A Laterally Acquired Galactose Oxidase-Like Gene Is Required for Aerial Development during Osmotic Stress in <em>Streptomyces coelicolor</em>

<div><p>Phylogenetic reconstruction revealed that most Actinobacterial orthologs of <em>S. coelicolor SCO2837</em>, encoding a metal-dependent galactose oxidase-like protein, are found within <em>Streptomyces</em> and were probably acquired by horizontal gene transfer from fungi. Disruption of <em>SCO2837</em> (<em>glxA)</em> caused a conditional <em>bld</em> phenotype that could not be reversed by extracellular complementation. Studies aimed at characterising the regulation of expression of <em>glxA</em> showed that it is not a target for other <em>bld</em> genes. We provide evidence that <em>glxA</em> is required for osmotic adaptation, although independently from the known osmotic stress response element SigB. <em>glxA</em> has been predicted to be part of an operon with the transcription unit comprising the upstream <em>cslA</em> gene and <em>glxA</em>. However, both phenotypic and expression studies indicate that it is also expressed from an independent promoter region internal to <em>cslA</em>. GlxA displays an <em>in situ</em> localisation pattern similar to that one observed for CslA at hyphal tips, but localisation of the former is independent of the latter. The functional role of GlxA in relation to CslA is discussed.</p> </div

Aerial development of <i>glxA</i>⁻ is impaired by hyper-osmotic stress in a medium dependent manner.

<p>All plates shown were incubated for 5 days. The plates shown on the right under the heading ‘KCl’ contain 250 mM KCl. The strains plated are: <i>S. coelicolor</i> M145 (1), <i>glxA</i>⁻ (2), <i>glxA^−/</i>pSH152 (4) and <i>glxA^−/</i>pREC2 (3, 5; two independent clones).</p

Subcellular localisation of GlxA.

<p>Immunomicroscopy showing preferential association of GlxA to growing hyphal tips (A, B) and sub-apical compartments in sporogenic hyphae (C). Bright light field and corresponding fluorescence field are shown. Insets in panel C highlights sporogenic hypha (brightfield) and the association of GlxA to sporulation septa (fluorescence). Panel D shows polar association of GlxA in detached spores. Panels E and F show <i>cslA</i> mutant displaying GlxA <i>in situ</i> localisation similar to that observed in the parental M145 strain. Panel G shows <i>glxA</i> mutant processed in similar manner (negative control). Bar: 10 µm. GlxA is not covalently associated to the cell surface (H). <i>S. coelicolor</i> M145 cells were grown on R5 for 24–30 h and total protein samples from sub-cellular compartments prepared. 1) Total protein, 2) Cell-membrane fraction, 3) Supernatant obtained from 1% SDS washing treatment of intact cells.</p

GlxA expression is not under the control of known developmental (A) or stress-response elements (B).

<p>Immunoblots showing GlxA protein abundance in various developmental mutants. Strains used (M145, <i>osaB</i>, <i>osaC</i> and <i>sigB</i>) are indicated on each gel lane. Ten micrograms of total protein were loaded in each lane.</p

Oligonucleotides.

<p>Oligonucleotides.</p

Only disruption of <i>glxA</i> abolishes production of GlxA.

<p>A- Parental and mutant strains were grown on R5 plates for the time indicated bellow and total protein samples prepared at the indicated times. Ten micrograms of total protein were loaded in each lane. Strains used were <i>S. coelicolor</i> M145 24 h, <i>glxA</i>⁻ 16 h, <i>glxA</i>⁻ 24 h, <i>cslA</i>::Tn<i>5062</i> 24 h and <i>cslA</i>::Tn<i>5062</i>b 24 h. B- A putative promoter internal to <i>cslA</i> supports expression of <i>glxA</i> and allows aerial development. Strains shown were grown on R5 plates for 4 days.</p

Amino acid alignment of <i>S. coelicolor</i> Dps proteins.

<p>Amino acid alignment of the three S. <i>coelicolor</i> Dps proteins showing the position of the five characteristic Dps helices along with the position of the different length N- and C-terminal tails.</p

<i>dps</i> location maps among 17 Streptomycete chromosomes.

<p>Approximate chromosome location of <i>dps</i> orthologs in 17 Streptomycete chromosomes. Species names are abbreviated to the left of the diagram: S. gha  =  <i>S. ghanaensis</i>; S. griseof  =  <i>S. griseoflavus</i>; S. prist  =  <i>S. pristinaespiralis</i>; S. co.  =  <i>S. coelicolor</i>; S. liv.  =  <i>S. lividans</i>; S. alb  =  <i>S. albus</i>; S. flavo  =  <i>S. flavogriseus</i>; S.gris  =  <i>S. griseus</i>; S. virido  =  <i>S.viridochromogenes</i>; S. scab  =  <i>S. scabies</i>; S. aver  =  <i>S. avermitilis</i>; S. clav  =  <i>S. clavuligerus</i>; S. gris. gris  =  <i>S. griseus</i> subsp. <i>griseus</i>; S. him  =  <i>S. himastatinicus</i>; S. svic  =  <i>S. sviceus</i>; S. griseoaur  =  <i>S. griseoaurantiacus</i>; S. venez  =  <i>S. venezuelae</i>. Chromosomes were orientated based on the centrally located initiator protein (black arrow). Light grey arrows represent orthologs of <i>dpsA_Sc</i>, clear arrows represent orthologs of <i>dpsC_Sc</i>, striped arrows represent orthologs of <i>dpsB_Sc</i>. Figures above arrows represent approximate chromosome location in Mb. Figures in parentheses  =  chromosome size. Asterisks indicate those genes where we identified <i>sigB-like</i> promoter motifs upstream of ORFs. Scale bar  =  1 Mb.</p

Schematic representation of the genomic neighbourhood of <i>dpsB_Sc</i> in <i>Streptomyces.</i>

<p>Consensus genomic neighbourhood around <i>dpsB_Sc</i> orthologs (without a <i>sigB</i> promoter) in completely sequenced and assembled <i>Streptomyces</i> genomes. The nomenclature of genes follows that of <i>S. coelicolor</i> orthologs. Numbers in parenthesis represents (as a percentage) the number of times that gene occurs in that position among all the <i>Streptomyces</i> tested. Genes that do not match 100% are not shown. H/p  =  hypothetical protein.</p

Majority rule consensus phylogenetic tree of Actinobacterial Dps orthologous proteins.

<p>Maximum-likelihood reconstructed phylogenetic tree of Actinobacterial Dps proteins. Indicated are three orthologous protein clusters (shaded boxes). DpsA_Sc, DpsB_Sc and DpsC_Sc indicates the position of <i>S. coelicolor</i> Dps in the tree. Paralogous gene pairs of DpsB in <i>Streptomyces</i> are indicated using matched Roman numerals. σB indicates those proteins where a putative <i>sigB-like</i> promoter was identified. Bootstrap values >60% are indicated next to major nodes.</p