No abstract

In this study, we redesigned and evaluated primers for the class Actinobacteria. In silico testing showed that the primers had a perfect match with 82% of genera in the class Actinobacteria, representing a 26-213% improvement over previously reported primers. Only 4% of genera that displayed mismatches did so in the terminal three bases of the 3' end, which is most critical for polymerase chain reaction success. The primers, designated S-C-Act-0235-a-S-20 and S-C-Act-0878-a-A-19, amplified an approximately 640 bp stretch of the 16S rRNA gene from all actinobacteria tested (except Rubrobacter radiotolerans) up to an annealing temperature of 72 degrees C. An Actinobacteria Amplification Resource (http://microbe2.ncl.ac.uk/MMB/AAR.htm) was generated to provide a visual guide to aid the amplification of actinobacterial 16S rDNA. Application of the primers to DNA extracted from marine and terrestrial samples revealed the presence of actinobacteria that have not been described previously. The use of 16S rDNA similarity and DNA-DNA pairing correlations showed that almost every actinomycete clone represented either a new species or a novel genus. The results of this study reinforce the proposition that current culture-based techniques drastically underestimate the diversity of Actinobacteria in the environment and highlight the need to evaluate taxon-specific primers regularly in line with improvements in databases holding 16S rDNA sequences.

To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance.

The Diels-Alder reaction, a [4 + 2] cycloaddition of a conjugated diene to a dienophile, is one of the most powerful reactions in synthetic chemistry. Biocatalysts capable of unlocking new and efficient Diels-Alder reactions would have major impact. Here we present a molecular-level description of the reaction mechanism of the spirotetronate cyclase AbyU, an enzyme shown here to be a bona fide natural Diels-Alderase. Using enzyme assays, X-ray crystal structures, and simulations of the reaction in the enzyme, we reveal how linear substrate chains are contorted within the AbyU active site to facilitate a transannular pericyclic reaction. This study provides compelling evidence for the existence of a natural enzyme evolved to catalyze a Diels-Alder reaction and shows how catalysis is achieved.

Thirty-eight actinomycetes were isolated from sediment collected from the Mariana Trench (10,898 m) using marine agar and media selective for actinomycetes, notably raffinose-histidine agar. The isolates were assigned to the class Actinobacteria using primers specific for members of this taxon. The phylogenetic analysis based on 16S rRNA gene sequencing showed that the isolates belonged to the genera Dermacoccus, Kocuria, Micromonospora, Streptomyces, Tsukamurella and Williamsia. All of the isolates were screened for genes encoding nonribosomal peptide and polyketide synthetases. Nonribosomal peptide synthetase sequences were detected in more than half of the isolates and polyketide synthases type I (PKS-I) were identified in five out of 38 strains. The Streptomyces isolates produced several unusual secondary metabolites, including a PKS-I associated product. In initial testing for piezotolerance, the Dermacoccus strain MT1.1 grew at elevated hydrostatic pressures.