensate for iron deprivation, likely substituting for iron in some enzymes in E. coli [137]. Organisms like Lactobacillaceae and the Lyme disease pathogen, Borrelia burgdorferi, appear to have dispensed with the absolute need for iron by increasing manganese accumulation [96,100].

Manganese is essential for Lactobacillaceae growth and is involved in a variety of metabolic pathways. Manganese is crucial for managing oxidative stress, especially for Lactobacillaceae grown under aerobic conditions, which produce reactive oxygen species as the byproducts of oxygen metabolism. Non-proteinaceous manganese antioxidants were first discovered by Archibald and Fridovich when they found that strains of L. plantarum that lacked SOD and accumulated high quantities of manganese retained resistance to superoxide [133]. The millimolar quantities of manganese accumulated by L. plantarum are needed for survival under aerobic conditions. Using extracts of L. plantarum, the authors demonstrated heat- and protease-resistance superoxide-scavenging activity that was due to dialyzable manganese [130]. There was also a correlation between aerobic growth and high manganese uptake or SOD activity in several lactic acid bacteria [133]. Strains that acquired high manganese concentrations did not have detectable SOD activity, but could grow at least as well aerobically as anaerobically. Strains that acquired less manganese and showed SOD activity could grow well aerobically. None of the Lactobacillaceae tested positive for SOD activity. Some strains showed no detectable SOD and low manganese acquisition, but these were highly oxygen sensitive. Manganese can efficiently scavenge superoxide when bound to small molecules like orthophosphate, lactate, and malate [138,139]. Among the organic acids, Mn-lactate showed the highest activity, which is of particular interest given the high levels of lactate produced by L. plantarum [138]. Later studies in other organisms showed that additional manganese complexes and mixtures can protect against radiation/oxidative damage and protein or DNA oxidative damage [135]. In addition to its superoxide-scavenging activity, manganese can disproportionate H₂O₂ in bicarbonate buffer at physiological pH [140,141]. Iron overload, however, can counteract the antioxidant benefits of manganese through Fenton chemistry and by outcompeting manganese for binding to MnSOD [135].

Investigations into the relationships between manganese, SOD, and catalase have remained limited to a few strains of lactic acid bacteria. Few Lactobacillaceae species have SOD activity or SOD genes although SOD genes engineered into these bacteria can confer protection against oxidative stress [142–144]. Some SOD genes are annotated in the reported genomes of species such as L. sakei and L. paracasei, but these have not been isolated and characterized. The oxygen-resistance of Lactobacillaceae seems to primarily be due to non-proteinaceous manganese, but some of these bacteria also have catalases. The catalases can be classified into heme- and Mn²⁺-dependent groups. For a long time, lactic acid bacteria were considered unable to produce heme catalase and cytochrome oxidase given that they lack the complete biosynthetic pathway for heme. Some Lactobacillaceae species have heme transport systems, however, and several studies show that some species can use exogenous heme to synthesize a heme-containing catalase and activate a minimal respiratory chain [113,145,146]. Non-heme catalase activity was also observed in some Lactobacillaceae and other lactic acid bacteria, but the Mn²⁺-catalase from L. plantarum ATCC 14431 is the only well-characterized enzyme from this group [136,147,148]. Some strains of Lactobacillaceae, such as L. casei N87, harbor the genes for both heme- and Mn²⁺-dependent catalases [149–151]. This discovery prompted a systematic study to confirm the occurrence of both enzymes and investigate the factors affecting gene expression and enzymatic functionality [146]. In a chemically defined medium supplemented with hemin, manganese, and/or iron, and using aerated or non-aerated growth conditions, hemin and manganese were required for transcription of the respective genes, while iron and oxygen were not required for gene expression but increased transcription of both catalases. Like manganese, hemin can improve the tolerance of Lactobacillaceae to oxidative stress [152,153]. The catalase activities were strongly boosted under aerobic growth conditions. A phylogenetic analysis of lactic acid bacteria revealed several other L. casei strains harboring both catalases, but the occurrence of both catalases in a single strain is rare. Lactic acid bacteria colonize a variety of environments in addition to humans and animals, so the presence of both catalases could contribute to adaptation to different niches. The antioxidant effects conferred by manganese, catalases, and SODs are considered a beneficial effect of probiotic Lactobacillaceae and studies have suggested that these effects can be beneficial for the host [154–156].

The discovery of the high manganese requirement of many Lactobacillaceae prompted early work investigating the properties of cellular manganese transporters, which provided evidence for an active Mn²⁺ uptake system in L. plantarum (Fig. 5) [96,157]. Of the several other d-block metals studied, only Cd²⁺ was able to effectively compete with Mn²⁺ for uptake and with a higher affinity [157]. A Mn²⁺- and Cd²⁺-specific P-type ATPase (MntA) was identified and characterized from L. plantarum ATCC 14917 [158]. The high affinity uptake system is induced by Mn²⁺ starvation. MntA expression in E. coli confers increased uptake and sensitivity to Cd²⁺. Protein sequences of known manganese transporters from other species, expanding beyond the P-type ATPase family to ABC transporters and Natural Resistance-Associated Macrophage Protein (NRAMP) transporters, were used to identify candidate manganese transport proteins in the L. plantarum WCFS1 genome [159]. MntA from strain WCFS1 showed 99% identity to MntA from L. plantarum ATCC 14917, suggesting it is highly conserved in this species. One complete ABC transporter (MtsCBA) with high homology to known Mn²⁺-specific ABC transporters and three proteins from the NRAMP family, designated MntH1, MntH2, and MntH3, were also found. Three of the systems (MtsCBA, MntH1, and MntH2) could be expressed under Mn²⁺ starvation conditions, but unlike for L. plantarum ATCC 14917 [158,160], MntA was not significantly expressed in this species for any Mn²⁺ concentration tested. These results support a role for MtsCBA, MntH1, and MntH2 in Mn²⁺ transport, but mutants (MtsA, MntH2, and MtsA-MntH2) did not lead to any growth defects or decreases in internal Mn²⁺ levels. Cross-regulation is a possible explanation since inactivation of mtsA and mntH2 moderately upregulates mntH2 and mtsCBA, respectively, but it remains unknown which Mn²⁺ transporter is induced for the double mutant since neither mntH1 nor mntA showed increased gene expression. Several other cation transporters are annotated in L. plantarum WCFS1 but predicting cation specificity without experimental verification is a challenge. The MntH2 promoter from a related strain, L. plantarum NC8, was used to develop manganese starvation-inducible expression [161]. Later, the response of another L. plantarum strain (CCFM436) to manganese starvation was investigated [162]. Here, MntH1–5 acted as potential importers of manganese and were negatively regulated by MntR. MntR is a transcriptional regulator that binds to manganese ions in manganese-replete cells, and then binds to MntH promoters to form MntR:Mn₂. MntH1 and 2 were most highly upregulated at ~20- and 25-fold, respectively. The same group investigated how these transporters responded to excess manganese, finding that MntH 1–3 continuously decreased, and MntH 4–5 increased, suggesting that the former are involved in intracellular uptake while the latter are involved in export of excess Mn²⁺ [163,164]. Beyond L. plantarum, few studies have investigated Mn²⁺ transport proteins in other Lactobacillaceae. MntH1 and MntH2 of L. plantarum WCFS1 share high identity with the hop-inducible cation transporter (HitA) protein from a beer spoilage isolate of L. brevis [159]. This protein is expressed with the addition of bitter hop compounds, one of which exchanges with extracellular protons for intracellular Mn²⁺ [165,166]. Recent work on different L. brevis isolates linked to beer spoilage revealed a manganese transporter (MntH₀₂₇₄) that is required for bacterial survival at low pH [167]. When the gene was silenced, the authors found that adding Mn²⁺ restored the growth, indicating the importance of Mn²⁺ for growth at low pH. MntH₀₂₇₄ shares ~50% amino acid sequence similarity with HitA, although HitA was not transcriptionally induced at low pH. Given the importance of manganese for Lactobacillaceae growth and oxygen tolerance, more studies to understand manganese transport and homeostasis in a variety of species are needed.

Fig. 5.

Manganese transporters identified in L. plantarum strains. MntA is a P-type ATPase induced by Mn²⁺ starvation in L. plantarum ATCC 14917 but not in strain WCFS1. MtsCBA is an ABC transporter expressed under Mn²⁺ starvation in strain WCFS1, along with NRAMP family proteins, MntH1 and MntH2. MntR negatively regulates MntH1–5 in strain CCFM436 by binding Mn²⁺ ions in Mn-replete cells. Under excess Mn²⁺ conditions, MntH1–3 are decreased and MntH4–5 are increased. The high intracellular Mn²⁺ contents found in Lactobacillaceae can bind to organic acids and scavenge superoxide.

5. Zinc

5.1. Zinc and Lactobacillaceae in the gut microbiota

Zinc deficiency is the second most widespread micronutrient deficiency after iron, affecting ~17% of the global population and causing symptoms ranging from diarrhea and chronic inflammation to immune system impairment, compromised physical growth and development, and neurological deficits [168–171]. Symptoms can be apparent even with mild zinc deficiency and supplementation can compensate for inadequate zinc uptake and increased zinc loss [172–174]. Zinc is important for the intestinal epithelial cell barrier and deficiency can lead to a failure of the barrier and increased intestinal permeability [175,176]. Zinc supplementation reverts intestinal permeability and reduces diarrhea [175,177]. Zinc is also important for Paneth cell function. Paneth cells are found in the small intestine and produce antimicrobial proteins and peptides and other proteins involved in inflammation and immunity. These cells accumulate zinc in the secretory granules and require zinc for antimicrobial function [178]. How zinc influences antimicrobial production or stability is not precisely known, but release of zinc from these cells could lead to local changes in the microbiome and mucus layer [179,180]. Furthermore, zinc has been linked to microbiome changes via Paneth cells. Knockout of the zinc transporter responsible for uptake of zinc into Paneth cells alters the microbiome and impairs the antimicrobial response [178].

Zinc deficiency and supplementation affect composition and function of the gut microbiota. Early work estimated that the intestinal microbiota uses ~20% of total dietary zinc [181]. Several studies have correlated changes in the gut microbiota with positive or negative impacts from varied levels of zinc (Table 3). These studies are primarily limited to animal models and clinical studies on humans are severely lacking. Weaned piglets are given zinc oxide supplements to mitigate post-weaning nutritional and growth deficits, and high levels (>2000 ppm) are beneficial for this purpose but frequently reduce beneficial bacteria like Lactobacillaceae and Bifidobacteria [182–188]. High zinc oxide often also increases Enterobacteriales and microbial diversity, which may increase interspecies competition against pathogenic E. coli [182–187]. Unlike humans, lactic acid bacteria are a major component of the pig small intestinal microbiota so their reduction could allow increase of a more diverse population of Enterobacteria better able to compete directly with pathogenic species. Conflicting results challenge these hypotheses. Some studies on weaned piglets show no effect on lactic acid bacteria, some have measured increases with high zinc, and others have found species-specific effects where L. reuteri decreases with zinc oxide supplementation and Lactobacillus amylovorus does not change [184,188–193]. Zinc also affects Lactobacillaceae in other animals. In chickens, several studies have found increases in beneficial Lactobacillaceae and decreases in E. coli and other pathogens upon zinc supplementation or biofortication [194–196], although Lactobacillaceae reduction has also been measured with zinc-bacitracin supplementation [197]. Zinc deficiency was correlated with an increase in Proteobacteria (Enterococcus and Enterobacteriaceae) and decrease in Firmicutes (the phylum that includes Lactobacillaceae) [198]. Some studies in mice found that zinc supplementation reduced Lactobacillaceae and others noted significant changes to the microbiota including reduced resistance to Clostridioides difficile infection but without specifically discussing Lactobacillaceae [199–203]. Conversely, other work found no notable differences in the microbiota composition for mice fed zinc- and protein-deficient diets [204].

Table 3.

Summary of studies on the effects of zinc on Lactobacillaceae.

	Research subject	Research design	Lactobacillaceae change	Reference
Zinc supplementation	Weaned piglets	2500 mg/kg ZnO compared to 100 mg/kg ZnO for 2 weeks	Decrease	[182]
	Weaned piglets	3000 mg/kg ZnO compared to 200 mg/kg ZnO for 2 weeks	Decrease	[183]
	Weaned piglets	3042 mg/kg ZnO compared to 142 mg/kg ZnO for 12–14 days	Species-specifica	[184]
	Weaned piglets	2500 mg/kg ZnO compared to 50 mg/kg ZnO for 2 weeks	No change	[190]
	Weaned piglets	2425 mg/kg ZnO compared to 57 mg/kg ZnO for 5 weeks	Decreaseb	[185]
	Broiler chickens	120 mg/kg ZnSO4 and challenged with S. typhimirium compared to no added zinc and challenged with S. typhimirium	Increase	[194]
	Weaned piglets	2250 mg/kg ZnO compared to 250 mg/kg ZnO for 2 weeks	Decrease	[186]
	Broiler chickens	500 ppm Zn (Zn bacitracin) compared to control for 10 days	Decrease	[197]
	Weaned piglets	150 mg/kg ZnSO4 (basal) and 3000 mg/kg ZnO (high)	Decrease	[187]
	Weaned piglets	600–2000 mg/kg ZnO nanoparticles for 14 days	Decrease (ileum) Increase (cecum, colon)	[188]
	Broiler chickens	Zn biofortified wheat (46.5 μg Zn/g) compared to standard wheat (32.8 μg Zn/g) for 42 days	Increase	[195]
	Weaned piglets	110 and 2400 mg/kg ZnO for 15 days	No change	[191]
	C57BL/6J mice	25 mM ZnSO4 in ultrapure water compared to no added zinc for 7 days	Decrease	[199]
	Female BALB/c mice	12, 50, 250 mg/kg ZnCl2 for 2 weeks	Decreasec	[200]
	Weaned piglets	40, 110, and 2500 mg/kg ZnO, and 110 mg/kg Zn-lysinate for 21 days	No change	[189]
	Broiler chickens	20–100 mg/kg Zn hydroxychloride for 35 days	Increase	[196]
Zinc deficiency	Broiler chickens	2.5 mg/g ZnCO3 (deficient) compared to 42 mg/g (adequate)	Decreased Firmicutesd	[198]
	Male C57BL/6 mice	<2 ppm Zn and 20% protein for 8 days	No change	[204]

^aDecreased L. reuteri, no change L. amylovorus.

^bDecreased for Lactobacillus species and for 3 of 5 specific species studied: L. acidophilus, Limosilactobacillus mucosae (formerly Lactobacillus mucosae), and L. amylovorus, but not L. johnsonii or L. reuteri.

^cDecreased Lactobacillaceae at the family level was subtle. A decrease at the Lactobacillus genus level was more pronounced.

^dDecreased in phylum Firmicutes, which includes Lactobacillaceae although Lactobacillaceae were not specifically discussed.

Although there are some consistencies, there is significant variability in correlating dietary zinc levels with microbiota composition. Differences can arise from the different animal models, different zinc sources which may have different bioavailabilities, different samples from various locations of the gastrointestinal tract, and different methods and sensitivities of detecting changes in microbiota members. Furthermore, few studies report changes on the species level, but those that do highlight how different species from the same family may respond distinctly [184]. These differences may be masked in studies that only measure changes in higher taxa. Whether species-specific responses are due to direct impacts on viability of the species (perhaps from insufficient regulatory mechanisms) or to indirect mechanisms is unknown and worthy of systematic studies. An in vitro study investigated the effect of zinc oxide on a broad range of intestinal bacteria species and found that the zinc resistance of commensal intestinal bacteria could not be grouped according to taxonomic origin [205]. Surprisingly, and in apparent opposition of some of the above in vivo results, 10 out of 11 Lactobacillaceae investigated showed high zinc resistance (except L. amylovorus). L. amylovorus may dominate the Lactobacillaceae in some of the in vivo studies, which would be more consistent with in vitro results, but more work is needed to understand how zinc is correlated with species-level changes in the microbiota.

The microbiota can also directly influence zinc bioavailability. Conventional and germ-free chickens (Gallus gallus) were used to show that the presence of the microbiota led to increased competition for zinc ions [206]. A chicken commensal organism, Campylobacter jejuni, requires a high affinity zinc uptake transporter, ZnuA, to survive in the presence of the microbiota. ZnuA is required for colonization in conventional but not germ-free chickens. Analysis of the intestinal contents from the conventional chickens revealed higher zinc concentrations and many zinc-binding proteins such as aminopeptidases that are involved in digestion. Most of these proteins were not found at all in the germ-free chickens. Different metabolic capabilities may occur with changes in the microbiota that can affect metal bioavailability and therefore also affect species competition.

Lactobacillaceae-based probiotics may also impact zinc status [207–210]. In rats, a combination of L. casei and zinc gluconate improved zinc bioavailability [207]. A prebiotic fiber fed to rats was associated with increased Lactobacillaceae and Bifidobacteria and increased bone (femur) zinc levels [208]. In one study on children, zinc supplementation with L. plantarum IS-10506 was associated with increased serum zinc levels [209]. On the other hand, some studies in children showed no significant effect on zinc status with Lactobacillaceae probiotics [211,212]. As for iron, one mechanism by which Lactobacillaceae may increase zinc bioavailability is to metabolize dietary zinc-binding compounds like phytic acid [82]. Other studies have found beneficial [213–215] or no beneficial [216] effects from co-administration of zinc and Lactobacillaceae, although these were not directly linked to changes in zinc availability.

5.2. Biochemistry and regulation of zinc in Lactobacillaceae

Few studies have investigated zinc homeostasis in Lactobacillaceae. Early work showed toxic effects from adding excess zinc to medium already containing zinc, which may be alleviated with addition of other metals like Mn²⁺ [217,218]. More recent work investigated effects of adding ZnO to growth medium of several intestinal bacteria, finding that 10 out of 11 Lactobacillaceae showed high zinc resistance (except L. amylovorus) [205]. Specific mechanisms underlying this resistance were not explored. Our group investigated the effects of zinc restriction and supplementation on growth of L. plantarum and L. acidophilus [95]. The growth rate and lag times of both species were affected by varied zinc in defined minimal medium but not in rich medium. Both zinc-deficient (20 nM) and high zinc (0.5–1 mM) conditions negatively affected growth. When bacteria were subcultured in various media prior to exposure to different zinc levels, different growth responses were observed. For example, growth of L. acidophilus in zinc-deficient medium was improved when the cells were first cultured in medium containing excess zinc (0.1 mM) or medium containing mucin, the glycoprotein that makes up much of the mucus layer in the gastrointestinal tract. No significant difference was observed for L. plantarum grown under the same conditions. More studies are needed to understand how sub-culturing cells in zinc or mucin helps L. acidophilus cells to better tolerate zinc-deficient conditions. Of particular importance would be to identify and study Zn²⁺ sensors and transporters in these species.

Some studies have investigated zinc transporters in lactic acid bacteria. A Lactococcus lactis mutant lacking the regulators for a putative low- and high-affinity zinc uptake system is more sensitive to H₂O₂ than the wild type [219]. L. lactis shares the same taxonomic order (Lactobacillales) but differs in family from Lactobacillaceae (Fig. 2). Transport systems for zinc uptake and efflux are reported for other gram-positive bacteria, many of which are ABC transporters [141,220]. In L. lactis, an ABC transporter putatively involved in high-affinity Zn²⁺ uptake was identified (ZitSQP) and is organized with an upstream repressor gene [220,221]. The promoter and repressor from the zit operon were used to develop a gene expression system highly induced by zinc starvation and strongly repressed by excess Zn²⁺ [222]. Later work studied regulation of the zit operon in response to Zn²⁺, finding that transcription was repressed across a wide concentration range from micromolar to nanomolar (Fig. 6) [223]. The ZitR regulator (homologous to streptococcal AdcR) is derepressed under starvation conditions (<nanomolar) and repressed at nontoxic micromolar levels. An additional low affinity zinc uptake transporter was proposed in addition to the high affinity ZitSQP transporter given the high intracellular zinc content detected at extracellular concentrations above the range of ZitSQP. No specific zinc regulatory or transport proteins have been studied in the Lactobacillaceae family, however, a BLAST search reveals ZitR in the genomes of many of these species. Zinc transporters have also not been studied among Lactobacillaceae but ZnuA, which is one component of the high affinity ZnuACB transporter, is found in Lactobacillus iners [224,225]. This organism is important to the vaginal microbiome and is the only Lactobacillaceae in that niche with this transporter annotated. The low affinity zinc transporter, ZupT, is found in several Lactobacillaceae, but has not been experimentally confirmed.

Fig. 6.

Model for zinc homeostasis in L. lactis. ZitR/AdcR is a repressor that controls transcription of the Zit operon for the ABC transporter ZitSQP/AdcACB. It is derepressed under Zn²⁺ starvation conditions (<nM) and repressed at low micromolar levels, possibly to save energy. Given high intracellular zinc levels in the presence of extracellular zinc >2 μM, the presence of another low affinity zinc uptake transporter is possible.

Probiotic microorganisms like Lactobacillaceae are proposed as micronutrient supplements given their metal bioabsorption capacities [226]. Some studies, therefore, have explored how Lactobacillaceae accumulate metals like zinc on their surfaces. One study investigated several strains of Lactobacillaceae and Bifidobacteria and found that all could accumulate zinc (11 to 135 μmol g⁻¹), but the highest amount was observed for a strain of L. acidophilus (WC 0203) [227]. Here, the authors compared the amount of cell-bound zinc for 48 hour cultures with 10 mM ZnSO₄ added at the start of growth (inoculation, 0 h), at the stationary phase (24 h), and at the stationary phase (24 h) for a pasteurized culture. Cell-bound zinc was measured at 48 h for each culture. Similar amounts of zinc were accumulated whether the cultures were viable or not and the highest levels of zinc were detected in the stationary phase cultures, suggesting that the mechanism is not metabolically mediated. The zinc detected in these cultures is likely associated with the cell surface and for L. acidophilus may be bound to S-layer proteins. Another study investigated zinc binding by several lactic acid bacteria, finding that the extent of binding was influenced by pH, ionic strength, and temperature, and concluded that both passive and active uptake of zinc ions contributed to binding [228]. Some studies have found that zinc biosorption by different Lactobacillaceae improves the bioavailability of zinc and can be beneficial for the host organism [229,230].

In sum, Lactobacillaceae are often correlated with zinc levels in the gut microbiome and harbor a zinc proteome, but much work remains to be done to understand zinc uptake, efflux, secretion, and the zinc requirements of these species. These biochemical studies are required to uncover the zinc-dependent mechanisms underlying the roles of Lactobacillaceae in the gut microbiota.

6. Outlook

Metal ion nutrients are crucial for the optimal function of all living systems, including the gut microbiota. As resident gut microbes and probiotics, Lactobacillaceae play numerous important roles in human health. These microorganisms are frequently impacted by changes in dietary metal nutrients, yet their metal homeostasis mechanisms are not well-defined. From a bioinorganic perspective, Lactobacillaceae are fascinating as early studies uncovered their unique lack of an iron requirement. Instead, these bacteria accumulate relatively high concentrations of manganese, which is documented to provide protective antioxidant effects, especially under aerobic conditions. As more and more genome sequences become available for these bacteria, it is apparent that although they are not negatively impacted by a lack of iron, they can use it. More investigations into the annotated iron regulators and transporters in Lactobacillaceae may help to better understand how they are affected in the gut microbiota in the face of altered iron availabilities. Similar studies on manganese and zinc homeostatic mechanisms would be valuable, as manganese transporters have been studied in few species and there are no studies directly on zinc transporters. Much of the work investigating metal homeostasis in intestinal microbes has focused on pathogens and their response to host-imposed metal ion limitation (nutritional immunity). Whether beneficial microorganisms such as Lactobacillaceae use similar or unique mechanisms is a highly underexplored area of research. With current genomic data, there are clearly many opportunities for future research, and a greater understanding of the metallobiology of Lactobacillaceae could lead to novel therapeutics for gastrointestinal infections and disease.

Abbreviations:

ABC

ATP-binding cassette

APT

All purpose Tween 80

ATCC

American type culture collection

BLAST

Basic local alignment search tool

CCFM

Culture collections of food microbiology

DcytB

Duodenal cytochrome b

DMT1

Divalent metal transporter

ECF

Energy coupling factor

LMG

Laboratorium voor microbiologie

Fpn1

Ferroportin

Fur

Ferric uptake regulator

HCP1

Heme carrier protein 1

HO

Heme oxygenase

HPLA

p-hydroxyphenyllactic acid

HIF2α

Hypoxia-inducible factor 2α

ICP-MS

Inductively coupled plasma-mass spectrometry

IRP2

Iron regulatory protein 2

MFS

Major facilitator superfamily

MntA

Mn²⁺- and Cd²⁺-specific P-type ATPase

MRS

De Man, Rogosa and Sharpe

MT

Metallothionein

NRAMP

Natural resistance-associated macrophage protein

SCFA

Short chain fatty acids

SOD

Superoxide dismutase

TNF

Tumor necrosis factor

TPY

Trypticase-phytone-yeast extract

WCFS

Wageningen centre for food sciences

ZIP

Zrt-/Irt-like protein

Data availability

No data was used for the research described in the article.