Micrococcus luteus strain CGK112 isolated from cow dung demonstrated efficient biofilm-forming ability and degradation potential toward high-density polyethylene (HDPE).

Biodegradation is the most promising environmentally sustainable method that offers a significant opportunity with minimal negative environmental consequences while searching for solutions to this global problem of plastic pollution that has now spread to almost everywhere in the entire world. In the present work, HDPE-degrading bacterial strain CGK112 was isolated from the fecal matter of a cow. The bacterial strain was identified as Micrococcus luteus CGK112 by 16S rRNA sequence coding analysis. Significant weight loss, i.e., 3.85% was recorded in the HDPE film treated with strain CGK112 for 90 days. The surface micromorphology was examined using FE-SEM, which revealed spectacular bacterial colonization as well as structural deformation. Furthermore, the EDX study indicated a significant decrease in the atomic percentage of carbon content, whereas FTIR analysis confirmed functional groups alternation as well as an increase in the carbonyl index which can be attributed to the metabolic activity of biofilm. Our findings provide insight into the capacity of our strain CGK112 to colonize and utilize HDPE as a single carbon source, thus promoting its degradation.

Comparative genomics reveals broad genetic diversity, extensive recombination and nascent ecological adaptation in Micrococcus luteus.

BACKGROUND: Micrococcus luteus is a group of actinobacteria that is widely used in biotechnology and is being thought as an emerging nosocomial pathogen. With one of the smallest genomes of free-living actinobacteria, it is found in a wide range of environments, but intraspecies genetic diversity and adaptation strategies to various environments remain unclear. Here, comparative genomics, phylogenomics, and genome-wide association studies were used to investigate the genomic diversity, evolutionary history, and the potential ecological differentiation of the species. RESULTS: High-quality genomes of 66 M. luteus strains were downloaded from the NCBI GenBank database and core and pan-genome analysis revealed a considerable intraspecies heterogeneity. Phylogenomic analysis, gene content comparison, and average nucleotide identity calculation consistently indicated that the species has diverged into three well-differentiated clades. Population structure analysis further suggested the existence of an unknown ancestor or the fourth, yet unsampled, clade. Reconstruction of gene gain/loss events along the evolutionary history revealed both early events that contributed to the inter-clade divergence and recent events leading to the intra-clade diversity. We also found convincing evidence that recombination has played a key role in the evolutionary process of the species, with upto two-thirds of the core genes having been affected by recombination. Furthermore, distribution of mammal-associated strains (including pathogens) on the phylogenetic tree suggested that the last common ancestor had a free-living lifestyle, and a few recently diverged lineages have developed a mammal-associated lifestyle separately. Consistently, genome-wide association analysis revealed that mammal-associated strains from different lineages shared genes functionally relevant to the host-associated lifestyle, indicating a recent ecological adaption to the new host-associated habitats. CONCLUSIONS: These results revealed high intraspecies genomic diversity of M. luteus and highlighted that gene gain/loss events and extensive recombination events played key roles in the genome evolution. Our study also indicated that, as a free-living species, some lineages have recently developed or are developing a mammal-associated lifestyle. This study provides insights into the mechanisms that drive the genome evolution and adaption to various environments of a bacterial species.

Phenotypic and genomic analysis of multiple heavy metal-resistant Micrococcus luteus strain AS2 isolated from industrial waste water and its potential use in arsenic bioremediation.

Multiple heavy metal-resistant bacterium, Micrococcus luteus strain AS2, was isolated from industrial waste water of District Sheikhupura, Pakistan. The isolated bacterium showed minimum inhibitory concentrations of 55 and 275 mM against arsenite and arsenate. The bacterial strain also showed resistance against other heavy metal ions, i.e., lead, cadmium, chromium, mercury, nickel, and zinc, apart from arsenic. The optimum temperature and pH were 37 °C and 7, respectively. The antioxidant enzymes such as catalase were significantly increased under arsenite stress. The increase in 43.9% of GSH/GSSG and 72.72% of non-protein thiol was determined under15 mM arsenite stress. Bacterial genome was sequenced through Illumina and Nanopore and genes related to arsenic and other heavy metals were identified and blast (tblastx) on NCBI. Through scanning electron microscopy, no morphological changes were observed in bacterial cells under arsenite stress. The peaks appeared in EDX showed that there is surface adsorption of arsenite in bacterial cell while it was confirmed from Fourier transformed infrared spectroscopy analysis that there is some interaction between arsenite and functional groups present on the surface of bacterial cell. The SDS-PAGE analysis of whole-cell proteins under 15 mM arsenite stress clearly revealed that there is upregulation of some proteins in ranged of 60 to 34 kDa. The bioremediation efficiency (E) of bacterial biomass was 72% after 2 h and 99% after 10 h. The bioremediation efficiency of bacterial biomass is an indicator for the isolated bacterium to employ as a potential candidate for the amelioration of sites contaminated with arsenic.

Interdependent Sequence Selectivity and Diastereoselectivity in the Alkylation of DNA by Decarbamoylmitomycin†C.

Mitomycin C (MC), an antitumor drug, and decarbamoylmitomycin C (DMC), a derivative of MC, alkylate DNA and form deoxyguanosine monoadducts and interstrand crosslinks (ICLs). Interestingly, in mammalian culture cells, MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-ß). The molecular basis for the stereochemical configuration exhibited by DMC has been investigated using biomimetic synthesis. Here, we present the results of our studies on the monoalkylation of DNA by DMC. We show that the formation of 1"-ß-deoxyguanosine adducts requires bifunctional reductive activation of DMC, and that monofunctional activation only produces 1"-α-adducts. The stereochemistry of the deoxyguanosine adducts formed is also dependent on the regioselectivity of DNA alkylation and on the overall DNA CG content. Additionally, we found that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by mitomycins: At 0 °C, both deoxyadenosine (dA) and deoxyguanosine (dG) alkylation occur whereas at 37 °C, mitomycins alkylate dG preferentially. The new reaction protocols developed in our laboratory to investigate DMC-DNA alkylation raise the possibility that oligonucleotides containing DMC 1"-ß-deoxyguanosine adducts at a specific site may be synthesized by a biomimetic approach.

pirA- and pirB-like gene identification in Micrococcus luteus strains in Mexico.

Acute hepatopancreatic necrosis disease (AHPND) was first reported in China in 2009 and afterwards in Mexico in 2013. AHPND is caused by Vibrio parahaemolyticus and affects Penaeus monodon and Litopenaeus vannamei shrimp cultures. The bacterium contains the pirA- and pirB-like genes in 69- to 70-Kb plasmids, which encode the toxins that produce the disease. The aim of this study was to determine whether pirA- and pirB-like genes existed in bacterial genera distinct from Vibrio before the first cases of AHPND were documented in Mexico. Two bacterial isolates were selected from shrimp farms in Nayarit in 2006 and analysed by nested-PCR to determine the presence of pirA- and pirB-like genes. The two isolates chosen did indeed show the presence of these genes, and those findings were confirmed by sequencing. Both strains matched to the bacterial species Micrococcus luteus. Results revealed two important situations: (a) the pirA- and pirB-like genes were present in a bacterial species that has not been reported previously (Micrococcus luteus); and (b) pirA- and pirB-like bacterial genes were present in Mexico before the first AHPND outbreak was reported in China.

A Proteomic Signature of Dormancy in the Actinobacterium Micrococcus luteus.

Dormancy is a protective state in which diverse bacteria, including Mycobacterium tuberculosis, Staphylococcus aureus, Treponema pallidum (syphilis), and Borrelia burgdorferi (Lyme disease), curtail metabolic activity to survive external stresses, including antibiotics. Evidence suggests dormancy consists of a continuum of interrelated states, including viable but nonculturable (VBNC) and persistence states. VBNC and persistence contribute to antibiotic tolerance, reemergence from latent infections, and even quorum sensing and biofilm formation. Previous studies indicate that the protein mechanisms regulating persistence and VBNC states are not well understood. We have queried the VBNC state of Micrococcus luteus NCTC 2665 (MI-2665) by quantitative proteomics combining gel electrophoresis, high-performance liquid chromatography, and tandem mass spectrometry to elucidate some of these mechanisms. MI-2665 is a nonpathogenic actinobacterium containing a small (2.5-Mb), high-GC-content genome which exhibits a well-defined VBNC state induced by nutrient deprivation. The MI-2665 VBNC state demonstrated a loss of protein diversity accompanied by increased levels of 18 proteins that are conserved across actinobacteria, 14 of which have not been previously identified in VNBC. These proteins implicate an anaplerotic strategy in the transition to VBNC, including changes in the glyoxylate shunt, redox and amino acid metabolism, and ribosomal regulatory processes. Our data suggest that MI-2665 is a viable model for dissecting the protein mechanisms underlying the VBNC stress response and provide the first protein-level signature of this state. We expect that this protein signature will enable future studies deciphering the protein mechanisms of dormancy and identify novel therapeutic strategies effective against antibiotic-tolerant bacterial infections.IMPORTANCE Dormancy is a protective state enabling bacteria to survive antibiotics, starvation, and the immune system. Dormancy is comprised of different states, including persistent and viable but nonculturable (VBNC) states that contribute to the spread of bacterial infections. Therefore, it is imperative to identify how bacteria utilize these different dormancy states to survive antibiotic treatment. The objective of our research is to eliminate dormancy as a route to antibiotic tolerance by understanding the proteins that control dormancy in Micrococcus luteus NCTC 2665. This bacterium has unique advantages for studying dormancy, including a small genome and a well-defined and reproducible VBNC state. Our experiments implicate four previously identified and 14 novel proteins upregulated in VBNC that may regulate this critical survival mechanism.

ß-Lactone Synthetase Found in the Olefin Biosynthesis Pathway.

The first ß-lactone synthetase enzyme is reported, creating an unexpected link between the biosynthesis of olefinic hydrocarbons and highly functionalized natural products. The enzyme OleC, involved in the microbial biosynthesis of long-chain olefinic hydrocarbons, reacts with syn- and anti-ß-hydroxy acid substrates to yield cis- and trans-ß-lactones, respectively. Protein sequence comparisons reveal that enzymes homologous to OleC are encoded in natural product gene clusters that generate ß-lactone rings, suggesting a common mechanism of biosynthesis.

Toxification of polycyclic aromatic hydrocarbons by commensal bacteria from human skin.

The ubiquitous occurrence of polycyclic aromatic hydrocarbons (PAHs) leads to constant human exposure at low levels. Toxicologically relevant are especially the high-molecular weight substances due to their (pro-)carcinogenic potential. Following ingestion or uptake, the eukaryotic phase I metabolism often activates these substances to become potent DNA binders, and unsurprisingly metabolism and DNA-adduct formation of model substances such as benzo[a]pyrene (B[a]P) are well studied. However, apart from being subjected to eukaryotic transformations PAHs are also carbon and energy sources for the myriads of commensal microbes inhabiting man's every surface. Yet, we know little about the microbiome's PAH-metabolism capacity and its potentially adverse impact on the human host. This study now shows that readily isolable skin commensals transform B[a]P into a range of highly cyto- and genotoxic metabolites that are excreted in toxicologically relevant concentrations during growth. The respective bacterial supernatants contain a mixture of established eukaryotic as well as hitherto unknown prokaryotic metabolites, the combination of which leads to an increased toxicity. Altogether we show that PAH metabolism of the microbiome has to be considered a potential hazard.

Radiation-Resistant Micrococcus luteus SC1204 and Its Proteomics Change Upon Gamma Irradiation.

To explore the radiation-resistance mechanisms in bacteria, a radiation-resistant strain SC1204 was isolated from the surrounding area of a (60)Co-γ radiation facility. SC1204 could survive up to 8 kGy dose of gamma irradiation and was identified as Micrococcus luteus by phylogenetic analysis of 16S rRNA gene sequences. Its proteomic changes under 2-kGy irradiation were examined by two-dimensional electrophoresis followed by MALDI-TOF-TOF/MS analysis. The results showed that at least 24 proteins displayed significant changes (p < 0.05) at expression level under the radiation stress, among which 22 were successfully identified and classified into the major functional categories of metabolism, energy production and conservation, translation, ribosomal structure, and biogenesis. Among these proteins, leucyl aminopeptidase involved in synthesis of glutathione was the most abundant induced protein during postirradiation recovery, indicating that anti-oxidation protection was the most important line of defense in SC1204 against radiation. The next abundant protein was phosphoribosyl aminoimidazole carboxamide formyltransferase/IMP cyclohydrolase (AICAR Tfase/IMPCH), the key enzyme in the biosynthetic pathway of purine that is anti-radiation compound. Other proteins changing significantly (p < 0.05) after radiation exposure included urocanate hydratase, dihydrolipoyl dehydrogenase, succinyl-CoA synthetase subunit alpha, phosphoglycerate kinase, cell division protein FtsZ, elongation factor Ts and Tu, translation elongation factor Tu and G, 30S ribosomal protein S1, histidyl-tRNA synthetase, and arginyl-tRNA synthetase, which were considered to be the key proteins in urocanate metabolism, tricarboxylic acid cycle, glycolysis, cell division process, and synthesis process of proteins. Therefore, these proteins may also play important roles in radiation resistance in M. luteus.

Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production.

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms.

Microbial production of aliphatic (S)-epoxyalkanes by using Rhodococcus sp. strain ST-10 styrene monooxygenase expressed in organic-solvent-tolerant Kocuria rhizophila DC2201.

We describe the development of biocatalysis for producing optically pure straight-chain (S)-epoxyalkanes using styrene monooxygenase of Rhodococcus sp. strain ST-10 (RhSMO). RhSMO was expressed in the organic solvent-tolerant microorganism Kocuria rhizophila DC2201, and the bioconversion reaction was performed in an organic solvent-water biphasic reaction system. The biocatalytic process enantioselectively converted linear terminal alkenes to their corresponding (S)-epoxyalkanes using glucose and molecular oxygen. When 1-heptene and 6-chloro-1-hexene were used as substrates (400 mM) under optimized conditions, 88.3 mM (S)-1,2-epoxyheptane and 246.5 mM (S)-1,2-epoxy-6-chlorohexane, respectively, accumulated in the organic phase with good enantiomeric excess (ee; 84.2 and 95.5%). The biocatalysis showed broad substrate specificity toward various aliphatic alkenes, including functionalized and unfunctionalized alkenes, with good to excellent ee. Here, we demonstrate that this biocatalytic system is environmentally friendly and useful for producing various enantiopure (S)-epoxyalkanes.

Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer-Villiger monooxygenase.

We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts.

Enzyme fusion for whole-cell biotransformation of long-chain sec-alcohols into esters.

Enzyme fusion was investigated as a strategy to improve productivity of a two-step whole-cell biocatalysis. The biotransformation of long-chain sec-alcohols into esters by an alcohol dehydrogenase (ADH) and Baeyer-Villiger monooxygenases (BVMOs) was used as the model reaction. The recombinant Escherichia coli, expressing the fusion enzymes between the ADH of Micrococcus luteus NCTC2665 and the BVMO of Pseudomonas putida KT2440 or Rhodococcus jostii RHA1, showed significantly greater bioconversion activity with long-chain sec-alcohols (e.g., 12-hydroxyoctadec-9-enoic acid (1a), 13-hydroxyoctadec-9-enoic acid (2a), 14-hydroxyicos-11-enoic acid (4a)) when compared to the recombinant E. coli expressing the ADH and BVMOs independently. For instance, activity of the recombinant E. coli expressing the ADH-Gly-BVMO, in which glycine-rich peptide was used as the linker, with 1a was increased up to 22 µmol g dry cells(-1) min(-1). This value is over 40 % greater than the recombinant E. coli expressing the ADH and BVMO independently. The substantial improvement appeared to be driven by an increase in the functional expression of the BVMOs and/or an increase in mass transport efficiency by localizing two active sites in close proximity.

Biotransformation of oleic acid into 10-ketostearic acid by recombinant Corynebacterium glutamicum-based biocatalyst.

OBJECTIVE: To produce 10-ketostearic acid from oleic acid. RESULTS: Oleic acid was converted to 10-ketostearic acid by a recombinant Corynebacterium glutamicum ATCC 13032 expressing oleate hydratase from Stenotrophomonas maltophilia and a secondary alcohol dehydrogenase from Micrococcus luteus under the control of a synthetic constitutive promoter. Optimal conditions for 10-ketostearic acid production were pH 7.5 and 30 °C with 5 g cells l(-1) and 2.5 g oleic acid l(-1). Under these conditions, the cells produced 1.96 g 10-ketostearic acid l(-1) from oleic acid in 6 h, with a conversion yield of 78 % (w) and a maximum volumetric productivity of 1.67 g l(-1) h(-1). CONCLUSION: This is the first report of 10-ketostearic acid production using a recombinant C. glutamicum.

An improved implementation of effective number of codons (nc).

The effective number of codons (N(c)) is a widely used index for characterizing codon usage bias because it does not require a set of reference genes as does codon adaptation index (CAI) and because of the freely available computational tools such as CodonW. However, N(c), as originally formulated has many problems. For example, it can have values far greater than the number of sense codons; it treats a 6-fold compound codon family as a single-codon family although it is made of a 2-fold and a 4-fold codon family that can be under dramatically different selection for codon usage bias; the existing implementations do not handle all different genetic codes; it is often biased by codon families with a small number of codons. We developed a new N(c) that has a number of advantages over the original N(c). Its maximum value equals the number of sense codons when all synonymous codons are used equally, and its minimum value equals the number of codon families when exactly one codon is used in each synonymous codon family. It handles all known genetic codes. It breaks the compound codon families (e.g., those involving amino acids coded by six synonymous codons) into 2-fold and 4-fold codon families. It reduces the effect of codon families with few codons by introducing pseudocount and weighted averages. The new N(c) has significantly improved correlation with CAI than the original N(c) from CodonW based on protein-coding genes from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Escherichia coli, Bacillus subtilis, Micrococcus luteus, and Mycoplasma genitalium. It also correlates better with protein abundance data from the yeast than the original N(c).

[Phylogenetic analysis of bacteria of extreme ecosystems].

Phylogenetic analysis of aerobic chemoorganotrophic bacteria of the two extreme regions (Dead Sea and West Antarctic) was performed on the basis of the nucleotide sequences of the 16S rRNA gene. Thermotolerant and halotolerant spore-forming bacteria 7t1 and 7t3 of terrestrial ecosystems Dead Sea identified as Bacillus licheniformis and B. subtilis subsp. subtilis, respectively. Taking into account remote location of thermotolerant strain 6t1 from closely related strains in the cluster Staphylococcus, 6t1 strain can be regarded as Staphylococcus sp. In terrestrial ecosystems, Galindez Island (Antarctic) detected taxonomically diverse psychrotolerant bacteria. From ornithogenic soil were isolated Micrococcus luteus O-1 and Microbacterium trichothecenolyticum O-3. Strains 4r5, 5r5 and 40r5, isolated from grass and lichens, can be referred to the genus Frondihabitans. These strains are taxonomically and ecologically isolated and on the tree diagram form the joint cluster with three isolates Frondihabitans sp., isolated from the lichen Austrian Alps, and psychrotolerant associated with plants F. cladoniiphilus CafT13(T). Isolates from black lichen in the different stationary observation points on the south side of a vertical cliff identified as: Rhodococcus fascians 181n3, Sporosarcina aquimarina O-7, Staphylococcus sp. 0-10. From orange biofilm of fouling on top of the vertical cliff isolated Arthrobacter sp. 28r5g1, from the moss-- Serratia sp. 6r1g. According to the results, Frondihabitans strains most frequently encountered among chemoorganotrophic aerobic bacteria in the Antarctic phytocenoses.

Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones.

Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative ß-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO2 and H2 as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones.

Development of an informatics platform for therapeutic protein and peptide analytics.

The momentum gained by research on biologics has not been met yet with equal thrust on the informatics side. There is a noticeable lack of software for data management that empowers the bench scientists working on the development of biologic therapeutics. SARvision|Biologics is a tool to analyze data associated with biopolymers, including peptides, antibodies, and protein therapeutics programs. The program brings under a single user interface tools to filter, mine, and visualize data as well as those algorithms needed to organize sequences. As part of the data-analysis tools, we introduce two new concepts: mutation cliffs and invariant maps. Invariant maps show the variability of properties when a monomer is maintained constant in a position of the biopolymer. Mutation cliff maps draw attention to pairs of sequences where a single or limited number of point mutations elicit a large change in a property of interest. We illustrate the program and its applications using a peptide data set collected from the literature.

Micrococcus luteus-derived extracellular vesicles attenuate neutrophilic asthma by regulating miRNAs in airway epithelial cells.

Bacterial extracellular vesicles (EVs) have been shown to regulate various pulmonary diseases, but their functions in asthma remain uncertain. To demonstrate the clinical significance of Micrococcus luteus-derived EVs (MlEVs) in asthma, we enrolled 45 asthmatic patients (20 patients with neutrophilic asthma [NA], 25 patients with eosinophilic asthma [EA]) and 40 healthy controls (HCs). When the prevalence of IgG1 and IgG4 specific to MlEVs was evaluated in serum by ELISA, lower levels of MlEV-specific IgG4 (but not IgG1) were noted in asthmatic patients than in HCs. Among asthmatic patients, significantly lower levels of MIEV-specific IgG4 were noted in patients with NA than in those with EA. Moreover, there was a positive correlation between serum MlEV-specific IgG4 levels and FEV1 (%) values. In asthmatic C57BL/6 mice, MlEVs significantly attenuated neutrophilic airway inflammation by reducing the production of IL-1ß and IL-17 in bronchoalveolar lavage fluid as well as the number of group 3 innate lymphoid cells (ILC3s) in lung tissues. To clarify the functional mechanism of MlEVs in NA, the effect of MlEVs on airway epithelial cells (AECs) and immune cells was investigated ex vivo. According to microarray analysis, MlEVs upregulated hsa-miR-4517 expression in AECs. Moreover, this miRNA could suppress IL-1ß production by monocytes, resulting in the inhibition of ILC3 activation and neutrophil recruitment. These findings suggest that MlEVs could be a novel therapeutic agent for managing unresolved NA by regulating miRNA expression in AECs.

[Cloning and expression of Micrococcus luteus IAM 14879 Rpf and its role in the recovery of the VBNC state in Rhodococcus sp. DS471].

OBJECTIVE: The purpose of the present study was to produce the Rpf (resuscitation promoting factor) protein by cloning and expressing the rpf gene, secreted by Micrococcus luteus IAM 14879, in Escherichia coli and to evaluate its role in the recovery of the VBNC (viable but non-culturable) state in high-GC Gram-positive bacteria. METHODS: Genomic DNA was extracted from Micrococcus luteus IAM 14879 and the rpf gene was amplified by PCR using specific primers. The PCR products was purified, cloned into a pET15b expression vector, and transformed into Escherichia coli BL21 (DE3). Then the pET15b plasmid expression vector was used to confirm the purification of the recombinant proteins via SDS-PAGE. The VBNC state cells from the high-GC Gram-positive bacteria, Rhodococcus sp. DS471, were used to confirm the promotion and recovery of growth capacity. Rhodococcus sp. DS471 were isolated from soil and closely related to Micrococcus luteus IAM 14879. RESULTS: The gene sequences confirmed that the rpf gene from Micrococcus luteus IAM 14879 that was expressed in Escherichia coli, was 672 bp. SDS-PAGE analysis showed that the recombinant Rpf protein was obtained successfully, and further studies showed it capable of promoting the recovery of the VBNC state by about 100-fold relative to the control. CONCLUSION: Rpf of Micrococus luteus IAM 14879 can be successfully cloned and expressed in Escherichia coli and shows a strong ability to promote the recovery of the VBNC state of cells of Rhodococcus sp. DS471.