Soils and sediments host Thermoplasmata archaea encoding novel copper membrane monooxygenases (CuMMOs)

Copper membrane monooxygenases (CuMMOs) play critical roles in the global carbon and nitrogen cycles. Organisms harboring these enzymes perform the first, and rate limiting, step in aerobic oxidation of ammonia, methane, or other simple hydrocarbons. Within archaea, only organisms in the order Nitrososphaerales (Thaumarchaeota) encode CuMMOs, which function exclusively as ammonia monooxygenases. From grassland and hillslope soils and aquifer sediments, we identified 20 genomes from distinct archaeal species encoding divergent CuMMO sequences. These archaea are phylogenetically clustered in a previously unnamed Thermoplasmatota order, herein named the Ca. Angelarchaeales. The CuMMO proteins in Ca. Angelarchaeales are more similar in structure to those in Nitrososphaerales than those of bacteria, and contain all functional residues required for general monooxygenase activity. Ca. Angelarchaeales genomes are significantly enriched in blue copper proteins (BCPs) relative to sibling lineages, including plastocyanin-like electron carriers and divergent nitrite reductase-like (nirK) 2-domain cupredoxin proteins co-located with electron transport machinery. Ca. Angelarchaeales also encode significant capacity for peptide/amino acid uptake and degradation and share numerous electron transport mechanisms with the Nitrososphaerales. Ca. Angelarchaeales are detected at high relative abundance in some of the environments where their genomes originated from. While the exact substrate specificities of the novel CuMMOs identified here have yet to be determined, activity on ammonia is possible given their metabolic and ecological context. The identification of an archaeal CuMMO outside of the Nitrososphaerales significantly expands the known diversity of CuMMO enzymes in archaea and suggests previously unaccounted organisms contribute to critical global nitrogen and/or carbon cycling functions.

The Inhibition Effect of Linezolid With Reyanning Mixture on MRSA and its Biofilm is More Significant than That of Linezolid Alone

Methicillin-resistant Staphylococcus aureus (MRSA) is a superbacterium, and when it forms biofilms, it is difficult to treat even with the first-line of antibiotic linezolid (LNZ). Reyanning mixture (RYN), a compound-based Chinese medicine formula, has been found to have inhibitory effects on biofilms. This study aims to explore the synergistic inhibitory effect and corresponding mechanisms of their (LNZ&RYN) combination on the planktonic as well as biofilm cells of MRSA. Broth microdilution and chessboard methods were employed for the determination of minimum inhibitory concentrations (MICs) and synergistic concentration of LNZ&RYN, respectively. The effect of the combined medication on biofilm and mature biofilm of MRSA were observed by biofilm morphology and permeability experiments, respectively. To unveil the molecular mechanism of action of the synergistic combination of LNZ and RYN, RT-PCR based biofilm-related gene expression analysis and ultra-high pressure liquid chromatography-time-of-flight mass spectrometry based endogenous metabonomic analysis were deployed. The results indicated that 1/16RYN as the best combined dose reduced LNZ (4 μg/ml) to 2 μg/ml. The combined treatment inhibited living MRSA before and after biofilm formation, removed the residual structure of dead bacteria in MRSA biofilms and affected the shape and size of bacteria, resulting in the improvement of biofilm permeability. The mechanism was that biofilm-related genes such as agrC, atlA, and sarA, as well as amino acid uptake associated with the metabolism of 3-dehydrocarnitine, kynurenine, L-leucine, L-lysine and sebacic acid were inhibited. This study provides evidence for the treatment of MRSA and its biofilms with LNZ combined with RYN.

Genomic Features and Construction of Streamlined Genome Chassis of Nisin Z Producer Lactococcus lactis N8

Lactococcus lactis is a commonly used fermenting bacteria in cheese, beverages and meat products. Due to the lack of simplified chassis strains, it has not been widely used in the fields of synthetic biology. Thus, the construction of lactic acid bacteria chassis strains becomes more and more important. In this study, we performed whole genome sequencing, annotation and analysis of L. lactis N8. Based on the genome analysis, we found that L. lactis N8 contains two large plasmids, and the function prediction of the plasmids shows that some regions are related to carbohydrate transport/metabolism, multi-stress resistance and amino acid uptake. L. lactis N8 contains a total of seven prophage-related fragments and twelve genomic islands. A gene cluster encoding a hybrid NRPS–PKS system that was found in L. lactis N8 reveals that the strain has the potential to synthesize novel secondary metabolites. Furthermore, we have constructed a simplified genome chassis of L. lactis N8 and achieved the largest amount of deletion of L. lactis so far. Taken together, the present study offers further insights into the function and potential role of L. lactis N8 as a model strain of lactic acid bacteria and lays the foundation for its application in the field of synthetic biology.

Amino Acid Sensing and Assimilation by the Fungal Pathogen Candida albicans in the Human Host

Nutrient uptake is essential for cellular life and the capacity to perceive extracellular nutrients is critical for coordinating their uptake and metabolism. Commensal fungal pathogens, e.g., Candida albicans, have evolved in close association with human hosts and are well-adapted to using diverse nutrients found in discrete host niches. Human cells that cannot synthesize all amino acids require the uptake of the “essential amino acids” to remain viable. Consistently, high levels of amino acids circulate in the blood. Host proteins are rich sources of amino acids but their use depends on proteases to cleave them into smaller peptides and free amino acids. C. albicans responds to extracellular amino acids by pleiotropically enhancing their uptake and derive energy from their catabolism to power opportunistic virulent growth. Studies using Saccharomyces cerevisiae have established paradigms to understand metabolic processes in C. albicans; however, fundamental differences exist. The advent of CRISPR/Cas9-based methods facilitate genetic analysis in C. albicans, and state-of-the-art molecular biological techniques are being applied to directly examine growth requirements in vivo and in situ in infected hosts. The combination of divergent approaches can illuminate the biological roles of individual cellular components. Here we discuss recent findings regarding nutrient sensing with a focus on amino acid uptake and metabolism, processes that underlie the virulence of C. albicans.

Type II porous ionic liquid based on metal-organic cage that enables L-tryptophan identification

Porous liquids with chemical separation properties are quite well-studied in general, but there is only a handful of reports in the context of identification and separation of non-gaseous molecules. Herein, we report the first example of Type-II porous ionic liquid that exhibits exceptional selectivity towards L-tryptophan (L-Trp) over other aromatic amino acids and the first in which coordination cages promote this selectivity. A previously known class of anionic organic-inorganic hybrid doughnut-like cage (HD) has been readily dissolved in trihexyltetradecylphosphonium chloride (THTP_Cl). The resulting liquid, HD/THTP_Cl, is thereby composed of common components, facile to prepare, and exhibit room temperature fluidity. The permanent porosity are manifested by the high-pressure isotherm for CH4 and modeling studies. With evidence from time-dependent amino acid uptake, competitive extraction studies and molecular dynamic simulations, HD/THTP_Cl exhibit better selectivity towards L-Trp than existing solid state sorbents, and we attribute it to not only the intrinsic porosity of HD but also the host-guest interactions between HD and L-Trp. Specifically, each HD unit is binded with nearly 5 L-Trp molecules, which is higher than the L-Trp occupation in the structure unit of other benchmark metal-organic frameworks.

The Amino Acid-mTORC1 Pathway Mediates APEC TW-XM-Induced Inflammation in bEnd.3 Cells

The blood–brain barrier (BBB) is key to establishing and maintaining homeostasis in the central nervous system (CNS); meningitis bacterial infection can disrupt the integrity of BBB by inducing an inflammatory response. The changes in the cerebral uptake of amino acids may contribute to inflammatory response during infection and were accompanied by high expression of amino acid transporters leading to increased amino acid uptake. However, it is unclear whether amino acid uptake is changed and how to affect inflammatory responses in mouse brain microvascular endothelial (bEnd.3) cells in response to Avian Pathogenic Escherichia coli TW-XM (APEC XM) infection. Here, we firstly found that APEC XM infection could induce serine (Ser) and glutamate (Glu) transport from extracellular into intracellular in bEnd.3 cells. Meanwhile, we also shown that the expression sodium-dependent neutral amino acid transporter 2 (SNAT2) for Ser and excitatory amino acid transporter 4 (EAAT4) for Glu was also significantly elevated during infection. Then, in amino acid deficiency or supplementation medium, we found that Ser or Glu transport were involving in increasing SNAT2 or EAAT4 expression, mTORC1 (mechanistic target of rapamycin complex 1) activation and inflammation, respectively. Of note, Ser or Glu transport were inhibited after SNAT2 silencing or EAAT4 silencing, resulting in inhibition of mTORC1 pathway activation, and inflammation compared with the APEC XM infection group. Moreover, pEGFP-SNAT2 overexpression and pEGFP-EAAT4 overexpression in bEnd.3 cells all could promote amino acid uptake, activation of the mTORC1 pathway and inflammation during infection. We further found mTORC1 silencing could inhibit inflammation, the expression of SNAT2 and EAAT4, and amino acid uptake. Taken together, our results demonstrated that APEC TW-XM infection can induce Ser or Glu uptake depending on amino acid transporters transportation, and then activate amino acid-mTORC1 pathway to induce inflammation in bEnd.3 cells.

The tea plant CsLHT1 and CsLHT6 transporters take up amino acids, as a nitrogen source, from the soil of organic tea plantations

Organic tea is more popular than conventional tea that originates from fertilized plants. Amino acids inorganic soils constitute a substantial pool nitrogen (N) available for plants. However, the amino-acid contents in soils of tea plantations and how tea plants take up these amino acids remain largely unknown. In this study, we show that the amino-acid content in the soil of an organic tea plantation is significantly higher than that of a conventional tea plantation. Glutamate, alanine, valine, and leucine were the most abundant amino acids in the soil of this tea plantation. When 15N-glutamate was fed to tea plants, it was efficiently absorbed and significantly increased the contents of other amino acids in the roots. We cloned seven CsLHT genes encoding amino-acid transporters and found that the expression of CsLHT1, CsLHT2, and CsLHT6 in the roots significantly increased upon glutamate feeding. Moreover, the expression of CsLHT1 or CsLHT6 in a yeast amino-acid uptake-defective mutant, 22∆10α, enabled growth on media with amino acids constituting the sole N source. Amino-acid uptake assays indicated that CsLHT1 and CsLHT6 are H+-dependent high- and low-affinity amino-acid transporters, respectively. We further demonstrated that CsLHT1 and CsLHT6 are highly expressed in the roots and are localized to the plasma membrane. Moreover, overexpression of CsLHT1 and CsLHT6 in Arabidopsis significantly improved the uptake of exogenously supplied 15N-glutamate and 15N-glutamine. Taken together, our findings are consistent with the involvement of CsLHT1 and CsLHT6 in amino-acid uptake from the soil, which is particularly important for tea plants grown inorganic tea plantations.

Coordination of the Uptake and Metabolism of Amino Acids in Mycobacterium tuberculosis-Infected Macrophages

Macrophage polarization to the M1-like phenotype, which is critical for the pro-inflammatory and antimicrobial responses of macrophages against intracellular pathogens, is associated with metabolic reprogramming to the Warburg effect and a high output of NO from increased expression of NOS2. However, there is limited understanding about the uptake and metabolism of other amino acids during M1 polarization. Based on functional analysis of a group of upregulated transporters and enzymes involved in the uptake and/or metabolism of amino acids in Mycobacterium tuberculosis-infected macrophages, plus studies of immune cell activation, we postulate a coherent scheme for amino acid uptake and metabolism during macrophage polarization to the M1-like phenotype. We describe potential mechanisms that the increased arginine metabolism by NOS2 is metabolically coupled with system L transporters LAT1 and LAT2 for the uptake of neutral amino acids, including those that drive mTORC1 signaling toward the M1-like phenotype. We also discuss the underappreciated pleiotropic roles of glutamine metabolism in the metabolic reprogramming of M1-like macrophages. Collectively, our analyses argue that a coordinated amino acid uptake and metabolism constitutes an integral component of the broad metabolic scheme required for macrophage polarization to M1-like phenotype against M. tuberculosis infection. This idea could stimulate future experimental efforts to elucidate the metabolic map of macrophage activation for the development of anti-tuberculosis therapies.