Porins Are Required for Uptake of Phosphates by Mycobacterium smegmatis

Abstract

Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membrane (OM) porin MspA also reduced the phosphatase activity in whole cells compared to that in lysed cells. A similar result was obtained for M. smegmatis that overexpressed endogenous alkaline phosphatase, indicating that PhoA is not a surface protein, contrary to a previous report. The uptake of phosphate by a mutant lacking the porins MspA and MspC was twofold lower than that by wild-type M. smegmatis. Strikingly, the loss of these porins resulted in a severe growth defect of M. smegmatis on low-phosphate plates. We concluded that the OM of M. smegmatis represents a permeability barrier for phosphates and that Msp porins are the only OM channels for the diffusion of phosphate in M. smegmatis. However, phosphate diffusion through Msp pores is rather inefficient as shown by the 10-fold lower permeability of M. smegmatis for phosphate compared to that for glucose. This is likely due to the negative charges in the constriction zone of Msp porins. The phosphatase activity in whole cells of Mycobacterium bovis BCG was significantly less than that in lysed cells, indicating a similar uptake pathway for phosphates in slow-growing mycobacteria. However, porins that could mediate the diffusion of phosphates across the OM of M. bovis BCG and Mycobacterium tuberculosis are unknown.Phosphorus is indispensable for the biosynthesis of nucleic acids and phospholipids and for the energy supply of any cell. Bacteria employ sophisticated transport mechanisms to acquire phosphorus-containing nutrients from the environment. In gramnegative bacteria, phosphates first need to cross the outer membrane (OM). To this end, Escherichia coli produces the two general porins, OmpF and OmpC, under conditions of phosphate excess. Under phosphate-limiting conditions, these porins are partially replaced by the pore protein PhoE (30), which preferentially allows the diffusion of anions (1), in contrast to the cation preference of OmpF and OmpC (29). Hence, the diffusion of phosphates through PhoE pores is more efficient and is the prevalent pathway for phosphates across the outer membrane under phosphate-limiting conditions (17). While inorganic phosphate is the preferred source of phosphorus, many bacteria can also take up organic phosphates and release phosphate by the action of periplasmic phosphatases such as PhoA. E. coli possesses four transport systems, Pst, Pit, GlpT, and UhpT, that translocate inorganic phosphate across the inner membrane (48). Part of the Pst system is the periplasmic protein PstS, which binds and transfers phosphate to the transmembrane components PstA and PstC. PstB hydrolyzes ATP and delivers energy for the translocation of phosphate across the inner membrane by PstA/PstC. Pst systems bind and transport phosphate with bindin...

Rv1698 of Mycobacterium tuberculosis Represents a New Class of Channel-forming Outer Membrane Proteins

“…smegmatis-The observation that Rv1698 forms an apparently nonspecific protein channel in a similar manner as MspA (9,10,31) raised the question why Rv1698 completely restored the susceptibility of the M. smegmatis mspA porin mutant to antibiotics (Fig. 1), whereas glucose diffusion was slow compared with MspA (Fig.…”

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

“…Hydrophilic substances cross this membrane via porins, whose importance in phosphate uptake was highlighted recently (530).…”

Nutrient acquisition by mycobacteria

“…Since MspA could not be expressed in M. smegmatis above wild-type levels (Stephan et al, 2005), it is also not clear whether the influx of nutrients really limits the growth rate of wild-type mycobacteria as suggested earlier (Jarlier & Nikaido, 1990). The fact that the lack of the MspA and MspC porins also caused a reduced uptake of phosphates and slower growth on low-phosphate plates (Wolschendorf et al, 2007) indeed suggested that the slow uptake of essential hydrophilic nutrients other than the carbon source may also contribute to the slow growth of M. smegmatis porin mutants.…”

“…Note that no experimental data exist for either the spatial distribution of the extractable lipids, or the confirmations and exact locations of the mycolic acids. The porin MspA mediates the uptake of small and hydrophilic nutrients such as sugars (Stephan et al, 2005) and phosphates (Wolschendorf et al, 2007) across the outer membrane of M. smegmatis. The representation of MspA is based on its crystal structure (Faller et al, 2004) and was created using the visualization software PyMol (DeLano Scientific).…”

“…However, recent progress has been made in understanding the uptake of some nutrients in both M. smegmatis and M. tuberculosis. For example, proteins have been identified and characterized that are involved in uptake of several nutrients such as phosphate (Gebhard et al, 2006;Peirs et al, 2005;Vyas et al, 2003;Webb, 2003;Wolschendorf et al, 2007), sulfate (Wooff et al, 2002) and some amino acids (Seth & Connell, 2000;Talaue et al, 2006). This review focuses primarily on the uptake of solutes that are considered to be major nutrients for mycobacteria.…”