Identification of the Leucine-to-2-Methylbutyric Acid Catabolic Pathway of Lactococcus lactis

Abstract

Nutrient starvation and nonculturability in bacteria lead to changes in metabolism not found during the logarithmic phase. Substrates alternate to those used during growth are metabolized in these physiological states, yielding secondary metabolites. In firmicutes and actinobacteria, amino acid catabolic pathways are induced during starvation and nonculturability. Examination of lactococci showed that the population entered a nonculturable state after carbohydrate depletion and was incapable of growth on solid media; however, the cells gained the ability to produce branched-chain fatty acids from amino acids. Gene expression profiling and in silico pathway analysis coupled with nuclear magnetic resonance spectroscopy were used to delineate the leucine catabolic pathway. Lactococci produced acetic and propionic acid during logarithmic growth and starvation. At the onset of nonculturability, 2-methylbutyric acid was produced via hydroxymethyl-glutarylcoenzyme A (CoA) and acetyl-CoA, along with ATP and oxidation/reduction precursors. Gene expression profiling and genome sequence analysis showed that lactococci contained redundant genes for branched-chain fatty acid production that were regulated by an unknown mechanism linked to carbon metabolism. This work demonstrated the ability of a firmicute to induce new metabolic capabilities in the nonculturable state for producing energy and intermediates needed for transcription and translation. Phylogenetic analyses showed that homologues of these enzymes and their functional motifs were widespread across the domains of life.Bacteria grow under many nutrient conditions throughout their life cycle. During logarithmic growth, an abundance of nutrients provide carbon and nitrogen to promote cell division and replication, whereas during stationary phase, the carbon and nitrogen sources are often exhausted. While nutrient limitation is one among many conditions that induce bacteria to sporulate (38), this ability is lacking in actinobacteria (e.g., Brevibacterium linens, Mycobacterium, Micrococcus, and Vibrio spp. [8,18,38]) and some firmicutes, such as Lactococcus and Listeria spp. These two groups of bacteria enter a metabolically active but quiescent state in response to the local nutrient availability (36).In this state lactococci metabolize amino acids (36). Normally, lactococci ferment sugars, producing lactic acid. When the cells enter stationary phase a portion of the population retains the ability to maintain an intact cell membrane and begins to use proteins for energy (36). Further incubation results in a nonculturable (NC) state for periods of at least 4 years (15). It is likely that these conditions lead to novel pathways that provide alternate carbon and energy sources and are of primary interest in lactococci.Catabolism of amino acids such as arginine and branchedchain amino acids (BCAAs) is implicated in long-term survival during starvation of lactococci (4,14). While addition of arginine extends survival during early post-carbohydrate depletion (4, 36), in th...

The d -2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis

“…However, based on their substrate specificities, we can suppose that they are involved in branched-chain amino acid catabolism. Recently, Ganesan et al (20) showed that during carbon starvation and nonculturability, L. lactis is capable of consuming leucine via a long metabolic route to produce ATP. This route is initiated by leucine transamination to KIC and KIC oxidative decarboxylation to isovaleryl coenzyme A (isovaleryl-CoA) and leads via many other steps to the production of 2-methylbutyric acid and 3 mol of ATP per mol of leucine.…”

Genomic Comparison of Campylobacter spp. and Their Potential for Zoonotic Transmission between Birds, Primates, and Livestock

“…DNA was extracted using whole-genome isolation kits (Qiagen, Valencia, CA, USA) with previously published modifications (46). The bacterial cells were processed using the protocols of the 100K Pathogen Genome Project (UC Davis, Weimer laboratory) as previously described (46)(47)(48). Briefly, bacteria were lysed with an enzyme preparation, vortexed, and processed according to the manufacturer's recommendations to obtain purified gDNA (Qiagen).…”

Acid Stress-Mediated Metabolic Shift in Lactobacillus sanfranciscensis LSCE1

“…However, according to the results obtained, in semisynthetic media with leucine added as the only source of BCAAs, and only in the presence of acid stress, leucine resulted in being a potential precursor of 2-methylbutanoic acid also and not only of 3-methylbutanoic acid. The rationale behind the production of 2-methylbutanoic acid from leucine under stress conditions in L. sanfranciscensis LSCE1 can be found in the new biosynthesis pathway regarding the biosynthesis of 2-methylbutanoic acid from leucine in L. lactis (18). Ganesan et al (18) showed that, during carbon starvation and onset of nonculturability, L. lactis is capable of consuming leucine via a long metabolic route to produce ATP.…”