1887

Abstract

is a common cause of disease in humans, particularly in hospitalized patients. This species needs to import several amino acids to survive, including proline. Previously, it was shown that an insertion mutation in the high-affinity proline uptake gene in strain RN6390 affected proline uptake by the bacteria as well as reducing their ability to survive . To further delineate the effect of the mutation on growth of strain RN6390, a proline uptake assay that spanned less than 1 min was done to measure transport. An eightfold difference in proline levels was observed between the wild-type strain and the high-affinity proline transport mutant strain after 15 s, indicating that the defect was only in proline transport and not a combination of proline transport, metabolism and accumulation that would have been assessed with longer assays. A mutant of strain RN4220 was then grown in minimal medium with different concentrations of proline. When compared to the wild-type strain, the mutant strain was significantly growth impaired when the level of proline was decreased to 1·74 μM. An assessment of proline concentrations in mouse livers and spleens showed proline concentrations of 7·5 μmol per spleen and 88·4 μmol per liver. To verify that the effects on proline transport and bacterial survival were indeed caused solely by a mutation in , the mutation was complemented by cloning a full-length gene on a plasmid that replicates in . Complementation of the mutant strains restored proline transport, growth in low-proline medium, and survival within mice. These results show that the mutation in led to attenuated growth in low-proline media and by corollary low-proline murine organ tissues due to less efficient transport of proline into the bacteria.

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2004-04-01
2020-04-10
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