ClpB1 overproduction in Synechocystis sp. strain PCC 6803 increases tolerance to rapid heat shock

Abstract

ClpB1 is a heat shock protein known to disaggregate large protein complexes. Constitutive, 16-fold ClpB1 overproduction in the cyanobacterium Synechocystis sp. strain PCC 6803 increased cell survival by 20-fold when cultures were heated quickly (1°C/s) to 50°C and delayed cell death by an average of 3 min during incubation at high temperatures (>46°C). Cooverexpression of ClpB1 and another heat shock protein, DnaK2, further increased cell survival. According to immunocytochemistry results, ClpB1 is dispersed throughout the cytoplasm but is concentrated in specific areas and is more prevalent near thylakoid membranes. However, ClpB1 overproduction does not lead to a change in the morphology, chlorophyll content, or photosystem ratio. Whereas electron microscopy demonstrated that apparent protein aggregation occurred after heat treatment in the control strain, protein aggregate size was maintained in the ClpB1 overexpresser. Constitutive ClpB1 overproduction allows an earlier response to heat shock and protects from rapid heating of cultures.

Fig 1

Features of the ClpB1-overproducing Slr1641+ strain relative to wild type. (A) Immunoblot of protein extracts from wild type and the Slr1641+ strain, both grown under normal conditions, probed with ClpB1 antibodies. Maximal loading (1) corresponds to 10 μg chlorophyll a per well. Loadings in other lanes have been reduced as indicated in order to allow a densitometric estimation of the level of ClpB1 overexpression in the Slr1641+ strain. A marker lane separates the wild-type and Slr1641+ lanes, and another marker lane is to the right. (B, C) Absorbance spectra (B) and fluorescence emission spectra at 77 K (C) of the wild-type and Slr1641+ strains (solid line, wild type; dashed line, Slr1641+; the lines are largely overlapping). AU, arbitrary units. (D) Doubling time and chlorophyll a (chl a) concentration in the wild-type and Slr1641+ strains. For all measurements, the OD₇₃₀ was 0.5 and the results are the averages of 3 experiments.

Fig 2

Survival of cells upon heat shock. (A) Comparison of cell survival upon linear heating from 30 to 50°C at the indicated ramp rates. White bars, wild type; gray bars, strain Slr1641+. (B) Survival of wild type (diamonds, solid line) and Slr1641+ (squares, dashed line) upon exposure to 50°C for the times indicated after having been heated linearly from 30 to 50°C (heating rate, 1.3°C/min). (C) Time of exposure at the temperatures indicated resulting in 50% survival in wild type (diamonds, solid line) and the Slr1641+ strain (squares, dashed line); an incubation of over 30 min was required for both strains at 46°C; points are calculated on the basis of survival (percentage of the numbers of CFU) versus time at different temperatures for wild type and the Slr1641+ strain and had an R² value of >90%. (D) Survival of wild type (diamonds, solid line), Slr1641+ (squares, dashed line), and Slr1641+/Sll0170+ (triangles, dotted line) upon exposure to 48°C for the times indicated after having been heated linearly from 30 to 48°C in 15 min; for all experiments, the results are averages of 3 experiments.

Fig 3

Transmission electron micrographs of Synechocystis. Wild-type (A, C) and Slr1641+ (B, D) cells from an exponential-phase culture that had not (A, B) or had (C, D) been exposed to heat stress (1 h at 46°C) are shown. White arrowheads, carboxysomes; black asterisks, electron-dense areas formed after heat shock, primarily in the wild type. Bars = 1 μm.

Fig 4

Size profile of total protein extracts from Synechocystis cells. Total proteins were separated by size exclusion chromatography, and eluted proteins were monitored by detection of the absorbance at 280 nm. Protein extracts were from the wild type (Wt) (A) and the Slr1641+ strain (B) from cultures with and without heat shock (HS); heat shock consisted of a 10-min ramp-up from 30 to 48°C and a 10-min incubation at this temperature. The average of three measurements is shown.

Fig 5

Localization of ClpB1. Anti-ClpB1 antibodies detected with secondary antibodies conjugated with 15-nm gold particles were monitored in 70-nm sections of wild-type (A, B) and Slr1641+ (C, D) cells. Cells had been grown under standard conditions before fixation and labeling (A, C) or had been exposed to 46°C for 1 h immediately before harvesting and fixation (B, D). Asterisk, example of a ClpB1-rich area. Bars = 0.2 μm.