Marine copepod culture as a potential source of bioplastic-degrading microbiome: The case of poly(butylene succinate-co-adipate).

The poly(butylene succinate-co-adipate) (PBSA) is emerging as environmentally sustainable polyester for applications in marine environment. In this work the capacity of microbiome associated with marine plankton culture to degrade PBSA, was tested. A taxonomic and functional characterization of the microbiome associated with the copepod Acartia tonsa, reared in controlled conditions, was analysed by 16S rDNA metabarcoding, in newly-formed adult stages and after 7 d of incubation. A predictive functional metagenomic profile was inferred for hydrolytic activities involved in bioplastic degradation with a particular focus on PBSA. The copepod-microbiome was also characterized in newly-formed carcasses of A. tonsa, and after 7 and 33 d of incubation in the plankton culture medium. Copepod-microbiome showed hydrolytic activities at all developmental stages of the alive copepods and their carcasses, however, the evenness of the hydrolytic bacterial community significantly increased with the time of incubation in carcasses. Microbial genera, never described in association with copepods: Devosia, Kordia, Lentibacter, Methylotenera, Rheinheimera, Marinagarivorans, Paraglaciecola, Pseudophaeobacter, Gaiella, Streptomyces and Kribbella sps., were retrieved. Kribbella sp. showed carboxylesterase activity and Streptomyces sp. showed carboxylesterase, triacylglycerol lipase and cutinase activities, that might be involved in PBSA degradation. A culturomic approach, adopted to isolate bacterial specimen from carcasses, led to the isolation of the bacterial strain, Vibrio sp. 01 tested for the capacity to promote the hydrolysis of the ester bonds. Granules of PBSA, incubated 82 d at 20 °C with Vibrio sp. 01, were characterized by scanning electron microscopy, infrared spectroscopy, thermogravimetric analysis, and differential scanning calorimetry, showing fractures compared to the control sample, and hydrolysis of ester bonds. These preliminary results are encouraging for further investigation on the ability of the microbiome associated with plankton to biodegrade polyesters, such as PBSA, and increasing knowledge on microorganisms involved in bioplastic degradation in marine environment.

Complete Genome Sequence Analysis of Kribbella sp. CA-293567 and Identification of the Kribbellichelins A & B and Sandramycin Biosynthetic Gene Clusters.

Minor genera actinomycetes are considered a promising source of new secondary metabolites. The strain Kribbella sp. CA-293567 produces sandramycin and kribbellichelins A & B In this work, we describe the complete genome sequencing of this strain and the in silico identification of biosynthetic gene clusters (BGCs), focusing on the pathways encoding sandramycin and kribbellichelins A-B. We also present a comparative analysis of the biosynthetic potential of 38 publicly available genomes from Kribbella strains.

Kribbellichelins A and B, Two New Antibiotics from Kribbella sp. CA-293567 with Activity against Several Human Pathogens.

Current needs in finding new antibiotics against emerging multidrug-resistant superbugs are pushing the scientific community into coming back to Nature for the discovery of novel active structures. Recently, a survey of halophilic actinomyectes from saline substrates of El Saladar del Margen, in the Cúllar-Baza depression (Granada, Spain), led us to the isolation and identification of 108 strains from the rhizosphere of the endemic plant Limonium majus. Evaluation of the potential of these strains to produce new anti-infective agents against superbug pathogens was performed through fermentation in 10 different culture media using an OSMAC approach and assessment of the antibacterial and antifungal properties of their acetone extracts. The study allowed the isolation of two novel antibiotic compounds, kribbellichelin A (1) and B (2), along with the known metabolites sandramycin (3), coproporphyrin III (4), and kribelloside C (5) from a bioassay-guided fractionation of scaled-up active extracts of the Kribbella sp. CA-293567 strain. The structures of the new molecules were elucidated by ESI-qTOF-MS/MS, 1D and 2D NMR, and Marfey's analysis for the determination of the absolute configuration of their amino acid residues. Compounds 1-3 and 5 were assayed against a panel of relevant antibiotic-resistant pathogenic strains and evaluated for cytotoxicity versus the human hepatoma cell line HepG2 (ATCC HB-8065). Kribbellichelins A (1) and B (2) showed antimicrobial activity versus Candida albicans ATCC-64124, weak potency against Acinetobacter baumannii MB-5973 and Pseudomonas aeruginosa MB-5919, and an atypical dose-dependent concentration profile against Aspergillus fumigatus ATCC-46645. Sandramycin (3) confirmed previously reported excellent growth inhibition activity against MRSA MB-5393 but also presented clear antifungal activity against C. albicans ATCC-64124 and A. fumigatus ATCC-46645 associated with lower cytotoxicity observed in HepG2, whereas Kribelloside C (5) displayed high antifungal activity only against A. fumigatus ATCC-46645. Herein, we describe the processes followed for the isolation, structure elucidation, and potency evaluation of these two new active compounds against a panel of human pathogens as well as, for the first time, the characterization of the antifungal activities of sandramycin (3).

Oxalate utilisation is widespread in the actinobacterial genus Kribbella.

The type strains of all 33 species in the genus Kribbella were tested for growth on oxalate (-OOC-COO-) as sole carbon source. Media were initially formulated to contain sodium oxalate, but even a concentration as low as 7.5 mM oxalate prevented growth. A modified medium based on calcium oxalate was very successful in characterising oxalate utilisation by Kribbella strains (metabolism of oxalate by oxalotrophic bacteria results in visible zones of clearing around the growth streaks on the opaque plates). To assess the variability of oxalate utilisation in Kribbella species, we also tested eight non-type strains for their ability to use oxalate. Thirty of 33 type strains (90.9%) and six of eight non-type strains (75%) were able to use oxalate as a sole carbon source. Based on these results, we propose that oxalate would be an excellent carbon source for the selective isolation of Kribbella strains. Based on the oxalate-utilisation phenotype and analyses of the 19 publicly available Kribbella type-strain genome sequences, we propose a pathway for oxalate metabolism in Kribbella. This pathway is significantly different from those previously proposed for oxalate metabolism in other bacteria, involving the indirect catabolism of oxalate to formate. Formate production is proposed to be involved in energy generation and to be crucial for oxalate import via an oxalate:formate antiporter. To our knowledge, this is the first report of an oxalate:formate antiporter in an aerobic, Gram-positive bacterium.

Novel South African Rare Actinomycete Kribbella speibonae Strain SK5: A Prolific Producer of Hydroxamate Siderophores Including New Dehydroxylated Congeners.

In this paper, we report on the chemistry of the rare South African Actinomycete Kribbella speibonae strain SK5, a prolific producer of hydroxamate siderophores and their congeners. Two new analogues, dehydroxylated desferrioxamines, speibonoxamine 1 and desoxy-desferrioxamine D1 2, have been isolated, together with four known hydroxamates, desferrioxamine D1 3, desferrioxamine B 4, desoxy-nocardamine 5 and nocardamine 6, and a diketopiperazine (DKP) 7. The structures of 1-7 were characterized by the analysis of HRESIMS and 1D and 2D NMR data, as well as by comparison with the relevant literature. Three new dehydroxy desferrioxamine derivatives 8-10 were tentatively identified in the molecular network of K. speibonae strain SK5 extracts, and structures were proposed based on their MS/MS fragmentation patterns. A plausible spb biosynthetic pathway was proposed. To the best of our knowledge, this is the first report of the isolation of desferrioxamines from the actinobacterial genus Kribbella.

Enrichment of novel Actinomycetales and the detection of monooxygenases during aerobic 1,4-dioxane biodegradation with uncontaminated and contaminated inocula.

1,4-Dioxane, a co-contaminant at many chlorinated solvent sites, is a problematic groundwater pollutant because of risks to human health and characteristics which make remediation challenging. In situ 1,4-dioxane bioremediation has recently been shown to be an effective remediation strategy. However, the presence/abundance of 1,4-dioxane degrading species across different environmental samples is generally unknown. Here, the objectives were to identify which 1,4-dioxane degrading functional genes are present and which genera may be using 1,4-dioxane and/or metabolites to support growth across different microbial communities. For this, laboratory sample microcosms and abiotic control microcosms (containing media) were inoculated with four uncontaminated soils and sediments from two contaminated sites. Live control microcosms were treated in the same manner, except 1,4-dioxane was not added. 1,4-Dioxane decreased in live microcosms with all six inocula, but not in the abiotic controls, suggesting biodegradation occurred. A comparison of live sample microcosms and live controls (no 1,4-dioxane) indicated nineteen genera were enriched following exposure to 1,4-dioxane, suggesting a growth benefit for 1,4-dioxane biodegradation. The three most enriched were Mycobacterium, Nocardioides, and Kribbella (classifying as Actinomycetales). There was also a higher level of enrichment for Arthrobacter, Nocardia, and Gordonia (all three classifying as Actinomycetales) in one soil, Hyphomicrobium (Rhizobiales) in another soil, Clavibacter (Actinomycetales) and Bartonella (Rhizobiales) in another soil, and Chelativorans (Rhizobiales) in another soil. Although Arthrobacter, Mycobacterium, and Nocardia have previously been linked to 1,4-dioxane degradation, Nocardioides, Gordonia, and Kribbella are potentially novel degraders. The analysis of the functional genes associated with 1,4-dioxane demonstrated three genes were present at higher relative abundance values, including Rhodococcus sp. RR1 prmA, Rhodococcus jostii RHA1 prmA, and Burkholderia cepacia G4 tomA3. Overall, this study provides novel insights into the identity of the multiple genera and functional genes associated with aerobic degradation of 1,4-dioxane in mixed communities.

Development of a Kribbella-specific isolation medium and description of Kribbella capetownensis sp. nov. and Kribbella speibonae sp. nov., isolated from soil.

Two actinobacterial strains were isolated from samples collected from the University of Cape Town, South Africa. A third actinobacterial strain was isolated from soil collected in the town of Stellenbosch, South Africa, using a newly-developed Kribbella-selective medium. Analysis of the 16S rRNA genes showed that the three strains belonged to the genus Kribbella. A multilocus sequence analysis using the concatenated gene sequences of the gyrB, rpoB, relA, recA and atpD genes showed that strains YM55T and SK5 were most closely related to the type strains of Kribbella sindirgiensis and Kribbella soli, while strain YM53T was most closely related to the type strain of Kribbella pittospori. Digital DNA-DNA hybridisation and Average Nucleotide Identity (ANI) analyses showed that strains YM55T and SK5 belong to the same genomic species (OrthoANI value = 98.4%), but are distinct from the genomic species represented by the type strains of K. sindirgiensis (OrthoANI values < 95.6%) and K. soli (OrthoANI values < 91.4%). Strain YM53T is distinct from the genomic species represented by the type strain of K. pittospori (OrthoANI value = 94.0%). Phenotypic comparisons showed that strains YM55T and SK5 are distinct from the type strains of K. sindirgiensis and K. soli and that strain YM53T is distinct from the type strain of K. pittospori. Strains YM53T and YM55T are thus presented as the type strains of novel species, for which the names Kribbella capetownensis sp. nov. (= DSM 29426T = NRRL B-65062T) and Kribbella speibonae sp. nov. (= DSM 29425T = NRRL B-59161T), respectively, are proposed.

Kribbella jiaozuonensis sp. nov., a novel actinomycete isolated from soil.

A novel actinobacterium, designated strain NEAU-THZ27T, was isolated from soil collected from the Cornel peak in Jiaozuo, Henan Province, PR China and characterized using a polyphasic approach. Morphological and chemotaxonomic characteristics of the strain coincided with those of members of the genusKribbella. The results of 16S rRNA gene sequence analysis showed that strain NEAU-THZ27T belongs to the genus Kribbella and was most closely related to Kribbella podocarpi YPL1T (98.96 %), Kribbella karoonensis Q41T (98.89 %), Kribbella aluminosa HKI 0478T (98.86%) and Kribbella hippodromi S1.4T (98.85 %), similarities to other type strains of species of the genus Kribbella were found to be less than 98.7 %. Phylogenetic analyses using the 16S rRNA gene sequence and multilocus sequence analysis using the concatenated gene sequences of the gyrB, rpoB, recA, relA and atpD genes all showed that the strain formed a separate branch in the genus Kribbella. The cell wall contained ll-diaminopimelic acid as the major diamino acid and the whole-cell hydrolysates were ribose and glucose. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol. The predominant menaquinone was MK-9(H4). Major fatty acids were iso-C16 : 0, iso-C14 : 0 and anteiso-C15 : 0, these chemotaxonomic data supported the affiliation of strain NEAU-THZ27T to the genus Kribbella. The DNA G+C content was 68.0 mol%. Furthermore, the strain could be clearly distinguished by concatenated gene genetic distances, the combination of DNA-DNA hybridization results and some phenotypic characteristics. Therefore, it is proposed that strain NEAU-THZ27T represents a novel species of the genus Kribbella, for which the name Kribbella jiaozuonensis sp. nov. is proposed. The type strain is NEAU-THZ27T (=CGMCC 4.7504T=DSM 105535T).

Kribbella turkmenica sp. nov., isolated from the Karakum Desert.

A novel actinobacterial strain, designated 16K104T, was isolated from desert soil collected from the Karakum Desert and characterized using a polyphasic approach to clarify its taxonomic position. Strain 16K104T was found to have chemotaxonomic and morphological properties consistent with classification in the genus Kribbella. The strain shared the highest 16S rRNA gene sequence similarity with Kribbella albertanoniae BC640T (99.2 %), and formed a branch with Kribbella antibiotica YIM 31530T in the 16S rRNA gene phylogenetic tree. Multilocus sequence analysis (MLSA) using five housekeeping genes (gyrB, rpoB, relA, recA and atpD) for comparing the strain with all Kribbella type strains showed that the MLSA distances of strain 16K104T to the closely related type strains of the genus were much higher than the 0.04 threshold. The organism was found to contain ll-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan. The whole-cell sugars were identified as ribose and glucose. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol and phosphatidylinositol. The predominant menaquinone was MK-9(H4). The major fatty acids were iso-C16 : 0, anteiso-C15:0, iso-C15 : 0 and iso-C17 : 0. The results of digital DNA-DNA hybridization and average nucleotide identity analyses, in addition to MLSA phylogenetic distances, confirmed that the strain represents a novel species of the genus Kribbella, for which the name Kribbella turkmenica sp. nov. is proposed. The type strain is 16K104T (=JCM 32914T=KCTC 49224T).

Kribbella monticola sp. nov., a novel actinomycete isolated from soil.

A novel actinobacterium, designated strain NEAU-SW521T, was isolated from soil collected from Xianglu Mountain, Heilongjiang province, north PR China. The results of analysis of the 16S rRNA gene indicated that NEAU-SW521T represented a member of the genus Kribbella. The results of phylogenetic analyses using the 16S rRNA gene and multilocus sequence analysis using the concatenated gene sequences of the gyrB, rpoB, relA, recA and atpD genes all indicated that the strain formed a clade with Kribbella alba DSM 15500T (99.16 %), Kribbella ginsengisoli JCM 16928T (98.96 %), Kribbella catacumbae JCM 14968T (98.82 %), Kribbella sancticallisti JCM 14969T (98.62 %), Kribbella qitaiheensis NEAU-GQTH2-3T (98.61 %) and Kribbella koreensis JCM 10977T (98.47 %). The cell wall contained ll-diaminopimelic acid as the major diamino acid and the whole-cell hydrolysates were ribose, glucose and galactose. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and an unidentified phospholipid. The predominant menaquinone was MK-9(H4). Major fatty acids were iso-C16 : 0 and anteiso-C15 : 0. These chemotaxonomic data supported the affiliation of NEAU-SW521T to the genus Kribbella. The DNA G+C content was 67.8 mol%. Furthermore, the strain could be clearly distinguished by concatenated gene genetic distances, the combination of DNA-DNA hybridization results and some phenotypic characteristics. Therefore, it is proposed that NEAU-SW521T represents a novel species of the genus Kribbella, for which the name Kribbellamonticola sp. nov. is proposed. The type strain is NEAU-SW521T (=CGMCC 4.7465T=DSM 105770T).

Isolation and screening of rare Actinobacteria, a new insight for finding natural products with antivascular calcification activity.

AIM: Vascular calcification (VC) is a significant pathological process in some life-threatening diseases. Several pathological mechanisms, including transdifferentiation of vascular smooth muscle cells to osteoblast-like cells and apoptosis are involved in VC. Compounds with an inhibitory effect on these processes are potentially efficient medications. In consideration of the multiple biological activities of Actinobacteria, this research was aimed at finding anti-VC metabolite-producing Actinobacteria. METHODS AND RESULTS: After the isolation and identification of Actinobacteria, the effect of their fermentation broth extracts on the apoptosis rate was measured using various methods, for example, ethidium bromide/acridine orange staining, DNA laddering and diphenylamine assays. The effect of the most effective fermentation broth extract of Actinobacteria (FBEA) on the mRNA expression of runt-related transcription factor 2 (Runx2) and osteopontin (OPN) was examined. Finally, the most effective FBEA was fractionated and the chemical composition of anti-VC fractions was analysed using GC-MS. Various VC inhibition rates were observed in the tested FBEA (20 µg ml-1 ; 17·9-60·15%). The inhibition of DNA fragmentation was 7-48%. The FBE with the greatest anticalcification activity belonged to Kribbella sp. UTMC 267 and, according to 16S rRNA analysis, Kribbella sancticallisti with a similarity of 98·53% is its nearest neighbour. The FBE of Kribbella sp. UTMC 267 reduced Runx2 mRNA expression by 2·95-fold and OPN mRNA expression by 28·57-fold, both of which are considered significant (P < 0·05). Finally, GC-MS analysis showed the existence of potent anti-oxidative and anti-inflammation agents in FBE of Kribbella sp. UTMC 267. CONCLUSIONS: Actinobacterial metabolites can provide a new strategy for treating VC diseases by reducing the expression of osteogenic genes, the apoptosis rate and oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the therapeutic potential of Kribbella sp. metabolites and Actinobacteria as a new natural source for drug discovery programs in the nonantibiotic bioactivity field.

Kribbella sindirgiensis sp. nov. isolated from soil.

A Kribbella strain FSN23T was isolated from soil sample which was collected from Caygoren Dam lakeside located in Sindirgi, Turkey. The isolate was investigated using a polyphasic approach consisting of numeric, chemotaxonomic and molecular analysis. The isolate indicated chemotaxonomic, morphological and phylogenetic properties associated with members of the genus Kribbella. Phylogenetic analysis based on the 16S rRNA sequence of the strain demonstrated that the strain forms a subclade with K. aluminosa HKI 0478T and K. jejuensis HD9T. The organism formed an extensively branched substrate and aerial hyphae which generated spiral chains of spores with smooth surfaces. The cell wall contained LL-diaminopimelic acid, and the whole cell sugars were glucose and ribose along with trace amounts of mannose. The polar lipids were identified as phosphatidylglycerol, diphosphatidylglycerol, four unidentified lipids and five unidentified polar lipids. The predominant menaquinone was MK-9(H4). The major cellular fatty acids were anteiso-C15:0 and iso-C16:0. Polyphasic taxonomy properties confirm that strain FSN23T represents a novel Kribbella taxon distinguished from closely related type strains. Hence, strain FSN23T (=KCTC 29220T = DSM 27082T) is proposed as the type strain of a novel species with the name Kribbella sindirgiensis sp. nov.

Isolation and screening of proangiogenic and antiangiogenic metabolites producing rare actinobacteria from soil.

AIMS: Angiogenesis is a physiological process that has important impacts on the pathology and healing of various diseases, and its induction or inhibition by bioactive actinobacterial metabolites can help the treatment of some diseases. In this study, the effects of actinobacterial extract in the process of angiogenesis have been explored. METHODS AND RESULTS: In this research, proangiogenic and antiangiogenic metabolites producing actinobacteria were isolated from soil samples and their fermentation broth were extracted and after evaluation of their toxicity by MTT assay, antiangiogenic and proangiogenic activities were screened against human umbilical vein endothelial cells (HUVECs) by in vitro tube formation and migration assay. Isolated strains were identified through molecular techniques. The results showed that Nocardiopsis arvandica UTMC 103 and Nonomuraea sp. UTMC 2180 extracts had a high potential of anti-angiogenic activity on HUVECs. CONCLUSIONS: For the first time proangiogenic potency of a rare actinobacterium, Kribbella sp. UTMC 522, was reported, and N. arvandica UTMC 103 and Nonomuraea sp. UTMC 2180 extracts inhibits the proliferation, migration and angiogenesis activity of HUVECs with reasonable potency. SIGNIFICANCE AND IMPACT OF THE STUDY: Metabolites of the introduced rare actinobacteria are potent proangiogenic and angiogenic inhibitors. Identification of angiogenic-antiangiogenic mechanisms and purification of the extracts would be useful in therapeutic angiogenesis.

Kribbella soli sp. nov., isolated from soil.

A novel actinobacterial strain, designated FMN22T, was isolated from soil and characterised using a polyphasic approach. Strain FMN22T showed high 16S rRNA gene sequence similarity to Kribbella karoonensis Q41T (99.3%), Kribbella shirazensis UTMC 693T (99.0%), Kribbella aluminosa HKI 0478T (98.9%) and Kribbella hippodromi S1.4T (98.6%). Phylogenetic analysis using the 16S rRNA and concatenated gene (gyrB, rpoB, relA, recA and atpD) sequences showed that strain FMN22T is closely related to the type strains of K. karoonensis DSM 17344T, K. shirazensis UTMC 693T, K. aluminosa HKI 0478T, K. hippodromi S1.4T, Kribbella jejuensis HD9T and Kribbella solani DSA1T. Based on concatenated gene genetic distances analysis, strain FMN22T is distinct from all Kribbella type strains. DNA-DNA hybridization experiments with closely related type strains, were found to be 59.2 ± 2.4, 54.8 ± 2.1, 16.4 ± 2.3 and 38.6 ± 2.5%, relatedness to K. karoonensis DSM 17344T, K. shirazensis DSM 45490T, K. aluminosa DSM 18824T and K. jejuensis DSM 17305T, respectively. The cell wall peptidoglycan contained LL-diaminopimelic acid, and whole cell sugars were glucose, mannose and ribose. The predominant menaquinone was MK-9(H4). The major phospholipids are diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol. Major fatty acids are anteiso-C15:0 and iso-C16:0. These chemotaxonomic traits are in good agreement with those known for representatives of the genus Kribbella. A combination of DNA-DNA hybridization results and phenotypic properties demonstrated that strain FMN22T can be clearly distinguished from all close phylogenetic relatives. Therefore, strain FMN22T (=DSM 27132T = KCTC 29219T) is considered to be the type strain of a novel species of the genus Kribbella, for which the name Kribbella soli is proposed.

Kribbella deserti sp. nov., isolated from rhizosphere soil of Ammopiptanthus mongolicus.

A Gram-stain-positive, aerobic bacterial strain, designated SL15-1T, was isolated from desert soil which was sampled from the rhizosphere of Ammopiptanthus mongolicus, Hangjin Banner, Ordos, Inner Mongolia, northern China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SL15-1T was clustered with Kribbella strains, sharing the highest similarity of 16S rRNA gene sequence (96.97 %) with Kribbella sandramycini DSM 15626T. Strain SL15-1T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, an unknown phospholipid, an unknown lipid and two unknown aminophospholipids as the major polar lipids. MK-9(H4) was the predominant menaquinone, while anteiso-C15 : 0, iso-C16 : 0, C17 : 1ω8c and iso-C14 : 0 were the major cellular fatty acids. Its genomic DNA G+C content was 65.3 mol%. The results of physiological and biochemical tests allowed the discrimination of strain SL15-1T from its phylogenetic relatives. Kribbella deserti sp. nov. is therefore proposed with strain SL15-1T (=CGMCC 1.15906T=KCTC 39825T) as the type strain.