Next Generation Sequencing of Actinobacteria for the Discovery of Novel Natural Products

Like many fields of the biosciences, actinomycete natural products research has been revolutionised by next-generation DNA sequencing (NGS). Hundreds of new genome sequences from actinobacteria are made public every year, many of them as a result of projects aimed at identifying new natural products and their biosynthetic pathways through genome mining. Advances in these technologies in the last five years have meant not only a reduction in the cost of whole genome sequencing, but also a substantial increase in the quality of the data, having moved from obtaining a draft genome sequence comprised of several hundred short contigs, sometimes of doubtful reliability, to the possibility of obtaining an almost complete and accurate chromosome sequence in a single contig, allowing a detailed study of gene clusters and the design of strategies for refactoring and full gene cluster synthesis. The impact that these technologies are having in the discovery and study of natural products from actinobacteria, including those from the marine environment, is only starting to be realised. In this review we provide a historical perspective of the field, analyse the strengths and limitations of the most relevant technologies, and share the insights acquired during our genome mining projects.     

Use of new immunoglobulin isotype-specific ELISA-systems to detect Salmonella infections in pigs

In Germany, the program for controlling salmonella infections in pigs is based on tests detecting salmonella-lipopolysaccharide (LPS) induced antibodies in meat-juice or blood. These conventional tests which are based on the technology of enzyme-linked immunosorbent assay (ELISA) detect exclusively or mainly immunoglobulin(lg)G antibodies. Meanwhile, novel ELISA systems (WCE-ELISA, 3-Isotype-Screening-ELISA) have been developed, which additionally detect the antibody classes IgM and IgA.This fact enables the registration of fresh salmonella infections (starting with day 5 p.i.) and thus, the distinction between early and older infections.The results show that animals with early salmonella infections appear significantly more often in herds with a high than with a low prevalence. With the newly developed tests this group of animals can be detected much more efficiently and precisely than with the tests used so far. Due to their clearly improved sensitivity the application of the WCE-ELISA and the 3-Isotype-Screening-ELISA in terms of the QS-Salmonella-Monitoring program can therefore significantly improve the selection of farms with potential salmonella excretors. Additionally, the WCE-ELISA can be applied very suitable for the examination of individual animals.     

The δ18O signatures of HCl‐extractable soil phosphates: methodological challenges and evidence of the cycling of biological P in arable soil

Soil phosphates exchange oxygen atoms rapidly with soil water once recycled by intracellular enzymes, thereby approaching an equilibrium δ¹⁸O_P signature that depends on ambient temperature and the δ¹⁸O_W signature of soil water. We hypothesized that in the topsoil, phosphates reach this equilibrium δ¹⁸O_P signature even if amended by different fertilizers. In the subsoil, however, there might be phosphates with a smaller δ¹⁸O_P value than that represented by the isotopic equilibrium value, a condition that could exist in the case of limited biological P cycling only. We tested these hypotheses for the HCl‐extractable P pool of the Hedley fractionation scheme of arable soil in Germany, which integrates over extended time‐scales of the soil P cycle. We sampled several types of fertilizer, the surface soil that received these fertilizer types and composites from a Haplic Luvisol depth profile under long‐term agricultural practice. Organic fertilizers had significantly smaller δ¹⁸O_P values than mineral fertilizers. Intriguingly, the fields fertilized organically also tended to have smaller δ¹⁸O_P signatures than other types of surface soil, which calls into question full isotopic equilibrium at all sites. At depths below 50 cm, the soil δ¹⁸O_P values were even depleted relative to the values calculated for isotopic equilibrium. This implies that HCl‐extractable phosphates in different soil horizons are of different origins. In addition, it supports the assumption that biological cycling of P by intracellular microbial enzymes might have been relatively inefficient in the deeper subsoil. At depths of 50–80 cm, there was a transition zone of declining δ¹⁸O_P values, which might be regarded as the first evidence that the degree of biological P cycling changed at this depth interval.     

Clinical and pathologic comparison of acute leptospirosis in dogs caused by two strains of Leptospira kirschneri serovar grippotyphosa

OBJECTIVE: To develop a method for inducing acute leptospirosis in dogs. ANIMALS: 31 nine-week-old female Beagles. PROCEDURE: Beagles were randomly assigned to 2 inoculation groups or a control group. Dogs were inoculated on 3 successive days by conjunctival instillation of 5 x 10(7) cells of Leptospira kirschneri serovar grippotyphosa strain 82 (12 dogs) or strain RM 52 (14 dogs). Control dogs (n = 5) were similarly inoculated with sterile leptospiral culture media. Clinical signs, clinicopathologic variables, anti-leptospiral antibody titers, and evidence of leptospires in tissues and body fluids were evaluated. Dogs were euthanatized and necropsied on days 7, 14, 22, or 28 after inoculation or as required because of severe illness. RESULTS: Clinical signs in infected dogs included conjunctivitis, lethargy, diarrhea, dehydration, vomiting, and icterus. Consistent clinicopathologic alterations included azotemia, hyperphosphatemia, increased anion gap, hyperbilirubinemia, and an increase in alkaline phosphatase activity. Leptospires were cultured from the kidneys (11/12), urine (6/9), aqueous humor (9/12), blood (12/12), and liver (12/12) of dogs inoculated with strain 82. Only 3 of 14 dogs became infected after inoculation with strain RM 52. Histopathologic lesions in infected dogs included interstitial nephritis, renal tubular degeneration and necrosis, pulmonary hemorrhage, and hepatic edema and perivasculitis. CONCLUSIONS AND CLINICAL RELEVANCE: Conjunctival exposure to L kirschneri serovar grippotyphosa strain 82 resulted in acute leptospirosis in all inoculated dogs, but only 3 of 14 dogs inoculated with strain RM 52 became infected. This method of infection by serovar grippotyphosa can be used to study the pathogenesis and prevention of leptospirosis in dogs.     

Development of chronic and acute golden Syrian hamster infection models with Leptospira borgpetersenii serovar Hardjo

The golden Syrian hamster (Mesocricetus auratus) is frequently used as a model to study virulence for several Leptospira species. Onset of an acute lethal infection following inoculation with several pathogenic Leptospira species has been widely adopted for pathogenesis studies. An important exception is the outcome following inoculation of hamsters with live L. borgpetersenii serovar Hardjo, the primary cause of bovine leptospirosis and a cause of human infections. Typically, inoculation of hamsters with L. borgpetersenii serovar Hardjo fails to induce clinical signs of infection. In this study, the authors defined LD(50) and ID(50) for 2 strains of L. borgpetersenii serovar Hardjo: JB197 and 203. Both strains infected hamsters with ID(50) values of approximately 1.5 × 10(2) bacteria yet differed in tissue invasion and interaction with leukocytes, resulting in widely divergent clinical outcomes. Hamsters infected with strain 203 established renal colonization within 4 days postinfection and remained asymptomatic with chronic renal infections similar to cattle infected with serovar Hardjo. In contrast, hamsters infected with strain JB197 developed a rapidly debilitating disease typical of acute leptospirosis common in accidental hosts (eg, humans) with an LD(50) of 3.6 × 10(4) bacteria. Evidence that strain JB197 resides in both extracellular and intracellular environments during hamster infection was obtained. Development of models that result in chronic and acute forms of leptospirosis provides a platform to study L. borgpetersenii pathogenesis and to test vaccines for the prevention of leptospirosis.     

Identification and characterization of two bovine spongiform encephalopathy cases diagnosed in the United States.

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy of cattle, first detected in 1986 in the United Kingdom and subsequently in other countries. It is the most likely cause of variant Creutzfeldt-Jakob disease (vCJD) in humans, but the origin of BSE has not been elucidated so far. This report describes the identification and characterization of two cases of BSE diagnosed in the United States. Case 1 (December 2003) exhibited spongiform changes in the obex area of the brainstem and the presence of the abnormal form of the prion protein, PrP(Sc), in the same brain area, by immunohistochemistry (IHC) and Western blot analysis. Initial suspect diagnosis of BSE for case 2 (November 2004) was made by a rapid ELISA-based BSE test. Case 2 did not exhibit unambiguous spongiform changes in the obex area, but PrP(Sc) was detected by IHC and enrichment Western blot analysis in the obex. Using Western blot analysis, PrP(Sc) from case 1 showed molecular features similar to typical BSE isolates, whereas PrP(Sc) from case 2 revealed an unusual molecular PrP(Sc) pattern: molecular mass of the unglycosylated and monoglycosylated isoform was higher than that of typical BSE isolates and case 2 was strongly labeled with antibody P4, which is consistent with a higher molecular mass. Sequencing of the prion protein gene of both BSE-positive animals revealed that the sequences of both animals were within [corrected] the range of the prion protein gene sequence diversity previously reported for cattle.     

Antibody class switching mediated by yeast endonuclease-generated DNA breaks

Antibody class switching in activated B cells uses class switch recombination (CSR), which joins activation-induced cytidine deaminase (AID)-dependent double-strand breaks (DSBs) within two large immunoglobulin heavy chain (IgH) locus switch (S) regions that lie up to 200 kilobases apart. To test postulated roles of S regions and AID in CSR, we generated mutant B cells in which donor Smu and accepter Sgamma1 regions were replaced with yeast I-SceI endonuclease sites. We found that site-specific I-SceI DSBs mediate recombinational IgH locus class switching from IgM to IgG1 without S regions or AID. We propose that CSR evolved to exploit a general DNA repair process that promotes joining of widely separated DSBs within a chromosome.