Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains

Background

Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity.

Methodology and Findings

We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer.

Conclusions

These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers {"type":"entrez-nucleotide","attrs":{"text":"CP001809","term_id":"340539261","term_text":"CP001809"}}CP001809 and {"type":"entrez-nucleotide","attrs":{"text":"CP001829","term_id":"302205157","term_text":"CP001829"}}CP001829.     

Mutations and genomic islands can explain the strain dependency of sugar utilization in 21 strains of Propionibacterium freudenreichii

Background

Propionibacterium freudenreichii (PF) is an actinobacterium used in cheese technology and for its probiotic properties. PF is also extremely adaptable to several ecological niches and can grow on a variety of carbon and nitrogen sources. The aim of this work was to discover the genetic basis for strain-dependent traits related to its ability to use specific carbon sources. High-throughput sequencing technologies were ideal for this purpose as they have the potential to decipher genomic diversity at a moderate cost.

Results

21 strains of PF were sequenced and the genomes were assembled de novo. Scaffolds were ordered by comparison with the complete reference genome CIRM-BIA1, obtained previously using traditional Sanger sequencing. Automatic functional annotation and manual curation were performed. Each gene was attributed to either the core genome or an accessory genome. The ability of the 21 strains to degrade 50 different sugars was evaluated. Thirty-three sugars were degraded by none of the sequenced strains whereas eight sugars were degraded by all of them. The corresponding genes were present in the core genome. Lactose, melibiose and xylitol were only used by some strains. In this case, the presence/absence of genes responsible for carbon uptake and degradation correlated well with the phenotypes, with the exception of xylitol. Furthermore, the simultaneous presence of these genes was in line the metabolic pathways described previously in other species. We also considered the genetic origin (transduction, rearrangement) of the corresponding genomic islands. Ribose and gluconate were degraded to a greater or lesser extent (quantitative phenotype) by some strains. For these sugars, the phenotypes could not be explained by the presence/absence of a gene but correlated with the premature appearance of a stop codon interrupting protein synthesis and preventing the catabolism of corresponding carbon sources.

Conclusion

These results illustrate (i) the power of correlation studies to discover the genetic basis of binary strain-dependent traits, and (ii) the plasticity of PF chromosomes, probably resulting from horizontal transfers, duplications, transpositions and an accumulation of mutations. Knowledge of the genetic basis of nitrogen and sugar degradation opens up new strategies for the screening of PF strain collections to enable optimum cheese starter, probiotic and white biotechnology applications.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1467-7) contains supplementary material, which is available to authorized users.     

The phage display technique: advantages and recent patents

Phage display technology has advanced considerably since its creation, and the number of research projects using this technique is constantly increasing, generating numerous antibody and antigen libraries. These libraries, besides expediting library screening, improving selection methods and allowing evaluation of novel applications, have great potential for the development of new vaccines, drugs and diagnosis tests. Consequently, patent registries for the protection of these sequences are essential.     

Tips and tricks for the assembly of a Corynebacterium pseudotuberculosis genome using a semiconductor sequencer

New sequencing platforms have enabled rapid decoding of complete prokaryotic genomes at relatively low cost. The Ion Torrent platform is an example of these technologies, characterized by lower coverage, generating challenges for the genome assembly. One particular problem is the lack of genomes that enable reference-based assembly, such as the one used in the present study, Corynebacterium pseudotuberculosis biovar equi, which causes high economic losses in the US equine industry. The quality treatment strategy incorporated into the assembly pipeline enabled a 16-fold greater use of the sequencing data obtained compared with traditional quality filter approaches. Data preprocessing prior to the de novo assembly enabled the use of known methodologies in the next-generation sequencing data assembly. Moreover, manual curation was proved to be essential for ensuring a quality assembly, which was validated by comparative genomics with other species of the genus Corynebacterium. The present study presents a modus operandi that enables a greater and better use of data obtained from semiconductor sequencing for obtaining the complete genome from a prokaryotic microorganism, C. pseudotuberculosis, which is not a traditional biological model such as Escherichia coli.     

Complete genome sequence of Corynebacterium pseudotuberculosis strain CIP 52.97, isolated from a horse in Kenya

In this work, we report the whole-genome sequence of Corynebacterium pseudotuberculosis bv. equi strain CIP 52.97 (Collection Institut Pasteur), isolated in 1952 from a case of ulcerative lymphangitis in a Kenyan horse, which has evidently caused significant losses to agribusiness. Therefore, obtaining this genome will allow the detection of important targets for postgenomic studies, with the aim of minimizing problems caused by this microorganism.     

Complete Genome Sequences of Corynebacterium pseudotuberculosis Strains 3/99-5 and 42/02-A, Isolated from Sheep in Scotland and Australia, Respectively

Here, we report the whole-genome sequences of two ovine-pathogenic Corynebacterium pseudotuberculosis isolates: strain 3/99-5, which represents the first C. pseudotuberculosis genome originating from the United Kingdom, and 42/02-A, the second from Australia. These genome sequences will contribute to the objective of determining the global pan-genome of this bacterium.     

Whole-genome sequence of Corynebacterium pseudotuberculosis PAT10 strain isolated from sheep in Patagonia, Argentina

In this work, we report the complete genome sequence of a Corynebacterium pseudotuberculosis PAT10 isolate, collected from a lung abscess in an Argentine sheep in Patagonia, whose pathogen also required an investigation of its pathogenesis. Thus, the analysis of the genome sequence offers a means to better understanding of the molecular and genetic basis of virulence of this bacterium.     

Rapid hybrid de novo assembly of a microbial genome using only short reads: Corynebacterium pseudotuberculosis I19 as a case study

Due to the advent of the so-called Next-Generation Sequencing (NGS) technologies the amount of monetary and temporal resources for whole-genome sequencing has been reduced by several orders of magnitude. Sequence reads can be assembled either by anchoring them directly onto an available reference genome (classical reference assembly), or can be concatenated by overlap (de novo assembly). The latter strategy is preferable because it tends to maintain the architecture of the genome sequence the however, depending on the NGS platform used, the shortness of read lengths cause tremendous problems the in the subsequent genome assembly phase, impeding closing of the entire genome sequence. To address the problem, we developed a multi-pronged hybrid de novo strategy combining De Bruijn graph and Overlap-Layout-Consensus methods, which was used to assemble from short reads the entire genome of Corynebacterium pseudotuberculosis strain I19, a bacterium with immense importance in veterinary medicine that causes Caseous Lymphadenitis in ruminants, principally ovines and caprines. Briefly, contigs were assembled de novo from the short reads and were only oriented using a reference genome by anchoring. Remaining gaps were closed using iterative anchoring of short reads by craning to gap flanks. Finally, we compare the genome sequence assembled using our hybrid strategy to a classical reference assembly using the same data as input and show that with the availability of a reference genome, it pays off to use the hybrid de novo strategy, rather than a classical reference assembly, because more genome sequences are preserved using the former.     

Linkage Replication for Chromosomal Region 13q32 in Schizophrenia: Evidence from a Brazilian Pilot Study on Early Onset Schizophrenia Families

We report analyses of a Brazilian study of early onset schizophrenia (BEOS) families. We genotyped 22 members of 4 families on a linkage SNP array and report here non-parametric linkage analyses using MERLIN® software. We found suggestive evidence for linkage on two chromosomal regions, 13q32 and 11p15.4. A LOD score of 2.71 was observed at 13q32 with a one LOD interval extending from 60.63–92.35 cM. From simulations, this LOD score gave a genome-wide empirical corrected p = 0.33, after accounting for all markers tested. Similarly 11p15.4 showed the same maximum LOD of 2.71 and a narrower one LOD interval of 4–14 cM. Of these, 13q32 has been reported to be linked to schizophrenia by multiple different studies. Thus, our study provides additional supporting evidence for an aetiological role of variants at 13q32 in schizophrenia.