Plasmid Effects on Escherichia coli Metabolism

ABSTRACT:?

The idea that plasmids replicate within hosts at the expense of cell metabolic energy and preformed cellular blocks depicts plasmids as a kind of molecular parasites that, even when they may eventually provide plasmid-carrying strains with growth advantages over plasmid-free strains, doom hosts to bear an unavoidable metabolic burden. Due to the consistency with experimental data, this idea was rapidly adopted and used as a basis of different hypotheses to explain plasmid-host interactions. In this article we critically discuss current ideas about plasmid effects on host metabolism, and present evidence suggesting that the complex interaction between plasmids and hosts is related to the alteration of the cellular regulatory status.     

Possible benefits of kalilo plasmids to their Neurospora hosts

Neurospora mitochondrial plasmids are ubiquitous in natural populations, yet many of them are lethal to their host strains or seem to impose a molecular genetic load. Five pairs of strains of Neurospora tetrasperma and N. crassa with and without kalilo-like plasmids were tested under a variety of situations. The purpose was to find possible beneficial effects of plasmids that might offset their disadvantages. We found that, in all cases tested, plasmids conferred an advantage to growth at temperatures close to the top of the range for this fungus. Also, the plasmids improved fertility, as measured by perithecial production. Negative results were obtained for heavy metal resistance and ascospore germination. The results generate the hypothesis that plasmids may have adaptive significance to their hosts.     

Gene and cell survival: lessons from prokaryotic plasmid R1

Plasmids are units of extrachromosomal genetic inheritance found in all kingdoms of life. They replicate autonomously and undergo stable propagation in their hosts. Despite their small size, plasmid replication and gene expression constitute a metabolic burden that compromises their stable maintenance in host cells. This pressure has driven the evolution of strategies to increase plasmid stability--a process accelerated by the ability of plasmids to transfer horizontally between cells and to exchange genetic material with their host and other resident episomal DNAs. These abilities drive the adaptability and diversity of plasmids and their host cells. Indeed, survival functions found in plasmids have chromosomal homologues that have an essential role in cellular responses to stress. An analysis of these functions in the prokaryotic plasmid R1, and of their intricate interrelationships, reveals remarkable overall similarities with other gene- and cell-survival strategies found within and beyond the prokaryotic world.     

A survey of Col plasmids in natural isolates of Escherichia coli and an investigation into the stability of Col-plasmid lineages.

A survey of colicins in the ECOR reference collection of Escherichia coli is presented. Twenty-five of the 72 ECOR strains exhibited a phenotype consistent with colicin production and E. coli isolated from human hosts were more likely to be colicinogenic than those from animal hosts. Multiple representatives of two Col plasmids, low-molecular-mass ColE1 plasmids and high-molecular-mass, conjugative ColIa plasmids were isolated from the ECOR collection and were examined with a combination of restriction fragment and Southern analysis. These data suggested that ColE1 plasmids comprise a stable (cohesive) plasmid lineage, while ColIa plasmids represent a family of distinct plasmid lineages united by the presence of the colicin Ia operon.     

Physical evidence for plasmids in autotrophic,especially hydrogen-oxidizing bacteria

Agarose gel electrophoresis of crude lysates from 23 species of autotrophic bacteria revealed plasmids of various sizes in 12 species. The plasmid pattern varied considerably. While the majority of the plasmid-bearing species harbored one or two plasmids, one species, Alcaligenes latus, exhibited more than six ccc-DNA bands. With one exception the molecular masses of the plasmids were 50×10⁶ or higher. In Achromobacter carboxydus, Alcaligenes latus, Derxia gummosa and three strains of Paracoccus denitrificans large plasmids of molecular masses higher than 300×10⁶ were resolved. The examination of Thiobacillus A2 resulted in the discovery of two plasmids while Pseudomonas oxalaticus was apparently free of resident plasmid DNA. So far these plasmids can only be characterized as cryptic. Future studies may allow to correlate them with specific metabolic activities of their hosts such as the ability to grow on carbon monoxide or thiosulfate, to fix molecular nitrogen and to form soluble NAD-reducing and/or membrane-bound hydrogenases.     

Transfer of Plasmids to an Antibiotic-Sensitive Mutant of Zymomonas mobilis

Wild-type strains of Zymomonas mobilis exhibit multiple antibiotic resistance and thus restrict the use of many broad-host-range plasmids in them as cloning vehicles. Antibiotic-sensitive mutants of Z. mobilis were isolated and used as hosts for the conjugal transfer of broad-host-range plasmids from Escherichia coli. Such antibiotic-sensitive strains can facilitate the application of broad-host-range plasmids to the study of Z. mobilis.     

Naturally Occurring Penicillinase Plasmids in Staphylococcus aureus

A series of plasmids harbored by naturally occurring penicillin-resistant strains of Staphylococcus aureus were surveyed with a view toward exploring the variability in plasmid-linked marker patterns. Plasmids were transduced from their natural hosts to either of two plasmid-negative laboratory strains by selection for cadmium resistance, and the transductants were tested for all other markers previously found to be plasmid-linked. All of the strains that were able to serve as genetic donors to one of the two stock strains could donate cadmium and lead resistance as linked, plasmid-borne markers. Among the other plasmid markers, a wide variety of patterns was found, including four plasmids that did not carry the penicillinase determinant. Each of the 26 plasmids studied, including the latter 4, was found to belong to one of the two incompatibility sets of penicillinase plasmids previously identified. With the exception of the penicillinase-negative plasmids, which were found in both sets, all the plasmids of incompatibility set I directed the production of penicillinase type A; those belonging to set II directed either type A or type C. Those of set II without exception increased the sensitivity of their host strains to bismuth ion; those of set I carried determinants of bismuth resistance or did not affect the sensitivity of their host to this ion. No other perfect correlations between markers were encountered; in particular, there was no correlation between penicillinase serotype and the excretion of the enzyme. This finding allows the prediction that there is, in addition to all of the markers thus far identified, a plasmid-linked determinant of penicillinase excretion.     

Broad diversity of conjugative plasmids in integron-carrying bacteria from wastewater environments

In this study we assessed the occurrence, diversity and conjugative potential of plasmids in integron-carrying Aeromonas and Enterobacteriaceae from wastewaters. Sixty-six strains were included as donors in mating assays using rifampicin-resistant Escherichia coli and Pseudomonas putida recipient strains. The diversity of plasmids from donors and transconjugants (resistant to tetracycline or streptomycin) was evaluated by restriction analysis and replicon typing targeting 19 incompatibility groups. Restriction patterns revealed a diverse plasmid pool present in these strains. Plasmids were assigned to FrepB (Aeromonas salmonicida, Aeromonas veronii, Aeromonas sp., E.?coli, Enterobacter sp.), FIC (A.?salmonicida, Aeromonas sp.), FIA (Shigella sp.), I1 (A.?veronii, Aeromonas sp., E.?coli), HI1 (E.?coli) and U (Aeromonas media) replicons. Nevertheless, 50% of the plasmids could not be assigned to any replicon type. Among integron-positive transconjugants, FrepB, I1 and HI1 replicons were detected. Results showed that wastewaters enclose a rich plasmid pool associated with integron-carrying bacteria, capable of conjugating to different bacterial hosts. Moreover, replicons detected in this study in Aeromonas strains expand our current knowledge of plasmid diversity in this genus.     

Homology among nearly all plasmids infecting three Bacillus species.

We have surveyed naturally occurring plasmids in strains of Bacillus subtilis and the closely related species B. mojavensis and B. licheniformis. Previous studies have failed to find host-benefitting functions for plasmids of these species, suggesting that these plasmids are nonmutualistic. Only one type of plasmid was found in each plasmid-bearing strain, suggesting that most of the plasmids infecting these Bacillus species are in the same incompatibility group. A sample of 18 plasmids from these species ranged in size from 6.9 to 16 kb, with all but 6 plasmids falling into three size groups. These groups differed in the sizes of their host ranges and geographical ranges. All but 1 of the 18 plasmids from these three host species are homologous with one another. The cryptic plasmids from these three species are far less diverse than are plasmids (from other species) that are known to benefit their bacterial hosts. The low-level diversity among these cryptic plasmids is consistent with the hypothesis that host-benefitting adaptations play an important role in fostering the coexistence of plasmid populations, but other explanations for the low-level plasmid diversity are possible. Comparison of the phylogenies of the plasmids with those of their hosts suggests that Bacillus plasmids are horizontally transferred in nature at a low rate similar to that found for the colicin plasmids of Escherichia coli.     

<Emphasis Type="Italic">Bacillus megaterium</Emphasis>—from simple soil bacterium to industrial protein production host

Bacillus megaterium has been industrially employed for more than 50 years, as it possesses some very useful and unusual enzymes and a high capacity for the production of exoenzymes. It is also a desirable cloning host for the production of intact proteins, as it does not possess external alkaline proteases and can stably maintain a variety of plasmid vectors. Genetic tools for this species include transducing phages and several hundred mutants covering the processes of biosynthesis, catabolism, division, sporulation, germination, antibiotic resistance, and recombination. The seven plasmids of B. megaterium strain QM B1551 contain several unusual metabolic genes that may be useful in bioremediation. Recently, several recombinant shuttle vectors carrying different strong inducible promoters and various combinations of affinity tags for simple protein purification have been constructed. Leader sequences-mediated export of affinity-tagged proteins into the growth medium was made possible. These plasmids are commercially available. For a broader application of B. megaterium in industry, sporulation and protease-deficient as well as UV-sensitive mutants were constructed. The genome sequence of two different strains, plasmidless DSM319 and QM B1551 carrying seven natural plasmids, is now available. These sequences allow for a systems biotechnology optimization of the production host B. megaterium. Altogether, a “toolbox” of hundreds of genetically characterized strains, genetic methods, vectors, hosts, and genomic sequences make B. megaterium an ideal organism for industrial, environmental, and experimental applications.     

Introduction of DNA into Actinomycetes by bacterial conjugation from E. coli--an evaluation of various transfer systems

Gene transfer is a basic requirement for optimizing bioactive natural substances produced by an increasing number of industrially used microorganisms. We have analyzed quantitatively horizontal gene transfer from Escherichia coli to Actinomycetes. The efficiencies of DNA transfer of four different systems were compared that consist of conjugative and mobilizable plasmids with a broad-host range. Three novel binary vector set-ups were constructed based on: (i) the IncQ group of mobilizable plasmids (RSF1010), (ii) IncQ-like pTF-FC2 and (iii) pSB102 that belongs to a new class of broad-host-range plasmids. The established system based on the IncPalpha group of conjugative plasmids served as the reference. For all plasmids constructed, we confirmed the functional integrity of the selected transfer machineries by intrageneric matings between E. coli strains. We demonstrate that the transfer systems introduced in this study are efficient in mediating gene transfer from E. coli to Actinomycetes and are possible alternatives for gene transfer into Actinomycetes for which the IncPalpha-based transfer system is not applicable. The use of plasmids that integrate into the recipients' chromosomes compared to that of plasmids replicating autonomously is shown to allow the access to a wider range of hosts.     

Host-vector systems for the thermotolerant methanol-utilizing bacteriaMethylophilus spp. KISRI-Strains

Fourteen recombinant plasmids were constructed by inserting fragments of pSAS, a naturally occurring plasmid ofMethylophilus spp. KISRI-5, into the multiple cloning sites of pUC19. Six recombinants and three knownEscherichia coli plasmids were used to transform three thermotolerant methylotrophic KISRI strains by use of an optimized protocol of electroporation. Analysis of transformants for plasmid DNA showed that all plasmids were stable in the methylotrophic hosts. These studies offer opportunities to developMethylophilus spp. as host-vector systems.     

Genome organization and localization of the pufLM genes of the photosynthesis reaction center in phylogenetically diverse marine Alphaproteobacteria

Genome organization, plasmid content and localization of the pufLM genes of the photosynthesis reaction center were studied by pulsed-field gel electrophoresis (PFGE) in marine phototrophic Alphaproteobacteria. Both anaerobic phototrophs (Rhodobacter veldkampii and Rhodobacter sphaeroides) and strictly aerobic anoxygenic phototrophs from the Roseobacter-Sulfitobacter-Silicibacter clade (Roseivivax halodurans, Roseobacter litoralis, Staleya guttiformis, Roseovarius tolerans, and five new strains isolated from dinoflagellate cultures) were investigated. The complete genome size was estimated for R. litoralis DSM6996(T) to be 4,704 kb, including three linear plasmids. All strains contained extrachromosomal elements of various conformations (linear or circular) and lengths (between 4.35 and 368 kb). In strain DFL-12, a member of a putative new genus isolated from a culture of the toxic dinoflagellate Prorocentrum lima, seven linear plasmids were found, together comprising 860 kb of genetic information. Hybridization with probes against the pufLM genes of the photosynthesis gene cluster after Southern transfer of the genomic DNAs showed these genes to be located on a linear plasmid of 91 kb in R. litoralis and on a linear plasmid of 120 kb in S. guttiformis, theoretically allowing their horizontal transfer. In all other strains, the pufLM genes were detected on the bacterial chromosome. The large number and significant size of the linear plasmids found especially in isolates from dinoflagellates might account for the metabolic versatility and presumed symbiotic association with eukaryotic hosts in these bacteria.     

Rapid evolution of novel traits in microorganisms

The use of natural microorganisms in biotransformations is frequently constrained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selection. This process of directed evolution can be accelerated in mutator strains, which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mutation rate of the selected organisms immediately after selection to prevent the accumulation of undesirable spontaneous mutations. To enable this process, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 20- to 4,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approach, we were able to increase the tolerance of three bacterial strains to dimethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo mutagenesis.     

Stable propagation of ‘selfish’ genetic elements

Extrachromosomal or chromosomally integrated genetic elements are common among prokaryotic and eukaryotic cells. These elements exhibit a variety of ‘selfish’ strategies to ensure their replication and propagation during the growth of their host cells. To establish long-term persistence, they have to moderate the degree of selfishness so as not to imperil the fitness of their hosts. Earlier genetic and biochemical studies together with more recent cell biological investigations have revealed details of the partitioning mechanisms employed by low copy bacterial plasmids. At least some bacterial chromosomes also appear to rely on similar mechanisms for their own segregation. The 2 μm plasmid ofSaccharomyces cerevisiae and related yeast plasmids provide models for optimized eukaryotic selfish DNA elements. Selfish DNA elements exploit the genetic endowments of their hosts without imposing an undue metabolic burden on them. The partitioning systems of these plasmids appear to make use of a molecular trick by which the plasmids feed into the segregation pathway established for the host chromosomes.     

Rapid Evolution of Novel Traits in Microorganisms

The use of natural microorganisms in biotransformations is frequently constrained by their limited tolerance to the high concentrations of metabolites and solvents required for effective industrial production. In many cases, more robust strains have to be generated by random mutagenesis and selection. This process of directed evolution can be accelerated in mutator strains, which carry defects in one or more of their DNA repair genes. However, in order to use mutator strains, it is essential to restore the normal low mutation rate of the selected organisms immediately after selection to prevent the accumulation of undesirable spontaneous mutations. To enable this process, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 20- to 4,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. By using this approach, we were able to increase the tolerance of three bacterial strains to dimethylformamide by 10 to 20 g/liter during only two subsequent transfers. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids. Our results demonstrate a new and efficient method for rapid strain improvement based on in vivo mutagenesis.     

Transfer of plasmids fromEscherichia coli andPseudomonas aeruginosa to methylotrophic bacteria and their detection in the new hosts

Several plasmids of incompatibility group P were transferred fromEscherichia coli andPseudomonas aeruginosa strains toMethylophilus methylotrophus and two other methylotrophs to test their recipient ability. The presence of plasmids in transconjugants was confirmed by electrophoretic analysis. Optimal conditions for detection of plasmid DNA in the strains tested based on alkaline lysis of cells at elevated temperature were established. Special behaviour of plasmids carrying the Mu phage in methylotrophic hosts is described.     

Rhizobial plasmids that cause impaired symbiotic nitrogen fixation and enhanced host invasion

The genetic rules that dictate legume-rhizobium compatibility have been investigated for decades, but the causes of incompatibility occurring at late stages of the nodulation process are not well understood. An evaluation of naturally diverse legume (genus Medicago) and rhizobium (genus Sinorhizobium) isolates has revealed numerous instances in which Sinorhizobium strains induce and occupy nodules that are only minimally beneficial to certain Medicago hosts. Using these ineffective strain-host pairs, we identified gain-of-compatibility (GOC) rhizobial variants. We show that GOC variants arise by loss of specific large accessory plasmids, which we call HR plasmids due to their effect on symbiotic host range. Transfer of HR plasmids to a symbiotically effective rhizobium strain can convert it to incompatibility, indicating that HR plasmids can act autonomously in diverse strain backgrounds. We provide evidence that HR plasmids may encode machinery for their horizontal transfer. On hosts in which HR plasmids impair N fixation, the plasmids also enhance competitiveness for nodule occupancy, showing that naturally occurring, transferrable accessory genes can convert beneficial rhizobia to a more exploitative lifestyle. This observation raises important questions about agricultural management, the ecological stability of mutualisms, and the genetic factors that distinguish beneficial symbionts from parasites.     

The conjugational transfer of plasmids to Legionella strains

In experiments on conjugation the transfer of a number of R-plasmids having a wide range of hosts, such as plasmids RP1, R68.45, RP4, N3, RK2, S-a, those having a narrow range of hosts, such as plasmid R64, to strains of different Legionella species was shown. The frequency of transfer varied from 3.1 X 10(-3) to 9.4 X 10(-7). The fact that the conjugation transfer was confirmed by the reverse transfer of plasmids from Legionella transconjugates to Escherichia coli strain K12, as well as by the detection of the DNA of the transferred plasmid by means of electrophoresis in agar gel. Plasmid RP1 showed different behavior in transconjugates of various Legionella species after several passages in a medium free of antibiotics. In the Legionella strain under study the unstable preservation of plasmid R64 was observed.