Single Carbon Source Research Articles

Thermococcus thermotolerans sp. nov., a hyperthermophilic archaeon isolated from a chimney in the Southwest Indian Ocean.

An anaerobic hyperthermophilic archaeon was isolated from a black smoker chimney with a snail attachment at a water depth of 2 739 m in the Southwest Indian Ocean. The sample was taken from the chimney exterior wall. The enrichment was conducted under a continuous culture with temperature fluctuation of 80-130 °C over 24 h for 42 days at 28 MPa. The isolation was performed at 90 °C at 0.1 MPa. Cells of the isolated strain 813A4T were irregular cocci. Strain 813A4T grew at 60-94 °C (optimal growth at 85 °C) at 0.1 MPa, and growth was detected at up to 99 °C at 28 MPa. At 85 °C, the strain was able to grow at pressures ranging from 0.1 to 110 MPa (optimal pressure, 0.1-40 MPa). At 85 °C, the cells of 813A4T grew at pH 5.5-9 (optimal, pH 7.0) and a NaCl concentration of 1.0-4.0 % (w/v; optimum concentration, 2.5 % NaCl). Strain 813A4T utilized yeast extract, tryptone and peptone as single carbon sources for growth. Elemental sulphur stimulated its growth. The G+C content of the complete genome was 53.48 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 813A4T belonged to the genus Thermococcus, with the highest sequence similarity to Thermococcus barossii SHCK-94T (99.73 %). The average nucleotide identity between strains 813A4T and SHCK-94T was 82.56 %. All these data indicated that strain 813A4T should be classified as representing a novel species of the genus Thermococcus, for which Thermococcus thermotolerans sp. nov. is proposed. The type strain is 813A4T (=JCM 39367T=MCCC M28628T).

The Halophilic Bacterium Paracoccus haeundaensis for the Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) from Single Carbon Sources.

The study objective was to evaluate the potential production of polyhydroxyalkanoates (PHAs), a biodegradable plastic material, by Paracoccus haeundaensis for which PHA production has never been reported. To identify the most effective nitrogen-limited culture conditions for PHAs production from this bacterium, batch fermentation using glucose concentrations ranging from 4 g l-1 to 20 g l-1 with a fixed ammonium concentration of 0.5 g l-1 was carried out at 30°C and pH 8.0. A glucose supplement of 12 g l-1 produced the highest PHA concentration (1.6 g l-1) and PHA content (0.63 g g-1) thereby identifying the optimal condition for PHA production from this bacterium. Gas chromatography-mass spectrometry analysis suggests that P. haeundaensis mostly produced copolymer types of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from glucose concentrations at 12 g l-1 or higher under the nitrogen-limited conditions. When several other single carbon sources were evaluated for the most efficient PHA production, fructose provided the highest biomass (2.8 g l-1), and PHAs (1.29 g l-1) concentrations. Results indicated that this bacterium mostly produced the copolymers P(3HB-co-3HV) from single carbon sources composing a range of 93-98% of 3-hydroxybutyrate and 2-7% of 3-hydroxyvalerate, whereas mannose-supplemented conditions produced the only homopolymer type of P(3HB). However, when propionic acid as a secondary carbon source were supplemented into the media, P. haeundaensis produced the copolymer P(3HB-co-3HV), composed of a 50% maximum monomeric unit of 3-hydroxyvaleric acid (3HV). However, as the concentration of propionic acid increased, cell biomass and PHAs concentrations substantially decreased due to cell toxicity.

In Vitro Evaluation of Intestinal Transport and High-Density Fermentation of Lactobacillus acidophilus.

Lactobacillus acidophilus strains have limiting factors such as low cell density and complex nutritional requirements in industrial production, which greatly restricts their industrial application. In this study, fermentation conditions for L. acidophilus were optimized and transcriptomic analysis used to understand growth mechanisms under high-density fermentation conditions. We found that L. acidophilus IMAU81186 has strong tolerance to gastrointestinal juice. In addition, its optimal culture conditions were 3% inoculum (v/v); culture temperature 37 °C; initial pH 6.5; and medium composition of 30.18 g/L glucose, 37.35 g/L soybean peptone, 18.68 g/L fish peptone, 2.46 g/L sodium citrate, 6.125 g/L sodium acetate, 2.46 g/L K2HPO4, 0.4 g/L MgSO4·7H2O,0.04 g/L MnSO4·5H2O, 0.01 g/L serine, and 0.3 g/L uracil. After optimization, viable counts of IMAU81186 increased by 7.03 times. Differentially expressed genes in IMAU81186 were analyzed at different growth stages using transcriptomics. We found that a single carbon source had limitations in improving the biomass of the strain, and terP and bfrA were significantly down-regulated in the logarithmic growth period, which may be due to the lack of extracellular sucrose. After optimizing the carbon source, we found that adding 12 g/L sucrose to the culture medium significantly increased cell density.

Biotransformation potential of the R-(+)-limonene by endophytic fungi strain associated with Amazonian fruit Malpighia emarginata DC

This study aims to offer an account of the biotransformation of limonene through the endophytic fungi strains associated with the Amazonian fruit Malpighia emarginata. The fungal biotransformation was executed in flasks containing the substrate R-(+)-limonene in a mineral medium, which was further incubated at 28 °C on a rotary shaker (120 rpm) for 120 h. Samples of each culture were taken every 24 hours, extracted with ethyl acetate, and analyzed using gas chromatography–mass spectrometry with the National Institute of Standards and Technology database. In this study with five endophytic fungi strains, only Penicillium sp. AF- LAB4 was considered a potential biocatalyst found in the screening due to its capacity to use the R-(+)-limonene as the single carbon and energy source in a mineral medium since they eventually accumulated interesting compound as limonene-1,2-diol (128.10 μL/L) after 48 to 120 h of reaction. This achievement is important and supports the development of the production of natural aromas and demonstrates the potential of using this endophytic fungus strain from the fruit M. emarginata, as a new biocatalyst.

Rational design of an acidic erythritol (ACER) medium for the enhanced isolation of the environmental pathogen Burkholderia pseudomallei from soil samples.

The soil bacterium Burkholderia pseudomallei causes melioidosis, a potentially fatal and greatly underdiagnosed tropical disease. Detection of B. pseudomallei in the environment is important to trace the source of infections, define risk areas for melioidosis and increase the clinical awareness. Although B. pseudomallei polymerase chain reaction (PCR)-based environmental detection provides important information, the culture of the pathogen remains essential but is still a methodological challenge. B. pseudomallei can catabolize erythritol, a metabolic pathway, which is otherwise rarely encountered among bacteria. We recently demonstrated that replacing threonine with erythritol as a single carbon source in the pH-neutral threonine-basal salt solution (TBSS-C50) historically used improved the isolation of B. pseudomallei from rice paddy soils. However, further culture medium parameters for an optimized recovery of B. pseudomallei strains from soils are still ill-defined. We, therefore, aimed to design a new erythritol-based medium by systematically optimizing parameters such as pH, buffer capacity, salt and nutrient composition. A key finding of our study is the enhanced erythritol-based growth of B. pseudomallei under acidic medium conditions. Our experiments with B. pseudomallei strains from different geographical origin led to the development of a phosphate-buffered acidic erythritol (ACER) medium with a pH of 6.3, higher erythritol concentration of 1.2%, supplemented vitamins and nitrate. This highly selective medium composition shortened the lag phase of B. pseudomallei cultures and greatly increased growth densities compared to TBSS-C50 and TBSS-C50-based erythritol medium. The ACER medium led to the highest enrichments of B. pseudomallei as determined from culture supernatants by quantitative PCR in a comparative validation with soil samples from the central part of Vietnam. Consequently, the median recovery of B. pseudomallei colony forming units on Ashdown's agar from ACER subcultures was 5.4 times higher compared to TBSS-C50-based erythritol medium (p = 0.005) and 30.7 times higher than TBSS-C50 (p < 0.001). In conclusion, our newly developed ACER medium significantly improves the isolation of viable B. pseudomallei from soils and, thereby, has the potential to reduce the rate of false-negative environmental cultures in melioidosis risk areas.

Designing glucose utilization "highway" for recombinant biosynthesis

cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRPmu9 mutant conferred both faster cell growth and a 133-fold improvement in expression level of lac promoter in presence of 2% glucose, compared with strain under regulation of CRPwild-type. Promoters free from “glucose repression” are advantageous for recombinant expression, as glucose is a frequently used inexpensive carbon source in high-cell-density fermentations. Transcriptome analysis demonstrated that the CRP mutant globally rewired cell metabolism, displaying elevated tricarboxylic acid cycle activity; reduced acetate formation; increased nucleotide biosynthesis; and improved ATP synthesis, tolerance, and stress-resistance activity. Metabolites analysis confirmed the enhancement of glucose utilization with the upregulation of glycolysis and glyoxylate-tricarboxylic acid cycle. As expected, an elevated biosynthetic capability was demonstrated with vanillin, naringenin and caffeic acid biosynthesis in strains regulated by CRPmu9. This study has expanded the significance of CRP optimization into glucose utilization and recombinant biosynthesis, beyond the conventionally designated carbon source utilization other than glucose. The Escherichiacoli cell regulated by CRPmu9 can be potentially used as a beneficial chassis for recombinant biosynthesis.

Effect of frequency on low temperature synthesis of diamond by pulsed discharge MPCVD

Low-temperature synthesis of diamond has broader application prospects compared to traditional diamond synthesis, but it faces issues such as low deposition rate and poor diamond quality. In this study, CH4–CO–H2 were used as reaction gases, and a pulsed discharge MPCVD apparatus was used to deposit diamond films at a substrate temperature of 400 °C and different frequencies were investigated. The results showed that the deposition rate of the pulse discharge was faster than that of continuous microwave discharge. The fastest deposition rate was observed at 500Hz, and crystal size became finer with increasing frequency, while the crystal orientation changed from (100) to (111). A diamond peak at 1333 cm−1 was observed in the Raman spectrum, and the diamond grain size was found to be biggest at 750Hz based on the measured half-width of the peak. Plasma OES analysis revealed that the brightness of CO increased gradually with increasing frequency, while the brightness of H showed an initial increase followed by a decrease. Comparison of emission spectra during low-temperature deposition with a single carbon source showed that CO participated in the reaction for low-temperature diamond synthesis.

Enhancing Lipid Production of Chlorella sp. by Mixotrophic Cultivation Optimization

Mixotrophic microalgal cultivation can utilize CO2 and organic carbon sources. This study optimized the cultivation nitrogen source (peptone, urea, yeast extract, NH4Cl, (NH4)2SO4, NH4NO3, NaNO3 and KNO3), carbon combination (glucose, glycerol, sucrose), (NH4)2SO4 nitrogen source and pH (6–11) in a local microalgae species with three characteristics (high pH-resistant, high growth rate and high lipid content). Chlorella sp. G3H3-1-2 biomass production and lipid accumulation were estimated using the fatty acid methyl esters (FAMEs) concentration. The Chlorella sp. G3H3-1-2 FAME content was strongly influenced by the carbon, nitrogen sources and pH variations. The pH ranged from 6.0 to 8.0, which produced the highest specific growth rate of 1.22 day−1 for Chlorella sp. G3H3-1-2 while using glucose as the single carbon source. However, the highest total FAMEs content of 59% in the Chlorella sp. G3H3-1-2 biomass of 1.75 g/L was obtained while using the combination of 1 g-glucose/L as the carbon source and 0.2 g-(NH4)2SO4/L as the nitrogen source at the high pH value of 10.

Comparative Characterization of Virulent and Less-Virulent Lasiodiplodia theobromae Isolates.

Lasiodiplodia theobromae attacks over 500 plant species and is an important pathogen of tropical and subtropical fruit. Due to global warming and climate change, the incidence of disease associated with L. theobromae is rising. Virulence tests performed on avocado and mango branches and fruit showed a large diversity of virulence of different L. theobromae isolates. Genome sequencing was performed for two L. theobromae isolates, representing more virulent (Avo62) and less-virulent (Man7) strains, to determine the cause of their variation. Comparative genomics, including orthologous and single-nucleotide polymorphism (SNP) analyses, identified SNPs in the less-virulent strain in genes related to secreted cell wall-degrading enzymes, stress, transporters, sucrose, and proline metabolism, genes in secondary metabolic clusters, effectors, genes involved in the cell cycle, and genes belonging to transcription factors that may contribute to the virulence of L. theobromae. Moreover, carbohydrate-active enzyme analysis revealed a minor increase in gene counts of cutinases and pectinases and the absence of a few glycoside hydrolases in the less-virulent isolate. Changes in gene-copy numbers might explain the morphological differences found in the in-vitro experiments. The more virulent Avo62 grew faster on glucose, sucrose, or starch as a single carbon source. It also grew faster under stress conditions, such as osmotic stress, alkaline pH, and relatively high temperature. Furthermore, the more virulent isolate secreted more ammonia than the less-virulent one both in vitro and in vivo. These study results describe genome-based variability related to L. theobromae virulence, which might prove useful for the mitigation of postharvest stem-end rot. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Lipid Accumulation by Snf-β Engineered Mucor circinelloides Strains on Glucose and Xylose.

Sucrose non-fermenting 1 (SNF1) protein kinase plays the regulatory roles in the utilization of selective carbon sources and lipid metabolism. Previously, the role of β subunit of SNF1 in lipid accumulation was evaluated by overexpression and knockout of Snf-β in oleaginous fungus M. circinelloides. In the present study, the growth and lipid accumulation of Snf-β overexpression and knockout strains were further analyzed and compared with glucose or xylose as a single or mixed carbon sources. The results showed that the lipid contents in Snf-β knockout strain improved by 23.2% (for glucose), 28.4% (for xylose), and 30.5% (for mixed glucose and xylose) compared with that of the control strain, respectively. The deletion of Snf-β subunit also altered the transcriptional level of acetyl-CoA carboxylase (ACC). The highest transcriptional levels of ACC1 in Snf-β knockout strain at 24h were increased by 2.4-fold (for glucose), 2.8-fold (for xylose), and 3.1-fold (for mixed glucose and xylose) compared with that of the control strain, respectively. Our results indicated that Snf-β subunit enhanced lipid accumulation through the regulation of ACC1 in response to xylose or mixed sugars of glucose and xylose more significantly than that of response to glucose. This is the first study to explore the effect of Snf-β subunit of M. circinelloides in regulating lipid accumulation responding to different carbon nutrient signals of glucose and xylose. This study provides a foundation for the future application of the Snf-β engineered strains in lipid production from lignocellulose.

Slow‐release fertilizers using lignin: Challenges and future prospects

AbstractLignin is an integral part of the plant cell wall and is the largest single source of aromatic carbon in the world, which makes it suitable for the production of feed, chemicals, fuels, and biomaterials. Total lignin production is greater than 50 million tons per annum; however, currently, less than 5% of the lignin is being valorized, and 95% is burnt to create steam and power. Burning aromatic carbons produces heat and power, making them low‐value materials. However, the macromolecules of lignin can be modified to use for several industrial applications, such as surfactants, paints, hydrogel, absorbents, and fertilizer. In this review, we provide a systematic and comprehensive summary of the last 10 years of the development of controlled release fertilizer (CRF) from lignin, which is a renewable material. Lignin‐based CRFs are green, environment friendly, and are considered efficient for agricultural use in place of urea and diammonium phosphate. Coating the fertilizer with lignin could increase fertilizer's 20–30 times slower release vis‐à‐vis uncoated fertilizer. However, the structure of lignin varies from one process to another process; it is therefore highly dependent upon the fractionation process and pulping methodologies adopted. A high degree of heterogeneity, complex structure, and low reactivity of the lignin results in an uneven coating, large surface porosity, and cracks in the coatings. The similarity of the lignin can be improved by chemical modifications in its molecule; however, any chemical processing increases the cost of slow‐release fertilizer. © 2023 Society of Industrial Chemistry and John Wiley &amp; Sons Ltd.

Method of selective isolation of &lt;i&gt;Acinetobacter baylyi&lt;/i&gt; from river water

The aim of the study is to increase the selectivity of isolating Acinetobacter baylyi from river water. For developing a selective culture medium, we used 5 strains of A. baylyi, 4 of which were isolated from the water of the river Neva, 1 from clinical material. A. baylyi strains were identified by a combination of phenotypic features and/or using the mass spectrometric method (MALDI-ToF MS). In preliminary studies we found that the bacteria A. baylyi utilizes no L-phenylalanine as a single source of carbon and nitrogen, as well as the only source of carbon in a medium with mineral nitrogen sources, but utilizes L-phenylalanine as the only source of nitrogen only if it is supplemented with ethanol as the only source of carbon. Based on this A. baylyi property, a selective medium was developed for the isolation of these bacteria from river water. Composition and preparation of liquid selective medium were as follows: 1 liter of distilled water added with L-phenylalanine (CAS 63-91-2) 2.0 (g/l); NaCI 5.0; Na2SO4 2.0; KH2PO4 1.0; K2HPO4 2.5; MgSO4 0.1; dissolved while heating, boiled for 5 minutes, 4 ml of 96-degree ethanol was added to a warm medium, pH 7.20.2 assessed; 40 ml were poured into sterile vials. Dense selective medium: the same ingredients and extra 15.0 g of agar are added to 1 liter of distilled water; with similar preparation technique, the medium is poured into sterile Petri dishes. Method of application of selective nutrient medium for isolation of A. baylyi from river water: water from the river Neva is seeded in a volume of 10 ml into a vial with 40 ml of liquid selective medium, incubated in aerobic conditions at 28С for 48 h, then the enriched material from the flask is placed with a bacteriological loop onto the surface of same dense selective medium in two flasks; incubated in aerobic conditions at 28С for 48 h. 10 isolated colonies are selected for subsequent identification, they are transplanted into sectors of nutrient agar, incubated at 28С 24 h and isolates are identified by a complex of phenotypic characteristic of A. baylyi, and/or by the MALDI-ToF MS method. In this case, 19 isolates from 20 were identified as A. baylyi (95% of isolates). A study using this technique of three more water samples from the river Neva showed the results of the same narrowly selective orientation 9095% of the isolates were bacteria A. baylyi.

Screening potential polyhydroxyalkanoate-producing bacteria from wastewater sludge.

We applied fluorescence staining of Nile red, polymerase chain reaction (PCR), and carbon substrate utilization and pressure tolerance analysis to execute three-stage screening for potential polyhydroxyalkanoate (PHA) producers in the sludge samples of 21 large-scale wastewater treatment plants of city and industrial parks in Taiwan area. Total 35,429 colonies were grown on 196 plates, the screened 30 strains were subjected to 16S rRNA analysis, and 18 identified genera belonged to Proteobacteria (67%), Firmicutes (17%), and Actinomycetota (16%). The PHA accumulation results revealed that nine genera (50% of 18 screened) produced PHAs by limiting the nitrogen source and excess single carbon sources of glucose in an aerobic status. The PHA accumulation percentage was 1.44-58.77% at dry cell weight, and the theoretical yield from glucose was 0.52-58.76%. Our results indicate that our triple-screening method is promising for identifying a high biodiversity of PHA-accumulating bacteria from activated sludge for future industrial applications.

Optimizing mixotrophic cultivation of oil-rich Tribonema minus using volatile fatty acids and glycerin: A promising approach for pH-controlling and enhancing lipid productivity

Volatile Fatty Acids (VFAs) have become a subject of significant interest recently, primarily because they can serve as a carbon source for microorganisms that produce bioactive compounds, biodegradable materials, and energy. VFAs can be produced from various waste streams via anaerobic fermentation (AF) in a cost-effective manner, providing a potential avenue for producing biofuels and biochemicals while reducing waste volume. In this study, different VFAs and glycerin individually or in combination were used as the sole carbon source in the mixotrophic cultivation of oil-rich filamentous Tribonema minus. Using different VFAs or glycerin individually caused pH fluctuation in the culture, which led to the accumulation of free ammonia, which produced protons, acidifying the environment and inhibiting microalgae growth. To minimize pH fluctuation, different proportions of organic carbon sources were combined. Under combined organic carbon sources, the growth and photosynthetic performance of T. minus were all higher than those of the single carbon source groups. When Cacetic acid:Cbutyric acid:Cglycerin was set to 0.5:0.5:1, pH in the culture remained close to neutrality, and the removal of NH4+-N and carbon reached the maximum of 81.17 and 97.79%, respectively. The microalgal biomass and lipid productivity reached the highest of 223.80 and 108.16 mg L−1 d−1, respectively. Under the optimized carbon source condition, the fatty acids and biodiesel-related qualities of T. minus were improved. This study provided a reference for the precise design of carbon sources to maximize the resource utilization of butyric acid-type AF effluent by oil-rich T. minus. These conditions also can be achieved on a large scale by adding glycerin wastes to butyric acid-type AF effluent, mainly composed of acetic and butyric acids to get the recommended ratio for microalgae cultivation.

Coupled mixotrophic denitrification and utilization of refractory organics driven by Mn redox circulation for significantly enhanced nitrogen removal

Coupled mixotrophic denitrification and degradation of organics driven by redox transition of Mn for nitrogen removal has attracted much attention. Herein, this study explored the removal performance and mechanisms for nitrogen and refractory organics from secondary effluent in up-flow MnOx biofilter. Results showed that the removal of organics and nitrate was significantly enhanced by the synergistic process of heterotrophic denitrification and Mn(II)-driven autotrophic denitrification (MnAD), which were originated from the facilitation of Mn circulation. But nitrate removal was closely related to the types of carbon source and Mn(II) concentration. Single small molecular carbon source (glucose) performed better than mixed carbon source (humic acid and glucose) in nitrate removal process (74.8% in stage 1–2 vs. 54.1% in stage 3–5). And raising external Mn(II) concentration increased the contribution of MnAD (60.2% in stage 5 vs. 46.5% in stage 3) to nitrate removal. Furthermore, the relationship between Mn/N transformation and microbial community structure shifts revealed that the redox transition between Mn(II) and Mn(IV) promoted the enrichment of denitrogenation bacteria and functional genes, thus contributing to pollutants removal. Our studies expand the knowledge of MnOx-mediated pollutants removal processes and support the potential application of MnOx for removal of residual refractory organics and nitrogen.

MRNA levels of tricarboxylic acid cycle genes in Streptomyces coelicolor M145 cultured on glucose.

Streptomyces strains degrade many complex organic compounds and produce secondary metabolites. In aerobic organisms such as Streptomyces species, the tricarboxylic acid (TCA) cycle represents an indispensable central carbon metabolic pathway for energy generation and metabolic intermediary replenishment. Although various precursors for antibiotic biosynthesis are derived from this cycle, relatively few studies have focused on determining how a single carbon source can impact this metabolic pathway at different growth phases. In this study, we identified chromosomal genes involved in the TCA cycle in Streptomyces coelicolor and determined their mRNA levels. We searched the genes involved in the TCA cycle in S. coelicolor through bioinformatic analysis. Growth, glucose concentration quantification and RNA isolation were made from cultures of S. coelicolor grown on minimal medium with glucose along 72h. mRNA levels of all identified genes were obtained by RT-qPCR. Five enzymes encoded by a single gene each were found, while for the rest at least two genes were found. The results showed that all the genes corresponding to the TCA enzymes were transcribed at very different levels and some of them displayed growth-phase dependent expression. All TCA cycle-associated genes, including paralog genes, were differentially transcribed in S. coelicolor grown in minimal medium with glucose as carbon source. Some of them, such as succinyl-CoA synthetase and succinate dehydrogenase, have low mRNA levels, which could limit the carbon flux through the TCA cycle. Our findings suggest that the genetic expansion of TCA cycle genes could confer to S. coelicolor the ability to adapt to diverse nutritional conditions and metabolic changes through different paralog genes expression.

Predicting the re‐distribution of antibiotic molecules caused by inter‐species interactions in microbial communities

Microbes associate in nature forming complex communities, but they are often studied in purified form. Here I show that neighbouring species enforce the re‐distribution of carbon and antimicrobial molecules, predictably changing drug efficacy with respect to standard laboratory assays. A simple mathematical model, validated experimentally using pairwise competition assays, suggests that differences in drug sensitivity between the competing species causes the re‐distribution of drug molecules without affecting carbon uptake. The re‐distribution of drug is even when species have similar drug sensitivity, reducing drug efficacy. But when their sensitivities differ the re‐distribution is uneven: The most sensitive species accumulates more drug molecules, increasing efficacy against it. Drug efficacy tests relying on samples with multiple species are considered unreliable and unpredictable, but study demonstrates that efficacy in these cases can be qualitatively predicted. It also suggests that living in communities can be beneficial even when all species compete for a single carbon source, as the relationship between cell density and drug required to inhibit their growth may be more complex than previously thought.

Genomic evidence for two pathways of formaldehyde oxidation and denitrification capabilities of the species Paracoccus methylovorus sp. nov.

Three strains (H4-D09T, S2-D11 and S9-F39) of a member of the genus Paracoccus attributed to a novel species were isolated from topsoil of temperate grasslands. The genome sequence of the type strain H4-D09T exhibited a complete set of genes required for denitrification as well as methylotrophy. The genome of H4-D09T included genes for two alternative pathways of formaldehyde oxidation. Besides the genes for the canonical glutathione (GSH)-dependent formaldehyde oxidation pathway, all genes for the tetrahydrofolate-formaldehyde oxidation pathway were identified. The strain has the potential to utilize methanol and/or methylamine as a single carbon source as evidenced by the presence of methanol dehydrogenase (mxaFI) and methylamine dehydrogenase (mau) genes. Apart from dissimilatory denitrification genes (narA, nirS, norBC and nosZ), genes for assimilatory nitrate (nasA) and nitrite reductases (nirBD) were also identified. The results of phylogenetic analysis based on 16S rRNA genes coupled with riboprinting revealed that all three strains represented the same species of genus Paracoccus. Core genome phylogeny of the type strain H4-D09T indicated that Paracoccus thiocyanatus and Paracoccus denitrificans are the closest phylogenetic neighbours. The average nucleotide index (ANI) and digital DNA-DNA hybridization (dDDH) with the closest phylogenetic neighbours revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. The major respiratory quinone is Q-10, and the predominant cellular fatty acids are C18 : 1ω7c, C19 : 0cyclo ω7c, and C16 : 0, which correspond to those detected in other members of the genus. The polar lipid profile consists of a diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), aminolipid (AL), glycolipid (GL) and an unidentified lipid (L).On the basis of our results, we concluded that the investigated isolates represent a novel species of the genus Paracoccus, for which the name Paracoccus methylovorus sp. nov. (type strain H4-D09T=LMG 31941T= DSM 111585T) is proposed.

Role of EmaSR in Ethanol Metabolism by Acinetobacter baumannii.

Acinetobacter baumannii is a well-known nosocomial pathogen that can survive in different environments through the use of intricate networks to regulate gene expression. Two-component systems (TCS) form an important part of such regulatory networks, and in this study, we describe the identification and characterization of a novel EmaSR TCS in A. baumannii. We constructed a Tn5-tagged mutagenesis library, from which an emaS sensor kinase gene and emaR response regulator gene were identified. We found that emaS/emaR single-mutants and double-mutants were unable to replicate in M9 medium with 1% ethanol as the single carbon source. Motility and biofilm formation were negatively affected in double-mutants, and transcriptomic analysis showed that mutation of emaSR dysregulated genes required for carbon metabolism. In addition, emaS/emaR single-mutants and double-mutants were unable to survive in acetic acid- and sodium acetate-containing medium, indicating that the EmaSR TCS is also important for acetate metabolism. Furthermore, virulence against Galleria mellonella was diminished in emaS/emaR single- and double-mutants. Taken together, these results show that this novel EmaSR TCS is involved in the regulation of A. baumannii ethanol metabolism and acetate metabolism, with important implications on motility, biofilm formation, and virulence if mutated. Further research on the underlying mechanisms is warranted.

Ppe51 Variants Enable Growth of Mycobacterium tuberculosis at Acidic pH by Selectively Promoting Glycerol Uptake.

In defined media supplemented with single carbon sources, Mycobacterium tuberculosis (Mtb) exhibits carbon source specific growth restriction. When supplied with glycerol as the sole carbon source at pH 5.7, Mtb establishes a metabolically active state of nonreplicating persistence known as acid growth arrest. We hypothesized that acid growth arrest on glycerol is not a metabolic restriction, but rather an adaptive response. To test this hypothesis, we selected for and identified several Mtb mutants that could grow under these restrictive conditions. All mutations were mapped to the ppe51 gene and resulted in variants with 3 different amino acid substitutions- S211R, E215K, and A228D. Expression of the ppe51 variants in Mtb promoted growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, Enhanced Acid Growth (EAG) phenotype. Testing growth on other single carbon sources showed the PPE51 variants specifically enhanced growth on glycerol, suggesting PPE51 plays a role in glycerol uptake. Using radiolabeled glycerol, enhanced glycerol uptake was observed in Mtb expressing the PPE51 (S211R) variant, with glycerol overaccumulation in triacylglycerol. Notably, the EAG phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication and reduced survival in activated macrophages compared to resting macrophages. Recombinant PPE51 protein exhibited differential thermostability in the wild type (WT) or S211R variants in the presence of glycerol, supporting the model that EAG substitutions alter PPE51-glycerol interactions. Together, these findings support that PPE51 variants selectively promote glycerol uptake and that slowed growth at acidic pH is an important adaptive mechanism required for macrophage pathogenesis. IMPORTANCE It is puzzling why Mycobacterium tuberculosis (Mtb) cannot grow on glycerol at acidic pH, as it has a carbon source and oxygen, everything it needs to grow. In this study, we found that Mtb limits uptake of glycerol at acidic pH to restrict its growth and that mutations in ppe51 promote uptake of glycerol at acidic pH and enable growth. That is, Mtb can grow well at acidic pH on glycerol, but has adapted instead to stop growth. Notably, ppe51 variants exhibit enhanced replication and reduced survival in activated macrophages, supporting a role for pH-dependent slowed growth during macrophage pathogenesis.