Identification of a lineage specific zinc responsive genomic island in Mycobacterium avium ssp. paratuberculosis

Background

Maintenance of metal homeostasis is crucial in bacterial pathogenicity as metal starvation is the most important mechanism in the nutritional immunity strategy of host cells. Thus, pathogenic bacteria have evolved sensitive metal scavenging systems to overcome this particular host defence mechanism. The ruminant pathogen Mycobacterium avium ssp. paratuberculosis (MAP) displays a unique gut tropism and causes a chronic progressive intestinal inflammation. MAP possesses eight conserved lineage specific large sequence polymorphisms (LSP), which distinguish MAP from its ancestral M. avium ssp. hominissuis or other M. avium subspecies. LSP14 and LSP15 harbour many genes proposed to be involved in metal homeostasis and have been suggested to substitute for a MAP specific, impaired mycobactin synthesis.

Results

In the present study, we found that a LSP14 located putative IrtAB-like iron transporter encoded by mptABC was induced by zinc but not by iron starvation. Heterologous reporter gene assays with the lacZ gene under control of the mptABC promoter in M. smegmatis (MSMEG) and in a MSMEG∆furB deletion mutant revealed a zinc dependent, metalloregulator FurB mediated expression of mptABC via a conserved mycobacterial FurB recognition site. Deep sequencing of RNA from MAP cultures treated with the zinc chelator TPEN revealed that 70 genes responded to zinc limitation. Remarkably, 45 of these genes were located on a large genomic island of approximately 90 kb which harboured LSP14 and LSP15. Thirty-five of these genes were predicted to be controlled by FurB, due to the presence of putative binding sites. This clustering of zinc responsive genes was exclusively found in MAP and not in other mycobacteria.

Conclusions

Our data revealed a particular genomic signature for MAP given by a unique zinc specific locus, thereby suggesting an exceptional relevance of zinc for the metabolism of MAP. MAP seems to be well adapted to maintain zinc homeostasis which might contribute to the peculiarity of MAP pathogenicity.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1076) contains supplementary material, which is available to authorized users.     

Biotransformation of biarylic compounds by yeasts of the genus trichosporon

The biotransformation of biphenyl, dibenzofuran, and diphenyl ether by 24 strains belonging to 18 species of the genus Trichosporon was investigated to assess the taxonomic relevance of this property at species and genus level. With the exceptions of T. brassicae and T. porosum CBS 2040, all other strains were able to transform the parent compounds to monohydroxylated intermediates. A second hydroxylation on the same aromatic ring was carried out by fewer strains and depended on the substrate. It appears that this step is the rate-limiting one in the biotransformation of the biarylic compounds tested. Ring fission of dihydroxylated derivatives of biphenyl was observed within 12 species. The aromatic ring system of dihydroxylated dibenzofuran was cleaved by strains of 5 species, while strains of 13 species were able to cleave the aromatic ring system of dihydroxylated diphenyl ether. Only 4 strains out of 18 species were able to cleave the aromatic ring system of all three parent compounds. These most active yeasts belong to the species T. coremiiforme, T. montevideense, T. mucoides, and T. sporotrichoides. In addition, strains of the species Cryptococcus curvatus and Cryptococcus humicola, closely related to the genus Trichosporon, were tested in parallel.     

Relative biological effectiveness of 25 and 10 kV X-rays for the induction of chromosomal aberrations in two human mammary epithelial cell lines

Administration of ionizing radiation for diagnostic purposes can be associated with a risk for the induction of tumors. Therefore, particularly with regard to general screening programs, e.g. with mammography, cost-benefit considerations must be discussed including risk estimation depending upon the radiation quality administered. The present study was initiated to investigate the in vitro X-ray energy dependence for the induction of chromosomal aberrations in the two mammary epithelial cell lines, 184A1 and MCF-12A. The induced excess fragments, dicentric chromosomes and centric rings were analyzed and the relative biological effectiveness (RBE) was determined for 10 and 25 kV X-rays relative to 200 kV X-rays. The assumed energy dependence with higher values for 10 kV X-rays was confirmed for the excess fragments, with RBE_M values of 1.92 ± 0.26 and 1.40 ± 0.12 for 10 kV X-rays and 1.17 ± 0.12 and 0.97 ± 0.10 for 25 kV photons determined for cell lines 184A1 and MCF-12A, respectively. Meaningful results for the induction of dicentric chromosomes and centric rings were obtained only for higher doses with RBE values of 1.31 ± 0.21 and 1.70 ± 0.29 for 184A1 and 1.08 ± 0.08 and 1.43 ± 0.12 for MCF-12A irradiated with 25 and 10 kV X-rays, respectively.     

Variation in flooding-induced morphological traits in natural populations of white clover (Trifolium repens) and their effects on plant performance during soil flooding

Background and Aims

Soil flooding leads to low soil oxygen concentrations and thereby negatively affects plant growth. Differences in flooding tolerance have been explained by the variation among species in the extent to which traits related to acclimation were expressed. However, our knowledge of variation within natural species (i.e. among individual genotypes) in traits related to flooding tolerance is very limited. Such data could tell us on which traits selection might have taken place, and will take place in future. The aim of the present study was to show that variation in flooding-tolerance-related traits is present among genotypes of the same species, and that both the constitutive variation and the plastic variation in flooding-induced changes in trait expression affect the performance of genotypes during soil flooding.

Methods

Clones of Trifolium repens originating from a river foreland were subjected to either drained, control conditions or to soil flooding. Constitutive expression of morphological traits was recorded on control plants, and flooding-induced changes in expression were compared with these constitutive expression levels. Moreover, the effect of both constitutive and flooding-induced trait expression on plant performance was determined.

Key Results

Constitutive and plastic variation of several morphological traits significantly affected plant performance. Even relatively small increases in root porosity and petiole length contributed to better performance during soil flooding. High specific leaf area, by contrast, was negatively correlated with performance during flooding.

Conclusions

The data show that different genotypes responded differently to soil flooding, which could be linked to variation in morphological trait expression. As flooded and drained conditions exerted different selection pressures on trait expression, the optimal value for constitutive and plastic traits will depend on the frequency and duration of flooding. These data will help us understanding the mechanisms affecting short- and long-term dynamics in flooding-prone ecosystems.Key words: Secondary roots, aerenchyma, genotypic variation, petiole length, plant performance, root porosity, selection, soil flooding, specific leaf area (SLA), Trifolium repens, white clover     

Modified tumour antigen-encoding mRNA facilitates the analysis of naturally occurring and vaccine-induced CD4 and CD8 T cells in cancer patients

The development of effective anti-cancer vaccines requires precise assessment of vaccine-induced immunity. This is often hampered by low ex vivo frequencies of antigen-specific T cells and limited defined epitopes. This study investigates the applicability of modified, in vitro-transcribed mRNA encoding a therapeutically relevant tumour antigen to analyse T cell responses in cancer patients. In this study transfection of antigen presenting cells, by mRNA encoding the tumour antigen NY-ESO-1, was optimised and applied to address spontaneous and vaccine-induced T cell responses in cancer patients. Memory CD8+ T cells from lung cancer patients having detectable humoral immune responses directed towards NY-ESO-1 could be efficiently detected in peripheral blood. Specific T cells utilised a range of different T cell receptors, indicating a polyclonal response. Specific killing of a panel of NY-ESO-1 expressing tumour cell lines indicates recognition restricted to several HLA allelic variants, including a novel HLA-B49 epitope. Using a modified mRNA construct targeting the translated antigen to the secretory pathway, detection of NY-ESO-1-specific CD4+ T cells in patients could be enhanced, which allowed the in-depth characterisation of established T cell clones. Moreover, broad CD8+ and CD4+ T cell responses covering multiple epitopes were detected following mRNA stimulation of patients treated with a recombinant vaccinia/fowlpox NY-ESO-1 vaccine. This approach allows for a precise monitoring of responses to tumour antigens in a setting that addresses the breadth and magnitude of antigen-specific T cell responses, and that is not limited to a particular combination of known epitopes and HLA-restrictions.     

Adaptation of the obligate CAM plant Clusia alata to light stress: Metabolic responses

In the Crassulacean acid metabolism (CAM) plants Clusia alata Triana and Planch., decarboxylation of citrate during phase III of CAM took place later than malate decarboxylation. The interdependence of these two CO₂ and NADPH sources is discussed. High light accelerated malate decarboxylation during the day and lowered citrate levels. Strong light stress also activated mechanisms that can protect the plant against oxidative stress. Upon transfer from low light (200 μmol m⁻² s⁻¹) to high light (650–740 μmol m⁻² s⁻¹), after 2 days, there was a transient increase of non-photochemical quenching (NPQ) of fluorescence of chlorophyll a of photosystem II. This indicated acute photoinhibition, which declined again after 7 days of exposure. Conversely, after 1 week exposure to high light, the mechanisms of interconversion of violaxanthin (V), antheraxanthin (A), zeaxanthin (Z) (epoxydation/de-epoxydation) were activated. This was accompanied by an increase in pigment levels at dawn and dusk.