Jakob Møller-Jensen

Plasmid and chromosome partitioning: surprises from phylogeny

Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chromosomes from prokaryotic organisms. All known plasmid‐encoded par loci specify three components: a cis‐acting centromere‐like site and two trans‐acting proteins that form a nucleoprotein complex at the centromere (i.e. the partition complex). The proteins are encoded by two genes in an operon that is autoregulated by the par‐encoded proteins. In all cases, the upstream gene encodes an ATPase that is essential for partitioning. Recent cytological analyses indicate that the ATPases function as adaptors between a host‐encoded component and the partition complex and thereby tether plasmids and chromosomal origin regions to specific subcellular sites (i.e. the poles or quarter‐cell positions). Two types of partitioning ATPases are known: the Walker‐type ATPases encoded by the par/sop gene family (type I partitioning loci) and the actin‐like ATPase encoded by the par locus of plasmid R1 (type II partitioning locus). A phylogenetic analysis of the large family of Walker type of partitioning ATPases yielded a surprising pattern: most of the plasmid‐encoded ATPases clustered into distinct subgroups. Surprisingly, however, the par loci encoding these distinct subgroups have different genetic organizations and thus divide the type I loci into types Ia and Ib. A second surprise was that almost all chromosome‐encoded ATPases, including members from both Gram‐negative and Gram‐positive Bacteria and Archaea, clustered into one distinct subgroup. The phylogenetic tree is consistent with lateral gene transfer between Bacteria and Archaea. Using database mining with the ParM ATPase of plasmid R1, we identified a new par gene family from enteric bacteria. These type II loci, which encode ATPases of the actin type, have a genetic organization similar to that of type Ib loci.

Prokaryotic DNA segregation by an actin-like filament

The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin‐like filaments with properties expected for a force‐generating protein. Filament formation depended on the other components encoded by par, ParR and the centromere‐like parC region to which ParR binds. Mutants defective in ParM ATPase exhibited hyperfilamentation and did not support plasmid partitioning. ParM polymerization was ATP dependent, and depolymerization of ParM filaments required nucleotide hydrolysis. Our in vivo and in vitro results indicate that ParM polymerization generates the force required for directional movement of plasmids to opposite cell poles and that the ParR–parC complex functions as a nucleation point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus.

F-actin-like filaments formed by plasmid segregation protein ParM

It was the general belief that DNA partitioning in prokaryotes is independent of a cytoskeletal structure, which in eukaryotic cells is indispensable for DNA segregation. Recently, however, immunofluorescence microscopy revealed highly dynamic, filamentous structures along the longitudinal axis of Escherichia coli formed by ParM, a plasmid‐encoded protein required for accurate segregation of low‐copy‐number plasmid R1. We show here that ParM polymerizes into double helical protofilaments with a longitudinal repeat similar to filamentous actin (F‐actin) and MreB filaments that maintain the cell shape of non‐spherical bacteria. The crystal structure of ParM with and without ADP demonstrates that it is a member of the actin family of proteins and shows a domain movement of 25° upon nucleotide binding. Furthermore, the crystal structure of ParM reveals major differences in the protofilament interface compared with F‐actin, despite the similar arrangement of the subunits within the filaments. Thus, there is now evidence for cytoskeletal structures, formed by actin‐like filaments that are involved in plasmid partitioning in E.coli.

Dysfunctional MreB inhibits chromosome segregation in Escherichia coli

The mechanism of prokaryotic chromosome segregation is not known. MreB, an actin homolog, is a shape‐determining factor in rod‐shaped prokaryotic cells. Using immunofluorescence microscopy we found that MreB of Escherichia coli formed helical filaments located beneath the cell surface. Flow cytometric and cytological analyses indicated that MreB‐depleted cells segregated their chromosomes in pairs, consistent with chromosome cohesion. Overexpression of wild‐type MreB inhibited cell division but did not perturb chromosome segregation. Overexpression of mutant forms of MreB inhibited cell division, caused abnormal MreB filament morphology and induced severe localization defects of the nucleoid and of the oriC and terC chromosomal regions. The chromosomal terminus regions appeared cohered in both MreB‐depleted cells and in cells overexpressing mutant forms of MreB. Our observations indicate that MreB filaments participate in directional chromosome movement and segregation.

Bacterial Mitosis: ParM of Plasmid R1 Moves Plasmid DNA by an Actin-like Insertional Polymerization Mechanism

Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

Bacterial mitotic machineries.

Here, we review recent progress that yields fundamental new insight into the molecular mechanisms behind plasmid and chromosome segregation in prokaryotic cells. In particular, we describe how prokaryotic actin homologs form mitotic machineries that segregate DNA before cell division. Thus, the ParM protein of plasmid R1 forms F actin-like filaments that separate and move plasmid DNA from mid-cell to the cell poles. Evidence from three different laboratories indicate that the morphogenetic MreB protein may be involved in segregation of the bacterial chromosome.

Switching off small RNA regulation with trap-mRNA

Small non‐coding regulatory RNAs in bacteria have been shown predominantly to be tightly regulated at the level of transcription initiation, and sRNAs that function by an antisense mechanism on trans‐encoded target mRNAs have been shown or predicted to act stoichiometrically. Here we show that MicM, which silences the expression of an outer membrane protein, YbfM under most growth conditions, does not become destabilized by target mRNA overexpression, indicating that the small RNA regulator acts catalytically. Furthermore, our regulatory studies suggested that control of micM expression is unlikely to operate at the level of transcription initiation. By employing a highly sensitive genetic screen we uncovered a novel RNA‐based regulatory principle in which induction of a trap‐mRNA leads to selective degradation of a small regulatory RNA molecule, thereby abolishing the sRNA‐based silencing of its cognate target mRNA. In the present case, antisense regulation by chb mRNA of the antisense regulator MicM by an extended complementary sequence element, results in induction of ybfM mRNA translation. This type of regulation is reminiscent of the regulation of microRNA activity through target mimicry that occurs in plants.

Regular cellular distribution of plasmids by oscillating and filament‐forming ParA ATPase of plasmid pB171

Centromere‐like loci from bacteria segregate plasmids to progeny cells before cell division. The ParA ATPase (a MinD homologue) of the par2 locus from plasmid pB171 forms oscillating helical structures over the nucleoid. Here we show that par2 distributes plasmid foci regularly along the length of the cell even in cells with many plasmids. In vitro, ParA binds ATP and ADP and has a cooperative ATPase activity. Moreover, ParA forms ATP‐dependent filaments and cables, suggesting that ParA can provide the mechanical force for the observed regular distribution of plasmids. ParA and ParB interact with each other in a bacterial two‐hybrid assay but do not interact with FtsZ, eight other essential cell division proteins or MreB actin. Based on these observations, we propose a simple model for how oscillating ParA filaments can mediate regular cellular distribution of plasmids. The model functions without the involvement of partition‐specific host cell receptors and is thus consistent with the striking observation that partition loci can function in heterologous host organisms.

Small regulatory RNAs control the multi‐cellular adhesive lifestyle of Escherichia coli

Small regulatory RNA molecules have recently been recognized as important regulatory elements of developmental processes in both eukaryotes and bacteria. We here describe a striking example in Escherichia coli that can switch between a single‐cell motile lifestyle and a multi‐cellular, sessile and adhesive state that enables biofilm formation on surfaces. For this, the bacterium needs to reprogramme its gene expression, and in many E. coli and Salmonella strains the lifestyle shift relies on control cascades that inhibit flagellar expression and activate the synthesis of curli, extracellular adhesive fibres important for co‐aggregation of cells and adhesion to biotic and abiotic surfaces. By combining bioinformatics, genetic and biochemical analysis we identified three small RNAs that act by an antisense mechanism to downregulate translation of CsgD, the master regulator of curli synthesis. Our demonstration that basal expression of each of the three RNA species is sufficient to downregulate CsgD synthesis and prevent curli formation indicates that all play a prominent role in the curli regulatory network. Our findings provide the first clue as to how the Rcs signalling pathway negatively regulates curli synthesis and increase the number of small regulatory RNAs that act directly on the csgD mRNA to five.

Novel Coiled-Coil Cell Division Factor Zapb Stimulates Z Ring Assembly and Cell Division.

Formation of the Z ring is the first known event in bacterial cell division. However, it is not yet known how the assembly and contraction of the Z ring are regulated. Here, we identify a novel cell division factor ZapB in Escherichia coli that simultaneously stimulates Z ring assembly and cell division. Deletion of zapB resulted in delayed cell division and the formation of ectopic Z rings and spirals, whereas overexpression of ZapB resulted in nucleoid condensation and aberrant cell divisions. Localization of ZapB to the divisome depended on FtsZ but not FtsA, ZipA or FtsI, and ZapB interacted with FtsZ in a bacterial two‐hybrid analysis. The simultaneous inactivation of FtsA and ZipA prevented Z ring assembly and ZapB localization. Time lapse microscopy showed that ZapB–GFP is present at mid‐cell in a pattern very similar to that of FtsZ. Cells carrying a zapB deletion and the ftsZ84ts allele exhibited a synthetic sick phenotype and aberrant cell divisions. The crystal structure showed that ZapB exists as a dimer that is 100% coiled‐coil. In vitro, ZapB self‐assembled into long filaments and bundles. These results raise the possibility that ZapB stimulates Z ring formation directly via its capacity to self‐assemble into larger structures.