Nonribosomal Peptide Synthetase Gene Research Articles

The microbiota of moon snail egg collars is shaped by host-specific factors.

Moon snails (Family: Naticidae) lay eggs using a mixture of mucus and sediment to form an egg mass commonly referred to as an egg collar. These egg collars do not appear to experience micro-biofouling or predation, and this observation led us to hypothesize that the egg collars possess a chemically rich microbiota that protect the egg collars from pathogens. Herein, we sought to gain an understanding of the bacterial composition of egg collars laid by a single species of moon snails, Neverita delessertiana, by amplifying and sequencing the 16S rRNA gene from the egg collar and sediment samples collected at four distinct geographical regions in southwest Florida. Relative abundance and non-metric multidimensional scaling plots revealed distinct differences in the bacterial composition between the egg collar and sediment samples. In addition, the egg collars had a lower α-diversity than the sediment, with specific genera being significantly enriched in the egg collars. Analysis of microorganisms consistent across two seasons suggests that Flavobacteriaceae make up a large portion of the core microbiota (36%-58% of 16S sequences). We also investigated the natural product potential of the egg collar microbiota by sequencing a core biosynthetic gene, the adenylation domains (ADs), within the gene clusters of non-ribosomal peptide synthetase (NRPS). AD sequences matched multiple modules within known NRPS gene clusters, suggesting that these compounds might be produced within the egg collar system. This study lays the foundation for future studies into the ecological role of the moon snail egg collar microbiota.IMPORTANCEAnimals commonly partner with microorganisms to accomplish essential tasks, including chemically defending the animal host from predation and/or infections. Understanding animal-microbe partnerships and the molecules used by the microbe to defend the animals from pathogens or predation has the potential to lead to new pharmaceutical agents. However, very few of these systems have been investigated. A particularly interesting system is nutrient-rich marine egg collars, which often lack visible protections, and are hypothesized to harbor beneficial microbes that protect the eggs. In this study, we gained an understanding of the bacterial strains that form the core microbiota of moon snail egg collars and gained a preliminary understanding of their natural product potential. This work lays the foundation for future work to understand the ecological role of the core microbiota and to study the molecules involved in chemically defending the moon snail eggs.

A Comparative Analysis of Different Xenorhabdus Strains Reveals a Virulent Factor, Cyclic Pro-Phe, Using a Differential Expression Profile Analysis of Non-Ribosomal Peptide Synthetases.

Entomopathogenic bacteria, classified into the genus Xenorhabdus, exhibit a dual lifestyle as mutualistic symbionts to Steinernema nematodes and as pathogens to a broad range of insects. Bacterial virulence depends on toxin proteins that induce toxemia and various immunosuppressive secondary metabolites that cause septicemia. Particularly, the immunosuppressive properties of Xenorhabdus bacteria determine the variability of their insecticidal activities. This study explored the role of peptide metabolites in virulence and its variation among six bacterial strains across three species: X. nematophila, X. bovienii, and X. hominickii. Initially, their virulence significantly varied against a susceptible lepidopteran host, Maruca vitrata, but showed less variation against a tolerant coleopteran host, Tenebrio molitor, with high median lethal bacterial doses. In M. vitrata, virulence was strongly correlated with bacterial growth rate and inhibitory activity against phospholipase A2. Secondly, the six strains differed in the compositions of their secreted secondary metabolites, analyzed by GC-MS following ethyl acetate extraction. Notably, there was significant variation in the production of di- or tetra-peptides. Highly virulent strains commonly produced the cyclic Pro-Phe (cPF). Thirdly, the expression of non-ribosomal peptide synthetase (NRPS) genes varied greatly among the strains. NRPS genes were minimally expressed in the tolerant T. molitor and highly expressed in the susceptible M. vitrata. In M. vitrata, specific NRPS genes were markedly expressed in the virulent strains. Finally, cPF demonstrated potent immunosuppressive activity against the cellular and humoral responses of M. vitrata. The addition of cPF significantly enhanced the virulence against the tolerant T. molitor. These findings suggest that immunosuppression is necessary for the pathogenicity of Xenorhabdus bacteria, wherein NRPS products play a critical role in suppressing immune-associated factors in target insects.

The impacts of ecological disturbances on the diversity of biosynthetic gene clusters in kauri (Agathis australis) soil

BackgroundThe ancient kauri (Agathis australis) dominated forests of Aotearoa New Zealand are under threat from a multitude of ecological disturbances such as forest fragmentation, biodiversity loss, climate change, and the spread of the virulent soil pathogen Phytophthora agathidicida. Taking a wider ecosystem-level approach, our research aimed to explore the impacts of forest disturbance and disease outbreaks on the biosynthetic potential and taxonomic diversity of the kauri soil microbiome. We explored the diversity of secondary metabolite biosynthetic gene clusters (BGCs) in soils from a range of kauri forests that varied according to historical disturbance and dieback expression. To characterise the diversity of microbial BGCs, we targeted the non-ribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) gene regions for sequencing using long-read PacBio® HiFi sequencing. Furthermore, the soil bacterial and fungal communities of each forest were characterized using 16 S rRNA and ITS gene region sequencing.ResultsWe identified a diverse array of naturally occurring microbial BGCs in the kauri forest soils, which may offer promising targets for the exploration of secondary metabolites with anti-microbial activity against P. agathidicida. We detected differences in the number and diversity of microbial BGCs according to forest disturbance history. Notably, soils associated with the most undisturbed kauri forest had a higher number and diversity of microbial NRPS-type BGCs, which may serve as a potential indicator of natural levels of microbiome resistance to pathogen invasion.ConclusionsBy linking patterns in microbial biosynthetic diversity to forest disturbance history, this research highlights the need for us to consider the influence of ecological disturbances in potentially predisposing forests to disease by impacting the wider health of forest soil ecosystems. Furthermore, by identifying the range of microbial BGCs present at a naturally high abundance in kauri soils, this research contributes to the future discovery of natural microbial compounds that may potentially enhance the disease resilience of kauri forests. The methodological approaches used in this study highlight the value of moving beyond a taxonomic lens when examining the response of microbial communities to ecosystem disturbance and the need to develop more functional measures of microbial community resilience to invasive plant pathogens.

Genomic analysis of Bacillus subtilis sub sp. subtilis GEB5 reveals its genetic assets for nematicidal and plant growth promoting mechanisms

The immense benefit of chemical nematicides in controlling plant parasitic nematodes in high-value crops is counterbalanced by the development of resistance in nematodes due to their overuse. Bacillus subtilis is a promising bacterium that not only confers resistance against plant parasitic nematodes but also promotes plant growth through nutrient acquisition. Considering the multifaceted action of B. subtilis attempts were made to sequence the whole genome of endophytic B. subtilis GEB5 to find the genome complexity of the potential bio-control agent isolated from the guava. The results confirmed the isolate as B. subtilis sub sp. subtilis with a circular genome of 8,341,276 bp (8.3 Mb) length and 41.8% GC content. The results of genome annotation revealed the presence of 8209 coding sequences (CDS) genes, 136 transfer RNA (tRNA) genes, and 6 ribosomal RNA (rRNA) genes. Nonribosomal peptide synthetase (NRPS) gene clusters in the genome of GEB5 included fengycin, bacillaene, plipastatin, subtilin, subtilosin A, bacilysin, surfactin, sporulation killing factor, bacillibactin. Moreover, the GEB5 genome assembly had 21 homologs corresponding to nematode-virulent proteases. Furthermore, the results of greenhouse experiments showcased that GEB5 quenched the population of M. enterolobii (72% reduction) and also boosted the plant growth of guava. A plurality of the putatively encoded nematode virulence protease and its ability to boost plant growth suggests its environmentally robust bio-control potential in protecting plants against invading plant parasitic nematodes.

Development of a CRISPR/Cas9-mediated gene knockout method for functional genomics of the barley spot blotch pathogen Bipolaris sorokiniana

Bipolaris sorokiniana (=Cochliobolus sativus) is an important fungal pathogen that causes spot blotch, common root rot and kernel blight in barley and wheat. In this study, we aimed to develop a CRISPR/Cas9-mediated approach for efficiently knocking out genes of B. sorokiniana. We first assessed the efficiency of Cas9/sgRNA combined with the split marker system for gene replacement. We designed sgRNAs to target a polyketide synthase gene (PKS1) that is required for melanin biosynthesis of the fungus. When the preassembled Cas9/gRNA ribonucleoproteins (RNPs) and the split hygromycin B resistance gene (HygB) fragments each harboring 449 bp upstream and 595 bp downstream flanking sequences of PKS1 were co-transformed into the protoplasts of B. sorokiniana, the number of transformants with the PKS1 gene replaced by the selection marker were significantly increased compared to the control without RNPs. We then used the RNPs with PCR-amplified HygB gene cassette carrying 40 bp or 60 bp arms homologous to the flanking sequences of the PKS1 target site for fungal transformation and showed that the RNPs significantly enhanced gene disruption efficiency through the short-homology recombination, and more gene knockout mutants were obtained with longer (60 bp) homologous arms than the shorter (40 bp) ones. Finally, we disrupted an uncharacterized non-ribosomal peptide synthetase (NRPS) gene (NPSx) in B. sorokiniana strain ND90Pr using the RNP-mediated gene knockout approach and showed the mutants lost virulence on barley cv. Bowman. The Cas9/sgRNA-mediated gene knockout method developed will facilitate large-scale functional genomics studies of B. sorokiniana.

Bacillus Paralicheniformis: A potential candidate for natural antibacterial agents from marine macro-algae associated bacteria.

Antibiotic resistance poses a major global health threat, necessitating new approaches to treat bacterial infections. We isolated bacteria from the brown macroalgae Sargassum swartzii collected along the Indian coast. Of 279 isolates, 47.6% showed antibacterial activity against Staphylococcus aureus and Vibrio parahaemolyticus when screened. Ten potent isolates underwent analysis of biosynthetic gene clusters and 16S rRNA sequencing. Isolate MP-99 carried polyketide synthase (pks) and non-ribosomal peptide synthetase (nrps) genes and exhibited antibacterial effects.

An efficient cre-based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum.

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis-independent recombinase-assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum. We then integrated a non-ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum, which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.

Orfamide production in Pseudomonas protegens CHA0T promotes rhizospheric colonization and influences assemblage of the bacterial community of wheat roots in soil.

Orfamides (Ofa) are a family of closely related cyclic lipopeptides (cLPs) produced by different soil-inhabiting and plant beneficial strains of the Pseudomonas protegens subgroup. Orfamides are required for swarming motility, they mediate toxicity on oomycetes, algae, and insects, and they can act as ISR elicitors. Ofa biosynthesis depends on a non-ribosomal peptide synthetase gene cluster whose expression is tightly controlled by the post-transcriptional regulatory cascade Gac-Rsm. Although the biological properties and physiological role of Ofa have been well characterized in vitro, the role of Ofa in the fitness of the producing bacterium in its niche has not been studied so far. Here, we used an Ofa biosynthesis-deficient mutant (ΔofaABC) derived from the biocontrol model strain P. protegens CHA0T to explore the relevance of this cLP in soil for the survival of strain CHA0T, its contribution to the bacterial competitiveness for colonization of wheat roots, and its impact on the corresponding rhizobacterial community structure. To facilitate CFU counts in non-sterile soil and root samples we used chromosomally tagged versions of CHA0T and its ΔofaABC mutant with mini-Tn7 constructs carrying antibiotic resistance markers. We confirmed that the tagged Ofa− mutant does not show drop-collapse activity neither it can swarm in semi-solid medium. We also confirmed that Ofa can be exploited in vitro as a social good by the Ofa− mutant to resume swarming in the presence of CHA0T cells. We found that Ofa production did not confer any advantage for bacterial survival along a period of 5 weeks after soil inoculation. However, we observed that Ofa production was associated with a higher competitiveness for colonization of the wheat rhizosphere. Expression of the ofa biosynthetic operon, by means of a transcriptional Pofa-gfp reporter fusion, was detected in the rhizoplane of colonized wheat roots, suggesting that Ofa is produced in situ on the surface of colonized wheat roots. Finally, we observed that seed inoculation with the wild type Ofa producer strain CHA0T resulted in a differential assemblage of the bacterial community of the wheat rhizosphere with respect to that of seeds bacterized with the Ofa deficient mutant. Overall, we provide evidence that Ofa is important for the competitiveness of P. protegens CHA0T cells to colonize the surface of wheat roots and that Ofa production in the rhizosphere can impact the structure of the assembled bacterial microbiome.

Isolation and identification of associated endophytic bacteria from barely seeds harbour non-ribosomal peptides and enhance tolerance to salinity stress

BackgroundBarely Hordeum vulgare L. is considered one of the most important cereal crops with economic and industrial importance in the world, but its productivity is affected by climate change and abiotic stresses. One of the most recent and important microbiological promising aspects is the use of associated microorganisms, especially the endophytic bacteria producers for non-ribosomal peptides which play an important role in promoting plant growth, productivity, and tolerance to biotic and abiotic stresses. This work aims to identify vertically transferred or inherited endophytic bacterial communities in barely seeds, detect the presence of non-ribosomal peptides from these isolated endophytic strains and study their effect on protein patterns as a response to salinity stress.ResultsFrom two different tolerant (Giza 126) and sensitive (Giza 123) barely seeds cultivars, six different endophytic bacterial strains were isolated and identified using 16S rRNA. Bacterial strains belonged to Bacillus, Paenibacillus, Staphylococcus and Acinetobacter genera. Three of them have been isolated from both sensitive and tolerant barely cultivar (Uncultured Staphylococcus, Acinetobacter and Priestia endophytica or Bacillus endophyticus), while the other three endophytes have been isolated uniquely from the tolerant barely cultivar (Paenibacillus glucanolyticus, Bacillus cereus and Bacillus sp.). Non-ribosomal peptide synthetases genes NRPs of two lipopeptide families; surfactins and kurstakins have been detected using both bioinformatic analysis and degenerate primers. On the other hand, fragments similar to NRPs genes might be considered new NRPS molecules in Paenibacillus glucanolyticus, Acinetobacter and Priestia endophytica which have been detected using degenerate primers and required whole genome sequencing. The effect of soaking barely seeds exposed to 2.5% NaCl using SDS-PAGE electrophoresis revealed the presence of 24 bands, 10 of them were monomorphic with 41.5%, and 14 were polymorphic with 58.5% polymorphism.ConclusionThe overnight soaking and co-cultivation of isolated endophytic strains with barely seeds before planting proved their capability in conferring salt stress tolerance to barely seedlings which appeared in protein patterns. We could consider these barely seeds endophytic among the PGPR strains promising to improve plant growth during abiotic stresses.

Decoding the chemical language of Suillus fungi: genome mining and untargeted metabolomics uncover terpene chemical diversity.

Ectomycorrhizal fungi establish mutually beneficial relationships with trees, trading nutrients for carbon. Suillus are ectomycorrhizal fungi that are critical to the health of boreal and temperate forest ecosystems. Comparative genomics has identified a high number of non-ribosomal peptide synthetase and terpene biosynthetic gene clusters (BGC) potentially involved in fungal competition and communication. However, the functionality of these BGCs is not known. This study employed co-culture techniques to activate BGC expression and then used metabolomics to investigate the diversity of metabolic products produced by three Suillus species (Suillus hirtellus EM16, Suillus decipiens EM49, and Suillus cothurnatus VC1858), core members of the pine microbiome. After 28 days of growth on solid media, liquid chromatography-tandem mass spectrometry identified a diverse range of extracellular metabolites (exometabolites) along the interaction zone between Suillus co-cultures. Prenol lipids were among the most abundant chemical classes. Out of the 62 unique terpene BGCs predicted by genome mining, 41 putative prenol lipids (includes 37 putative terpenes) were identified across the three Suillus species using metabolomics. Notably, some terpenes were significantly more abundant in co-culture conditions. For example, we identified a metabolite matching to isomers isopimaric acid, sandaracopimaric acid, and abietic acid, which can be found in pine resin and play important roles in host defense mechanisms and Suillus spore germination. This research highlights the importance of combining genomics and metabolomics to advance our understanding of the chemical diversity underpinning fungal signaling and communication.IMPORTANCEUsing a combination of genomics and metabolomics, this study's findings offer new insights into the chemical diversity of Suillus fungi, which serve a critical role in forest ecosystems.

Cyclo-diphenylalanine production in Aspergillus nidulans through stepwise metabolic engineering

Cyclo-diphenylalanine (cFF) is a symmetrical aromatic diketopiperazine (DKP) found wide-spread in microbes, plants, and resulting food products. As different bioactivities continue being discovered and relevant food and pharmaceutical applications gradually emerge for cFF, there is a growing need for establishing convenient and efficient methods to access this type of compound. Here, we present a robust cFF production system which entailed stepwise engineering of the filamentous fungal strain Aspergillus nidulans A1145 as a heterologous expression host. We first established a preliminary cFF producing strain by introducing the heterologous nonribosomal peptide synthetase (NRPS) gene penP1 to A. nidulans A1145. Key metabolic pathways involving shikimate and aromatic amino acid biosynthetic support were then engineered through a combination of gene deletions of competitive pathway steps, over-expressing feedback-insensitive enzymes in phenylalanine biosynthesis, and introducing a phosphoketolase-based pathway, which diverted glycolytic flux toward the formation of erythrose 4-phosphate (E4P). Through the stepwise engineering of A. nidulans A1145 outlined above, involving both heterologous pathway addition and native pathway metabolic engineering, we were able to produce cFF with titers reaching 611 mg/L in shake flask culture and 2.5 g/L in bench-scale fed-batch bioreactor culture. Our study establishes a production platform for cFF biosynthesis and successfully demonstrates engineering of phenylalanine derived diketopiperazines in a filamentous fungal host.

PCR Optimization for Polyketide Synthase (PKS) and Non-Ribosomal Peptide Synthetase (NRPS) Gene Detection in Actinomycetes

PCR Optimization for Polyketide Synthase (PKS) and Non-Ribosomal Peptide Synthetase (NRPS) Gene Detection in Actinomycetes

Total substitution and partial modification of the set of non-ribosomal peptide synthetases clusters lead to pyoverdine diversity in the Pseudomonas fluorescens complex.

Pyoverdines are high affinity siderophores produced by most Pseudomonas with a wide role in microbial interspecies interactions. They are primarily composed of a conserved chromophore moiety, an acyl side chain and a peptide backbone which may be highly variable among strains. Upon ferric iron sequestration, pyoverdines are internalized through specialized receptors. The peptide precursor of pyoverdine, termed ferribactin, is synthesized by a set of non-ribosomal peptide synthetase (NRPS) enzymes and further modified by tailoring enzymes. While PvdL, the NRPS responsible for the synthesis of the peptide moiety that derives into the chromophore is conserved, the NRPSs for the peptide backbone are different across fluorescent Pseudomonas. Although the variation of pyoverdine is a widely recognized characteristic within the genus, the evolutionary events associated with the diversity and distribution of this trait remain mostly unknown. This study analyzed the NRPSs clusters for the biosynthesis of the peptide backbone of ferribactin in the genomes of a representative subset of strains of the Pseudomonas fluorescens complex. Bioinformatic analysis of the specificity of adenylation domains of the NRPSs allowed the prediction of 30 different pyoverdine variants. Phylogenetic reconstruction and mapping of the NRPS clusters pinpointed two different general levels of modifications. In the first level, a complete replacement of the set of NRPRs by horizontal transfer occurs. In the second level, the original set of NRPSs is modified through different mechanisms, including partial substitution of the NRPS genes by horizontal transfer, adenylation domain specificity change or NRPS accessory domain gain/loss.

Effect of growth medium composition on the efficiency of non-ribosomal synthesis in bacteria of the genus Bacillus

Bacteria of the genus Bacillus are known for their ability to suppress a wide range of pathogenic microflora through the production of a variety of secondary metabolites, a significant proportion of which are non-ribosomal peptides. The importance of selecting conditions for the most efficient synthesis of non-ribosomal peptides is related to this. The influence of cultivation conditions can be assessed by analyzing the gene expression of non-ribosomal peptide synthetases of target NRPs and studying the mechanisms of its regulation. The aim of this study is to investigate the influence of growth medium composition on the expression of non-ribosomal peptide synthetase genes in Bacillus bacteria.

Complete Genome Sequence and Pan-Genome Analysis of Shewanella oncorhynchi Z-P2, a Siderophore Putrebactin-Producing Bacterium.

In this study, we reported the complete genome sequence of Shewanella oncorhynchi for the first time. S. oncorhynchi Z-P2 is a bacterium that produces the siderophore putrebactin. Its genome consists of a circular chromosome of 5,034,612 bp with a G + C content of 45.4%. A total of 4544 protein-coding genes, 109 tRNAs and 31 rRNAs were annotated by the RAST. Five non-ribosomal peptide synthetase (NRPS) and polyketide synthetase (PKS) gene clusters were identified by the antiSMASH analysis. The pan-genome analysis of Z-P2 and 10 Shewanella putrefaciens revealed 9228 pan-gene clusters and 2681 core gene clusters, with Z-P2 having 618 unique gene clusters. Additionally, the gene cluster involved in putrebactin biosynthesis in Z-P2 was annotated, and the mechanism of putrebactin biosynthesis was analyzed. The putrebactin produced by Z-P2 was detected using UPLC-MS analysis, with an [M + H]+ molecular ion at m/z 373.21. These findings provide valuable support for further research on the genetic engineering of putrebactin biosynthetic genes of Z-P2 and their potential applications.

A small step to discover candidate biological control agents from preexisting bioresources by using novel nonribosomal peptide synthetases hidden in activated sludge metagenomes.

Biological control agents (BCAs), beneficial organisms that reduce the incidence or severity of plant disease, have been expected to be alternatives to replace chemical pesticides worldwide. To date, BCAs have been screened by culture-dependent methods from various environments. However, previously unknown BCA candidates may be buried and overlooked because this approach preferentially selects only easy-to-culture microbial lineages. To overcome this limitation, as a small-scale test case, we attempted to explore novel BCA candidates by employing the shotgun metagenomic information of the activated sludge (AS) microbiome, which is thought to contain unutilized biological resources. We first performed genome-resolved metagenomics for AS taken from a municipal sewage treatment plant and obtained 97 nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS)-related gene sequences from 43 metagenomic assembled bins, most of which were assigned to the phyla Proteobacteria and Myxococcota. Furthermore, these NRPS/PKS-related genes are predicted to be novel because they were genetically dissimilar to known NRPS/PKS gene clusters. Of these, the condensation domain of the syringomycin-related NRPS gene cluster was detected in Rhodoferax- and Rhodocyclaceae-related bins, and its homolog was found in previously reported AS metagenomes as well as the genomes of three strains available from the microbial culture collections, implying their potential BCA ability. Then, we tested the antimicrobial activity of these strains against phytopathogenic fungi to investigate the potential ability of BCA by in vitro cultivation and successfully confirmed the actual antifungal activity of three strains harboring a possibly novel NRPS gene cluster. Our findings provide a possible strategy for discovering novel BCAs buried in the environment using genome-resolved metagenomics.

The Lrp transcriptional factor of an entomopathogenic bacterium, Xenorhabdus hominickii, activates non-ribosomal peptide synthetases to suppress insect immunity

Two bacterial genera, Xenorhabdus and Photorhabdus, are mutually symbiotic to the entomopathogenic nematodes, Steinernema and Heterorhabditis, respectively. The infective juveniles deliver the symbiotic bacteria to the hemocoel of target insects, in which the bacteria proliferate and help the development of the host nematode. The successful parasitism of the nematode-bacterial complex depends on host immunosuppression by the bacteria via their secondary metabolites. Leucine-responsive regulatory protein (Lrp) is a global bacterial transcriptional factor that plays a crucial role in parasitism. However, its regulatory targets to suppress insect immunity are not clearly understood. This study investigated the bacterial genes regulated by Lrp and the subsequent production of secondary metabolites in Xenorhabdus hominickii. Lrp expression occurred at the early infection stage of the bacteria in a target insect, Spodoptera exigua. A preliminary in silico screening indicated that 3.7% genes among 4075 predicted genes encoded in X. hominickii had the Lrp-response element on their promoters, including two non-ribosomal peptide synthetases (NRPSs). Eight NRPS (NRPS1-NRPS8) genes were predicted in the bacterial genome, in which six NRPS (NRPS3-NRPS8) expressions were positively correlated with Lrp expression in the infected larvae of S. exigua. Exchange of the Lrp promoter with an inducible promoter altered the production of the secondary metabolites and the NRPS expression levels. The immunosuppressive activities of X. hominickii were dependent on the Lrp expression level. The metabolites produced by Lrp expression included the eicosanoid-biosynthesis inhibitors and hemolytic factors. A cyclic dipeptide (=cPF) was produced by the bacteria at high Lrp expression and inhibited the phospholipase A2 activity of S. exigua in a competitive inhibitory manner. These results suggest that Lrp is a global transcriptional factor of X. hominickii and plays a crucial role in insect immunosuppression by modulating NRPS expression.

Profile of PKS and NRPS Gene Clusters in the Genome of Streptomyces cellostaticus NBRC 12849T

Polyketides and nonribosomal peptides are major secondary metabolites in members of the genus Streptomyces. Streptomyces cellostaticus is a validly recognized species and the type strain produces cellostatin. However, little is known about whether it has the potential to produce diverse polyketides and nonribosomal peptides. Here, we sequenced the whole genome of S. cellostaticus NBRC 12849T and surveyed polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters in the genome. The genome encoded 12 PKS, one NRPS and eight hybrid PKS/NRPS gene clusters. Among the 21 gene clusters, products of 10 gene clusters were annotated to be an annimycin congener, fuelimycins, lankamycin, streptovaricin, spore pigment, flaviolin, foxicin, blasticidin, lankacidin and an incarnatapeptine congener via our bioinformatic analysis. Although the other clusters were orphan and their products were unknown, five of them were predicted to be compounds derived from two independent diketides, a tridecaketide, a triketide and a tetraketide with a cysteine residue, respectively. These results suggest that S. cellostaticus is a source of diverse polyketides and hybrid polyketide/nonribosomal peptides, including unknown and new secondary metabolites.

A Staphylococcus capitis strain with unusual bacteriocin production.

Staphylococcus capitis is a member of the human and mammal skin microbiomes and is considered less harmful than Staphylococcus aureus. S. capitis subsp. urealyticus BN2 was isolated from a cat and expressed strong antibacterial activity against a range of Gram-positive species, most notably including S. aureus strains with resistance to methicillin (MRSA) and strains with intermediate resistance to vancomycin (VISA). These latter strains are normally relatively resistant to bacteriocins, due to cell wall and cell membrane modifications. Genomic sequencing showed that the strain harboured at least two complete gene clusters for biosynthesis of antagonistic substances. The complete biosynthetic gene cluster of the well-known lantibiotic gallidermin was encoded on a large plasmid and the mature peptide was present in isopropanol cell extracts. In addition, a chromosomal island contained a novel non-ribosomal peptide synthetase (NRPS) gene cluster. Accidental deletion of two NRPS modules and partial purification of the anti-VISA activity showed that this novel bacteriocin represents a complex of differently decorated, non-ribosomal peptides. Additionally, a number of phenol-soluble modulins (PSMs) was detected by mass spectrometry of whole cells. Producing these compounds, the strain was able to outcompete several S. aureus strains, including MRSA and VISA, in tube cultures.

Global distribution and diversity of antimicrobial genes across subsurface bacterial and archaeal metagenome assembled genomes

Microorganisms have the capability to produce antimicrobial compounds through secondary metabolism, which are not essential within their natural environments, but have been found to have many effects on the ecosystem. Antimicrobial production genes have been identified in a wide range of microorganisms; however, research into natural ecosystems has historically been limited to continental soil environments. Antimicrobial production research has been limited in the deep continental subsurface and marine environments, especially deeply buried marine sediments. We analyzed 466 high-quality metagenome assembled genomes (MAGs) collected from continental and marine subsurface environments through the Deep Carbon Observatory’s Census of Deep Life. A total of 383 MAGs contained biosynthetic gene clusters, namely Type I and Type III polyketide synthase genes, non-ribosomal peptide synthetase genes, and other unspecified ribosomally synthesized and post-translationally modified peptide products. All of these genes were found across continental mines, subglacial lakes, hotsprings, and serpentinizing environments. These environments have previously not been investigated via metagenomics for antimicrobial gene diversity, which may be produced for competition or communication purposes. All other biosynthetic genes identified in this study were less than 50% similar to reference biosynthesis genes indicating the novelty of secondary metabolism in subsurface microorganisms. The majority of predicted antimicrobial products were found to be produced exergonically, which could indicate microbial populations use energy-conserving mechanisms to produce compounds that could offer a competitive advantage.