Putative Gene Cluster Research Articles

Genomic Analysis of a Novel Antarctic Bacterium, Cyrobacterium sp.SO2 Provides Insights into its Genomic Potential for Production of Antimicrobial Compounds

A novel strain of Cryobacterium designated as SO2, was isolated from the Antarctic. Hence, this study was undertaken to gain further insight into the antimicrobial compounds and secondary metabolites produced by Cryobacterium sp. SO2. It was found that strain SO2 is a Gram-positive that exhibits an irregular rod shape, which formed yellow to orange pigmented colonies on semi-solid media. Strain SO2 grows at temperatures ranging from 4 to 25 ºC. It has a complete genomic size of 4.097 Mb. SO2 has a DNA G+C content of 68.43%, and genomic annotation showed that the genome contained 3,862 CDS, 10 rRNA, 55 tRNA and 1 tm-RNA. Phylogenetic and OrthoANI analysis suggested Cryobacterium sp. strains SO1, N22, TMB1-8, LW097, TMN39-1, C. zongtaiiTMN-42, C. arcticumPAMC27867 and C. soli GCJ02 as its closest phylogenetic neighbour. Genome annotation shows that strain SO2 confers β-lactamase class A, cephalosporin-C deacetylases, and 27 drug-resistance encoding genes, and allows resistance to ceftazidime. Functional annotation identifies 28.74% of predicted genes are of unknown functions. Genome mining indicates that there are six putative secondary metabolite gene clusters in strain SO2. They are made up of RRE-containing, terpene, beta-lactone, T3PKS, NAPAA, and 2dos. This finding shows strain SO2 harbours genes that may be involved in the production of compounds with antibacterial and antioxidant activities.

Genomic and phenotypic characterization of a red-pigmented strain of Massilia frigida isolated from an Antarctic microbial mat.

The McMurdo Dry Valleys of Antarctica experience a range of selective pressures, including extreme seasonal variation in temperature, water and nutrient availability, and UV radiation. Microbial mats in this ecosystem harbor dense concentrations of biomass in an otherwise desolate environment. Microbial inhabitants must mitigate these selective pressures via specialized enzymes, changes to the cellular envelope, and the production of secondary metabolites, such as pigments and osmoprotectants. Here, we describe the isolation and characterization of a Gram-negative, rod-shaped, motile, red-pigmented bacterium, strain DJPM01, from a microbial mat within the Don Juan Pond Basin of Wright Valley. Analysis of strain DJMP01's genome indicates it can be classified as a member of the Massilia frigida species. The genome contains several genes associated with cold and salt tolerance, including multiple RNA helicases, protein chaperones, and cation/proton antiporters. In addition, we identified 17 putative secondary metabolite gene clusters, including a number of nonribosomal peptides and ribosomally synthesized and post-translationally modified peptides (RiPPs), among others, and the biosynthesis pathway for the antimicrobial pigment prodigiosin. When cultivated on complex agar, multiple prodiginines, including the antibiotic prodigiosin, 2-methyl-3-propyl-prodiginine, 2-methyl-3-butyl-prodiginine, 2-methyl-3-heptyl-prodiginine, and cycloprodigiosin, were detected by LC-MS. Genome analyses of sequenced members of the Massilia genus indicates prodigiosin production is unique to Antarctic strains. UV-A radiation, an ecological stressor in the Antarctic, was found to significantly decrease the abundance of prodiginines produced by strain DJPM01. Genomic and phenotypic evidence indicates strain DJPM01 can respond to the ecological conditions of the DJP microbial mat, with prodiginines produced under a range of conditions, including extreme UV radiation.

Iodate respiration by Azoarcus sp. DN11 and its potential use for removal of radioiodine from contaminated aquifers.

Azoarcus sp. DN11 was previously isolated from gasoline-contaminated groundwater as an anaerobic benzene-degrading bacterium. Genome analysis of strain DN11 revealed that it contained a putative idr gene cluster (idrABP1P2 ), which was recently found to be involved in bacterial iodate (IO3 -) respiration. In this study, we determined if strain DN11 performed iodate respiration and assessed its potential use to remove and sequester radioactive iodine (129I) from subsurface contaminated aquifers. Strain DN11 coupled acetate oxidation to iodate reduction and grew anaerobically with iodate as the sole electron acceptor. The respiratory iodate reductase (Idr) activity of strain DN11 was visualized on non-denaturing gel electrophoresis, and liquid chromatography-tandem mass spectrometry analysis of the active band suggested the involvement of IdrA, IdrP1, and IdrP2 in iodate respiration. The transcriptomic analysis also showed that idrA, idrP1 , and idrP2 expression was upregulated under iodate-respiring conditions. After the growth of strain DN11 on iodate, silver-impregnated zeolite was added to the spent medium to remove iodide from the aqueous phase. In the presence of 200 μM iodate as the electron acceptor, more than 98% of iodine was successfully removed from the aqueous phase. These results suggest that strain DN11 is potentially helpful for bioaugmentation of 129I-contaminated subsurface aquifers.

Characterization of the heteropolysaccharides produced by Liquorilactobacillus sicerae CUPV261 and Secundilactobacillus collinoides CUPV237 isolated from cider

Some lactic acid bacteria (LAB) strains isolated from alcoholic beverages are able to produce exopolysaccharides (EPS). The present work focuses on the physico-chemical characterization of the heteropolysaccharides (HePS) produced by Liquorilactobacillus sicerae CUPV261T (formerly known as Lactobacillus sicerae) and Secundilactobacillus collinoides CUPV237 (formerly known as Lactobacillus collinoides) strains isolated from cider. Genome sequencing and assembly enabled the identification of at least four putative HePS gene clusters in each strain, which correlated with the ability of both strains to secrete EPS. The crude EPS preparation from CUPV261T contained glucose, galactose and rhamnose, and that of CUPV237 was composed of glucose, galactose and N-acetylglucosamine. Both EPS were mixtures of HePS of different composition, with two major soluble components of average molecular weights (Mw) in the range of 106 and 104 g.mol−1. These HePS were resistant to gastric stress conditions in an in vitro model, and they significantly reduced zebrafish larvae mortality in an in vivo model of inflammatory bowel disease.

Vibrio type III secretion system 2 is not restricted to the Vibrionaceae and encodes differentially distributed repertoires of effector proteins.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. A distinctive feature of the O3:K6 pandemic clone, and its derivatives, is the presence of a second, phylogenetically distinct, type III secretion system (T3SS2) encoded within the genomic island VPaI-7. The T3SS2 allows the delivery of effector proteins directly into the cytosol of infected eukaryotic cells to subvert key host-cell processes, critical for V. parahaemolyticus to colonize and cause disease. Furthermore, the T3SS2 also increases the environmental fitness of V. parahaemolyticus in its interaction with bacterivorous protists; hence, it has been proposed that it contributed to the global oceanic spread of the pandemic clone. Several reports have identified T3SS2-related genes in Vibrio and non-Vibrio species, suggesting that the T3SS2 gene cluster is not restricted to the Vibrionaceae and can mobilize through horizontal gene transfer events. In this work, we performed a large-scale genomic analysis to determine the phylogenetic distribution of the T3SS2 gene cluster and its repertoire of effector proteins. We identified putative T3SS2 gene clusters in 1130 bacterial genomes from 8 bacterial genera, 5 bacterial families and 47 bacterial species. A hierarchical clustering analysis allowed us to define six T3SS2 subgroups (I-VI) with different repertoires of effector proteins, redefining the concepts of T3SS2 core and accessory effector proteins. Finally, we identified a subset of the T3SS2 gene clusters (subgroup VI) that lacks most T3SS2 effector proteins described to date and provided a list of 10 novel effector candidates for this subgroup through bioinformatic analysis. Collectively, our findings indicate that the T3SS2 extends beyond the family Vibrionaceae and suggest that different effector protein repertories could have a differential impact on the pathogenic potential and environmental fitness of each bacterium that has acquired the Vibrio T3SS2 gene cluster.

RNA-seq analysis revealed considerable genetic diversity and enabled the development of specific KASP markers for Psathyrostachys huashanica.

Psathyrostachys huashanica, which grows exclusively in Huashan, China, is an important wild relative of common wheat that has many desirable traits relevant for wheat breeding. However, the poorly characterized interspecific phylogeny and genomic variations and the relative lack of species-specific molecular markers have limited the utility of P. huashanica as a genetic resource for enhancing wheat germplasm. In this study, we sequenced the P. huashanica transcriptome, resulting in 50,337,570 clean reads that were assembled into 65,617 unigenes, of which 38,428 (58.56%) matched at least one sequence in public databases. The phylogenetic analysis of P. huashanica, Triticeae species, and Poaceae species was conducted using 68 putative orthologous gene clusters. The data revealed the distant evolutionary relationship between P. huashanica and common wheat as well as the substantial diversity between the P. huashanica genome and the wheat D genome. By comparing the transcriptomes of P. huashanica and Chinese Spring, 750,759 candidate SNPs between P. huashanica Ns genes and their common wheat orthologs were identified. Among the 90 SNPs in the exon regions with different functional annotations, 58 (64.4%) were validated as Ns genome-specific SNPs in the common wheat background by KASP genotyping assays. Marker validation analyses indicated that six specific markers can discriminate between P. huashanica and the other wheat-related species. In addition, five markers are unique to P. huashanica, P. juncea, and Leymus species, which carry the Ns genome. The Ns genome-specific markers in a wheat background were also validated regarding their specificity and stability for detecting P. huashanica chromosomes in four wheat-P. huashanica addition lines. Four and eight SNP markers were detected in wheat-P. huashanica 2Ns and 7Ns addition lines, respectively, and one marker was specific to both wheat-P. huashanica 3Ns, 4Ns, and 7Ns addition lines. These markers developed using transcriptome data may be used to elucidate the genetic relationships among Psathyrostachys, Leymus, and other closely-related species. They may also facilitate precise introgressions and the high-throughput monitoring of P. huashanica exogenous chromosomes or segments in future crop breeding programs.

Secondary Metabolic Potential of Kutzneria.

Kutzneria is a rare genus of Actinobacteria that harbors a variety of secondary metabolite gene clusters and produces several interesting types of bioactive secondary metabolites. Recent efforts have partially elucidated the biosynthetic pathways of some of these bioactive natural products, suggesting the diversity and specificity of secondary metabolism within this genus. Here, we summarized the chemical structures, biosynthetic pathways, and key metabolic enzymes of the secondary metabolites isolated from Kutzneria strains. In-depth comparative genomic analysis of all six available high-quality Kutzneria genomes revealed that the majority (77%) of the biosynthetic gene cluster families of Kutzneria were untapped and identified homologues of key metabolic enzymes in the putative gene clusters, including cytochrome P450s, halogenases, and flavin-dependent N-hydroxylases. The present study suggests that Kutzneria exhibits great potential to synthesize novel secondary metabolites, encodes a variety of valuable metabolic enzymes, and also provides valuable information for the targeted discovery and biosynthesis of novel natural products from Kutzneria.

Thermophilin 13: In Silico Analysis Provides New Insight in Genes Involved in Bacteriocin Production.

Bacteriocins are a large family of ribosomally synthesised proteinaceous toxins that are produced by bacteria and archaea and have antimicrobial activity against closely related species to the producer strain. Antimicrobial proteinaceous compounds are associated with a wide range of applications, including as a pathogen inhibitor in food and medical use. Among the several lactic acid bacteria (LAB) commonly used in fresh and fermented food preservation, Streptococcus thermophilus is well known for its importance as a starter culture for yoghurt and cheese. Previous studies described the bacteriocin thermophilin 13 exclusively in S. thermophilus SFi13 and the genes encoding its production as an operon consisting of two genes (thmA and thmB). However, the majority of bacteriocins possess a complex production system, which involves several genes encoding dedicated proteins with relatively specific functions. Up to now, far too little attention has been paid to the genes involved in the synthesis, regulation and expression of thermophilin 13. The aim of the present study, using in silico gene mining, was to investigate the presence of a regulation system involved in thermophilin 13 production. Results revealed the dedicated putative bacteriocin gene cluster (PBGC), which shows high similarity with the class IIb bacteriocins genes. This newly revealed PBGC, which was also found within various strains of Streptococcus thermophilus, provides a new perspective and insights into understanding the mechanisms implicated in the production of thermophilin 13.

Mucilaginibacter straminoryzae sp. nov., isolated from rice straw used for growing periphyton.

A rod-shaped, non-motile, Gram-negative bacterium, strain RS28T, was isolated from rice straw used as material for periphyton growth. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain RS28T was affiliated with the genus Mucilaginibacter and had the highest sequence similarity to Mucilaginibacter ginkgonis HMF7856T (96.47 %) and Mucilaginibacter polytrichastri DSM 26907T (96.12 %). Strain RS28T was found to grow at pH 5.5-8.0, 17-40 °C and in the presence of 0-1.5 % (w/v) NaCl. Strain RS28T contained summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C15 : 0 and iso-C17 : 0 3-OH as the major fatty acids (> 10.0 %). The major polar lipids were phosphatidylethanolamine, two unidentified phospholipids, two unidentified aminophospholipids, three unidentified aminolipids and one unidentified lipid. The respiratory quinone was menaquinone 7. The genomic DNA G+C content was 44.7 mol%. Strain RS28T possessed six putative secondary metabolite gene clusters involved in the synthesis of resorcinol, NRPS-like, terpene, lassopeptide, T3PKS and arylpolyene. On the basis of the phenotypic, chemotaxonomic, and phylogenetic characteristics, strain RS28T represents a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter straminoryzae sp. nov. is proposed. The type strain is RS28T (=KCTC 92039T=LMG 32424T).

Identification and heterologous expression of the globomycin biosynthetic gene cluster

Globomycin is a cyclic lipodepsipeptide originally isolated from several Streptomyces species which displays strong and selective antibacterial activity against Gram-negative pathogens. Its mode of action is based on the competitive inhibition of the lipoprotein signal peptidase II (LspA), which is absent in eukaryotes and considered an attractive target for the development of new antibiotics. Despite its interesting biological properties, the gene cluster encoding its biosynthesis has not yet been identified. In this study we employed a genome-mining approach in the globomycin-producing Streptomyces sp. CA-278952 to identify a candidate gene cluster responsible for its biosynthesis. A null mutant was constructed using CRISPR base editing where production was abolished, strongly suggesting its involvement in the biosynthesis. The putative gene cluster was then cloned and heterologously expressed in Streptomyces albus J1074 and Streptomyces coelicolor M1146, therefore unambiguously linking globomycin and its biosynthetic gene cluster. Our work paves the way for the biosynthesis of new globomycin derivatives with improved pharmacological properties.

Discovery of dual PKS involved in sclerotiorin biosynthesis in Penicillium meliponae using genome mining and gene knockout.

Fungi of the genus Penicillium section Sclerotiora have as their main characteristic the presence of orange-pigmented mycelium, which is associated with sclerotiorin, a chlorinated secondary metabolite of the azaphilone subclass of polyketides. Sclerotiorin presents anti-diabetes, antioxidant, anti-inflammatory, anti-Alzheimer, antiviral, and antimicrobial activities, which has always attracted the attention of researchers worldwide. During our ongoing search for azaphilone-producing Amazonian fungi, the strain of Penicillium MMSRG-058 was isolated as an endophyte from the roots of Duguetia stelechantha and showed great capacity for producing sclerotiorin-like metabolites. Using multilocus phylogeny, this strain was identified as Penicillium meliponae. Moreover, based on the genome mining of this strain through the reverse approach, a cluster of putative biosynthetic genes (BGC) responsible for the biosynthesis of sclerotiorin-like metabolites (scl cluster) was identified. The knockout of the sclA (highly reducing PKS) and sclI (non-reducing PKS) genes resulted in mutants with loss of mycelial pigmentation and terminated the biosynthesis of sclerotiorin-like metabolites: geumsanol B, chlorogeumsanol B, 7-deacetylisochromophilone VI, isochromophilone VI, ochrephilone, isorotiorin, and sclerotiorin. Based on these results, a biosynthetic pathway was proposed considering the homology of BGC scl genes with the azaphilone BGCs that have already been functionally characterized.

Microbial Contributions to Iodide Enrichment in Deep Groundwater in the North China Plain.

Microorganisms play crucial roles in the global iodine cycling through iodine oxidation, reduction, volatilization, and deiodination. In contrast to iodate formation in radionuclide-contaminated groundwater by the iodine-oxidizing bacteria, microbial contribution to the formation of high level of iodide in geogenic high iodine groundwater is poorly understood. In this study, our results of comparative metagenomic analyses of deep groundwater with typical high iodide concentrations in the North China Plain revealed the existence of putative dissimilatory iodate-reducing idrABP1P2 gene clusters in groundwater. Heterologous expression and characterization of an identified idrABP1P2 gene cluster confirmed its functional role in iodate reduction. Thus, microbial dissimilatory iodate reduction could contribute to iodide formation in geogenic high iodine groundwater. In addition, the identified iron-reducing, sulfur-reducing, sulfur-oxidizing, and dehalogenating bacteria in the groundwater could contribute to the release and production of iodide through the reductive dissolution of iron minerals, abiotic iodate reduction of derived ferrous iron and sulfide, and dehalogenation of organic iodine, respectively. These microbially mediated iodate reduction and organic iodine dehalogenation processes may also result in the transformation among iodine species and iodide enrichment in other geogenic iodine-rich groundwater systems worldwide.

Stereodivergent Hydroxylation of Berkeleylactones by Penicillium turbatum.

Penicillium turbatum has previously been reported to produce A26771B, a 16-membered macrocyclic polyketide with activity against Gram-positive bacteria, mycoplasma, and fungi, as well as the structurally related compounds berkeleylactone E and berkeleylactones I-O. In this work, large-scale cultivation of P. turbatum NRRL 5630 on rice yielded seven new berkeleylactone analogues, berkeleylactone E methyl ester, 14-epi-berkeleylactone F, berkeleylactones P-R, 12-epi-berkeleylactone Q, and 13-epi-berkeleylactone R, and six previously reported analogues, A26771B and berkeleylactones E-G and J-K. The structures of the berkeleylactones were elucidated by detailed analysis of spectroscopic data, molecular modeling, and comparison with literature values. Interestingly, six of the berkeleylactone analogues were isolated as pairs of hydroxy epimers, highlighting how Nature can exploit stereodivergence in biosynthetic pathways to increase chemical diversity. The genome of P. turbatum was sequenced, and a putative gene cluster (bekl) responsible for the biosynthesis of the berkeleylactones was identified. The new berkeleylactone analogues exhibited no significant biological activity against a panel of bacteria, fungi, the parasite Giardia duodenalis, or NS-1 murine myeloma cells, suggesting a hitherto undiscovered biological role.

Genetic potential for exopolysaccharide synthesis in activated sludge bacteria uncovered by genome-resolved metagenomics

A good floc formation of activated sludge (AS) is crucial for solid-liquid separation and production of clean effluent during wastewater treatment. Floc formation is partly controlled by self-produced extracellular polymeric substances (EPS) such as exopolysaccharides, proteins, and nucleic acids. Little is known about the composition, structure, and function of EPS in AS and which bacteria produce them. To address this knowledge gap for the exopolysaccharides, we took advantage of 1083 high-quality metagenome-assembled genomes (MAGs) obtained from 23 Danish wastewater treatment plants. We investigated the genomic potential for exopolysaccharide biosynthesis in bacterial species typical in AS systems based on genome mining and gene synteny analyses. Putative gene clusters associated with the biosynthesis of alginate, cellulose, curdlan, diutan, hyaluronic acids, Pel, poly-β-1,6-N-acetyl-d-glucosamine (PNAG), Psl, S88 capsular polysaccharide, salecan, succinoglycan, and xanthan were identified and linked to individual MAGs, providing a comprehensive overview of the genome-resolved potential for these exopolysaccharides in AS bacteria. The approach and results provide a starting point for a more comprehensive understanding of EPS composition in wastewater treatment systems, which may facilitate a more refined regulation of the activated sludge process for improved stability.

Transcription Factor VdCf2 Regulates Growth, Pathogenicity, and the Expression of a Putative Secondary Metabolism Gene Cluster in Verticillium dahliae.

Transcription factors (TFs) bind to the promoters of target genes to regulate gene expression in response to different stimuli. The functions and regulatory mechanisms of transcription factors (TFs) in Verticillium dahliae are, however, still largely unclear. This study showed that a C2H2-type zinc finger TF, VdCf2 (V. dahliae chorion transcription factor 2), plays key roles in V. dahliae growth, melanin production, and virulence. Transcriptome sequencing analysis showed that VdCf2 was involved in the regulation of expression of genes encoding secreted proteins, pathogen-host interaction (PHI) homologs, TFs, and G protein-coupled receptors (GPCRs). Furthermore, VdCf2 positively regulated the expression of VdPevD1 (VDAG_02735), a previously reported virulence factor. VdCf2 thus regulates the expression of several pathogenicity-related genes that also contribute to virulence in V. dahliae. VdCf2 also inhibited the transcription of the Vd276-280 gene cluster and interacted with two members encoding proteins (VDAG_07276 and VDAG_07278) in the gene cluster. IMPORTANCE Verticillium dahliae is an important soilborne phytopathogen which can ruinously attack numerous host plants and cause significant economic losses. Transcription factors (TFs) were reported to be involved in various biological processes, such as hyphal growth and virulence of pathogenic fungi. However, the functions and regulatory mechanisms of TFs in V. dahliae remain largely unclear. In this study, we identified a new transcription factor, VdCf2 (V. dahliae chorion transcription factor 2), based on previous transcriptome data, which participates in growth, melanin production, and virulence of V. dahliae. We provide evidence that VdCf2 regulates the expression of the pathogenicity-related gene VdPevD1 (VDAG_02735) and Vd276-280 gene cluster. VdCf2 also interacts with VDAG_07276 and VDAG_07278 in this gene cluster based on a yeast two-hybrid and bimolecular fluorescence complementation assay. These results revealed the regulatory mechanisms of a pivotal pathogenicity-related transcription factor, VdCf2 in V. dahliae.

Draft Genome Sequence of Pseudomonas sp. Strain MWU13-3659, Isolated from Commercial Cranberry Bog Soil in Massachusetts, USA.

Pseudomonas sp. strain MWU13-3659 was isolated from cultivated cranberry bog soil in Massachusetts, USA. Its closest known relative is Pseudomonas entomophila (digital DNA-DNA hybridization [d4 formula] value of 57.2% and average nucleotide identity based on BLAST value of 93.90), and its genome contains putative gene clusters for the production of polyketides, siderophores, and cyclic lipopeptides that have insecticidal activity in other proteobacteria.

First Finding of a Methicillin-Resistant Staphylococcus aureus (MRSA) t304/ST6 from Bovine Clinical Mastitis.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) comprises a global threat to humans and animals. Here, we report and characterize the MRSA t304/ST6 variant which, to our knowledge, represents the first case found in bovine clinical mastitis. In general, the MRSA t304/ST6 variant is rarely described in livestock, contrary to humans where it is widely recognized. Phenotypic and genotypic resistance profiling showed that the bovine-MRSA t304/ST6 isolate expressed low susceptibility toward cefoxitin (MICcefoxitin = 16 µg/mL) and carried the mecA resistance gene in the SCCmec IVa. The bovine-MRSA t304/ST6 isolate carried a plasmid similar to that which has been frequently observed among human-MRSA t304/ST6 isolates in Denmark (GenBank accession no. NZ_CP047022). In addition, a Staphylococcus prophage 3 (ϕSA3) was detected, encoding an immune evasion cluster (IEC) of putative virulence genes associated with human host-specificity (sea, sak, and scn). Taken together, these findings suggest that the MRSA t304/ST6 found in this study represents a recent host-jump event, with human to cow transmission. This study emphasizes the importance of and the need for performance of antimicrobial resistance surveillance among bovine mastitis pathogens, including S. aureus and MRSA.

Draft Genome Sequence of Pseudomonas sp. Strains MWU12-2319 and MWU12-2311, Isolated from a Wild Cranberry Bog in the Cape Cod National Seashore.

Pseudomonas sp. strains MWU12-2319 and MWU12-2311 were isolated from the soil of a wild cranberry bog in the Cape Cod National Seashore as part of a culture-dependent bacterial population survey. The genomes exceed 7 Mbp and contain putative gene clusters for the biosurfactant orfamides A and C.

Four Novel Leaderless Bacteriocins, Bacin A1, A2, A3, and A4 Exhibit Potent Antimicrobial and Antibiofilm Activities against Methicillin-Resistant Staphylococcus aureus.

ABSTRACTMethicillin-resistant Staphylococcus aureus (MRSA) is a major bacterial pathogen that causes hospital- and community-acquired infections. Owing to its multidrug resistance, it is imperative to develop new antimicrobial agents to treat MRSA infections. In this study, using genome mining analysis and a culture-based screening method to detect bacteriocin activity, we screened a strain, Bacillus sp. TL12, which harbored a putative leaderless bacteriocin gene cluster (bac gene cluster) and exhibited potent anti-MRSA activity. The antimicrobial agents, products of the bac gene cluster, were purified and identified as four novel leaderless bacteriocins: bacin A1, A2, A3, and A4. Bacin A2 was evaluated as a representative antimicrobial agent and showed remarkable antimicrobial activity against S. aureus, MRSA, and the foodborne pathogens Listeria monocytogenes and Bacillus cereus. Mechanistic experiments revealed that bacin A2 damaged cell membranes and exhibited bactericidal activity against MRSA. Bacin A2 effectively inhibited the formation of S. aureus and MRSA biofilms (>0.5× MIC) and killed the cells in their established biofilms (>4× MIC). The hemolytic and NIH/3T3 cytotoxicity assay results for bacin A2 confirmed its biosafety. Thus, bacins have potential as alternative antimicrobial agents for treating MRSA infections.IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that is difficult to treat because of its resistance to several widely used antibiotics. The present study aimed to identify novel anti-MRSA bacteriocins in a prominent producer of bacteriocins, Bacillus cereus group. Four novel leaderless bacteriocins, bacin A1, A2, A3, and A4, which show potent bactericidal effect against S. aureus and MRSA, were identified in Bacillus sp. TL12. Moreover, bacins inhibited biofilm formation and killed cells in the established biofilms of S. aureus and MRSA. These findings suggest that bacins are promising alternatives to treat MRSA infections.

Genome-Based Analysis of Verticillium Polyketide Synthase Gene Clusters.

Simple SummaryFungi can produce many types of secondary metabolites, including mycotoxins. Poisonous mushrooms and mycotoxins that cause food spoilage have been known for a very long time. For example, Aspergillus flavus, which can grow on grains and nuts, produces highly toxic substances called Aflatoxins. Despite their menace to other living organisms, mycotoxins can be used for medicinal purposes, i.e., as antibiotics, growth-promoting compounds, and other kinds of drugs. These and other secondary metabolites produced by plant-pathogenic fungi may cause host plants to display disease symptoms and may play a substantial role in disease progression. Therefore, the identification and characterization of the genes involved in their biosynthesis are essential for understanding the molecular mechanism involved in their biosynthetic pathways and further promoting sustainable knowledge-based crop production. Polyketides are structurally diverse and physiologically active secondary metabolites produced by many organisms, including fungi. The biosynthesis of polyketides from acyl-CoA thioesters is catalyzed by polyketide synthases, PKSs. Polyketides play roles including in cell protection against oxidative stress, non-constitutive (toxic) roles in cell membranes, and promoting the survival of the host organisms. The genus Verticillium comprises many species that affect a wide range of organisms including plants, insects, and other fungi. Many are known as causal agents of Verticillium wilt diseases in plants. In this study, a comparative genomics approach involving several Verticillium species led us to evaluate the potential of Verticillium species for producing polyketides and to identify putative polyketide biosynthesis gene clusters. The next step was to characterize them and predict the types of polyketide compounds they might produce. We used publicly available sequences from ten species of Verticillium including V. dahliae, V. longisporum, V. nonalfalfae, V. alfalfae, V. nubilum, V. zaregamsianum, V. klebahnii, V. tricorpus, V. isaacii, and V. albo-atrum to identify and characterize PKS gene clusters by utilizing a range of bioinformatic and phylogenetic approaches. We found 32 putative PKS genes and possible clusters in the genomes of Verticillium species. All the clusters appear to be complete and functional. In addition, at least five clusters including putative DHN-melanin-, cytochalasin-, fusarielien-, fujikurin-, and lijiquinone-like compounds may belong to the active PKS repertoire of Verticillium. These results will pave the way for further functional studies to understand the role of these clusters.