Polyphosphate (poly-P) is a polymer of phosphate residues synthesized and in some cases accumulated by microorganisms, where it plays crucial physiological roles such as the participation in the response to nutritional stringencies and environmental stresses. Poly-P metabolism has received little attention in Lactobacillus, a genus of lactic acid bacteria of relevance for food production and health of humans and animals. We show that among 34 strains of Lactobacillus, 18 of them accumulated intracellular poly-P granules, as revealed by specific staining and electron microscopy. Poly-P accumulation was generally dependent on the presence of elevated phosphate concentrations in the culture medium, and it correlated with the presence of polyphosphate kinase (ppk) genes in the genomes. The ppk gene from Lactobacillus displayed a genetic arrangement in which it was flanked by two genes encoding exopolyphosphatases of the Ppx-GppA family. The ppk functionality was corroborated by its disruption (LCABL_27820 gene) in Lactobacillus casei BL23 strain. The constructed ppk mutant showed a lack of intracellular poly-P granules and a drastic reduction in poly-P synthesis. Resistance to several stresses was tested in the ppk-disrupted strain, showing that it presented a diminished growth under high-salt or low-pH conditions and an increased sensitivity to oxidative stress. These results show that poly-P accumulation is a characteristic of some strains of lactobacilli and may thus play important roles in the physiology of these microorganisms.

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...

Saccharomyces cerevisiae contains two genes (DOGR1 and DOGR2) that are able to confer 2-deoxyglucose resistance when they are overexpressed. These genes are very similar, sharing 92% identity at the protein level. They code for two isoenzymes with 2-deoxyglucose-6 phosphate (2-DOG-6P) phosphatase activity. These enzymes have been purified and characterized. DogR1p shows an optimum pH of 6, an optimum temperature of 30 degrees C and a KM on 2-DOG-6P of 17 mM. DogR2p shows a similar optimum pH, but the optimum temperature is 40 degrees C and it exhibits a KM on 2-DOG-6P of 41 mM. Both enzymes require 10 mM-MgCl2 for maximal activity and they are inhibited by inorganic phosphate.