Background Alkanes have already been hypothesized to do something as common

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Background Alkanes have already been hypothesized to do something as common inducers of bacterial cytochrome P450 gene manifestation. the CYP110 proteins utilizing a spectroscopic spin-shift assay, but alkanes didn’t bind. CYP110 proteins was recognized in vegetative cells but not in differentiated heterocysts where nitrogen fixation occurs. Conclusion Hexadecane treatment was an effective inducer of CYP110 expression in cyanobacteria. Based on substrate binding profiles and amino acid sequence similarities it is hypothesized that CYP110 is a fatty acid -hydroxylase in photosynthetic cells. CYP110 was found associated with membrane fractions unlike other soluble microbial P450 proteins, and in this regard CYP110 more closely resembles eukarytotic P450s. Substrate stablization is an unlikely mechanism for alkane induction because alkanes did not bind to purified CYP110 protein. Background em Anabaena /em sp. strain PCC 7120 ( em Anabaena /em 7120) is an obligate photoautotrophic cyanobacterium that reduces atmospheric nitrogen in terminally differentiated cells known as heterocysts [1]. Approximately every tenth cell along a filament differentiates into a heterocyst under nitrogen-fixing conditions. Three developmentally regulated DNA rearrangements occur within the heterocyst chromosome which excise DNA elements integrated within the em nifD /em , em fdxN /em and em hupL /em genes [2,3]. The element interrupting the em nifD /em gene is 11,268 base pairs in length and carries its own site-specific recombinase, the em xisA /em gene [4]. The em nifD /em element is found in most heterocyst forming cyanobacteria integrated within the MK-0822 ic50 em nifD /em gene [5,6]. It is absent from all non-heterocystous strains examined to date [7,8]. While factors driving the evolution of the em nifD /em element and its associated genes remain obscure, an essential role in diazotrophic growth can be ruled out since an em Anabaena /em strain cured of the em nifD /em element was shown to fix nitrogen under aerobic conditions in heterocysts of normal morphology [9]. The complete DNA sequence of the em nifD /em element reveals little about the biological functions it encodes. Nine open reading frames are known (see Fig. ?Fig.1)1) only one of which can be identified by sequence homology: the first reported cyanobacterial cytochrome P450 gene, em cyp110 /em [10]. The em cyp110 /em gene is widely conserved in the heterocyst forming cyanobacteria including em Anabaena /em , em Nostoc /em MK-0822 ic50 and em Calothrix/Fremyella /em . The amino acid sequence of CYP110 is most similar to the mammalian P450 family 4 fatty acid omega-hydroxylases and P450BM3 from em Bacillus megatarium /em , another fatty acid omega hydroxylase. However, the metabolic significance of fatty acid omega hydroxylation in bacteria remains obscure. Even for the well-characterized P450BM3 enzyme, the identity of the em in vivo /em substrate(s) of this protein and the pathway involved are not known [11]. Studies suggest n-alkanes act as multi-purpose microbial P450 inducers and our preliminary data suggested this was also true for em cyp110 /em [12,13]. Moreover, a biosensor for hexadecane detection based on changes in em cyp110 /em mRNA from em Anabeana variabilias /em has been described [14]. Here we report on the pattern of expression of the em cyp110 /em mRNA and protein in em Anabaena /em 7120, substrate binding characteristics of CYP110, the effects of alkanes on cellular morphology, and the distribution of CYP110 in vegetative cells versus heterocysts using electron microscopy. Open in a separate window Figure 1 Genetic Map of the em nif D /em Element. The positions of known em nif D /em element genes and other open reading frames are indicated with arrows showing the orientation. LB = left border, RB = right border, H = em Hin /em dIII sites. The numbers inside the double lines designate the em Hin /em dIII fragment subclones of the em nifD /em component produced from a 17 kbp em Eco /em MK-0822 ic50 RI fragment, An 207. The em cyp110 /em probe was created from the An207.4 em Hin /em dIII fragment. The entire sequence from the em nif D /em component can be transferred in Genbank (“type”:”entrez-nucleotide”,”attrs”:”text message”:”U38537″,”term_id”:”3953452″,”term_text message”:”U38537″U38537). Outcomes Hexadecane-dependent BMP8B induction of em cyp110 /em transcripts in nitrogen-fixing and ammonia-supplemented ethnicities We first examined the consequences of hexane, octane, hexadecane and dodecane for the development of em Anabaena /em 7120 ethnicities. All alkane improvements with this research were carried out at 0.2% (v/v), the recommended concentration for microbial P450 induction [12]. In preliminary experiments, we determined that em Anabaena /em 7120 exhibited rapid chlorosis with 0.2% hexane MK-0822 ic50 and octane. These treatments yielded only highly degraded RNA and were not further analyzed (data not shown). RNA samples from control, dodecane and hexadecane treated cultures were size fractionated and hybridized with em cyp110 /em . A em psbA1 /em specific probe was used to assess RNA quality and for normalization. The em cyp110 /em probe identified a range of transcript sizes with a maximum of 9 kb seen in mRNA from both nitrogen-fixing and ammonia supplemented em Anabaena /em 7120 cultures Fig. 2(a) and 2(c). The size of the transcript identified by the em cyp110 /em probe and the genetic organization of the em nifD /em -element suggest that all of the open reading frames to the right of em xisA /em on the em nifD /em element are coordinately transcribed.