Genome evolution and the emergence of fruiting body development in Myxococcus xanthus

Background

Lateral gene transfer (LGT) is thought to promote speciation in bacteria, though well-defined examples have not been put forward.

Methodology/Principle Findings

We examined the evolutionary history of the genes essential for a trait that defines a phylogenetic order, namely fruiting body development of the Myxococcales. Seventy-eight genes that are essential for Myxococcus xanthus development were examined for LGT. About 73% of the genes exhibit a phylogeny similar to that of the 16S rDNA gene and a codon bias consistent with other M. xanthus genes suggesting vertical transmission. About 22% have an altered codon bias and/or phylogeny suggestive of LGT. The remaining 5% are unique. Genes encoding signal production and sensory transduction were more likely to be transmitted vertically with clear examples of duplication and divergence into multigene families. Genes encoding metabolic enzymes were frequently acquired by LGT. Myxobacteria exhibit aerobic respiration unlike most of the δ Proteobacteria. M. xanthus contains a unique electron transport pathway shaped by LGT of genes for succinate dehydrogenase and three cytochrome oxidase complexes.

Conclusions/Significance

Fruiting body development depends on genes acquired by LGT, particularly those involved in polysaccharide production. We suggest that aerobic growth fostered innovation necessary for development by allowing myxobacteria access to a different gene pool from anaerobic members of the δ Proteobacteria. Habitat destruction and loss of species diversity could restrict the evolution of new bacterial groups by limiting the size of the prospective gene pool.     

Maize protein phosphatase gene family: identification and molecular characterization

Background

Protein phosphatases (PPs) play critical roles in various cellular processes through the reversible protein phosphorylation that dictates many signal transduction pathways among organisms. Recently, PPs in Arabidopsis and rice have been identified, while the whole complement of PPs in maize is yet to be reported.

Results

In this study, we have identified 159 PP-encoding genes in the maize genome. Phylogenetic analyses categorized the ZmPP gene family into 3 classes (PP2C, PTP, and PP2A) with considerable conservation among classes. Similar intron/exon structural patterns were observed in the same classes. Moreover, detailed gene structures and duplicative events were then researched. The expression profiles of ZmPPs under different developmental stages and abiotic stresses (including salt, drought, and cold) were analyzed using microarray and RNA-seq data. A total of 152 members were detected in 18 different tissues representing distinct stages of maize plant developments. Under salt stress, one gene was significantly up-expressed in seed root (SR) and one gene was down-expressed in primary root (PR) and crown root (CR), respectively. As for drought stress condition, 13 genes were found to be differentially expressed in leaf, out of which 10 were up-regulated and 3 exhibited down-regulation. Additionally, 13 up-regulated and 3 down-regulated genes were found in cold-tolerant line ETH-DH7. Furthermore, real-time PCR was used to confirm the expression patterns of ZmPPs.

Conclusions

Our results provide new insights into the phylogenetic relationships and characteristic functions of maize PPs and will be useful in studies aimed at revealing the global regulatory network in maize abiotic stress responses, thereby contributing to the maize molecular breeding with enhanced quality traits.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-773) contains supplementary material, which is available to authorized users.     

BEACON: automated tool for Bacterial GEnome Annotation ComparisON

Background

Genome annotation is one way of summarizing the existing knowledge about genomic characteristics of an organism. There has been an increased interest during the last several decades in computer-based structural and functional genome annotation. Many methods for this purpose have been developed for eukaryotes and prokaryotes. Our study focuses on comparison of functional annotations of prokaryotic genomes. To the best of our knowledge there is no fully automated system for detailed comparison of functional genome annotations generated by different annotation methods (AMs).

Results

The presence of many AMs and development of new ones introduce needs to: a/ compare different annotations for a single genome, and b/ generate annotation by combining individual ones. To address these issues we developed an Automated Tool for Bacterial GEnome Annotation ComparisON (BEACON) that benefits both AM developers and annotation analysers. BEACON provides detailed comparison of gene function annotations of prokaryotic genomes obtained by different AMs and generates extended annotations through combination of individual ones. For the illustration of BEACON’s utility, we provide a comparison analysis of multiple different annotations generated for four genomes and show on these examples that the extended annotation can increase the number of genes annotated by putative functions up to 27 %, while the number of genes without any function assignment is reduced.

Conclusions

We developed BEACON, a fast tool for an automated and a systematic comparison of different annotations of single genomes. The extended annotation assigns putative functions to many genes with unknown functions. BEACON is available under GNU General Public License version 3.0 and is accessible at: http://www.cbrc.kaust.edu.sa/BEACON/.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1826-4) contains supplementary material, which is available to authorized users.     

Diversity of 23S rRNA Genes within Individual Prokaryotic Genomes

Background

The concept of ribosomal constraints on rRNA genes is deduced primarily based on the comparison of consensus rRNA sequences between closely related species, but recent advances in whole-genome sequencing allow evaluation of this concept within organisms with multiple rRNA operons.

Methodology/Principal Findings

Using the 23S rRNA gene as an example, we analyzed the diversity among individual rRNA genes within a genome. Of 184 prokaryotic species containing multiple 23S rRNA genes, diversity was observed in 113 (61.4%) genomes (mean 0.40%, range 0.01%–4.04%). Significant (1.17%–4.04%) intragenomic variation was found in 8 species. In 5 of the 8 species, the diversity in the primary structure had only minimal effect on the secondary structure (stem versus loop transition). In the remaining 3 species, the diversity significantly altered local secondary structure, but the alteration appears minimized through complex rearrangement. Intervening sequences (IVS), ranging between 9 and 1471 nt in size, were found in 7 species. IVS in Deinococcus radiodurans and Nostoc sp. encode transposases. T. tengcongensis was the only species in which intragenomic diversity >3% was observed among 4 paralogous 23S rRNA genes.

Conclusions/Significance

These findings indicate tight ribosomal constraints on individual 23S rRNA genes within a genome. Although classification using primary 23S rRNA sequences could be erroneous, significant diversity among paralogous 23S rRNA genes was observed only once in the 184 species analyzed, indicating little overall impact on the mainstream of 23S rRNA gene-based prokaryotic taxonomy.     

Population structure of mitochondrial genomes in Saccharomyces cerevisiae

Background

Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences.

Results

To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae.

Conclusions

Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1664-4) contains supplementary material, which is available to authorized users.     

Vitamins A & D inhibit the growth of mycobacteria in radiometric culture

Background

The role of vitamins in the combat of disease is usually conceptualized as acting by modulating the immune response of an infected, eukaryotic host. We hypothesized that some vitamins may directly influence the growth of prokaryotes, particularly mycobacteria.

Methods

The effect of four fat-soluble vitamins was studied in radiometric Bactec® culture. The vitamins were A (including a precursor and three metabolites,) D, E and K. We evaluated eight strains of three mycobacterial species (four of M. avium subspecies paratuberculosis (MAP), two of M. avium and two of M. tb. complex).

Principal Findings

Vitamins A and D cause dose-dependent inhibition of all three mycobacterial species studied. Vitamin A is consistently more inhibitory than vitamin D. The vitamin A precursor, β-carotene, is not inhibitory, whereas three vitamin A metabolites cause inhibition. Vitamin K has no effect. Vitamin E causes negligible inhibition in a single strain.

Significance

We show that vitamin A, its metabolites Retinyl acetate, Retinoic acid and 13-cis Retinoic acid and vitamin D directly inhibit mycobacterial growth in culture. These data are compatible with the hypothesis that complementing the immune response of multicellular organisms, vitamins A and D may have heretofore unproven, unrecognized, independent and probable synergistic, direct antimycobacterial inhibitory activity.     

Developmental morphology of strap-shaped gametophytes of Colysis decurrens: a new look at meristem development and function in fern gametophytes

Background and Aims

The gametophytes of most homosporous ferns are cordate–thalloid in shape. Some are strap- or ribbon-shaped and have been assumed to have evolved from terrestrial cordate shapes as an adaptation to epiphytic habitats. The aim of the present study was to clarify the morphological evolution of the strap-shaped gametophyte of microsoroids (Polypodiaceae) by precise analysis of their development.

Methods

Spores of Colysis decurrens collected in Kagoshima, Japan, were cultured and observed microscopically. Epi-illuminated micrographs of growing gametophytes were captured every 24 h, allowing analysis of the cell lineage of meristems. Light microscopy of resin-sections and scanning electron microscopy were also used.

Key Results

Contrary to previous assumptions that strap-shaped Colysis gametophytes have no organized meristem, three different types of meristems are formed during development: (1) apical-cell based – responsible for early growth; (2) marginal – further growth, including gametophyte branching; and (3) multicellular – formation of cushions with archegonia. The cushion is two or three layers thick and intermittent. The apical-cell and multicellular meristems are similar to those of cordate gametophytes of other ferns, but the marginal meristem is unique to the strap-shaped gametophyte of this fern.

Conclusions

The strap-shaped gametophytes of C. decurrens may have evolved from ancestors with a cordate shape by insertion of the marginal meristem phase between the first apical-cell-based meristem and subsequent multicellular meristem phases. Repeated retrieval of the marginal meristem at the multicellular meristem phase would result in indefinite prolongation of gametophyte growth, an ecological adaptation to epiphytic habitats.     

Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity

Background

So-called 936-type phages are among the most frequently isolated phages in dairy facilities utilising Lactococcus lactis starter cultures. Despite extensive efforts to control phage proliferation and decades of research, these phages continue to negatively impact cheese production in terms of the final product quality and consequently, monetary return.

Results

Whole genome sequencing and in silico analysis of three 936-type phage genomes identified several putative (orphan) methyltransferase (MTase)-encoding genes located within the packaging and replication regions of the genome. Utilising SMRT sequencing, methylome analysis was performed on all three phages, allowing the identification of adenine modifications consistent with N-6 methyladenine sequence methylation, which in some cases could be attributed to these phage-encoded MTases. Heterologous gene expression revealed that M.Phi145I/M.Phi93I and M.Phi93DAM, encoded by genes located within the packaging module, provide protection against the restriction enzymes HphI and DpnII, respectively, representing the first functional MTases identified in members of 936-type phages.

Conclusions

SMRT sequencing technology enabled the identification of the target motifs of MTases encoded by the genomes of three lytic 936-type phages and these MTases represent the first functional MTases identified in this species of phage. The presence of these MTase-encoding genes on 936-type phage genomes is assumed to represent an adaptive response to circumvent host encoded restriction-modification systems thereby increasing the fitness of the phages in a dynamic dairy environment.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-831) contains supplementary material, which is available to authorized users.     

Genotype-phenotype correlation in Japanese patients with familial Mediterranean fever: differences in genotype and clinical features between Japanese and Mediterranean populations

Introduction

Familial Mediterranean fever (FMF) is a hereditary autoinflammatory disease characterized by recurrent self-limiting fever and serositis that mainly affects Mediterranean populations. Many patients with FMF have been reported in Japan due to increasing recognition of this condition and the availability of genetic analysis for the gene responsible, MEFV. The present study was performed to elucidate the clinical characteristics of Japanese FMF patients and to examine the precise genotype-phenotype correlation in a large cohort of Japanese FMF patients.

Methods

We analyzed the MEFV genotypes and clinical manifestations in 116 patients clinically diagnosed as having FMF and with at least one mutation.

Results

The most frequent mutation in Japanese patients was E148Q (40.2%), followed by M694I (21.0%), L110P (18.8%), P369S (5.4%), and R408Q (5.4%). In contrast, common mutations seen in Mediterranean patients, such as M694V, V726A, and M680I, were not detected in this population. The clinical features with M694I were associated with more severe clinical course compared to those seen with E148Q. P369S/R408Q showed variable phenotypes with regard to both clinical manifestations and severity. Patients with M694I showed a very favorable response to colchicine therapy, while those with P369S and R408Q did not.

Conclusions

Clinical features and efficacy of treatment in Japanese FMF patients vary widely according to the specific MEFV gene mutation, and therefore genetic analysis should be performed for diagnosis in cases of Japanese FMF.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0439-7) contains supplementary material, which is available to authorized users.     

Glycine Betaine Biosynthesized from Glycine Provides an Osmolyte for Cell Growth and Spore Germination during Osmotic Stress in Myxococcus xanthus

Glycine sarcosine methyltransferase (Gsm) and sarcosine dimethylglycine methyltransferase (Sdm) catalyze glycine betaine synthesis from glycine. Disruption of the M. xanthus gsmA (MXAN 7068) or sdmA (MXAN 3190) gene, encoding Gsm or Sdm homologue proteins, respectively, generated mutants that exhibited a longer lag period of growth and delayed spore germination under osmostress.Myxococcus xanthus is a Gram-negative bacterium that exhibits a complex multicellular developmental cycle (6, 7). These bacteria live in soil, where they prey on other microbes for food. In response to nutritional stress, hundreds of thousands of vegetative cells aggregate to form multicellular fruiting bodies containing differentiated myxospores. Once conditions become favorable for growth, the desiccation- and heat-resistant spores can germinate and initiate vegetative growth.It was reported previously that the receptor-type adenylyl cyclases CyaA and CyaB of M. xanthus act as osmosensors during spore germination and growth, respectively (8, 9). Glycine betaine is a very efficient osmolyte found in a wide range of prokaryotic and eukaryotic organisms, where it is accumulated at high cytoplasmic concentrations in response to osmotic stress (4, 16). In this study, it is reported that in M. xanthus glycine betaine can be biosynthesized from glycine and mainly functions as an osmoprotectant for cell growth and spore germination under osmotic stress conditions.     

A novel mitochondrial genome architecture in thrips (Insecta: Thysanoptera): extreme size asymmetry among chromosomes and possible recent control region duplication

Background

Multipartite mitochondrial genomes are very rare in animals but have been found previously in two insect orders with highly rearranged genomes, the Phthiraptera (parasitic lice), and the Psocoptera (booklice/barklice).

Results

We provide the first report of a multipartite mitochondrial genome architecture in a third order with highly rearranged genomes: Thysanoptera (thrips). We sequenced the complete mitochondrial genomes of two divergent members of the Scirtothrips dorsalis cryptic species complex. The East Asia 1 species has the single circular chromosome common to animals while the South Asia 1 species has a genome consisting of two circular chromosomes. The fragmented South Asia 1 genome exhibits extreme chromosome size asymmetry with the majority of genes on the large, 14.28 kb, chromosome and only nad6 and trnC on the 0.92 kb mini-circle chromosome. This genome also features paralogous control regions with high similarity suggesting a very recent origin of the nad6 mini-circle chromosome in the South Asia 1 cryptic species.

Conclusions

Thysanoptera, along with the other minor paraenopteran insect orders should be considered models for rapid mitochondrial genome evolution, including fragmentation. Continued use of these models will facilitate a greater understanding of recombination and other mitochondrial genome evolutionary processes across eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1672-4) contains supplementary material, which is available to authorized users.     

Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus

New natural products for drug discovery may be accessed by heterologous expression of bacterial biosynthetic pathways in metagenomic DNA libraries. However, a “universal” host is needed for this experiment. Herein, we show that Myxococcus xanthus is a potential “universal” host for heterologous expression of polyketide biosynthetic gene clusters.Bacterial natural products are excellent lead compounds for drug discovery and have played major roles in the development of pharmaceutical agents in nearly all therapeutic areas (1, 7, 9). Unfortunately, the rate of discovery of new bacterial natural products has decreased, due in part to frequent rediscovery of known compounds (7). An enormous and currently inaccessible reservoir of new natural products is located in the biosynthetic pathways found in the genomes of uncultivated bacteria (18). Heterologous expression of these biosynthetic gene clusters represents a powerful tool for discovering new natural products (20, 21). Herein, we demonstrate that the deltaproteobacterium Myxococcus xanthus is an effective host for heterologous expression of aromatic polyketide biosynthetic pathways. This work expands the scope of polyketide biosynthetic pathways which can be heterologously expressed in M. xanthus and suggests that M. xanthus may be a suitable general host for heterologous expression.Molecular phylogenetic studies have shown that bacterial diversity is enormous, and the vast majority of the diversity is found in uncultivated bacterial species (18). Estimates suggest that 99% of bacteria from the environment are uncultivatable using standard techniques (2, 15, 16). Culture-independent analyses of metagenomic DNA libraries from soil and marine environments indicate that there is a wealth of natural product diversity in these uncultivated strains. For example, analysis of a soil metagenome for a highly conserved region of polyketide synthase genes showed that none of the sequences found were present in the known public databases (5). Polyketide synthases are key enzymes responsible for the production of the polyketide family of natural products in proteobacteria, actinobacteria, and “low-G+C Gram-positive bacteria” (4, 12, 19). Polyketide natural products have been developed into antibiotic, anticancer, and immunosuppressant clinical agents (1, 6, 8). Based on these observations, metagenomic DNA libraries are expected to possess a large number of new polyketide biosynthetic pathways, representing substantial new chemical diversity for drug discovery.Heterologous expression of biosynthetic pathways can play a major role in interrogating metagenomic DNA libraries for new polyketide biosynthetic pathways. Heterologous production of polyketides in hosts such as Streptomyces coelicolor and Streptomyces lividans is an important tool in the identification and characterization of these pathways (6, 8, 17). Results from these studies have shown that Streptomyces strains are good hosts for heterologous production of many polyketides, particularly those from actinomycetes. However, Streptomyces strains have proved to be poor hosts for expression of deltaproteobacterial polyketide biosynthetic pathways, such as those in myxobacteria (10, 17). As polyketide biosynthetic pathways in metagenomic DNA libraries contain both actinomycete- and deltaproteobacterium-derived pathways, a heterologous expression host competent to express pathways of both origins is needed.We examined the ability of the deltaproteobacterium M. xanthus to act as a general heterologous expression host. M. xanthus is a predatory bacterium that undergoes multicellular development in response to nutrient starvation. During development, M. xanthus is known to be an effective host for the heterologous expression of the deltaproteobacterium-derived epothilone D biosynthetic pathway and has been used for the production of epothilone D for clinical trials (17). M. xanthus has also been shown to be an excellent host for the heterologous expression of several other myxobacterial metabolites, including myxothiazol and myxochromide S (3, 11, 22). We demonstrate that M. xanthus can also heterologously express the Streptomyces rimosus oxytetracycline biosynthetic pathway, producing oxytetracycline. This is the first example of a polyketide from a nonmyxobacterial species heterologously expressed in a myxobacterium.To generate an M. xanthus strain capable of heterologously expressing oxytetracycline, the Streptomyces rimosus oxytetracycline biosynthetic pathway (Fig. ​(Fig.1)1) was inserted via homologous recombination into the asgE locus of M. xanthus. The asgE locus of M. xanthus was amplified and inserted into the BglII site of pET28b (Novagen) to produce pMRH02. The oligonucleotides used for the amplification of the asgE locus were 5′-GACGAGATCTGTTGGAAGGTCGGCAACTGG-3′ and 5′-CTTAAGATCTTCCGTGAAGTACTGGCGCAC-3′. The asgE locus provides a chromosomal region for single-crossover homologous recombination into the M. xanthus chromosome. The 32-kb oxytetracycline pathway in S. rimosus was excised from pYT264 (24) and cloned into the EcoRI site of pMRH02 to produce pMRH08. M. xanthus DK1622 was electroporated under standard conditions (13) with pMRH08 to provide an M. xanthus ΔasgE Kan^r mutant. Positive selection for the chromosomal insertion was maintained throughout all experiments by use of kanamycin supplementation (40 μg/ml). This large genomic insertion significantly increased the doubling time for the strain (doubling time, ≈10 h).Open in a separate windowFIG. 1.Oxytetracycline biosynthetic pathway. (A) Enzymatic pathway responsible for formation of oxytetracycline. (B) Oxytetracycline biosynthesis gene cluster from S. rimosus.Oxytetracycline was heterologously produced in M. xanthus under standard rich medium culture conditions and detected in culture broth by liquid chromatography-mass spectrometry (LC-MS). A liquid culture of the mutant strain containing the oxytetracycline gene cluster was cultured for 10 days at 33°C in CTTYE (1.0% Casitone, 0.5% yeast extract, 10.0 mM Tris-HCl, 1.0 mM KH₂PO₄, and 8.0 mM MgSO₄; 100 ml). Acetone (10%, vol/vol) was added to the culture and vigorously mixed. The resulting mixture was extracted with 3 volumes of ethyl acetate to remove the organic soluble materials, including oxytetracycline. The organic extracts were concentrated in vacuo and resuspended in methanol (100 μl). LC-MS analyses were carried out using an Altima Hypersil C₁₈ column (3-μm particle size; 150 mm by 2.1 mm) with a linear gradient of water-acetonitrile (5 to 95%) with 0.05% formic acid over 90 min (0.20 ml/min), followed by positive-ion electrospray ionization (5,500 V) and analysis with a Shimadzu 2010A single quadrupole mass spectrometer. LC-MS analysis indicated that oxytetracycline was present in the fermentation broth (Fig. ​(Fig.2).2). The titer of oxytetracycline was determined to be approximately 10 mg per liter of fermentation broth. Quantification was performed in triplicate by LC-MS analysis using a standard curve generated from commercial oxytetracycline. Negative controls of M. xanthus DK1622 cultures processed under identical conditions did not contain detectable levels of oxytetracycline.Open in a separate windowFIG. 2.LC-MS ion extraction analysis of the molecular ion [M+H]⁺ of standard and culture extracts. (A) Oxytetracycline standard. (B) M. xanthus ΔasgE Kan^r mutant containing the oxytetracycline biosynthetic pathway. (C) Wild-type M. xanthus DK1622.These data indicate that M. xanthus can heterologously express the oxytetracycline polyketide synthase biosynthetic pathway in S. rimosus. Several factors affect the successful heterologous production of polyketide synthase pathways, including codon usage, mRNA stability, functionality of regulatory elements, and the presence of all necessary starter and extender units (14). As codon usages between M. xanthus and the genus Streptomyces are very similar and myxobacteria are known to produce polyketide products requiring a wide diversity of starter and extender units, neither codon usage nor starter and extender unit availability was considered likely to affect the ability of M. xanthus to heterologously express streptomycete biosynthetic pathways. As Streptomyces strains do not appear to be effective at heterologous expression of myxobacterial biosynthetic pathways, we were concerned that Myxococcus and Streptomyces strains may possess substantially different regulatory elements. Our data indicate that the regulatory elements present in streptomycete-derived biosynthetic pathways are sufficient to enable expression of the biosynthetic genes in M. xanthus. Further work exploring the regulatory elements present in myxobacterial polyketide biosynthetic gene clusters is needed to evaluate this hypothesis.This study demonstrates that M. xanthus can heterologously express streptomycete-derived polyketide biosynthetic pathways in addition to myxobacterial polyketide biosynthetic pathways. The observed titer of 10 mg/liter of culture broth is comparable to titers reported for the heterologous expression of myxobacterial polyketide biosynthetic pathways in myxobacteria (11) and streptomycete-derived polyketide biosynthetic pathways in Streptomyces (14, 23) and is sufficient for characterization of the polyketide product. Pseudomonas putida, which has a more favorable growth profile, has been shown to be a good host for heterologous expression of myxobacterial polyketide biosynthetic pathways, with product titers in the range of 0.6 to 40 mg/liter of culture broth (14, 21, 23). The observed breadth of polyketide pathways accessible and the titers of the polyketide products produced make M. xanthus an attractive potential candidate for a “universal” host for facilitating heterologous expression of polyketide biosynthetic pathways derived from environmental samples of metagenomic DNA.     

Studies on the antibacterial effects of statins--in vitro and in vivo

Background

Statin treatment has been associated with a beneficial outcome on respiratory tract infections. In addition, previous in vitro and in vivo experiments have indicated favorable effects of statins in bacterial infections.

Aim

The aim of the present study was to elucidate possible antibacterial effects of statins against primary pathogens of the respiratory tract.

Methods

MIC-values for simvastatin, fluvastatin and pravastatin against S. pneumoniae, M. catarrhalis and H. influenzae were determined by traditional antibacterial assays. A BioScreen instrument was used to monitor effects of statins on bacterial growth and to assess possible synergistic effects with penicillin. Bacterial growth in whole blood and serum from healthy volunteers before and after a single dose of simvastatin, fluvastatin and penicillin (positive control) was determined using a blood culture system (BactAlert).

Findings

The MIC-value for simvastatin against S pneumoniae and M catarrhalis was 15 µg/mL (36 mmol/L). Fluvastatin and Pravastatin showed no antibacterial effect in concentrations up to 100 µg/mL (230 µmol/L). Statins did not affect growth or viability of H influenzae. Single doses of statins given to healthy volunteers did not affect growth of pneumococci, whereas penicillin efficiently killed all bacteria.

Conclusions

Simvastatin at high concentrations 15 µg/mL (36 µmol/L) rapidly kills S pneumoniae and M catarrhalis. However, these concentrations by far exceed the concentrations detected in human blood during simvastatin therapy (1–15 nmol/L) and single doses of statins given to healthy volunteers did not improve antibacterial effects of whole blood. Thus, a direct bactericidal effect of statins in vivo is probably not the mechanism behind the observed beneficial effect of statins against various infections.     

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

Background

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species.

Methods and Findings

We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity.

Conclusions

The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.     

Dissemination of Periodontal Pathogens in the Bloodstream after Periodontal Procedures: A Systematic Review

Background

To date, there is no compilation of evidence-based information associating bacteremia and periodontal procedures. This systematic review aims to assess magnitude, duration, prevalence and nature of bacteremia caused by periodontal procedures.

Study Design

Systematic Review

Types of Studies Reviewed

MEDLINE, EMBASE and LILACS databases were searched in duplicate through August, 2013 without language restriction. Observational studies were included if blood samples were collected before, during or after periodontal procedures of patients with periodontitis. The methodological quality was assessed in duplicate using the modified Newcastle-Ottawa scale (NOS).

Results

Search strategy identified 509 potentially eligible articles and nine were included. Only four studies demonstrated high methodological quality, whereas five were of medium or low methodological quality. The study characteristics were considered too heterogeneous to conduct a meta-analysis. Among 219 analyzed patients, 106 (49.4%) had positive bacteremia. More frequent bacteria were S. viridans, A. actinomycetemcomitans P. gingivalis, M. micros and species Streptococcus and Actinomyces, although identification methods of microbiologic assays were different among studies.

Clinical Implications

Although half of the patients presented positive bacteremia after periodontal procedures, accurate results regarding the magnitude, duration and nature of bacteremia could not be confidentially assessed.