Manganese acquisition is facilitated by PilA in the cyanobacterium Synechocystis sp. PCC 6803

Abstract

Manganese is an essential element required by cyanobacteria, as it is an essential part of the oxygen-evolving center of photosystem II. In the presence of atmospheric oxygen, manganese is present as manganese oxides, which have low solubility and consequently provide low bioavailability. It is unknown if cyanobacteria are able to utilize these manganese sources, and what mechanisms may be employed to do so. Recent evidence suggests that type IV pili in non-photosynthetic bacteria facilitate electron donation to extracellular electron acceptors, thereby enabling metal acquisition. Our present study investigates whether PilA1 (major pilin protein of type IV pili) enables the cyanobacterium Synechocystis PCC 6808 to access to Mn from manganese oxides. We present physiological and spectroscopic data, which indicate that the presence of PilA1 enhances the ability of cyanobacteria to grow on manganese oxides. These observations suggest a role of PilA1-containing pili in cyanobacterial manganese acquisition.

Fig 1. Synechocystis sp. PCC 6803 operon containing pilA1 deletion schematic.

The genomic organization of the pilA1-containing operon housed between sll1693 and slr1816 in the native wild type. The relative positions of primers used for strain construction are shown, with the number corresponding to the specific primer in Table 1.

Fig 2. Growth of various BG11 media.

Photoautotrophic growth characteristics of wild type and the Δsll1694 strain grown on agar plates containing BG11 with Mn(II) chloride (A), Mn(III) (B), pyrolusite (C), and Mn(II, III) (D) as the exclusive manganese sources (Table 2). The exponential doubling time of these growth conditions for both wild type and the Δsll1694 strain are shown in Table 3. Trend shown is indicative of nine separate measurements (three strains with three replicates each). Error bars showing the standard error of the biological replicates.

Fig 3. Synechocystis sp. PCC 6803 growth phenotype observed on agar plate.

Images of wild type and Δsll1694 strains on petri dishes containing agar-solidified BG11 medium with Mn(II) chloride (A), Mn(III) (B), pyrolusite (C), and Mn(II, III) (D) as the exclusive manganese sources. The extracellular protein harvested from the strains and normalized to chlorophyll content before analyzed by gel electrophoresis. The PilA protein (encoded by sll1694) is present in wild type strain where sll1694 is intact, but not in the Δsll1694 strain where sll1694 has been interrupted.

Fig 4. Oxygen evolution of Synechocystis sp. PCC 6803 grown on agar plates.

The wild type and Δsll1694 strain were grown using BG11 agar media with Mn(II) chloride (A & C) or pyrolusite (B &D) as the exclusive Mn source. The oxygen evolution was measured in μmol O₂・mg Chl^-1・h^-1 (A & B), and as a percentage of the wild type oxygen evolution (C & D). Trend shown is indicative of nine separate measurements (three strains with three replicates each). Error bars showing the standard error of the biological replicates.

Fig 5. Absorption spectra of a photoautotrophically grown Synechocystis. sp. PCC 6803 agar plate cultures.

Wild type and Δsll1694 strains were grown on agar plates containing BG11 with Mn(II) chloride (A), Mn(III) (B), pyrolusite (C), and Mn(II, III) (D) as the exclusive manganese sources (Table 2). Samples were standardized to an OD750 of 0.3, then traces normalized to 700 nm. Trend shown is indicative of nine separate measurements (three strains with three replicates each). The standard error of these replicates was calculated. Wild type standard error: ±3.9×10⁻² (A), ±4.1×10⁻² (B), ±2.8×10⁻² (C), ±4.5×10⁻² (D). Δsll1694 standard error: ±3.2×10⁻² (A), ±5.1×10⁻² (B), ±4.2×10⁻² (C), ±5.2×10⁻² (D).

Fig 6. Synechocystis sp. PCC 6803 77 K fluorescence emission as a function of pyrolusite concentration.

77 K fluorescence emission characteristics of the wild type (A) and the Δsll1694 (B) strain were obtained in response to varying pyrolusite concentrations. Images representing the growth characteristics of the wild type (C) and the Δsll1694 (D) strain were also acquired in response to the varying pyrolusite concentrations. Concentration series were 14.4 μM, 144 nM, 14.4 nM and 0 nM of pyrolusite. The samples were excited by a 440 nm LED light source. Traces were normalized to the PS I peak at 725 nm. Trend shown is indicative of three separate biological replicates. The standard error of these replicates was calculated. Wild type standard error: ±2.7×10⁻² (14.4 μM pyrolusite), ±3.1×10⁻² (144 nM pyrolusite), ±3.3×10⁻² (14.4 nM pyrolusite), ±3.8×10⁻² (0 nM pyrolusite). Δsll1694 standard error: ±3.6×10⁻² (14.4 μM pyrolusite), ±5.6×10⁻² (144 nM pyrolusite), ±6.2×10⁻² (14.4 nM pyrolusite), ±6.9×10⁻² (0 nM pyrolusite).