subtilis strain 168. Results show an increase in the generation time of strain NF54 during growth in LB medium: NF54 has a doubling time of ~31 minutes while that of wild-type strain 168 is ~22 minutes under these conditions. However strain NF54 does not grow in minimal medium whereas wild-type strain 168 has a generation time of ~76 minutes in this medium. To establish whether this growth phenotype was due to reduced tRNALys charging, the P lysK(T box) lacZ was introduced into strain NF54 generating strain NF206. Reduced charging of

tRNALys in strain NF206 will result in increased β-galactosidase accumulation when compared with strain this website BCJ363 that has the P lysK(T box) lacZ contruct in an otherwise wild-type background (ie. with the endogenous class II lysS). Results show that 250-300 units of β-galactosidase accumulate during exponential growth of strain NF206, an ~20-fold increase over that observed in the control strain BCJ363. We conclude T box control of LysR1 expression is compatible with FK506 concentration viability

of B. subtilis. However such strains have a reduced growth rate in rich medium and cannot be propagated in minimal medium probably due to reduced tRNALys charging. A B. subtilis strain with expression of the endogenous class II lysS under the control of the T box regulatory element is viable and indistinguishable from wild-type in terms of growth and tRNALys charging While T box control of LysRS1 expression supports growth of B. subtilis, the level of charged tRNALys is reduced and there is a growth phenotype. However it is unclear whether this phenotype is caused by T box regulation of LysRS expression or is due next to the B. cereus derived

class I LysRS1 enzyme that is reported to be less efficient catalytically than its class II counterpart [21]. To distinguish between these possibilities and to further address the issue of T box regulation of LysRS, we constructed strain NF113 (lysS::P lysK(T box) lysS) that placed expression of the endogenous B. subtilis lysS gene under the control of the lysK promoter and T box element from B. cereus strain 14579. It is important to note that in strain NF113 the P lysK(T box) lysS cassette is flanked by transcriptional terminators ensuring that lysS expression is solely dependent on the P lysK(T box) promoter. Strain NF113 was successfully constructed and the relevant chromosomal regions verified by PCR and Southern blotting (data not shown) confirming that T box regulation of LysRS2 expression supports growth of B. subtilis. Importantly growth of strain NF113 in rich (LB) and minimal media (Spizizen salts) was indistinguishable from wild-type strain 168 (data not shown). The level of charged tRNALys was assessed in strain NF113 by introducing the P lysK(T box) lacZ transcriptional fusion to generate strain NF205. Approximately 10 units of β-galactosidase accumulated during exponential growth of strain NF205 similar to control strain BCJ363 (data not shown).