Analysis of DNA processing reactions in bacterial conjugation by using suicide oligonucleotides

Protein TrwC is the conjugative relaxase responsible for DNA processing in plasmid R388 bacterial conjugation. TrwC has two catalytic tyrosines, Y18 and Y26, both able to carry out cleavage reactions using unmodified oligonucleotide substrates. Suicide substrates containing a 30-Sphosphorothiolate linkage at the cleavage site displaced TrwC reaction towards covalent adducts and thereby enabled intermediate steps in relaxase reactions to be investigated. Two distinct covalent TrwC–oligonucleotide complexes could be separated from noncovalently bound protein by SDS–PAGE. As observed by mass spectrometry, one complex contained a single, cleaved oligonucleotide bound to Y18, whereas the other contained two cleaved oligonucleotides, bound to Y18 and Y26. Analysis of the cleavage reaction using suicide substrates and Y18F or Y26F mutants showed that efficient Y26 cleavage only occurs after Y18 cleavage. Strand-transfer reactions carried out with the isolated Y18–DNA complex allowed the assignment of specific roles to each tyrosine. Thus, only Y18 was used for initiation. Y26 was specifically used in the second transesterification that leads to strand transfer, thus catalyzing the termination reaction that occurs in the recipient cell.

Incorporation of chitosan in acrylic bone cement: Effect on antibiotic release, bacterial biofilm formation and mechanical properties

Bacterial infection remains a significant problem following total joint replacement. Efforts to prevent recurrent implant infection, including the use of antibiotic-loaded bone cement for implant fixation at the time of revision surgery, are not always successful. In this in vitro study, we investigated whether the addition of chitosan to gentamicin-loaded Palacos® R bone cement increased antibiotic release and prevented bacterial adherence and biofilm formation by Staphylococcus spp. clinical isolates. Furthermore, mechanical tests were performed as a function of time post-polymerisation in pseudo-physiological conditions. The addition of chitosan to gentamicin-loaded Palacos® R bone cement significantly decreased gentamicin release and did not increase the efficacy of the bone cement at preventing bacterial colonisation and biofilm formation. Moreover, the mechanical performance of cement containing chitosan was significantly reduced after 28 days of saline degradation with the compressive and bending strengths not in compliance with the minimum requirements as stipulated by the ISO standard for PMMA bone cement. Therefore, incorporating chitosan into gentamicin-loaded Palacos® R bone cement for use in revision surgery has no clinical antimicrobial benefit and the detrimental effect on mechanical properties could adversely affect the longevity of the prosthetic joint.

New ways to break an old bond: the bacterial carbon-phosphorus hydrolases and their role in biogeochemical phosphorus cycling

Phosphonates are organophosphorus molecules that contain the highly stable C-P bond, rather than the more common, and more labile, C-O-P phosphate ester bond. They have ancient origins but their biosynthesis is widespread among more primitive organisms and their importance in the contemporary biosphere is increasingly recognized; for example phosphonate-P is believed to play a particularly significant role in the productivity of the oceans. The microbial degradation of phosphonates was originally thought to occur only under conditions of phosphate limitation, mediated exclusively by the poorly characterized C-P lyase multienzyme system, under Pho regulon control. However, more recent studies have demonstrated the Pho-independent mineralization by environmental bacteria of three of the most widely distributed biogenic phosphonates: 2-aminoethylphosphonic acid (ciliatine), phosphonoacetic acid, and 2-amino-3-phosphonopropionic acid (phosphonoalanine). The three phosphonohydrolases responsible have unique specificities and are members of separate enzyme superfamilies; their expression is regulated by distinct members of the LysR family of bacterial transcriptional regulators, for each of which the phosphonate substrate of the respective degradative operon serves as coinducer. Previously no organophosphorus compound was known to induce the enzymes required for its own degradation. Whole-genome and metagenome sequence analysis indicates that the genes encoding these newly described C-P hydrolases are distributed widely among prokaryotes. As they are able to function under conditions in which C-P lyases are inactive, the three enzymes may play a hitherto-unrecognized role in phosphonate breakdown in the environment and hence make a significant contribution to global biogeochemical P-cycling.

Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera

The economically most important honey bee species, Apis mellifera, was formerly considered to be parasitized by one microsporidian, Nosema apis. Recently, [Higes, M., Martin, R., Meana, A., 2006. Nosema ceranae, a new microsporidian parasite in honeybees in Europe, J. Invertebr. Pathol. 92, 93-95] and [Huang, W.-F., Jiang, J.-H., Chen, Y.-W., Wang, C.-H., 2007. A Nosema ceranae isolate from the honeybee Apis mellifera. Apidologie 38, 30-37] used 16S (SSU) rRNA gene sequences to demonstrate the presence of Nosema ceranae in A. mellifera from Spain and Taiwan, respectively. We developed a rapid method to differentiate between N. apis and N. ceranae based on PCR-RFLPs of partial SSU rRNA. The reliability of the method was confirmed by sequencing 29 isolates from across the world (N = 9 isolates gave N. apis RFLPs and sequences, N = 20 isolates gave N. ceranae RFLPs and sequences; 100%, correct classification). We then employed the method to analyze N = 115 isolates from across the world. Our data, combined with N = 36 additional published sequences demonstrate that (i) N. ceranae most likely jumped host to A. mellifera, probably within the last decade, (ii) that host colonies and individuals may be co-infected by both microsporidia species, and that (iii) N. ceranae is now a parasite of A. mellifera across most of the world. The rapid, long-distance dispersal of N. ceranae is likely due to transport of infected honey bees by commercial or hobbyist beekeepers. We discuss the implications of this emergent pathogen for worldwide beekeeping. (c) 2007 Elsevier Inc. All rights reserved.