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.

Effects of Abolishing Whi2 on the Proteome and Nitrogen Catabolite Repression-Sensitive Protein Production

In yeast physiology, a commonly used reference condition for many experiments, including those involving Nitrogen Catabolite Repression (NCR), is growth in Synthetic Complete (SC) medium. Four SC formulations, SCCSH, 1990, SCCSH, 1994, SCCSH, 2005 and SCME, have been used interchangeably as the nitrogen-rich medium of choice (Cold Spring Harbor Yeast Course Manuals, SCCSH, and a formulation in The Methods in Enzymology SCME). It has been tacitly presumed that all of these formulations support equivalent responses. However, Chen et al. (2018) concluded that: (i) TorC1 activity is down regulated by the lower concentration of primarily leucine in SCME relative to SCCSH. (ii) The Whi2-Psr1/2 complex is responsible for this down regulation. TorC1 is a primary nitrogen-responsive regulator in yeast. Among its downstream targets is control of NCR-sensitive transcription activators Gln3 and Gat1. They in turn control production of catabolic transporters and enzymes needed to scavenge poor nitrogen sources (e.g., Proline) and activate autophagy (ATG14). One of the reporters used in Chen et al. was an NCR-sensitive DAL80-GFP promoter fusion. This intrigued us because we expected minimal if any DAL80 expression in SC medium. Therefore, we investigated the source of the Dal80-GFP production and the proteomes of wild type and whi2Δ cells cultured in SCCSH and SCME. We found a massive and equivalent reorientation of amino acid biosynthetic proteins in both wild type and whi2Δ cells even though both media contained high overall concentrations of amino acids. Gcn2 appears to play a significant regulatory role in this reorientation. NCR-sensitive DAL80 expression and overall NCR-sensitive protein production were only marginally affected by the whi2Δ. In contrast, the levels of 58 proteins changed by an absolute value of log2 between 3 and 8) when Whi2 was abolished relative to wild type. Surprisingly, with only two exceptions could those proteins be related in GO analyses, i.e., GO terms associated with carbohydrate metabolism and oxidative stress after shifting a whi2Δ from SCCSH to SCME for 6 hours. What was conspicuously missing were proteins related by TorC1- and NCR-associated GO terms.

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.

Regulation of ytfK by cAMP-CRP Contributes to SpoT-Dependent Accumulation of (p)ppGpp in Response to Carbon Starvation YtfK Responds to Glucose Exhaustion

Guanosine penta- or tetraphosphate (known as (p)ppGpp) serves as second messenger to respond to nutrient downshift and other environmental stresses, a phenomenon called stringent response. Accumulation of (p)ppGpp promotes the coordinated inhibition of macromolecule synthesis, as well as the activation of stress response pathways to cope and adapt to harmful conditions. In Escherichia coli, the (p)ppGpp level is tightly regulated by two enzymes, the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified the small protein YtfK as a key regulator of SpoT-mediated activation of stringent response in E. coli. Here, we further characterized the regulation of ytfK. We observed that ytfK is subjected to catabolite repression and is positively regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Importantly, YtfK contributes to SpoT-dependent accumulation of (p)ppGpp and cell survival in response to glucose starvation. Therefore, regulation of ytfK by the cAMP-CRP appears important to adjust (p)ppGpp level and coordinate cellular metabolism in response to glucose availability.