Abstract
Rifampin resistant (RifR) mutants of the insect pathogenic bacterium Photorhabdus luminescens LN2 from entomopathogenic nematode Heterorhabditis indica LN2 were genetically and proteomically characterized. The RifR mutants showed typical phase one characters of Photorhabdus bacteria, and insecticidal activity against Galleria mellonella larvae, but surprisingly influenced their nematicidal activity against axenic infective juveniles (IJs) of H. bacteriophora H06, an incompatible nematode host. 13 out of 34 RifR mutants lost their nematicidal activity against H06 IJs but supported the reproduction of H06 nematodes. 7 nematicidal-producing and 7 non-nematicidal-producing RifR mutants were respectively selected for rpoB sequence analysis. rpoB mutations were found in all 14 RifR mutants. The rpoB (P564L) mutation was found in all 7 mutants which produced nematicidal activity against H06 nematodes, but not in the mutants which supported H06 nematode production. Allelic exchange assays confirmed that the Rif-resistance and the impact on nematicidal activity of LN2 bacteria were conferred by rpoB mutation(s). The non-nematicidal-producing RifR mutant was unable to colonize in the intestines of H06 IJs, but able to colonize in the intestines of its indigenous LN2 IJs. Proteomic analysis revealed different protein expression between wild-type strain and RifR mutants, or between nematicidal-producing and non nematicidal-producing mutants. At least 7 putative proteins including DsbA, HlpA, RhlE, RplC, NamB (a protein from T3SS), and 2 hypothetical proteins (similar to unknown protein YgdH and YggE of Escherichia coli respectively) were probably involved in the nematicidal activity of LN2 bacteria against H06 nematodes. This hypothesis was further confirmed by creating insertion-deletion mutants of three selected corresponding genes (the downregulated rhlE and namB, and upregualted dsbA). These results indicate that the rpoB mutations greatly influence the symbiotic association between the symbionts and their entomopathogenic nematode hosts.
Highlights
Rifampin (Rif), first introduced in 1972 as an antitubercular drug, was initially extremely effective against Mycobacterium tuberculosis, and other bacteria [1,2]
Most Rif-resistance mutations in M. tuberculosis as well as in Escherichia coli and Staphylococcus aureus were conferred by a set of restrictive mutations in the rpoB gene, which encoded the bsubunit of RNA polymerase (RNAP) in bacteria [3,4]
The recombinant LN2-WDrpoB-LR31 showed Rif resistance and lost the nematicidal activity against H06 infective juveniles (IJs), while LN2R31DrpoB-LW was sensitive to Rif and restored the nematicidal activity. These results clearly indicated that rpoB mutation was responsible for the Rif-resistance and the absence of nematicidal activity of LN2-R31
Summary
Rifampin (Rif), first introduced in 1972 as an antitubercular drug, was initially extremely effective against Mycobacterium tuberculosis, and other bacteria [1,2]. Most Rif-resistance mutations in M. tuberculosis as well as in Escherichia coli and Staphylococcus aureus were conferred by a set of restrictive mutations in the rpoB gene, which encoded the bsubunit of RNA polymerase (RNAP) in bacteria [3,4]. Besides Rif-resistance, the rpoB mutation (A621E) conferred dual heteroresistance to daptomycin and vancomycin in Staphylococcus aureus [17], but most rpoB mutations were involved in reduced vancomycin susceptibility [18]. It suggested that different rpoB mutations may have different effect on bacteria
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