Escherichia coli strains were grown in batch cultures in different media, and cell size and DNA content were analyzed by flow cytometry. Steady-state growth required large dilutions and incubation for many generations at low cell concentrations. In rich media, both cell size and DNA content started to decrease at low cell concentrations, long before the cultures left the exponential growth phase. Stationary-phase cultures contained cells with several chromosomes, even after many days, and stationary-phase populations exclusively composed of cells with a single chromosome were never observed, regardless of growth medium. The cells usually contained only one nucleoid, as visualized by phase and fluorescence microscopy. The results have implications for the use of batch cultures to study steady-state and balanced growth and to determine mutation and recombination frequencies in stationary phase.

No abstract

The frequency of replication of IncFII plasmids is regulated by the availability of a rate‐limiting protein, RepA. This protein acts to promote initiation of replication and its synthesis is negatively controlled both at the transcriptional and translational level. The translational control is exerted by the binding of a small antisense RNA, CopA RNA, to its target, CopT, which is located in the leader region of the RepA mRNA. As a consequence, formation of RepA is inhibited. Here we demonstrate the binding of CopA RNA to CopT RNA in vitro; the rate constant of binding was determined to be approximately 1 X 10(6) M‐1 s‐1 at 37 degrees C. We have also shown that in vitro synthesized RepA mRNA molecules differing in length, but which contain the whole CopT region, are able to bind CopA RNA with similar rates. Analysis of the binding of CopA/CopT molecules derived from a copy‐number mutant plasmid showed that the effect of the mutation on the rate of in vitro binding correlates well with its phenotypic effect in vivo, i.e. the binding rate constant is lowered in proportion to the increase in copy number. Likewise, the result of an in vitro incompatibility test is in agreement with in vivo data.

The replication frequency of IncFH plasmids is regulated through the availability of a rate-limiting protein, RepA.

No abstract

SummaryA strain of Escherichia coli in which both the seqA and mukB genes were inactivated displayed partial suppressions of their individual phenotypes. Temperature sensitivity, anucleate cell production and poor nucleoid folding seen in the mukB strain were suppressed by the seqA null mutation, whereas ®la-mentation, asymmetric septation and compact folding of the nucleoids observed in the seqA strain were suppressed by inactivation of the mukB gene function. However, the asynchronous initiation of chromosome replication in the seqA strain was not reversed in the mukBseqA double mutant. Membrane-associated nucleoids were isolated from the wild-type, mukB, seqA and mukBseqA strains and their sedimentation rates were compared under identical conditions. Whereas the mukB mutation caused unfolding of the nucleoid, the seqA mutation led to a more compact packaging of the chromosome. The mukBseqA double mutant regained the wild-type nucleoid organization as revealed from its rate of sedimentation. Microscopic appearances of the nucleoids were consistent with the sedimentation pro®les. The mukB mutant was oversensitive to novobiocin and this susceptibility was suppressed in the mukBseqA strain, suggesting possible roles of MukB and SeqA in maintaining chromosome topology. The mutual phenotypic suppression of mukB and seqA alleles thus suggests that these genes have opposing in¯u-ences on the organization of the bacterial nucleoid.

No abstract

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.