ClpB in a cyanobacterium: predicted structure, phylogenetic relationships, and regulation by light and temperature

Abstract

The sequence of a genomic clone encoding a 100-kDa stress protein of Plectonema boryanum (p-ClpB) was determined. The predicted polypeptide contains two putative ATPase regions located within two highly conserved domains (N1 and N2), a spacer region that likely forms a coiled-coil domain, and a highly conserved consensus CK2 phosphorylation domain. The coiled-coil region and the putative site of phosphorylation are not unique to p-ClpB; they are present in all ClpB sequences examined and are absent from the ClpB paralogs ClpA, ClpC, ClpX, and ClpY. Small quantities of a 4.5-kb p-clpB transcript and 110-kDa cytosolic p-ClpB protein were detected in cells grown under optimal conditions; however, increases in the quantities of the transcript and protein were observed in cells grown under excess light and low temperature conditions. Finally, we analyzed ClpA, ClpB, and ClpC sequences from 27 organisms in order to predict phylogenetic relationships among the homologs. We have used this information, along with an identity alignment, to redefine the Clp subfamilies.

FIG. 1

Cultures of P. boryanum grown under optimal (A) and ELLT (B) conditions.

FIG. 2

Southern blot analysis of the clpB gene in P. boryanum. In all panels, genomic DNA was digested with BglI (B), EcoRI (E), and HindIII (H). The blot was probed with the Sau3A fragment of the PCR product (A), the 8.8-kb HindIII genomic DNA fragment (B), and the 1.7-kb ClaI-AccI and 1.4-kb ClaI-ClaI subfragments (C and D, respectively). The hybridization pattern of clpB is indicated by the size markers 4.9 (BglII), 4.1 (EcoRI), and 8.8 (HindIII) kb. (The stronger bands, i.e., 8.1 [BglII], 5.6 [EcoRI], and 4.5 [HindIII] kb, are the hybridization pattern for p-KLC [9].)

FIG. 3

Amino acid sequence identities of the ClpABC family of proteins. Sequences were aligned to the profile which was generated from all ClpABC sequences used in this study. Species’ amino acid similarities to the profile are indicated by white (gaps), shades of gray (similarity with the profile), and black (complete identity with the profile). Major structural regions including the N1 and N2 regions that contain the ATP-binding sites are identified above the alignment. The accession numbers for the sequences used in the alignment are as follows: Swiss-PROT P03815, P05444, P15716, P31541, P17422, P31539, P31542, P31543, P33416, P35100, P37571, P42730, P44403, P49574, P53532, P53533, P53533, and P77686; and GenBank 511145, 755163, 1001555, 1171612, 1314297, 1563721, 1653543, 1653648, 1705923, 1705925, 1946209, 2058336, 2113980, 2146076, L35272, M67479, and U46549.

FIG. 4

Aligned amino acids proximal to the predicted coiled-coil domain. The conserved serine residue (aa 420), within a consensus CK2 phosphorylation domain, is highlighted.

FIG. 5

Predictions of coiled-coil structures in ClpB from P. boryanum. Probabilities in excess of 90% were obtained with weighted and unweighted scoring matrices, predicting strongly that the region does form a coiled coil in p-ClpB. The greatest percent difference between matrices was observed when comparison was made of unweighted MTIDK and MTK (5.5%), a value that is still far below the critical maximum (20 to 30%) to confidently assign the region as being predicted to form a coiled coil (37).

FIG. 6

Deduced phylogeny of ClpABC amino acid sequences. The numbers at the nodes are percentages of puzzling steps which support the branch in this maximum likelihood analysis.

FIG. 7

(A) Northern blot analysis of clpB from P. boryanum UTEX 485. Total RNA was isolated from P. boryanum cells grown under optimal (lanes 1) and ELLT (lanes 2) conditions. The gels were stained with ethidium bromide, and RNA samples were loaded equally, based on the quantity of rRNA present (a). The p-clpB transcript was detected on Northern blots by using radiolabelled 1.7-kb ClaI-AccI genomic DNA fragment of p-clpB (b). Large arrowhead indicates the 4.5-kb transcript. Smaller arrowheads indicate two additional transcripts of approximately 8 and 10 kb detected in the RNA of cells grown under high light and low temperature. (B) Characterization and immunolocalization of the p-ClpB protein. (I) Analysis of total proteins from P. boryanum by SDS-PAGE and stained with Coomassie G-250. Lanes 1 and 2, cells grown under ELLT and optimal conditions, respectively. ST, protein standards. (II) Western blot of gel (corresponding to gel in panel BI) probed with the polyclonal antibody anti-hsp78 (34).