Alteration of RNA I metabolism in a temperature-sensitive Escherichia coli rnpA mutant strain.

E. coli strain A49 carries the themosensitive mutation in the rnpA gene encoding the protein component of RNase P, a tRNA-processing enzyme. Two small RNAs were highly accumulated in the A49 carrying derivatives of ColE1-type plasmids, at nonpermissive temperature. Characterization of these RNAs showed that they were the processed or degraded products derived from RNA I, which is the negative controller of ColE1-type plasmid replication. These derivatives of RNA I only differ in size at the 5' ends. The data of their degradation and synthesis kinetics suggest that they are intermediates of RNA I metabolism.     

Discriminatory function of ribonuclease H in the selective initiation of plasmid DNA replication.

The initiation stage of ColE1-type plasmid replication was reconstituted with purified protein fractions from Escherichia coli. The reconstituted system included DNA polymerase I, DNA ligase, RNA polymerase, DNA gyrase, and a discriminating activity copurifying with RNAase H (but free of RNAase III). Initiation of DNA synthesis in the absence of RNAase H did not occur at the normal replication origin and was non-selective with respect to the plasmid template. In the presence of RNAase H the system was selective for ColE1-type plasmids and could not accept the DNA of non-amplifiable plasmids. Electron microscopic analysis of the reaction product formed under discriminatory conditions indicated that origin usage and directionally of ColE1, RSF1030, and CloDF13 replication were consistent with the normal replication pattern of these plasmids. It is proposed that the initiation of ColE1-type replication depends on the formation of an extensive secondary structure in the origin primer RNA that prevents its degradation by RNAase H.     

Growth-rate dependence reveals design principles of plasmid copy number control

Genetic circuits in bacteria are intimately coupled to the cellular growth rate as many parameters of gene expression are growth-rate dependent. Growth-rate dependence can be particularly pronounced for genes on plasmids; therefore the native regulatory systems of a plasmid such as its replication control system are characterized by growth-rate dependent parameters and regulator concentrations. This natural growth-rate dependent variation of regulator concentrations can be used for a quantitative analysis of the design of such regulatory systems. Here we analyze the growth-rate dependence of parameters of the copy number control system of ColE1-type plasmids in E. coli. This analysis allows us to infer the form of the control function and suggests that the Rom protein increases the sensitivity of control.     

A segregated model for plasmid content and product synthesis in unstable binary fission recombinant organisms

Plasmid propagation in populations of unstable, binary fission recombinant organisms has been studied using a segregated, population balance mathematical model. Segregated models have the advantage of direct incorporation of basic information on mechanisms and kinetics of plasmid replication and segregation at the single-cell level. The distribution of cellular plasmid content and specific rates of plasmid gene expression have been obtained for several single-cell models of plasmid replication, partition, and gene expression. Plasmid replication kinetics during cell growth significantly influence the plasmid content distribution. In the case of transient growth of plasmid-containing and plasmid-free cells in partially selective medium, the degree of selection required for stable maintenance of plasmid-containing cells has been determined. Guidelines are presented for applicability of simpler, nonsegregated models and for evaluation of the parameters in these models based on single-cell mechanisms and associated parameters.     

Characterization of a cryptic plasmid from Bacillus sphaericus strain LP1-G

A cryptic plasmid from Bacillus sphaericus strain LP1-G, designated as pLG, was sequenced and characterized. It was an 11,066bp circular molecule, with G+C content of 37%. The plasmid pLG was predicted to encode 23 putative ORFs, and ORF 21 shared the highest identity with Rep of pGI1 and pBMB9741, members of rolling-circle replication (RCR) pC194-family. Sequence analysis revealed a pC194-type double strand origin (dso) and a single strand origin (sso) like sequence located upstream and downstream of ORF 21, respectively. Moreover, Mung bean nuclease analysis and Southern hybridization confirmed the existence of single stranded DNA (ssDNA) intermediates, indicating that pLG belongs to the RCR pC194-family. Accumulation of multiple ssDNA intermediates in native strain LP1-G and decline of ssDNA and supercoiled DNA in rifampicin-treated strain implied that a special mechanism might be employed by pLG. Furthermore, the copy number of pLG in its original host was determined and about 58 copies of the plasmid exist in each cell. Subcloning and transformation experiments proved that the minimal replicon of pLG was within a 1.6-kb fragment, which was composed of rep gene and dso. These data are a good basis for the understanding of replication mechanisms and genetics of this B. sphaericus plasmid.     

Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli.

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.     

Cell cycle analysis of plasmid-containing Escherichia coli HB101 populations with flow cytometry

The relationship between cell mass and cell number dynamics for bacteria such as Escherichia coli depends on the cell cycle parameters C and D. Effects of plasmid copy number on these cell cycle parameters have been studied for Escherichia coli HB101 containing pMB1 plasmids propagated at different copy numbers ranging from 12 to 122. Determination of cell cycle and cell size parameters was accomplished using flow cytometry data on single-cell light scattering and DNA content frequency functions in conjunction with a mathematical model of cell population statistics. Two independent methods for estimating C and D intervals based on flow cytometry were developed and applied with essentially identical results. The presence of plasmids decreases the C and D periods, mean cell sizes, and initiation masses for chromosome replication by 14, 24, 38, and 18%, respectively, relative to corresponding values for plasmid-free host cells. Plasmid copy number has a negligible influence on these parameters, suggesting that host-plasmid inter actions which determine these properties are centered on the single plasmid selected for replication according to the random selection model established for ColE1-type plasmids.     

DnaG-dependent priming signals can substitute for the two essential DNA initiation signals in oriV of the broad host-range plasmid RSF1010.

Broad host-range plasmid RSF1010 contains in the oriV region two DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010), which are essential for plasmid replication. Each of ssiA and ssiB could be substituted functionally by either of the two G4-type (DnaG-dependent) priming signals, the oric of bacteriophage G4 and an ssi signal from plasmid pSY343 (an R1 plasmid derivative). Functions of the chimeric oriVs of RSF1010 thus constructed were dependent on the RSF1010-specific replication proteins, RepA, RepB' and RepC. When both of ssiA and ssiB were replaced by the G4-type ssi signals, functions of the chimeric oriVs were no longer dependent on RepB' (RSF1010-specific DNA primase). The replication activities of the chimeric oriVs of RSF1010 were not influenced markedly by the type of heterologous priming signals they contained. It is conceivable that DNA replication of RSF1010 does not need the priming mechanism for lagging strand synthesis and proceeds by the strand displacement mechanism.     

pIH01, a small cryptic plasmid from Leuconostoc citreum IH3

A small cryptic plasmid pIH01 from Leuconostoc citreum IH3 was characterized. This 1.8-kb sized plasmid contains single open reading frame that encodes a RepC class protein (342 amino acids) and a conserved pT181-type double strand origin, suggesting a rolling circle replication mode. This putative replicase protein shows the highest similarity to a replicase from pFR18 plasmid of Leuconostoc mesenteroides FR52 (64% identity), one of the pT181-type rolling circle plasmid family and contains a strictly conserved RepC-type active site sequence of pT181 family. A shuttle vector that was developed on the basis of this cryptic plasmid by insertion of both erythromycin resistance gene (ermC) from pE194 and Escherichia coli ColE1 origin was able to transform Leuconostoc strains, Lactobacillus plantarum, and Lactococcus lactis. Therefore, pIH01 derivative plasmids might be useful for the manipulation of Leuconostoc strains.     

Analysis of the physiological control of replication of ColE1-type plasmids

The physiology of ColE1-type plasmid replication in a growing host has been examined both theoretically, using computer simulation, and experimentally, by observing replication of the plasmid pBR322 after a nutritional shift-up from glycerol minimal medium (doubling time 71 min) to LB medium (doubling time 24 min). The theory was based on a negative control model and uses three rate equations: for the accumulation of cell mass, for the accumulation of the replication inhibitor, and for the rate of plasmid synthesis. The implications of the theory were explored by simulating the effects of changes in the expression of replication control genes. The nutritional shift-up experiment showed that plasmid replication was blocked immediately after the shift for about half a mass doubling time; after that time, replication rapidly increased until plasmid numbers per unit volume of culture parallelled the increase in culture mass. After the establishment of steady-state growth in the post-shift medium, the plasmid concentration (plasmids per cell mass) was reduced in comparison to pre-shift growth in the same proportion as the culture doubling time. The results showed that plasmid replication factors are under metabolic control and that the changes in the control of these factors compensate one another during steady-state growth, but not immediately after the medium shift.     

Isolation and sequence analysis of a small cryptic plasmid pRK10 from a corrosion inhibitor degrading strain Serratia marcescens ACE2

The nucleotide sequence of a smallest cryptic plasmid pRK10 of Serratia marcescens ACE2 was determined. When compared to the all other plasmids reported so far from S. marcescens in sizes of over 70 kb, pRK10 is only 4241 bp long with 53% G + C content and has five coding sequences representing a coding percentage of 65.41. This small plasmid consists of one Tdh gene, four mobilization genes, mobCABD, and an origin of replication homologous to those of ColE1-type plasmids. Analysis of the five open reading frames identified on the plasmid suggests the presence of genes involved in replication and mobilization containing sequences homologous to the bom region and mobCABD genes of ColE1 and Tdh from Acinetobacter baumannii str. AYE. Results also indicate that pRK10 does not encode any gene for antibiotic/heavy metal resistance. Copy number and incompatibility of the plasmid with plasmids of ColE1 origin of replication was determined and it is quite stable in its natural host as well as in Escherichia coli DH5α. This relatively small plasmid will be useful for construction of shuttle vectors to facilitate the genetic analysis.     

Replication of the multicopy mini-R1 plasmid Rsc11 in Escherichia coli K12: Possible nonrandom selection of participants

The kinetics of replication of the multicopy mini-R1 plasmid Rsc11, in the Escherichia coli K12 strain 1100 were determined by density-shift analysis. One generation after the shift, 7% of the plasmid population remained unreplicated, 86% had replicated once, and only 7% twice. Half a generation later the values were 4, 64, and 32%, respectively. This pattern is unlike that previously reported for the Rsc11 progenitor molecule, pKN102 (R1drd19-B2), and other multicopy plasmids, but closely resembles that of the chromosome and therefore suggests that Rsc11 copies are selected nonrandomly for replication. Another interpretation is discussed. Precursor incorporation into plasmid DNA is sharply curtailed upon transition to the stationary phase of growth. It is possible that Rsc11 replication is stringently controlled.     

Identification of a replicon from pTXL1, a small cryptic plasmid from Leuconostoc mesenteroides subsp. mesenteroides Y110, and development of a food-grade vector

A 2,665-bp cryptic plasmid, pTXL1, isolated from Leuconostoc mesenteroides subsp. mesenteroides Y110 was identified. This plasmid harbors a replicon localized on a 1,300-bp fragment. Two observations suggested that pTXL1 does not belong to rolling-circle replication (RCR)-type plasmids and most likely replicates via a theta mechanism. These hypotheses are supported by the observation that no detectable single-stranded intermediate was found for the replicon and that, unlike in RCR-type plasmids, the pTXL1 replicon sequence lacks an open reading frame encoding a replicase. The small-sized pTXL1 plasmid is stable and, according to its origin, can be considered in the "generally recognized as safe" category. Its ability to replicate in several lactic acid bacteria was exploited to develop a vector producing mesentericin Y105, a class II anti-Listeria bacteriocin. With this new vector, a recombinant industrial Leuconostoc cremoris strain able to produce mesentericin Y105 was constructed.     

Nucleotide sequences required for a ColE1-type plasmid to replicate in Escherichia coli cells with or without RNase H

To elucidate the replication mechanism of a ColE1-type plasmid in RNase H-deficient (rnh-) strains of Escherichia coli, we constructed plasmid derivatives that deleted the whole, or a part, of the 5'-AAAAA-3' sequence (positions -3 to +2) that acts as the origin of replication in vivo and in vitro in the presence of RNase H. The activity of plasmid replication in rnh+ cells was found to be reduced by alterations of the AAAAA sequence. The activity could be restored when the derivatives, retaining the upstream sequence down to -8, regained a sequence containing at least two A residues in the region from -3 to +2. By contrast, replication in rnh- cells was maintained at high levels even when the deletion included the AAAAA sequence and extended up to position -7. The activity in rnh- cells decreased as deletions proceeded to -8 and further up to -17, and was abolished completely by further upward deletions. We concluded that in rnh- cells the plasmid replicates by a mechanism that operates only when RNase H is inactive. This RNase H-sensitive replication in rnh- cells seems to require the RNA-DNA hybrid formation that is also required for RNase H-dependent replication in rnh+ cells. The hybrid formation probably contributes by unwinding a portion of DNA from which replication can be initiated.