Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Oct 15;10(10):1623.
doi: 10.3390/antiox10101623.

Influence of Redox Imbalances on the Transposition of Insertion Sequences in Deinococcus geothermalis

Qianying Ye  1 , Chanjae Lee  1 , Eunjung Shin  1 , Sung-Jae Lee  1
Affiliations

Influence of Redox Imbalances on the Transposition of Insertion Sequences in Deinococcus geothermalis

Qianying Ye et al. Antioxidants (Basel). .

Abstract

The transposition of insertion sequence elements was evaluated among different Deinococcus geothermalis lineages, including the wild-type, a cystine importer-disrupted mutant, a complemented strain, and a cystine importer-overexpressed strain. Cellular growth reached early exponential growth at OD600 2.0 and late exponential growth at OD600 4.0. Exposing the cells to hydrogen peroxide (80-100 mM) resulted in the transposition of insertion sequences (ISs) in genes associated with the carotenoid biosynthesis pathway. Particularly, ISDge7 (an IS5 family member) and ISDge5 (an IS701 family member) from the cystine importer-disrupted mutant were transposed into phytoene desaturase (dgeo_0524) via replicative transposition. Further, the cystine importer-overexpressed strain Δdgeo_1985R showed transposition of both ISDge2 and ISDge5 elements. In contrast, IS transposition was not detected in the complementary strain. Interestingly, a cystine importer-overexpressing strain exhibited streptomycin resistance, indicating that point mutation occurred in the rpsL (dgeo_1873) gene encoding ribosomal protein S12. qRT-PCR analyses were then conducted to evaluate the expression of oxidative stress response genes, IS elements, and low-molecular-weight thiol compounds such as mycothiol and bacillithiol. Nevertheless, the mechanisms that trigger IS transposition in redox imbalance conditions remain unclear. Here, we report that the active transposition of different IS elements was affected by intracellular redox imbalances caused by cystine importer deficiencies or overexpression.

Keywords: Deinococcus geothermalis; cystine importer; insertion sequences; oxidative stress; redox-balance; transcriptomic analysis; transposition.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
PCR confirmed both parent strains ∆dgeo_1986-87 and ∆dgeo_1985R, and their non-pigmented colonies induced by hydrogen peroxide, as well as the wild-type strain. Schematic illustration of mutant construction (A). Phenotypic difference between parent (p) and non-pigment strains (w) and PCR confirmation with primer set encompassing target genes for dgeo_1986-87 (B) and dgeo_1985R (C). Lanes: M, size marker; WT, wild-type; P, parent strain; w, non-pigment strains; s, streptomycin-resistant strain.
Figure 2
Figure 2
PCR detection of transposition loci on four target genes for carotenoid biosynthesis within dgeo_0523 (1.87 kb), dgeo_0524 (2.09 kb), dgeo_0857 (1.85 kb), and dgeo_2309 (1.86 kb). Lanes: M, size marker; 1 and 9, wild-type; 2 and 10, ∆dgeo_1986-87; 3 and 11, ∆dgeo_1986-87_w1; 4 and 12, ∆dgeo_1986-87_w2; 5 and 13, ∆dgeo_1986-87_w3; 6 and 14, ∆dgeo_1985R; 7 and 15, ∆dgeo_1985R_w1; 8 and 16, ∆dgeo_1985R_w2. Arrow indicates the IS integrated samples: 7, 2.62 kb; 11 and 16, 3.24 kb; 12 and 13, 2.96 kb.
Figure 3
Figure 3
Detection of IS integration loci in three non-pigmented ∆dgeo_1986-87 mutant strains (w1-w3). (A) There were two IS element integration sites of ISDge5 and ISDge7 on dgeo_0524 encoding a phytoene desaturase. (B) PCR detection of four ISDge7 copies in the genome using the target gene primer sets from the wild-type, a parent strain, and ∆dgeo_1986-87_w2: dgeo_1042 (2.0 kb), dgeo_1699 (2.37 kb), dgeo_2208 (2.03 kb), and dgeo_2276 (2.11 kb). Lanes: M, size marker; 1, 4, 7, and 10, wild-type; 2, 5, 8, and 11, parent strain; 3, 6, 9, and 12, ∆dgeo_1986-87_w2 mutant. PCR detection of ten ISDge5 copies in the genome indicated in Figure S3.
Figure 4
Figure 4
Detection of IS integration sites in two non-pigment ∆dgeo_1985R mutant strains (w1 and w2). (A) There are two IS element integration sites of ISDge5 and ISDge2 on dgeo_0524 and dgeo_0523, respectively, which encode phytoene synthase. (B) PCR detection of nine ISDge2 copies in the genome was amplified using the target gene primer sets from a parent strain and ∆dgeo_1985R_w1: dgeo_1673 (0.98 kb), dgeo_2377 (0.76 kb), dgeo_2446 (0.75 kb), dgeo_2436 (0.94 kb), dgeo_2587 (0.75 kb), dgeo_2700 (1.05 kb), dgeo_2795 (0.88 kb), dgeo_2987 (0.98 kb), and dgeo_3100 (0.85 kb). Lanes: M, size marker; odd numbers, parent strain; even numbers, ∆dgeo_1985R_w1 mutant.
Figure 5
Figure 5
Identification of a streptomycin-resistant mutant from ∆dgeo_1985R. (A) Measurement of streptomycin MIC values between a parent strain and a SmR mutant (s1). (B) PCR detection of four SmR related genes (dgeo_0447 (0.9 kb), dgeo_0776 (1.05 kb), dgeo_1873 (0.9 kb), and dgeo_2335 (0.92 kb)) in a genome created using a parent strain and a SmR mutant. Lanes: M, size marker; 1,4,7, and 10, wild-type; 2,5,8, and 11, ∆dgeo_1985R parent strain; 3,6,9, and 12, ∆dgeo_1985R_s1 mutant.
Figure 6
Figure 6
qRT-PCR analysis of four mycothiol biosynthesis genes (A) and three IS elements during active transposition (B) at two different growth phases in the presence and absence of hydrogen peroxide (50 mM). Pairwise comparisons were conducted using Student’s t-test to identify differences between the samples using the PrismTM software. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).
Figure 6
Figure 6
qRT-PCR analysis of four mycothiol biosynthesis genes (A) and three IS elements during active transposition (B) at two different growth phases in the presence and absence of hydrogen peroxide (50 mM). Pairwise comparisons were conducted using Student’s t-test to identify differences between the samples using the PrismTM software. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Winterbourn C.C., Hampton M.B. Thiol chemistry and specificity in redox signaling. Free Radic. Biol. Med. 2008;45:549–561. doi: 10.1016/j.freeradbiomed.2008.05.004. - DOI - PubMed
    1. Helmann J.D. Bacillithiol, a new player in bacterial redox homeostasis. Antioxid. Redox. Signal. 2011;15:123–133. doi: 10.1089/ars.2010.3562. - DOI - PMC - PubMed
    1. Hung J., Cooper D., Turner M.S., Walsh T., Giffard P.M. Cystine uptake prevents production of hydrogen peroxide by Lactobacillus fermentum BR11. FEMS Microbiol. Lett. 2003;227:93–99. doi: 10.1016/S0378-1097(03)00653-0. - DOI - PubMed
    1. Lo R., Turner M.S., Barry D.G., Sreekumar R., Walsh T.P., Giffard P.M. Cystathionine gamma-lyase is a component of cystine-mediated oxidative defense in Lactobacillus reuteri BR11. J. Bacteriol. 2009;191:1827–1837. doi: 10.1128/JB.01553-08. - DOI - PMC - PubMed
    1. Ohtsu I., Kawano Y., Suzuki M., Morigasaki S., Saiki K., Yamazaki S., Nonaka G., Takagi H. Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli. PLoS ONE. 2015;10:e0120619. doi: 10.1371/journal.pone.0120619. - DOI - PMC - PubMed