Malic Enzyme and Malolactic Enzyme Pathways Are Functionally Linked but Independently Regulated in Lactobacillus casei BL23

Abstract

bLactobacillus casei is the only lactic acid bacterium in which two pathways for L-malate degradation have been described: the malolactic enzyme (MLE) and the malic enzyme (ME) pathways. Whereas the ME pathway enables L. casei to grow on L-malate, MLE does not support growth. The mle gene cluster consists of three genes encoding MLE (mleS), the putative L-malate transporter MleT, and the putative regulator MleR. The mae gene cluster consists of four genes encoding ME (maeE), the putative transporter MaeP, and the two-component system MaeKR. Since both pathways compete for the same substrate, we sought to determine whether they are coordinately regulated and their role in L-malate utilization as a carbon source. Transcriptional analyses revealed that the mle and mae genes are independently regulated and showed that MleR acts as an activator and requires internalization of L-malate to induce the expression of mle genes. Notwithstanding, both L-malate transporters were required for maximal L-malate uptake, although only an mleT mutation caused a growth defect on L-malate, indicating its crucial role in Lmalate metabolism. However, inactivation of MLE resulted in higher growth rates and higher final optical densities on L-malate. The limited growth on L-malate of the wild-type strain was correlated to a rapid degradation of the available L-malate to L-lactate, which cannot be further metabolized. Taken together, our results indicate that L. casei L-malate metabolism is not optimized for utilization of L-malate as a carbon source but for deacidification of the medium by conversion of L-malate into L-lactate via MLE. Lactobacillus casei is a facultatively heterofermentative lactic acid bacterium (LAB) isolated from a wide variety of habitats, including raw and fermented milk, the gastrointestinal tracts of animals, and plant materials (1). Lb. casei strains are used as cheese starter cultures, but a major interest in this species has arisen from the probiotic properties of some strains (2). Lb. casei is also remarkable because is the only LAB in which both the malic enzyme and the malolactic enzyme L-malate dissimilation pathways have been demonstrated (3, 4). Most LAB decarboxylate L-malate to L-lactate by a NAD ϩ and Mn 2ϩ -dependent malolactic enzyme (MLE). A few of them, however, can convert L-malate into pyruvate by the action of a malic enzyme (ME). This pathway was first detected in Enterococcus faecalis (5) and later in Lb. casei (4, 6) and Streptococcus bovis (7). Although there is evidence showing that some LAB strains can utilize lactate as a carbon source (8-12), most LAB cannot channel lactate into the gluconeogenic pathway. For this reason, the utilization of L-malate through MLE cannot sustain their growth, whereas the utilization of the ME pathway enables these organisms to grow with L-malate as a carbon source (3, 13).The metabolism of L-malic acid by LAB has led to considerable interest because of its relevance in winemaking (14), since the degradation of L-malate leads to a reduction in the acidity ...

Streptococcus pyogenes Malate Degradation Pathway Links pH Regulation and Virulence

“…These include the maePE operon that encodes the transmembrane permease (maeP) and cytosolic malic enzyme (maeE). Expression of these genes is dependent on the adjacent two-component system (TCS), which includes a sensor histidine kinase (maeK) and response regulator (maeR) (15,17,18). This similar organization is observed in the S. pyogenes chromosome (Fig.…”

“…1B). A unique feature of ME is that, unlike MLF, it enables cells to utilize malate as a carbon source for growth (16,18). However, while the MLF system has been extensively studied, the regulation and physiological significance of the ME pathway are not as well understood.…”

“…The most common of these is malolactic fermentation (MLF), which allows for the conversion of malate into lactate through the function of the malolactic enzyme (MLE). Typically, MLF does not contribute to growth yields but does play an important role in maintenance of ATP pools during starvation and in protection from acid killing (18)(19)(20)(21). Since malate is a stronger acid than lactate, its decarboxylation by MLF results in alkalinization of the cytoplasm, and the resulting pH gradient drives the malate/lactate antiporter coupled to ATP synthesis (7,(18)(19)(20)(21).…”

“…Less commonly found is an alternative degradation pathway that converts malate to pyruvate and carbon dioxide (18) that is known as the malic enzyme (ME) pathway (Fig. 1B).…”

Characterization of Two Component Systems of Lactobacillus casei BL23 and their involvement in stress response

“…Its origin is uncertain (Acedo-Félix and Pérez-Martínez, 2003) however its genome has been recently sequenced (3.1 Mb) and it has been used by several laboratories in genetic and physiological studies for many years (Rochat et al, 2007;Muñoz-Provencio et al, 2010;Watterlot et al, 2010;Bourand et al, 2013;Landete et al, 2013;.…”