Part:BBa_K2586001

GltT: glutamate and glyphosate transporter in B. subtilis

This part is coding for a glutamate and glyphosate transporter. The DNA sequence of the gltT gene is coding for the GltT transporter, which is responsible for glutamate uptake into the cell. We found that GltT is also involved in glyphosate uptake in the Gram-positive model bacterium Bacillus subtilis.

Bacteria lacking the gltT gene are highly resistant to glyphosate because the herbicide is not transported into the cell. This part could be useful to engineer bacteria for the uptake and degradation of the weedkiller.

Deletion of gltT confers glyphosate tolerance

We tested whether the clean deletion of the gltT gene sufficient to confer high-level resistance of B. subtilis to glyphosate. For this purpose, we constructed the mutant strain BP233 (gltT). To assess the glyphosate resistance of the gltT mutant, we cultivated the bacteria in CS-Glc minimal medium supplemented with increasing amounts of glyphosate. As shown in Figure 1A, growth of the wild type was inhibited by 5 mM glyphosate. The transporter with a high-affinity for glyphosate seems to be in fact GltT because the deletion of the gltT gene conferred high-level resistance to the herbicide (Figure 1C). Moreover, the growth rates of the strain BP233 (gltT) were reduced by 50% at herbicide concentrations of 6.1 mM (Figure 1B). Thus, in comparison to the wild type strain, 6-fold higher glyphosate concentration is needed to reduce the growth rate of the strain BP233 (gltT) by 50%.

Double mutant gltT gltP shows even higher glyphosate tolerance

In order to test whether the deletion of both gltT and gltP confers an even higher glyphosate tolerance, we constructed the mutant strain BP235 (gltT gltP). We cultivated the bacteria in CS-Glc minimal medium supplemented with increasing amounts of glyphosate. As shown in Figure 1D, the double deletion increased the glyphosate tolerance to 8.0 mM. Therefore, an 8-fold higher glyphosate concentration is needed to reduce the growth rate of the strain BP235 (gltT gltP) by 50%.

Fig. 1. TESTESTEST

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 742
  Illegal NgoMIV site found at 1123
  
• 1000
  COMPATIBLE WITH RFC[1000]