Abstract
The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.
Highlights
Bacillus subtilis is a Gram-positive aerobic bacterium, it can grow anaerobically under some conditions [1, 2]
Cytochrome bd can be found in B. subtilis cells grown with glucose [11, 16, 19] and is a quinol oxidases (QOX) that does not pump protons [11]
LUH27 grown without glucose at high aeration (A) or with glucose at low aeration (B)
Summary
Chemicals—Aurachin D was a kind gift of Prof. Peter Rich (University College, London) obtained through the courtesy of Dr Jeff Osborne (University of Illinois, Urbana-Champain, IL). Construction of Strain LUH27—B. subtilis strain 168 was transformed to phleomycin resistance with chromosomal DNA from strain JO1, which contains a ⌬ctaCD::ble deletion [30]. The resulting strain, LUH15 (trpC2 ⌬ctaCD::ble), was transformed to chloramphenicol resistance with strain 3G18⌬12 DNA containing a ⌬sdhCAЈ::cat mutation [31]. The triple respiratory deficient mutant LUH27 (trpC2 ⌬ctaCD::ble ⌬sdhCAЈ::cat ⌬qoxABCD::kan) was obtained by the transformation of strain LUH19 to neomycin resistance with chromosomal DNA from a strain containing a ⌬qoxABCD::kan mutation [12]. Strain LUW123 (trpC2 ⌬ythAB::tet ⌬ctaCD::ble ⌬sdhCA::cat ⌬qoxABCD::kan) was obtained by transforming LUH27 with chromosomal DNA isolated from LUW122. Transformation of B. subtilis Strains with Chromosomal or Plasmid DNA—Transformations were performed essentially as described by Hoch [33], and transformants were selected on tryptose blood agar base plates containing chloramphenicol (4 or 5 mg/liter), phleomycin (3 mg/ liter), neomycin (5 mg/liter), or tetracyclin (15 mg/liter) as appropriate. Proton pumping was assayed using the “oxygen pulse” method as described in Ref. 12, except that KSCN was replaced by valinomycin
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