The effects of RNA degradation enzymes on antisense RNAI controlling ColE2 plasmid copy number

Replication of the ColE2 plasmid requires a plasmid-coded initiator protein (Rep). Rep expression is controlled by antisense RNA (RNAI), which prevents the Rep mRNA translation. In this paper, we examined the effects of RNA degradation enzymes on the degradation pathways of RNAI of the ColE2 plasmid. In the DeltapcnB strain lacking the poly(A) polymerase I (PAP I) the RNAI degradation intermediate (RNAI(*)) accumulates much more than that in the wt strain. RNAI(*) is produced by the RNase E cleavage. RNase II and PNPase are involved in further degradation of RNAI(*) and PAP I is necessary for efficient degradation. The degradation process of ColE2 RNAI is similar to those of R1 CopA RNA and ColE1 RNAI, although the nucleotide sequences and fine secondary structures of these three RNAs are different. ColE2 RNAI is cleaved at multiple positions in the 5' end region by RNase E. The degradation pathway of ColE2 RNAI shown here is quite different from that of the ColE2 Rep mRNA which we have previously reported. In the DeltapcnB strain used for RNA analysis the copy number of the ColE2 plasmid decreases to about a half as compared with that in the isogenic wt strain.     

PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of CoIE1-related plasmids

The replication of CoIE1-related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for pcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5′-end processing by RNAse E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST- PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.     

Origin-specific reduction of ColE1 plasmid copy number due to mutations in a distinct region of the Escherichia coli RNA polymerase

Mutations affecting a region of the Escherichia coli RNA polymerase have been isolated that specifically reduce the copy number of ColE1-type plasmids. The mutations, which result in a single amino acid alteration (G1161R) or a 41-amino acid deletion (Delta1149-1190) are located near the 3'-terminal region in the rpoC gene, which encodes the largest subunit (beta ') of the RNA polymerase. The rpoC deletion and the point mutation cause over 20- and 10-fold reductions, respectively, in the copy number of ColE1. ColE1 plasmid numbers are regulated by two plasmid-encoded RNAs: RNA II, which acts as a preprimer for the DNA polymerase I to start initiation of replication, and RNA I, its antisense inhibitor. Altered expression from the RNA I and RNA II promoters in vivo was observed in the RNA polymerase mutants. The RNA I/RNA II ratio is higher in the mutants than in the wild-type strain and this is most probably the main reason for the reduction in the ColE1 copy number in the two rpoC mutants.     

DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number.

The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. Here we report that the location of origin-opening, initiation site of replication forks and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. However, origins with DnaA boxes proximal to the origin-opening location opened more efficiently and maintained plasmids at higher copy numbers. Origins with the distal set were inactive unless the adjacent P1 DNA sequences beyond the boxes were included. At either end, phasing of the boxes with respect to the remainder of the origin influenced the copy number. Thus, although the boxes can be at either end, their precise context is critical for efficient origin function.     

An evaluation of models for the effect of plasmid copy number on bacterial growth rate

The rates of growth of recombinant bacteria depend on their plasmid content. This is modelled by expressing the specific growth rate in terms of the number of copies of the plasmid per cell. Three models in common use have been tested with different Escherichia coli strains and one strain of Bacillus stearothermophilus containing different plasmids. While no particular model was decisively better than others for all data, that of Bentley & Quiroga (Biotechnol. Bioeng. 1993, 42: 222–234) was the best for specific growth rates which vary inversely with the plasmid copy number, and a modified form of the model of Satyagal & Agarwal (Biotechnol. Bioeng. 1989, 33: 1135–1144) was the best for growth rates which increase with the copy number. The differences appear to be linked to the plasmid replication mechanisms. Contrary to some claims, no model portrayed the experimentally observed inflection points.     

The effect of dnaA protein and n' sites on the replication of plasmid ColE1

The role of the dna A protein in the replication of plasmid ColE1 and its derivatives was examined. Wild-type and mutant ColE1 plasmids were compared as to their ability to replicate in an in vitro replication system supplemented with ammonium sulfate fractionated extracts from a dnaA-overproducing strain. Synthesis on plasmid templates containing the wild-type origin of replication was stimulated 1.3-fold by addition of the dnaA-overproducing extract. A larger effect was observed after deletion of the primosome assembly site, the n' site, on the leading strand. On the latter template, synthesis was only about one-half that observed with the wild-type templates, but synthesis could be restored to normal levels by addition of the dnaA-overproducing fractions. When the n' site on the lagging strand of pBR322 was deleted, synthesis in the in vitro replication system was reduced to less than 10% of levels seen with intact templates. dnaA-overproducing extract did not restore activity since the dnaA site was also deleted on these plasmids. To verify that the observed stimulation of wild-type and leading strand n' site mutants was due to the dnaA protein, dnaA protein was purified to greater than 50% homogeneity, and antiserum was prepared. The purified protein stimulated synthesis on the plasmid templates to the same extent as the overproducing extracts, and dnaA antiserum blocked stimulation both by extracts and by the purified protein. Thus, dnaA protein, and, by inference, the dnaA recognition site at the ColE1 origin of replication seem to be important for ColE1 replication. The effect of dnaA protein is enhanced when the n'site is defective, suggesting that the dnaA protein plays a role similar to that of the proteins i, n, n', and n' in directing primosome assembly, as proposed by Seufert, W., and Messer, W. ((1987) Cell 48, 73-78).     

Replication control of plasmid pLS1: efficient regulation of plasmid copy number is exerted by the combined action of two plasmid components, CopG and RNA II

Two elements, the products of genes copG and rnall , are involved in the copy-number control of plasmid pLS1. RNA II is synthesized in a dosage-dependent manner. Mutations in both components have been characterized. To determine the regulatory role of the two genes, we have cloned copG , rnall or both elements at various gene dosages into pLS1-compatible plasmids. Assays of incompatibility towards wild-type or mutant pLS1 plasmids showed that: (i) the rnall gene product, rather than the DNA sequence encoding it, is responsible for the incompatibility, and (ii) CopG and RNA II act in trans and are able to correct up fluctuations in pLS1 copy number. A correlation between the gene dosage at which the regulatory elements were supplied and the incompatibility effect on the resident plasmid was observed. The entire copG-rnall circuit has a synergistic effect when compared with any of its components in the correction of pLS1 copy-number fluctuations, indicating that, in the homoplasmid steady-state situation, the control of pLS1 replication is exerted by the co-ordinate action of CopG and RNA II.     

Nature of the compatible inheritance of hybrid plasmid pAS8 and its deletion mutants with replicon ColE1]

Hybrid plasmid pAS8, that consists of RP4 and ColE1 replicons, is incompatible with RP4 but co-exists with ColE1 replicon. The deletion mutants of pAS8, that replicates under the control of ColE1 replicon only, are incompatible with ColE1 derivatives, although the copy number of pAS8 and its deletion mutants in the cell is the same (1-3 per the chromosome). Incompatibility effect of plasmids is expressed in a sharp decrease in transformant's yield during selection of incoming and resident plasmids markers. In this case incompatibility is a very fast process, that leads to the elimination of resident or incoming plasmid just before plating on the selective medium. On the base of negative control theory, the repressors yield, synthesized in the presence of 1-3 copies of the deletion mutant is enough for the expression of ColE1-specific incompatibility. This ColE1 incompatibility is probably connected with the functional activity of ColE1 replicon. When ColE1 factor replicates under the control of RP4 replicon the expression of ColE1-specific incompatibility does not occur. Possibly, the incompatibility effect takes place when pAS8 deletion mutants cause the synthesis of ColE1-specific repressor. Also, the replicons of ColE1 may competein the membrane attachment site.     

Replication of the broad-host-range plasmid RK2: direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number.

The trfA gene of the broad-host-range plasmid RK2 is essential for initiation of plasmid replication. Two related TrfA proteins of 43 and 32 kilodaltons (kDa) are produced by independent translation initiation at two start codons within the trfA open reading frame. These proteins were o overproduced in Escherichia coli and partially purified. Rabbit antisera raised against the 32-kDa TrfA protein (TrfA-32) and cross-reacting with the 43-kDa protein (TrfA-43) were used in Western blotting (immunoblotting) assays to measure intracellular TrfA levels. In logarithmically growing E. coli HB101, RK2 produced 4.6 +/- 0.6 ng of TrfA-32 and 1.8 +/- 0.2 ng of TrfA-43 per unit of optical density at 600 nm (mean +/- standard deviation). On the basis of determinations of the number of cells per unit of optical density at 600 nm, this corresponds to about 220 molecules of TrfA-32 and 80 molecules of TrfA-43 per cell. Dot blot hybridizations showed that plasmid RK2 is present in about 15 copies per E. coli cell under these conditions. Using plasmid constructs that produce different levels of TrfA proteins, the effect of excess TrfA on RK2 replication was tested. A two- to threefold excess of total TrfA increased the copy number of RK2 by about 30%. Additional increases in TrfA protein concentration had no further effect on copy number, even at levels 170-fold above normal. An RK2 minimal origin plasmid showed a similar response to intracellular TrfA concentration. These results demonstrate that TrfA protein concentration is not strictly rate limiting for RK2 replication and that a mechanism that is independent of TrfA concentration functions to limit RK2 copy number in the presence of excess TrfA.     

Acquisition of resistance genes by the IncQ plasmid R1162 is limited by its high copy number and lack of a partitioning mechanism.

R1162 is a representative member of the broad-host-range IncQ group of multicopy plasmids. Lower-copy-number derivatives of R1162 were constructed in vitro and shown to be unstable, indicating that partitioning of plasmid copies at cell division is due to random distribution and not to an active partitioning mechanism. However, the normal copy number of R1162 reduces cell fitness during growth in broth and favors the emergence of unstable, lower-copy-number variants. As a result, plasmid-borne antibiotic resistance genes active at a low copy number eventually result in plasmid loss during periods of no selection. We argue that the maintenance of R1162 in a population requires a gene that is selected only at high levels. This reduces the potential for acquiring genes from other R factors and could explain the limited variety of antibiotic resistance genes contained by naturally occurring IncQ plasmids.     

The genetic effect of copy number variations on the risk of type 2 diabetes in a Korean population

Background

Unlike Caucasian populations, genetic factors contributing to the risk of type 2 diabetes mellitus (T2DM) are not well studied in Asian populations. In light of this, and the fact that copy number variation (CNV) is emerging as a new way to understand human genomic variation, the objective of this study was to identify type 2 diabetes–associated CNV in a Korean cohort.

Methodology/Principal Findings

Using the Illumina HumanHap300 BeadChip (317,503 markers), genome-wide genotyping was performed to obtain signal and allelic intensities from 275 patients with type 2 diabetes mellitus (T2DM) and 496 nondiabetic subjects (Total n = 771). To increase the sensitivity of CNV identification, we incorporated multiple factors using PennCNV, a program that is based on the hidden Markov model (HMM). To assess the genetic effect of CNV on T2DM, a multivariate logistic regression model controlling for age and gender was used. We identified a total of 7,478 CNVs (average of 9.7 CNVs per individual) and 2,554 CNV regions (CNVRs; 164 common CNVRs for frequency>1%) in this study. Although we failed to demonstrate robust associations between CNVs and the risk of T2DM, our results revealed a putative association between several CNVRs including chr15:45994758–45999227 (P = 8.6E-04, P^corr = 0.01) and the risk of T2DM. The identified CNVs in this study were validated using overlapping analysis with the Database of Genomic Variants (DGV; 71.7% overlap), and quantitative PCR (qPCR). The identified variations, which encompassed functional genes, were significantly enriched in the cellular part, in the membrane-bound organelle, in the development process, in cell communication, in signal transduction, and in biological regulation.

Conclusion/Significance

We expect that the methods and findings in this study will contribute in particular to genome studies of Asian populations.