cereus strains§ lysS   II No   lysK   I Yes B. thuringiensis Konkukian lysS BT9727_0072 II No   lysK BT9727_2375 I Yes B. thuringiensis

Al Hakam lysS BALH_0075 II No   lysK BALH_2333 I Yes Clostridium S63845 molecular weight beijerinckii lysS1 Cbei_0105 II No   lysS2 Cbei_3591 II Yes Symbiobacterium thermophilum lysS STH525 II Yes   lysK STH208 I No The T-box element controlling expression of lysK in B. cereus strain 14579 is functional The T-box element in the B. cereus and B. thuringiensis strains has a canonical structure [8], is highly conserved and controls expression of a class I LysRS (encoded by the lysK gene) of Pyrococcal origin [20]. Interestingly, the lysK gene is see more expressed predominantly during stationary phase

in B. cereus strain 14579, whereas the class II LysRS is expressed during exponential growth of this bacterium [8]. To ascertain whether this T-box element is functional, expression of a P lysK(T box) lacZ transcriptional fusion (present in single copy at the amyE locus of the B. subtilis chromosome) was established under conditions of lysine starvation (strain NF33 is a lysine auxotroph) and LysRS2 depletion Emricasan chemical structure (strain BCJ367 has the endogenous lysS gene under the control of the IPTG-inducible Pspac promoter). The results are shown in Figure 1. When strain NF33 is grown in lysine replete medium, only a low level of P lysK(T box) lacZ expression (~10 units of β-galactosidase activity) is observed (Figure 1A, squares). However growth in a lysine depleted medium (growth cessation occurs at ~ OD600 1 due to lysine deficiency) results in a high level of P lysK(T box) lacZ expression, with accumulation of ~1200 units of β-galactosidase activity. Importantly P lysK(T box) lacZ induction is coincident with the point

of growth cessation due to lysine deficiency (Figure 1A). To confirm that increased P lysK(T box) lacZ expression is associated with increased levels of uncharged tRNALys, strain BCJ367 (Pspac lysS P lysK(T box) lacZ) was grown in the presence FER of 1 mM IPTG, 250 μM IPTG and 100 μM IPTG. Growth of the cultures containing 1 mM and 250 μM IPTG was similar to that of wild-type strain 168 while growth of the cultures with 100 μM IPTG was reduced, presumably due to a decreased level of charged lysyl-tRNALys (Figure 1B). Expression of P lysK(T box) lacZ is low (~10 units β-galactosidase activity) in cultures containing 1 mM IPTG. P lysK(T box) lacZ expression is initially low in cultures containing 250 μM IPTG but gradually increases with accumulation of ~200 units of β-galactosidase activity at the onset of stationary phase. However in cultures with 100 μM IPTG, P lysK(T box) lacZ expression increases throughout exponential growth with accumulation of more than 800 units of β-galactosidase during this period (Figure 1B).