Expression of distinct maternal and somatic 5.8S, 18S, and 28S rRNA types during zebrafish development.

There is mounting evidence that the ribosome is not a static translation machinery, but a cell-specific, adaptive system. Ribosomal variations have mostly been studied at the protein level, even though the essential transcriptional functions are primarily performed by rRNAs. At the RNA level, oocyte-specific 5S rRNAs are long known for Xenopus. Recently, we described for zebrafish a similar system in which the sole maternal-type 5S rRNA present in eggs is replaced completely during embryonic development by a somatic-type. Here, we report the discovery of an analogous system for the 45S rDNA elements: 5.8S, 18S, and 28S. The maternal-type 5.8S, 18S, and 28S rRNA sequences differ substantially from those of the somatic-type, plus the maternal-type rRNAs are also replaced by the somatic-type rRNAs during embryogenesis. We discuss the structural and functional implications of the observed sequence differences with respect to the translational functions of the 5.8S, 18S, and 28S rRNA elements. Finally, in silico evidence suggests that expansion segments (ES) in 18S rRNA, previously implicated in ribosome-mRNA interaction, may have a preference for interacting with specific mRNA genes. Taken together, our findings indicate that two distinct types of ribosomes exist in zebrafish during development, each likely conducting the translation machinery in a unique way.

Linking maternal and somatic 5S rRNA types with different sequence-specific non-LTR retrotransposons.

5S rRNA is a ribosomal core component, transcribed from many gene copies organized in genomic repeats. Some eukaryotic species have two 5S rRNA types defined by their predominant expression in oogenesis or adult tissue. Our next-generation sequencing study on zebrafish egg, embryo, and adult tissue identified maternal-type 5S rRNA that is exclusively accumulated during oogenesis, replaced throughout the embryogenesis by a somatic-type, and thus virtually absent in adult somatic tissue. The maternal-type 5S rDNA contains several thousands of gene copies on chromosome 4 in tandem repeats with small intergenic regions, whereas the somatic-type is present in only 12 gene copies on chromosome 18 with large intergenic regions. The nine-nucleotide variation between the two 5S rRNA types likely affects TFIII binding and riboprotein L5 binding, probably leading to storage of maternal-type rRNA. Remarkably, these sequence differences are located exactly at the sequence-specific target site for genome integration by the 5S rRNA-specific Mutsu retrotransposon family. Thus, we could define maternal- and somatic-type MutsuDr subfamilies. Furthermore, we identified four additional maternal-type and two new somatic-type MutsuDr subfamilies, each with their own target sequence. This target-site specificity, frequently intact maternal-type retrotransposon elements, plus specific presence of Mutsu retrotransposon RNA and piRNA in egg and adult tissue, suggest an involvement of retrotransposons in achieving the differential copy number of the two types of 5S rDNA loci.

Analysis of novel kitasatosporae reveals significant evolutionary changes in conserved developmental genes between Kitasatospora and Streptomyces.

Actinomycetes are antibiotic-producing filamentous bacteria that have a mycelial life style. The members of the three genera classified in the family Streptomycetaceae, namely Kitasatospora, Streptacidiphilus and Streptomyces, are difficult to distinguish using phenotypic properties. Here we present biochemical and genetic evidence that helps underpin the case for the continued recognition of the genus Kitasatospora and for the delineation of additional Kitasatospora species. Two novel Kitasatospora strains, isolates MBT63 and MBT66, and their genome sequences are presented. The cell wall of the Kitasatospora strains contain a mixture of meso-and LL-diaminopimelic acid (A2pm), whereby a single DapF surprisingly suffices to incorporate both components into the Kitasatospora cell wall. The availability of two new Kitasatospora genome sequences in addition to that of the previously sequenced Kitasatospora setae KM-6054(T) allows better phylogenetic comparison between kitasatosporae and streptomycetes. This showed that the developmental regulator BldB and the actin-like protein Mbl are absent from kitasatosporae, while the cell division activator SsgA and its transcriptional activator SsgR have been lost from some Kitasatospora species, strongly suggesting that Kitasatospora have evolved different ways to control specific steps in their development. We also show that the tetracycline-producing strain "Streptomyces viridifaciens" DSM 40239 not only has properties consistent with its classification in the genus Kitasatospora but also merits species status within this taxon.

Natural product proteomining, a quantitative proteomics platform, allows rapid discovery of biosynthetic gene clusters for different classes of natural products.

Information on gene clusters for natural product biosynthesis is accumulating rapidly because of the current boom of available genome sequencing data. However, linking a natural product to a specific gene cluster remains challenging. Here, we present a widely applicable strategy for the identification of gene clusters for specific natural products, which we name natural product proteomining. The method is based on using fluctuating growth conditions that ensure differential biosynthesis of the bioactivity of interest. Subsequent combination of metabolomics and quantitative proteomics establishes correlations between abundance of natural products and concomitant changes in the protein pool, which allows identification of the relevant biosynthetic gene cluster. We used this approach to elucidate gene clusters for different natural products in Bacillus and Streptomyces, including a novel juglomycin-type antibiotic. Natural product proteomining does not require prior knowledge of the gene cluster or secondary metabolite and therefore represents a general strategy for identification of all types of gene clusters.

Eliciting antibiotics active against the ESKAPE pathogens in a collection of actinomycetes isolated from mountain soils.

The rapid emergence of multidrug-resistant (MDR) bacterial pathogens poses a major threat for human health. In recent years, genome sequencing has unveiled many poorly expressed antibiotic clusters in actinomycetes. Here, we report a well-defined ecological collection of >800 actinomycetes obtained from sites in the Himalaya and Qinling mountains, and we used these in a concept study to see how efficiently antibiotics can be elicited against MDR pathogens isolated recently from the clinic. Using 40 different growth conditions, 96 actinomycetes were identified - predominantly Streptomyces - that produced antibiotics with efficacy against the MDR clinical isolates referred to as ESKAPE pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and/or Enterobacter cloacae. Antimicrobial activities that fluctuated strongly with growth conditions were correlated with specific compounds, including borrelidin, resistomycin, carbomethoxy-phenazine, and 6,7,8- and 5,6,8-trimethoxy-3-methylisocoumarin, of which the latter was not described previously. Our work provided insights into the potential of actinomycetes as producers of drugs with efficacy against clinical isolates that have emerged recently and also underlined the importance of targeting a specific pathogen.

Streptomyces leeuwenhoekii sp. nov., the producer of chaxalactins and chaxamycins, forms a distinct branch in Streptomyces gene trees.

A polyphasic study was carried out to establish the taxonomic status of an Atacama Desert isolate, Streptomyces strain C34(T), which synthesises novel antibiotics, the chaxalactins and chaxamycins. The organism was shown to have chemotaxonomic, cultural and morphological properties consistent with its classification in the genus Streptomyces. Analysis of 16S rRNA gene sequences showed that strain C34(T) formed a distinct phyletic line in the Streptomyces gene tree that was very loosely associated with the type strains of several Streptomyces species. Multilocus sequence analysis based on five house-keeping gene alleles underpinned the separation of strain C34(T) from all of its nearest phylogenetic neighbours, apart from Streptomyces chiangmaiensis TA-1(T) and Streptomyces hyderabadensis OU-40(T) which are not currently in the MLSA database. Strain C34(T) was distinguished readily from the S. chiangmaiensis and S. hyderabadensis strains by using a combination of cultural and phenotypic data. Consequently, strain C34(T) is considered to represent a new species of the genus Streptomyces for which the name Streptomyces leeuwenhoekii sp. nov. is proposed. The type strain is C34(T) (= DSM 42122(T) = NRRL B-24963(T)). Analysis of the whole-genome sequence of S. leeuwenhoekii, with 6,780 predicted open reading frames and a total genome size of around 7.86 Mb, revealed a high potential for natural product biosynthesis.

A novel taxonomic marker that discriminates between morphologically complex actinomycetes.

In the era when large whole genome bacterial datasets are generated routinely, rapid and accurate molecular systematics is becoming increasingly important. However, 16S ribosomal RNA sequencing does not always offer sufficient resolution to discriminate between closely related genera. The SsgA-like proteins are developmental regulatory proteins in sporulating actinomycetes, whereby SsgB actively recruits FtsZ during sporulation-specific cell division. Here, we present a novel method to classify actinomycetes, based on the extraordinary way the SsgA and SsgB proteins are conserved. The almost complete conservation of the SsgB amino acid (aa) sequence between members of the same genus and its high divergence between even closely related genera provides high-quality data for the classification of morphologically complex actinomycetes. Our analysis validates Kitasatospora as a sister genus to Streptomyces in the family Streptomycetaceae and suggests that Micromonospora, Salinispora and Verrucosispora may represent different clades of the same genus. It is also apparent that the aa sequence of SsgA is an accurate determinant for the ability of streptomycetes to produce submerged spores, dividing the phylogenetic tree of streptomycetes into liquid-culture sporulation and no liquid-culture sporulation branches. A new phylogenetic tree of industrially relevant actinomycetes is presented and compared with that based on 16S rRNA sequences.

The genome sequence of Streptomyces lividans 66 reveals a novel tRNA-dependent peptide biosynthetic system within a metal-related genomic island.

The complete genome sequence of the original isolate of the model actinomycete Streptomyces lividans 66, also referred to as 1326, was deciphered after a combination of next-generation sequencing platforms and a hybrid assembly pipeline. Comparative analysis of the genomes of S. lividans 66 and closely related strains, including S. coelicolor M145 and S. lividans TK24, was used to identify strain-specific genes. The genetic diversity identified included a large genomic island with a mosaic structure, present in S. lividans 66 but not in the strain TK24. Sequence analyses showed that this genomic island has an anomalous (G + C) content, suggesting recent acquisition and that it is rich in metal-related genes. Sequences previously linked to a mobile conjugative element, termed plasmid SLP3 and defined here as a 94 kb region, could also be identified within this locus. Transcriptional analysis of the response of S. lividans 66 to copper was used to corroborate a role of this large genomic island, including two SLP3-borne "cryptic" peptide biosynthetic gene clusters, in metal homeostasis. Notably, one of these predicted biosynthetic systems includes an unprecedented nonribosomal peptide synthetase--tRNA-dependent transferase biosynthetic hybrid organization. This observation implies the recruitment of members of the leucyl/phenylalanyl-tRNA-protein transferase family to catalyze peptide bond formation within the biosynthesis of natural products. Thus, the genome sequence of S. lividans 66 not only explains long-standing genetic and phenotypic differences but also opens the door for further in-depth comparative genomic analyses of model Streptomyces strains, as well as for the discovery of novel natural products following genome-mining approaches.

Identification and isolation of lantibiotics from culture: a bioorthogonal chemistry approach.

A distinguishing feature of the lantibiotic family of cyclic peptides is the presence of thioethers. Treatment of a lantibiotic with an alkaline solution at high pH gives rise to a ß-elimination reaction yielding the corresponding ring opened precursor, containing a dehydro-amino acid residue. We here reveal in a proof-of-concept study that a ring opened lantibiotic (mersacidin) can be captured for pull-down from a culture broth, subsequently released and identified by mass spectrometry.

Upstream start codon in segment 4 of North American H2 avian influenza A viruses.

H2N2 influenza A virus was the cause of the 1957 pandemic. Due to its constant presence in birds, the H2 subtype remains a topic of interest. In this work, comparison of H2 leader sequences of influenza A segment 4 revealed the presence of an upstream in-frame start codon in a majority of North American avian strains. This AUG is located seven codons upstream of the conventional start codon and is in a good Kozak context. In vivo experiments, using a luciferase reporter gene fused to leader sequences derived from North American avian H2 strains, support the efficient use of the upstream start codon. These results were corroborated by in vitro translation data using full-length segment 4 mRNA. Phylogenic analyses indicate that the upstream AUG, first detected in 1976, is stably nested in the North American avian lineage of H2 strains nowadays. The possible consequences of the upstream AUG are discussed.

Role of the phenazine-inducing protein Pip in stress resistance of Pseudomonas chlororaphis.

The triggering of antibiotic production by various environmental stress molecules can be interpreted as bacteria's response to obtain increased fitness to putative danger, whereas the opposite situation - inhibition of antibiotic production - is more complicated to understand. Phenazines enable Pseudomonas species to eliminate competitors for rhizosphere colonization and are typical virulence factors used for model studies. In the present work, we have investigated the negative effect of subinhibitory concentrations of NaCl, fusaric acid and two antibiotics on quorum-sensing-controlled phenazine production by Pseudomonas chlororaphis. The selected stress factors inhibit phenazine synthesis despite sufficient cell density. Subsequently, we have identified connections between known genes of the phenazine-inducing cascade, including PsrA (Pseudomonas sigma regulator), RpoS (alternative sigma factor), Pip (phenazine inducing protein) and PhzI/PhzR (quorum-sensing system). Under all tested conditions, overexpression of Pip or PhzR restored phenazine production while overexpression of PsrA or RpoS did not. This forced restoration of phenazine production in strains overexpressing regulatory genes pip and phzR significantly impairs growth and stress resistance; this is particularly severe with pip overexpression. We suggest a novel physiological explanation for the inhibition of phenazine virulence factors in pseudomonas species responding to toxic compounds. We propose that switching off phenazine-1-carboxamide (PCN) synthesis by attenuating pip expression would favour processes required for survival. In our model, this 'decision' point for promoting PCN production or stress resistance is located downstream of rpoS and just above pip. However, a test with the stress factor rifampicin shows no significant inhibition of Pip production, suggesting that stress factors may also target other and so far unknown protagonists of the PCN signalling cascade.

Central role of quorum sensing in regulating the production of pathogenicity factors in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic human pathogen, causing various infections that are often very persistent. P. aeruginosa infections are the major cause of death in cystic fibrosis patients. Infections are difficult to treat since P. aeruginosa is resistant to most antibiotics and its antibiotic susceptibility is decreased when it is present in biofilms. P. aeruginosa produces many exoproducts (including toxins and hydrolytic enzymes) that are involved in virulence. Recent research has elucidated many mechanisms and pathways that regulate the production of these virulence factors. The regulation is extremely complex and many components are influenced by environmental conditions. Quorum sensing is a key regulatory system, which itself is affected by many other regulators. Targeting the regulation of pathogenicity factors provides a novel strategy for combating P. aeruginosa infections. Degradation of acyl homoserine lactones, the signaling molecules of the quorum-sensing system, is a promising therapeutic treatment option.

Pip, a novel activator of phenazine biosynthesis in Pseudomonas chlororaphis PCL1391.

Secondary metabolites are important factors for interactions between bacteria and other organisms. Pseudomonas chlororaphis PCL1391 produces the antifungal secondary metabolite phenazine-1-carboxamide (PCN) that inhibits growth of Fusarium oxysporum f. sp. radius lycopersici the causative agent of tomato foot and root rot. Our previous work unraveled a cascade of genes regulating the PCN biosynthesis operon, phzABCDEFGH. Via a genetic screen, we identify in this study a novel TetR/AcrR regulator, named Pip (phenazine inducing protein), which is essential for PCN biosynthesis. A combination of a phenotypical characterization of a pip mutant, in trans complementation assays of various mutant strains, and electrophoretic mobility shift assays identified Pip as the fifth DNA-binding protein so far involved in regulation of PCN biosynthesis. In this regulatory pathway, Pip is positioned downstream of PsrA (Pseudomonas sigma factor regulator) and the stationary-phase sigma factor RpoS, while it is upstream of the quorum-sensing system PhzI/PhzR. These findings provide further evidence that the path leading to the expression of secondary metabolism gene clusters in Pseudomonas species is highly complex.

Regulatory roles of psrA and rpoS in phenazine-1-carboxamide synthesis by Pseudomonas chlororaphis PCL1391.

Production of the secondary metabolite phenazine-1-carboxamide (PCN) by Pseudomonas chlororaphis PCL1391 is crucial for biocontrol activity against the phytopathogen Fusarium oxysporum f. sp. radicis lycopersici on tomato. Regulation of PCN production involves the two-component signalling system GacS/GacA, the quorum-sensing system PhzI/PhzR and the regulator PsrA. This paper reports that a functional rpoS is required for optimal PCN and N-hexanoyl-L-homoserine lactone (C(6)-HSL) production. Constitutive expression of rpoS is able to complement partially the defect of a psrA mutant for PCN and N-acylhomoserine lactone production. Western blotting shows that rpoS is regulated by gacS. Altogether, these results suggest the existence of a cascade consisting of gacS/gacA upstream of psrA and rpoS, which influence expression of phzI/phzR. Overproduction of phzR complements the effects on PCN and C(6)-HSL production of all mutations tested in the regulatory cascade, which shows that a functional quorum-sensing system is essential and sufficient for PCN synthesis. In addition, the relative amounts of PCN, phenazine-1-carboxylic acid and C(6)-HSL produced by rpoS and psrA mutants harbouring a constitutively expressed phzR indicate an even more complex network of interactions, probably involving other genes. Preliminary microarray analyses of the transcriptomics of the rpoS and psrA mutants support the model of regulation described in this study and allow identification of new genes that might be involved in secondary metabolism.

Influence of fusaric acid on phenazine-1-carboxamide synthesis and gene expression of Pseudomonas chlororaphis strain PCL1391.

Production of the antifungal metabolite phenazine-1-carboxamide (PCN) by Pseudomonas chlororaphis strain PCL1391 is essential for the suppression of tomato foot and root rot caused by the soil-borne fungus F. oxysporum f. sp. radicis-lycopersici. The authors have shown previously that fusaric acid (FA), a phytotoxin produced by Fusarium oxysporum, represses the production of PCN and of the quorum-sensing signal N-hexanoyl-l-homoserine lactone (C(6)-HSL). Here they report that PCN repression by FA is maintained even during PCN-stimulating environmental conditions such as additional phenylalanine, additional ferric iron and a low Mg(2+) concentration. Constitutive expression of phzI or phzR increases the production of C(6)-HSL and abolishes the repression of PCN production by FA. Transcriptome analysis using P. chlororaphis PCL1391 microarrays showed that FA represses expression of the phenazine biosynthetic operon (phzABCDEFGH) and of the quorum-sensing regulatory genes phzI and phzR. FA does not alter expression of the PCN regulators gacS, rpoS and psrA. In conclusion, reduction of PCN levels by FA is due to direct or indirect repression of phzR and phzI. Microarray analyses identified genes of which the expression is strongly influenced by FA. Genes highly upregulated by FA are also upregulated by iron starvation in Pseudomonas aeruginosa. This remarkable overlap in the expression profile suggests an overlapping stress response to FA and iron starvation.

Structural motifs in the RNA encoded by the early nodulation gene enod40 of soybean.

The plant gene enod40 is highly conserved among legumes and also present in various non-legume species. It is presumed to play a central regulatory role in the Rhizobium-legume interaction, being expressed well before the initiation of cortical cell divisions resulting in nodule formation. Two small peptides encoded by enod40 mRNA as well as its secondary structure have been shown to be key elements in the signalling processes underlying nodule organogenesis. Here results concerning the secondary structure of mRNA of enod40 in soybean are presented. This study combined a theoretical approach, involving structure prediction and comparison, as well as structure probing. Our study indicates five conserved domains in enod40 mRNA among numerous leguminous species. Structure comparison suggests that some domains are also conserved in non-leguminous species and that an additional domain exists that was found only in leguminous species developing indeterminate nodules. Enzymatic and chemical probing data support the structure for three of the domains, and partially for the remaining two. The rest of the molecule appears to be less structured. Some of the domains include motifs, such as U-containing internal loops and bulges, which seem to be conserved. Therefore, they might be involved in the regulatory role of enod40 RNA.