Growth of <i>E. coli</i> MKH13::pCI372 and <i>E. coli</i> MKH13 carrying a plasmid encoded copy of either <i>brpA_L</i>, <i>brpA_S</i> or <i>brpAatfA</i> in (A) LB broth or (B) LB+3% NaCl.

<p>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).</p

Overview of SMG 6 fosmid insert and features of specific genes.

<p>(<b>A</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103318#pone-0103318-t002" target="_blank">Table 2</a> and are drawn approximately to scale. (<b>B</b>) Focus on genes 25 (<i>atfA</i>) and 26 (<i>brpA</i>), showing the regions cloned for each construct. (<b>C</b>) Detailed view of putative ATG and TTG start codons of <i>brpA</i>, including upstream regions, as well as predicted promoter regions (highlighted in bold) and transcription factor binding site sequences (blue and orange boxes). (<b>D</b>) TMHMM prediction of seven transmembrane regions in BrpA.</p

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.

<p>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.</p

EZTn<i>5</i> transposon mutagenesis of SMG 6 was performed to identify mutants lacking pigmentation when grown in the presence of β-carotene.

<p>(<b>A</b>) 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>B</b>) Approximate locations of transposon insertions in relation to <i>brpA</i> and neighbouring genes. <b>(C)</b> 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.</p

List of putative proteins encoded on SMG 6 fosmid insert.

<p><b>Abbreviations:</b> aa = amino acid; % ID = % identity at amino acid level over the entire length of the protein; % G+C = Percentage guanine and cytosine content;</p><p>DUF = Domain of Unknown Function.</p

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.

<p>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.</p

Possible mechanism(s) of action of BrpA (A) Representation of the known reaction for the formation of retinal.

<p>B-carotene is cleaved at its central 15,15′ bond by <i>brp</i> 15,15′- β-carotene monooxygenase to form two molecules of <i>­all-trans</i> retinal (Vitamin A aldehyde). We propose that <i>brpA</i> 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>B</b>) Pigmentation phenotype: regulation of <i>brpA</i> from promoter 1 (upstream of ATG start codon) under “normal” cellular conditions, or possibly by β-carotene, could result in (<b>B1</b>) 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 (<b>B2</b>) BrpA may cleave β-carotene to retinal and subsequently bind the derived retinal anchoring it in the cell membrane. (<b>C</b>) Stress response: regulation of <i>brpA</i> 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.</p

16S phylogenetic composition of the bacterial component of the kefir grain (A) and kefir fermented milk (B) at genus level.

<p>16S phylogenetic composition of the bacterial component of the kefir grain (A) and kefir fermented milk (B) at genus level.</p

List of fungal species identified in the study, listed in teleomorph form with anamorph or synonym names and previous kefir association.

<p>List of fungal species identified in the study, listed in teleomorph form with anamorph or synonym names and previous kefir association.</p

Procrustes imaging of unweighted UniFrac distance matrices highlight the diversity amongst the 16S bacterial component (A) and fungal component (B) of the different kefir samples.

<p>The two different sample types are linked with a bar (white represents grain flora; red represents milk flora). The direction of each axis is arbitrary.</p