Construction And Application Of Plasmid Cenetic Manipulating System In Lactobacillus Casei

Many lactic acid bacteria（LAB） carry different plasmids, particularly those that replicate via a theta mechanism. In this study, several LAB strains were isolated from a yoghurt starter, most of which carried at least one plasmid. One strain containing two different plasmids was chosen for further analysis. The strain was characterized by 16 Sr RNA sequencing and physiological and biochemical studies, and classified as Lactobacillus casei MCJ（CCTCC AB20130356）. Since plasmids are fundamental elements for vector construction, the smaller plasmid of < 15 kb was isolated from L. casei MCJ and analyzed by restriction digestion. Several DNA fragments were then purified and cloned to p MD18 T for DNA sequencing. The obtained sequences were then used to design primers for reverse PCR to yield DNA fragments the entire plasmid. These DNA fragments were then ligated to a T-vector and prepared for DNA sequencing. Raw data were assembled by Seqman of DNAstar 7.1 software, yielding a circular plasmid sequence. The plasmid was 11274 bp in size with G+C percentage 43.89%, and contained nine open reading frames（ORFs）, including a complete trehalose operon with tre R, tre B and tre C genes. The plasmid was named p MC11, carrying two distinct theta-type replicons rep1 and rep2. rep1 was composed of a 3.5 interon of direct repeats（DR） as the putative replication origin ori V1 and putative replication protein genes rep A₁ and rep B, whereas rep2 contained a 4.5 DRs as the putative replication origin ori V2 and putative replication proteins rep B₁ and rep A with which two Escherichia coli/Lactobacillus shuttle vectors, p EL5.7 and p EL5.6 were constructed. Furthermore, a p MC11-minus strain L. casei MCJΔ1 was obtained by continuous incubation of L. casei MCJ at 42℃, which was the host for genetic analysis of the plasmid. Truncation analysis were used to define minimal replicons for both plasmid replicons and these analyses revealed that the rep1 replicon was composed of ori V2 and rep A₁ in which rep B was not essential,whereas the minimal replicon of rep2 was composed of ori V1 and two putative replication protein genes rep B₁ and rep A. After transformation of different host strains with each plasmid, we found that p EL5.7 was capable of replicating in L. casei MCJΔ1 and L. delbrueckii subsp. lactic LBCH-1 but failed to do so in two other tested lactobacilli strains whereas p EL5.6 replicated in three different strains: L. casei MCJΔ1, L. casei NJ, L. paracasei LPC-37 and L. delbrueckii subsp. lactic LBCH-1. Moreover, both p EL5.6 and p EL5.7 were very stably maintained in L. casei, as the loss rate was lower than 1% per generation. p EL5.7 was also stable in L. delbrueckii subsp. lactic LBCH-1 with the loss rate estimated to be 3%. These vectors were then employed to express a green fluorescent protein（GFP） using the promoter of S-layer protein Slp A from L. acidophilus. And a growth-phase regulated expression of GFP was observed in different strains. The fluorenscent was strongest in exponential growth stage, while decreased though protein accumulated in the later stage, and this indicated that the slp A promoter is functional in L. casei.The unknown ORF8 in p MC11 was found in different lactobacilli strains is turned out as a trans membrane protein. The C-terminal of ORF8 was tested via fusion expression with GFP. The localization was determined by western blotting analysis after lysozyme and proteinase K digestion. Results showed that C-terminal of residue 125 a.a and the 161 a.a full length ORF8 were outside the cell membrane, which provided a reasonable leading sequence candidate for membrane-anchoring expression.In conclusion, we have developed efficient host-vector expression systems for Lactobacillus casei using p MC11 as the backbone, which constitute efficient genetic tools for DNA cloning and heterologous gene expression in lactobacilli.