Candidate Genes That May Be Responsible for the Unusual Resistances Exhibited by Bacillus pumilus SAFR-032 Spores

Abstract

The spores of several Bacillus species, including Bacillus pumilus SAFR-032 and B. safensis FO-36b, which were isolated from the spacecraft assembly facility at NASA's Jet Propulsion Laboratory, are unusually resistant to UV radiation and hydrogen peroxide. In order to identify candidate genes that might be associated with these resistances, the whole genome of B. pumilus SAFR-032, and the draft genome of B. safensis FO-36b were compared in detail with the very closely related type strain B. pumilus ATCC7061T. 170 genes are considered characteristic of SAFR-032, because they are absent from both FO-36b and ATCC7061T. Forty of these SAFR-032 characteristic genes are entirely unique open reading frames. In addition, four genes are unique to the genomes of the resistant SAFR-032 and FO-36b. Fifty three genes involved in spore coat formation, regulation and germination, DNA repair, and peroxide resistance, are missing from all three genomes. The vast majority of these are cleanly deleted from their usual genomic context without any obvious replacement. Several DNA repair and peroxide resistance genes earlier reported to be unique to SAFR-032 are in fact shared with ATCC7061T and no longer considered to be promising candidates for association with the elevated resistances. Instead, several SAFR-032 characteristic genes were identified, which along with one or more of the unique SAFR-032 genes may be responsible for the elevated resistances. These new candidates include five genes associated with DNA repair, namely, BPUM_0608 a helicase, BPUM_0652 an ATP binding protein, BPUM_0653 an endonuclease, BPUM_0656 a DNA cytosine-5- methyltransferase, and BPUM_3674 a DNA helicase. Three of these candidate genes are in immediate proximity of two conserved hypothetical proteins, BPUM_0654 and BPUM_0655 that are also absent from both FO-36b and ATCC7061T. This cluster of five genes is considered to be an especially promising target for future experimental work.

Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase-like reactivity ofBacillus pumiluscatalase monitored by X-ray crystallography and EPR spectroscopy

“…Not surprisingly, this spectrum is very similar to the BLC EPR spectrum at pH 8.0 (Fig. bottom, dark red trace) and to those of other bacterial catalases previously reported …”

“…The enzyme was first purified directly from extracts of BPC B6033 yielding a protein >85% pure by SDS PAGE with a subunit size of ∼58 kDa and a UV–visible electronic absorption spectrum typical for heme b‐containing catalases . Expression of the cloned katX2 gene in E. coli and subsequent purification yielded a protein with similar subunit size with an apparent K M for H 2 O 2 of 65 m M and a maximal turnover rate of 339,000 s −1 , which is among the highest so far determined for a catalase .…”

“…To achieve binding of the substrates to the heme site, 4 μL substrate at 1 M concentration was added to 60 mL enzyme at 0.3 m M concentration (a 200 fold‐excess of substrate, prepared in the same buffer and pH as the enzyme) and incubated for 2 h at room temperature (20 °C) followed by 12 h incubation in the fridge (8 °C). Samples of commercial catalase from bovine liver, further purified as previously described, were used for comparisons of pH and substrate binding effect on the EPR ferric signal. EPR samples of BLC were prepared in identical experimental conditions as described above for the BPC catalase.…”

Germination of Spores of Astrobiologically RelevantBacillusSpecies in High-Salinity Environments

“…Its spores have an outstandingly high resistance against UV radiation and H 2 O 2 , enabling this strain to withstand decontamination procedures (Link et al, 2004;Tirumalai et al, 2013). Bacillus pumilus SAFR-032 spores have previously been used for astrobiological experiments including prolonged exposure to real space conditions on board the International Space Station Nicholson et al, 2012;Vaishampayan et al, 2012).…”

Complete genome sequence of Bacillus pumilus W3: A strain exhibiting high laccase activity

“…Then a draft genome with 15 scaffolds (no gaps in scaffolds) was achieved. The order of scaffolds was determined by a similarity search among published references (Gioia et al, 2007;Tirumalai et al, 2013). Gaps between scaffolds were closed by longrange PCR using TaKaRa LA Taq TM with GC Buffer (Dalian, China) and subsequent Sanger sequencing using an ABI 3730 capillary sequencer.…”