Structural and Physiological Analyses of the Alkanesulphonate-Binding Protein (SsuA) of the Citrus Pathogen Xanthomonas citri

Background

The uptake of sulphur-containing compounds plays a pivotal role in the physiology of bacteria that live in aerobic soils where organosulfur compounds such as sulphonates and sulphate esters represent more than 95% of the available sulphur. Until now, no information has been available on the uptake of sulphonates by bacterial plant pathogens, particularly those of the Xanthomonas genus, which encompasses several pathogenic species. In the present study, we characterised the alkanesulphonate uptake system (Ssu) of Xanthomonas axonopodis pv. citri 306 strain (X. citri), the etiological agent of citrus canker.

Methodology/Principal Findings

A single operon-like gene cluster (ssuEDACB) that encodes both the sulphur uptake system and enzymes involved in desulphurisation was detected in the genomes of X. citri and of the closely related species. We characterised X. citri SsuA protein, a periplasmic alkanesulphonate-binding protein that, together with SsuC and SsuB, defines the alkanesulphonate uptake system. The crystal structure of SsuA bound to MOPS, MES and HEPES, which is herein described for the first time, provides evidence for the importance of a conserved dipole in sulphate group coordination, identifies specific amino acids interacting with the sulphate group and shows the presence of a rather large binding pocket that explains the rather wide range of molecules recognised by the protein. Isolation of an isogenic ssuA-knockout derivative of the X. citri 306 strain showed that disruption of alkanesulphonate uptake affects both xanthan gum production and generation of canker lesions in sweet orange leaves.

Conclusions/Significance

The present study unravels unique structural and functional features of the X. citri SsuA protein and provides the first experimental evidence that an ABC uptake system affects the virulence of this phytopathogen.     

Potential mineralization of nitrogen from organic wastes to ryegrass and wheat crops

Two-pot experiments with ryegrass and wheat plants were conducted in a Cambic Arenosol to test the reliability of N fate predicted by incubation experiments previously performed, with the same soil, to assess potentially mineralizable nitrogen from six organic wastes (municipal solid waste compost, secondary pulp mill sludge, horn meal, poultry manure, solid phase from pig slurry and composted pig manure). Two treatments, corresponding to 80 and 160 kgN/ha were tested, with or without mineral N fertilization. Experimental data obtained in the pot trials was consistent with nitrogen net mineralization trend observed in the aerobic incubations with all the wastes tested. Values of potentially mineralizable nitrogen (N(0)) from the equations obtained by model fitting, to the incubation data, were well correlated to ryegrass and wheat N uptake. Poultry manure was the most efficient N supplier to crops.     

Breeding mute swan habitat selection when accounting for detectability: a plastic behaviour consistent with rapidly expanding populations

A number of native and exotic animal species show dramatic population increases in terms of both numbers and geographic range. Understanding the habitat selection processes behind such increases is crucial to implement adequate management measures. Mute swan (Cygnus olor) populations have experienced a tremendous demographic and geographic expansion in Western Europe during the twentieth century, colonizing a wide variety of aquatic habitats. We aimed at assessing how swans select nesting sites during the pre-laying and laying periods on medium to large fishponds (from 10 to 50 ha) in Eastern France, while accounting for detectability biases and testing for the effects of fishpond spatial configuration, vegetation resources, human disturbance and habitat management. Our results demonstrate that the mute swan is a non-selective species regarding its nesting habitat among such fishponds, using these independently from the parameters considered although fishpond characteristics varied. Although mute swan is one of the least cryptic Anatidae, owing to its white colour and large size, detection of breeding pairs remained imperfect for each over several sampling occasions. However, because we repeated the sampling sessions, detection of swan pairs by the end of the monitoring period was as high as 0.94. These results are consistent with previous assertions that the mute swan is a species of high ecological plasticity, which may partly explain its recent colonization rates. Given that even swan breeding events were imperfectly detected on each occasion, we highlight the fact that most studies of breeding ducks (which are more cryptic) would be considerably improved by better considering detection biases.     

Effect of goniothalamin on the development of Ehrlich solid tumor in mice

In this work the antiproliferative activity of goniothalamin (1), both in racemic and in its enantiomeric pure forms, in a solid tumor experimental model using laboratory animals is described. The antiedematogenic activity displayed by racemic 1 in the carrageenan edema model in mice together with the reduction of Ehrlich solid tumor model suggest a relationship between anticancer and antiinflammatory activities with the antiinflammatory activity favoring the antiproliferative activity itself.     

Lipase Maturation Factor LMF1, Membrane Topology and Interaction with Lipase Proteins in the Endoplasmic Reticulum

Lipase maturation factor 1 (LMF1) is predicted to be a polytopic protein localized to the endoplasmic reticulum (ER) membrane. It functions in the post-translational attainment of enzyme activity for both lipoprotein lipase and hepatic lipase. By using transmembrane prediction methods in mouse and human orthologs, models of LMF1 topology were constructed and tested experimentally. Employing a tagging strategy that used insertion of ectopic glycan attachment sites and terminal fusions of green fluorescent protein, we established a five-transmembrane model, thus dividing LMF1 into six domains. Three domains were found to face the cytoplasm (the amino-terminal domain and loops B and D), and the other half was oriented to the ER lumen (loops A and C and the carboxyl-terminal domain). This representative model shows the arrangement of an evolutionarily conserved domain within LMF1 (DUF1222) that is essential to lipase maturation. DUF1222 comprises four of the six domains, with the two largest ones facing the ER lumen. We showed for the first time, using several naturally occurring variants featuring DUF1222 truncations, that Lmf1 interacts physically with lipoprotein lipase and hepatic lipase and localizes the lipase interaction site to loop C within DUF1222. We discuss the implication of our results with regard to lipase maturation and DUF1222 domain structure.     

Single-fluorophore monitoring of DNA hybridization for investigating the effect of secondary structure on the nucleation step

Nucleic acid hybridization is one of the essential biological processes involved in storage and transmission of genetic information. Here we quantitatively determined the effect of secondary structure on the hybridization activation energy using structurally defined oligonucleotides. It turned out that activation energy is linearly proportional to the length of a single-stranded region flanking a nucleation site, generating a 0.18 kcal/mol energy barrier per nucleotide. Based on this result, we propose that the presence of single-stranded segments available for non-productive base pairing with a nucleation counterpart extends the searching process for nucleation sites to find a perfect match. This result may provide insights into rational selection of a target mRNA site for siRNA and antisense gene silencing.     

Identification of trophic interactions between <Emphasis Type="Italic">Macrolophus caliginosus</Emphasis> (Heteroptera: Miridae) and <Emphasis Type="Italic">Myzus persicae</Emphasis> (Homoptera: Aphididae) using real time PCR

Understanding food web interactions in native or agricultural ecosystems is an important step towards establishing sustainable pest management strategies. While the role of generalist predators as biological control agents is increasingly appreciated, the study of trophic interactions between individual predator species and their prey provides practical difficulties. Recently, different approaches have been suggested to determine prey items from predator guts using molecular methods. Macrolophus caliginosus is a generalist predator active in herbaceous agro-ecosystems. We developed a system to identify the DNA of its prey after ingestion, using Myzus persicae as a model. Esterase (MpEST) and cytochrome oxidase I (MpCOI) genes were targeted in the aphid, while M. caliginosus COI gene was used as control for predator DNA. Real time PCR proved to be specific and sensitive enough to detect prey DNA upon ingestion after feeding experiments. The system provided a linear amplification response with only 10 fg of prey genomic DNA as template. The detection system of MpCOI gene was more sensitive than MpEST, while the detection period was similar for both genes. Possibilities for using the system in ecological and biosafety studies with regard to sustainable pest management are discussed.