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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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Ferric exochelin biosynthesis (Msmeg0015) and uptake genes (Msmeg0011–0014) are required for biofilm formation but not for planktonic growth. A. Mutants defective in exochelin biosynthesis (ΔMsmeg0015, fxbA), exochelin uptake (ΔMsmeg0011–0014, fxuABC) and mycobactin biosynthesis (ΔMsmeg4509, mbtB) were generated by recombineering (van Kessel and Hatfull, 2007) and tested for biofilm growth. Growth after 4 days of biofilm development is shown using standard media containing a 2 μM iron supplement. All strains, including the wild-type control (mc2155), carry the pJV53 recombineering plasmid. B. Suppression of the biofilm defect of the mutants ΔMsmeg0011–0014 and ΔMsmeg0015 by addition of 50 μM iron to the growth medium. C. CAS-agar assay to test the synthesis and secretion of siderophore by ΔMsmeg0011–0014, ΔMsmeg0015, ΔMsmeg4509 and mc2155: pJV53 was used as a control. The orange halo around the colony is indicative of siderophore-mediated chelation of iron from CAS–iron complexes. D. Planktonic growth of the mutants described in A, compared with the parental M. smegmatis mc2155 strain. Cultures were grown in biofilm medium containing a 2 μM iron supplement.
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fig03: Ferric exochelin biosynthesis (Msmeg0015) and uptake genes (Msmeg0011–0014) are required for biofilm formation but not for planktonic growth. A. Mutants defective in exochelin biosynthesis (ΔMsmeg0015, fxbA), exochelin uptake (ΔMsmeg0011–0014, fxuABC) and mycobactin biosynthesis (ΔMsmeg4509, mbtB) were generated by recombineering (van Kessel and Hatfull, 2007) and tested for biofilm growth. Growth after 4 days of biofilm development is shown using standard media containing a 2 μM iron supplement. All strains, including the wild-type control (mc2155), carry the pJV53 recombineering plasmid. B. Suppression of the biofilm defect of the mutants ΔMsmeg0011–0014 and ΔMsmeg0015 by addition of 50 μM iron to the growth medium. C. CAS-agar assay to test the synthesis and secretion of siderophore by ΔMsmeg0011–0014, ΔMsmeg0015, ΔMsmeg4509 and mc2155: pJV53 was used as a control. The orange halo around the colony is indicative of siderophore-mediated chelation of iron from CAS–iron complexes. D. Planktonic growth of the mutants described in A, compared with the parental M. smegmatis mc2155 strain. Cultures were grown in biofilm medium containing a 2 μM iron supplement.

Mentions: The requirement for the addition of 2 μM iron along with the induction of iron-acquisition genes suggests that iron plays a role in biofilm formation that is distinct from that in planktonic growth. It thus seems likely that one or more of the iron-acquisition systems play a critical role in biofilm formation. To test this, we constructed a series of M. smegmatis mutants in which we deleted those genes involved in siderophore synthesis or other putative iron uptake systems, and examined their ability to form mature biofilms. Interestingly, mutants defective in mycobactin biosynthesis (ΔMsmeg4509; mbtB), iron ABC transporters (ΔMsmeg6006–6007; ΔMsmeg6024–6025) or the iron-utilization gene ΔMsmeg5028 have similar biofilm growth as the parent M. smegmatis strain (Table 1; Fig. 3). Thus, even though these genes are all strongly upregulated in the 4 day biofilm, none are required for biofilm maturation under these conditions. In contrast, mutants defective in either ferric exochelin biosynthesis, ΔfxbA (ΔMsmeg0015), or uptake ΔfxuABC (ΔMsmeg0011–0014) are strongly defective in biofilm formation; loss of the exochelin uptake system has the strongest biofilm defect among all the mutants we have analysed (Table 1) – including the previously described ΔgroEL1 mutant (Ojha et al., 2005) – whereas the mutant defective in exochelin biosynthesis has a relatively milder defect (and behaves similarly to the ΔgroEL1 mutant) (Table 1; Fig. 3A). However, both the exochelin synthesis and uptake mutants grow normally in planktonic growth in the presence of 2 μM iron (see Fig. 3D), as well as without addition of any supplemental iron (data not shown). We have constructed and tested seven additional mutants (ΔMsmeg0396, ΔMsmeg0923, ΔMsmeg1739, ΔMsmeg3411, ΔMsmeg5088, ΔMsmeg6030 and ΔMsmeg6214) that are defective in biofilm-regulated genes, most of which form normal biofilms; only the ΔMsmeg6030 mutant is mildly defective (Table 1). These observations are consistent with the interpretation that much of the global change in gene expression patterns is in response to the changing environment cells experience within a biofilm, and that few of these are involved in the biofilm-development pathway directly. The exochelin biosynthesis and uptake genes are clearly thus distinct in being not only induced but also required for biofilm growth.


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)

Ferric exochelin biosynthesis (Msmeg0015) and uptake genes (Msmeg0011–0014) are required for biofilm formation but not for planktonic growth. A. Mutants defective in exochelin biosynthesis (ΔMsmeg0015, fxbA), exochelin uptake (ΔMsmeg0011–0014, fxuABC) and mycobactin biosynthesis (ΔMsmeg4509, mbtB) were generated by recombineering (van Kessel and Hatfull, 2007) and tested for biofilm growth. Growth after 4 days of biofilm development is shown using standard media containing a 2 μM iron supplement. All strains, including the wild-type control (mc2155), carry the pJV53 recombineering plasmid. B. Suppression of the biofilm defect of the mutants ΔMsmeg0011–0014 and ΔMsmeg0015 by addition of 50 μM iron to the growth medium. C. CAS-agar assay to test the synthesis and secretion of siderophore by ΔMsmeg0011–0014, ΔMsmeg0015, ΔMsmeg4509 and mc2155: pJV53 was used as a control. The orange halo around the colony is indicative of siderophore-mediated chelation of iron from CAS–iron complexes. D. Planktonic growth of the mutants described in A, compared with the parental M. smegmatis mc2155 strain. Cultures were grown in biofilm medium containing a 2 μM iron supplement.
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Related In: Results  -  Collection

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

fig03: Ferric exochelin biosynthesis (Msmeg0015) and uptake genes (Msmeg0011–0014) are required for biofilm formation but not for planktonic growth. A. Mutants defective in exochelin biosynthesis (ΔMsmeg0015, fxbA), exochelin uptake (ΔMsmeg0011–0014, fxuABC) and mycobactin biosynthesis (ΔMsmeg4509, mbtB) were generated by recombineering (van Kessel and Hatfull, 2007) and tested for biofilm growth. Growth after 4 days of biofilm development is shown using standard media containing a 2 μM iron supplement. All strains, including the wild-type control (mc2155), carry the pJV53 recombineering plasmid. B. Suppression of the biofilm defect of the mutants ΔMsmeg0011–0014 and ΔMsmeg0015 by addition of 50 μM iron to the growth medium. C. CAS-agar assay to test the synthesis and secretion of siderophore by ΔMsmeg0011–0014, ΔMsmeg0015, ΔMsmeg4509 and mc2155: pJV53 was used as a control. The orange halo around the colony is indicative of siderophore-mediated chelation of iron from CAS–iron complexes. D. Planktonic growth of the mutants described in A, compared with the parental M. smegmatis mc2155 strain. Cultures were grown in biofilm medium containing a 2 μM iron supplement.
Mentions: The requirement for the addition of 2 μM iron along with the induction of iron-acquisition genes suggests that iron plays a role in biofilm formation that is distinct from that in planktonic growth. It thus seems likely that one or more of the iron-acquisition systems play a critical role in biofilm formation. To test this, we constructed a series of M. smegmatis mutants in which we deleted those genes involved in siderophore synthesis or other putative iron uptake systems, and examined their ability to form mature biofilms. Interestingly, mutants defective in mycobactin biosynthesis (ΔMsmeg4509; mbtB), iron ABC transporters (ΔMsmeg6006–6007; ΔMsmeg6024–6025) or the iron-utilization gene ΔMsmeg5028 have similar biofilm growth as the parent M. smegmatis strain (Table 1; Fig. 3). Thus, even though these genes are all strongly upregulated in the 4 day biofilm, none are required for biofilm maturation under these conditions. In contrast, mutants defective in either ferric exochelin biosynthesis, ΔfxbA (ΔMsmeg0015), or uptake ΔfxuABC (ΔMsmeg0011–0014) are strongly defective in biofilm formation; loss of the exochelin uptake system has the strongest biofilm defect among all the mutants we have analysed (Table 1) – including the previously described ΔgroEL1 mutant (Ojha et al., 2005) – whereas the mutant defective in exochelin biosynthesis has a relatively milder defect (and behaves similarly to the ΔgroEL1 mutant) (Table 1; Fig. 3A). However, both the exochelin synthesis and uptake mutants grow normally in planktonic growth in the presence of 2 μM iron (see Fig. 3D), as well as without addition of any supplemental iron (data not shown). We have constructed and tested seven additional mutants (ΔMsmeg0396, ΔMsmeg0923, ΔMsmeg1739, ΔMsmeg3411, ΔMsmeg5088, ΔMsmeg6030 and ΔMsmeg6214) that are defective in biofilm-regulated genes, most of which form normal biofilms; only the ΔMsmeg6030 mutant is mildly defective (Table 1). These observations are consistent with the interpretation that much of the global change in gene expression patterns is in response to the changing environment cells experience within a biofilm, and that few of these are involved in the biofilm-development pathway directly. The exochelin biosynthesis and uptake genes are clearly thus distinct in being not only induced but also required for biofilm growth.

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