Microbial ecology of sauerkraut fermentation and genome analysis of lactic acid bacterium Leuconostoc mesenteroides ATCC 8293

by 1966- Plengvidhya, Vethachai

Plengvidhya Vethachai. Microbial ecology of sauerkraut fermentation and genome analysis of lactic acid bacterium Leuconostoc mesenteroides ATCC 8293. (Under the direction of Fred Breidt) Previous studies using traditional biochemical methods to study the ecology of commercial sauerkraut fermentations revealed that four major lactic acid bacteria species, namely, Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis were involved. However, molecular fingerprinting techniques can lead to a better understanding of the ecology. The objective of this research was to study the microbial diversity and community structure of commercial sauerkraut fermentations by using nucleic acid-based detection methods. Brine samples from commercial fermentation tanks were sampled several times, over a period of 14 days. The samples were plated on MRS agar and selected numbers of isolates for each time point were recovered. DNA was isolated for PCR fingerprinting and 16S rDNA sequence analysis. A total of six hundred eighty-six isolates were collected for analysis. A database of PCR fragment patterns has been generated by amplifying the intergenic transcribed spacer regions between the 16S and 23S rDNA genes. The 16S rDNA gene has been sequenced to further identify selected isolates. Some of the isolates recovered had never been reported to be present in sauerkraut fermentations, including Leuconostoc citreum, Leuconostoc argentinum, Lactobacillus paraplantarum, Lactobacillus coryniformis, and Weissella sp. The newly identified lactic acid bacteria species, Leuconostoc fallax, was also present at day three and seven of the fermentations. We recovered only a small number of isolates belonging to Lactobacillus brevis and Pediococcus pentosaceus. According to ITS-PCR banding patterns, the number of bacterial species involved in the fermentations were highest on days seven and nine. The use of molecular techniques to complement biochemical identification methods has revealed the presence of species not previously reported to be present in sauerkraut fermentations. The ecology of these commercial fermentations has been shown to be more complex than previously reported. L. mesenteroides is perhaps the most predominant lactic acid bacteria (LAB) species found on fruits and vegetables and is responsible for initiating the sauerkraut and other vegetable fermentations. L. mesenteroides starter cultures also used in some dairy and bread dough fermentations. More recently, L. mesenteroides is being used in the production of industrial chemical and biological products including biopolymers. Realizing L. mesenteroidesc practical significance in fermentation, bioprocesssing, agriculture, food, and medicine, the whole genome sequence of L. mesenteroides ATCC 8293 originally isolated from olive fermentation has been determined. The chromosome has 2,038,395 bp and contains predicted 1,924 protein-encoding genes. Putative biological functions could be assigned to 54% of the predicted proteins. Consistent with the classification of L. mesenteroides as heterofermentative lactic acid bacterium (LAB), the genome encoded all enzymes required for the 6- phosphogluconate/phosphoketolase pathway. Furthermore, L. mesenteroides encodes a number of pyruvate dissipating enzymes that are predicted to catalyze the production of many metabolites leading to various end products of fermentation. A large proportion of the genes is encoded for carbohydrate transport and utilization. The chromosome also containing genes belonging to phage remnants and mobile genetic elements. The genomic sequence also indicated a potential horizontal transfer of genetic information between L. mesenteroides and gram-negative bacteria including Salmonella-Escherichia group. The data presented here can help facilitate evaluation of L. mesenteroides as a model organism for studies of heterofermentative LAB metabolism, physiology, and regulation. Compared to the well established Gram-positive model of, B. subtilis, L. mesenteroides represents more simplified but also several unique and interesting characteristics.