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

Xiao, Fengxu; Li, Youran; Zhang, Yupeng; Wang, Hanrong; Zhang, Liang; Ding, Zhongyang; Gu, Zhenghua; Xu, Sha; Shi, Guiyang

doi:10.1016/j.isci.2021.102400

Cited by 19 publications

(11 citation statements)

References 42 publications

(42 reference statements)

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“…Notably, the underlying molecular basis of the CcpA regulon in Firmicutes has not been sufficiently investigated. For example, novel CcpA-binding motifs were identified in Lactobacillus plantarum ( Chen et al., 2018 ) and Bacillus licheniformis ( Xiao et al., 2021 ). Moreover, HPr, HPrK, and glycolytic intermediates (fructose-1,6-bisphosphate and glucose-6-phosphate), except for CcpA, play key roles in CCR and CCA; and HPr(Ser-P), a cofactor for CcpA, is also competitively inhibited by HPr(His-P) ( Gorke and Stulke, 2008 ).…”

Section: Discussionmentioning

confidence: 99%

Exogenous glycogen utilization effects the transcriptome and pathogenicity of Streptococcus suis serotype 2

Tan

Tan²,

Zhang³

et al. 2022

Front. Cell. Infect. Microbiol.

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Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes severe infections in humans and the swine industry. Acquisition and utilization of available carbon sources from challenging host environments is necessary for bacterial pathogens to ensure growth and proliferation. Glycogen is abundant in mammalian body and may support the growth of SS2 during infection in hosts. However, limited information is known about the mechanism between the glycogen utilization and host adaptation of SS2. Here, the pleiotropic effects of exogenous glycogen on SS2 were investigated through transcriptome sequencing. Analysis of transcriptome data showed that the main basic metabolic pathways, especially the core carbon metabolism pathways and virulence-associated factors, of SS2 responded actively to glycogen induction. Glycogen induction led to the perturbation of the glycolysis pathway and citrate cycle, but promoted the pentose phosphate pathway and carbohydrate transport systems. Extracellular glycogen utilization also promoted the mixed-acid fermentation in SS2 rather than homolactic fermentation. Subsequently, apuA, a gene encoding the unique bifunctional amylopullulanase for glycogen degradation, was deleted from the wild type and generated the mutant strain ΔapuA. The pathogenicity details of the wild type and ΔapuA cultured in glucose and glycogen were investigated and compared. Results revealed that the capsule synthesis or bacterial morphology were not affected by glycogen incubation or apuA deletion. However, extracellular glycogen utilization significantly enhanced the hemolytic activity, adhesion and invasion ability, and lethality of SS2. The deletion of apuA also impaired the pathogenicity of bacteria cultured in glucose, indicating that ApuA is indeed an important virulence factor. Our results revealed that exogenous glycogen utilization extensively influenced the expression profile of the S. suis genome. Based on the transcriptome response, exogenous glycogen utilization promoted the carbon adaption and pathogenicity of SS2.

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Section: Discussionmentioning

confidence: 99%

Exogenous glycogen utilization effects the transcriptome and pathogenicity of Streptococcus suis serotype 2

Tan

Tan²,

Zhang³

et al. 2022

Front. Cell. Infect. Microbiol.

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“…The reagents and medium for Bacillus transformation were prepared according to Li [ 33 ]. E. coli and Bacillus were grown in terrific broth (TB) based on Xiao’s methods [ 40 ]. Ampicillin (100 μg/mL) was added when necessary to maintain the plasmids in E. coli .…”

Section: Methodsmentioning

confidence: 99%

Engineering of Bacillus Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit

Zhang

et al. 2022

IJMS

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Bacillus genetics need more versatile promoters for gene circuit engineering. UP elements are widely distributed in noncoding regions and interact with the α-subunit of RNA polymerase (RNAP). They can be applied as a standard element for synthetic biology. Characterization of the binding motif between UP elements and RNAP may assist with rational and effective engineering. In this study, 11 Bacillus constitutive promoters were screened for strength in Bacillus licheniformis. The motif in UP elements from a strong native promoter, PLan, was characterized. The influence of specific sequences on RNAP binding and expression strength was investigated both in vitro and in vivo. It was found that sequences up to 50 base pairs upstream of the consensus motif significantly contributed to α-CTD (the alpha subunit carboxy-terminal domain) association. Meanwhile, two repeats of a proximal subsite were able to more strongly activate the expression (by 8.2-fold) through strengthening interactions between UP elements and RNAP. Based the above molecular basis, a synthetic UP element, UP5-2P, was constructed and applied to nine wild-type promoters. Fluorescence polarization results demonstrated that it had an apparent effect on promoter–α-CTD interactions, and elevated expression strength was observed for all the engineered promoters. The highest improved core promoter, Pacpp, was more strongly activated by 7.4-fold. This work thus develops a novel strategy for Bacillus promoter engineering.

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“…The samples were electroblotted from the acrylamide gels onto a nylon membrane (Beyotime, FFN15) and immobilized by UV cross-linking. The subsequent procedures were performed according to the manufacturer’s instructions with cautionary notes. , …”

Section: Methodsmentioning

confidence: 99%

“…The subsequent procedures were performed according to the manufacturer's instructions with cautionary notes. 44,45 ■ CONCLUSION…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

Engineering of a Biosensor in Response to Malate in Bacillus licheniformis

Zhang

Xiao

et al. 2021

ACS Synth. Biol.

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Malate is an essential intermediate in the tricarboxylic acid (TCA) cycle; it also has valuable uses in medicine and food. The production of malate with a microbial synthesis method is still in its early stages. One of the key problems in metabolic engineering is that the dynamic and subtle changes in malate are difficult to detect. It remains critical to develop techniques with direct and precise detection of malate in microbial metabolism, which facilitates high-throughput screening of the engineered strains. In this study, a genetically encoded biosensor in response to malate was constructed in B. licheniformis. Key regulator MalR and the action site of the biosensor were first identified. Then, the output of the reporter gene expression was amplified by introducing a strong constitutive promoter and iteratively tuning the action sites. The engineered biosensor can respond to malate from 5 to 15 g/L; within this range, it shows a linear correlation between eGFP fluorescence and malate concentration. This biosensor enrich our toolbox of synthetic biology in pathway engineering for malate production in microorganisms.

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

Cited by 19 publications

References 42 publications

Exogenous glycogen utilization effects the transcriptome and pathogenicity of Streptococcus suis serotype 2

Exogenous glycogen utilization effects the transcriptome and pathogenicity of Streptococcus suis serotype 2

Engineering of Bacillus Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit

Engineering of a Biosensor in Response to Malate in Bacillus licheniformis

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