Inspection of the amino acid sequence revealed six trans-membrane spanning regions reminiscent of a membrane solute uptake system (Additional file 1, Figure S1 and discussed below). To extend
these MA4008 gene expression findings, quantitative PCR experiments were performed (Methods, Figure 6A). MA4008 was expressed at a 125-fold higher level during acetate versus methanol cell growth conditions. Interestingly, when methanol was also present in the culture medium in addition to acetate, MA4008 expression was suppressed selleck to a level seen when only methanol was present (ca. by 215 fold). This indicates that the MA4008 gene is expressed only when the energetically superior carbon substrate is absent, consistent with a proposed role in acetate uptake. The M. acetivorans MA4008 orf is designated aceP for its role in an acetate-dependent membrane function. Two other genes required for acetate utilization are ack (MA3606) and pta (MA3607) that encode acetate kinase and phosphoacetyl transferase, 3-MA clinical trial respectively ( Table 1). Quantitative PCR experiments (Figure 6A) established that both genes were highly expressed and at levels similar to aceP when acetate was the sole substrate. The 11-18-fold Lonafarnib ic50 Differential pta and ack gene expression findings are similar to previous reports in M. acetivorans and M. thermophila [6, 16]. Figure 6 Differential
expression of genes induced in presence of acetate. Panel A) The indicated genes include ack (acetate kinase), pta (phosphoacetyl transferase), and a gene designated aceP encoding a putative acetate uptake system. The RT-PCR data were
determined as described in Materials. Panel B) Transcript abundance for aceP from cells grown in the presence or absence of the methanogenic substrate, methanol with the indicated amounts of acetate present. Location of the fpoP, hdrE, hdrA1, mrpA, pta, aceP, and ahaA promoters The mRNA 5′ ends of the fpoPABCDHIJJKLMNO, hdrED1, hdrA1-pfd, mrpABCDEFG, pta ack, aceP and ahaHIKECFABD genes/clusters were determined to locate their corresponding Tyrosine-protein kinase BLK promoter elements. Using primer extension methods (Figure 7A), all but one of the promoter elements were demonstrated to have long un-translated regions (UTR’s) that range from 51 to 137 nucleotides in length. For example, the aceP 5′ mRNA end is located 104 nucleotides upstream of the translational start site. Similar findings were seen for the mrpA, fpoP, ahaH, hdrE, and hdrA genes. Only the pta gene had a relatively short UTR (i.e., 27 nt). We did not detect mRNA 5′ ends for either rnfX or hdrC1. Alignment of all the upstream regions of these promoter elements (Figure 7A) revealed the highly conserved sequence present in other archaeal promoters, the TATA box (Figure 7B) located approximately 20-30 nt upstream of the +1 mRNA start site (discussed below). This site is bound by the TBP protein that aids RNA polymerase binding . In contrast, the BRE box elements were not well conserved.