Nickel-inducible lysis system in Synechocystis sp. PCC 6803

Abstract

We designed and constructed a controllable inducing lysis system in Synechocystis sp. PCC 6803 to facilitate extracting lipids for biofuel production. Several bacteriophage-derived lysis genes were integrated into the genome and placed downstream of a nickel-inducible signal transduction system. We applied 3 strategies: (i) directly using the phage lysis cassette, (ii) constitutively expressing endolysin genes while restricting holin genes, and (iii) combining lysis genes from different phages. Significant autolysis was induced in the Synechocystis sp. PCC 6803 cells with this system by the addition of NiSO(4). Our inducible cyanobacterial lysing system eliminates the need for mechanical or chemical cell breakage and could facilitate recovery of biofuel from cyanobacteria.

Fig. 1.

The envelope layers of wild-type Synechocystis cell. The S-layer (asterisk), outer membrane (white arrowhead), peptidoglycan layer (arrow), and cytoplasmic membrane (black arrowhead) are indicated. (Scale bar, 50 nm.) (This picture was kindly provided by Robert Roberson, School of Life Sciences, Arizona State University, Tempe, AZ).

Fig. 2.

The functions of holin and endolysins in degrading the cell wall. Holin (H) makes non-specific lesions in the cytoplasmic membrane so that the endolysins (A, amidase; M, muramidase; T, transglycosylase; and E, endopeptidase) can reach the peptidoglycan layer and break the glycosidic bonds between N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) (by M or T), and also break the peptide cross-bridges (P-P) that link the rows of sugars together (by A or E).

Fig. 3.

The strains and strategies used in this study. Km^R, kanamycin-resistance cassette; nrsBACD, nickel-resistance genes; nrsRS, nickel-sensing and -responding genes; P_psbAII, promoter of Synechocystis gene psbAII; P_nrsB, the nickel inducible promoter; S, R, and Rz, coliphage λ genes: holin (S), endolysin (R), and auxiliary lysis enzyme (Rz); sacB, sacB gene, which is lethal for cyanobacteria in the presence of sucrose; TT, transcriptional terminator from cyanophage Pf-WMP4; 13, 19, and 15, Salmonella phage P22 genes: holin (13), endolysin (19), and auxiliary lysis enzyme (15).

Fig. 4.

The lysis response of SD123 after addition of 7 μM Ni²⁺. The value of viable cell density (cfa) and protein, DNA, and phycocyanin concentrations in the supernatant were normalized into percentage by their maximum values (i.e., 2.25 × 10⁷ cells/mL for the cell density, 0.20 mg/mL for proteins, 13.4 mg/mL for DNA, and 22.5 emission intensity at 650 nm for phycocyanin). The percentage of permeable cells of the total cells was not normalized.

Fig. 5.

Transmission electron microscopy images of the SD121 cells before and after the addition of 7 μM Ni²⁺. (A) SD121 cells before Ni²⁺ addition; (B) 6 h after Ni²⁺ addition; (C) 12 h after Ni²⁺ addition; (D) 24 h after Ni²⁺ addition.