Genome and phylogenetic analysis of strain DZ-12
We sequenced the genome of the maize bacterial pathogen, strain DZ-12. The genome consisted of a chromosome of 4.85 Mb (average G+C content 54.72%), and a large plasmid, pDZ-12, of 304 kb (average G+C content) 53.61% (Fig. 1a). There are 4,367 predicted coding sequences (CDSs) with an average length of 901 nucleotides in the chromosome, and 297 predicted CDSs with an average length of 871 nucleotides in the plasmid. Putative functions have been assigned to 72% of the encoded proteins in the chromosome, and 58% in the plasmid.
A phylogenetic dendrogram based on GyrB amino acid sequences indicated that strain DZ-12 was most closely related to P. ananatis strain LMG 20103 (Fig. 1b), suggesting that strain DZ-12 belongs to Pantoea ananatis.
Construction and verification of P. ananatis DZ-12 endogenous plasmid-curing mutant and complemented strains
Many pathogens, including P. ananatis, harbor plasmids that may contain virulence determinants [8, 13]. Hence, we used the λ-Red-recombineering technique  to cure the endogenous plasmid of P. ananatis strain DZ-12. The repA (WP_013028019.1) gene encodes a replication protein located on pDZ-12, the endogenous plasmid of strain DZ-12. We substituted it with the temperature-sensitive repA101(Ts) gene, amplified from pCas (15) using primers Frep101-F/Frep101-R (Table 2). The resulting strain was grown at high temperature (37 °C), yielding mutant ∆p lacking pDZ-12 (Fig. 2). The pDZ-12 deletion was complemented by electroplating pDZ-12 into the ∆p strain, yielding strain ∆p-c.
P.ananatis produces a yellow pigment via phytoene synthase, the product of the crtB gene [16, 17]. The ctrB gene is present on pDZ-12. We detected a yellow pigment in the wild-type (WT) strain. However, the ∆p mutant no longer produced this pigment (its colonies appeared white) (Fig. 3a). Furthermore, Southern blot analysis showed that pDZ-12 was cured (Fig. 3b). Loss of the plasmid did not affect the growth of P. ananatis (Fig. 3c).
The endogenous plasmid of P. ananatis DZ-12 is important for pathogenesis in maize
We determined the ability of WT DZ-12, ∆p and ∆p-c strains of P. ananatis to infect maize plants by monitoring disease symptoms. The WT and complemented strains showed similar pathogenicity, whereas the ∆p strain was significantly reduced in pathogenicity compared with the WT (p ≤ 0.05) (Figs. 4a, 4b). The results suggest that pDZ-12 is important for pathogenesis in maize.
The endogenous plasmid of P. ananatis DZ-12 is important for biofilm formation
Biofilms are considered virulence determinants of many bacterial pathogens and are important for colonization of plants . Therefore, we investigated P. ananatis biofilm assembly by confocal scanning laser microscopy (CSLM). The ∆p mutant had impaired ability to form biofilms, while the complemented strain performed similarly to the WT (Figs. 4c, 4d). We conclude that pDZ-12 is important for biofilm formation in P. ananatis DZ-12.
The endogenous plasmid elimination strategy is also applicable to Bacillus subtilis
To evaluate the broader applicability of our protocol for curing endogenous plasmids, Gram-positive Bacillus subtilis strain NCIB 3610, harboring the endogenous plasmid pBS32 , was selected. RepN (WP_020846123.1) is a replication protein located on pBS32, thus we replaced the native repN with repPE194(Ts) from pJOE8999 . The endogenous plasmid pBS32 was cured by growing at high temperature (50 °C), yielding strain ∆pBS32. The observed efficient curing of the endogenous plasmid indicated a possible broader applicability of this methodology in both Gram-negative and -positive bacteria.