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.
Yin
Sheng
S
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
Hao
Yanling
Y
Zhai
Zhengyuan
Z
Li
Ruoyu
R
Huang
Ying
Y
Tian
Hongtao
H
Luo
Yunbo
Y
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