HatA and HatR, implicated in the uptake of inorganic carbon in Synechocystis PCC6803, contain WD40 domains

In a recent publication, Bédu et al. (1995, Mol Microbiol 18: 559–568) identified two adjacent genes (designated hatA and hatR) believed to function in the uptake of inorganic carbon (CO2 HCO3 ) in the cyanobacterium Synechocystis PCC6803. Inactivation of hatA yielded a clone that was unable to grow with limiting amounts of inorganic carbon, while inactivation of hatR yielded a strain with nearly normal growth properties but lacking the high-affinity inorganiccarbon-uptake system. The HatA protein was reported to lack homology with all proteins in the protein sequence databases but to possess three hydrophobic domains including a putative N-terminal signal peptide. The deduced HatR protein was reported to exhibit the features characteristic of a response regulator of a bacterial two-component regulatory system (Stock et al., 1990, Nature 344: 395–400). It was postulated that HatR might control the activity of an inorganic-carbon-transport system. Various potential roles for HatA were proposed. We have examined the HatA and HatR amino acid sequences and report here that: (i) HatR lacks significant sequence similarity to response regulators; (ii) HatA possesses only one sequence that appears to be sufficiently long and hydrophobic to span the membrane as an a-helix; and (iii) HatA and HatR both possess at least two recognizable WD40 domains (Neer et al., 1994, Nature 371: 297–300). Janda et al. (1996, J Bacteriol 178: 1487–1489) recently reported the presence of WD40 repeats in a chromosomally encoded protein from the actinomycete, Thermomonospora curvata. This protein also possesses a domain that exhibits sequence similarity to eukaryotic serine/threonine protein kinases, and it was therefore designated PkwA. WD40 domains were also noted in a putative Synechocystis protein with GenBank Accession Number D63999. These were the first WD40 domains to be identified in prokaryotic proteins. BLAST searches (Altschul et al., 1990, J Mol Biol 215: 403–410) of both HatA and HatR revealed that these proteins exhibit domains that are similar in sequence to the bacterial WD40 domains noted above, as well as to many eukaryotic WD40 sequences. No response-regulator protein in the database exhibited significant sequence similarity to HatR. Two putative WD40 domains of HatA and those of HatR are shown multiply aligned in Fig. 1 with two WD40 sequences of PkwA, two WD40 domains of the Synechocystis protein noted by Janda et al. (1996, ibid.) and two recognized eukaryotic WD40 sequences, one of bovine origin and the other of yeast origin. All 10 sequences align without the introduction of a single gap. RDF2 comparison scores (500 random shuffles; Pearson and Lipman, 1988, Proc Natl Acad Sci USA 85: 2444–2448) were calculated for the domains shown in Fig. 1. The N-terminal WD40 sequence of HatA exhibits comparison scores of 9.0 and 10.4 SD with the first and second WD40 repeats of PkwA, respectively, thereby establishing homology. The C-terminal WD40 sequence of HatA exhibits a value of 7.3 SD with the PkwA second repeat. The HatR first and second tandem Molecular Microbiology (1997) 24(1), 229–232