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Abstract

Porins mediate the diffusion of hydrophilic solutes across the outer membrane of mycobacteria, but the efficiency of this pathway is very low compared to Gram-negative bacteria. To examine the importance of porins in slow-growing mycobacteria, the major porin MspA of was expressed in and . Approximately 20 and 35 MspA molecules per μm cell wall were observed in and BCG, respectively, by electron microscopy and quantitative immunoblot experiments. Surface accessibility of MspA in was demonstrated by flow cytometry. Glucose uptake was twofold faster, indicating that the outer membrane permeability of BCG to small and hydrophilic solutes was increased by MspA. This significantly accelerated the growth of BCG, identifying very slow nutrient uptake as one of the determinants of slow growth in mycobacteria. The susceptibility of both BCG and to zwitterionic -lactam antibiotics was substantially enhanced by MspA, decreasing the minimal inhibitory concentration up to 16-fold. Furthermore, became significantly more susceptible to isoniazid, ethambutol and streptomycin. Fluorescence with the nucleic acid binding dye SYTO 9 was 10-fold increased upon expression of . These results indicated that MspA not only enhanced the efficiency of the porin pathway, but also that of pathways mediating access to large and/or hydrophobic agents. This study provides the first experimental evidence that porins are important for drug susceptibility of .

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2004-04-01
2019-08-17
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vol. , part 4, pp. 853 - 864

Supplementary Fig. S1. Fluorescence microscopy of BCG

The viability and aggregation of bacteria in liquid cultures was checked routinely using the LIVE/DEAD kit (Molecular Probes). A sample of the cell suspension was mixed with an equal volume of a fluorescence dye mixture containing equimolar amounts of propidium iodide and SYTO 9 and was incubated in the dark for 1 min. The stained cells were examined using a Zeiss Axioskop2 fluorescence microscope. Pictures were analysed by counting live (green) and dead (red) bacteria. The -expressing BCG (A) showed on average 98 % viable and single cells, the wild-type strain (B) 97 %.



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