Bacillus endospores isolated from granite: close molecular relationships to globally distributed Bacillus spp. from endolithic and extreme environments

Abstract

As part of an ongoing effort to catalog spore-forming bacterial populations in environments conducive to interplanetary transfer by natural impacts or by human spaceflight activities, spores of Bacillus spp. were isolated and characterized from the interior of near-subsurface granite rock collected from the Santa Catalina Mountains, AZ. Granite was found to contain approximately 500 cultivable Bacillus spores and approximately 10(4) total cultivable bacteria per gram. Many of the Bacillus isolates produced a previously unreported diffusible blue fluorescent compound. Two strains of eight tested exhibited increased spore UV resistance relative to a standard Bacillus subtilis UV biodosimetry strain. Fifty-six isolates were identified by repetitive extragenic palindromic PCR (rep-PCR) and 16S rRNA gene analysis as most closely related to B. megaterium (15 isolates), B. simplex (23 isolates), B. drentensis (6 isolates), B. niacini (7 isolates), and, likely, a new species related to B. barbaricus (5 isolates). Granite isolates were very closely related to a limited number of Bacillus spp. previously found to inhabit (i) globally distributed endolithic sites such as biodeteriorated murals, stone tombs, underground caverns, and rock concretions and (ii) extreme environments such as Antarctic soils, deep sea floor sediments, and spacecraft assembly facilities. Thus, it appears that the occurrence of Bacillus spp. in endolithic or extreme environments is not accidental but that these environments create unique niches excluding most Bacillus spp. but to which a limited number of Bacillus spp. are specifically adapted.

FIG. 1.

Numbers of total viable bacteria (solid bars) and heat-resistant spores (open bars) per gram of granite, grown on solid LB medium. Numbers are averages ± standard deviations (n = 2 or 3).

FIG. 2.

Blue fluorescence exhibited by Bacillus spp. from granite. (A) Colonies of strains WN612 (fluorescent granite isolate; top) and WN613 (nonfluorescent granite isolate; bottom) after 2 days on solid LB medium. (B) Cell-free culture supernatants of 2-day cultures in liquid LB medium of B. subtilis HA101 (nonfluorescent reference strain; left), strain WN572 (fluorescent granite isolate; center), and strain WN613 (nonfluorescent granite isolate; right). Photographs were taken under medium-wavelength UV illumination.

FIG. 3.

rep-PCR analysis of 56 endolithic Bacillus sp. isolates from granite. The rep-PCR gel image is shown to the right of the corresponding strain number. Bracketed groups I to XII denote strains sharing >93% homology in their rep-PCR patterns based on cluster analysis. Strains with asterisks were chosen as group representatives for 16S rRNA gene sequence determination. The production of blue fluorescence after 48 h on LB solid medium is denoted as follows: −, no fluorescence; +, weak to moderate fluorescence; ++, strong fluorescence.

FIG. 4.

rep-PCR analysis of seven endolithic Bacillus sp. isolates from basalt (3). Strain WN696 was most closely related to B. subtilis, and strains WN691-695 and WN697 were most closely related to B. pumilus by 16S rRNA gene analysis (3). Isolates WN570 (B. simplex) and WN611 (B. megaterium) from granite and type strains B. subtilis 168 and B. pumilus ATCC 7061 were included for comparison.

FIG. 5.

Spore UV resistance. The graphs show comparisons of LD₉₀ values (A) and D values (B) of granite isolates (white bars) (numbers denote strain numbers; see Table 1) and the UV dosimetry strain B. subtilis ATCC 6633 (6633; shaded bars). Bars and error bars represent averages and standard deviations, respectively (n = 3 or 4). Strains with P values of <0.05 by analysis of variance were judged as belonging to separate groups (indicated with lowercase letters).