Susceptibility of staphylococcal biofilms to enzymatic treatments depends on their chemical composition

Bacterial infections are serious complications after orthopaedic implant surgery. Staphylococci, with Staphylococcus epidermidis as a leading species, are the prevalent and most important species involved in orthopaedic implant-related infections. The biofilm mode of growth of these bacteria on an implant surface protects the organisms from the host’s immune system and from antibiotic therapy. Therapeutic agents that disintegrate the biofilm matrix would release planktonic cells into the environment and therefore allow antibiotics to eliminate the bacteria. An addition of a biofilm-degrading agent to a solution used for washing–draining procedures of infected orthopaedic implants would greatly improve the efficiency of the procedure and thus help to avoid the removal of the implant. We have previously shown that the extracellular staphylococcal matrix consists of a poly-N-acetylglucosamine (PNAG), extracellular teichoic acids (TAs) and protein components. In this study, we accessed the sensitivity of pre-formed biofilms of five clinical staphylococcal strains associated with orthopaedic prosthesis infections and with known compositions of the biofilm matrix to periodate, Pectinex Ultra SP, proteinase K, trypsin, pancreatin and dispersin B, an enzyme with a PNAG-hydrolysing activity. We also tested the effect of these agents on the purified carbohydrate components of staphylococcal biofilms, PNAG and TA. We found that the enzymatic detachment of staphylococcal biofilms depends on the nature of their constituents and varies between the clinical isolates. We suggest that a treatment with dispersin B followed by a protease (proteinase K or trypsin) could be capable to eradicate biofilms of a variety of staphylococcal strains on inert surfaces.     

Biofilms of clinical strains of Staphylococcus that do not contain polysaccharide intercellular adhesin

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. The capacity of S. epidermidis to form biofilms, allowing it to evade host immune defence mechanisms and antibiotic therapy, is considered to be crucial in colonizing the surfaces of medical implants and dissemination of infection. It has previously been demonstrated that the biofilm of a model strain S. epidermidis RP62A comprises two carbohydrate-containing moieties, a polysaccharide having a structure of a linear poly-N-acetyl-(1-->6)-beta-D-glucosamine and teichoic acid. In the present paper we show that, unlike this model strain, certain clinical isolates of coagulase-negative staphylococci produce biofilms that do not contain detectable amounts of poly-N-acetyl-(1-->6)-beta-D-glucosamine. In contrast to that of S. epidermidis RP62A, these biofilms are not detached with metaperiodate, while proteinase K causes their partial dispersal.     

Purification and identification of a Bacillus nitroreductase: Potential use in 3,5-DNBTF biosensoring system

We have investigated a specific enzymatic biosensor for detecting target pollutant 3,5-dinitro-trifluoromethylbenzene (3,5-DNBTF). The predicted enzyme is a nitroreductase that catalyzes the total nitroreduction of 3,5-DNBTF to its corresponding diamine. The photo-activation of this diamine offers a large panel of detection tools. After broad screening of microorganisms, only the strains belonging to the genus Bacillus were able to reduce the two nitro groups of 3,5-DNBTF. Among them, Bacillus LMA, isolated from explosives-polluted effluents, was the most efficient in reducing this compound. The involved nitroreductase was identified by 2D gel electrophoresis coupled to mass spectrometry, as the Bacillus subtilis oxygen-insensitive nitroreductase NfrA. The enzyme was purified by mono-P chromatofocusing.     

Carbohydrate-containing components of biofilms produced in vitro by some staphylococcal strains related to orthopaedic prosthesis infections

The capacity of coagulase-negative staphylococci to colonize implanted medical devices is generally attributed to their ability to produce biofilms. Biofilm of the model strain of Staphylococcus epidermidis RP62A was shown to contain two carbohydrate-containing moieties, a linear poly-beta-(1-->6)-N-acetyl-D-glucosamine (PNAG) and teichoic acid. In the present study, we investigated several biofilm-producing staphylococci isolated from infected orthopaedic implants and characterized the composition of the laboratory-grown biofilms using chemical analysis and 1H nuclear magnetic resonance spectroscopy. Extracellular teichoic acid was produced by all strains studied. Some of the clinical strains were shown to produce biofilms with compositions similar to that of the model strain, containing a varying amount of PNAG. The chemical structure of PNAG of the clinical strains was similar to that previously described for the model strains S. epidermidis RP62A and Staphylococcus aureus MN8m, differing only in the amount of charged groups. Biofilms of the strains producing a substantial amount of PNAG were detached by dispersin B, a PNAG-degrading enzyme, while being unsusceptible to proteinase K treatment. On the other hand, some strains produced biofilms without any detectable amount of PNAG. The biofilms of these strains were dispersed by proteinase K, but not by dispersin B.     

Gene targeting in maize by somatic ectopic recombination

Low transformation efficiency and high background of non‐targeted events are major constraints to gene targeting in plants. We demonstrate here applicability in maize of a system that reduces the constraint from transformation efficiency. The system requires regenerable transformants in which all of the following elements are stably integrated in the genome: (i) donor DNA with the gene of interest adjacent to sequence for repair of a defective selectable marker, (ii) sequence encoding a rare‐cutting endonuclease such as I‐SceI, (iii) a target locus (TL) comprising the defective selectable marker and I‐SceI cleavage site. Typically, this requires additional markers for the integration of the donor and target sequences, which may be assembled through cross‐pollination of separate transformants. Inducible expression of I‐SceI then cleaves the TL and facilitates homologous recombination, which is assayed by selection for the repaired marker. We used bar and gfp markers to identify assembled transformants, a dexamethasone‐inducible I‐SceI::GR protein, and selection for recombination events that restored an intact nptII. Applying this strategy to callus permitted the selection of recombination into the TL at a frequency of 0.085% per extracted immature embryo (29% of recombinants). Our results also indicate that excision of the donor locus (DL) through the use of flanking I‐SceI cleavage sites may be unnecessary, and a source of unwanted repair events at the DL. The system allows production, from each assembled transformant, of many cells that subsequently can be treated to induce gene targeting. This may facilitate gene targeting in plant species for which transformation efficiencies are otherwise limiting.     

Identification of structural and molecular determinants of the tyrosine‐kinase Wzc and implications in capsular polysaccharide export

Capsular polysaccharides are well‐established virulence factors of pathogenic bacteria. Their biosynthesis and export are regulated within the transmembrane polysaccharide assembly machinery by the autophosphorylation of atypical tyrosine‐kinases, named BY‐kinases. However, the accurate functioning of these tyrosine‐kinases remains unknown. Here, we report the crystal structure of the non‐phosphorylated cytoplasmic domain of the tyrosine‐kinase Wzc from Escherichia coli in complex with ADP showing that it forms a ring‐shaped octamer. Mutational analysis demonstrates that a conserved EX₂RX₂R motif involved in subunit interactions is essential for polysaccharide export. We also elucidate the role of a putative internal regulatory tyrosine and we show that BY‐kinases from proteobacteria autophosphorylate on their C‐terminal tyrosine cluster via a single‐step intermolecular mechanism. This structure‐function analysis also allows us to demonstrate that two different parts of a conserved basic region called the RK‐cluster are essential for polysaccharide export and for kinase activity respectively. Based on these data, we revisit the dichotomy made between BY‐kinases from proteobacteria and firmicutes and we propose a unique process of oligomerization and phosphorylation. We also reassess the function of BY‐kinases in the capsular polysaccharide assembly machinery.     

Copper bioavailability and rhizosphere pH changes as affected by nitrogen supply for tomato and oilseed rape cropped on an acidic and a calcareous soil

Vineyard soils have been contaminated by long-term applications of copper salts as fungicides against mildew, raising the question of the bioavailability (and toxicity) of such accumulated Cu to cultivated plants which can replace vines. The aim of this study was to assess, in an acidic and a calcareous Cu-contaminated soil, how the extractability and bioavailability of soil Cu was affected by pH changes in the rhizosphere of two plant species (oilseed rape and tomato), in response to various forms of nitrogen supply (nitrate only or both nitrate and ammonium). Besides shoot analysis, the experimental approach used in the present work provided an easy access to both roots and rhizosphere soil. Roots of tomato and rape induced a systematic acidification in the calcareous soil while root-induced alkalinization occurred in the acidic soil. Whilst few differences were found between treatments in the calcareous soil, oilseed rape took up more Cu and also alkalinized its rhizosphere more strongly than tomato in the acidic soil. The growth of tomato roots was restricted in the acidic soil, while that of oilseed rape was not, suggesting that tomato was either more sensitive to soil acidity and/or Cu toxicity. A major finding was that, in the acidic soil, Cu bioavailability increased with increasing rhizosphere pH. This was largely due to the enhanced accumulation of Cu in the root compartment of both species with increasing rhizosphere pH. The hypothetical explanation proposed here is that Cu binding to root cell walls played a major role in the accumulation of Cu into the plant. Apoplasmic Cu (Cu bound to cell walls) would indeed be expected to increase with increasing pH as a consequence of the pH-dependency of the charges of cell wall constituents.     

Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize

Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.