Extent of Genetic Lesions of the Arginine and Pyrimidine Biosynthetic Pathways in Lactobacillus plantarum , L. paraplantarum, L. pentosus , and L. casei : Prevalence of CO ₂ -Dependent Auxotrophs and Characterization of Deficient arg Genes in L. plantarum

Abstract

Lactic acid bacteria require rich media since, due to mutations in their biosynthetic genes, they are unable to synthesize numerous amino acids and nucleobases. Arginine biosynthesis and pyrimidine biosynthesis have a common intermediate, carbamoyl phosphate (CP), whose synthesis requires CO 2 . We investigated the extent of genetic lesions in both the arginine biosynthesis and pyrimidine biosynthesis pathways in a collection of lactobacilli, including 150 strains of Lactobacillus plantarum, 32 strains of L. pentosus, 15 strains of L. paraplantarum, and 10 strains of L. casei. The distribution of prototroph and auxotroph phenotypes varied between species. All L. casei strains, no L. paraplantarum strains, two L. pentosus strains, and seven L. plantarum strains required arginine for growth. Arginine auxotrophs were more frequently found in L. plantarum isolated from milk products than in L. plantarum isolated from fermented plant products or humans; association with dairy products might favor arginine auxotrophy. In L. plantarum the argCJBDF genes were functional in most strains, and when they were inactive, only one gene was mutated in more than one-half of the arginine auxotrophs. Random mutation may have generated these auxotrophs since different arg genes were inactivated (there were single point mutations in three auxotrophs and nonrevertible genetic lesions in four auxotrophs). These data support the hypothesis that lactic acid bacteria evolve by progressively loosing unnecessary genes upon adaptation to specific habitats, with genome evolution towards cumulative DNA degeneration. Although auxotrophy for only uracil was found in one L. pentosus strain, a high CO 2 requirement (HCR) for arginine and pyrimidine was common; it was found in 74 of 207 Lactobacillus strains tested. These HCR auxotrophs may have had their CP cellular pool-related genes altered or deregulated.Lactic acid bacteria (LAB) are gram-positive bacteria that have adapted to rich environments. As a result, in addition to sugars as energy and carbon sources these organisms require nucleobases, vitamins, cations, and amino acids (18). For example, some LAB associated with particular fermented foods have developed auxotrophies for specific growth factors, including orotic acid present in milk for Lactobacillus delbrueckii subsp. bulgaricus (32), a small peptide present in freshly prepared yeast extracts for the sourdough bacterium Lactobacillus sanfrancisco (1), and D-mevalonic acid for rice wine spoilage lactobacilli (33). Some authors (21) consider LAB to be a highly specialized form of life in view of their complex nutritional requirements and restricted habitats. The complex nutritional needs of LAB may be the result of two opposing evolutionary processes. A primitive LAB may have had restricted metabolism and gradually acquired new enzymatic activities. Alternatively, a chemoorganotrophic ancestor with many biochemical abilities may have evolved by progressively loosing unnecessary genes upon closer association with plants, animals, or hu...

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“…The effect of inorganic carbon concentrations on two L. plantarum strains was tested. Strain NCIMB 8826, the L. plantarum whose genome has been sequenced, requires CO 2 -enriched air to grow on defined media plates when arginine and pyrimidines are absent [4]. The other strain, CCM 1904 (equivalent to ATCC 8014), is prototrophic for arginine and pyrimidines and grew in normal air.…”

“…High-CO 2 -requiring natural auxotrophs are prevalent in natural isolates of L. plantarum without arginine and pyrimidine supplements [4]. This suggests that inorganic carbon is required as a substrate of CPS.…”

“…In E. faecalis and L. mesenteroides, some auxotrophies were suppressed when strains were incubated in a CO 2 -enriched atmosphere [24,25]. In L. plantarum, auxotrophies for phenylalanine, arginine, tyrosine and pyrimidines were observed in several strains incubated under normal air but not when incubated in a CO 2 -enriched air [4,25,29]. CO 2 may be required in these organisms for carboxylation reactions like in E. coli.…”

Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac− to Lac+

“…Earlier studies (Bringel & Hubert, 2003 have suggested that LAB evolve by progressively losing unnecessary genes upon adaptation to some specific habitats. DNA degeneration by spontaneous mutation may inactivate unnecessary genes during their adaptation to specific habitats in order to improve the growth or cell viability under the stressful or unusual conditions.…”

“…Once LAB have adapted to some rich environments, they may lose the ability to synthesize many essential amino acids and vitamins. Most of these genetic lesions have been found to be located in genes rather than in the promoter regions (Delorme et al, 1993;Godon et al, 1993;Cavin et al, 1999;Nomura et al, 2000;Bringel & Hubert, 2003. A systematic attempt to isolate mutants that no longer require each of the essential amino acids has been undertaken for several lactobacilli (Morishita et al, 1974(Morishita et al, , 1981Bringel & Hubert, 2003, including Enterococcus, Pediococcus and Lactococcus species (Deguchi & Morishita, 1992).…”