Computational protocol: Presence of a Haloarchaeal Halorhodopsin-Like Cl− Pump in Marine Bacteria

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Protocol publication

[…] Multiple alignments of amino acid sequences were performed using the CLUSTALW option in MEGA 6.0 software (). Phylogenetic trees were constructed using the maximum-likelihood (ML) method () with 1,000 bootstrap replications (). The nucleotide sequences of 16S rRNA and the amino acid sequences of rhodopsins, 15,15′-β-carotene dioxygenase (blh), and lycopene cyclase (crtY and crtYcd) genes were collected from the NCBI database and RAST annotated sequences for the genera Rubricoccus and Rubrivirga. In order to compare gene synteny around the Cl−-pumping rhodopsin gene and blh gene, we acquired the genomes of H. salinarum R1, S. ruber M31T, Synechocystis sp. PCC 7506, Mastigocladopsis repens PCC 10914, Salisaeta longa DSM 21114T, and Rhodothermus marinus DSM 4252T from the NCBI RefSeq database. The ortholog clustering of the CDSs of each genome was performed using eggNOG-mapper () and the bactNOG dataset in the eggNOG database, version 4.5 (). We visualized the operon structure and colored each gene based on each eggNOG ortholog cluster using R software. In order to evaluate the potential LGT, a BLAST analysis was performed on the genomes of H. salinarum R1, genes with an e-value less than 1e-10 were counted, and a threshold with a sequence identity >50% was established. We used the genome family Rhodothermaceae, Cytophaga hutchinsonii ATCC 33406T, which is a type species of the class Cytophagia, Indibacter alkaliphilus LW1T, Spirosoma linguale DSM 74T, N. marinus S1-08T, and Flavobacterium aquatile LMG 4008T. Since I. alkaliphilus and S. linguale, belonging to the class Cytophagia, have a rhodopsin gene, these genome data were used for this assay. Furthermore, because N. marinus is a marine flavobacterium with the ClR gene and F. aquatile is a type species of the class Flavobacteriia, these genome data were also used. […]

Pipeline specifications

Software tools Clustal W, MEGA, eggNOG-mapper
Applications Genome annotation, Phylogenetics
Organisms Escherichia coli, Bacteria
Chemicals Amino Acids, Retinaldehyde