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The role of iron in Mycobacterium smegmatis biofilm formation: the exochelin siderophore is essential in limiting iron conditions for biofilm formation but not for planktonic growth.

Ojha A, Hatfull GF - Mol. Microbiol. (2007)

Bottom Line: In contrast, although the expression of mycobactin and iron ABC transport operons is highly upregulated during biofilm formation, mutants in these systems form normal biofilms in low-iron (2 microM) conditions.A close correlation between iron availability and matrix-associated fatty acids implies a possible metabolic role in the late stages of biofilm maturation, in addition to the early regulatory role.M. smegmatis surface motility is similarly dependent on iron availability, requiring both supplemental iron and the exochelin pathway to acquire it.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.

ABSTRACT
Many species of mycobacteria form structured biofilm communities at liquid-air interfaces and on solid surfaces. Full development of Mycobacterium smegmatis biofilms requires addition of supplemental iron above 1 microM ferrous sulphate, although addition of iron is not needed for planktonic growth. Microarray analysis of the M. smegmatis transcriptome shows that iron-responsive genes - especially those involved in siderophore synthesis and iron uptake - are strongly induced during biofilm formation reflecting a response to iron deprivation, even when 2 microM iron is present. The acquisition of iron under these conditions is specifically dependent on the exochelin synthesis and uptake pathways, and the strong defect of an iron-exochelin uptake mutant suggests a regulatory role of iron in the transition to biofilm growth. In contrast, although the expression of mycobactin and iron ABC transport operons is highly upregulated during biofilm formation, mutants in these systems form normal biofilms in low-iron (2 microM) conditions. A close correlation between iron availability and matrix-associated fatty acids implies a possible metabolic role in the late stages of biofilm maturation, in addition to the early regulatory role. M. smegmatis surface motility is similarly dependent on iron availability, requiring both supplemental iron and the exochelin pathway to acquire it.

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Regulation of genes involved in siderophore synthesis and uptake. A. Analysis of expression of exochelin biosynthesis, fxbA (Msmeg0015), exochelin uptake, fxuC (Msmeg0012) and mycobactin synthesis, mbtB (Msmeg4509) genes by real-time RT-PCR. Expression levels were measured in samples grown for 3 or 4 days under biofilm conditions, and in early stationary phase, and represented as the relative log2 change in expression level compared with early planktonic growth. Similar samples were tested under the same growth conditions but in the presence of either 0, 2 or 50 μM iron as shown. B. Organization of the exochelin synthesis and uptake genes. The positions of gene between Msmeg0007 and Msmeg0019 are shown, along with their gene designations; Msmeg0008 and Msmeg0010 encode tRNAile and tRNAala genes respectively. Note that the organization differs somewhat from that described previously by Yu et al. (1998) in that Msmeg0011 is clearly part of an operon with Msmeg0012 (fxuC), Msmeg0013 (fxuB) and Msmeg0014 (fxuA). Putative IdeR iron boxes are shown as grey boxes. The lower panel shows the intergenic sequence between Msmeg0010 and Msmeg0011 that contains the putative regulatory features. The iron box consensus recognized by the IdeR regulator is shown above the location of a putative IdeR binding site (bold) upstream of Msmeg0011. The correspondence of this putative site to the consensus sequence (13 of the 19 positions are conserved) is similar to that for the known IdeR binding site upstream of fxbA (Msmeg0015) (Dussurget et al., 1996), and the position relative to the gene start site and putative −10 and −35 promoter is very similar. Arrows indicate the location of an imperfect inverted repeat that is a potential binding site for a second regulatory protein.
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fig04: Regulation of genes involved in siderophore synthesis and uptake. A. Analysis of expression of exochelin biosynthesis, fxbA (Msmeg0015), exochelin uptake, fxuC (Msmeg0012) and mycobactin synthesis, mbtB (Msmeg4509) genes by real-time RT-PCR. Expression levels were measured in samples grown for 3 or 4 days under biofilm conditions, and in early stationary phase, and represented as the relative log2 change in expression level compared with early planktonic growth. Similar samples were tested under the same growth conditions but in the presence of either 0, 2 or 50 μM iron as shown. B. Organization of the exochelin synthesis and uptake genes. The positions of gene between Msmeg0007 and Msmeg0019 are shown, along with their gene designations; Msmeg0008 and Msmeg0010 encode tRNAile and tRNAala genes respectively. Note that the organization differs somewhat from that described previously by Yu et al. (1998) in that Msmeg0011 is clearly part of an operon with Msmeg0012 (fxuC), Msmeg0013 (fxuB) and Msmeg0014 (fxuA). Putative IdeR iron boxes are shown as grey boxes. The lower panel shows the intergenic sequence between Msmeg0010 and Msmeg0011 that contains the putative regulatory features. The iron box consensus recognized by the IdeR regulator is shown above the location of a putative IdeR binding site (bold) upstream of Msmeg0011. The correspondence of this putative site to the consensus sequence (13 of the 19 positions are conserved) is similar to that for the known IdeR binding site upstream of fxbA (Msmeg0015) (Dussurget et al., 1996), and the position relative to the gene start site and putative −10 and −35 promoter is very similar. Arrows indicate the location of an imperfect inverted repeat that is a potential binding site for a second regulatory protein.

Mentions: All three operons are iron-responsive, and in planktonic growth, are further repressed (2- to 3-fold) in high-iron conditions (50 μM) relative to either of the low-iron (0 or 2 μM) conditions (Fig. 4A). The overall patterns of response of the three operons during biofilm growth are similar, although the levels of induction are different. Maximal induction is seen at the 4 day point, and when grown in 2 μM iron, Msmeg0012 is induced about 4-fold, Msmeg0015 about 6-fold, and Msmeg4509 about 14-fold. These induction levels are similar in the absence of supplemental iron, although we note that in each case, the induction is somewhat higher in the 3 day samples in the absence of iron than with 2 μM iron (Fig. 4A). Perhaps the most striking observation is that high-iron conditions (50 μM) leads to strong repression of all three operons during biofilm growth (Fig. 4A), consistent with the suppression of the ΔMsmeg0011–0014 and ΔMsmeg0015 mutant defects in high iron (Fig. 3B).


The role of iron in Mycobacterium smegmatis biofilm formation: the exochelin siderophore is essential in limiting iron conditions for biofilm formation but not for planktonic growth.

Ojha A, Hatfull GF - Mol. Microbiol. (2007)

Regulation of genes involved in siderophore synthesis and uptake. A. Analysis of expression of exochelin biosynthesis, fxbA (Msmeg0015), exochelin uptake, fxuC (Msmeg0012) and mycobactin synthesis, mbtB (Msmeg4509) genes by real-time RT-PCR. Expression levels were measured in samples grown for 3 or 4 days under biofilm conditions, and in early stationary phase, and represented as the relative log2 change in expression level compared with early planktonic growth. Similar samples were tested under the same growth conditions but in the presence of either 0, 2 or 50 μM iron as shown. B. Organization of the exochelin synthesis and uptake genes. The positions of gene between Msmeg0007 and Msmeg0019 are shown, along with their gene designations; Msmeg0008 and Msmeg0010 encode tRNAile and tRNAala genes respectively. Note that the organization differs somewhat from that described previously by Yu et al. (1998) in that Msmeg0011 is clearly part of an operon with Msmeg0012 (fxuC), Msmeg0013 (fxuB) and Msmeg0014 (fxuA). Putative IdeR iron boxes are shown as grey boxes. The lower panel shows the intergenic sequence between Msmeg0010 and Msmeg0011 that contains the putative regulatory features. The iron box consensus recognized by the IdeR regulator is shown above the location of a putative IdeR binding site (bold) upstream of Msmeg0011. The correspondence of this putative site to the consensus sequence (13 of the 19 positions are conserved) is similar to that for the known IdeR binding site upstream of fxbA (Msmeg0015) (Dussurget et al., 1996), and the position relative to the gene start site and putative −10 and −35 promoter is very similar. Arrows indicate the location of an imperfect inverted repeat that is a potential binding site for a second regulatory protein.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2170428&req=5

fig04: Regulation of genes involved in siderophore synthesis and uptake. A. Analysis of expression of exochelin biosynthesis, fxbA (Msmeg0015), exochelin uptake, fxuC (Msmeg0012) and mycobactin synthesis, mbtB (Msmeg4509) genes by real-time RT-PCR. Expression levels were measured in samples grown for 3 or 4 days under biofilm conditions, and in early stationary phase, and represented as the relative log2 change in expression level compared with early planktonic growth. Similar samples were tested under the same growth conditions but in the presence of either 0, 2 or 50 μM iron as shown. B. Organization of the exochelin synthesis and uptake genes. The positions of gene between Msmeg0007 and Msmeg0019 are shown, along with their gene designations; Msmeg0008 and Msmeg0010 encode tRNAile and tRNAala genes respectively. Note that the organization differs somewhat from that described previously by Yu et al. (1998) in that Msmeg0011 is clearly part of an operon with Msmeg0012 (fxuC), Msmeg0013 (fxuB) and Msmeg0014 (fxuA). Putative IdeR iron boxes are shown as grey boxes. The lower panel shows the intergenic sequence between Msmeg0010 and Msmeg0011 that contains the putative regulatory features. The iron box consensus recognized by the IdeR regulator is shown above the location of a putative IdeR binding site (bold) upstream of Msmeg0011. The correspondence of this putative site to the consensus sequence (13 of the 19 positions are conserved) is similar to that for the known IdeR binding site upstream of fxbA (Msmeg0015) (Dussurget et al., 1996), and the position relative to the gene start site and putative −10 and −35 promoter is very similar. Arrows indicate the location of an imperfect inverted repeat that is a potential binding site for a second regulatory protein.
Mentions: All three operons are iron-responsive, and in planktonic growth, are further repressed (2- to 3-fold) in high-iron conditions (50 μM) relative to either of the low-iron (0 or 2 μM) conditions (Fig. 4A). The overall patterns of response of the three operons during biofilm growth are similar, although the levels of induction are different. Maximal induction is seen at the 4 day point, and when grown in 2 μM iron, Msmeg0012 is induced about 4-fold, Msmeg0015 about 6-fold, and Msmeg4509 about 14-fold. These induction levels are similar in the absence of supplemental iron, although we note that in each case, the induction is somewhat higher in the 3 day samples in the absence of iron than with 2 μM iron (Fig. 4A). Perhaps the most striking observation is that high-iron conditions (50 μM) leads to strong repression of all three operons during biofilm growth (Fig. 4A), consistent with the suppression of the ΔMsmeg0011–0014 and ΔMsmeg0015 mutant defects in high iron (Fig. 3B).

Bottom Line: In contrast, although the expression of mycobactin and iron ABC transport operons is highly upregulated during biofilm formation, mutants in these systems form normal biofilms in low-iron (2 microM) conditions.A close correlation between iron availability and matrix-associated fatty acids implies a possible metabolic role in the late stages of biofilm maturation, in addition to the early regulatory role.M. smegmatis surface motility is similarly dependent on iron availability, requiring both supplemental iron and the exochelin pathway to acquire it.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.

ABSTRACT
Many species of mycobacteria form structured biofilm communities at liquid-air interfaces and on solid surfaces. Full development of Mycobacterium smegmatis biofilms requires addition of supplemental iron above 1 microM ferrous sulphate, although addition of iron is not needed for planktonic growth. Microarray analysis of the M. smegmatis transcriptome shows that iron-responsive genes - especially those involved in siderophore synthesis and iron uptake - are strongly induced during biofilm formation reflecting a response to iron deprivation, even when 2 microM iron is present. The acquisition of iron under these conditions is specifically dependent on the exochelin synthesis and uptake pathways, and the strong defect of an iron-exochelin uptake mutant suggests a regulatory role of iron in the transition to biofilm growth. In contrast, although the expression of mycobactin and iron ABC transport operons is highly upregulated during biofilm formation, mutants in these systems form normal biofilms in low-iron (2 microM) conditions. A close correlation between iron availability and matrix-associated fatty acids implies a possible metabolic role in the late stages of biofilm maturation, in addition to the early regulatory role. M. smegmatis surface motility is similarly dependent on iron availability, requiring both supplemental iron and the exochelin pathway to acquire it.

Show MeSH
Related in: MedlinePlus