Comparison of sapwood invasion by three Phytophthora spp.in different hosts

Many Phytophthora spp. have recently been isolated from native vegetation in Western Australia. As their pathogenicity is often unknown, it is not possible to provide advice to land managers on the impact of site infestation on native plants and how these infestations should be managed. We describe a rapid screening method based on sapwood invasion that has been used to compare the pathogenicity of Phytophthora arenaria, P. cinnamomi and P. multivora. Radial invasion into the xylem of six banksias and three eucalypts was assessed in an excised branch assay in summer and winter. Branches were wound inoculated and invasion was assessed by plating from a strip of tissue cut across the stem at the inoculation point and at 40 mm above and below. A symptomless infection had established in both the bark and sapwood within 6 days. P. arenaria was only isolated from the strip of tissue at the inoculation point. P. cinnamomi was isolated from the sapwood of Banksia attenuata, B. burdettii, B. menziesii and B. speciosa 40 mm above or below the inoculation point in some experiments. P. multivora was isolated from B. speciosa 40 mm below the inoculation point in one experiment. Hyphae of both species were seen in both ray parenchyma cells and xylem vessels. The invasiveness of the Phytophthora spp. was compared on the two groups of hosts using scores for sapwood invasion at the inoculation point. For banksias, P. cinnamomi and P. multivora had significantly higher invasion scores on banksias than P. arenaria but were not significantly different to one another. There was no significant difference between the three Phytophthora spp. on the eucalypt hosts. Assessing sapwood invasion provides a rapid, inexpensive and biologically meaningful way of screening the many Phytophthora spp. that have been isolated from native vegetation

Inverse Correlation between Promoter Strength and Excision Activity in Class 1 Integrons

Class 1 integrons are widespread genetic elements that allow bacteria to capture and express gene cassettes that are usually promoterless. These integrons play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. They typically consist of a gene (intI) encoding an integrase (that catalyzes the gene cassette movement by site-specific recombination), a recombination site (attI1), and a promoter (Pc) responsible for the expression of inserted gene cassettes. The Pc promoter can occasionally be combined with a second promoter designated P2, and several Pc variants with different strengths have been described, although their relative distribution is not known. The Pc promoter in class 1 integrons is located within the intI1 coding sequence. The Pc polymorphism affects the amino acid sequence of IntI1 and the effect of this feature on the integrase recombination activity has not previously been investigated. We therefore conducted an extensive in silico study of class 1 integron sequences in order to assess the distribution of Pc variants. We also measured these promoters' strength by means of transcriptional reporter gene fusion experiments and estimated the excision and integration activities of the different IntI1 variants. We found that there are currently 13 Pc variants, leading to 10 IntI1 variants, that have a highly uneven distribution. There are five main Pc-P2 combinations, corresponding to five promoter strengths, and three main integrases displaying similar integration activity but very different excision efficiency. Promoter strength correlates with integrase excision activity: the weaker the promoter, the stronger the integrase. The tight relationship between the aptitude of class 1 integrons to recombine cassettes and express gene cassettes may be a key to understanding the short-term evolution of integrons. Dissemination of integron-driven drug resistance is therefore more complex than previously thought

Characterisation of a chloramphenicol acetyltransferase determinant found in the chromosome of Pseudomonas aeruginosa

The open reading frame (ORF) in the Pseudomonas aeruginosa chromosome, whose product resembles the chloramphenicol acetyltransferases (CAT) belonging to the CATB family, was cloned and shown to confer resistance to chloramphenicol (Cm) in Escherichia coli. The determinant was therefore named catB7 and the corresponding protein CATB7. When the copy number and expression signals were identical, the catB7 gene conferred resistance to Cm at a level slightly lower than those of three other catB genes. CATB7 resembles other CATBs in that it acetylates Cm but not 1-acetoxy-Cm. For CATB7, the Km values for acetyl-CoA and Cm were 5.05.4-fold higher than the corresponding values for each of the three other CATB proteins (CATB1, CATB3 and CATB5) examined and the Vmax was 56 fold lower. Using PCR, the catB7 gene was found in all six P. aeruginosa strains examined but not in any other species of pseudomonad tested. Weak CAT activity was detected in crude cell extracts from five of the six P. aeruginosa strains. However, this activity did not correlate with the Cm susceptibility of the strains, indicating that catB7 is not likely to be the major determinant of intrinsic Cm resistance in P. aeruginosa

Impacts of anthropogenic activity on the ecology of class 1 integrons and integron-associated genes in the environment

The impact of human activity on the selection for antibiotic resistance in the environment is largely unknown, although considerable amounts of antibiotics are introduced through domestic wastewater and farm animal waste. Selection for resistance may occur by exposure to antibiotic residues or by co-selection for mobile genetic elements (MGEs) which carry genes of varying activity. Class 1 integrons are genetic elements that carry antibiotic and quaternary ammonium compound (QAC) resistance genes that confer resistance to detergents and biocides. This study aimed to investigate the prevalence and diversity of class 1 integron and integron-associated QAC resistance genes in bacteria associated with industrial waste, sewage sludge and pig slurry. We show that prevalence of class 1 integrons is higher in bacteria exposed to detergents and/or antibiotic residues, specifically in sewage sludge and pig slurry compared with agricultural soils to which these waste products are amended. We also show that QAC resistance genes are more prevalent in the presence of detergents. Studies of class 1 integron prevalence in sewage sludge amended soil showed measurable differences compared with controls. Insertion sequence elements were discovered in integrons from QAC contaminated sediment, acting as powerful promoters likely to upregulate cassette gene expression. On the basis of this data, > 1 x 10(19) bacteria carrying class 1 integrons enter the United Kingdom environment by disposal of sewage sludge each year. The ISME Journal (2011) 5, 1253-1261; doi:10.1038/ismej.2011.15; published online 3 March 201