6 research outputs found
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
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
Aerial development of <i>glxA</i><sup>β</sup> 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 βKClβ contain 250 mM KCl. The strains plated are: <i>S. coelicolor</i> M145 (1), <i>glxA</i><sup>β</sup> (2), <i>glxA<sup>β/</sup></i>pSH152 (4) and <i>glxA<sup>β/</sup></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 Β΅m. GlxA is not covalently associated to the cell surface (H). <i>S. coelicolor</i> M145 cells were grown on R5 for 24β30 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
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><sup>β</sup> 16 h, <i>glxA</i><sup>β</sup> 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