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Polyhydroxyalkanoates (PHAs) are a well-known group of biodegradable and biocompatible bioplastics that are synthesised and stored by microorganisms as carbon and energy reservoirs. Extracellular PHA depolymerases (ePhaZs), secreted by a limited range of microorganisms, are the main hydrolytic enzymes responsible for their environmental degradation. Pseudomonas sp. GK13, initially identified as P. fluorescens GK13, produces PHA and a prototypic ePhaZ that specifically degrades mcl-PHA. In this study, a comprehensive characterization of strain GK13 was performed. The whole genomic sequence of GK13 was consolidated into one complete chromosome, leading to its reclassification as P. solani GK13. We conducted a detailed in silico examination of the bacteria genomic sequence, specifically targeting PHA metabolic functions. From the different growth conditions explored, PHA accumulation occurred only under carbon/nitrogen (C/N) imbalance, whereas ePhaZ production was induced even at balanced C/N ratios in mineral media. We extend our study to other bacteria belonging to the Pseudomonas genus revealing that the ePhaZ production capacity is closely associated with mcl-PHA synthesis capacity, as also suggested by metagenomic samples. This finding suggests that these types of microorganisms could contribute to the carbon economy of the microbial community, by storing PHA in carbon-rich times, and sharing it with the rest of the population during times of carbon scarcity through PHA hydrolysis. The conclusion pointed that carbon cycle metabolism performed by P. solani GK13 may contribute to the environmental circular economy at a microscopic scale., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Environmental concerns about residues and the traditional disposal methods are driving the search for more environmentally conscious processes, such as pyrolysis and gasification. Their main final product is synthesis gas (syngas) composed of CO, CO 2 , H 2 , and methane. Syngas can be converted into various products using CO-tolerant microorganisms. Among them, Rhodospirillum rubrum is highlighted for its biotechnological potential. However, the extent to which high doses of CO affect its physiology is still opaque. For this reason, we have studied R. rubrum behavior under high levels of this gas (up to 2.5 bar), revealing a profound dependence on the presence or absence of light. In darkness, the key variable affected was the lag phase, where the highest levels of CO retarded growth to more than 20 days. Under light, R. rubrum ability to convert CO into CO 2 and H 2 depended on the presence of an additional carbon source, such as acetate. In those conditions where CO was completely exhausted, CO 2 fixation was unblocked, leading to a diauxic growth. To enhance R. rubrum tolerance to CO in darkness, a UV-accelerated adaptive laboratory evolution (UVa-ALE) trial was conducted to isolate clones with shorter lag phases, resulting in the isolation of clones 1.4-2B and 1.7-2A. The adaptation of 1.4-2B was mainly based on mutated enzymes with a metabolic function, while 1.7-3A was mostly affected at regulatory genes, including the anti-repressor PpaA/AerR. Despite these mutations having slight effects on biomass and pigment levels, they successfully provoked a significant reduction in the lag phase (-50%). KEYPOINTS: • CO affects principally R. rubrum lag phase (darkness) and growth rate (light) • CO is converted to CO 2 /H 2 during acetate uptake and inhibits CO 2 fixation (light) • UVa-ALE clones showed a 50% reduction in the lag phase (darkness)., (© 2024. The Author(s).)

Biodegradable polyesters, such as polyhydroxyalkanoates (PHAs), are having a tremendous impact on biomedicine. However, these polymers lack functional moieties to impart functions like targeted delivery of molecules. Inspired by native GAPs, such as phasins and their polymer-binding and surfactant properties, we generated small material binding peptides (MBPs) for polyester surface functionalization using a rational approach based on amphiphilicity. Here, two peptides of 48 amino acids derived from phasins PhaF and PhaI from Pseudomonas putida, MinP and the novel-designed MinI, were assessed for their binding towards two types of PHAs, PHB and PHOH. In vivo, fluorescence studies revealed selective binding towards PHOH, whilst in vitro binding experiments using the Langmuir-Blodgett technique coupled to ellipsometry showed K D in the range of nM for all polymers and MBPs. Marked morphological changes of the polymer surface upon peptide adsorption were shown by BAM and AFM for PHOH. Moreover, both MBPs were successfully used to immobilize cargo proteins on the polymer surfaces. Altogether, this work shows that by redesigning the amphiphilicity of phasins, a high affinity but lower specificity to polyesters can be achieved in vitro. Furthermore, the MBPs demonstrated binding to PET, showing potential to bind cargo molecules also to synthetic polyesters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Importance: Rhodospirillum rubrum vast metabolic versatility places it as a remarkable model bacterium and an excellent biotechnological chassis. The key component of photosynthesis (PS) studied in this work (HP1) stands out among the other members of PpaA/AerR anti-repressor family since it lacks the motif they all share: the cobalamin B-12 binding motif. Despite being reduced and poorly conserved, HP1 stills controls PS as the other members of the family, allowing a fast response to changes in the redox state of the cell. This work also shows that HP1 absence affects genes from relevant biological processes other than PS, including nitrogen fixation and stress response. From a biotechnological perspective, HP1 could be manipulated in approaches where PS is not necessary, such as hydrogen or polyhydroxyalkanoates production, to save energy., Competing Interests: The authors declare no conflict of interest.

Background: Infections by SARS-CoV-2 in liver transplant recipients (LT) patients are of particular concern, notably due to perceived added risks related to immunosuppression and comorbidity burden. Current literature on this topic often relies on small, non-standardized, and geographically limited studies. This manuscript describes COVID-19 presentations and causes for elevated mortality in a large cohort of LT recipients., Methods: This study was designed as a multicentric historical cohort, including LT recipient patients with COVID-19 in 25 study centers, with the primary endpoint being COVID-related death. We also collected demographic, clinical, and laboratory data regarding presentation and disease progression., Results: Two hundred and thirty-four cases were included. The study population was predominantly male and White and had a median age of 60 years. The median time from transplantation was 2.6 years (IQR 1-6). Most patients had at least one comorbidity (189, 80.8%). Patient age (P = .04), dyspnea (P < .001), intensive care unit admission (P < .001), and mechanical ventilation (P < .001) were associated with increased mortality. Modifications of immunosuppressive therapy (P < .001), specifically the suspension of tacrolimus, maintained significance in multivariable analysis., Conclusions: Attention to risk factors and the individualization of patient care, especially regarding immunosuppression management, is crucial for delivering more precise interventions to these individuals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Microbially produced bioplastics are specially promising materials since they can be naturally synthesized and degraded, making its end-of-life management more amenable to the environment. A prominent example of these new materials are polyhydroxyalkanoates. These polyesters serve manly as carbon and energy storage and increase the resistance to stress. Their synthesis can also work as an electron sink for the regeneration of oxidized cofactors. In terms of biotechnological applications, the co-polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), or PHBV, has interesting biotechnological properties due to its lower stiffness and fragility compared to the homopolymer poly(3-hydroxybutyrate) (P3HB). In this work, we explored the potentiality of Rhodospirillum rubrum as a producer of this co-polymer, exploiting its metabolic versatility when grown in different aeration conditions and photoheterotrophically., Results: When shaken flasks experiments were carried out with limited aeration using fructose as carbon source, PHBV production was triggered reaching 29 ± 2% CDW of polymer accumulation with a 75 ± 1%mol of 3-hydroxyvalerate (3HV) (condition C2). Propionate and acetate were secreted in this condition. The synthesis of PHBV was exclusively carried out by the PHA synthase PhaC2. Interestingly, transcription of cbbM coding RuBisCO, the key enzyme of the Calvin-Benson-Bassham cycle, was similar in aerobic and microaerobic/anaerobic cultures. The maximal PHBV yield (81% CDW with 86%mol 3HV) was achieved when cells were transferred from aerobic to anaerobic conditions and controlling the CO 2 concentration by adding bicarbonate to the culture. In these conditions, the cells behaved like resting cells, since polymer accumulation prevailed over residual biomass formation. In the absence of bicarbonate, cells could not adapt to an anaerobic environment in the studied lapse., Conclusions: We found that two-phase growth (aerobic-anaerobic) significantly improved the previous report of PHBV production in purple nonsulfur bacteria, maximizing the polymer accumulation at the expense of other components of the biomass. The presence of CO 2 is key in this process demonstrating the involvement of the Calvin-Benson-Bassham in the adaptation to changes in oxygen availability. These results stand R. rubrum as a promising producer of high-3HV-content PHBV co-polymer from fructose, a PHBV unrelated carbon source., (© 2023. The Author(s).)

Manipulation of global regulators is one of the strategies used for the construction of bacterial strains suitable for the synthesis of bioproducts. However, the pleiotropic effects of these regulators can vary under different conditions and are often strain dependent. This study analyzed the effects of ArcA, CreC, Cra, and Rob using single deletion mutants of the well-characterized and completely sequenced Escherichia coli strain BW25113. Comparison of the effects of each regulator on the synthesis of major extracellular metabolites, tolerance to several compounds, and synthesis of native and nonnative bioproducts under different growth conditions allowed the discrimination of the particular phenotypes that can be attributed to the individual mutants and singled out Cra and ArcA as the regulators with the most important effects on bacterial metabolism. These data were used to identify the most suitable backgrounds for the synthesis of the reduced bioproducts succinate and 1,3-propanediol (1,3-PDO). The Δ cra mutant was further modified to enhance succinate synthesis by the addition of enzymes that increase NADH and CO 2 availability, achieving an 80% increase compared to the parental strain. Production of 1,3-PDO in the Δ arcA mutant was optimized by overexpression of PhaP, which increased more than twice the amount of the diol compared to the wild type in a semidefined medium using glycerol, resulting in 24 g · liter -1 of 1,3-PDO after 48 h, with a volumetric productivity of 0.5 g · liter -1 h -1 IMPORTANCE Although the effects of many global regulators, especially ArcA and Cra, have been studied in Escherichia coli , the metabolic changes caused by the absence of global regulators have been observed to differ between strains. This scenario complicates the identification of the individual effects of the regulators, which is essential for the design of metabolic engineering strategies. The genome of Escherichia coli BW25113 has been completely sequenced and does not contain additional mutations that could mask or interfere with the effects of the global regulator mutations. The uniform genetic background of the Keio collection mutants enabled the characterization of the physiological consequences of altered carbon and redox fluxes caused by each global regulator deletion, eliminating possible strain-dependent results. As a proof of concept, Δ cra and Δ arcA mutants were subjected to further manipulations to obtain large amounts of succinate and 1,3-PDO, demonstrating that the metabolic backgrounds of the mutants were suitable for the synthesis of bioproducts., (Copyright © 2018 American Society for Microbiology.)

Aims: The aim of this work is to analyse the effect of pH, fungal identity and P chemical nature on microbial development and phosphatase release, discussing solubilization and mineralization processes in P cycling., Methods and Results: P solubilizing fungi (Talaromyces flavus, T. helicus L, T. helicus N, T. diversus and Penicillium purpurogenum) were grown under three pH conditions (6, 6·5 and 8·5) and with different inorganic (calcium, iron, aluminium and rock) and organic (lecithin and phytate) P sources. P solubilization, mineralization, growth and phosphatase production were recorded. Acid and neutral environments maximized fungal development and P recycling. P chemical nature changed the phosphatases release pattern depending on the fungal identity. Acid phosphatase activity was higher than alkaline phosphatases, regardless of pH or sample times. Alkaline phosphatases were affected by a combination of those factors., Conclusions: P chemical nature and pH modify fungal growth, P mineralization and solubilization processes. The underlying fungal identity-dependent metabolism governs the capacity and efficiency of P solubilization and mineralization. P solubilization and mineralization processes are interrelated and simultaneously present in soil fungi., Significance and Impact of the Study: This study constitutes a reference work to improve the selection of fungal bioinoculants in different environmental conditions, highlighting their role in P cycling., (© 2017 The Society for Applied Microbiology.)

Human activity has been altering many ecological cycles for decades, disturbing the natural mechanisms which are responsible for re-establishing the normal environmental balances. Probably, the most disrupted of these cycles is the cycle of carbon. In this context, many technologies have been developed for an efficient CO 2 removal from the atmosphere. Once captured, it could be stored in large geological formations and other reservoirs like oceans. This strategy could present some environmental and economic problems. Alternately, CO 2 can be transformed into carbonates or different added-value products, such as biofuels and bioplastics, recycling CO 2 from fossil fuel. Currently different methods are being studied in this field. We classified them into biological, inorganic and hybrid systems for CO 2 transformation. To be environmentally compatible, they should be powered by renewable energy sources. Although hybrid systems are still incipient technologies, they have made great advances in the recent years. In this scenario, biotechnology is the spearhead of ambitious strategies to capture CO 2 and reduce global warming., (© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

The aim of this work was to study phosphate (P) solubilization (and the processes involved in this event) by Talaromyces flavus (BAFC 3125) as a function of carbon and/or nitrogen sources. P solubilization was evaluated in NBRIP media supplemented with different carbon (glucose, sorbitol, sucrose, and fructose) and nitrogen (L-asparagine, urea, ammonium sulfate (AS), and ammonium nitrate (AN) combinations. The highest P solubilization was related to the highest organic acid production (especially gluconic acid) and pH drop for those treatments where glucose was present. Also P solubilization was higher when an inorganic nitrogen source was supplemented to the media when compared to an organic one. Although not being present an organic P source, phosphatase activity was observed. This shows that P mineralization and P solubilization can occur simultaneously, and that P mineralization is not induced by the enzyme substrate. The combination that showed highest P solubilization was for AN-glucose. The highest acid phosphatase activity was for AS-fructose, while for alkaline phosphatase were for AS-fructose and AN-fructose. Acid phosphatase activity was higher than alkaline. P solubilization and phosphatase activity (acid and alkaline) were influenced by the different carbon-nitrogen combinations. A better understanding of phosphate-solubilizing fungi could bring a better use of soil P.

The CreBC (carbon source-responsive) two-component regulation system of Escherichia coli affects a number of functions, including intermediary carbon catabolism. The impacts of different creC mutations (a ΔcreC mutant and a mutant carrying the constitutive creC510 allele) on bacterial physiology were analyzed in glucose cultures under three oxygen availability conditions. Differences in the amounts of extracellular metabolites produced were observed in the null mutant compared to the wild-type strain and the mutant carrying creC510 and shown to be affected by oxygen availability. The ΔcreC strain secreted more formate, succinate, and acetate but less lactate under low aeration. These metabolic changes were associated with differences in AckA and LdhA activities, both of which were affected by CreC. Measurement of the NAD(P)H/NAD(P)(+) ratios showed that the creC510 strain had a more reduced intracellular redox state, while the opposite was observed for the ΔcreC mutant, particularly under intermediate oxygen availability conditions, indicating that CreC affects redox balance. The null mutant formed more succinate than the wild-type strain under both low aeration and no aeration. Overexpression of the genes encoding phosphoenolpyruvate carboxylase from E. coli and a NADH-forming formate dehydrogenase from Candida boidinii in the ΔcreC mutant further increased the yield of succinate on glucose. Interestingly, the elimination of ackA and adhE did not significantly improve the production of succinate. The diverse metabolic effects of this regulator on the central biochemical network of E. coli make it a good candidate for metabolic-engineering manipulations to enhance the formation of bioproducts, such as succinate., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Bats can be monoestrous or polyestrous, and seasonal or non-seasonal in their reproductive patterns. The strategy adopted by each species or population depends on the regional climate. The objective this study was to analyze reproductive data of Sturnira lilium from long-term sampling carried out in several sites in Rio de Janeiro states, southeastern Brazil. We carried out sampling in 42 sites (with altitudes ranging from sea level to 1300 m a.s.l.) from May 1989 to December 2011. In total, we obtained 2602 captures of S. lilium: 1242 captures of adult females, 1225 captures of adult males, and 136 captures of subadults. The sex ratio was 0.99 males: 1 female. The reproductive season varied from eight to twelve months a year, and it was not related to the total accumulated rainfall. Sturnira lilium have continuously polyestrous reproduction with postpartum estrus and pregnant females can be observed in all months except July. In the present study, the highest proportions of pregnant females were observed in the months with the highest rainfall.

Digestive proteases of the digestive tract of the apple snail Pomacea canaliculata were studied. Luminal protease activity was found in the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Several protease bands and their apparent molecular weights were identified in both tissue extracts and luminal contents by gel zymography: (1) a 125 kDa protease in salivary gland extracts and in the crop content; (2) a 30 kDa protease throughout all studied luminal contents and in extracts of the midgut gland and of the endosymbionts isolated from this gland; (3) two proteases of 145 and 198 kDa in the coiled gut content. All these proteases were inhibited by aprotinin, a serine-protease inhibitor, and showed maximum activity between 30°C and 35°C and pH between 8.5 and 9.5. Tissue L-alanine-N-aminopeptidase activity was determined in the wall of the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Our findings show that protein digestion in P. canaliculata is carried out through a battery of diverse proteases originated from the salivary glands and the endosymbionts lodged in the midgut gland and by proteases of uncertain origin that occur in the coiled gut lumen.

Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative aerobe Escherichia coli, the microbial cell factory par excellence, has elaborate sensing and signal transduction mechanisms that respond to the availability of electron acceptors and alternative carbon sources in the surrounding environment. In particular, the ArcBA and CreBC two-component signal transduction systems are largely responsible for the metabolic regulation of redox control in response to O2 availability and carbon source utilization, respectively. Significant advances in the understanding of the biochemical, genetic, and physiological duties of these regulatory systems have been achieved in recent years. This situation allowed to rationally-design novel engineering approaches that ensure optimal carbon and energy flows within central metabolism, as well as to manipulate redox homeostasis, in order to optimize the production of industrially-relevant metabolites. In particular, metabolic flux analysis provided new clues to understand the metabolic regulation mediated by the ArcBA and CreBC systems. Genetic manipulation of these regulators proved useful for designing microbial cells factories tailored for the synthesis of reduced biochemicals with added value, such as poly(3-hydroxybutyrate), under conditions with restricted O2 supply. This network-wide strategy is in contrast with traditional metabolic engineering approaches, that entail direct modification of the pathway(s) at stake, and opens new avenues for the targeted modulation of central catabolic pathways at the transcriptional level.

Pigments present in the brown-greenish C morph of an intracellular endosymbiont of Pomacea canaliculata were investigated. Acetone extracts of the endosymbiotic corpuscles showed an absorption spectrum similar to that of chlorophylls. Three fractions obtained from silica gel column chromatography of the acetone extracts (C(I), C(II), and C(III)), were studied by positive ion fast atom bombardment-mass spectrometry (FAB-MS) and hydrogen-nuclear magnetic resonance (H-NMR). Results indicated the presence of (1) a sterol in the yellow colored C(I) fraction; (2) a mixture ofpheophorbides a and b in the major green fraction, C(II); and (3) a modified pheophorbide a in the smaller green fraction, C(III). Aqueous extracts of the C endosymbiont did not show evidence of the occurrence of C-phycocyanin, allophycocyanin or phycoerithrin (light absorption, fluorescence emission, and electrophoresis of the protein moieties) while cyanobacterial cells (Nostoc sp.) showed evidence of C-phycocyanin and allophycocyanin. The possible phylogenetic and functional significance of the pigments present in the C endosymbiont is discussed.

Aims: To study phosphate solubilization in Penicillium purpurogenum as function of medium pH, and carbon and nitrogen concentrations., Methods and Results: Tricalcium phosphate (CP) solubilization efficiency of P. purpurogenum was evaluated at acid or alkaline pH using different C and N sources. Glucose- and (NH(4) )(2) SO(4) -based media showed the highest P solubilization values followed by fructose. P. purpurogenum solubilizing ability was higher in cultures grown at pH 6·5 than cultures at pH 8·5. Organic acids were detected in both alkaline and neutral media, but the relative percentages of each organic acid differed. Highest P release coincided with the highest organic acids production peak, especially gluconic acid. When P. purpurogenum grew in alkaline media, the nature and concentration of organic acids changed at different N and C concentrations. A factorial categorical experimental design showed that the highest P-solubilizing activity, coinciding with the highest organic acid production, corresponded to the highest C concentration and lowest N concentration., Conclusions: The results described in the present study show that medium pH and carbon and nitrogen concentrations modulate the P solubilization efficiency of P. purpurogenum through the production of organic acids and particularly that of gluconic acid. In the P solubilization optimization studies, glucose and (NH(4) )(2) SO(4) as C and N sources allowed a higher solubilization efficiency at high pH., Significance and Impact of the Study: This organism is a potentially proficient soil inoculant, especially in P-poor alkaline soils where other P solubilizers fail to release soluble P. Further work is necessary to elucidate whether these results can be extrapolated to natural soil ecosystems, where different pH values are present. Penicillium purpurogenum could be used to develop a bioprocess for the manufacture of phosphatic fertilizer with phosphate calcium minerals., (© 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.)

The effect of eliminating D-lactate synthesis in poly(3-hydroxybutyrate) (PHB)-accumulating recombinant Escherichia coli (K24K) was analyzed using glycerol as a substrate. K24KL, an ldhA derivative, produced more biomass and had altered carbon partitioning among the metabolic products, probably due to the increased availability of carbon precursors and reducing power. This resulted in a significant increase of PHB and ethanol synthesis and a decrease in acetate production. Cofactor measurements revealed that cultures of K24K and K24KL had a high intracellular NADPH content and that the NADPH/NADP(+) ratio was higher than the NADH/NAD(+) ratio. The ldhA mutation affected cofactor distribution, resulting in a more reduced intracellular state, mainly due to a further increase in NADPH/NADP(+). In 60-h fed-batch cultures, K24KL reached 41.9 g·liter⁻¹ biomass and accumulated PHB up to 63% ± 1% (wt/wt), with a PHB yield on glycerol of 0.41 ± 0.03 g·g⁻¹, the highest reported using this substrate.