Discovery of the Autonomously Replicating Plasmid pMF1 from Myxococcus fulvus and Development of a Gene Cloning System in Myxococcus xanthus

Abstract

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G؉C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies (ϳ10 5 CFU/g DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.Myxobacteria are gram-negative gliding bacteria that are phylogenetically located in the delta division of the Proteobacteria (29,34,41). The two most intriguing characteristics of myxobacteria are their complicated multicellular social behavior, which provides an excellent model for studies of cell-to-cell communication and evolution (6,18,39,47), and their excellent capacity for production of diverse and novel bioactive secondary metabolites. Their production of bioactive secondary metabolites makes myxobacteria an important source of potential new drugs, although this possibility has not been well explored (36). The study and utilization of myxobacteria have been limited by the formidable isolation and culture techniques required (35) and the difficulty of performing genetic manipulations. In the past few decades, genetic studies of myxobacteria were performed mainly with the model species Myxococcus xanthus using transduction (7, 23) and the more efficient electroporation protocols (19). Besides these studies, Sorangium strains were also studied using conjugation protocols (13,14,22,31,33). Sorangium is a special cellulose degrader among the 17 myxobacterial genera (34, 50) and produces almost one-half of the known secondary metabolites produced by myxobacteria (8). Because no naturally occurring self-replicating plasmid has been discovered previously and no broad-hostrange vectors can replicate in myxobacterial cells, all the genetic transfer systems used have been based on integration of introduced plasmids or phages into the recipient chromosomes. Consequently, some genetic manipulations are hard to perform in myxobacteria or are not v...

Natural Transformation of Myxococcus xanthus

“…The existence of a complicated R/M system in M. xanthus limits the source of donor DNA to similar bacterial species, which is consistent with the finding that much of the genome expansion of M. xanthus is specific to the lineage of myxobacteria (21). In this study, only the plasmid pZJY41 has yielded efficient transformation, and it is important to note that the existence of an autonomously replicating plasmid in Myxococcus is very rare (69). The donor DNA that can partake in M. xanthus transformation in natural environments is still unknown.…”

“…The donor DNA that integrated into the genome by homologous or site-specific recombination did not generate any transformants under the conditions tested, and only the CFE-modified sitespecific recombinant plasmid transformed at a very low frequency. In contrast, plasmid pZJY41 generated a relatively high frequency of transformation, which could be due to its ability to replicate in M. xanthus (69) and therefore avoid recombination into a replicon like the chromosome (59).…”

“…Under our transformation conditions, only the autonomously replicating plasmid pZJY41 could be efficiently transformed into EPS Ϫ M. xanthus cells (Table 3). Many plasmids carry R/M systems (34) that potentially affect transfer to new hosts (9), but proteins related to the R/M system were not identified among the products encoded by all 23 predicted open reading frames (ORFs) on pMF1 (parent plasmid of pZJY41) (69). At the same time, a remarkable difference was observed in the transformation frequencies with pZJY41 isolated from E. coli and M. xanthus (Table 3).…”

“…Transformation of pZJY41 was further confirmed by colony PCR with the pair of primers PMF-rep1 and PMF-rep2 (Table 1), which specifically target the rep region on the plasmid (670 bp). At the same time, we performed plasmid extractions from all the putative pZJY41 transformants as previously described (69). PCR products and extracted plasmids were visualized by gel electrophoresis in 1% Tris-acetate-EDTA (TAE)-agarose with ethidium bromide.…”

“…Plasmid DNA from E. coli DH5␣ was prepared with the SV Minipreps DNA purification system (Promega). Plasmid pZJY41 was isolated from SW810-41 according to the published protocol (69), and genomic DNA of SW505 (difA::Tn5) was prepared as described previously (3). The SW505 genome (1 g/l) was partially digested by DNase I (2 ϫ 10 Ϫ5 U/l) at 37°C for 5 min, 10 min, and 15 min to obtain fragments of genomic DNA at Ͼ20 kb, 5 to 20 kb, and Ͻ5 kb in size, respectively, and fragment size was confirmed by 1% agarose gel electrophoresis.…”