The Carbohydrate Metabolism of Lactiplantibacillus plantarum

Lactiplantibacillus plantarum has a strong carbohydrate utilization ability. This characteristic plays an important role in its gastrointestinal tract colonization and probiotic effects. L. plantarum LP-F1 presents a high carbohydrate utilization capacity. The genome analysis of 165 L. plantarum strains indicated the species has a plenty of carbohydrate metabolism genes, presenting a strain specificity. Furthermore, two-component systems (TCSs) analysis revealed that the species has more TCSs than other lactic acid bacteria, and the distribution of TCS also shows the strain specificity. In order to clarify the sugar metabolism mechanism under different carbohydrate fermentation conditions, the expressions of 27 carbohydrate metabolism genes, catabolite control protein A (CcpA) gene ccpA, and TCSs genes were analyzed by quantitative real-time PCR technology. The correlation analysis between the expressions of regulatory genes and sugar metabolism genes showed that some regulatory genes were correlated with most of the sugar metabolism genes, suggesting that some TCSs might be involved in the regulation of sugar metabolism.

Transcriptome Analysis Reveals Catabolite Control Protein A Regulatory Mechanisms Underlying Glucose-Excess or -Limited Conditions in a Ruminal Bacterium, Streptococcus bovis

Ruminants may suffer from rumen acidosis when fed with high-concentrate diets due to the higher proliferation and overproduction of lactate by Streptococcus bovis. The catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase (ldh) and pyruvate formate-lyase (pfl) in S. bovis, but its role in response to different carbon concentrations remains unclear. To characterize the regulatory mechanisms of CcpA in S. bovis S1 at different levels of carbon, herein, we analyzed the transcriptomic and physiological characteristics of S. bovis S1 and its ccpA mutant strain grown in glucose-excess and glucose-limited conditions. A reduced growth rate and a shift in fermentation pattern from homofermentation to heterofermentation were observed under glucose-limited condition as compared to glucose-excess condition, in S. bovis S1. Additionally, the inactivation of ccpA significantly affected the growth and end metabolites in both conditions. For the glycolytic intermediate, fructose 1,6-bisphosphate (FBP), the concentration significantly reduced at lower glucose conditions; its concentration decreased significantly in the ccpA mutant strain. Transcriptomic results showed that about 46% of the total genes were differentially transcribed between the wild-type strain and ccpA mutant strain grown in glucose-excess conditions; while only 12% genes were differentially transcribed in glucose-limited conditions. Different glucose concentrations led to the differential expression of 38% genes in the wild-type strain, while only half of these were differentially expressed in the ccpA-knockout strain. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the substrate glucose concentration significantly affected the gene expression in histidine metabolism, nitrogen metabolism, and some carbohydrate metabolism pathways. The deletion of ccpA affected several genes involved in carbohydrate metabolism, such as glycolysis, pyruvate metabolism, fructose and mannose metabolism, as well as in fatty acid biosynthesis pathways in bacteria grown in glucose-excess conditions; this effect was attenuated under glucose-limited conditions. Overall, these findings provide new information on gene transcription and metabolic mechanisms associated with substrate glucose concentration and validate the important role of CcpA in the regulation of carbon metabolism in S. bovis S1 at differential glucose availability.

Ribozyme-Mediated Downregulation Uncovers DNA Integrity Scanning Protein A (DisA) as a Solventogenesis Determinant in Clostridium beijerinckii

Carbon catabolite repression (CCR) limits microbial utilization of lignocellulose-derived pentoses. To relieve CCR in Clostridium beijerinckii NCIMB 8052, we sought to downregulate catabolite control protein A (CcpA) using the M1GS ribozyme technology. A CcpA-specific ribozyme was constructed by tethering the catalytic subunit of Escherichia coli RNase P (M1 RNA) to a guide sequence (GS) targeting CcpA mRNA (M1GSCcpA). As negative controls, the ribozyme M1GSCcpA–Sc (constructed with a scrambled GSCcpA) or the empty plasmid pMTL500E were used. With a ∼3-fold knockdown of CcpA mRNA in C. beijerinckii expressing M1GSCcpA (C. beijerinckii_M1GSCcpA) relative to both controls, a modest enhancement in mixed-sugar utilization and solvent production was achieved. Unexpectedly, C. beijerinckii_M1GSCcpA–Sc produced 50% more solvent than C. beijerinckii_pMTL500E grown on glucose + arabinose. Sequence complementarity (albeit suboptimal) suggested that M1GSCcpA–Sc could target the mRNA encoding DNA integrity scanning protein A (DisA), an expectation that was confirmed by a 53-fold knockdown in DisA mRNA levels. Therefore, M1GSCcpA–Sc was renamed M1GSDisA. Compared to C. beijerinckii_M1GSCcpA and _pMTL500E, C. beijerinckii_M1GSDisA exhibited a 7-fold decrease in the intracellular c-di-AMP level after 24 h of growth and a near-complete loss of viability upon exposure to DNA-damaging antibiotics. Alterations in c-di-AMP-mediated signaling and cell cycling likely culminate in a sporulation delay and the solvent production gains observed in C. beijerinckii_M1GSDisA. Successful knockdown of the CcpA and DisA mRNAs demonstrate the feasibility of using M1GS technology as a metabolic engineering tool for increasing butanol production in C. beijerinckii.

Modulating the selective utilization of carbon sources by engineering the 3rd and 4th helices of the DNA-binding domain of catabolite control protein A (CcpA) in Bacillus licheniformis

The gram-positive bacterium Bacillus licheniformis exhibits obvious selective utilization on carbon sources. This process is mainly governed by the global regulator catabolite control protein A (CcpA), which can recognize and bind to multiple target genes widely distributed in metabolic pathways. Although the DNA-binding domain of CcpA has been predicted, the influence of key amino acids on target gene recognition and binding remains elusive. In this study, the impact of Lys31, Ile42 and Leu56 on in vitro protein-DNA interactions and in vivo carbon source selective utilization was investigated. The results showed that alanine substitution of Lys31 and Ile42, located within the 3rd helices of the DNA-binding domain, significantly weakened the binding strength between CcpA and target genes. These mutations also lead to alleviated repression of xylose utilization in the presence of glucose. On the other hand, the Leu56Arg mutant in the 4th helices exhibited enhanced binding affinity compared with that of the wild-type one. When this mutant was used to replace the native one in B. licheniformis cells, the selective utilization of glucose over xylose increased. The above research results are helpful for a deep understanding of how microorganisms can flexibly sense and adapt to changes in the external environment. Additionally, they can provide important theoretical basis for the rational design of biomass utilization and environmental adaptability of B. licheniformiscell factories.

Identification of a chromosomally-encoded sucrose operon-like gene cluster in Vibrio parahaemolyticus strain PH05 isolated from Negros Island, Philippines

ABSTRACTThe ability of bacteria to metabolize a wide variety of carbon sources has been known to aid in their ability for efficient colonization. Vibrio parahaemolyticus, a known aquatic pathogen has been reported to have the ability to metabolize a number of carbohydrates including D-glucose, D-galactose, L-arabinose, D-mannose, and D-ribose to name a few. Classical isolation of V. parahaemolyticus from other members of the family Vibrionaceae relies on its carbon utilization pattern. Conventionally, V. parahaemolyticus lacks the ability to utilize sucrose and this has been the basis for its isolation using the Thiosulfate-citrate-bile salts-sucrose (TCBS) agar. Reports of V. parahaemolyticus having the ability to utilize sucrose have been presented yet there is paucity of information and detailed study on this phenotype. In this study, we report the V. parahaemolyticus strain PH05 that has the ability to metabolize sucrose. Phenotypic and genotypic characterization of this V. parahaemolyticus strain isolated from Negros Island, Philippines, revealed that V. parahaemolyticus strain PH05 is atypical appearing yellow on TCBS agar plates. It is capable of utilizing sucrose, unlike the majority of V. parahaemolyticus isolates. Genome analyses of this strain revealed the presence of a chromosomally encoded sucrose operon-like gene cluster encoded in chromosome 2 with the following sucrose-utilization associated genes: scrY, ccpA, treP, scrK, and scrB genes coding for sucrose porin, catabolite control protein A, PTS System sucrose-specific EIIBC component, fructokinase, and sucrose-6-phosphate hydrolase. The mode of transmission of these genes to V. parahaemolyticus strain PH05 is still unknown. However, the presence of insertion sequences (IS) and phage elements in the same chromosome suggests horizontal gene transfer events. Taken together, our results point to the possibility that acquired sucrose utilization genes may contribute to the fitness of V. parahaemolyticus strain PH05 in the environment.

Impact of the Histidine-Containing Phosphocarrier Protein HPr on Carbon Metabolism and Virulence in Staphylococcus aureus

Carbon catabolite repression (CCR) is a common mechanism pathogenic bacteria use to link central metabolism with virulence factor synthesis. In gram-positive bacteria, catabolite control protein A (CcpA) and the histidine-containing phosphocarrier protein HPr (encoded by ptsH) are the predominant mediators of CCR. In addition to modulating CcpA activity, HPr is essential for glucose import via the phosphotransferase system. While the regulatory functions of CcpA in Staphylococcus aureus are largely known, little is known about the function of HPr in CCR and infectivity. To address this knowledge gap, ptsH mutants were created in S. aureus that either lack the open reading frame or harbor a ptsH variant carrying a thymidine to guanosine mutation at position 136, and the effects of these mutations on growth and metabolism were assessed. Inactivation of ptsH altered bacterial physiology and decreased the ability of S. aureus to form a biofilm and cause infections in mice. These data demonstrate that HPr affects central metabolism and virulence in S. aureus independent of its influence on CcpA regulation.

Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group A Streptococcus

SummaryCatabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in non-pathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIPseq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIPseq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.Data sharing and data availabilityThe data that support the findings of this study are available from the corresponding author upon reasonable request.

Enhanced Production of Polymyxin E in Paenibacillus polymyxa by Replacement of Glucose by Starch

Polymyxin E or colistin, produced by Paenibacillus polymyxa, is an important antibiotic against Gram-negative pathogens. The objective of this study is to evaluate the effect of starch in fermentation medium on colistin biosynthesis in P. polymyxa. The results indicated that replacement of glucose by starch stimulated colistin production and biosynthesis rate. Overall, the stimulation extent was starch concentration-dependent. As expected, addition of starch induced the expression of amyE encoding amylase and increased amylase activity in fermentation solution. Additionally, replacement of glucose by starch resulted in residue reducing sugar and pH of fermentation mixture low relative to glucose as the sole sugar source. At the molecular level, it was found that replacement of glucose by starch has enhanced the relative expression level of ccpA encoding catabolite control protein A. Therefore, the repression of starch utilization by glucose could be probably relieved. In addition, use of starch stimulated the expression of regulatory gene spo0A but repressed the expression of another regulatory gene abrB. As a result, the expression of genes directly involved in colistin biosynthesis and secretion increased, indicating that at the transcriptional level spo0A and abrB played opposite roles in regulating colistin biosynthesis in P. polymyxa. Taken together, our data demonstrated that starch instead of glucose can promote colistin production probably by affecting the expression of colistin biosynthesis-related genes, as well as reducing the repression of glucose to a secondary metabolic product.