The expression of glycosyltransferases sdgA and sdgB in Staphylococcus epidermidis depends on the conditions of biofilm formation.

The Staphylococcus aureus SdrG protein is glycosylated by SdgA and SdgB for protection against its degradation by the neutrophil cathepsin G. So far, there is no information about the role of Staphylococcus epidermidis SdgA or SdgB in biofilm-forming; therefore, the focus of this work was to determine the distribution and expression of the sdrG, sdgA and sdgB genes in S. epidermidis under in vitro and in vivo biofilm conditions. The frequencies of the sdrG, sdgA and sdgB genes were evaluated by PCR in a collection of 75 isolates. Isolates were grown in dynamic (non-biofilm-forming) or static (biofilm-forming) conditions. The expression of sdrG, sdgA and sdgB was determined by RT-qPCR in cells grown under dynamic conditions (CGDC), as well as in planktonic and sessile cells from a biofilm and cells adhered to a catheter implanted in Balb/c mice. The sdrG and sdgB genes were detected in 100% of isolates, while the sdgA gene was detected in 71% of the sample (p < 0.001). CGDC did not express sdrG, sdgA and sdgB mRNAs. Planktonic and sessile cells expressed sdrG and sdgB, and the same was observed in cells adhered to the catheter. In particular, one isolate, capable of inducing a biofilm under treatment with cathepsin G, expressed sdrG and sdgB in planktonic and sessile cells and cells adhering to the catheter. This suggests that bacteria require biofilm conditions as an important factor for the transcription of the sdgA, sdgB and sdrG genes.

Deciphering the mechanism by which the yeast Phaffia rhodozyma responds adaptively to environmental, nutritional, and genetic cues.

Phaffia rhodozyma is a basidiomycetous yeast that synthesizes astaxanthin (ASX), which is a powerful and highly valuable antioxidant carotenoid pigment. P. rhodozyma cells accrue ASX and gain an intense red-pink coloration when faced with stressful conditions such as nutrient limitations (e.g., nitrogen or copper), the presence of toxic substances (e.g., antimycin A), or are affected by mutations in the genes that are involved in nitrogen metabolism or respiration. Since cellular accrual of ASX occurs under a wide variety of conditions, this yeast represents a valuable model for studying the growth conditions that entail oxidative stress for yeast cells. Recently, we proposed that ASX synthesis can be largely induced by conditions that lead to reduction-oxidation (redox) imbalances, particularly the state of the NADH/NAD+ couple together with an oxidative environment. In this work, we review the multiple known conditions that elicit ASX synthesis expanding on the data that we formerly examined. When considered alongside the Mitchell's chemiosmotic hypothesis, the study served to rationalize the induction of ASX synthesis and other adaptive cellular processes under a much broader set of conditions. Our aim was to propose an underlying mechanism that explains how a broad range of divergent conditions converge to induce ASX synthesis in P. rhodozyma. The mechanism that links the induction of ASX synthesis with the occurrence of NADH/NAD+ imbalances may help in understanding how other organisms detect any of a broad array of stimuli or gene mutations, and then adaptively respond to activate numerous compensatory cellular processes.

Differential Expression of the apsXRS System by Antimicrobial Peptide LL-37 in Commensal and Clinical Staphylococcus epidermidis Isolates.

The three-component apsXRS system senses and responds to cationic antimicrobial peptides (CAMPs), which induces the expression of the dlt operon and the genes mprF and vrafG, modifying the surface net charge in Staphylococcus epidermidis, resulting in the repulsion of CAMPs. The apsXRS system has been only studied in the S. epidermidis 1457 strain, and there are no studies of prevalence and level of expression of apsXRS in commensal and clinical isolates. From 60 isolates, those selected from commensal healthy skin (n = 20), commensal healthy conjunctive (n = 10), and clinical ocular infection (n = 30) presented the apsX, apsR, and apsS genes in their genomes. Constitutive expression of apsX, apsR, and apsS genes was determined by RT-qPCR in all isolates. It was found that expression of apsX, apsR, and apsS was 3.3-5.9-fold higher in commensal isolates stimulated with LL-37 (15 µg/mL) than in clinical isolates. Similarly, expression of the dlt operon and the genes mprF, and vraFG was 8-10-fold higher in commensal isolates than in clinical. However, LL-37 did not increase the addition of lysine in the phospholipids of the cytoplasmic membrane in any of the isolates. Mutations in the apsS loop region, apsR, and their promoter sequence were not found. These results demonstrated that apsXRS system is essential in all isolates for its constitutive expression; however, LL-37 caused an increase of apsXRS expression in commensal isolates, suggesting that S. epidermidis isolates do not respond in the same way to the presence of LL-37.

A common mechanism explains the induction of aerobic fermentation and adaptive antioxidant response in Phaffia rhodozyma.

BACKGROUND: Growth conditions that bring about stress on Phaffia rhodozyma cells encourage the synthesis of astaxanthin, an antioxidant carotenoid, which protects cells against oxidative damage. Using P. rhodozyma cultures performed with and without copper limitation, we examined the kinetics of astaxanthin synthesis along with the expression of asy, the key astaxanthin synthesis gene, as well as aox, which encodes an alternative oxidase protein. RESULTS: Copper deficiency had a detrimental effect on the rates of oxygen consumption and ethanol reassimilation at the diauxic shift. In contrast, copper deficiency prompted alcoholic fermentation under aerobic conditions and had a favorable effect on the astaxanthin content of cells, as well as on aox expression. Both cultures exhibited strong aox expression while consuming ethanol, but particularly when copper was absent. CONCLUSION: We show that the induction of either astaxanthin production, aox expression, or aerobic fermentation exemplifies the crucial role that redox imbalance plays in triggering any of these phenomena. Based on our own results and data from others, we propose a mechanism that rationalizes the central role played by changes of respiratory activity, which lead to redox imbalances, in triggering both the short-term antioxidant response as well as fermentation in yeasts and other cell types.

Molecular cloning and characterization of the ATP citrate lyase from carotenogenic yeast Phaffia rhodozyma.

ATP citrate lyase (ACL), is a key cytosolic source of acetyl-CoA for fatty acid and sterol biosynthesis and appear to be involved in carotenoid biosynthesis in yeasts. Three homologous DNA sequences encoding ACLs in Phaffia rhodozyma were isolated i.e two genes and one cDNA. The two genes were multi-intronic, with 3450-bp-coding sequences and both genes, as the cDNA, encoded identical 120.1-kDa polypeptides. Full-length amino acid sequences of these ACLs showed the two multidomains, PLN02235 and PLN02522, which are necessary for activity. The ACLs showed 82-87% similarity to putative ACLs from other basidiomycetes and 71% similarity to human ACL. The acl cDNA was used to express the heterologous ACL 6XHis-tagged which was identified using MALDI-TOF-MS. The sequenced peptides with 42.2% coverage showed 100% identity to the amino acid sequence generated in silico. The recombinant ACL purified to homogeneity showed an activity of 2 U. This is the first study to characterize a recombinant ACL from a carotenogenic yeast. The present study provides a key foundation for future studies to assess (a) the possible occurrence of alternative splicing, (b) identify the promoter(s) sequence(s) and (c) the involvement of ACL in the differential regulation of fatty acid and carotenoid biosynthesis in yeasts.

The potential of cold-shock promoters for the expression of recombinant proteins in microbes and mammalian cells.

BACKGROUND: Low-temperature expression of recombinant proteins may be advantageous to support their proper folding and preserve bioactivity. The generation of expression vectors regulated under cold conditions can improve the expression of some target proteins that are difficult to express in different expression systems. The cspA encodes the major cold-shock protein from Escherichia coli (CspA). The promoter of cspA has been widely used to develop cold shock-inducible expression platforms in E. coli. Moreover, it is often necessary to employ expression systems other than bacteria, particularly when recombinant proteins require complex post-translational modifications. Currently, there are no commercial platforms available for expressing target genes by cold shock in eukaryotic cells. Consequently, genetic elements that respond to cold shock offer the possibility of developing novel cold-inducible expression platforms, particularly suitable for yeasts, and mammalian cells. CONCLUSIONS: This review covers the importance of the cellular response to low temperatures and the prospective use of cold-sensitive promoters to direct the expression of recombinant proteins. This concept may contribute to renewing interest in applying white technologies to produce recombinant proteins that are difficult to express.

ATP-citrate lyase activity and carotenoid production in batch cultures of Phaffia rhodozyma under nitrogen-limited and nonlimited conditions.

ATP-citrate lyase (ACL) is the key cytoplasmic enzyme which supplies acetyl-CoA for fatty acids in oleaginous yeast. Although it has been suggested that fatty acid and carotenoid biosynthesis may have a common source of acetyl-CoA in Phaffia rhodozyma, the source for carotenoids is currently unknown. The purpose of this work was to analyze the development of ACL activity during batch cultures of P. rhodozyma under ammonium-limited and nonammonium-limited conditions and study its possible relationship with carotenoid synthesis. Every experiment showed carotenoid accumulation linked to an increasing ACL activity. Moreover, the ACL activity increased with dissolved oxygen (DO), i.e., ACL responded to DO in a similar way as carotenoid synthesis. Additionally, in the ammonium-limited culture, ACL activity increased upon ammonium depletion. However, the contribution to carotenoid accumulation in that case was negligible. This suggests that P. rhodozyma has developed two components of ACL, each one responsive to a different environmental stimulus, i.e., DO and ammonium depletion. The role of each component is still unknown; however, considering that the former responds to DO and the known role of carotenoids as antioxidants, it may be a provider of acetyl-CoA for carotenoid synthesis.

Correction to: Autolysis of Pichia pastoris induced by cold.

The original version of this article (Bartolo-Aguilar et al. 2017) was written and published including the first construction strategy of pLGC09, but not the final one. This error was pointed out by a reader and an analysis of sequences of parts of the plasmid corroborated this. The final construction strategy was reanalysed and confirmed the error. This error affected the text, Table 2, Fig. 1 and Additional files, but did not affect the results and conclusions stated in the paper. The authors regret that this error occurred in the original publication of the article. The corrected text, Table 2 and Fig. 1, and Additional files (Additional file 1. Construction strategy of pLGC09 and Additional file 2. Plasmid pLGC09) are given in this correction.

Autolysis of Pichia pastoris induced by cold.

The production of recombinant biopharmaceutical proteins is a multi-billion dollar market. Protein recovery represents a major part of the production costs. Pichia pastoris is one of the microbial systems most used for the production of heterologous proteins. The use of a cold-induced promoter to express lytic enzymes in the yeast after the growth stage could reduce protein recovery costs. This study shows that a cold-shock can be applied to induce lysis of the yeast cells. A strain of P. pastoris was constructed in which the endogenous eng gene encoding a putative endo-ß-1,3-glucanase was overexpressed using the cold-shock induced promoter of the cctα gene from Saccharomyces cerevisiae. In the transgenic P. pastoris, the expression of eng increased 3.6-fold after chilling the cells from 30 to 4 °C (cold-shock stage) followed by incubation for 6 h (eng expression stage). The culture was heated to 30 °C for 6 h (ENG synthesis stage) and kept at 37 °C for 24 h (lysis stage). After this procedure the cell morphology changed, spheroplasts were obtained and cellular lysis was observed. Thus, a clone of P. pastoris was obtained, which undergoes autolysis after a cold-shock.