Table 1.
Bacterial strains and plasmids.
Figure 1.
Growth of metagenomic clones SMG 1, 6 and 52 compared to EPI300 carrying an empty fosmid vector (pCC1FOS) in (A) LB broth and (B) LB broth supplemented with 7% NaCl.
Table 2.
List of putative proteins encoded on SMG 6 fosmid insert.
Figure 2.
Overview of SMG 6 fosmid insert and features of specific genes.
(A) Gene map of SMG 6 insert, displaying gene orientation and individual %G+C content indicated with a gradient colour bar. Gene numbers correspond to those in Table 2 and are drawn approximately to scale. (B) Focus on genes 25 (atfA) and 26 (brpA), showing the regions cloned for each construct. (C) Detailed view of putative ATG and TTG start codons of brpA, including upstream regions, as well as predicted promoter regions (highlighted in bold) and transcription factor binding site sequences (blue and orange boxes). (D) TMHMM prediction of seven transmembrane regions in BrpA.
Figure 3.
BrpA homologues identified when BLAST searched against Human Microbiome Project (HMP) datasets from 17 body sites at maximum e-value cut-off of (A) 1e−50 and (B) 1e−05.
Figure 4.
Growth of E. coli MKH13::pCI372 and E. coli MKH13 carrying a plasmid encoded copy of either brpAL, brpAS or brpAatfA in (A) LB broth or (B) LB+3% NaCl.
All of the genes confer a significant salt tolerance phenotype to MKH13 relative to cells with an empty plasmid vector. All values are the average of triplicate experiments and error bars are representative of the standard error of the mean (SEM).
Figure 5.
Pigmentation observed in cell pellets.
(A) Appearance of cell pellets grown in LB supplemented with β-carotene. From left to right: E. coli MKH13::pCI372, MKH13::pCI372-brpAL, MKH13::pCI372-brpAS and MKH13::pCI372-brpAatfA. (B) Appearance of cell pellets of clones grown in LB supplemented with β-carotene and Copy Control Induction solution (L-arabinose). From left to right: E. coli EPI300::pCC1FOS, SMG 1, SMG 6 and SMG 52. (C) Appearance of cell pellets grown in LB supplemented with β-carotene and L-arabinose. From left to right: E. coli EPI300::pBAD, EPI300::pBAD-brpAS, EPI300::pBAD-brpAL, and EPI300::pBAD-brpAatfA.
Figure 6.
EZTn5 transposon mutagenesis of SMG 6 was performed to identify mutants lacking pigmentation when grown in the presence of β-carotene.
(A) Clones positive for a transposon in this region of SMG 6 fosmid insert were identified by PCR, with amplicons of ∼3.3 kb indicative of an insertion event. (B) Approximate locations of transposon insertions in relation to brpA and neighbouring genes. (C) Appearance of cell pellets of SMG 6 and transposon insertion mutants (EZTn #24, #26, #34 and #38) following growth in the presence of β-carotene.
Figure 7.
Possible mechanism(s) of action of BrpA (A) Representation of the known reaction for the formation of retinal.
B-carotene is cleaved at its central 15,15′ bond by brp 15,15′- β-carotene monooxygenase to form two molecules of all-trans retinal (Vitamin A aldehyde). We propose that brpA may be regulated from two promoters, with translation being initiated from one of two potential start codons (ATG and TTG), depending on environmental conditions. While speculative, we illustrate some possibilities discussed in the text. (B) Pigmentation phenotype: regulation of brpA from promoter 1 (upstream of ATG start codon) under “normal” cellular conditions, or possibly by β-carotene, could result in (B1) BrpA adding an acyl group to β-carotene, allowing it to interact with phosphate head groups of lipids and anchoring it in the hydrophobic core of the lipid membrane or (B2) BrpA may cleave β-carotene to retinal and subsequently bind the derived retinal anchoring it in the cell membrane. (C) Stress response: regulation of brpA from promoter 2 (upstream of TTG start codon), may be initiated by environmental signals such as changes in external osmolarity, resulting in increased tolerance or resistance to environmental stress, such as increased NaCl concentrations by an as yet unknown mechanism. Alternative start codons, such as TTG, have been found in a number of stress response genes.