Genome mining reveals unlocked bioactive potential of marine Gram-negative bacteria

Background

Antibiotic resistance in bacteria spreads quickly, overtaking the pace at which new compounds are discovered and this emphasizes the immediate need to discover new compounds for control of infectious diseases. Terrestrial bacteria have for decades been investigated as a source of bioactive compounds leading to successful applications in pharmaceutical and biotech industries. Marine bacteria have so far not been exploited to the same extent; however, they are believed to harbor a multitude of novel bioactive chemistry. To explore this potential, genomes of 21 marine Alpha- and Gammaproteobacteria collected during the Galathea 3 expedition were sequenced and mined for natural product encoding gene clusters.

Results

Independently of genome size, bacteria of all tested genera carried a large number of clusters encoding different potential bioactivities, especially within the Vibrionaceae and Pseudoalteromonadaceae families. A very high potential was identified in pigmented pseudoalteromonads with up to 20 clusters in a single strain, mostly NRPSs and NRPS-PKS hybrids. Furthermore, regulatory elements in bioactivity-related pathways including chitin metabolism, quorum sensing and iron scavenging systems were investigated both in silico and in vitro. Genes with siderophore function were identified in 50% of the strains, however, all but one harboured the ferric-uptake-regulator gene. Genes encoding the syntethase of acylated homoserine lactones were found in Roseobacter-clade bacteria, but not in the Vibrionaceae strains and only in one Pseudoalteromonas strains. The understanding and manipulation of these elements can help in the discovery and production of new compounds never identified under regular laboratory cultivation conditions. High chitinolytic potential was demonstrated and verified for Vibrio and Pseudoalteromonas species that commonly live in close association with eukaryotic organisms in the environment. Chitin regulation by the ChiS histidine-kinase seems to be a general trait of the Vibrionaceae family, however it is absent in the Pseudomonadaceae. Hence, the degree to which chitin influences secondary metabolism in marine bacteria is not known.

Conclusions

Utilizing the rapidly developing sequencing technologies and software tools in combination with phenotypic in vitro assays, we demonstrated the high bioactive potential of marine bacteria in an efficient, straightforward manner – an approach that will facilitate natural product discovery in the future.     

Genomic sequence of the aflatoxigenic filamentous fungus Aspergillus nomius

Background

Aspergillus nomius is an opportunistic pathogen and one of the three most important producers of aflatoxins in section Flavi. This fungus has been reported to contaminate agricultural commodities, but it has also been sampled in non-agricultural areas so the host range is not well known. Having a similar mycotoxin profile as A. parasiticus, isolates of A. nomius are capable of secreting B- and G- aflatoxins.

Results

In this study we discovered that the A. nomius type strain (NRRL 13137) has a genome size of approximately 36 Mb which is comparable to other Aspergilli whose genomes have been sequenced. Its genome encompasses 11,918 predicted genes, 72 % of which were assigned GO terms using BLAST2GO. More than 1,200 of those predicted genes were identified as unique to A. nomius, and the most significantly enriched GO category among the unique genes was oxidoreducatase activity. Phylogenomic inference shows NRRL 13137 as ancestral to the other aflatoxigenic species examined from section Flavi. This strain contains a single mating-type idiomorph designated as MAT1-1.

Conclusions

This study provides a preliminary analysis of the A. nomius genome. Given the recently discovered potential for A. nomius to undergo sexual recombination, and based on our findings, this genome sequence provides an additional evolutionary reference point for studying the genetics and biology of aflatoxin production.     

Comparative genome analysis of pathogenic and non-pathogenic Clavibacter strains reveals adaptations to their lifestyle

Background

The genus Clavibacter harbors economically important plant pathogens infecting agricultural crops such as potato and tomato. Although the vast majority of Clavibacter strains are pathogenic, there is an increasing number of non-pathogenic isolates reported. Non-pathogenic Clavibacter strains isolated from tomato seeds are particularly problematic because they affect the current detection and identification tests for Clavibacter michiganensis subsp. michiganensis (Cmm), which is regulated with a zero tolerance in tomato seed. Their misidentification as pathogenic Cmm hampers a clear judgment on the seed quality and health.

Results

To get more insight in the genetic features linked to the lifestyle of these bacteria, a whole-genome sequence of the tomato seed-borne non-pathogenic Clavibacter LMG 26808 was determined. To gain a better understanding of the molecular determinants of pathogenicity, the genome sequence of LMG 26808 was compared with that of the pathogenic Cmm strain (NCPPB 382). The comparative analysis revealed that LMG 26808 does not contain plasmids pCM1 and pCM2 and also lacks the majority of important virulence factors described so far for pathogenic Cmm. This explains its apparent non-pathogenic nature in tomato plants. Moreover, the genome analysis of LMG 26808 detected sequences from a plasmid originating from a member of Enterobacteriaceae/Klebsiella relative. Genes received that way and coding for antibiotic resistance may provide a competitive advantage for survival of LMG 26808 in its ecological niche. Genetically, LMG 26808 was the most similar to the pathogenic Cmm NCPPB 382 but contained more mobile genetic elements. The genome of this non-pathogenic Clavibacter strain contained also a high number of transporters and regulatory genes.

Conclusions

The genome sequence of the non-pathogenic Clavibacter strain LMG 26808 and the comparative analyses with other pathogenic Clavibacter strains provided a better understanding of the genetic bases of virulence and adaptation mechanisms present in the genus Clavibacter.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-392) contains supplementary material, which is available to authorized users.     

Genomic and experimental evidence for a potential sexual cycle in the pathogenic thermal dimorphic fungus Penicillium marneffei

All meiotic genes (except HOP1) and genes encoding putative pheromone processing enzymes, pheromone receptors and pheromone response pathways proteins in Aspergillus fumigatus and Aspergillus nidulans and a putative MAT-1 alpha box mating-type gene were present in the Penicillium marneffei genome. A putative MAT-2 high-mobility group mating-type gene was amplified from a MAT-1 alpha box mating-type gene-negative P. marneffei strain. Among 37 P. marneffei patient strains, MAT-1 alpha box and MAT-2 high-mobility group mating-type genes were present in 23 and 14 isolates, respectively. We speculate that P. marneffei can potentially be a heterothallic fungus that does not switch mating type.     

Surface ultrastructure of the uredinial stage ofCerotelium fici and its infection process on mulberry

Surface morphology of uredinia and urediniospores ofCerotelium fici (Cast.) Arth., and its infection process in mulberry (Morus alba L.) have been described using the scanning electron microscope. The uredinia ofC. fici are paraphysate and bear pedicellate urediniospores. The surface morphology of urediniospore is similar to most of the rust fungi which have pedicellate urediniospores. The infection process ofC. fici on mulberry leaves differs from other rust fungi in not forming appressoria over the stomates. Further, the germ tube of the urediniospore crosses over the stomata, and sometimes forms an appressorium close to the stoma rather than forming over it. Thus, the present study indicates that the formation of appressoria byC. fici on mulberry leaves is not site specific but an independent, specialized and inherent mechanism required byC. fici to penetrate the mulberry leaf cuticle and epidermis.     

Full genome SNP-based phylogenetic analysis reveals the origin and global spread of Brucella melitensis

Background

Brucellosis is an important zoonotic disease that affects both humans and animals. We sequenced the full genome and characterised the genetic diversity of two Brucella melitensis isolates from Malaysia and the Philippines. In addition, we performed a comparative whole-genome single nucleotide polymorphism (SNP) analysis of B. melitensis strains collected from around the world, to investigate the potential origin and the history of the global spread of B. melitensis.

Results

Single sequencing runs of each genome resulted in draft genome sequences of MY1483/09 and Phil1136/12, which covered 99.85% and 99.92% of the complete genome sequences, respectively. The B. melitensis genome sequences, and two B. abortus strains used as the outgroup strains, yielded a total of 13,728 SNP sites. Phylogenetic analysis using whole-genome SNPs and geographical distribution of the isolates revealed spatial clustering of the B. melitensis isolates into five genotypes, I, II, III, IV and V. The Mediterranean strains, identified as genotype I, occupied the basal node of the phylogenetic tree, suggesting that B. melitensis may have originated from the Mediterranean regions. All of the Asian B. melitensis strains clustered into genotype II with the SEA strains, including the two isolates sequenced in this study, forming a distinct clade denoted here as genotype IId. Genotypes III, IV and V of B. melitensis demonstrated a restricted geographical distribution, with genotype III representing the African lineage, genotype IV representing the European lineage and genotype V representing the American lineage.

Conclusion

We showed that SNPs retrieved from the B. melitensis draft full genomes were sufficient to resolve the interspecies relationships between B. melitensis strains and to discriminate between the vaccine and endemic strains. Phylogeographic reconstruction of the history of B. melitensis global spread at a finer scale by using whole-genome SNP analyses supported the origin of all B. melitensis strains from the Mediterranean region. The possible global distribution of B. melitensis following the ancient trade routes was also consistent with whole-genome SNP phylogeny. The whole genome SNP phylogenetics analysis, hence is a powerful tool for intraspecies discrimination of closely related species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1294-x) contains supplementary material, which is available to authorized users.     

Antifungal and cytotoxic activities of the secondary metabolites from endophytic fungus Massrison sp.

Three novel compounds with spiro-5, 6-lactone ring skeleton has been isolated from the fermentation broth of Massrison sp. which could be isolated repeatedly from wild Rehmannia glutinosa. Psetariae oryza P-2b was applied to guide fractionation of bioactive compounds produced by Massrison sp. The molecular structures were established by a variety of one- and two-dimensional NMR experiments and the compounds with similar skeleton were reported for the first time from endophytic fungi of terraneous plant. Antifungal and cytotoxic activities of the compounds were tested, compounds 2 and 3 displayed stronger antifungal and cytotoxic activities. The compounds have the potential to be antibiotic against fungal pathogens and tumor cells.     

Metabolites from the endophytic fungus Phomopsis sp. PSU-D15

From the endophytic fungus Phomopsis sp. PSU-D15, three metabolites named as phomoenamide (1), phomonitroester (2) and deacetylphomoxanthone B (3), were isolated together with three known compounds, dicerandrol A (4), (1S,2S,4S)-p-menthane-1,2,4-triol (5) and uridine. Their structures were elucidated by spectroscopic methods. Phomoenamide (1) exhibited moderate in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra.     

RNAseq analysis of Aspergillus fumigatus in blood reveals a just wait and see resting stage behavior

Background

Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long.

Results

We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes.

Conclusions

We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage. This might reflect the harsh conditions in blood where A. fumigatus cannot take up sufficient nutrients to establish self-defense mechanisms combined with significant growth.

Electronic supplementary material

The online version of this article (doi10.1186/s12864-015-1853-1) contains supplementary material, which is available to authorized users.     

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress

The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. In addition, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated, and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. We found that Wolbachia responded not only by modulating expression of the translation machinery, but also by upregulating nucleotide synthesis and energy metabolism, while downregulating outer membrane proteins. Moreover, Wolbachia increased the expression of a key component of the twin-arginine translocase (tatA) and a phosphate ABC transporter ATPase (PstB); the latter is associated with decreased susceptibility to antimicrobials in free-living bacteria. Finally, the downregulation of 6S RNA during translational inhibition suggests that this small RNA is involved in growth rate control. Despite its highly reduced genome, Wolbachia shows a surprising ability to regulate gene expression during exposure to a potent stressor. Our findings have general relevance for the chemotherapy of obligate intracellular bacteria and the mechanistic basis of persistence in the Rickettsiales.     

Genomic analysis of the emergence of 20th century epidemic dysentery

Background

Shigella dysenteriae type 1 (Sd1) causes recurrent epidemics of dysentery associated with high mortality in many regions of the world. Sd1 infects humans at very low infectious doses (10 CFU), and treatment is complicated by the rapid emergence of antibiotic resistant Sd1 strains. Sd1 is only detected in the context of human infections, and the circumstances under which epidemics emerge and regress remain unknown.

Results

Phylogenomic analyses of 56 isolates collected worldwide over the past 60 years indicate that the Sd1 clone responsible for the recent pandemics emerged at the turn of the 20^th century, and that the two world wars likely played a pivotal role for its dissemination. Several lineages remain ubiquitous and their phylogeny indicates several recent intercontinental transfers. Our comparative genomics analysis reveals that isolates responsible for separate outbreaks, though closely related to one another, have independently accumulated antibiotic resistance genes, suggesting that there is little or no selection to retain these genes in-between outbreaks. The genomes appear to be subjected to genetic drift that affects a number of functions currently used by diagnostic tools to identify Sd1, which could lead to the potential failure of such tools.

Conclusions

Taken together, the Sd1 population structure and pattern of evolution suggest a recent emergence and a possible human carrier state that could play an important role in the epidemic pattern of infections of this human-specific pathogen. This analysis highlights the important role of whole-genome sequencing in studying pathogens for which epidemiological or laboratory investigations are particularly challenging.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-355) contains supplementary material, which is available to authorized users.     

Genome analysis of Flaviramulus ichthyoenteri Th78T in the family Flavobacteriaceae: insights into its quorum quenching property and potential roles in fish intestine

Background

Intestinal microbes play significant roles in fish and can be possibly used as probiotics in aquaculture. In our previous study, Flaviramulus ichthyoenteri Th78^T, a novel species in the family Flavobacteriaceae, was isolated from fish intestine and showed strong quorum quenching (QQ) ability. To identify the QQ enzymes in Th78^T and explore the potential roles of Th78^T in fish intestine, we sequenced the genome of Th78^T and performed extensive genomic analysis.

Results

An N-acyl homoserine lactonase FiaL belonging to the metallo-β-lactamase superfamily was identified and the QQ activity of heterologously expressed FiaL was confirmed in vitro. FiaL has relatively little similarity to the known lactonases (25.2 ~ 27.9% identity in amino acid sequence). Various digestive enzymes including alginate lyases and lipases can be produced by Th78^T, and enzymes essential for production of B vitamins such as biotin, riboflavin and folate are predicted. Genes encoding sialic acid lyases, sialidases, sulfatases and fucosidases, which contribute to utilization of mucus, are present in the genome. In addition, genes related to response to different stresses and gliding motility were also identified. Comparative genome analysis shows that Th78^T has more specific genes involved in carbohydrate transport and metabolism compared to other two isolates in Flavobacteriaceae, both isolated from sediments.

Conclusions

The genome of Th78^T exhibits evident advantages for this bacterium to survive in the fish intestine, including production of QQ enzyme, utilization of various nutrients available in the intestine as well as the ability to produce digestive enzymes and vitamins, which also provides an application prospect of Th78^T to be used as a probiotic in aquaculture.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1275-0) contains supplementary material, which is available to authorized users.     

Genomic comparison of 93 Bacillus phages reveals 12 clusters, 14 singletons and remarkable diversity

Background

The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains.

Results

Whole genome nucleotide and proteome comparison of the 93 extant Bacillus phages revealed 12 distinct clusters, 28 subclusters and 14 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member of the group. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,922 protein families (phams) of which only 951 (19%) had a predicted function. In addition, 3,058 (62%) of phams were orphams (phams containing a gene product from a single phage). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains.

Conclusions

This analysis provides a basis for understanding and characterizing Bacillus phages and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.     

The genome sequence of the biocontrol fungus Metarhizium anisopliae and comparative genomics of Metarhizium species

Background

Metarhizium anisopliae is an important fungal biocontrol agent of insect pests of agricultural crops. Genomics can aid the successful commercialization of biopesticides by identification of key genes differentiating closely related species, selection of virulent microbial isolates which are amenable to industrial scale production and formulation and through the reduction of phenotypic variability. The genome of Metarhizium isolate ARSEF23 was recently published as a model for M. anisopliae, however phylogenetic analysis has since re-classified this isolate as M. robertsii. We present a new annotated genome sequence of M. anisopliae (isolate Ma69) and whole genome comparison to M. robertsii (ARSEF23) and M. acridum (CQMa 102).

Results

Whole genome analysis of M. anisopliae indicates significant macrosynteny with M. robertsii but with some large genomic inversions. In comparison to M. acridum, the genome of M. anisopliae shares lower sequence homology. While alignments overall are co-linear, the genome of M. acridum is not contiguous enough to conclusively observe macrosynteny. Mating type gene analysis revealed both MAT1-1 and MAT1-2 genes present in M. anisopliae suggesting putative homothallism, despite having no known teleomorph, in contrast with the putatively heterothallic M. acridum isolate CQMa 102 (MAT1-2) and M. robertsii isolate ARSEF23 (altered MAT1-1). Repetitive DNA and RIP analysis revealed M. acridum to have twice the repetitive content of the other two species and M. anisopliae to be five times more RIP affected than M. robertsii. We also present an initial bioinformatic survey of candidate pathogenicity genes in M. anisopliae.

Conclusions

The annotated genome of M. anisopliae is an important resource for the identification of virulence genes specific to M. anisopliae and development of species- and strain- specific assays. New insight into the possibility of homothallism and RIP affectedness has important implications for the development of M. anisopliae as a biopesticide as it may indicate the potential for greater inherent diversity in this species than the other species. This could present opportunities to select isolates with unique combinations of pathogenicity factors, or it may point to instability in the species, a negative attribute in a biopesticide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-660) contains supplementary material, which is available to authorized users.     

Genome sequencing of two Neorhizobium galegae strains reveals a noeT gene responsible for the unusual acetylation of the nodulation factors

Background

The species Neorhizobium galegae comprises two symbiovars that induce nodules on Galega plants. Strains of both symbiovars, orientalis and officinalis, induce nodules on the same plant species, but fix nitrogen only in their own host species. The mechanism behind this strict host specificity is not yet known. In this study, genome sequences of representatives of the two symbiovars were produced, providing new material for studying properties of N. galegae, with a special interest in genomic differences that may play a role in host specificity.

Results

The genome sequences confirmed that the two representative strains are much alike at a whole-genome level. Analysis of orthologous genes showed that N. galegae has a higher number of orthologs shared with Rhizobium than with Agrobacterium. The symbiosis plasmid of strain HAMBI 1141 was shown to transfer by conjugation under optimal conditions. In addition, both sequenced strains have an acetyltransferase gene which was shown to modify the Nod factor on the residue adjacent to the non-reducing-terminal residue. The working hypothesis that this gene is of major importance in directing host specificity of N. galegae could not, however, be confirmed.

Conclusions

Strains of N. galegae have many genes differentiating them from strains of Agrobacterium, Rhizobium and Sinorhizobium. However, the mechanism behind their ecological difference is not evident. Although the final determinant for the strict host specificity of N. galegae remains to be identified, the gene responsible for the species-specific acetylation of the Nod factors was identified in this study. We propose the name noeT for this gene to reflect its role in symbiosis.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-500) contains supplementary material, which is available to authorized users.