Kaliningrad, Kalinigrad, Russian Federation
Kemerovo, Kemerovo, Russian Federation
Universal genus-specific primers for comparative analysis of two aligned 16S rRNA gene nucleotide sequences of lactic acid bacteria were constructed. The method to identify lactic acid bacteria and a comprehensive plan for their genus and species identification may be used to characterize isolated strains of the Lactobacillus genus bacteria and in quality control of foodstuffs enriched with Lactobacillus.
lactic acid bacteria, primers, nucleotide sequence, 16S rRNA gene, identification, PCR, phylogenetic analysis, construction
INTRODUCTION
Strains of the Lactobacillus genus, most subspecies of which transform lactose and other carbohydrates into lactic acid, are industrially important lactic acid bacteria.
This genus of microorganisms is most frequently used in production of foodstuffs of animal and plant origin by fermentation. The Lactobacillus genus is divided into three subspecies by the niche they occupy and their ability to ferment substances, for example, L. delbruekcii subsp. delbrueckii are usually found in vegetables treated with enzymes while L. delbrueckii subsp. bulgaris and L. delbrueckii subsp. lactis, are typically present in dairy products with addition of wide variety of carbohydrates. Specificity of industrial characteristics and vital processes makes strains of the Lactobacillus subspecies convenient and widely used in industry both individually and in consortium with other species to produce lactic acid dietary products, including cheese and yogurt [1, 3].
Simplicity and reliability of L. delbrueckii identification at various levels, that is level of strain and level of subspecies, makes them interesting not only for fundamental knowledge but for practical applications as well. Identification of microorganisms indicated on the label of a product by the manufacturer should correspond to those microorganisms that are used at a particular plant to that are applied for obtain cheese and various kinds of fermented milk products in reality.
Today, application of taxonometric identification of L. delbrueckii becomes urgent since there is a chance of mistake in case of utilization of phenotypic methods. These methods are based on utilization of genetic methods using strain-specific oligonucleotide probes upon blot-hybridization, DNA fingerprinting, or ribosomal DNA restriction. Today, precise and rapid identification of lactic acid bacteria is possible owing to development of methods based on polymerase chain reaction (PCR) [2, 3].
Therefore, in case rapid result is needed, methods based on PCR will be considered an alternative to microbiology tests and find wide application. Application of PCR methods allows for high-precision identification and determination of genus and species.
In the process of construction of genus- and species-specific primers, typically, 16S or 23S rRNA gene fragments, specific for each species and genus, and hypervariable internal transcribed regions (ITS), separating the above-mentioned loci, are chosen [4]. Also, transaldolase gene, small recA protein gene involved in recombination of homologous DNA, or tuf-gene coding for Tu elongation factor may serve as templates [5, 6].
In various species of Lactobacillus, the 16S rRNA genetic determinant has approximately the same size of about 1500 bp and is present in bacterial genome in several copies. Spacer regions of various size serve as separators between the copies of bacterial genes. Presence of two flanked spacer regions different in size between 16 and 23S rRNA genes is typical of the Lactobacillus genus. The 16S rRNA gene, containing both variable and conservative regions in the nucleotide sequence, is the most appropriate for determination of genus and species affiliation of a bacterium.
Therefore, the aim of the work was to construct genus- and species-specific primers to detect and identify industrially important strains of the Lactobacillus genus.
1. Tochilina, A.G., Indikatsyya i identifikatsyya bakterii roda lactobacillus s ispol’zovaniem polimeraznoi tsepnoi reaktsii (Detection and identification of bacteria of the Lactobacillus genus using polymerase chain reaction), Mikrobiologiya, epidemiologiya i immunobiologiya (Microbiology, Epidemiology, and Immunobiology), 2008, no. 3, pp. 69-73.
2. Botina, S.G., Identifikatsyya promyshlennykh shtammov molochnokislykh bakterii metodami molekulyarno-geneticheskogo tipirovaniya (Identification of industrial strains of lactic acid bacteria by methods of molecular genetics typing), Genetika (Genetics), 2006, vol. 42, no. 12, pp. 1621-1635.
3. Torriani, S., Zapparoli, F., and Dellaglio, F., Use of PCR-Based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis, Appl. Environ. Microbiol., 1999, vol. 65, no. 10, pp. 4351-4356.
4. Heilig, H.G., Zoetendal, E.G., Marteau, P, et al., Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA, Appl. Environ. Microbiol., 2002, vol. 68, no. 1, pp. 114-123.
5. Torriani, S., Felis, G.E., Dellaglio, F., Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers, Appl. Environ. Microbiol., 2001, vol. 67, no. 8, pp. 3450-3454.
6. Ventura, M., Canchaya, C., Meylan, V., et al., Analysis, characterization, and loci of the tuf genes in Lactobacillus and Bifidobacterium species and their direct application for species identification, Appl. Environ. Microbiol., 2003, vol. 69, no. 11, pp. 6908-6922.
