Characterization of bacterial operons consisting of two tubulins and a kinesin-like gene by the novel Two-Step Gene Walking method

Abstract

Tubulins are still considered as typical proteins of Eukaryotes. However, more recently they have been found in the unusual bacteria Prosthecobacter (btubAB). In this study, the genomic organization of the btub-genes and their genomic environment were characterized by using the newly developed Two-Step Gene Walking method. In all investigated Prosthecobacters, btubAB are organized in a typical bacterial operon. Strikingly, all btub-operons comprise a third gene with similarities to kinesin light chain sequences. The genomic environments of the characterized btub-operons are always different. This supports the hypothesis that this group of genes represents an independent functional unit, which was acquired by Prosthecobacter via horizontal gene transfer. The newly developed Two-Step Gene Walking method is based on randomly primed polymerase chain reaction (PCR). It presents a simple workflow, which comprises only two major steps—a Walking-PCR with a single specific outward pointing primer (step 1) and the direct sequencing of its product using a nested specific primer (step 2). Two-Step Gene Walking proved to be highly efficient and was successfully used to characterize over 20 kb of sequence not only in pure culture but even in complex non-pure culture samples.

Four-stranded mini microtubules formed by Prosthecobacter BtubAB show dynamic instability

“…1F, Right). Given the organization of btubABC into a likely operon (6,16), the fact that BtubC binds to BtubAB filaments (10) and the misleading appearance of the term kinesin in its previous BKLC name, we decided to rename the protein BtubC.…”

A Novel 3-Sulfinopropionyl Coenzyme A (3SP-CoA) Desulfinase from Advenella mimigardefordensis Strain DPN7 T Acting as a Key Enzyme during Catabolism of 3,3′-Dithiodipropionic Acid Is a Member of the Acyl-CoA Dehydrogenase Superfamily

“…Flanking regions of the Tn5::mob insertion loci were sequenced by a PCR-based two-step genome walking method (40). IS50 walking and IS50 sequencing primers were constructed as described previously by Pilhofer et al (40).…”

“…Flanking regions of the Tn5::mob insertion loci were sequenced by a PCR-based two-step genome walking method (40). IS50 walking and IS50 sequencing primers were constructed as described previously by Pilhofer et al (40). Genomic DNA of the Tn5::mob-induced mutant was isolated according to methods described previously by Marmur (36) (primers are listed in Table S1 in the supplemental material).…”

Bacterial Tubulins A and B Exhibit Polarized Growth, Mixed-Polarity Bundling, and Destabilization by GTP Hydrolysis

“…They interact to form the BtubA/B heterodimer that polymerizes in the presence of GTP (2,3). btubA and btubB genes were discovered, along with a kinesin light chain homolog (bklc), in the genomes of four Prosthecobacter bacterial species (1) to form the bacterial tubulin operon (btub operon) (4). Because of this high sequence identity and structural similarity, it has been proposed that Prosthecobacter acquired the btub operon by horizontal gene transfer (1-7) from an unknown species.…”