Transcription of the Lysine-2,3-Aminomutase Gene in the kam Locus of Bacillus thuringiensis subsp. kurstaki HD73 Is Controlled by Both σ ⁵⁴ and σ ^K Factors

Abstract

Reverse transcription-PCR (RT-PCR) analysis revealed that this locus forms two operons:yodT (yodT-yodS-yodR-yodQ-yodPkamR) and kamA (kamA-yokU-yozE). The transcriptional start sites (TSSs) of the kamA gene were determined using 5= rapid amplification of cDNA ends (RACE). A typical ؊12/؊24 54 binding site was identified in the promoter P kamA , which is located upstream of the kamA gene TSS. A ␤-galactosidase assay showed that P kamA , which directs the transcription of the kamA operon, is controlled by the 54 factor and is activated through the 54 -dependent transcriptional regulator KamR. The kamA operon is also controlled by K and regulated by the GerE protein in the late stage of sporulation. kamR and kamA mutants were prepared by homologous recombination to examine the role of the kam locus. The results showed that the sporulation rate in B. thuringiensis HD(⌬kamR) was slightly decreased compared to that in HD73, whereas that in HD(⌬kamA) was similar to that in HD73. This means that other genes regulated by KamR are important for sporulation. Bacteria synthesize a number of different sigma factors that allow the coordinated expression of groups of genes due to the ability of sigma factors to confer promoter-specific transcriptional initiation of RNA polymerase (RNAP) (1). Most of these sigma factors belong to a single family of proteins that appear to be related structurally and functionally to the major Escherichia coli sigma factor, 70 . A second class of these factors is represented solely by the alternative factor 54 , which is widely present in prokaryotes (2, 3). Studies of 54 have showed that this sigma factor is quite distinct both structurally and functionally from the 70 family: (i) unlike the 70 factor, which recognizes Ϫ10/Ϫ35 regions (4), 54 directs its RNAP to promoters characterized by conserved sequences located at Ϫ24 and Ϫ12 bp upstream from the transcriptional start site (TSS) (5), and (ii) the activator ATPase approaches the leading edge of the closed RNAP-promoter complex, and a regulatory protein is required for 54 to stimulate the isomerization of the closed complexes to the corresponding open complexes (1, 3). 54 participates in the regulation of many metabolic pathways in bacteria, such as the arginine degradation pathway, the isoleucine and valine degradation pathway, and the acetoin catabolic pathway in Bacillus subtilis (6-8). We previously found that the gab gene cluster involved in the GABA shunt for the utilization of GABA is controlled by 54 in Bacillus thuringiensis (9), a process that is not 54 dependent in B. subtilis (10). However, other metabolic pathways controlled by the 54 factor in B. thuringiensis remain unknown.L-Lysine is first converted to L-␤-lysine by a lysine-2,3-aminomutase (KAM; EC 5.4.3.2) in the lysine degradation pathway (11), and this intermediate is then acetylated to N ε -acetyl-␤-lysine by the action of an acetyltransferase (12). Genes potentially encoding lysine-2,3-aminomutase (ablA) and ␤-lysine acetyltransferase (ablB) have been identified on th...

Identification of metabolism pathways directly regulated by sigma54 factor in Bacillus thuringiensis

“…To validate the results of the transcriptome and −12/−24 region analyses, 21 promoter fusions with the lacZ gene were constructed and analyzed by evaluating β-galactosidase activity. The activation of 16 promoters was abolished and decreased in the Δ sigL mutants containing the gabT (Zhu et al, 2010 ) and kamA (Zhang et al, 2014 ) promoters, these promoters are regulated by σ 54 (Figures 1A–F , 2A–H ). Three of these (HD73_3213, HD73_4468, and HD73_5614) were regulated by sequences that were highly similar to B. subtilis SigL promoter elements (Debarbouille et al, 1991a , 1999 ; Ali et al, 2001 ).…”

“…The activation of the kamA promoter—which is a typical −12/−24 sequence—was abolished in the Δ sigL mutant from T 0 to T 4 and was reduced from T 4 to T 7 as compared to the HD73 strain. The promoter was activated by KamR prior to T 7 and controlled by σ K and GerE after T 10 , the late stage of sporulation (Zhang et al, 2014 ).…”

“…Bt forms parasporal crystals during the stationary phase of growth that are toxic to a wide variety of insect larvae (Schnepf et al, 1998 ), making Bt strains the most commonly used biological pesticide worldwide. Among these species, only two metabolic pathways are known to be controlled by σ 54 : the γ-aminobutyric acid (GABA) (Zhu et al, 2010 ) and l-lysine metabolism (Zhang et al, 2014 ) pathways in the Bt HD73 strain. Little is known about the roles of σ 54 and its regulons in the B. cereus group.…”

Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis

“…To do so, we incubated a mixture of purified CwlC-His protein and the B. thuringiensis cell wall on ice. The SigK-His protein was used as a control to eliminate possible interference from the histidine tag (20). The CwlC-His protein alone was incubated in distilled water without addition of the cell wall as another control to determine whether CwlC-His itself would precipitate.…”

“…Cell samples were analyzed with a BX61 optical microscope (Olympus, Japan). The sporulation frequency was calculated as described previously (18,20). Briefly, the total number of cells in the sample taken at T 1 was determined.…”