Antibiotic resistance mutations induced in growing cells of Bacillus-related thermophiles

Abstract

Stress-induced mutagenesis can assist pathogens in generating drug-resistant cells during antibiotic therapy; however, if and how antibiotics induce mutagenesis in microbes remains poorly understood. A non-pathogenic thermophile, Geobacillus kaustophilus HTA426, efficiently produces derivative cells resistant to rifampicin and streptomycin via rpoB and rpsL mutations, respectively. Here, we examined this phenomenon to suggest a novel mutagenic mode induced by antibiotics. Fluctuation analysis indicated that mutations occurred via spontaneous mutations during culture. However, mutations were much more frequent in growing cells than stationary cells, and mutation sites were varied through cell growth. These observations suggested that growing cells induced mutagenesis in response to antibiotics. An in-frame deletion of mfd, which governs transcription-coupled repair to correct DNA lesions on the transcribed strand, caused mutations that were comparable between growing and stationary cells; therefore, the mutagenic mechanism was attributable to DNA repair defects where growing cells depressed mfd function. Mutations occurred more frequently at optimal growth temperatures for G. kaustophilus than at a higher growth temperature, suggesting that the mutagenesis relies on active cellular activities rather than high temperature-associated DNA damage. In addition, the mutagenesis may involve a mutagenic factor targeting these sites, in addition to mfd depression, because rpoB and rpsL mutations were dominant at thymine and guanine sites on the transcribed strand. A similar mutagenic profile was observed for other Geobacillus and thermophilic Bacillus species. This suggests that Bacillus-related thermophiles commonly induce mutagenesis in response to rifampicin and streptomycin to produce resistant cells.

A novel thermostable TP-84 capsule depolymerase: a method for rapid polyethyleneimine processing of a bacteriophage-expressed proteins

“…Nevertheless, the bacteriophage yields and morphology of its plaques varies with changes in temperature and humidity. To avoid contaminations as well as for a future scaled-up biotechnology applications, the streptomycin-resistant mutant [ 30 ] G. stearothermophilus 10 was generated. Bacterial colonies, cells morphology, culture growth rate, metabolism did not show any differences and the mutant was used for the studies.…”

Frequent Transposition of Multiple Insertion Sequences in Geobacillus kaustophilus HTA426

“…can form robust endospores in the stationary phase (Suzuki, 2018); therefore, IS transposition may be enhanced as an adaptation strategy specific to the proliferative phase. We previously observed that exposure of G. kaustophilus to rifampicin apparently induced mutagenesis to produce rifampicin-resistant mutants and the induction was stronger in proliferative cells than in stationary cells (Suzuki et al, 2018). Although this manner implies that mutagenesis may be also governed by SigX in parallel to IS transposition, rifampicinresistant mutants were comparably generated between MK536 p74 and MK536 sigX ( Figure 5B); therefore, mutagenesis is not under the SigX control.…”

“…Geobacillus spp. apparently induce mutagenesis when proliferative cells are exposed to rifampicin and efficiently generate rifampicin-resistant cells via rpoB mutations (Suzuki et al, 2018). To see whether the mutagenesis depends on sigX-dependent stress responses, MK536 derivatives (MK536 p74 , MK536 rsbV , MK536 sigB , and MK536 sigX ) were assessed by generation frequency assay of rifampicin-resistant mutants.…”

“…can also rapidly generate mutant genes for thermostable variants from thermolabile protein genes (Liao et al, 1986;Suzuki et al, 2015;Kobayashi et al, 2015a,b;Wada et al, 2016). In the species, mutagenesis is apparently induced when proliferative cells are faced with growth inhibition by antibiotics (Suzuki et al, 2018). These observations suggest that Geobacillus spp.…”

Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae

“…A vector carrying a tetracycline resistance marker has been successfully introduced into a moderately thermophilic strain, and the spectinomycin resistance marker was shown to work with two thermophilic strains [ 119 ]. However, the typically necessary use of low antibiotic concentrations can lead to spontaneous antibiotic-resistant colonies [ 120 , 121 ]. Antibiotics and resistance mechanisms used under mesophilic conditions are thus not directly applicable for work with thermophilic microorganisms due to thermal instability [ 122 ].…”