Further evidence to justify reassignment of Mycoplasma mycoides subspecies mycoides Large Colony type to Mycoplasma mycoides subspecies capri

Analysis, using the polymerase chain reaction (PCR), restriction enzyme endonuclease analysis (REA), protein profile patterns, random amplification of polymorphic DNA (RAPD) fingerprinting, 16S rRNA gene sequencing and antisera growth inhibition tests, of 22 strains of Mycoplasma mycoides subsp. mycoides Large Colony type (MmmLC) and eight strains of M. mycoides subsp. capri (Mmc) are presented, along with a summary of comparative data from the literature for over 100 strains, all of which supports the reclassification of the MmmLC and Mmc strains into the single subspecies, M. mycoides subspecies capri.     

丝状支原体山羊亚种PG3株的全基因组序列测定与分析

【目的】全面了解丝状支原体山羊亚种PG3株的全基因组序列信息,寻找该病原体的主要保护性抗原基因。【方法】利用高通量Illumina Hi Seq 2000测序技术对丝状支原体山羊亚种PG3株的全基因组进行测序与拼接,借助软件和数据库对全基因组序列所承载的遗传信息进行注释和分析。【结果】丝状支原体山羊亚种PG3株基因组大小为1 025 065 bp,G+C%为23.6%,预测含有846个编码基因。根据COG分类和KEGG代谢通路分类,基因组中绝大多数基因主要与蛋白翻译、核糖体结构与合成、DNA复制与修复、糖代谢和环境信号传递与转换方面有关。该菌株具有丝状支原体山羊亚种特有的麦芽糊精/麦芽糖代谢途径。与Mmc str.95010的基因组的比对结果显示二者具有良好的共线性关系。在基因组中发现3个串联排列的可变表面脂蛋白基因,即GL000459、GL000461和GL000462。【结论】获得丝状支原体山羊亚种PG3株的全基因组序列,分析基因组基本特征,初步解析3个串联排列的可变表面脂蛋白基因,为进一步研究支原体可变表面脂蛋白的功能和研制生物工程疫苗奠定基础。     

TREC-IN: gene knock-in genetic tool for genomes cloned in yeast

Background

With the development of several new technologies using synthetic biology, it is possible to engineer genetically intractable organisms including Mycoplasma mycoides subspecies capri (Mmc), by cloning the intact bacterial genome in yeast, using the host yeast’s genetic tools to modify the cloned genome, and subsequently transplanting the modified genome into a recipient cell to obtain mutant cells encoded by the modified genome. The recently described tandem repeat coupled with endonuclease cleavage (TREC) method has been successfully used to generate seamless deletions and point mutations in the mycoplasma genome using the yeast DNA repair machinery. But, attempts to knock-in genes in some cases have encountered a high background of transformation due to maintenance of unwanted circularization of the transforming DNA, which contains possible autonomously replicating sequence (ARS) activity. To overcome this issue, we incorporated a split marker system into the TREC method, enabling seamless gene knock-in with high efficiency. The modified method is called TREC-assisted gene knock-in (TREC-IN). Since a gene to be knocked-in is delivered by a truncated non-functional marker, the background caused by an incomplete integration is essentially eliminated.

Results

In this paper, we demonstrate applications of the TREC-IN method in gene complementation and genome minimization studies in Mmc. In the first example, the Mmc dnaA gene was seamlessly replaced by an orthologous gene, which shares a high degree of identity at the nucleotide level with the original Mmc gene, with high efficiency and low background. In the minimization example, we replaced an essential gene back into the genome that was present in the middle of a cluster of non-essential genes, while deleting the non-essential gene cluster, again with low backgrounds of transformation and high efficiency.

Conclusion

Although we have demonstrated the feasibility of TREC-IN in gene complementation and genome minimization studies in Mmc, the applicability of TREC-IN ranges widely. This method proves to be a valuable genetic tool that can be extended for genomic engineering in other genetically intractable organisms, where it may be implemented in elucidating specific metabolic pathways and in rationale vaccine design.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1180) contains supplementary material, which is available to authorized users.     

Host specificity of mollicutes oriC plasmids: functional analysis of replication origin

Recently, artificial oriC plasmids containing the chromosomal dnaA gene and surrounding DnaA box sequences were obtained for the mollicutes Spiroplasma citri and Mycoplasma pulmonis. In order to study the specificity of these plasmids among mollicutes, a set of similar oriC plasmids was developed for three mycoplasmas belonging to the mycoides cluster, Mycoplasma mycoides subsp. mycoides LC (MmmLC), M.mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subsp. capricolum. Mycoplasmas from the mycoides cluster, S.citri and M.pulmonis were used as recipients for transformation experiments by homologous and heterologous oriC plasmids. All five mollicutes were successfully transformed by homologous plasmids, suggesting that the dnaA gene region represents the functional replication origin of the mollicute chromosomes. However, the ability of mollicutes to replicate heterologous oriC plasmids was found to vary noticeably with the species. For example, the oriC plasmid from M.capricolum did not replicate in the closely related species MmmSC and MmmLC. In contrast, plasmids harbouring the oriC from MmmSC, MmmLC and the more distant species S.citri were all found to replicate in M.capricolum. Our results suggest that the cis-elements present in oriC sequences are not the only determinants of this host specificity.     

Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains

Background

Francisella tularensis subspecies tularensis and holarctica are pathogenic to humans, whereas the two other subspecies, novicida and mediasiatica, rarely cause disease. To uncover the factors that allow subspecies tularensis and holarctica to be pathogenic to humans, we compared their genome sequences with the genome sequence of Francisella tularensis subspecies novicida U112, which is nonpathogenic to humans.

Results

Comparison of the genomes of human pathogenic Francisella strains with the genome of U112 identifies genes specific to the human pathogenic strains and reveals pseudogenes that previously were unidentified. In addition, this analysis provides a coarse chronology of the evolutionary events that took place during the emergence of the human pathogenic strains. Genomic rearrangements at the level of insertion sequences (IS elements), point mutations, and small indels took place in the human pathogenic strains during and after differentiation from the nonpathogenic strain, resulting in gene inactivation.

Conclusion

The chronology of events suggests a substantial role for genetic drift in the formation of pseudogenes in Francisella genomes. Mutations that occurred early in the evolution, however, might have been fixed in the population either because of evolutionary bottlenecks or because they were pathoadaptive (beneficial in the context of infection). Because the structure of Francisella genomes is similar to that of the genomes of other emerging or highly pathogenic bacteria, this evolutionary scenario may be shared by pathogens from other species.     

Animal model of Mycoplasma fermentans respiratory infection

Background

Mycoplasma fermentans has been associated with respiratory, genitourinary tract infections and rheumatoid diseases but its role as pathogen is controversial. The purpose of this study was to probe that Mycoplasma fermentans is able to produce respiratory tract infection and migrate to several organs on an experimental infection model in hamsters. One hundred and twenty six hamsters were divided in six groups (A-F) of 21 hamsters each. Animals of groups A, B, C were intratracheally injected with one of the mycoplasma strains: Mycoplasma fermentans P 140 (wild strain), Mycoplasma fermentans PG 18 (type strain) or Mycoplasma pneumoniae Eaton strain. Groups D, E, F were the negative, media, and sham controls. Fragments of trachea, lungs, kidney, heart, brain and spleen were cultured and used for the histopathological study. U frequency test was used to compare recovery of mycoplasmas from organs.

Results

Mycoplasmas were detected by culture and PCR. The three mycoplasma strains induced an interstitial pneumonia; they also migrated to several organs and persisted there for at least 50 days. Mycoplasma fermentans P 140 induced a more severe damage in lungs than Mycoplasma fermentans PG 18. Mycoplasma pneumoniae produced severe damage in lungs and renal damage.

Conclusions

Mycoplasma fermentans induced a respiratory tract infection and persisted in different organs for several weeks in hamsters. This finding may help to explain the ability of Mycoplasma fermentans to induce pneumonia and chronic infectious diseases in humans.     

Evolutionary History of Contagious Bovine Pleuropneumonia Using Next Generation Sequencing of Mycoplasma mycoides Subsp. mycoides “Small Colony”

Mycoplasma mycoides subsp. mycoides “Small Colony” (MmmSC) is responsible for contagious bovine pleuropneumonia (CBPP) in bovidae, a notifiable disease to the World Organization for Animal Health (OIE). Although its origin is not documented, the disease was known in Europe in 1773. It reached nearly world-wide distribution in the 19^th century through the cattle trade and was eradicated from most continents by stamping-out policies. During the 20^th century it persisted in Africa, and it reappeared sporadically in Southern Europe. Yet, classical epidemiology studies failed to explain the re-occurrence of the disease in Europe in the 1990s. The objectives of this study were to obtain a precise phylogeny of this pathogen, reconstruct its evolutionary history, estimate the date of its emergence, and determine the origin of the most recent European outbreaks. A large-scale genomic approach based on next-generation sequencing technologies was applied to construct a robust phylogeny of this extremely monomorphic pathogen by using 20 representative strains of various geographical origins. Sixty two polymorphic genes of the MmmSC core genome were selected, representing 83601 bp in total and resulting in 139 SNPs within the 20 strains. A robust phylogeny was obtained that identified a lineage specific to European strains; African strains were scattered in various branches. Bayesian analysis allowed dating the most recent common ancestor for MmmSC around 1700. The strains circulating in Sub-Saharan Africa today, however, were shown to descend from a strain that existed around 1810. MmmSC emerged recently, about 300 years ago, and was most probably exported from Europe to other continents, including Africa, during the 19^th century. Its diversity is now greater in Africa, where CBPP is enzootic, than in Europe, where outbreaks occurred sporadically until 1999 and where CBPP may now be considered eradicated unless MmmSC remains undetected.     

Controlling contagious agalactia in artificial insemination centers for goats and detection of Mycoplasma mycoides subspecies capri in semen

Many goat artificial insemination (AI) centers in Spain have adopted new measures to control contagious agalactia (CA). To avoid the introduction of male goats carrying mycoplasma organisms subclinically in their external ear canal (auricular carriers) in these centers, two ear swabs and a blood sample are obtained from all candidate animals for polymerase chain reaction (PCR), culture (swabs) and serologic tests to detect the presence of mycoplasmas. In addition, the semen produced at these centers is routinely cultured and PCR tested also to detect the presence of mycoplasmas. One y after the introduction of this program, we tested 48 ear swabs and 24 blood samples from 24 candidates for admission to these AI Centers. Three of these ear swab samples (3/48, 6.25%) scored positive for the presence of mycoplasmas; Mycoplasma agalactiae (Ma) was detected in two samples and Mycoplasma mycoides subsp. capri (Mmc) in one. All animals were serologically negative for Ma. Also, out of 173 semen samples obtained from 137 admitted animals (2 and 3 samples were obtained in 16 and 10 bucks, respectively), one (1/173, 0.56%) was positive for Mmc. Our findings suggest that ear swab and semen samples are useful tools to control CA at AI Centers. The introduction of this program has also resulted in the first detection of Mmc in semen from a naturally infected goat, confirming the ability of this mycoplasma to colonize the reproductive tract of male goats. These results highlight the need to improve control measures in semen producing centers to minimize the risk of CA transmission.     

Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus

Background

A low genetic diversity in Francisella tularensis has been documented. Current DNA based genotyping methods for typing F. tularensis offer a limited and varying degree of subspecies, clade and strain level discrimination power. Whole genome sequencing is the most accurate and reliable method to identify, type and determine phylogenetic relationships among strains of a species. However, lower cost typing schemes are necessary in order to enable typing of hundreds or even thousands of isolates.

Results

We have generated a high-resolution phylogenetic tree from 40 Francisella isolates, including 13 F. tularensis subspecies holarctica (type B) strains, 26 F. tularensis subsp. tularensis (type A) strains and a single F. novicida strain. The tree was generated from global multi-strain single nucleotide polymorphism (SNP) data collected using a set of six Affymetrix GeneChip^® resequencing arrays with the non-repetitive portion of LVS (type B) as the reference sequence complemented with unique sequences of SCHU S4 (type A). Global SNP based phylogenetic clustering was able to resolve all non-related strains. The phylogenetic tree was used to guide the selection of informative SNPs specific to major nodes in the tree for development of a genotyping assay for identification of F. tularensis subspecies and clades. We designed and validated an assay that uses these SNPs to accurately genotype 39 additional F. tularensis strains as type A (A1, A2, A1a or A1b) or type B (B1 or B2).

Conclusion

Whole-genome SNP based clustering was shown to accurately identify SNPs for differentiation of F. tularensis subspecies and clades, emphasizing the potential power and utility of this methodology for selecting SNPs for typing of F. tularensis to the strain level. Additionally, whole genome sequence based SNP information gained from a representative population of strains may be used to perform evolutionary or phylogenetic comparisons of strains, or selection of unique strains for whole-genome sequencing projects.     

Local hopping mobile DNA implicated in pseudogene formation and reductive evolution in an obligate cyanobacteria-plant symbiosis

Background

Insertion sequences (ISs) are approximately 1 kbp long “jumping” genes found in prokaryotes. ISs encode the protein Transposase, which facilitates the excision and reinsertion of ISs in genomes, making these sequences a type of class I (“cut-and-paste”) Mobile Genetic Elements. ISs are proposed to be involved in the reductive evolution of symbiotic prokaryotes. Our previous sequencing of the genome of the cyanobacterium ‘Nostoc azollae’ 0708, living in a tight perpetual symbiotic association with a plant (the water fern Azolla), revealed the presence of an eroding genome, with a high number of insertion sequences (ISs) together with an unprecedented large proportion of pseudogenes. To investigate the role of ISs in the reductive evolution of ‘Nostoc azollae’ 0708, and potentially in the formation of pseudogenes, a bioinformatic investigation of the IS identities and positions in 47 cyanobacterial genomes was conducted. To widen the scope, the IS contents were analysed qualitatively and quantitatively in 20 other genomes representing both free-living and symbiotic bacteria.

Results

Insertion Sequences were not randomly distributed in the bacterial genomes and were found to transpose short distances from their original location (“local hopping”) and pseudogenes were enriched in the vicinity of IS elements. In general, symbiotic organisms showed higher densities of IS elements and pseudogenes than non-symbiotic bacteria. A total of 1108 distinct repeated sequences over 500 bp were identified in the 67 genomes investigated. In the genome of ‘Nostoc azollae’ 0708, IS elements were apparent at 970 locations (14.3%), with 428 being full-length. Morphologically complex cyanobacteria with large genomes showed higher frequencies of IS elements, irrespective of life style.

Conclusions

The apparent co-location of IS elements and pseudogenes found in prokaryotic genomes implies earlier IS transpositions into genes. As transpositions tend to be local rather than genome wide this likely explains the proximity between IS elements and pseudogenes. These findings suggest that ISs facilitate the reductive evolution in for instance in the symbiotic cyanobacterium ‘Nostoc azollae’ 0708 and in other obligate prokaryotic symbionts.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1386-7) contains supplementary material, which is available to authorized users.     

Pharmacodynamics of Antimicrobials against Mycoplasma mycoides mycoides Small Colony, the Causative Agent of Contagious Bovine Pleuropneumonia

Background

Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC) is the causative agent of Contagious Bovine Pleuropneumonia (CBPP), a disease of substantial economic importance in sub-Saharan Africa. Failure of vaccination to curtail spread of this disease has led to calls for evaluation of the role of antimicrobials in CBPP control. Three major classes of antimicrobial are effective against mycoplasmas, namely tetracyclines, fluoroquinolones and macrolides. Therefore, the objectives of this study were to determine the effector kinetics of oxytetracycline, danofloxacin and tulathromycin against two MmmSC field strains in artificial medium and adult bovine serum.

Methods

Minimum inhibitory concentrations (MIC) were determined for oxytetracycline, danofloxacin and tulathromycin against MmmSC strains B237 and Tan8 using a macrodilution technique, and time-kill curves were constructed for various multiples of the MIC over a 24 hour period in artificial medium and serum. Data were fitted to sigmoid E_max models to obtain 24 hour-area under curve/MIC ratios for mycoplasmastasis and, where appropriate, for mycoplasmacidal activity and virtual mycoplasmal elimination.

Results

Minimum inhibitory concentrations against B237 were 20-fold higher, 2-fold higher and approximately 330-fold lower in serum than in artificial medium for oxytetracycline, danofloxacin and tulathromycin, respectively. Such differences were mirrored in experiments using Tan8. Oxytetracycline was mycoplasmastatic against both strains in both matrices. Danofloxacin elicited mycoplasmacidal activity against B237 and virtual elimination of Tan8; similar maximum antimycoplasmal effects were observed in artificial medium and serum. Tulathromycin effected virtual elimination of B237 but was mycoplasmastatic against Tan8 in artificial medium. However, this drug was mycoplasmastatic against both strains in the more physiologically relevant matrix of serum.

Conclusions

Oxytetracycline, danofloxacin and tulathromycin are all suitable candidates for further investigation as potential treatments for CBPP. This study also highlights the importance of testing drug activity in biological matrices as well as artificial media.     

Influence of cohabitation between domestic goat (Capra aegagrus hircus) and Iberian ibex (Capra pyrenaica hispanica) on seroprevalence of infectious diseases

We have investigated the prevalence of infection with different pathogens in domestic goat (Capra hircus) and Iberian ibex (Capra pyrenaica hispanica) populations from two neighbouring geographical zones (with or without cohabitation between wild and domestic species) in Spain. No ibexes were found to be seropositive for Brucella melitensis, Mycoplasma mycoides supspecies mycoides (Mycoplasma mycoides), bovine leukaemia virus, and Chlamydophila abortus or bovine herpes virus type 1 (BHV-1). Domestic goats from both zones were detected with antibodies to Chlamydophila abortus and BHV-1. The percentage seroprevalence against Coxiella burnetii, Mycoplasma agalactiae, Mycobacterium avium subspecies paratuberculosis (MAP), caprine arthritis encephalitis virus (CAEV) and bluetongue virus (BTV) was clearly higher in domestic goats compared to wild ibexes in both zones, although CAEV showed negative results in a zone where species were isolated from each other. Moreover, Borrelia burgdorferi and Pestivirus infection showed moderate seroprevalence differences between domestic and wild goats in cohabitation zones, with the highest prevalence found in wild ibexes from a non-cohabitation zone. Our results showed that cohabitation between species was a risk factor (P?<?0.05) in Q fever infection.     

Large-scale genomic correlations in Arabidopsis thaliana relate to chromosomal structure

Background

The genome of classical laboratory strains of mice is an artificial mosaic of genomes originated from several mouse subspecies with predominant representation (>90%) of the Mus m. domesticus component. Mice of another subspecies, East European/Asian Mus m. musculus, can interbreed with the classical laboratory strains to generate hybrids with unprecedented phenotypic and genotypic variations. To study these variations in depth we prepared the first genomic large insert BAC library from an inbred strain derived purely from the Mus m. musculus-subspecies. The library will be used to seek and characterize genomic sequences controlling specific monogenic and polygenic complex traits, including modifiers of dominant and recessive mutations.

Results

A representative mouse genomic BAC library was derived from a female mouse of the PWD/Ph inbred strain of Mus m. musculus subspecies. The library consists of 144 768 primary clones from which 97% contain an insert of 120 kb average size. The library represents an equivalent of 6.7 × mouse haploid genome, as estimated from the total number of clones carrying genomic DNA inserts and from the average insert size. The clones were arrayed in duplicates onto eight high-density membranes that were screened with seven single-copy gene probes. The individual probes identified four to eleven positive clones, corresponding to 6.9-fold coverage of the mouse genome. Eighty-seven BAC-ends of PWD/Ph clones were sequenced, edited, and aligned with mouse C57BL/6J (B6) genome. Seventy-three BAC-ends displayed unique hits on B6 genome and their alignment revealed 0.92 single nucleotide polymorphisms (SNPs) per 100 bp. Insertions and deletions represented 0.3% of the BAC end sequences.

Conclusion

Analysis of the novel genomic library for the PWD/Ph inbred strain demonstrated coverage of almost seven mouse genome equivalents and a capability to recover clones for specific regions of PWD/Ph genome. The single nucleotide polymorphism between the strains PWD/Ph and C57BL/6J was 0.92/100 bp, a value significantly higher than between classical laboratory strains. The library will serve as a resource for dissecting the phenotypic and genotypic variations between mice of the Mus m. musculus subspecies and classical laboratory mouse strains.     

Highly Dynamic Genomic Loci Drive the Synthesis of Two Types of Capsular or Secreted Polysaccharides within the Mycoplasma mycoides Cluster

Mycoplasmas of the Mycoplasma mycoides cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides is responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role for Mycoplasma mycoides subsp. mycoides polysaccharides in pathogenicity. Mycoplasma mycoides subsp. mycoides-secreted EPS was recently characterized as a β(1→6)-galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of the M. mycoides cluster: M. capricolum subsp. capripneumoniae, M. capricolum subsp. capricolum, M. leachii, and M. mycoides subsp. capri (including the LC and Capri serovars). Extracted EPS was characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of β(1→2)-glucopyranose (glucan) in M. capricolum subsp. capripneumoniae and M. leachii. Monoclonal antibodies specific for this glucan and for the Mycoplasma mycoides subsp. mycoides-secreted galactan were used to detect the two polysaccharides. While M. mycoides subsp. capri strains of serovar LC produced only capsular galactan, no polysaccharide could be detected in strains of serovar Capri. All strains of M. capricolum subsp. capripneumoniae and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among M. capricolum subsp. capricolum strains. Genes associated with polysaccharide synthesis and forming a biosynthetic pathway were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hot spots. Phylogenetic analysis showed that some of these genes, notably galE and glf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides.     

Relationship between homoeologous regulatory and structural genes in allopolyploid genome – A case study in bread wheat

Background

Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a Vitis vinifera 'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed.

Results

The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome.

Conclusion

Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance loci in grapevine.     

Zinc accumulation patterns in four Anthyllis vulneraria subspecies supplemented with mineral nitrogen or grown in the presence of their symbiotic bacteria

Aims

This work examines Zn accumulation in four Anthyllis vulneraria subspecies supplemented with mineral nitrogen or grown in the presence of their symbiotic bacteria.

Methods

Anthyllis vulneraria subspecies were grown hydroponically in the presence of high levels of ZnSO₄. The plants were either grown in symbiosis with one of two non-metallicolous or metallicolous Mesorhizobium inoculants or in the presence of KNO₃.

Results

When exposed to 1,000 μM Zn, shoot and root biomass of three out of our four Anthyllis subspecies cultivated with NO₃ dropped significantly by about 24–28 %; carpatica, the fourth subspecies, was not affected. Subspecies carpatica Zn tolerance was confirmed when in symbiosis with the metallicolous strain. In the presence of 1,000 μM Zn, the different Anthyllis subspecies concentrated more Zn in their roots than in their shoots and only subsp. carpatica accumulated a significant amount of Zn in its shoots. The most remarkable feature was the drastic decrease in Zn concentration in both roots (up to 2.5–3 fold) and shoots (2.6-fold) of subsp. carpatica exposed to 1,000 μM Zn and nodulated whatever the Mesorhizobium strain used, compared to the N-grown plants.

Conclusions

Our results bring new perspectives as regards phytostabilization, with the potential use of a rhizobium-inoculated leguminous subspecies displaying unusual Zn tolerance.     

Genomic maps of some strains within the Mycoplasma mycoides cluster

Genomic restriction maps for the small colony (SC) strains (PG1, KH3J, Gladysdale, and V5) of Mycoplasma mycoides subsp. mycoides (the agent of contagious bovine pleuropneumonia) and for Mycoplasma strain PG50 (classified as bovine serogroup 7), with respective sizes of 1,280, 1,280, 1,260, 1,230, and 1,040 kbp, were compared with the map (1,200 kbp) for a large colony strain (Y goat) of M. mycoides subsp. mycoides. The number and order of all mapped restriction sites were fully conserved in the SC genomes, as were the approximate positions of mapped loci. A number of these restriction sites in the Y genome and some, but fewer, in the PG50 genome appeared to be conserved. The SC and large colony strains shared conservation in the relative positions of the mapped loci, except for rpoC.     

Caprine mycoplasmal mastitis in Nigeria

In a study to investigate the current status of intramammary mycoplasmosis in caprine udders in Nigeria, a total of 57 and 24 milk samples were collected from udders of goats affected by mastitis and from apparently normal goats’ udders, respectively. Acute and chronic mastitis were more commonly observed in goats between 1 and 3 years old. Mycoplasma agalactiae and Mycoplasma capricolum occurred at a significantly higher rate (p<0.05) in udders affected by mastitis than in normal healthy udders. Other mycoplasma occurring in low prevalence include Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides LC. It is concluded that cultural (microbiological) surveillance is necessary for effective treatment and control of the disease in Nigeria.     

Whole Genome Sequence of the Treponema pallidum subsp. endemicum Strain Bosnia A: The Genome Is Related to Yaws Treponemes but Contains Few Loci Similar to Syphilis Treponemes

Background

T. pallidum subsp. endemicum (TEN) is the causative agent of bejel (also known as endemic syphilis). Clinical symptoms of syphilis and bejel are overlapping and the epidemiological context is important for correct diagnosis of both diseases. In contrast to syphilis, caused by T. pallidum subsp. pallidum (TPA), TEN infections are usually spread by direct contact or contaminated utensils rather than by sexual contact. Bejel is most often seen in western Africa and in the Middle East. The strain Bosnia A was isolated in 1950 in Bosnia, southern Europe.

Methodology/Principal Findings

The complete genome of the Bosnia A strain was amplified and sequenced using the pooled segment genome sequencing (PSGS) method and a combination of three next-generation sequencing techniques (SOLiD, Roche 454, and Illumina). Using this approach, a total combined average genome coverage of 513× was achieved. The size of the Bosnia A genome was found to be 1,137,653 bp, i.e. 1.6–2.8 kbp shorter than any previously published genomes of uncultivable pathogenic treponemes. Conserved gene synteny was found in the Bosnia A genome compared to other sequenced syphilis and yaws treponemes. The TEN Bosnia A genome was distinct but very similar to the genome of yaws-causing T. pallidum subsp. pertenue (TPE) strains. Interestingly, the TEN Bosnia A genome was found to contain several sequences, which so far, have been uniquely identified only in syphilis treponemes.

Conclusions/Significance

The genome of TEN Bosnia A contains several sequences thought to be unique to TPA strains; these sequences very likely represent remnants of recombination events during the evolution of TEN treponemes. This finding emphasizes a possible role of repeated horizontal gene transfer between treponemal subspecies in shaping the Bosnia A genome.     

Decoding the massive genome of loblolly pine using haploid DNA and novel assembly strategies

Background

The size and complexity of conifer genomes has, until now, prevented full genome sequencing and assembly. The large research community and economic importance of loblolly pine, Pinus taeda L., made it an early candidate for reference sequence determination.

Results

We develop a novel strategy to sequence the genome of loblolly pine that combines unique aspects of pine reproductive biology and genome assembly methodology. We use a whole genome shotgun approach relying primarily on next generation sequence generated from a single haploid seed megagametophyte from a loblolly pine tree, 20-1010, that has been used in industrial forest tree breeding. The resulting sequence and assembly was used to generate a draft genome spanning 23.2 Gbp and containing 20.1 Gbp with an N50 scaffold size of 66.9 kbp, making it a significant improvement over available conifer genomes. The long scaffold lengths allow the annotation of 50,172 gene models with intron lengths averaging over 2.7 kbp and sometimes exceeding 100 kbp in length. Analysis of orthologous gene sets identifies gene families that may be unique to conifers. We further characterize and expand the existing repeat library based on the de novo analysis of the repetitive content, estimated to encompass 82% of the genome.

Conclusions

In addition to its value as a resource for researchers and breeders, the loblolly pine genome sequence and assembly reported here demonstrates a novel approach to sequencing the large and complex genomes of this important group of plants that can now be widely applied.