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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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Biofilm and planktonically grown cells used for microarray analyses. A. Biofilm cultures of wild-type strain of M. smegmatis mc2155 were grown in a modified M63 medium, either with or without supplemental iron (2 μM FeSO4) as shown, for 3, 4 or 5 days. RNA samples for microarray analysis were harvested from the 3 and 4 day plates grown in iron-supplemented medium. B. Planktonic growth of wild-type M. smegmatis mc2155 in no iron (blue), 2 μM iron (purple) or 50 μM iron (red). C. Growth curves of M. smegmatis mc2155 in liquid medium showing the increase in cell density (OD600; purple) and viable colony counts (cfu ml−1; blue). Arrows indicate the points at which cells were harvested for the exponentially growing planktonic and stationary phase to prepare samples for microarray analysis.
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fig01: Biofilm and planktonically grown cells used for microarray analyses. A. Biofilm cultures of wild-type strain of M. smegmatis mc2155 were grown in a modified M63 medium, either with or without supplemental iron (2 μM FeSO4) as shown, for 3, 4 or 5 days. RNA samples for microarray analysis were harvested from the 3 and 4 day plates grown in iron-supplemented medium. B. Planktonic growth of wild-type M. smegmatis mc2155 in no iron (blue), 2 μM iron (purple) or 50 μM iron (red). C. Growth curves of M. smegmatis mc2155 in liquid medium showing the increase in cell density (OD600; purple) and viable colony counts (cfu ml−1; blue). Arrows indicate the points at which cells were harvested for the exponentially growing planktonic and stationary phase to prepare samples for microarray analysis.

Mentions: The modified M63 medium that supports robust growth of pellicle-like M. smegmatis biofilms contains a relatively low supplemental iron concentration of 2 μM ferrous sulphate (Ojha et al., 2005). Inclusion of iron at this concentration is important for normal biofilm maturation, and omission leads to the inability to form mature biofilms, even though planktonic growth is unaffected (Ojha et al., 2005). In the absence of the iron supplement, surface growth is observed but without the reticulated appearance associated with formation of the extracellular matrix (Fig. 1A). Planktonic growth, however, is normal over a wide range of iron concentrations (0–50 μM), and because no specific addition of iron is required for planktonic growth, we assume that residual iron contaminants in other media components can satisfy any iron demands (Fig. 1B). Because these observations show that there is apparently a specific requirement for iron to support biofilm formation, we performed microarray analysis to determine how the iron regulons respond during biofilm development.


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)

Biofilm and planktonically grown cells used for microarray analyses. A. Biofilm cultures of wild-type strain of M. smegmatis mc2155 were grown in a modified M63 medium, either with or without supplemental iron (2 μM FeSO4) as shown, for 3, 4 or 5 days. RNA samples for microarray analysis were harvested from the 3 and 4 day plates grown in iron-supplemented medium. B. Planktonic growth of wild-type M. smegmatis mc2155 in no iron (blue), 2 μM iron (purple) or 50 μM iron (red). C. Growth curves of M. smegmatis mc2155 in liquid medium showing the increase in cell density (OD600; purple) and viable colony counts (cfu ml−1; blue). Arrows indicate the points at which cells were harvested for the exponentially growing planktonic and stationary phase to prepare samples for microarray analysis.
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Related In: Results  -  Collection

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fig01: Biofilm and planktonically grown cells used for microarray analyses. A. Biofilm cultures of wild-type strain of M. smegmatis mc2155 were grown in a modified M63 medium, either with or without supplemental iron (2 μM FeSO4) as shown, for 3, 4 or 5 days. RNA samples for microarray analysis were harvested from the 3 and 4 day plates grown in iron-supplemented medium. B. Planktonic growth of wild-type M. smegmatis mc2155 in no iron (blue), 2 μM iron (purple) or 50 μM iron (red). C. Growth curves of M. smegmatis mc2155 in liquid medium showing the increase in cell density (OD600; purple) and viable colony counts (cfu ml−1; blue). Arrows indicate the points at which cells were harvested for the exponentially growing planktonic and stationary phase to prepare samples for microarray analysis.
Mentions: The modified M63 medium that supports robust growth of pellicle-like M. smegmatis biofilms contains a relatively low supplemental iron concentration of 2 μM ferrous sulphate (Ojha et al., 2005). Inclusion of iron at this concentration is important for normal biofilm maturation, and omission leads to the inability to form mature biofilms, even though planktonic growth is unaffected (Ojha et al., 2005). In the absence of the iron supplement, surface growth is observed but without the reticulated appearance associated with formation of the extracellular matrix (Fig. 1A). Planktonic growth, however, is normal over a wide range of iron concentrations (0–50 μM), and because no specific addition of iron is required for planktonic growth, we assume that residual iron contaminants in other media components can satisfy any iron demands (Fig. 1B). Because these observations show that there is apparently a specific requirement for iron to support biofilm formation, we performed microarray analysis to determine how the iron regulons respond during biofilm development.

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