Staphylococcus aureus does not synthesize arginine from proline under physiological conditions

The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway, despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC-deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA-deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway.

Mechanism of differential expression of β-glucosidase genes in functional microbial communities in response to carbon catabolite repression

Background β-Glucosidase is the rate-limiting enzyme of cellulose degradation. It has been stipulated and established that β-glucosidase-producing microbial communities differentially regulate the expression of glucose/non-glucose tolerant β-glucosidase genes. However, it is still unknown if this differential expression of functional microbial community happens accidentally or as a general regulatory mechanism, and of what biological significance it has. To investigate the composition and function of microbial communities and how they respond to different carbon metabolism pressures and the transcriptional regulation of functional genes, the different carbon metabolism pressure was constructed by setting up the static chamber during composting. Results The composition and function of functional microbial communities demonstrated different behaviors under the carbon metabolism pressure. Functional microbial community up-regulated glucose tolerant β-glucosidase genes expression to maintain the carbon metabolism rate by enhancing the transglycosylation activity of β-glucosidase to compensate for the decrease of hydrolysis activity under carbon catabolite repression (CCR). Micrococcales play a vital role in the resistance of functional microbial community under CCR. The transcription regulation of GH1 family β-glucosidase genes from Proteobacteria showed more obvious inhibition than other phyla under CCR. Conclusion Microbial functional communities differentially regulate the expression of glucose/non-glucose tolerant β-glucosidase genes under CCR, which is a general regulatory mechanism, not accidental. Furthermore, the differentially expressed β-glucosidase gene exhibited species characteristics at the phylogenetic level.

Effect of Glucose on Endo-xylanase and β-xylosidase Production by Fungi Isolated in Indonesia

Xylanases are widely produced by fungi, and the production of polysaccharide-degrading enzymes, in general, are usually subjected to carbon catabolite repression. In this work, the ability of several Indonesian indigenous fungi to produce endo-xylanase and β-xylosidase and their responses to glucose as a repressor were determined. Ten fungi were grown in a liquid medium supplemented with glucose as the repressor (0, 1%, 3%, and 5%), and the endo-xylanase and β-xylosidase productions were assayed. Aspergillus aculeatus FIG1 and A. oryzae KKB4 produced 3.85 and 0.70 U/mL of endo-xylanase, respectively, compared with other strains (0.22 U/mL or less). Trichoderma asperellum PK1J2, T. virens MLT2J2, A. aculeatus FIG1, T. asperellum MLT5J1, A. oryzae KKB4, and T. asperellum MLT3J2 produced 0.021–0.065 U/mL of β-xylosidase, whereas the other strains produced 0.013 U/mL or less of β-xylosidase. Adding 1% glucose to the growth medium can partially repress endo-xylanase production in A. aculeatus FIG1, T. asperellum PK1J2, and T. virens MLT4J1 and completely repress other strains. By adding 1% glucose, strains FIG1, PK1J2, and MLT4J1 suffered almost complete repression of β-xylosidase production, although such strains exhibited partial repression of endo-xylanase production. β-Xylosidase produced by the other strains showed complete repression by adding 1% glucose, except for A. aculeatus FIG1, A. tamarii FNCC 6151, and T. asperellum MLT1J1, which showed partial repression. Therefore, adding 3% glucose to the growth medium can result in complete repression of endo-xylanase and β-xylosidase productions in all strains examined.

Carbon catabolite repression involves physical interaction of the transcription factor CRE1/CreA and the Tup1–Cyc8 complex in Penicillium oxalicum and Trichoderma reesei

Background Cellulolytic enzyme production in filamentous fungi requires a release from carbon catabolite repression (CCR). The protein CRE1/CreA (CRE = catabolite responsive element) is a key transcription factor (TF) that is involved in CCR and represses cellulolytic gene expression. CRE1/CreA represents the functional equivalent of Mig1p, an important Saccharomyces cerevisiae TF in CCR that exerts its repressive effect by recruiting a corepressor complex Tup1p–Cyc8p. Although it is known from S. cerevisiae that CRE1/CreA might repress gene expression via interacting with the corepressor complex Tup1–Cyc8, this mechanism is unconfirmed in other filamentous fungi, since the physical interaction has not yet been verified in these organisms. The precise mechanism on how CRE1/CreA achieves transcriptional repression after DNA binding remains unknown. Results The results from tandem affinity purification and bimolecular fluorescence complementation revealed a direct physical interaction between the TF CRE1/CreA and the complex Tup1–Cyc8 in the nucleus of cellulolytic fungus Trichoderma reesei and Penicillium oxalicum. Both fungi have the ability to secrete a complex arsenal of enzymes to synergistically degrade lignocellulosic materials. In P. oxalicum, the protein PoCyc8, a subunit of complex Tup1–Cyc8, interacts directly with TF PoCreA and histone H3 lysine 36 (H3K36) methyltransferase PoSet2 in the nucleus. The di-methylation level of H3K36 in the promoter of prominent cellulolytic genes (cellobiohydrolase-encoding gene Pocbh1/cel7A and endoglucanase-encoding gene Poegl1/cel7B) is positively correlated with the expression levels of TF PoCreA. Since the methylation of H3K36 was also demonstrated to be a repression marker of cellulolytic gene expression, it appears feasible that the cellulolytic genes are repressed via PoCreA-Tup1–Cyc8-Set2-mediated transcriptional repression. Conclusion This study verifies the long-standing conjecture that the TF CRE1/CreA represses gene expression by interacting with the corepressor complex Tup1–Cyc8 in filamentous fungi. A reasonable explanation is proposed that PoCreA represses gene expression by recruiting complex PoTup1–Cyc8. Histone methyltransferase Set2, which methylates H3K36, is also involved in the regulatory network by interacting with PoCyc8. The findings contribute to the understanding of CCR mechanism in filamentous fungi and could aid in biotechnologically relevant enzyme production.

When metabolic prowess is too much of a good thing: how carbon catabolite repression and metabolic versatility impede production of esterified α,ω-diols in Pseudomonas putida KT2440

Background Medium-chain-length α,ω-diols (mcl-diols) are important building blocks in polymer production. Recently, microbial mcl-diol production from alkanes was achieved in E. coli (albeit at low rates) using the alkane monooxygenase system AlkBGTL and esterification module Atf1. Owing to its remarkable versatility and conversion capabilities and hence potential for enabling an economically viable process, we assessed whether the industrially robust P. putida can be a suitable production organism of mcl-diols. Results AlkBGTL and Atf1 were successfully expressed as was shown by oxidation of alkanes to alkanols, and esterification to alkyl acetates. However, the conversion rate was lower than that by E. coli, and not fully to diols. The conversion was improved by using citrate instead of glucose as energy source, indicating that carbon catabolite repression plays a role. By overexpressing the activator of AlkBGTL-Atf1, AlkS and deleting Crc or CyoB, key genes in carbon catabolite repression of P. putida increased diacetoxyhexane production by 76% and 65%, respectively. Removing Crc/Hfq attachment sites of mRNAs resulted in the highest diacetoxyhexane production. When the intermediate hexyl acetate was used as substrate, hexanol was detected. This indicated that P. putida expressed esterases, hampering accumulation of the corresponding esters and diesters. Sixteen putative esterase genes present in P. putida were screened and tested. Among them, Est12/K was proven to be the dominant one. Deletion of Est12/K halted hydrolysis of hexyl acetate and diacetoxyhexane. As a result of relieving catabolite repression and preventing the hydrolysis of ester, the optimal strain produced 3.7 mM hexyl acetate from hexane and 6.9 mM 6-hydroxy hexyl acetate and diacetoxyhexane from hexyl acetate, increased by 12.7- and 4.2-fold, respectively, as compared to the starting strain. Conclusions This study shows that the metabolic versatility of P. putida, and the associated carbon catabolite repression, can hinder production of diols and related esters. Growth on mcl-alcohol and diol esters could be prevented by deleting the dominant esterase. Carbon catabolite repression could be relieved by removing the Crc/Hfq attachment sites. This strategy can be used for efficient expression of other genes regulated by Crc/Hfq in Pseudomonas and related species to steer bioconversion processes.

Effect of deregulation of repressor-specific carbon catabolite repression on carbon source consumption in Escherichia coli

Escherichia coli has been used as a host to construct the cell factory for biobased production of chemicals from renewable feedstocks. Because galactose is found in marine biomass as a major component, the strategy for galactose utilization in E. coli has been gained more attention. Although galactose and glucose co-fermentation has been reported using the engineered E. coli strain, few reports have covered fermentation supplemented with galactose as a sole carbon source in the mutant lacking the repressor-specific carbon catabolite repression (CCR). Here, we report the effects of the deregulation of the repressor-specific CCR (galR− and galS−) in fermentation supplemented with galactose as a sole carbon source, using the engineered E. coli strains. In the fermentation using the galR− and galS− double mutant (GR2 strain), an increase of rates in sugar consumption and cell growth was observed compared to the parent strain. In the glucose fermentation, wild-type W3110 and its mutant GR2 and GR2PZ (galR−, galS−, pfkA−, and zwf−) consumed sugar at a higher rate than those values obtained from galactose fermentation. However, the GR2P strain (galR−, galS−, and pfkA−) showed no difference between fermentations using glucose and galactose as a sole carbon source. This study provides essential information for galactose fermentation using the CCR-deregulated E. coli strains.