A new CcpA binding site plays a bidirectional role in carbon catabolism in Bacillus licheniformis

Abstract

Bacillus licheniformis is widely used to produce various valuable products, such as food enzymes, industrial chemicals, and biocides. The carbon catabolite regulation process in the utilization of raw materials is crucial to maximizing the efficiency of this microbial cell factory. The current understanding of the molecular mechanism of this regulation is based on limited motif patterns in protein-DNA recognition, where the typical catabolite-responsive element (CRE) motif is "TGWNANCGNTNWCA". Here, CRE_Tre is identified and characterized as a new CRE. It consists of two palindrome arms of 6 nucleotides (AGCTTT/AAAGCT) and an intermediate spacer. CRE_Tre is involved in bidirectional regulation in a glucose stress environment. When AGCTTT appears in the 5' end, the regulatory element exhibits a carbon catabolite activation effect, while AAAGCT in the 5' end corresponds to carbon catabolite repression. Further investigation indicated a wide occurrence of CRE_Tre in the genome of B. licheniformis.

Keywords: Microbial Metabolism; Microbiology; Structural Biology.

Graphical abstract

Figure 1

Identification of CcpA binding sites in trehalose operon (A) The non-coding region of the trehalose operon was divided into two fragments ([fragment A] and [fragment B]). The CcpA binding region is shown in red, and the TreR binding region is shown in blue. The −10 region and −35 region are shown in green. The transcription start site is shown in purple. The SD sequence is shown in orange. (B) EMSA of CcpA protein binding to three fragments (fragment A, fragment B, and fragment C) labeled with 5′ biotin. (C) The region of treR gene was divided into four fragments (fragment D, fragment E, fragment F, and fragment G). The fragment D of the treR gene was further divided into six fragments (fragment D, fragment D1, fragment D2, fragment D3, fragment D4, and fragment D5). The fragment E of the treR gene was further divided into six fragments (fragment E, fragment E1, fragment E2, fragment E3, fragment E4, and fragment E5). The CcpA binding site in fragment D or fragment E is shown in red. (D) EMSA of CcpA protein binding to four fragments (fragment D, fragment E, fragment F, and fragment G) labeled with 5′ biotin, six fragments labeled with 5′ biotin (fragment D, fragment D1, fragment D2, fragment D3, fragment D4, and fragment D5) that were derived from fragment D, and six fragments labeled with 5′ biotin (fragment E, fragment E1, fragment E2, fragment E3, fragment E4, and fragment E5) that were derived from fragment E.

Figure 2

Influence of the 12-bp symmetrical region and the intermediate spacer region with CRE_Tre (AGCTTT-Yx-AAAGCT) on CcpA protein regulation (A) Construction of recombination fragments (fragment H1, fragment H2, fragment H3, fragment H4, fragment H5, and fragment H6) harboring the CRE_Tre with different intermediate spacers length or different 12-bp symmetrical region. (B) Two fragments that change the intermediate spacer region and three fragments that change the 12-bp symmetrical region are derived from tre-26-1 fragment, the intermediate spacer region with black and the 12-bp symmetrical region with red. (C) EMSA of CcpA protein binding to six fragments (fragment H1, fragment H2, fragment H3, fragment H4, fragment H5, and fragment H6) that carrying different CRE_Tre sites.

Figure 3

Influence of single point mutation of the 12-bp symmetrical region in CRE_Tre for CcpA protein binding (A) Single point mutation of the 12-bp symmetrical region of CRE_Tre. (B) EMSA of CcpA protein for 12 mutants (5′ biotin), 12 mutants (T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, and T12) derived from CRE_Tre, with concentrations of 0.9 μM–2.0 μM of CcpA protein. (C) The ratio of protein-bound probe/total probe for 12 mutants that derived from CRE_Tre at the 0.9 μM CcpA protein was shown. Statistical significance was determined by Student's t-test (∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001).

Figure 4

Characterization of the CcpA regulation in vivo for trehalose-inducible system (A) The fluorescence intensity and OD 600 were measured for the strain carrying pBLTE plasmid under ten conditions (control: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% fructose, 1.5% trehalose + 1.5% mannitol, 1.5% trehalose + 1.5% glycerol, 1.5% trehalose + 1.5% glycerol, 1.5% trehalose + 1.5% saccharose, 1.5% trehalose + 1.5% mannose, 1.5% trehalose + 1.5% sorbitol, 1.5% trehalose + 1.5% arabinose, 1.5% trehalose + 1.5% xylose). Data are shown as means ± SD, n = 3. (B) Quantify the CCR/CCA effect with the formula I=(FI₁-FI₂)/FI₁×100% due to different carbohydrates (FI₁ represents the fluorescence intensity when only trehalose is added. FI₂ represents the fluorescence intensity when trehalose is added and another carbohydrate is also added. Glucose, fructose, mannitol, glycerol, sucrose, mannose, sorbitol, arabinose, and xylose added at a concentration of 1.5% while adding 1.5% trehalose. Data are shown as means ± SD, n = 3. (C) The fluorescence intensity and OD 600 were measured for trehalose promoter in both the strain BlspTE (control 1: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol) and CcpA-deletion strain BlspTE1 (control 2: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol). Data are shown as means ± SD, n = 3. (D) The fluorescence intensity and OD 600 were measured for trehalose promoter whose CRE site was replaced by CRE_Tre site in both the strain BlspT1E (control 3: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol) and CcpA-deletion strain BlspT1E1 (control 4: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol). Data are shown as means ± SD, n = 3. (E) The fluorescence intensity and OD 600 were measured for trehalose promoter whose CRE site was replaced by CRE_Tre(R) site in both the strain BlspT2E (control 5: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol) and CcpA-deletion strain BlspT2E1 (control 6: 1.5% trehalose, 1.5% trehalose + 1.5% glucose, 1.5% trehalose + 1.5% glycerol). Data are shown as means ± SD, n = 3. (F) Compared three CRE sites by using formula I=(FI₁-FI₂)/FI₁×100% while extra adding glucose or glycerol. Data are shown as means ± SD, n = 3.

Figure 5

Influence of the 12-bp symmetrical region and the intermediate spacer region with CRE_Tre(R) (AAAGCT-Yx-AGCTTT) on CcpA protein regulation (A) Construction of recombination fragments (fragment I1, fragment I2, fragment I3, fragment I4, fragment I5, and fragment I6) harboring the CRE_Tre(R) with different intermediate spacer length or different 12-bp symmetrical region. (B) Two fragments that change the intermediate spacer region and three fragments that change the 12-bp symmetrical region are derived from tre(R)-26-1 fragment, the intermediate spacer region with black and the 12-bp symmetrical region with red. (C) EMSA of CcpA protein binding to six fragments (fragment I1, fragment I2, fragment I3, fragment I4, fragment I5, and fragment I6) that carry different CRE_Tre(R) sites.

Figure 6

Influence of single point mutation of the 12-bp symmetrical region in CRE_Tre(R) for CcpA protein binding (A) Single point mutation of the 12-bp symmetrical region of CRE_Tre(R). (B) EMSA of CcpA protein for 12 mutants (5′ biotin), 12 mutants (TR1, TR2, TR3, TR4, TR5, TR6, TR7, TR8, TR9, TR10, TR11, and TR12) derived from CRE_Tre(R), with concentrations of 1.2 μM–2.0 μM of CcpA protein. (C) The ratio of protein-bound probe/total probe for 12 mutants that derived from CRE_Tre(R) at the 1.2 μM CcpA protein were shown. Statistical significance was determined by Student's t-test (∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001).

Figure 7

Exploring the property of CRE_Tre in Pdanp, the CRE_Tre(R) in P5A2 (A) The sequences of the PdnaP and P5A2 from Bacillus licheniformis are shown, and the CRE_Tre or CRE_Tre(R) is shown in red, −10 region is shown in green, and −35 region is shown in blue. (B) EMSA of CcpA protein binding to two fragments (PdnaP, P5A2) labeled with 5′ biotin, while not binding to two fragments (PdnaPΔCRE_Tre, P5A2ΔCRE_Tre(R)) labeled with 5′ biotin. (C) The OD 600 and the fluorescence intensity were measured in both the Bacillus licheniformis CICIM B1391 and CcpA-deletion strain when using PdnaP or P5A2 as the expression element. Data are shown as means ± SD, n = 3. Statistical significance was determined by Student's t-test (∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗P ≤ 0.001).

Figure 8

Searching Bacillus licheniformis genome for CRE_Tre and CRE_Tre(R)