Genomic analyses of bacterial porin-cytochrome gene clusters

Abstract

The porin-cytochrome (Pcc) protein complex is responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III) by the dissimilatory metal-reducing bacterium Geobacter sulfurreducens PCA. The identified and characterized Pcc complex of G. sulfurreducens PCA consists of a porin-like outer-membrane protein, a periplasmic 8-heme c-type cytochrome (c-Cyt) and an outer-membrane 12-heme c-Cyt, and the genes encoding the Pcc proteins are clustered in the same regions of genome (i.e., the pcc gene clusters) of G. sulfurreducens PCA. A survey of additionally microbial genomes has identified the pcc gene clusters in all sequenced Geobacter spp. and other bacteria from six different phyla, including Anaeromyxobacter dehalogenans 2CP-1, A. dehalogenans 2CP-C, Anaeromyxobacter sp. K, Candidatus Kuenenia stuttgartiensis, Denitrovibrio acetiphilus DSM 12809, Desulfurispirillum indicum S5, Desulfurivibrio alkaliphilus AHT2, Desulfurobacterium thermolithotrophum DSM 11699, Desulfuromonas acetoxidans DSM 684, Ignavibacterium album JCM 16511, and Thermovibrio ammonificans HB-1. The numbers of genes in the pcc gene clusters vary, ranging from two to nine. Similar to the metal-reducing (Mtr) gene clusters of other Fe(III)-reducing bacteria, such as Shewanella spp., additional genes that encode putative c-Cyts with predicted cellular localizations at the cytoplasmic membrane, periplasm and outer membrane often associate with the pcc gene clusters. This suggests that the Pcc-associated c-Cyts may be part of the pathways for extracellular electron transfer reactions. The presence of pcc gene clusters in the microorganisms that do not reduce solid-phase Fe(III) and Mn(IV) oxides, such as D. alkaliphilus AHT2 and I. album JCM 16511, also suggests that some of the pcc gene clusters may be involved in extracellular electron transfer reactions with the substrates other than Fe(III) and Mn(IV) oxides.

Keywords: c-type cytochromes with multiple hemes; extracellular electron transfer; metal reduction; outer membrane; porin-cytochrome protein complex.

Figure 1

Identified pcc gene cluster in Geobacter spp. (A) and other bacteria (B). The genes encoding different type of proteins are labeled with different colors: green/the Pcc porin-like outer-membrane proteins; red/the Pcc periplasmic c-type cytochromes (c-Cyts); purple/the Pcc outer-membrane c-Cyts; black/the cytoplasmic membrane c-Cyts; pink/the periplasmic c-Cyts; yellow/the porin-like outer-membrane c-Cyt; light blue/transcriptional factors; dark blue/chitinase and orange/hypothetic proteins. The numbers displayed above the gene clusters are part of their locus tags whose letter parts are displayed at left side of the gene clusters with exception that PCA is displayed for the gene clusters identified from G. sulfurreducens PCA. The numbers for the pcc genes are in red, green, blue or purple, while the numbers for the gene associated with pcc genes are in black. Gbem: Geobacter bemidjiensis Bem; Geob: Geobacter sp. FRC-32; Glov: Geobacter lovleyi SZ; Gmet: Geobacter metallireducens GS-15; Gura: Geobacter uraniireducens Rf4; GM18: Geobacter sp. M18; GM21: Geobacter sp. M21; A2cp1: Anaeromyxobacter dehalogenans 2CP-1; Adeh: A. dehalogenans 2CP-C; AnaeK: Anaeromyxobacter sp. K; Kuster: Candidatus Kuenenia stuttgartiensis; Dacet: Denitrovibrio acetiphilus DSM 12809; Selin: Desulfurispirillum indicum S5; DaAHT2: Desulfurivibrio alkaliphilus AHT2; Dester: Desulfurobacterium thermolithotrophum DSM 11699; Dace: Desulfuromonas acetoxidans DSM 684; IALB: Ignavibacterium album JCM 16511; and Theam: Thermovibrio ammonificans HB-1.

Figure 2

Phylogenetic analyses of the Pcc porin-like outer-membrane proteins (A) and periplasmic c-type cytochromes (B). The phylogenetic trees were constructed with MEGA6 and confidence levels are indicated in the major nodes by the bootstrap values (%) in red. The numbers in the parenthesis next to the locus tags are the numbers of their predicted trans-outer membrane motifs of the porin-like outer-membrane proteins (A) and heme-binding motifs of the periplasmic c-type cytochromes (B). The phylogenetic groups of the Pcc porin-like outer-membrane proteins (A) and periplasmic c-type cytochromes (B) are indicated by Roman numerals. The trees are not drawn to scale.

Figure 3

Phylogenetic analyses of the Pcc outer-membrane c-type cytochromes. The phylogenetic trees were constructed with MEGA6 and confidence levels are indicated in the major nodes by the bootstrap values (%) in red. The numbers in the parenthesis next to the locus tags are the numbers of their predicted typical (CX₂CH)/atypical (CX_3-15CH) heme-binding motifs of the c-type cytochromes. The tree is not drawn to scale.

Figure 4

The amino acid sequence of GM18_3465 of Geobacter sp. M18. The N-terminus and predicted short solvent-exposed loops are in green, the predicted trans-outer membrane motifs are in red, and predicted long solvent-exposed loops are in blue. The 21 trans-outer membrane motifs are numbered sequentially and the numbers are displayed in red and Arabic numerals and above the amino acid sequence. The long solvent-exposed loops are numbered sequentially and numbers are displayed in blue and Roman numerals and above the amino acid sequence. The heme-binding motifs are underlined and in italic.