A Family of Single Copy repABC-Type Shuttle Vectors Stably Maintained in the Alpha-Proteobacterium Sinorhizobium meliloti

Abstract

A considerable share of bacterial species maintains segmented genomes. Plant symbiotic α-proteobacterial rhizobia contain up to six repABC-type replicons in addition to the primary chromosome. These low or unit-copy replicons, classified as secondary chromosomes, chromids, or megaplasmids, are exclusively found in α-proteobacteria. Replication and faithful partitioning of these replicons to the daughter cells is mediated by the repABC region. The importance of α-rhizobial symbiotic nitrogen fixation for sustainable agriculture and Agrobacterium-mediated plant transformation as a tool in plant sciences has increasingly moved biological engineering of these organisms into focus. Plasmids are ideal DNA-carrying vectors for these engineering efforts. On the basis of repABC regions collected from α-rhizobial secondary replicons, and origins of replication derived from traditional cloning vectors, we devised the versatile family of pABC shuttle vectors propagating in Sinorhizobium meliloti, related members of the Rhizobiales, and Escherichia coli. A modular plasmid library providing the elemental parts for pABC vector assembly was founded. The standardized design of these vectors involves five basic modules: (1) repABC cassette, (2) plasmid-derived origin of replication, (3) RK2/RP4 mobilization site (optional), (4) antibiotic resistance gene, and (5) multiple cloning site flanked by transcription terminators. In S. meliloti, pABC vectors showed high propagation stability and unit-copy number. We demonstrated stable coexistence of three pABC vectors in addition to the two indigenous megaplasmids in S. meliloti, suggesting combinability of multiple compatible pABC plasmids. We further devised an in vivo cloning strategy involving Cre/lox-mediated translocation of large DNA fragments to an autonomously replicating repABC-based vector, followed by conjugation-mediated transfer either to compatible rhizobia or E. coli.

Front-propagation in bacterial inter-colony communication

“…For the sender, this construct was cloned into the suicide plasmid pK18mob2 Km also carrying a wgeA promoter-cerulean fusion -the wgeA promoter drives expression of genes essential for exopolysaccharide production and served as the integration site for the plasmid. The receiver strain with the sinI promoter-mVenus fusion carried only the synthetic trp promoter-mCherry fusion cloned into the single copy plasmid pABC1mob [38].…”

Tol-Pal system and Rgs proteins interact to promote unipolar growth and cell division inSinorhizobium meliloti

“…To enable RgsA depletion, the chromosomal rgsA gene was deleted in presence of a plasmid-borne copy of this gene controlled by its native promoter in Rm2011 rgsP-egfp . Replication of this single copy plasmid pGCH14 was mediated by a repABC operon whose expression was dependent on IPTG (18). Omitting IPTG from the growth medium of this RgsA depletion strain resulted in loss of normal RgsP localization and growth inhibition (Fig.…”

Synthetische Biologie

“…Hierzu zählen ihre Größe, eine strikte Kontrolle ihrer einfachen Kopienzahl sowie die stabile Weitergabe an die Tochterzellen. Die nur wenige Kilobasen große repABC‐Region wurde als funktionelle Einheit identifiziert, die die stabile Vererbung und einfache Kopienzahl der Megaplasmide vermittelt . Um das Potenzial der Megaplasmide für synthetisch‐biologische Forschungsansätze nutzbar zu machen, wurde auf Basis einer Kollektion von repABC‐Regionen verschiedener α‐Rhizobien die Familie der pABC‐Vektoren entwickelt .…”

“…Die nur wenige Kilobasen große repABC‐Region wurde als funktionelle Einheit identifiziert, die die stabile Vererbung und einfache Kopienzahl der Megaplasmide vermittelt . Um das Potenzial der Megaplasmide für synthetisch‐biologische Forschungsansätze nutzbar zu machen, wurde auf Basis einer Kollektion von repABC‐Regionen verschiedener α‐Rhizobien die Familie der pABC‐Vektoren entwickelt . Ein modularer Aufbau aus fünf Elementen und ein standardisiertes Assemblierungssystem ermöglichen die maßgeschneiderte Konstruktion einer großen Vielfalt an Varianten dieser pABC‐Vektoren aus einer umfangreichen Baustein‐Bibliothek (Abbildung ).…”