Abstract
Bacteria belonging to the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) superphylum are of interest for biotechnology, evolutionary cell biology, ecology, and human health. Some PVC species lack a number of typical bacterial features while others possess characteristics that are usually more associated to eukaryotes or archaea. For example, the Planctomycetes phylum is atypical for the absence of the FtsZ protein and for the presence of a developed endomembrane system. Studies of the cellular and molecular biology of these infrequent characteristics are currently limited due to the lack of genetic tools for most of the species. So far, genetic manipulation in Planctomycetes has been described in Planctopirus limnophila only. Here, we show a simple approach that allows mutagenesis by homologous recombination in three different planctomycetes species (i.e., Gemmata obscuriglobus, Gimesia maris, and Blastopirellula marina), in addition to P. limnophila, thus extending the repertoire of genetically modifiable organisms in this superphylum. Although the Planctomycetes show high resistance to most antibiotics, we have used kanamycin resistance genes in G. obscuriglobus, P. limnophila, and G. maris, and tetracycline resistance genes in B. marina, as markers for mutant selection. In all cases, plasmids were introduced in the strains by mating or electroporation, and the genetic modification was verified by Southern Blotting analysis. In addition, we show that the green fluorescent protein (gfp) is expressed in all four backgrounds from an Escherichia coli promoter. The genetic manipulation achievement in four phylogenetically diverse planctomycetes will enable molecular studies in these strains, and opens the door to developing genetic approaches not only in other planctomycetes but also other species of the superphylum, such as the Lentisphaerae.
Highlights
The PVC superphylum comprises the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) superphylum comprises the Planctomycetes, the Verrucomicrobia and the Chlamydiae phyla, and several others, including the Lentisphaerae (Wagner and Horn, 2006; Fuerst, 2013)
Taking into account that those bacteria are fastidious, i.e., their doubling times are in the order of a few hours (Jetten et al, 1998), they tend to aggregate into big clamps and they are naturally resistant to many antibiotics (Cayrou et al, 2010), it is not surprising that little is known about their genetic manipulation
Saturated cultures of G. obscuriglobus were spotted onto Luria–Bertani medium (LB) NaCl-free agar plates supplemented with different concentrations of the antibiotic (5, 10, 20, and 30 μg ml−1)
Summary
The PVC superphylum comprises the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) superphylum comprises the Planctomycetes, the Verrucomicrobia and the Chlamydiae phyla, and several others, including the Lentisphaerae (Wagner and Horn, 2006; Fuerst, 2013). Bacteria belonging to the Planctomycetes present peculiar features that are rare in bacteria (Devos, 2013; Fuerst, 2013; Devos and Ward, 2014), and some of which are more frequent in archaea or eukaryotes (Devos and Reynaud, 2010; Reynaud and Devos, 2011). Despite the interest in the study of these and other peculiar features, their detailed characterization has so far been limited, mainly to computational genomes and proteomes analyses, and microscopy, due to the lack of genetic tools to manipulate the organisms. Genetic manipulation of P. limnophila by electroporation of circular or linear DNA, containing homologous sequence of the chromosome, has been achieved (Jogler et al, 2011; Erbilgin et al, 2014), and by transposon mutagenesis using the EZ-Tn5 transposome (Epicenter) (Schreier et al, 2012)
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