Characterization Of Maltocin P28and Regulator Genes Of Its Biosynthesis

Stenotrophomonas maltophilia is an important global opportunistic pathogen for which limited therapeutics are available because of the emergence of multidrug-resistant strains. It is urgently needed to develop novel therapeutic agents against S. maltophilia infections.Both the filamentous phage ?SHP2and maltocin P28could be produced by the environmental S. maltophilia strain P28. The separated phage and maltocin particles were obtained using DEAE-cellulose column chromatography. The phage particles were filamentous in shape and about800x10nm in size. The major coat protein of ?SHP2is approximate3.0kDa. It was proved that a high-copy plasmid harbored in S. maltophilia strain P28was the replicative form (RF) of phage ?SHP2. The genome of filamentous phage ?SHP2was5817nt in length. Sequence analyses showed that it contains9putative Open Reading Frames (ORFs). orf1to orf7were transcribed in one direction, while orf8and orf9in the opposite direction. The hypothetical products of orfl, orf2and orf7were replication initiation factor (Rep), single-stranded DNA binding protein (SSB) and Zot-like protein, respectively. According to the size and genomic position of the genes coding for capsid proteins of other inoviruses, orf3-orf6were likely to constitute the structure module. The two genes, orf8and orf9, transcribed in the opposite direction may participate in the regulation of cpSHP2biosynthesis. In the phylogenetic trees, phage ?SHP2had the closest relationship with another Stenotrophomonas phage cpSHPl.A novel bacteriocin, maltocin P28, is the first-identified bacteriocin from S. maltophilia. Maltocin P28resembles a contractile but non-flexible phage tail structure based on electron microscopy, and it is sensitive to trypsin, proteinase K and heat. Maltocin P28had bactericidal activity against38(19environmental strains and19 clinical strains) of80(37environmental strains and43clinical strains) tested S. maltophilia strains.SDS-PAGE analysis of maltocin P28revealed two major protein bands of approximately43and20kDa. The N-terminal amino acid residues of these two major subunits were sequenced, and the maltocin P28gene cluster was located on the S. maltophilia P28chromosome. Sequence analysis results indicate that this maltocin gene cluster consists of23ORFs, and that its gene organization is similar to the P2phage genome and R2pyocin gene cluster. ORF17and ORF18encode the two major structural proteins, which correspond to gpFI (tail sheath) and gpF ? (tail tube) of P2phage, respectively, In addition, we prepared the polyclonal antibody for ORF17product.Either lexA or recA gene in S. maltophilia genome was deleted by knock-out method. The results indicated that LexA and RecA, the major proteins in SOS repair system, did not affect maltocin P28production. It suggested that there be a novel mechanism for the regulation of maltocin P28biosynthesis. At the same time, the deletion strains were constructed by knocking out ORF1-ORF7one by one. Antimicrobial activity, Western blot and RT-PCR analyses showed that ORF3, ORF5and ORF6might participate in the regulation of maltocin P28biosynthesis,In conclusion, we identified the first Stenotrophomonas bacteriocin, maltocin P28, as well as a novel filamentous phage ?SHP2. Although the gene organization of structural genes of maltocin P28is similar to those of P2phage genome and R2pyocin gene cluster, there is a distinctive mechanism for regulation of maltocin P28production. Considering its advantage of well biosecurity (proteinaceous compounds and absence of nucleic acid), high production and restricted bactericidal spectrum, maltocin P28may be a promising therapeutic substitute to antibiotics for S. maltophilia infections.