Diversity and strength of internal outward-oriented promoters in group IIC-attC introns

Integrons are genetic elements that incorporate mobile gene cassettes by site-specific recombination and express them as an operon from a promoter (Pc) located upstream of the cassette insertion site. Most gene cassettes found in integrons contain only one gene followed by an attC recombination site. We have recently shown that a specific lineage of group IIC introns, named group IIC-attC introns, inserts into the bottom strand sequence of attC sites. Here, we show that S.ma.I2, a group IIC-attC intron inserted in an integron cassette array of Serratia marcescens, impedes transcription from Pc while allowing expression of the following antibiotic resistance cassette using an internal outward-oriented promoter (Pout). Bioinformatic analyses indicate that one or two putative Pout, which have sequence similarities with the Escherichia coli consensus promoters, are conserved in most group IIC-attC intron sequences. We show that Pout with different versions of the −35 and −10 sequences are functionally active in expressing a promoterless chloramphenicol acetyltransferase (cat) reporter gene in E. coli. Pout in group IIC-attC introns may therefore play a role in the expression of one or more gene cassettes whose transcription from Pc would otherwise be impeded by insertion of the intron

A rapid and sensitive system for recovery of nucleic acids from Mycobacteria sp. on archived glass slides

The field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM coupled with PCR amplification of a suitable target. Mycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM. This study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system

Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cu^r Sm^r), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cu^s Sm^r strains contained a 68-kb conjugative plasmid. Cu^r Sm^s, strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Sm^r genes from the broad-host-range plasmid RSF1010. The Sm^r determinant was subsequently cloned from a 68-kb Cu^r Sm^r plasmid designated pPSR1. A restriction map detailing the organization of the homologous Sm^r genes from pPSR1 and RSF1010 and cloned Sm^r genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cu^r genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cu^r plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.Peer reviewedPlant Patholog

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure

Aims: To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs). Methods and Results: The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random amplification of polymorphic DNA (RAPD). Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure. Conclusions: Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups. Significance and Impact of the Study: This information should help in an understanding of genetic exchanges in lactic acid bacteria