Knock-out of SO1377 gene, which encodes the member of a conserved hypothetical bacterial protein family COG2268, results in alteration of iron metabolism, increased spontaneous mutation and hydrogen peroxide sensitivity in Shewanella oneidensis MR-1

Abstract

Background: Shewanella oneidensis MR-1 is a facultative, gram-negative bacterium capable of coupling the oxidation of organic carbon to a wide range of electron acceptors such as oxygen, nitrate and metals, and has potential for bioremediation of heavy metal contaminated sites. The complete 5-Mb genome of S. oneidensis MR-1 was sequenced and standard sequence-comparison methods revealed approximately 42% of the MR-1 genome encodes proteins of unknown function. Defining the functions of hypothetical proteins is a great challenge and may need a systems approach. In this study, by using integrated approaches including whole genomic microarray and proteomics, we examined knockout effects of the gene encoding SO1377 (gi24372955), a member of the conserved, hypothetical, bacterial protein family COG2268 (Clusters of Orthologous Group) in bacterium Shewanella oneidensis MR-1, under various physiological conditions.

Results: Compared with the wild-type strain, growth assays showed that the deletion mutant had a decreased growth rate when cultured aerobically, but not affected under anaerobic conditions. Whole-genome expression (RNA and protein) profiles revealed numerous gene and protein expression changes relative to the wild-type control, including some involved in iron metabolism, oxidative damage protection and respiratory electron transfer, e. g. complex IV of the respiration chain. Although total intracellular iron levels remained unchanged, whole-cell electron paramagnetic resonance (EPR) demonstrated that the level of free iron in mutant cells was 3 times less than that of the wild-type strain. Siderophore excretion in the mutant also decreased in iron-depleted medium. The mutant was more sensitive to hydrogen peroxide and gave rise to 100 times more colonies resistant to gentamicin or kanamycin.

Conclusion: Our results showed that the knock-out of SO1377 gene had pleiotropic effects and suggested that SO1377 may play a role in iron homeostasis and oxidative damage protection in S. oneidensis MR-1.

Figure 1

Schematic presentation of generation of in-frame deletion mutant of SO1377 gene A. Generation of a deletion construct using asymmetric crossover PCR; B. Integration of the suicide plasmid during a single crossover event; C. Generation of in-frame deletion during the excision of the suicide plasmid. More detail about this construction process was described under "methods".

Figure 2

Growth curve of both SO1377 wild-type strain DSP-10 and deletion mutant WG3 in medium MR2A under aerobic conditions at 30°C. Note that the maximum growth peak reached by the wild-type control is around 1.2 while the mutant is 1.0.

Figure 3

Protein profile differentiation between the SO1377 wild-type strain DSP-10 and deletion mutant WG3. Numbers indicated proteins showing statistically significant (P < 0.001) difference in abundance; Note that protein #433 and #333 are absent from the mutant protein pattern. Approximate MW in KDa × 10E-10 is shown on the Y-axis; approximate isoelectric point is shown on the X-axis.

Figure 4

Iron EPR signal from whole-cell preparations. Both SO1377 wild-type strain (DSP-10) and mutant (WG3) were cultured in LB liquid for 24 h at 30°C, and EPR samples were prepared as described under "methods". Cell density was adjusted to equal before filling in the quartz EPR tubes according to measured OD_{600 nm} Value of each strain. The major signal appears at g = 4.3.

Figure 5

Intracellular iron concentration in both SO1377 wild-type strain DSP-10 and mutant WG3 after overnight growth in LB medium supplemented with iron (as ferric citrate). The implemented iron concentration in the medium is shown on the X-axis while the determined intracellular iron is shown in the Y-axis.

Figure 6

Siderophore production comparison between the SO1377 wild-type strain DSP-10 and mutant WG3 on a CAS siderophore testing agar. The chelator-iron (III) complex tints the agar with a rich blue background. The orange halo surrounding the colony indicates the excretion of siderophore and its dimension approximates the amount of siderphore excreted.

Figure 7

Survival curve of the SO1377 wild-type strain DSP-10 and mutant WG3 after subjected to H₂O₂ treatment. Note that almost 100% mutant cells cannot survive after 2 mM H₂O₂ treatment while about 30 % wild-type cells can survive under the same conditions.