Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea

The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus–Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the β-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this β-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.     

Genomic evidence of rapid,global-scale gene flow in a Sulfolobus species

Local populations of Sulfolobus islandicus diverge genetically with geographical separation, and this has been attributed to restricted transfer of propagules imposed by the unfavorable spatial distribution of acidic geothermal habitat. We tested the generality of genetic divergence with distance in Sulfolobus species by analyzing genomes of Sulfolobus acidocaldarius drawn from three populations separated by more than 8000 km. In sharp contrast to S. islandicus, the geographically diverse S. acidocaldarius genomes proved to be nearly identical. We could not link the difference in genome conservation between the two species to a corresponding difference in genome stability or ecological factors affecting propagule dispersal. The results provide the first evidence that genetic isolation of local populations does not result primarily from properties intrinsic to Sulfolobus and the severe discontinuity of its geothermal habitat, but varies with species, and thus may reflect biotic interactions that act after propagule dispersal.     

Genomic and proteomic analyses of the coral pathogen Vibrio coralliilyticus reveal a diverse virulence repertoire

Vibrio coralliilyticus has been implicated as an important pathogen of coral species worldwide. In this study, the nearly complete genome of Vibrio coralliilyticus strain P1 (LMG23696) was sequenced and proteases implicated in virulence of the strain were specifically investigated. The genome sequence of P1 (5 513 256 bp in size) consisted of 5222 coding sequences and 58 RNA genes (53 tRNAs and at least 5 rRNAs). Seventeen metalloprotease and effector (vgrG, hlyA and hcp) genes were identified in the genome and expressed proteases were also detected in the secretome of P1. As the VcpA zinc-metalloprotease has been considered an important virulence factor of V. coralliilyticus, a vcpA deletion mutant was constructed to evaluate the effect of this gene in animal pathogenesis. Both wild-type and mutant (ΔvcpA) strains exhibited similar virulence characteristics that resulted in high mortality in Artemia and Drosophila pathogenicity bioassays and strong photosystem II inactivation of the coral dinoflagellate endosymbiont (Symbiodinium). In contrast, the ΔvcpA mutant demonstrated higher hemolytic activity and secreted 18 proteins not secreted by the wild type. These proteins included four types of metalloproteases, a chitinase, a hemolysin-related protein RbmC, the Hcp protein and 12 hypothetical proteins. Overall, the results of this study indicate that V. coralliilyticus strain P1 has a diverse virulence repertoire that possibly enables this bacterium to be an efficient animal pathogen.     

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous,multidrug resistant,biofilm producing isolate from India

Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniaespecies from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1,blaSHV-28, aac(6’)1b-cr,catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1,aac(6’)-Ib, aac(3)-IId,sul1,2, blaTEM-1A,1B,blaOXA-9, blaCTX-M-15,blaSHV-11, cmlA1, erm(B),mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniaestrains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13 was found inserted.     

Differential detection of transposable elements between Saccharum species

Cultivars of sugarcane (Saccharum) are hybrids between species S. officinarum (x = 10, 2n = 8x = 80) and S. spontaneum (x = 8, 2n = 5 – 16x = 40 – 128). These accessions have 100 to 130 chromosomes, 80–85% of which are derived from S. officinarum, 10–15% from S. spontaneum, and 5–10% are possible recombinants between the two genomes. The aim of this study was to analyze the repetition of DNA sequences in S. officinarum and S. spontaneum. For this purpose, genomic DNA from S. officinarum was digested with restriction enzymes and the fragments cloned. Sixty-eight fragments, approximately 500 bp, were cloned, sequenced and had their identity analyzed in NCBI, and in the rice, maize, and sorghum genome databases using BLAST. Twelve clones containing partial transposable elements, one single-copy control, one DNA repetitive clone control and two genome controls were analyzed by DNA hybridization on membrane, using genomic probes from S. officinarum and S. spontaneum. The hybridization experiment revealed that six TEs had a similar repetitive DNA pattern in the genomes of S. officinarum and S. spontaneum, while six TEs were more abundant in the genome of S. officinarum. We concluded that the species S. officinarum and S. spontaneum have differential accumulation LTR retrotransposon families, suggesting distinct insertion or modification patterns.     

Comparative genomic analysis reveals occurrence of genetic recombination in virulent Cryptosporidium hominis subtypes and telomeric gene duplications in Cryptosporidium parvum

Background

Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis–associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent yearsIaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome.

Results

Altogether, 8.59-9.05 Mb of Cryptosporidium sequences in 45–767 assembled contigs were obtained from the four specimens, representing 94.36-99.47% coverage of the expected genome. These genomes had complete synteny in gene organization and 96.86-97.0% and 99.72-99.83% nucleotide sequence similarities to the published genomes of C. parvum and C. hominis, respectively. Several major insertions and deletions were seen between C. hominis and C. parvum genomes, involving mostly members of multicopy gene families near telomeres. The four C. hominis genomes were highly similar to each other and divergent from the reference IaA25R3 genome in some highly polymorphic regions. Major sequence differences among the four specimens sequenced in this study were in the 5′ and 3′ ends of chromosome 6 and the gp60 region, largely the result of genetic recombination.

Conclusions

The sequence similarity among specimens of the two dominant outbreak subtypes and genetic recombination in chromosome 6, especially around the putative virulence determinant gp60 region, suggest that genetic recombination plays a potential role in the emergence of hyper-transmissible C. hominis subtypes. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of MEDLE family secreted proteins and insulinase-like proteases indicate that telomeric gene duplications could potentially contribute to host expansion in C. parvum.

Electronic supplementary material

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Rendering the Intractable More Tractable: Tools from Caenorhabditis elegans Ripe for Import into Parasitic Nematodes

Recent and rapid advances in genetic and molecular tools have brought spectacular tractability to Caenorhabditis elegans, a model that was initially prized because of its simple design and ease of imaging. C. elegans has long been a powerful model in biomedical research, and tools such as RNAi and the CRISPR/Cas9 system allow facile knockdown of genes and genome editing, respectively. These developments have created an additional opportunity to tackle one of the most debilitating burdens on global health and food security: parasitic nematodes. I review how development of nonparasitic nematodes as genetic models informs efforts to import tools into parasitic nematodes. Current tools in three commonly studied parasites (Strongyloides spp., Brugia malayi, and Ascaris suum) are described, as are tools from C. elegans that are ripe for adaptation and the benefits and barriers to doing so. These tools will enable dissection of a huge array of questions that have been all but completely impenetrable to date, allowing investigation into host–parasite and parasite–vector interactions, and the genetic basis of parasitism.     

An Archaeal Immune System Can Detect Multiple Protospacer Adjacent Motifs (PAMs) to Target Invader DNA

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.     

A Site-Specific Integrative Plasmid Found in Pseudomonas aeruginosa Clinical Isolate HS87 along with A Plasmid Carrying an Aminoglycoside-Resistant Gene

Plasmids play critical roles in bacterial fitness and evolution of Pseudomonas aeruginosa. Here two plasmids found in a drug-resistant P. aeruginosa clinical isolate HS87 were completely sequenced. The pHS87b plasmid (11.2 kb) carries phage-related genes and function-unknown genes. Notably, pHS87b encodes an integrase and has an adjacent tRNA^Thr-associated attachment site. A corresponding integrated form of pHS87b at the tRNA^Thr locus was identified on the chromosome of P. aeruginosa, showing that pHS87b is able to site-specifically integrate into the 3’-end of the tRNA^Thr gene. The pHS87a plasmid (26.8 kb) displays a plastic structure containing a putative replication module, stability factors and a variable region. The RepA of pHS87a shows significant similarity to the replication proteins of pPT23A-family plasmids. pHS87a carries a transposon Tn6049, a truncated insertion sequence ΔIS1071 and a Tn402-like class 1 integron which contains an aacA4 cassette that may confer aminoglycoside resistance. Thus, pHS87b is a site-specific integrative plasmid whereas pHS87a is a plastic antibiotic resistance plasmid. The two native plasmids may promote the fitness and evolution of P. aeruginosa.     

Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia

Background

Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes.

Results

Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30 % of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome.

Conclusions

Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20 %.

Electronic supplementary material

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Identification of three extra-chromosomal replicons in Leptospira pathogenic strain and development of new shuttle vectors

Background

The genome of pathogenic Leptospira interrogans contains two chromosomes. Plasmids and prophages are known to play specific roles in gene transfer in bacteria and can potentially serve as efficient genetic tools in these organisms. Although plasmids and prophage remnants have recently been reported in Leptospira species, their characteristics and potential applications in leptospiral genetic transformation systems have not been fully evaluated.

Results

Three extrachromosomal replicons designated lcp1 (65,732 bp), lcp2 (56,757 bp), and lcp3 (54,986 bp) in the L. interrogans serovar Linhai strain 56609 were identified through whole genome sequencing. All three replicons were stable outside of the bacterial chromosomes. Phage particles were observed in the culture supernatant of 56609 after mitomycin C induction, and lcp3, which contained phage-related genes, was considered to be an inducible prophage. L. interrogans–Escherichia coli shuttle vectors, constructed with the predicted replication elements of single rep or rep combined with parAB loci from the three plasmids were shown to successfully transform into both saprophytic and pathogenic Leptospira species, suggesting an essential function for rep genes in supporting auto-replication of the plasmids. Additionally, a wide distribution of homologs of the three rep genes was identified in L. interrogans isolates, and correlation tests showed that the transformability of the shuttle vectors in L. interrogans isolates depended, to certain extent, on genetic compatibility between the rep sequences of both plasmid and host.

Conclusions

Three extrachromosomal replicons co-exist in L. interrogans, one of which we consider to be an inducible prophage. The vectors constructed with the rep genes of the three replicons successfully transformed into saprophytic and pathogenic Leptospira species alike, but this was partly dependent on genetic compatibility between the rep sequences of both plasmid and host.

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The online version of this article (doi:10.1186/s12864-015-1321-y) contains supplementary material, which is available to authorized users.     

Unusual genome complexity in Lactobacillus salivarius JCM1046

Background

Lactobacillus salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable and is often mediated by plasmids or integrative and conjugative elements. L. salivarius strains typically have multireplicon genomes including circular megaplasmids that encode strain-specific traits for intestinal survival and probiotic activity. Linear plasmids are less common in lactobacilli and show a very limited distribution in L. salivarius. Here we present experimental evidence that supports an unusually complex multireplicon genome structure in the porcine isolate L. salivarius JCM1046.

Results

JCM1046 harbours a 1.83 Mb chromosome, and four plasmids which constitute 20% of the genome. In addition to the known 219 kb repA-type megaplasmid pMP1046A, we identified and experimentally validated the topology of three additional replicons, the circular pMP1046B (129 kb), a linear plasmid pLMP1046 (101 kb) and pCTN1046 (33 kb) harbouring a conjugative transposon. pMP1046B harbours both plasmid-associated replication genes and paralogues of chromosomally encoded housekeeping and information-processing related genes, thus qualifying it as a putative chromid. pLMP1046 shares limited sequence homology or gene synteny with other L. salivarius plasmids, and its putative replication-associated protein is homologous to the RepA/E proteins found in the large circular megaplasmids of L. salivarius. Plasmid pCTN1046 harbours a single copy of an integrated conjugative transposon (Tn6224) which appears to be functionally intact and includes the tetracycline resistance gene tetM.

Conclusion

Experimental validation of sequence assemblies and plasmid topology resolved the complex genome architecture of L. salivarius JCM1046. A high-coverage draft genome sequence would not have elucidated the genome complexity in this strain. Given the expanding use of L. salivarius as a probiotic, it is important to determine the genotypic and phenotypic organization of L. salivarius strains. The identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications.

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Role of the Yeast Gin4p Protein Kinase in Septin Assembly and the Relationship between Septin Assembly and Septin Function

To identify septin-interacting proteins in Saccharomyces cerevisiae, we screened for mutations that are synthetically lethal with a cdc12 septin mutation. One of the genes identified was GIN4, which encodes a protein kinase related to Hsl1p/Nik1p and Ycl024Wp in S. cerevisiae and to Nim1p/Cdr1p and Cdr2p in Schizosaccharomyces pombe. The Gin4p kinase domain displayed a two-hybrid interaction with the COOH-terminal portion of the Cdc3p septin, and Gin4p colocalized with the septins at the mother–bud neck. This localization depended on the septins and on the COOH-terminal (nonkinase) region of Gin4p, and overproduction of this COOH-terminal region led to a loss of septin organization and associated morphogenetic defects. We detected no effect of deleting YCL024W, either alone or in combination with deletion of GIN4. Deletion of GIN4 was not lethal but led to a striking reorganization of the septins accompanied by morphogenetic abnormalities and a defect in cell separation; however, remarkably, cytokinesis appeared to occur efficiently. Two other proteins that localize to the neck in a septin-dependent manner showed similar reorganizations and also appeared to remain largely functional. The septin organization observed in gin4Δ vegetative cells resembles that seen normally in cells responding to mating pheromone, and no Gin4p was detected in association with the septins in such cells. The organization of the septins observed in gin4Δ cells and in cells responding to pheromone appears to support some aspects of the model for septin organization suggested previously by Field et al. (Field, C.M., O. Al-Awar, J. Rosenblatt, M.L. Wong, B. Alberts, and T.J. Mitchison. 1996. J. Cell Biol. 133:605–616).     

Comparative genomics of two ‘Candidatus Accumulibacter' clades performing biological phosphorus removal

Members of the genus Candidatus Accumulibacter are important in many wastewater treatment systems performing enhanced biological phosphorus removal (EBPR). The Accumulibacter lineage can be subdivided phylogenetically into multiple clades, and previous work showed that these clades are ecologically distinct. The complete genome of Candidatus Accumulibacter phosphatis strain UW-1, a member of Clade IIA, was previously sequenced. Here, we report a draft genome sequence of Candidatus Accumulibacter spp. strain UW-2, a member of Clade IA, assembled following shotgun metagenomic sequencing of laboratory-scale bioreactor sludge. We estimate the genome to be 80–90% complete. Although the two clades share 16S rRNA sequence identity of >98.0%, we observed a remarkable lack of synteny between the two genomes. We identified 2317 genes shared between the two genomes, with an average nucleotide identity (ANI) of 78.3%, and accounting for 49% of genes in the UW-1 genome. Unlike UW-1, the UW-2 genome seemed to lack genes for nitrogen fixation and carbon fixation. Despite these differences, metabolic genes essential for denitrification and EBPR, including carbon storage polymer and polyphosphate metabolism, were conserved in both genomes. The ANI from genes associated with EBPR was statistically higher than that from genes not associated with EBPR, indicating a high selective pressure in EBPR systems. Further, we identified genomic islands of foreign origins including a near-complete lysogenic phage in the Clade IA genome. Interestingly, Clade IA appeared to be more phage susceptible based on it containing only a single Clustered Regularly Interspaced Short Palindromic Repeats locus as compared with the two found in Clade IIA. Overall, the comparative analysis provided a genetic basis to understand physiological differences and ecological niches of Accumulibacter populations, and highlights the importance of diversity in maintaining system functional resilience.     

Genome sequence and comparative analysis of a putative entomopathogenic Serratia isolated from Caenorhabditis briggsae

Background

Entomopathogenic associations between nematodes in the genera Steinernema and Heterorhabdus with their cognate bacteria from the bacterial genera Xenorhabdus and Photorhabdus, respectively, are extensively studied for their potential as biological control agents against invasive insect species. These two highly coevolved associations were results of convergent evolution. Given the natural abundance of bacteria, nematodes and insects, it is surprising that only these two associations with no intermediate forms are widely studied in the entomopathogenic context. Discovering analogous systems involving novel bacterial and nematode species would shed light on the evolutionary processes involved in the transition from free living organisms to obligatory partners in entomopathogenicity.

Results

We report the complete genome sequence of a new member of the enterobacterial genus Serratia that forms a putative entomopathogenic complex with Caenorhabditis briggsae. Analysis of the 5.04 MB chromosomal genome predicts 4599 protein coding genes, seven sets of ribosomal RNA genes, 84 tRNA genes and a 64.8 KB plasmid encoding 74 genes. Comparative genomic analysis with three of the previously sequenced Serratia species, S. marcescens DB11 and S. proteamaculans 568, and Serratia sp. AS12, revealed that these four representatives of the genus share a core set of ~3100 genes and extensive structural conservation. The newly identified species shares a more recent common ancestor with S. marcescens with 99 % sequence identity in rDNA sequence and orthology across 85.6 % of predicted genes. Of the 39 genes/operons implicated in the virulence, symbiosis, recolonization, immune evasion and bioconversion, 21 (53.8 %) were present in Serratia while 33 (84.6 %) and 35 (89 %) were present in Xenorhabdus and Photorhabdus EPN bacteria respectively.

Conclusion

The majority of unique sequences in Serratia sp. SCBI (South African Caenorhabditis briggsae Isolate) are found in ~29 genomic islands of 5 to 65 genes and are enriched in putative functions that are biologically relevant to an entomopathogenic lifestyle, including non-ribosomal peptide synthetases, bacteriocins, fimbrial biogenesis, ushering proteins, toxins, secondary metabolite secretion and multiple drug resistance/efflux systems. By revealing the early stages of adaptation to this lifestyle, the Serratia sp. SCBI genome underscores the fact that in EPN formation the composite end result – killing, bioconversion, cadaver protection and recolonization- can be achieved by dissimilar mechanisms. This genome sequence will enable further study of the evolution of entomopathogenic nematode-bacteria complexes.

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