A novel membrane-anchored cytochrome c-550 of alkaliphilic Bacillus clarkii K24-1U: expression, molecular features and properties of redox potential

Abstract

A membrane-anchored cytochrome c-550, which is highly expressed in obligately alkaliphilic Bacillus clarkii K24-1U, was purified and characterized. The protein contained a conspicuous sequence of Gly(22)-Asn(34), in comparison with the other Bacillus small cytochromes c. Analytical data indicated that the original and lipase-treated intermediate forms of cytochrome c-550 bind to fatty acids of C(15), C(16) and C(17) chain lengths and C(15) chain length, respectively, and it was considered that these fatty acids are bound to glycerol-Cys(18). Since there was a possibility that the presence of a diacylglycerol anchor contributed to the formation of dimeric states of this protein (20 and 17 kDa in SDS-PAGE), a C18M (Cys(18) --> Met)-cytochrome c-550 was constructed. The molecular mass of the C18M-cytochrome c-550 was determined as 15 and 10 kDa in SDS-PAGE and 23 kDa in blue native PAGE. The C18M-cytochrome c-550 bound with or without Triton X-100 formed a tetramer as the original cytochrome c-550 bound with Triton X-100, as determined by gel filtration. The midpoint redox potential of cytochrome c-550 as determined by redox titration was +83 mV, while that determined by cyclic voltammetric measurement was +7 mV. The above results indicate that cytochrome c-550 is a novel cytochrome c.

Relationship between rates of respiratory proton extrusion and ATP synthesis in obligately alkaliphilic Bacillus clarkii DSM 8720T

“…From our results, the growth characteristics of B. clarkii DSM 8720 T and B. subtilis IAM 1026 (Table 1) are comparable to those previously reported results in alkaliphiles and neutralophiles (Guffanti and Hicks 1991;Ogami et al 2009). These findings suggest that alkaliphilic B. clarkii DSM 8720 T possesses certain features advantageous for energy production rather than the presence of a disadvantage in terms of ΔpH in their physiological mechanism.…”

“…This function of cytochrome c has also been reported in the study of alkaliphilic Pseudomonas alcaliphila (Matsuno et al 2012). On the other hand, acidic membrane-anchored cytochromes c have been isolated from alkaliphilic Bacillus species (Hicks and Krulwich 1995;Quirk et al 1993;Ogami et al 2009). It is considered that the presence of acidic cytochromes c may attract protons translocated by the respiratory complexes.…”

“…The generation time for a culture pH of 10.6 (38 min) is shorter than that for pH 7.5 (54 min) (Sturr et al 1994). On the other hand, the obligately alkaliphilic Bacillus clarkii strain K24-1U exhibits a higher specific growth rate (μ max 00.33 h −1 ) than neutralophilic Bacillus subtilis IAM 1026 (μ max 00.26 h −1 ) cultured in a fermenter at an agitation speed of 106 rpm and an air flow rate of 20 L min −1 (Ogami et al 2009). The above-mentioned experimental data indicate that even though it has been considered that an alkaline condition is disadvantageous for production of energy theoretically, it seems that the growth of alkaliphilic microorganisms utilizes a specific advantageous feature under alkaline condition for their energy production.…”

Bioenergetics and the Role of Soluble Cytochromescfor Alkaline Adaptation in Gram-Negative AlkaliphilicPseudomonas

“…Generation times of 54 and 38 min were obtained at medium pHs controlled at 7.5 and 10.6, respectively, in the facultatively alkaliphilic bacterium Bacillus pseudofirmus OF4 [27]. The obligate alkaliphilic bacterium Bacillus clarkii strain K24-IU exhibits a higher growth rate ( μ max⁡ = 0.33 h −1 ) than the neutralophilic bacterium Bacillus subtilis IAM 1026 ( μ max⁡ = 0.26 h −1 ) in batch culture [28]. These growth characteristics of alkaliphilic Bacillus species contradict the deficiency of bioenergetics parameter owing to the reversed transmembrane pH gradient in alkaliphilic Bacillus in alkaline conditions.…”

“…Although several diacylglyceryl-cysteine moieties attached to cytochromes c have been isolated from several Gram-positive bacteria, there has been no report on the diacylglyceryl-cysteine moiety attached to cytochromes c from alkaliphilic Bacillus . Ogami et al purified and characterized a cytochrome c with a diacylglyceryl-cysteine moiety attached, namely, cytochrome c -550, from an obligate alkaliphilic Bacillus clarkii K24-1U [28]. Cytochrome c -550 binds to fatty acids of C 15 , C 16 , and C 17 chain lengths via glycerol-Cys 18 .…”