A Flexible Binding Site Architecture Provides New Insights into CcpA Global Regulation in Gram-Positive Bacteria

Yang, Yunpeng; Zhang, Lu; Huang, He; Yang, Chen; Yang, Sheng; Gu, Yang; Jiang, Weihong

doi:10.1128/mbio.02004-16

Cited by 45 publications

(46 citation statements)

References 34 publications

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“…It is likely that CcpA regulates pil expression indirectly, similar to the regulation reported for C. difficile (32) and others (33). We also cannot rule out the possibility that an atypical cre is recognized by CcpA in S. sanguinis SK36, as seen in Streptococcus suis (34) and Clostridium acetobutylicum (35). Comparable levels of p pilB _WT-derived CAT-specific activity were observed between glucose-grown and galactose-grown cultures (data not shown), suggesting that CcpA exerts its regulation in a glucose-independent manner.…”

Section: Discussionmentioning

confidence: 62%

Molecular and Functional Analysis of the Type IV Pilus Gene Cluster in Streptococcus sanguinis SK36

Chen

Chiang

Tseng

et al. 2019

Appl Environ Microbiol

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Streptococcus sanguinis, dominant in the oral microbiome, is the only known streptococcal species possessing a pil gene cluster for the biosynthesis of type IV pili (Tfp). Although this cluster is commonly present in the genome of S. sanguinis, most of the strains do not express Tfp-mediated twitching motility. Thus, this study was designed to investigate the biological functions encoded by the cluster in the twitching-negative strain S. sanguinis SK36. We found that the cluster was transcribed as an operon, with three promoters located 5= to the cluster and one in the intergenic region between SSA_2307 and SSA_2305. Studies using promoter-cat fusion strains revealed that the transcription of the cluster was mainly driven by the distal 5= promoter, which is located more than 800 bases 5= to the first gene of the cluster, SSA_2318. Optimal expression of the cluster occurred at the early stationary growth phase in a CcpA-dependent manner, although a CcpA-binding consensus is absent in the promoter region. Expression of the cluster resulted in a short hairlike surface structure under transmission electron microscopy. Deletion of the putative pilin genes (SSA_2313 to SSA_2315) abolished the biosynthesis of this structure and significantly reduced the adherence of SK36 to HeLa and SCC-4 cells. Mutations in the pil genes downregulated biofilm formation by S. sanguinis SK36. Taken together, the results demonstrate that Tfp of SK36 are important for host cell adherence, but not for motility, and that expression of the pil cluster is subject to complex regulation. IMPORTANCE The proteins and assembly machinery of the type IV pili (Tfp) are conserved throughout bacteria and archaea, and yet the function of this surface structure differs from species to species and even from strain to strain. As seen in Streptococcus sanguinis SK36, the expression of the Tfp gene cluster results in a hairlike surface structure that is much shorter than the typical Tfp. This pilus is essential for the adherence of SK36 but is not involved in motility. Being a member of the highly diverse dental biofilm, perhaps S. sanguinis could more effectively utilize this structure to adhere to host cells and to interact with other microbes within the same niche.

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Section: Discussionmentioning

confidence: 62%

Molecular and Functional Analysis of the Type IV Pilus Gene Cluster in Streptococcus sanguinis SK36

Chen

Chiang

Tseng

et al. 2019

Appl Environ Microbiol

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“…CcpA directly or indirectly controls the expression levels of enzymes involved in the pyruvate pathway, which facilitate the utilization of available carbohydrates [9,30] Pyruvate plays a very important role in growth and metabolism since it affects the energy and redox status of the cells [30]. In this work, inactivation of ccpA caused pyruvate flux away from lactate production in L. bulgaricus under aerobic conditions.…”

Section: Discussionmentioning

confidence: 87%

“…The role for CcpA in control of carbohydrate metabolism has been reported in some LAB [12,29]. The regulatory pathways differ among microorganisms, although all are based on PTS/CcpA-mediated signal transduction [30]. In L. lactis and L. plantarum, the pfk-pyk operon is positively regulated by CcpA, which activates gene transcription [9,14].…”

Section: Discussionmentioning

confidence: 99%

Effects of ccpA gene deficiency in Lactobacillus delbrueckii subsp. bulgaricus under aerobic conditions as assessed by proteomic analysis

Zhang

Liu

2020

Microb Cell Fact

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Background: Aerobic growth provides benefits in biomass yield and stress tolerance of Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). Catabolite control protein A (CcpA) is a master regulator involved in the aerobic and anaerobic growth, metabolic production and stress response in L. bulgaricus, but its potential molecular mechanisms remains unclear. The aim of this study is to elucidate the role of CcpA in L. bulgaricus in aerobic growth at the proteomic perspective. Results: The differential proteomic analysis was performed on the L. bulgaricus ATCC11842 and its ccpA inactivated mutant strain using iTRAQ technology. A total of 132 differentially expressed proteins were obtained, among which 58 were up-regulated and 74 were down-regulated. These proteins were mainly involved in the cellular stress response, carbohydrate and energy metabolism, amino acid transport and protein synthesis, genetic information processing. Moreover, inactivation of ccpA negatively affected the expression of key enzymes involved in glycolysis pathway, while it enhanced the expression of proteins related to the pyruvate pathway, supporting the conclusion that CcpA mediated the shift from homolactic fermentation to mixed acid fermentation in L. bulgaricus. Conclusions: Overall, these results showed that the role of CcpA in L. bulgaricus as a pleiotropic regulator in aerobic metabolism and stress response. This proteomic analysis also provide new insights into the CcpA-mediated regulatory network of L. bulgaricus and potential strategies to improve the production of starter and probiotic cultures based on the metabolic engineering of global regulators.

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“…2D). The cre P site is quite different from the typical cre site as well as the newly identified, flexible cre var architecture in C. acetobutylicum (15,19). To further examine whether cre P is a CcpA-binding site, it was mutated, which resulted in a derived P-I fragment (P-I-mu) that was used as the probe in EMSAs ( Fig.…”

Section: Resultsmentioning

confidence: 99%

A Novel Dual- cre Motif Enables Two-Way Autoregulation of CcpA in Clostridium acetobutylicum

Zhang

Liu

Yang

et al. 2018

Appl Environ Microbiol

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The master regulator CcpA (catabolite control protein A) manages a large and complex regulatory network that is essential for cellular physiology and metabolism in Gram-positive bacteria. Although CcpA can affect the expression of target genes by binding to a -acting catabolite-responsive element (), whether and how the expression of CcpA is regulated remain poorly explored. Here, we report a novel dual- motif that is employed by the CcpA in , a typical solventogenic species, for autoregulation. Two sites are involved in CcpA autoregulation, and they reside in the promoter and coding regions of CcpA. In this dual- motif, , in the promoter region, positively regulates transcription, whereas , in the coding region, negatively regulates this transcription, thus enabling two-way autoregulation of CcpA. Although CcpA bound more strongly than , the assay showed that -based repression dominates CcpA autoregulation during the entire fermentation. Finally, a synonymous mutation of was made within the coding region, achieving an increased intracellular CcpA expression and improved cellular performance. This study provides new insights into the regulatory role of CcpA in and, moreover, contributes a new engineering strategy for this industrial strain. CcpA is known to be a key transcription factor in Gram-positive bacteria. However, it is still unclear whether and how the intracellular CcpA level is regulated, which may be essential for maintaining normal cell physiology and metabolism. We discovered here that CcpA employs a dual- motif to autoregulate, enabling dynamic control of its own expression level during the entire fermentation process. This finding answers the questions above and fills a void in our understanding of the regulatory network of CcpA. Interference in CcpA autoregulation leads to improved cellular performance, providing a new useful strategy in genetic engineering of Since CcpA is widespread in Gram-positive bacteria, including pathogens, this dual--based CcpA autoregulation would be valuable for increasing our understanding of CcpA-based global regulation in bacteria.

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A Flexible Binding Site Architecture Provides New Insights into CcpA Global Regulation in Gram-Positive Bacteria

Cited by 45 publications

References 34 publications

Molecular and Functional Analysis of the Type IV Pilus Gene Cluster in Streptococcus sanguinis SK36

Molecular and Functional Analysis of the Type IV Pilus Gene Cluster in Streptococcus sanguinis SK36

Effects of ccpA gene deficiency in Lactobacillus delbrueckii subsp. bulgaricus under aerobic conditions as assessed by proteomic analysis

A Novel Dual- cre Motif Enables Two-Way Autoregulation of CcpA in Clostridium acetobutylicum

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