Don B. Clewell

Unconstrained bacterial promiscuity: the Tn916–Tn1545 family of conjugative transposons

Conjugative transposons are highly ubiquitous elements found throughout the bacterial world. Members of the Tn916-Tn1545 family carry the widely disseminated tetracycline-resistance determinant Tet M, as well as additional resistance genes. They have been found naturally in, or been introduced into, over 50 different species and 24 genera of bacteria. Recent investigations have led to insights into the molecular basis of movement of these interesting mobile elements.

A cloning vector able to replicate in Escherichia coli and Streptococcus sanguis

A plasmid that is able to replicate in both Escherichia coli and Streptococcus sanguis has been constructed by the in vitro joining of the pACYC184 (Cmr Tcr) and pVA749 (Emr) replicons. This plasmid, designated pVA838, is 9.2 kb in size and expresses Emr in both E. coli and S. sanguis. Its Cmr marker is expressed only in E. coli and may be inactivated by addition of DNA inserts at its internal EcoRI or PvuII sites. The pVA838 molecule also contains unique SalI, SphI, BamHI, NruI and XbaI cleavage sites suitable for molecular cloning. pVA838 may be amplified in E. coli but not in S. sanguis. We have used the pVA838 plasmid as a shuttle vector to clone streptococcal plasmid fragments in E. coli. Such chimeras isolated from E. coli were readily introduced into S. sanguis by transformation.

Plasmid transfer in Streptococcus faecalis: Production of multiple sex pheromones by recipients

In a previous report (1978, Proc. Nat. Acad. Sci. USA 75, 3479-3483), we showed that recipient strains of Streptococcusfaecalis excrete a heat-stable substance (sex pheromone) which induces donor cells carrying certain conjugative plasmids to become adherent, generating the cell-to-cell contact necessary for plasmid transfer. Since donors themselves could be induced to aggregate or “clump” by recipient filtrates, the substance was referred to as “clumping-inducing agent” (CIA). In this report, we present a simplified assay for CIA and determine the level of activity in filtrates prepared at various stages of growth. We also present evidence that recipient cells produce multiple pheromones, each specific for donors harboring a particular class of plasmids. Whereas a recipient that acquires a conjugative plasmid no longer produces the corresponding CIA, it still produces CIAs specific for donors with different conjugative plasmids. In addition, an analysis of 100 clinical isolates of S. faecalis showed that drug-resistant strains are significantly more likely to respond to and produce CIA activities than drug-sensitive strains. A model is discussed describing the relationships of sex pheromones to the mating process.

Movable genetic elements and antibiotic resistance in enterococci.

The enterococci possess genetic elements able to move from one strain to another via conjugation. Certain enterococcal plasmids exhibit a broad host range among gram-positive bacteria, but only when matings are performed on solid surfaces. Other plasmids are more specific to enterococci, transfer efficiently in broth, and encode a response to recipient-produced sex phermones. Transmissible non-plasmid elements, the conjugative transposons, are widespread among the enterococci and determine their own fertility properties. Drug resistance, hemolysin, and bacteriocin determinants are commonly found on the various transmissible enterococcal elements. Examples of the different systems are discussed in this review.

Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning

A novel plasmid vector that is able to replicate both in Escherichia coli and in Streptococcus sanguis is described. This 9.2-kb plasmid, designated pVA856, carries Cmr, Tcr, and Emr determinants that are expressed in E. coli. Only the Emr determinant is expressed in S. sanguis. Both the Cmr and the Tcr of pVA856 may be insertionally inactivated. This plasmid affords several different cleavage-ligation strategies for cloning in E. coli followed by subsequent introduction of chimeras into S. sanguis. In addition, we have modified a previously described E. coli-S. sanguis shuttle plasmid [pVA838; Macrina et al., Gene 19 (1982) 345-353], so that it is unable to replicate in S. sanguis. The utility of such a plasmid for cloning and selecting sequences enabling autonomous replication in S. sanguis is demonstrated.

Plasmids in bacteria

Structure and Evolution.- Plasmids as Organisms.- Report on a Workshop: Structure and Function.- Evolutionary Relevance of Genetic Rearrangements Involving Plasmids.- Mechanisms of Transposition in Bacteria.- Insertion of Transcriptional Elements Outside the Replication Region Can Interfere with Replication, Maintenance, and Stability of ColE1-Derived Plasmids.- Studies on the Transposition of IS1.- On the Transposition and Evolution of Tn1721 and its Relatives.- Repeated DNA Sequences Recombine 1,000 Times More Frequently in a Plasmid Than in the Chromosome of BacillusSubtilis.- Mercury Resistance Transposon Tn813 Mediates Chromosome Transfer in Rhodopseudomonas sphaeroides and Intergeneric Transfer of pBR322.- Report on a Workshop: Plasmids in Unusual Systems.- Replication, Incompatibility, and Partition.- Chairmans Introduction: Replication, Incompatibility, and Partition.- Role of the ? Initiation Protein and Direct Nucleotide Sequence Repeats in the Regulation of Plasmid R6K Replication.- Initiation of Replication of the EscherichiaColi Chromosomal Origin Reconstituted with Purified Enzymes.- Origin and Initiation Sites of ?dv DNA Replication In Vitro.- Broad Host-Range Plasmid R1162: Replication, Incompatibility, and Copy-Number Control.- Control of Plasmid Replication: Theoretical Considerations and Practical Solutions.- The Partition Functions of PI, P7, and F Miniplasmids.- Genetic Interactions of Broad Host-Range Plasmid RK2: Evidence for a Complex Replication Regulon.- Replication Determinants of the Broad Host Range Plasmid RSF1010.- Regulation of Replication and Maintenance Functions of Broad Host-Range Plasmid RK2.- Control of Chromosome Replication in Bacteria.- Stable Maintenance of Plasmid CLO DF13: Structural and Functional Relationships Between Replication, Control, Partitioning, and Incompatibility.- Replication Control for PT181, An Indirectly Regulated Plasmid.- Construction of ColE1 RNA1 Mutants and Analysis of their Function In Vivo.- Incompatibility and INCFII Plasmid Replication Control.- P1 Plasmid Maintenance: A Paradigm of Precise Control.- Partitioning of the pSC101 Plasmid During Cell Division.- DNA-Protein Interaction at the Replication Origins of Plasmid Chromosomes.- Positive Regulation and Transcription Initiation of xy1 Operons on TOL Plasmid.- Plasmid Transfer.- Chairmens Introduction: Plasmid Transfer.- Linear Plasmids with Terminal Inverted Repeats Obtained from Streptomycesrochei and Kluyveromyceslactis.- Conjugal Plasmid Transfer in BacillusThuringiensis.- Mechanisms Essential for Stable Inheritance of Mini-F Plasmid.- Sex Pheromones and Plasmid Transfer in StreptococcusFaecalis: A Pheromone, cAM373, Which is Also Excreted by StaphylococcusAureus.- General Genetic Recombination of Bacterial Plasmids.- The Origin of Plasmid DNA Transfer During Bacterial Conjugation.- Genes and Gene Products Involved in the Synthesis of F-PILI.- Genetics of Clindamycin Resistance in Bacteroides.- Effect of Chromosome Homology of Plasmid Transformation and Plasmid Conjugal Transfer in Haemophilus Influenzae.- Plasmid DNA Primases and Their Role in Bacterial Conjugation.- Promoters in the Transfer Region of Plasmid F.- Conjugative Sex Plasmids of Streptomyces.- Specialized Functions, Structure, and Evolution.- The Structure and Source of Plasmid DNA Determine the Cloning Properties of Vectors for BacillusSubtilis.- Shuttle Vector for EscherichiaColi, PseudomonasPutida, and PseudomonasAeruginosa.- Report on a Workshop: Plasmid Cloning Vehicles.- Plasmids in the Degradation of Chlorinated Aromatic Compounds.- Notes on Metabolic Plasmid Organization and Expression.- The Structure of the mer Operon.- Analysis and Manipulation of Plasmid-Encoded Pathways for the Catabolism of Aromatic Compounds by Soil Bacteria.- The Plasmid-Specified Aerobactin Iron Uptake System of EscherichiaColi.- Plasmid-Mediated Iron Sequestering Systems in Pathogenic Strains of Vibrioanguillarum and EscherichiaColi.- Chairmans Introduction: Specialized Functions.- Cloning of an Enzymatically Active Segment of the Exotoxin-A Gene of PseudomonasAeruginosa Ill.- Structure, Function, and Regulation of the Plasmid-Encoded Hemolysin Determinant of EscherichiaColi.- pIAA, A Virulence Plasmid in PseudomonasSavastanoi.- The Molecular Basis of Plant Cell Transformation by AgrobacteriumTumefaciens.- Post-Transcriptional Regulation of Chloramphenicol Acetyl Transferase.- Plasmid and Larval Toxin of Bacilluslaterosporus.- Posters (Poster abstracts have been reproduced as submitted).- Replication, Incompatibility, Partition.- Specialized Functions.- Structure and Evolution.- Transfer.- Vectors.- Rosters.- Speakers.- Participants.- Index of Poster Authors.

Plasmid Content of a Vancomycin-Resistant Enterococcus faecalis Isolate from a Patient Also Colonized by Staphylococcus aureus with a VanA Phenotype

ABSTRACT Vancomycin-resistant Enterococcus faecalis coisolated with vancomycin-resistant (VanA) Staphylococcus aureus was found to contain two plasmids, designated pAM830 (45 kb) and pAM831 (95 kb). pAM830, found to be conjugative and closely related to the Inc18 family of broad-host-range conjugative plasmids, encodes resistances to vancomycin (via a Tn1546-like element) and erythromycin; pAM831 encodes resistances to gentamicin, streptomycin, and erythromycin.

Enterococcal plasmid transfer: sex pheromones, transfer origins, relaxases, and the Staphylococcus aureus issue.

Certain conjugative plasmids in Enterococcus faecalis encode a mating response to peptide sex pheromones encoded on the chromosome of potential recipient (plasmid-free) strains. The pheromone precursors correspond to the precursors of surface lipoproteins with the mature peptides coming from the last 7-8 residues of the related signal sequences. Processing that gives rise to the pAD1-related peptide involves a chromosome-encoded metalloprotease (Eep) that is believed to operate within the cytoplasmic membrane. Mutations in the determinants for cAD1 and cAM373, cad and camE, respectively, do not affect cell viability; and when the related plasmid is present, the pheromone response is normal. A cAM373-like activity is produce by Staphylococcus aureus, but the corresponding lipoprotein determinant (camS) is unrelated to the enterococcal determinant (camE). pAD1 has two origins of transfer, oriT1 and oriT2 and encodes a relaxase (TraX), which has been shown to specifically nick in oriT2. pAM373 has a site, oriT, that is similar to oriT2 of pAD1. Both sites (oriT2 of pAD1 and oriT of pAM373) have a series of short direct repeats (5-6 bp with 5-6 bp-spacings) adjacent to a long inverted repeat (140 bp). The direct repeats differ significantly and confer specificity to the two systems. pAD1 and pAM373 are both able to mobilize the nonconjugative plasmid pAMalpha1, which encodes two relaxases that are involved in transfer. Relevant information concerning the possible movement of vancomycin resistance from E. faecalis to S. aureus in a clinical environment is discussed.

The Conjugative Transposons of Gram-Positive Bacteria

Conjugative transposons are characterized by their ability to move from one bacterial cell to another by a process requiring cell-to-cell contact. Evidence for such elements became apparent about 13 years ago from studies of Enterococcus (formerly Streptococcus) faecalis strain DS16, a clinical isolate obtained in 1975 from St. Joseph’s Mercy Hospital in Ann Arbor, Michigan (38, 39). DS16 was of interest at that time because of its multiple antibiotic resistance and hemolytic properties. It was found to harbor a hemolysin/bacteriocin plasmid, pAD1, and a resistance plasmid, pAD2 (26, 112). pAD1 was conjugative and conferred a mating response to a peptide sex pheromone secreted by potential recipient (pAD1-free) cells, whereas pAD2 conferred resistance to erythromycin, streptomycin, and kanamycin and was nonconjugative. The erythromycin resistance determinant (erm) of pAD2 was associated with a transposon designated Tn917 (111). Derivatives of DS16 cured of both pAD1 and pAD2 maintained a resistance to tetracycline (Tc), indicating that a Tc-resistance determinant (tet) was located on the bacterial chromosome. A series of filter membrane mating experiments designed to examine transfer of the various resistance determinants of DS16 showed that tet could be mobilized at frequencies of 10−5 per donor (38). The majority of transconjugants (about 90%) harbored pAD1 and had tet on the chromosome. Among most of the remaining transconjugants, tet was linked with pAD1, and this correlated with insertion of a 16-kb segment of DNA. A surprising result arose when certain “control” experiments were performed using a plasmid-free (cured) derivative of DS16 as a donor. As reported by Franke and Clewell (38), tet was able to transfer from such strains at a frequency of about 10−8 per donor, and transconjugants could pass on the trait in subsequent matings. Intercellular transfer was DNase resistant and was not affected by the presence of a Rec−allele in either the donor or the recipient. In addition, donor filtrates did not transfer tet to recipients, nor did donor cells exposed to chloroform prior to mating. Because cell contact appeared necessary, the term “conjugative transposon,” was adopted, and the element was designated Tn916. Additional studies showing interspecies transfer added strong support that the transposon encoded its own fertility functions.

Identification of the cAD1 Sex Pheromone Precursor in Enterococcus faecalis

The Enterococcus faecalis virulence plasmid pAD1 encodes a mating response induced by exposure to an octapeptide sex pheromone, cAD1, secreted by plasmid-free enterococci. The determinant for the pheromone in E. faecalis FA2-2, designated cad, was found to encode a 309-amino-acid lipoprotein precursor with the last 8 residues of its 22-amino acid signal sequence representing the cAD1 moiety. The lipoprotein moiety contained two 77-amino-acid repeats (70% identity) separated by 45 residues. The nonisogenic E. faecalis strain V583 determinant encodes a homologous precursor protein, but it differs at two amino acid positions, both of which are located within the pheromone peptide moiety (positions 2 and 8). Construction of a variant of strain FA2-2 containing the differences present in V583 resulted in cells that did not produce detectable cAD1. The mutant appeared normal under laboratory growth conditions, and while significantly reduced in recipient potential, when carrying pAD1 it exhibited a normal mating response. A mutant of FA2-2 with a truncated lipoprotein moiety appeared normal with respect to recipient potential and, when carrying plasmid DNA, donor potential. A gene encoding a protein designated Eep, believed to be a zinc metalloprotease, had been previously identified as required for pheromone biosynthesis and was believed to be involved in the processing of a pheromone precursor. Our new observation that the pAD1-encoded inhibitor peptide, iAD1, whose precursor is itself a signal sequence, is also dependent on Eep is consistent with the likelihood that such processing occurs at the amino terminus of the cAD1 moiety.