Insights into the phylogenetic diversity, biological activities, and biosynthetic potential of mangrove rhizosphere Actinobacteria from Hainan Island.

Mangrove rhizosphere soils host diverse Actinobacteria tolerant to numerous stresses and are inevitably capable of exhibiting excellent biological activity by producing impressive numbers of bioactive natural products, including those with potential medicinal applications. In this study, we applied an integrated strategy of combining phylogenetic diversity, biological activities, and biosynthetic gene clusters (BGCs) screening approach to investigate the biotechnological importance of Actinobacteria isolated from mangrove rhizosphere soils from Hainan Island. The actinobacterial isolates were identifified using a combination of colony morphological characteristics and 16S rRNA gene sequence analysis. Based on the results of PCR-detected BGCs screening, type I and II polyketide synthase (PKS) and non-ribosomal synthetase (NRPS) genes were detected. Crude extracts of 87 representative isolates were subjected to antimicrobial evaluation by determining the minimum inhibitory concentration of each strain against six indicator microorganisms, anticancer activities were determined on human cancer cell lines HepG2, HeLa, and HCT-116 using an MTT colorimetric assay, and immunosuppressive activities against the proliferation of Con A-induced T murine splenic lymphocytes in vitro. A total of 287 actinobacterial isolates affiliated to 10 genera in eight families of six orders were isolated from five different mangrove rhizosphere soil samples, specififically, Streptomyces (68.29%) and Micromonospora (16.03%), of which 87 representative strains were selected for phylogenetic analysis. The crude extracts of 39 isolates (44.83%) showed antimicrobial activity against at least one of the six tested indicator pathogens, especially ethyl acetate extracts of A-30 (Streptomyces parvulus), which could inhibit the growth of six microbes with MIC values reaching 7.8 µg/mL against Staphylococcus aureus and its resistant strain, compared to the clinical antibiotic ciproflfloxacin. Furthermore, 79 crude extracts (90.80%) and 48 (55.17%) of the isolates displayed anticancer and immunosuppressive activities, respectively. Besides, four rare strains exhibited potent immunosuppressive activity against the proliferation of Con A-induced T murine splenic lymphocyte in vitro with an inhibition rate over 60% at 10 µg/mL. Type I and II polyketide synthase (PKS) and non-ribosomal synthetase (NRPS) genes were detected in 49.43, 66.67, and 88.51% of the 87 Actinobacteria, respectively. Signifificantly, these strains (26 isolates, 29.89%) harbored PKS I, PKS II, and NRPS genes in their genomes. Nevertheless, their bioactivity is independent of BGCs in this study. Our findings highlighted the antimicrobial, immunosuppressive, and anticancer potential of mangrove rhizosphere Actinobacteria from Hainan Island and the biosynthetic prospects of exploiting the corresponding bioactive natural product.

Environmental selection and evolutionary process jointly shape genomic and functional profiles of mangrove rhizosphere microbiomes.

Mangrove reforestation with introduced species has been an important strategy to restore mangrove ecosystem functioning. However, how such activities affect microbially driven methane (CH4), nitrogen (N), and sulfur (S) cycling of rhizosphere microbiomes remains unclear. To understand the effect of environmental selection and the evolutionary process on microbially driven biogeochemical cycles in native and introduced mangrove rhizospheres, we analyzed key genomic and functional profiles of rhizosphere microbiomes from native and introduced mangrove species by metagenome sequencing technologies. Compared with the native mangrove (Kandelia obovata, KO), the introduced mangrove (Sonneratia apetala, SA) rhizosphere microbiome had significantly (p < 0.05) higher average genome size (AGS) (5.8 vs. 5.5 Mb), average 16S ribosomal RNA gene copy number (3.5 vs. 3.1), relative abundances of mobile genetic elements, and functional diversity in terms of the Shannon index (7.88 vs. 7.84) but lower functional potentials involved in CH4 cycling (e.g., mcrABCDG and pmoABC), N2 fixation (nifHDK), and inorganic S cycling (dsrAB, dsrC, dsrMKJOP, soxB, sqr, and fccAB). Similar results were also observed from the recovered Proteobacterial metagenome-assembled genomes with a higher AGS and distinct functions in the introduced mangrove rhizosphere. Additionally, salinity and ammonium were identified as the main environmental drivers of functional profiles of mangrove rhizosphere microbiomes through deterministic processes. This study advances our understanding of microbially mediated biogeochemical cycling of CH4, N, and S in the mangrove rhizosphere and provides novel insights into the influence of environmental selection and evolutionary processes on ecosystem functions, which has important implications for future mangrove reforestation.

Two new unsaturated fatty acids from the mangrove rhizosphere soil-derived fungus Penicillium javanicum HK1-22.

Two new unsaturated fatty acids, 6R,8R-dihydroxy-9Z,12Z-octadecadienoic acid (1) and methyl-6R,8R-dihydroxy-9Z,12Z-octadecadienoate (2), and two known 9Z,12Z-octadecadienoic acid analogues (3, 4) together with a known sesquiterpenoid (5) were isolated from the mangrove rhizosphere soil-derived fungus Penicillium javanicum HK1-22. An acetonide derivative (1a) from 1 was also prepared. The relative configuration of 1 was determined by analysis of the 1D and 2D NOE spectra of 1a. The absolute configuration of 1 was assigned on the basis of biogenetic considerations. The antifungal activity of the high yield compound 5 was evaluated against four strains of crop pathogens and it showed significant antifungal activities against all the tested strains.

Agromyces mangrovi sp. nov., a novel actinobacterium isolated from the rhizosphere of a mangrove.

A novel actinobacterium, designated HIr16-25T, was isolated from the rhizosphere soil of a mangrove growing on Iriomote Island in Japan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain HIr16-25T fell within the cluster of the members of the genus Agromyces and the highest sequence similarity value was observed with Agromyces rhizospherae IFO 16236T (98.6%). Strain HIr16-25T possessed L-2,4-diaminobutyric acid as a diagnostic diamino acid of the peptidoglycan, and MK-12 and MK-11 as the predominant menaquinones. The major fatty acids were identified as iso-C16:0, anteiso-C15:0 and anteiso-C17:0 and the principal polar lipids were phosphatidylglycerol and one glycolipid. These chemotaxonomic features matched well those described for the members of the genus Agromyces. Meanwhile, the result of DNA-DNA hybridization and the presence of differential phenotypic characteristics between strain HIr16-25T and the type strain of A. rhizospherae indicated that strain HIr16-25T be classified as a novel species of the genus Agromyces. Therefore, we propose strain HIr16-25T to represent a novel species of the genus Agromyces, with the name Agromyces mangrovi sp. nov. The type strain is HIr16-25T (= NBRC 112812T = TBRC 7760T).

Purification, characterization and production optimization of a vibriocin produced by mangrove associated Vibrio parahaemolyticus.

OBJECTIVE: To identify a potential bacterium which produces antimicrobial peptide (vibriocin), and its purification, characterization and production optimization. The bacteria subjected in the study were isolated from a highly competitive ecological niche of mangrove ecosystem. METHODS: The bacterium was characterized by phenotype besides 16S rRNA gene sequence analysis. The antibacterial activity was recognised by using agar well diffusion method. The vibriocin was purified using ammonium sulphate precipitation, butanol extraction, gel filtration chromatography, ion-exchange chromatography and subsequently, by HPLC. Molecular weight of the substance identified in SDS-PAGE. Production optimization performed according to Taguchi's mathematical model using 6 different nutritional parameters as variables. RESULTS: The objective bacterium was identified as Vibrio parahaemolyticus. The vibriocin showed 18 KDa of molecular mass with mono peptide in nature and highest activity against pathogenic Vibrio harveyi. The peptide act stable in a wide range of pH, temperature, UV radiation, solvents and chemicals utilized. An overall ∼20% of vibriocin production was improved, and was noticed that NaCl and agitation speed played a vital role in secretion of vibriocin. CONCLUSION: The vibriocin identified here would be an effective alternative for chemically synthesized drugs for the management of Vibrio infections in mariculture industry.

Inhibitory efficacy of cyclo(L-leucyl-L-prolyl) from mangrove rhizosphere bacterium-Bacillus amyloliquefaciens (MMS-50) toward cariogenic properties of Streptococcus mutans.

Since Streptococcus mutans is the principal etiologic agent causing dental caries, by encompassing an array of unique virulence traits, emerging treatment strategies that specifically target the virulence of this pathogen may be promising as alternative approaches compared to conventional antibiotic therapy. In this perspective, we investigated chloroform extract of cell-free culture supernatant from mangrove rhizosphere bacterium Bacillus amyloliquefaciens (MMS-50) in terms of anticariogenic properties of S. mutans, without suppressing its viability. Crude chloroform extract of MMS-50 was subjected to column and high performance liquid chromatographic techniques to obtain the active fraction (AF), and MMS-50 AF was used for all further assays. GC-MS and FT-IR were carried out to identify the major components present in MMS-50 AF. Comparative gene expression analysis of some biofilm-forming and virulence genes (vicR, comDE, gtfC, and gbpB) was done by real-time PCR. Cyclo(L-leucyl-L-prolyl) was found to be the chief compound in MMS-50 AF responsible for bioactivity. The minimum and maximum inhibitory concentrations of MMS-50 AF against S. mutans were found to be 100 and 250 µg/mL, respectively. Anti-virulence assays performed using below-sub-MIC levels of MMS-50 AF (30 µg/mL) resulted in significant reduction in adherence (68%), acid production, acid tolerance, glucan synthesis (32%), biofilm formation (53.5%) and cell surface hydrophobicity, all devoid of affecting its viability. The micrographs of CLSM and SEM further confirmed the antibiofilm and anti-virulence efficacies of MMS-50 AF. Expression data showed significant reduction in expression of all studied virulence genes. Thus, the current study unveils the anticariogenic potential of cyclo(L-leucyl-L-prolyl) from B. amyloliquefaciens, as well as its suitability as a novel and alternative anticariogenic agent against dental caries.

Purification, characterization and production optimization of a vibriocin produced by mangrove associated Vibrio parahaemolyticus

<p><b>OBJECTIVE</b>To identify a potential bacterium which produces antimicrobial peptide (vibriocin), and its purification, characterization and production optimization. The bacteria subjected in the study were isolated from a highly competitive ecological niche of mangrove ecosystem.</p><p><b>METHODS</b>The bacterium was characterized by phenotype besides 16S rRNA gene sequence analysis. The antibacterial activity was recognised by using agar well diffusion method. The vibriocin was purified using ammonium sulphate precipitation, butanol extraction, gel filtration chromatography, ion-exchange chromatography and subsequently, by HPLC. Molecular weight of the substance identified in SDS-PAGE. Production optimization performed according to Taguchi's mathematical model using 6 different nutritional parameters as variables.</p><p><b>RESULTS</b>The objective bacterium was identified as Vibrio parahaemolyticus. The vibriocin showed 18 KDa of molecular mass with mono peptide in nature and highest activity against pathogenic Vibrio harveyi. The peptide act stable in a wide range of pH, temperature, UV radiation, solvents and chemicals utilized. An overall ∼20% of vibriocin production was improved, and was noticed that NaCl and agitation speed played a vital role in secretion of vibriocin.</p><p><b>CONCLUSION</b>The vibriocin identified here would be an effective alternative for chemically synthesized drugs for the management of Vibrio infections in mariculture industry.</p>

Purification, characterization and production optimization of a vibriocin produced by mangrove associated Vibrio parahaemolyticus

Objective: To identify a potential bacterium which produces antimicrobial peptide (vibriocin), and its purification, characterization and production optimization. The bacteria subjected in the study were isolated from a highly competitive ecological niche of mangrove ecosystem. Methods:The bacterium was characterized by phenotype besides 16S rRNA gene sequence analysis.The antibacterial activity was recognised by using agar well diffusion method. The vibriocin was purified using ammonium sulphate precipitation, butanol extraction, gel filtration chromatography, ion-exchange chromatography and subsequently, by HPLC. Molecular weight of the substance identified in SDS-PAGE. Production optimization performed according to Taguchi’s mathematical model using 6 different nutritional parameters as variables. Results:The objective bacterium was identified as Vibrio parahaemolyticus. The vibriocin showed 18 KDa of molecular mass with mono peptide in nature and highest activity against pathogenic Vibrio harveyi. The peptide act stable in a wide range of pH, temperature, UV radiation, solvents and chemicals utilized. An overall ~20% of vibriocin production was improved, and was noticed that NaCl and agitation speed played a vital role in secretion of vibriocin. Conclusion: The vibriocin identified here would be an effective alternative for chemically synthesized drugs for the management of Vibrio infections in mariculture industry.