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Evolution of a bacteria/plasmid association.

Associations between bacteria and their accessory elements (viruses, plasmids and transposons) range from antagonistic to mutualistic. A number of previous studies have demonstrated that plasmid carriage reduces bacterial fitness in the absence of selection for specific functions such as antibiotic resistance. Many studies have demonstrated increased fitness of evolving microbial populations in laboratory environments, but we are aware of only one study in which fitness gains were partitioned between a plasmid and its host. Here, we examine the evolution of an association between a plasmid and its bacterial host. Carriage of the non-conjugative plasmid pACYC184 initially reduced the fitness of Escherichia coli B in the absence of antibiotic. We then cultured plasmid-bearing bacteria for 500 generations in the presence of antibiotic. The fitness of each combination of host and plasmid, with and without the culture history, was determined by competing it against a baseline strain. The results indicate adaptation by the host genome, but no plasmid adaptation. We also competed the evolved host, transformed with the baseline plasmid, against its isogenic plasmid-free counterpart. The plasmid now increased the fitness of its host.

Adaptation, Physiological↗

Monitoring genome evolution ex vivo: reversible chromosomal integration of a 106 kb plasmid at two tRNA(Lys) gene loci in sequential Pseudomonas aeruginosa airway isolates.

The genome rearrangements in sequential Pseudomonas aeruginosa clone K isolates from the airways of a patient with cystic fibrosis were determined by an integrated approach of mapping, sequencing and bioinformatics. Restriction mapping uncovered an 8.9 kb deletion of PAO sequence between phnAB and oprL in clone K, and two 106 kb insertions either adjacent to this deletion or several hundred kilobases away, close to the pilA locus. These 106 kb blocks of extra DNA also co-existed as the circular plasmid pKLK106 in several clone K isolates and were found to be closely related to plasmid pKLC102 in P. aeruginosa clone C isolates. The breakpoints of the deletion in clone K and the attB-attP sequences for the reversible integration of the plasmid in clones C and K were located within the 3' end of the lysine tRNA structural genes (att site). pKLK106 sequentially recombined with either of the two tRNA(Lys) genes in clone K isolates. The att site of the pilA hypervariable region has been utilized by clone C to target its plasmid pKLC102 into the chromosome; the att site of the phnAB-oprL region has been employed by strain PAO to incorporate a DNA block encoding pyocin, transposases and IS elements. The use of typical phage attachment sites by conjugative genetic elements could be one of the major mechanisms used by P. aeruginosa to generate the mosaic genome structure of blocks of species-, clone- and strain-specific DNA. The example described here demonstrates the potential impact of systematic genome analysis of sequential isolates from the same habitat on our understanding of the evolution of microbial genomes.

Base Sequence↗

Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3.

Colicin E8-J and its immunity protein were characterized with regard to their activities and gene structures. Colicin E8 is a complex of proteins A and B; protein A (the naked E8) exhibits an apparently nonspecific DNase activity that is inhibited by protein B (the immunity protein), as in the case of colicin E2. The nucleotide sequence of the downstream half of the colicin operon of ColE8-J was determined to be highly homologous to that of ColE2-P9, with the exception of the hot spot region of the 3'-terminal segment of the colicin gene and the adjacent immunity gene. The immE2-like gene of ColE3-CA38 was, as assumed previously, extensively homologous to the immE8 gene of ColE8-J, and thus, ColE8-J was shown to be situated between ColE2-P9 and ColE3-CA38 in the evolution of the E-group Col plasmids.

Amino Acid Sequence↗

Acquisition of stcE, a C1 esterase inhibitor-specific metalloprotease, during the evolution of Escherichia coli O157:H7.

Escherichia coli O157:H7 is a source of foodborne illness, causing diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. E. coli O157:H7 secretes, via the etp type II secretion system, a metalloprotease, StcE, that specifically cleaves the serpin C1 esterase inhibitor. We determined by hybridization techniques the prevalence of stcE and etpD, a type II secretion gene, among diarrheagenic E. coli strains. stcE and etpD are ubiquitous among the O157:H7 serotype and are found in some enteropathogenic E. coli O55:H7 strains but are absent from other diarrheagenic E. coli. stcE was acquired on a large plasmid early in the evolution of E. coli O157:H7, before the inheritance of the Shiga toxin prophage. Other plasmidborne virulence factors, such as ehxA, katP, and espP, were acquired later by the enterohemorrhagic E. coli 1 complex in a stepwise manner. These data refine the sequential model of E. coli O157:H7 evolution proposed elsewhere.

Complement C1 Inactivator Proteins↗

Natural instability of Agrobacterium vitis Ti plasmid due to unusual duplication of a 2.3-kb DNA fragment.

The octopine/cucumopine (o/c) Ti plasmids of Agrobacterium vitis carry two T regions, TA and TB. The TA region resembles the octopine TL region. The TB region contains the auxin synthesis genes TB-iaaM and TB-iaaH and the cucumopine synthesis gene cus. Within the group of o/c isolates, strains 2608 and 2641 are closely related. However, 2641 lacks the TB region. The restriction maps of pTi2608 and pTi2641 were established and showed that the TB deletion resulted from intramolecular recombination between two directly repeated sequences separated by 66 kb in pTi2608. The 2,294-bp repeated sequence lacks inverted repeats and does not duplicate its target site, indicating that it is not a classical bacterial insertion sequence (IS element). It was therefore called an RSAv element (repeated sequence of A. vitis). The RSAv element carries two open reading frames: ORF234 is homologous to the traR gene of the A. tumefaciens nopaline Ti plasmid pTiC58; ORF488 is homologous to the sucrose phosphorylase gene of Leuconostoc mesenteroides and the glucosyl transferase A gene of Streptococcus mutans. The RSAv repeat starts precisely at the start codon of ORF488 and ends two base pairs 3' of the stop codon of ORF234. The structural organization of the RSAv element suggests that the amplification event did not result from a random amplification process. A study of the distribution of the two RSAv copies (RSAv-1 and RSAv-2) in pTi2608 and other o/c isolates indicates that the ancestor o/c Ti plasmid contained only RSAv-1 and that this sequence was duplicated at one point during the divergent evolution of the o/c Ti plasmids.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Distribution of restriction enzyme recognition sequences on broad host range plasmid RP4: molecular and evolutionary implications.

IncP alpha plasmids, exemplified by RP4, are remarkable for their broad host range. They contain strikingly few cleavage sites for many commonly used type II restriction enzymes but an overabundance of sites for certain enzymes that target G + C-rich sequences. To identify factors responsible for these distributions, the recently compiled nucleotide sequence of RP4 was analysed to determine the frequency of tetra- and hexanucleotide motifs in the 49 kb plasmid backbone. This is defined as the sectors encoding basic plasmid functions. The overabundant restriction targets in RP4 are concentrated in the backbone and contain overlapping copies of CGGC/GCCG, identified as the most abundant tetranucleotide motif in the plasmid. Motif frequencies in the RP4 backbone are shown to be similar to those in Pseudomonas aeruginosa, a natural host of RP4, with the notable exception that a number of 6-bp palindromes are underrepresented in the plasmid. It is proposed that 6-bp palindromes were counterselected as type II restriction enzyme recognition sequences. Conjugative transfer of RP4 and R751 (IncP beta) is unusually sensitive to restriction compared to enterobacterial plasmids of the IncFII and IncI1 groups, implying that IncP plasmids experienced particularly strong selection for loss of restriction targets. Pseudomonas spp. of rRNA homology group I specify many type II restriction enzymes that target 6-bp palindromes and are candidates for the evolutionary hosts of IncP alpha plasmids.

Base Composition↗

Variations in R-plasmid DNA concentrations of Escherichia coli during starvation in sewage and brackish waters.

Cell culturability and plasmid stability in Escherichia coli containing plasmids RP1, R388 and pUB824 were studied in raw and treated wastewater, and in brackish water. The E. coli strain survived well in the three samples of water employed. Moreover, the three plasmids were maintained under all conditions studied. Interestingly, plasmid DNA concentration of individual plasmids followed the same evolution as the culturable bacteria in the corresponding selective medium when the bacteria grew in raw or treated wastewater. In contrast, in brackish water, the stress due to the oligotrophic and salinity conditions of the medium produced an initial paradoxical increase in plasmid DNA concentration, followed by a decrease in the number of culturable bacteria in the corresponding selective medium. Maintenance of RP1 (56 kbp) and R388 (33 kbp) was markedly influenced by nutritive conditions, which caused a segregation of the plasmids from cells. The results of the present study suggest that variations in plasmid DNA concentrations in an aquatic environment depend on the quality of the water and also on the molecular weight of the plasmid considered.

DNA, Bacterial↗

Evolution of the replication regions of IncI alpha and IncFII plasmids by exchanging their replication control systems.

The basic replicons of bacterial plasmids consist of two sets of genetic systems, the replication-structural system and the replication control system. Comparison of nucleotide sequences suggested that the basic replicons of plasmids P307 (IncFI) and pMU2200 (IncZ) were generated by reciprocal recombination between ancestors of R100 (IncFII) and ColIb-P9 (IncI alpha), or vice versa. The plasmids of each pair, P307/pMU2200 and R100/ColIb-P9, are structurally unrelated to each other. Based on this information, we constructed in vitro and analyzed P307-like chimeric replicons from ColIb-P9 and R100. When the replication-structural region of ColIb-P9 was combined with the whole replication control region of R100, the resultant replicon replicated stably as R100 did. These results revealed that the basic replicons of the plasmids diverged by exchanging their replication control systems. Thus, we propose that the replication control systems of plasmids, in some cases, evolved independently of their structural systems, although these two systems work together to maintain the replication functions. We also showed that the reciprocal recombination was specified by the unique secondary structures of RNA involved in the control of expression of the genes encoding the replication initiator proteins.

Amino Acid Sequence↗

Characterization of plasmids in aminocyclitol-resistant Staphylococcus aureus: electron microscopic and restriction endonuclease analysis.

Plasmid species isolated from aminocyclitol-resistant Staphylococcus aureus have been analyzed by restriction endonuclease digestion and electron microscopy. These plasmids can be divided into two interrelated groups; intergroup variability is due to the gain or loss of defined DNA sequences. Plasmids pSJ1 and pSJ24 are related to staphylococcal penicillinase plasmid pI524 which was first described over 20 years ago. Both pSJ1 and pSJ24 differ from pI524 by the acquisition of 8 and 4 kbp, respectively, and encode additional resistance to the antibiotics erythromycin and kanamycin. The gain of these resistance determinants suggests that the evolution of staphylococcal resistance plasmids parallels that observed for plasmids of gram-negative bacteria and has serious implications for the spread of antibiotic resistance among the staphylococci.

Aminoglycosides↗

A hitchhiker's guide to survival finally makes CENs.

Most strains of the yeast Saccharomyces cerevisiae contain many copies of a 2-microm plasmid, a selfish autonomously replicating DNA that relies on two different mechanisms to ensure its survival. One of these mechanisms involves the high fidelity segregation of the plasmids to daughter cells during cell division, a property that is starkly reminiscent of centromeres. A new study reported in this issue (see Hajra et al. on p. 779) demonstrates that this high fidelity is achieved by the 2-microm plasmid, effectively recruiting the centromeric histone Cse4 from its host yeast cell to forge its own centromere and finally revealing how the 2-microm plasmid has survived in budding yeasts over millions of years.

Cell Division↗

Bacterial fitness and plasmid loss: the importance of culture conditions and plasmid size.

Several pBluescript-derived plasmids of various sizes were constructed to study the effects of multicopy plasmid size on bacterial fitness and plasmid loss. Transformed and untransformed bacterial clones were grown in media with or without ampicillin. Bacterial fitness (measured by growth rate), plasmid presence or absence, and plasmid copy number were assessed during successive subculturings. In selective media (minimal medium or Luria Broth plus ampicillin), the clone transformed with the largest plasmid (pBluescript with a 9000-bp insert) had a significantly longer lag phase than all other clones. In nonselective media the rate of plasmid loss during successive subculturings was greatest in the clone with the largest insert. The clone with the largest insert displayed a lower plasmid copy number than clones with a small insert or no insert at all. Plasmid loss in the form of segregational instability and plasmid copy number reduction in nonselective environments are important to the understanding of the evolution of the bacteria-plasmid associations and the appreciation of the potential for altering the genetic properties of a clone maintained or subcultured on a standard medium.

Ampicillin↗

Presence of a group II intron in a multiresistant Serratia marcescens strain that harbors three integrons and a novel gene fusion.

We analyzed the role of integrons in the dissemination of antibiotic resistance in a recent multiresistant clinical isolate, Serratia marcescens SCH88050909 (SCH909). This isolate harbors three integrons, all on a 60-kb conjugative plasmid. By PCR, hybridization, and sequencing analyses, we found that integron 1 has the dfrA1 and ant(3")-Ia cassettes. The first cassette in integron 2 contains the ant(2")-Ia gene, separated from its attC site (59-base element) by a 1,971-bp insert containing a group II intron; this intron codes for a putative maturase-reverse transcriptase on the complementary strand and is the first such intron to be found associated with an integron. The attC site is followed by a novel aminoglycoside resistance gene, ant(3")-Ii-aac(6')-IId, which has been characterized for its bifunctional ANT(3")-I and AAC(6')-II activities. DNA sequence analysis of this fused cassette suggests that insertion and excision due to the integrase activity could have an important role in the evolution of aminoglycoside resistance genes. This gene is followed by an unknown open reading frame with a typical attC site and a partial cassette composed of the beginning of the bla(OXA-10) cassette interrupted by IS1. The sequence downstream of IS1 revealed that the bla(OXA-10) cassette is incomplete and that the 3' conserved segment of this integron is absent. Integron 3 is in a Tn1696-like transposon with the aac(3)-Ia cassette followed by three unknown cassettes and ant(3")-Ia. The presence of the group II intron and the relationship of group II introns in eubacteria with mobile elements suggest a possible role of this element in events such as cassette formation and/or plasmid evolution.

Amino Acid Sequence↗

What does "plasmid biology" currently mean? Summary of the Plasmid Biology 2004 Meeting.

Almost 200 scientists from America, Europe, Asia, Australia, and Africa participated in the Plasmid Biology 2004 meeting, which was organized between 15th and 21st September 2004 in Kanoni (Corfu island), Greece. Various aspects of biology of plasmids and other mobile genetic elements were discussed during the meeting, including problems of replication, transfer, stable inheritance, and evolution. Medical and veterinary aspects of plasmids were highlighted as well as other applications of these replicons. It appears that plasmids and other mobile genetic elements are still excellent models in studies of basic biological problems at the molecular level, and their role in medicine and genetic engineering can be enormous. Moreover, studies on ecology of plasmids provide extremely important data that can be used in environment protection as well as in biotechnology. Understanding the importance of studies on plasmids and other mobile genetic elements, participants of the meeting decided to establish the International Society for Plasmid Biology.

Biological Evolution↗

Discovery and molecular characterization of a plasmid localized in Buchnera sp. bacterial endosymbiont of the aphid Rhopalosiphum padi.

We have identified and completely sequenced a novel plasmid isolated from the aphid Rhopalosiphum padi. Evidence which suggests that the plasmid occurs localized within the bacterial endosymbionts is presented. The plasmid contains the four genes that constitute the entire leucine operon. This fact makes it really unique since most plasmids are dispensable and lack genes that encode essential anabolic functions. Four more phloem-feeding aphid species also seem to contain homologous plasmids. Although further work is necessary, we hypothesize that this plasmid has appeared during the evolution of the symbiotic association between the aphid and the bacterial endosymbiont. The fact that this plasmid contains the entire leucine operon can be related to physiological evidence showing that the aphid host's diet of plant phloem is deficient in essential amino acids.

Amino Acid Sequence↗

Microbial degradation of synthetic recalcitrant compounds.

Synthetic compounds, particularly highly chlorinated aromatics, comprise the bulk of the environmental pollutants that somehow must be removed from the environment. Microbial degradation of such compounds is usually very slow, making them highly persistent in nature. Some synthetic compounds, with a lower degree of chlorination are, however, biodegradable; biochemical, genetic, and molecular studies demonstrate the evolution of new plasmid-encoded enzymatic activities specifically designed for the chlorinated substrates. Nucleotide sequences of many of the genes encoding such enzymatic activities demonstrate considerable homology either near the active sites or throughout the molecules with the chromosomal genes encoding enzymes catalyzing analogous reactions. In some cases, unique repeated sequences, reminiscent of prokaryotic insertion sequence elements, are present at or near the newly evolved genes. This suggests gene duplication and divergence as well as recombinational events mediated by transposable type elements as key ingredients in the evolution of new degradative functions. An understanding of such evolutionary processes is an essential feature for the development of genetically-improved bacteria capable of utilizing and thereby removing highly chlorinated environmental pollutants from our environment.

Journal Article↗

Agrobacterium vitis nopaline Ti plasmid pTiAB4: relationship to other Ti plasmids and T-DNA structure.

The Ti plasmid of the Agrobacterium vitis nopaline-type strain AB4 was subcloned and mapped. Several regions of the 157 kb Ti plasmid are similar or identical to parts of the A. vitis octopine/cucumopine (o/c)-type Ti plasmids, and other regions are homologous to the nopaline-type Ti plasmid pTiC58. The T-DNA of pTiAB4 is a chimaeric structure of recent origin: the left part is 99.2% homologous to the left part of the TA-DNA of the o/c-type Ti plasmids, while the right part is 97.1% homologous to the right part of an unusual nopaline T-DNA recently identified in strain 82.139, a biotype II strain from wild cherry. The 3' noncoding regions of the ipt genes from pTiAB4 and pTi82.139 are different from those of other ipt genes and contain a 62 bp fragment derived from the coding sequence of an ipt gene of unknown origin. A comparison of different ipt gene sequences indicates that the corresponding 62 bp sequence within the coding region of the AB4 ipt gene has been modified during the course of its evolution, apparently by sequence transfer from the 62 bp sequence in the 3' non-coding region. In pTi82.139 the original coding region of the ipt gene has remained largely unmodified. The pTiAB4 6b gene differs from its pTi82.139 counterpart by the lack of a 12 bp repeat in the 3' part of the coding sequence. This leads to the loss of four glutamic acid residues from a series of ten. In spite of these differences, the ipt and 6b genes of pTiAB4 are functional. Our results provide new insight into the evolution of Agrobacterium Ti plasmids and confirm the remarkable plasticity of these genetic elements. Possible implications for the study of bacterial phylogeny are discussed.

Amino Acid Sequence↗

Escherichia coli in disguise: molecular origins of Shigella.

Shigella, which still stands as a genus with four species today, in reality belongs to the extremely diverse species Escherichia coli. There are several lineages of Shigella strains derived through independent acquisition of the pINV virulence plasmid. The chromosomally determined phenotypic properties of Shigella result from convergent evolution during niche adaptation, most due to loss of function, some from negative selection pressure.

Adaptation, Biological↗