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. 2020 Oct 16;45(51):26840-26854.
doi: 10.1016/j.ijhydene.2020.07.108.

The adherence-associated Fdp fasciclin I domain protein of the biohydrogen producer Rhodobacter sphaeroides is regulated by the global Prr pathway

E-L Jeong  1 , S J Broad  1 , R G Moody  1   2 , M K Phillips-Jones  3
Affiliations

The adherence-associated Fdp fasciclin I domain protein of the biohydrogen producer Rhodobacter sphaeroides is regulated by the global Prr pathway

E-L Jeong et al. Int J Hydrogen Energy. .

Abstract

Expression of fdp, encoding a fasciclin I domain protein important for adherence in the hydrogen-producing bacterium Rhodobacter sphaeroides, was investigated under a range of conditions to gain insights into optimization of adherence for immobilization strategies suitable for H2 production. The fdp promoter was linked to a lacZ reporter and expressed in wild type and in PRRB and PRRA mutant strains of the Prr regulatory pathway. Expression was significantly negatively regulated by Prr under all conditions of aerobiosis tested including anaerobic conditions (required for H2 production), and aerobically regardless of growth phase, growth medium complexity or composition, carbon source, heat and cold shock and dark/light conditions. Negative fdp regulation by Prr was reflected in cellular levels of translated Fdp protein. Since Prr is required directly for nitrogenase expression, we propose optimization of Fdp-based adherence in R. sphaeroides for immobilized biohydrogen production by inactivation of the PrrA binding site(s) upstream of fdp.

Keywords: Biohydrogen; Fasciclin domain; Gene regulation; Green energy; Prr two-component system; Rhodobacter sphaeroides.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Alignment of R. sphaeroides Fdp with fasciclin-1 proteins. Amino acid residues that are identical to Fdp are in bold and grey shading; similar amino acid residues are in bold. Sinorhizobium meliloti Nex18, symbiotically-induced conserved protein Nex18 of Sinorhizobium meliloti (pir|F95334) 60/74% identical/similar; Trichodesmium erythraeum hypothetical protein (gb|ZP00071101.1) 60/72%; Synech TGF-ip, Synechocystis transforming growth factor-induced protein (pir|S76811) 56/73%; Nostoc, hypothetical protein all 4894 of Nostoc sp. PCC7120 (pir|AF2417) 57/74%; Synech MPB70-like, Synechocystis secreted protein MPB70-like slll735 (pir|S77329) 47/63%; M. tuberculosis MPT70, Mycobacterium tuberculosis major secreted MPT70 protein (gb|AAF13402.1/AF189006) 39/55%; M. bovis MPB70, Mycobacterium bovis major secreted protein MPB70 precursor (pir|A37195) 39/55%; Human βIG-H3, human transforming growth factor β-induced protein BIG-H3 (pir|I52996) 36/58%; Pig βIG-H3, pig transforming growth factor β-induced protein (kerato epithelin)(RGD-CAP)(sp|O11780|BGH3 PIG) 36/58%; Human FEEL-1, human FEEL-1 protein (dbj|BAC15606.1) 35/52%; Human stabilin, human stabilin 1 protein (emb|CAB61827.1) 35/52%; Mouse stabilin, mouse stabilin 1 protein (gb|AAL91671.2/AF2909141) 34/53%; Chicken RGD-CAP, RGD-containing collagen-associated protein (βIG-H3)(kerato epithelin) (dbj|BAA21479.1) 33/59%; Mouse OSF-2, mouse osteoblast-specific factor 2 (pir|S36109) 34/52%; Human OSF-2, human osteoblast-specific factor 2 (pir|S36110) 32/52%; Anthocidaris EBP-α, Anthocidaris crassispina EBP-α protein (dbj|BAA82956.1) 31/53%; Echinoid HLC-32, Echinoidea HLC-32 protein (gb|AAB32327.1) 30/50%; Human FEEL-2, human FEEL-2 protein (dbj|BAC15608.1) 29/49%; Drosophila FAS1-4, Drosophila melanogaster FAS1 4th fasciclin domain (pir|B29900) 29/52%; Grasshopper FAS1, grasshopper FAS1 (pir|A29900) 24/44%. Secondary structural data is derived from the structure of domain pair 3 and 4 of Drosophila FAS1 and is shown below the alignment (α-helix: ∗∗∗∗∗; β-strand: ≡≡≡≡). The conserved HI and H2 regions identified as protein interaction sites in several fasciclin I proteins are shown (residues 37-46 and 124-133 in Fdp, respectively).
Fig. 2
Fig. 2
Activity of the fdp promoter in R. sphaeroides wild type and PRRA strains during aerobic, semi-aerobic and anaerobic batch growth. The fdp promoter region was inserted upstream of lacZ in reporter plasmid pSDP1 as described in Methods, resulting in pSDP-FDPP. A promoter-less control was also included throughout all growth experiments; reporter levels remained at the expected very low levels throughout these experiments. Plasmids were introduced into R. sphaeroides wild type and PRRA strains and maintained as described in Methods. Growth experiments were performed at 34 °C in M22 media under (a) aerobic/dark, (b) semi-aerobic/dark and (c) anaerobic/dark (in the presence of 60 mM DMSO) conditions for both wild type- and PRRA-transformed strains. Samples (1–50 ml) were taken for measurements of growth (absorbance at 680 nm, A680) (−■−) and duplicate β-galactosidase measurements (shown by the blue bars), as described in Methods. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3
Fig. 3
Separation of soluble proteins of wild type, PRRA and PRRB strains of R. sphaeroides by two-dimensional SDS-polyacrylamide gel electrophoresis. Extracts (40 μg protein) from mid-exponential phase cells cultured under semi-aerobic/dark conditions at 34 °C were separated as described in Methods. Proteins were extracted from: (A) wild type; (B) PRRA (an insertionally inactivated prrA mutant). The boxed area is the region showing the Fdp protein (indicated by an arrow). This boxed area is expanded for comparisons of: (C) wild type and (D) PRRB (an insertionally inactivated prrB mutant) extracts. Typical results from five comparisons of wild type versus PRRA, and one comparison of wild type versus PRRB.
Fig. 4
Fig. 4
Production and verification of purified His-tagged Fdp. (A) SDS-polyacrylamide gel (6% stacking and 15% resolving gel) showing 4 μg purified recombinant Fdp (predicted molecular mass 16004.8 Da) with apparent molecular mass of 20,100 Da, visualised using Coomassie blue staining. (B) Western blot using 4 μg of purified Fdp with an INDIA His probe to detect the presence of the N-terminal hexahistidine tag. Arrow denotes the position of the Fdp protein. The positions of molecular mass markers are indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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