Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4.
A cryptic plasmid from Lactobacillus plantarum M4 isolated from fresh milk, designated as pM4, was sequenced and characterized. It was 3320 bp in length with a G+C content of 38.73 mol%. The plasmid pM4 was predicted to encode three putative ORFs, in which ORF1 shared 99% and 98% homology, respectively, with the Rep proteins of reported plasmids pWCFS101 and pF8801, members of the rolling circle replication (RCR) pC194 family. Sequence analysis revealed a typical pC194 family double strand origin (dso) and a putative single strand origin (sso) located upstream of the rep gene. Mung bean nuclease analysis and Southern hybridization confirmed the presence of single-stranded DNA (ssDNA) intermediates, suggesting that pM4 belongs to the RCR pC194 family. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA. Furthermore, the relative copy number of pM4 was estimated to be about 25 in each cell by real-time PCR. The new RCR plasmid would be valuable in constructing cloning vectors for application in the food industry.
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
Sheng
S
Yin
Yanling
Y
Hao
Zhengyuan
Z
Zhai
Ruoyu
R
Li
Ying
Y
Huang
Hongtao
H
Tian
Yunbo
Y
Luo
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001482:Base Composition; D001483:Base Sequence; D015139:Blotting, Southern; D004265:DNA Helicases / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D018628:Gene Dosage; D005822:Genetic Vectors; D048191:Lactiplantibacillus plantarum / Q000235:genetics* / Q000302:isolation & purification; D008892:Milk / Q000382:microbiology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D018741:Replication Origin; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D015534:Trans-Activators; C045264:replication initiator protein; D012321:DNA-Directed RNA Polymerases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D004265:DNA Helicases