The Capability of Utilizing Abiotic Enantiomers of Amino Acids by Halomonas sp. LMO_D1 Derived From the Mariana Trench

D-amino acids (D-AAs) have been produced both in organisms and in environments via biotic or abiotic processes. However, the existence of these organic materials and associated microbial degradation activity has not been previously investigated in subduction zones where tectonic activities result in the release of hydrothermal organic matter. Here, we isolated the bacterium Halomonas sp. LMO_D1 from a sample obtained from the Mariana trench, and we determined that this isolate utilized 13 different D-AAs (D-Ala, D-Glu, D-Asp, D-Ser, D-Leu, D-Val, D-Tyr, D-Gln, D-Asn, D-Pro, D-Arg, D-Phe, and D-Ile) in the laboratory and could grow on D-AAs under high hydrostatic pressure (HHP). Moreover, the metabolism of L-AAs was more severely impaired under HHP conditions compared with that of their enantiomers. The essential function gene (Chr_2344) required for D-AA catabolism in strain LMO_D1 was identified and confirmed according to the fosmid library method used on the D-AAs plate. The encoded enzyme of this gene (DAADH_2344) was identified as D-amino acid dehydrogenase (DAADH), and this gene product supports the catabolism of a broad range of D-AAs. The ubiquitous distribution of DAADHs within the Mariana Trench sediments suggests that microorganisms that utilize D-AAs are common within these sediments. Our findings provide novel insights into the microbial potential for utilizing abiotic enantiomers of amino acids within the subduction zone of the Mariana trench under HHP, and our results provide an instructive significance for understanding these abiotic enantiomers and allow for insights regarding how organisms within extraterrestrial HHP environments can potentially cope with toxic D-AAs.

Functional Characterisation of Bile Metagenome: Study of Metagenomic Dark Matter

Gallbladder metagenome involves a wide range of unidentified sequences comprising the so-called metagenomic dark matter. Therefore, this study aimed to characterise three gallbladder metagenomes and a fosmid library with an emphasis on metagenomic dark matter fraction. For this purpose, a novel data analysis strategy based on the combination of remote homology and molecular modelling has been proposed. According to the results obtained, several protein functional domains were annotated in the metagenomic dark matter fraction including acetyltransferases, outer membrane transporter proteins, membrane assembly factors, DNA repair and recombination proteins and response regulator phosphatases. In addition, one deacetylase involved in mycothiol biosynthesis was found in the metagenomic dark matter fraction of the fosmid library. This enzyme may exert a protective effect in Actinobacteria against bile components exposure, in agreement with the presence of multiple antibiotic and multidrug resistance genes. Potential mechanisms of action of this novel deacetylase were elucidated by molecular simulations, highlighting the role of histidine and aspartic acid residues. Computational pipelines presented in this work may be of special interest to discover novel microbial enzymes which had not been previously characterised.

A novel oxidase from Alcaligenes sp. HO-1 oxidizes hydroxylamine to N2

Hydroxylamine is a key intermediate of microbial ammonia oxidation and plays an important role in the biogeochemical cycling of N-compounds. Hydroxylamine is oxidized to NO or N2O by hydroxylamine oxidases or cytochrome P460 from heterotrophic or autotrophic bacteria, but its enzymatic oxidation to N2 has not yet been observed. Here, we report on the discovery of a novel oxidase that converts hydroxylamine to N2 from the newly isolated heterotrophic nitrifier Alcaligenes strain HO-1. Strain HO-1 accumulated hydroxylamine and produced N2 from ammonia oxidation. Using transcriptome analysis and heterologous expression via fosmid library screening, we identified three genes (dnfABC) of strain HO-1 that enabled E. coli cells not only to produce hydroxylamine from 15N-labelled ammonium but also to further convert it to 15N2. The three genes were individually cloned and expressed, and their translational products DnfA, DnfB, and DnfC were purified. In vitro DnfA bound to hydroxylamine and catalyzed the conversion of hydroxylamine to N2 in the presence of FAD, NADH and O2. Thus, DnfA was identified as a novel hydroxylamine oxidase and catalyzed a previously unknown N-N bond forming reaction with a yet-to-be discovered mechanism. DnfA homologs were detected in different bacterial groups, suggesting that hydroxylamine oxidation to nitrogen might occur in additional microbial taxa.

Generation and Immunogenicity of a Recombinant Pseudorabies Virus Co-Expressing Classical Swine Fever Virus E2 Protein and Porcine Circovirus Type 2 Capsid Protein Based on Fosmid Library Platform

Pseudorabies (PR), classical swine fever (CSF), and porcine circovirus type 2 (PCV2)-associated disease (PCVAD) are economically important infectious diseases of pigs. Co-infections of these diseases often occur in the field, posing significant threat to the swine industry worldwide. gE/gI/TK-gene-deleted vaccines are safe and capable of providing full protection against PR. Classical swine fever virus (CSFV) E2 glycoprotein is mainly used in the development of CSF vaccines. PCV2 capsid (Cap) protein is the major antigen targeted for developing PCV2 subunit vaccines. Multivalent vaccines, and especially virus-vectored vaccines expressing foreign proteins, are attractive strategies to fight co-infections for various swine diseases. The gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live virus-vectored vaccines. Herein, we constructed a gE/gI/TK-gene-deleted PRV co-expressing E2 of CSFV and Cap of PCV2 by fosmid library platform established for PRV, and the expression of E2 and Cap proteins was confirmed using immunofluorescence assay and western blotting. The recombinant virus propagated in porcine kidney 15 (PK-15) cells for 20 passages was genetically stable. The evaluation results in rabbits and pigs demonstrate that rPRVTJ-delgE/gI/TK-E2-Cap elicited detectable anti-PRV antibodies, but not anti-PCV2 or anti-CSFV antibodies. These findings provide insights that rPRVTJ-delgE/gI/TK-E2-Cap needs to be optimally engineered as a promising trivalent vaccine candidate against PRV, PCV2 and CSFV co-infections in future.

PPE37 Is Essential forMycobacterium tuberculosisHeme-Iron Acquisition (HIA), and a Defective PPE37 inMycobacterium bovisBCG Prevents HIA

ABSTRACTMycobacterium tuberculosis, one of the world’s leading causes of death, must acquire nutrients, such as iron, from the host to multiply and cause disease. Iron is an essential metal andM. tuberculosispossesses two different systems to acquire iron from its environment: siderophore-mediated iron acquisition (SMIA) and heme-iron acquisition (HIA), involving uptake and degradation of heme to release ferrous iron. We have discovered thatMycobacterium bovisBCG, the tuberculosis vaccine strain, is severely deficient in HIA, and we exploited this phenotypic difference between BCG andM. tuberculosisto identify genes involved in HIA by complementing BCG’s defect with a fosmid library. We identifiedppe37, an iron-regulated PPE family gene, as being essential for HIA. BCG complemented withM. tuberculosisppe37exhibits HIA as efficient as that ofM. tuberculosis, achieving robust growth with <0.2 µM hemin. Conversely, deletion ofppe37fromM. tuberculosisresults in a strain severely attenuated in HIA, with a phenotype nearly identical to that of BCG, requiring a 200-fold higher concentration of hemin to achieve growth equivalent to that of its parental strain. A nine-amino-acid deletion near the N terminus of BCG PPE37 (amino acids 31 to 39 of theM. tuberculosisPPE37 protein) underlies BCG’s profound defect in HIA. Significant genetic variability exists inppe37genes across differentM. tuberculosisstrains, with more than 60% of sequences from completely sequencedM. tuberculosisgenomes having mutations that result in altered PPE37 proteins; furthermore, these altered PPE37 proteins are nonfunctional in HIA. Our findings should allow delineation of the relative roles of HIA and SMIA inM. tuberculosispathogenesis.