Identification of Brevibacterium flavum genes related to receptors involved in bacteriophage BFK20 adsorption.

Phage infection of bacterial cells is a process requiring the interaction between phage receptor binding proteins and receptors on the bacterial cell surface. We prepared a Brevibacterium flavum CCM 251 EZ-Tn5 transposon insertional library and isolated phage-resistant mutants. Analysis of the DNA fragments produced by single-primer PCR was used to determine the EZ-Tn5 transposon insertion sites in the genomes of phage-resistant B. flavum mutants. Seven disrupted genes were identified in forty B. flavum mutants. The phage resistance of these mutants was demonstrated by cultivation analysis in the presence of BFK20, and the adsorption rate of BFK20 to these mutants was tested. B. flavum mutants displayed significantly reduced adsorption rates; the lowest rate was observed for mutants containing interrupted major facilitator superfamily (MFS) protein and glycosyltransferase genes. Uninterrupted forms of these genes were cloned into corynebacterial vector pJUP06 and used for in trans complementation of the corresponding B. flavum mutants. The growth of these complemented mutants when infected with BFK20 closely resembled that of wild-type B. flavum. These complemented mutants also exhibited similar BFK20 adsorption as the wild-type control. We infer that the disrupted MFS protein and glycosyltransferase genes are responsible for the phage-resistant phenotype of these B. flavum transposition mutants.

The BFK20 phage replication origin confers a phage-encoded resistance phenotype to the industrial strain Brevibacterium flavum.

The phage BFK20 replication origin was identified using bioinformatics tools and a fragment with the origin nucleotide sequence was cloned into the tetracycline resistance gene of Escherichia coli vector pBR328, to make the plasmid pBOS. After transformation into the host strain Brevibacterium flavum CCM 251, pBOS was able to replicate, showing that the cloned region may function as a replication origin. The presence of the BFK20 origin sequence in a pBOS plasmid isolated from B. flavum CCM 251 was confirmed by Southern hybridisation. Monitoring pBOS stability in corynebacterial hosts showed that pBOS was stable in Corynebacterium glutamicum RM3 for 20 generations and in B. flavum CCM 251 for 10 generations. The effect of the cloned BFK20 replication origin on host resistance to BFK20 infection was tested. Growth of a B. flavum CCM 251 strain harbouring pBOS stopped after phage infection, but without complete lysis. Five hours after infection, the viability of the modified strain was about five times higher than the viability of wild-type B. flavum CCM 251. Thus, the ability of the BFK20 replication origin to confer the origin-derived phage-encoded resistance phenotype to B. flavum CCM 251 was confirmed.

Gp41, a superfamily SF2 helicase from bacteriophage BFK20.

Gp41 is one of two helicases encoded by the genome of bacteriophage BFK20. The gp41 sequence contains conserved motifs from the SF2 family of helicases. We prepared and studied three recombinant proteins: gp41HN, a wild type-like protein with an N-terminal His-Tag; gp41HC, with an S2A mutation and a C-terminal His-Tag; and gp41dC, a mutant protein with a deleted C-terminal region and His-Tags on both N- and C-termini. We tested the enzymatic activities and DNA binding abilities of these isolated proteins. We found that both gp41HN and gp41HC had strong DNA-dependent ATPase activities, but that the ATPase activity of gp41dC was significantly lower regardless of the presence of DNA. The preferred substrates for the NTP hydrolysis reactions were ATP and dATP. gp41HC and gp41HN exhibited a low helicase activity in a fluorescence-based assay using dsDNA substrates with a 3' overhang and with a forked end in the presence of ATP. We infer that the C-terminal region of gp41 may be involved in DNA binding, since removing this region in gp41dC reduced the protein's DNA binding ability.