Requirement of theLactobacillus caseiMaeKR Two-Component System forl-Malic Acid Utilization via a Malic Enzyme Pathway

Abstract

Lactobacillus casei can metabolize L-malic acid via malolactic enzyme (malolactic fermentation [MLF]) or malic enzyme (ME). Whereas utilization of L-malic acid via MLF does not support growth, the ME pathway enables L. casei to grow on L-malic acid. In this work, we have identified in the genomes of L. casei strains BL23 and ATCC 334 a cluster consisting of two diverging operons, maePE and maeKR, encoding a putative malate transporter (maeP), an ME (maeE), and a two-component (TC) system belonging to the citrate family (maeK and maeR). Homologous clusters were identified in Enterococcus faecalis, Streptococcus agalactiae, Streptococcus pyogenes, and Streptococcus uberis. Our results show that ME is essential for L-malic acid utilization in L. casei. Furthermore, deletion of either the gene encoding the histidine kinase or the response regulator of the TC system resulted in the loss of the ability to grow on L-malic acid, thus indicating that the cognate TC system regulates and is essential for the expression of ME. Transcriptional analyses showed that expression of maeE is induced in the presence of L-malic acid and repressed by glucose, whereas TC system expression was induced by L-malic acid and was not repressed by glucose. DNase I footprinting analysis showed that MaeR binds specifically to a set of direct repeats [5-TTATT(A/T)AA-3] in the mae promoter region. The location of the repeats strongly suggests that MaeR activates the expression of the diverging operons maePE and maeKR where the first one is also subjected to carbon catabolite repression.The metabolism of L-malic acid by lactic acid bacteria (LAB) has brought about considerable interest because of its relevance in winemaking (24). The degradation of L-malate to L-lactate leads to a reduction of the acidity of wine, and it provides microbiological stability by preventing the secondary growth of LAB after bottling. Most LAB decarboxylate Lmalate to L-lactate by an NAD ϩ -and Mn 2ϩ -dependent malolactic enzyme (MLE) (Fig. 1); nevertheless, a few LAB species can also degrade L-malate to pyruvate by a malic enzyme (ME) (Fig. 1). This pathway was first detected in Enterococcus faecalis (20) and later in Lactobacillus casei (23,33) and Streptococcus bovis (14). In contrast to the utilization of L-malate through MLE, the utilization of the ME pathway enables these organisms to grow with L-malate as a carbon source (22). However, whereas MLE has been the focus of an extensive research effort, the physiological role and the regulation of ME remain largely unknown.L. casei is a facultative heterofermentative lactic acid bacterium frequently used as a cheese starter culture and which is also employed as a probiotic. Extensive research has been carried out on the study of sugar catabolism (28, 39-41); however, the knowledge of the utilization of organic acids has received less attention. As previously indicated, physiological and biochemical studies identified two L-malate dissimilation pathways in L. casei. Furthermore, these studies showed that ME expression...

Discovery and Characterization of Human-Urine Utilization by Asymptomatic-Bacteriuria-Causing Streptococcus agalactiae

“…Therefore, we analyzed whether the growth of ABSA and UPSA strains would differ in a synthetic medium that contained a defined amount of malate. For this, we used SHU as defined in previous studies (56)(57)(58) supplemented with 40 mM malic acid, a concentration consistent with prior studies that have used 30 to 50 mM malic acid in vitro (51,(53)(54)(55)(59)(60)(61)(62). We observed that the growth of ABSA 834 and ABSA 729 was significantly higher than the growth of UPSA 058 and UPSA 714 in SHU (Fig.…”

“…2). The mae gene clusters from ABSA 1014 and UPSA 807 shared identical structural organization with the homologous mae cluster from Lactobacillus casei BL23 (55). Notably, however, a frameshift mutation in a poly(A) sequence at the 5= end of maeK of UPSA 807 was identified (Fig.…”

Proteomic and transcriptomic analysis of the response to bile stress of Lactobacillus casei BL23

“…RT-qPCR analyses and primer design were carried out essentially as previously described (Landete et al, 2010). Primers used are listed in Supplementary Table S1available with the online version of this paper.…”

“…Primers used are listed in Supplementary Table S1available with the online version of this paper. The lepA, ileS, pyrG and pcrA sequences were selected from a set of 10 reference genes (Landete et al, 2010) by using the geNorm application (Vandesompele et al, 2002). The relative expression based on the expression ratio between the target genes and reference genes was calculated using the software tool REST (Pfaffl et al, 2002).…”

Comparative Genomic Analysis of Two-Component Signal Transduction Systems in Probiotic Lactobacillus casei

“…L. casei requires a complex array of TCS proteins to cope with diverse human hosts, host responses, and environmental conditions. The TCS MaeKR belonging to the citrate family is essential for the expression of malic enzyme of L. casei strains BL23 and ATCC 334, and MaeKR expression was induced by L-malic acid [9]. The genome sequences of L. casei strains BL23 and ATCC 334 harbor 17 putative TCSs, among which the role of three TCSs involved in bile response, cell envelope stress response, oxidative stress tolerance, and acid tolerance [10].…”