Carrier Plasmid Research Articles

Random Insertion of Transposon Tn4001in the Genome ofSpiroplasma citriStrain GII3

Electroporation ofSpiroplasma citristrain GII3 with plasmid pMUT containing theStaphylococcus aureustransposon Tn4001resulted in random insertion of Tn4001into the spiroplasmal genome. Transformation frequencies reached 10−8per colony-forming unit (CFU) when 100 μg of plasmid DNA and 3 × 109S. citriCFU were used. Three other strains ofS. citrifailed to be transformed under the same conditions. In most cases Tn4001was randomly inserted in the genome ofS. citristrain GII3, without insertion of the carrier plasmid. For most transformed spiroplasmas, Tn4001was stably maintained in the absence of antibiotic selection for at least 80 bacterial generations, making Tn4001a potential tool forS. citrimutagenesis.

Two hundred years of vaccination

There are real or potential difficulties with all vaccines. There may be problems with attenuation of live vaccines, with possible risks for vaccinees or their contacts. With non-infectious vaccines, there may be problems in identifying and preserving the immunogenic components and of ensuring correct presentation of the antigens so that the appropriate immune response is stimulated. With all vaccines, there are potential problems of delivery and storage, particularly in tropical climates. In this respect, the WHO Programme of Expanded Immunization is building on experience gained during the Smallpox Eradication Campaign to facilitate widespread use of vaccines. There are still problems caused by, for example, influenza virus, human immunodeficiency virus (HIV), and various respiratory and enteric pathogens which have variable and/or multiple serotypes, and also by malaria, but progress is being made. Various vaccines are being tested against typhoid, cholera and rotavirus gastroenteritis [[1]xPlotkin, S and Fantini, B eds. See all References, [8]xThe legacy of Jenner. Levine, MM. Brit Med J. 1996; 312: 1177–1178Crossref | PubMedSee all References], and considerable attention is being paid to a synthetic subunit vaccine for malaria [9xMalaria – advances in vaccines. Targett, GAT. Curr Opin Infect Dis. 1995; 8: 322–327Crossref | Scopus (6)See all References[9]. Various HIV vaccines are being developed, and the recent isolation of apparently naturally attenuated mutants of HIV which lack the nef gene suggests they may be developed into attenuated vaccines [10xReconsidering an attenuated vaccine for AIDS. Beale, AJ. Lancet. 1996; 347: 344–345Abstract | PubMed | Scopus (3)See all References[10].Recombinant DNA technology has already made a considerable impact through the successful yeast recombinant hepatitis B vaccine [[1]xPlotkin, S and Fantini, B eds. See all References, [11]xProgress on the control of hepatitis B infection through immunization. Kane, MA. Gut. 1993; 34 (suppl): 10–12Crossref | Scopus (40)See all References], and it is appropriate that smallpox vaccine still has a role to play. Infectious vaccinia recombinant vaccines which express the immunogenic glycoprotein of rabies virus are proving very successful in Europe for oral immunization of foxes, the principal wildlife reservoir of rabies [12xThe development and use of a vaccinia–rabies recombinant oral vaccine for the control of wildlife rabies. Pastoret, PP and Brochier, B. Epidemiol Infect. 1996; 116: 235–240Crossref | PubMedSee all References[12]. Although there are reservations about the widespread use of recombinant vaccinia vaccines, particularly in humans, the technology is being transferred to other poxviruses. Candidate mammalian vaccines are being developed using recombinant canarypox vectors which induce good immune responses although they do not replicate in mammalian cells [[1]xPlotkin, S and Fantini, B eds. See all References, [13]xNon-replicating expression vectors : applications in vaccine development and gene therapy. Limbach, K and Paoletti, E. Epidemiol Infect. 1996; 116: 241–256Crossref | PubMedSee all References].Considerable interest is also being shown in ‘DNA vaccines’ [14xDNA vaccines. McDonnell, WM and Askari, FK. New Eng J Med. 1996; 334: 42–45Crossref | PubMed | Scopus (155)See all References[14]. These comprise the appropriate gene inserted into a plasmid carrier which, when injected into the body, is taken up by host cells. The viral gene is expressed and the antigen preferentially stimulates cell-mediated immunity. By appropriate use of reverse transcriptase, recombinant DNA vaccines are being developed for RNA virus infections, particularly influenza.Jenner's pioneer work on smallpox vaccine was not immediately extended to the control of other infections. However, vaccines have made a significant impact in developed countries, through the control of, for example, measles, diphtheria, tetanus and polio, and the widespread use of these and other vaccines in developing countries should have similar effects. There are still problems with infectious diseases, however, and current vaccine research may help to solve these. Increasing concern about antibiotic-resistant bacteria and the slow rate of development of antiviral drugs emphasizes the ‘truism’ that prevention is better than cure; the ultimate goal should be eradication. This has been achieved for smallpox [5xFenner, F, Henderson, DA, Arita, I, Jezek, Z, and Ladnyi, ID. See all References[5], and is achievable for polio by the year 2000 by mass vaccination [15xMass polio vaccination; eradication by 2000 is a realistic goal. Chander, J and Subrahmanyan, S. Brit Med J. 1996; 312: 1178–1179Crossref | PubMedSee all References[15].

Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure.

An improved lithium acetate (LiAc)/single-stranded DNA (SS-DNA)/polyethylene glycol (PEG) protocol which yields > 1 x 10(6) transformants/micrograms plasmid DNA and the original protocol described by Schiestl and Gietz (1989) were used to investigate aspects of the mechanism of LiAc/SS-DNA/PEG transformation. The highest transformation efficiency was observed when 1 x 10(8) cells were transformed with 100 ng plasmid DNA in the presence of 50 micrograms SS carrier DNA. The yield of transformants increased linearly up to 5 micrograms plasmid per transformation. A 20-min heat shock at 42 degrees C was necessary for maximal yields. PEG was found to deposit both carrier DNA and plasmid DNA onto cells. SS carrier DNA bound more effectively to the cells and caused tighter binding of 32P-labelled plasmid DNA than did double-stranded (DS) carrier. The LiAc/SS-DNA/PEG transformation method did not result in cell fusion. DS carrier DNA competed with DS vector DNA in the transformation reaction. SS plasmid DNA transformed cells poorly in combination with both SS and DS carrier DNA. The LiAc/SS-DNA/PEG method was shown to be more effective than other treatments known to make cells transformable. A model for the mechanism of transformation by the LiAc/SS-DNA/PEG method is discussed.

Recombination‐Based Assay (RBA) for Screening Bacteriophage Lambda Libraries

The recombination-based assay represents a convenient way to screen a complex library constructed in bacteriophage lambda for homology to a given sequence cloned into a specially designed plasmid. The technique serves to screen a bacteriophage library rapidly and efficiently with a sequence cloned into a plasmid; counterselection then yields the gene product of interest with its plasmid carrier deleted. Because 10(6) to 10(7) plaque-forming units (pfu) may be screened using several petri dishes, and the homology for crossing-over need only be greater than 25 bp, the RBA represents an efficient way to screen complex lambda libraries rapidly for homology to a given sequence. In this procedure, a lambda library is screened using a specially designed plasmid carrying the desired target sequence. Recombinants arising from cross-over events between the plasmid and a bacteriophage carrying a corresponding region of homology are selected by their ability to grow on strain DM21. Growth of lambda on DM21 requires the presence of an allele encoded on the plasmid to suppress an amber mutation in the host strain that prevents lambda propagation. Recovery of the original phage carrying the target sequence requires a reversal of the homologous recombination event. This reversal occurs spontaneously, and is detected by PCR amplification using primers that flank the cloning site in the lambda vector.

High-level heterologous gene expression in Saccharomyces cerevisiae from a stable 2μm plasmid system

The best candidate for a high-copy-number and mitotic stability expression system in yeast is the endogenous 2μm plasmid. Nevertheless, derivatives of the 2μm plasmid typically exhibit lower copy numbers and require selection for adequate maintenance within cells. We report the construction and utilization of an efficient heterologous gene expression system containing a 4.5-kb inducible expression cassette inserted into the 2μm plasmid and selected in cells utilizing a carrier plasmid which is subsequently lost via FRT/Flp recombination. The non-selectable 2μm plasmid, containing the cassette, was found to be stably maintained in cells, without selection, at high copy number. The dynamics of resolution and partitioning of this plasmid were analyzed during the course of 50 generations of growth under non-selective conditions. The heterologous lacZ reporter gene coding for β-galactosidase (βGal) is driven by the hybrid, galactoseinducible promoter GAL10::pMFα 1. Upon induction, βGal was secreted into the periplasm and culture supernatant at levels which could be detected directly from Coomassie blue-stained SDS-PAGE. Furthermore, plasmid-containing cells could be maintained directly on rich YPD medium and identified either by utilizing XGal or by observing inhibition of colony growth on YPGal solid medium. The cassette was designed for direct, high-level, inducible expression of cloned genes downstream from the MFα1 signal sequence, with or without a C-terminal lacZ fusion. This vector represents the first demonstration of a non-selectable, mitotically stable, episomal plasmid system capable of expressing recombinant proteins at high levels. By supplanting the need for synthetic medium, this system could provide both an efficient and cost-effective means of generating recombinant protein at either the laboratory or large-scale level.

Introducing DNA into Yeast by Transformation

Transformation and electroporation protocols that yield more than one million yeast colonies per microgram of plasmid DNA have been developed. In the LiAc/SS-DNA/PEG protocol, cells are treated with lithium acetate, incubated with plasmid and single-stranded carrier DNA in the presence of PEG 3350, heat-shocked, and plated onto selective medium. The electroporation protocol involves the administration of a high-voltage pulse to cells that have been heat-shocked in the presence of single-stranded carrier DNA, plasmid DNA, and PEG. This protocol is rapid and can be completed in approximately 30 min. Spheroplast transformation requires the removal of the yeast cell wall with β-glucanase or β-glucuronidase. The spheroplasts are incubated with plasmid DNA and PEG and plated in osmotically stabilized agar. We also describe a protocol for the preparation of transformation-competent cells that can be stored at −70°C. The frozen cells are transformed with plasmid DNA in the presence of single-stranded carrier DNA and PEG. The numbers of transformants obtained with these protocols are sufficient for complex genomic, cDNA, and gene fusion libraries, facilitating the molecular analysis of the structure and function of genes from higher eukaryotes.

Electroporation-mediated gene transfer efficiency is reduced by linear plasmid carrier DNAs

Carrier DNA has generally been found to stimulate DNA-mediated gene transfer of Chinese hamster ovary (CHO) cells by calcium phosphate coprecipitation. In studies employing electroporation, however, we observed that linear plasmid DNA was inhibitory to the transfection of CHO cells. This unexpected result prompted us to explore the effects of various types and forms of plasmid, cosmid, and chromosomal DNAs on transfection efficiencies. Both carrier DNA form and type were found to influence transfection efficiencies. Circular and linear forms of plasmid carrier DNA had opposite effects: circular plasmids increased and linear plasmids decreased transfection efficiencies. These effects were independent of homology with the selected plasmid and are probably independent of homologous recombination mechanisms. Bacterial genomic DNA failed to stimulate transfection, while calf thymus and cosmid DNA consisting primarily of human sequences stimulated transfection significantly. Our results have importance for plasmid-based experiments in mammalian cells such as those involving the induction of interplasmid homologous recombination.

Expression of Streptomyces sp. cholesterol oxidase in Lactobacillus casei

Three strains of Lactobacillus casei were electrotransformed with pNCO937, an 8.1-kb recombinant plasmid carrier of a Streptomyces sp. cholesterol oxidase gene. Transformation frequency was generally low and strain-dependent, ranging from 6 to 40 transformants/μg DNA. L. casei transformants stably maintained pNCO937 with no indication of deletion mutational events. Transformants produced active cholesterol oxidase and sonicated cells formed 4-cholesten-3-one from both free and lipoprotein-bound cholesterol. L. casei shows promise as a host suitable for studying heterologous gene expression in lactobacilli.

Characterization of multiple molecular interactions between human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1)

In transient gene expression assays we observed an increase in expression of the bacterial chloramphenicol acetyl-transferase (CAT) gene, under the transcriptional control of the HIVA LTR (pLTR-CAT), when this plasmid was cotransfected into Vero or MRC-5 cells with a plasmid containing either the HCMV immediate early 1 and 2 (E 1, IE 2) genes (pRL43a) or just the IEZ gene (pMP18). When the HCMV IE, gene (pMP12) was cotransfected with pLTR-CAT into Vero cells the level of measurable CAT gene activity was below the level observed when pLTR-CAT was cotransfected with a nonspecific carrier plasmid (pGEM3). The negative influence of the HCMV IE 1, gene product on the HIVA LTR in Vero cells was also observed when the HIVA tat gene (pLTR-TAT) was cotransfected into Vero cells with pLTR-CAT and pMP12. However, when the HCMV IE 1 gene was cotransfected into rhabdomyosarcoma (RD) cells with proviral HIV-1 DNA, an increase in viral production, as monitored by measurement of HIV-1 reverse transcriptase activity, was observed. In electrophoretic mobility shift assays, nuclear extracts obtained 15 hr post-HCMV infection (hpi) were found to contain a lower level of interaction with an oligonucleotide which corresponded to the HIVA LTR Sp-1 binding motif. Nuclear extracts obtained 40 hpi of MRC-5 cells had a greater level of interaction with, and changed the mobility of, the Sp-1 oligonucleotide relative to the uninfected nuclear extracts. HCMV-infected MRC-5 cell nuclear extracts also contain a factor(s) which interacted with the HIV-1 LTR between nucleotide positions −15 to −2 relative to the HIV-1 mRNA Start Site.

Nucleotide sequence and cloning in Bacillus subtilis of the Bacillus stearothermophilus pleiotropic regulatory gene degT

The regulatory gene (degT) from Bacillus stearothermophilus NCA1503 which enhanced production of extracellular alkaline protease (Apr) was cloned in Bacillus subtilis with pTB53 as a vector. When B. subtilis MT-2 (Npr- [deficiency of neutral protease] Apr+) was transformed with the recombinant plasmid, pDT145, the plasmid carrier produced about three times more alkaline protease than did the wild-type strain. In contrast, when B. subtilis DB104 (Npr- Apr-) was used as a host, the transformant with pDT145 could not exhibit any protease activity. After construction of the deletion plasmids, DNA sequencing was done. A large open reading frame was found, and nucleotide sequence analysis showed that the degT gene was composed of 1,116 bases (372 amino acid residues, molecular weight of 41,244). A Shine-Dalgarno sequence was found nine bases upstream from the open reading frame. A B. subtilis strain carrying degT showed the following pleiotropic phenomena: (i) enhancement of production of extracellular enzymes such as alkaline protease and levansucrase, (ii) repression of autolysin activity, (iii) decrease of transformation efficiency for B. subtilis (competent cell procedure), (iv) altered control of sporulation, (v) loss of flagella, and (vi) abnormal cell division. When B. stearothermophilus SIC1 was transformed with the recombinant plasmid carrying degT, the transformants exhibited abnormal cell division. These phenomena are similar to those of the phenotypes of degSU(Hy) (hyperproduction), degQ(Hy), and degR mutants of B. subtilis. However, the amino acid sequence of the degT product (DegT) is different from those of the reported gene products. Furthermore, DegT includes a hydrophobic core region in the N-terminal portion (amino acid numbers 50 to 160), a consensus sequence for a DNA binding region (amino acid numbers 160 to 179), and a region homologous to transcription activator proteins (amino acid numbers 351 to 366). We discuss the possibility that the membrane protein DegT functions as a sensor protein and transfers the signal of environmental stimuli to the regulatory region of target genes to activate or repress transcription of the genes.

Functional and structural map of pLST1000: a multiresistance plasmid widely distributed in Enterobacteriaceae.

pLST1000, an 80-kb plasmid found in Enterobacteriaceae in North and South America, harbors the aadB and several other resistance genes. We suggested earlier that, because of its widespread distribution, pLST1000 could act as a carrier plasmid, bringing the aadB gene to new locations. This paper presents the restriction enzyme recognition site and functional map of the plasmid. The resistance genes lie in a discreet region. The aadB and aadA genes form an operon with the aadB gene promoter proximal. This operon is flanked by bla-TEM and bla-OXA2 genes, the former located in a functional Tn3-like transposon. This arrangement is similar to that of relatives of the transposon TN21, where additional resistance genes are precisely inserted in recombinational "hot spot" sequences that flank the aadA gene. We were not able to demonstrate transposition of the aadB gene in Escherichia coli. A sul gene and mer operon lie beyond the bla-OXA2 gene. The transfer genes form a single region, defined by insertions of Tn5-132 that give the Tra- phenotype.

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis.

A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.

First position wobble in codon-anticodon pairing: amber suppression by a yeast glutamine tRNA

A 2.4-kb fragment of DNA isolated from the Saccharomyces cerevisiae genome was found to suppress amber mutations when its carrier plasmid was present in high copy number. A 1.2-kb subclone of this fragment was sufficient to confer suppressor activity. Sequencing has established that this fragment carries a normal glutamine tRNA gene. Deletion of this tRNA gene from the subclone resulted in the loss of suppressor activity. The tRNA Gln has the anticodon CUG that normally recognizes the glutamine codon CAG. We propose that suppression occurs via an inefficient readthrough of the UAG amber stop codons during translation. Such readthrough requires wobble in the first position of the codon.

Control of pT181 replication I. The pT181 copy control function acts by inhibiting the synthesis of a replication protein.

pT181 is a fully sequenced 4.4-kb 20 copy Tcr plasmid from Staphylococcus aureus. Its replication system involves a unique unidirectional origin embedded in the coding sequence for a plasmid-determined protein, RepC, that is required for initiation. When joined to a 55 copy carrier plasmid, pE194, pT181 excludes autonomous isologous replicons by inhibiting their replication. Two types of spontaneous pT181 copy mutants have been isolated, one that eliminates sensitivity to this inhibition and another that does not. A spontaneous 180-bp deletion, delta 144, eliminates both the inhibitory activity and sensitivity to it. This deletion increases copy number by 50-fold and RepC production by at least 10-fold. It is located directly upstream from the repC coding sequence and the deletion-bearing plasmid supports the replication of inhibitor-sensitive plasmids in cells containing active inhibitor. This effect is probably due to the overproduction of RepC by the delta 144 plasmid. On the basis of these results, it is suggested that RepC synthesis is negatively controlled by an inhibitor that is encoded directly upstream from the repC coding sequence and acts as a tareget set in the same region. It is likely, therefore, that pT181 replication rate is determined by the level of RepC.

How the R1 replication control system responds to copy number deviations

The copy number of R1 and of a repA- lacZ gene fusion was increased above normal by coupling to a plasmid which is present at a fivefold higher copy number at 30 °C. This carrier plasmid is deficient in replication at 42 °C, and it was thus possible after a temperature shift to analyze the response to the increased plasmid concentration of the R1 replication control system. Both the frequency of replication per plasmid molecule and the rate of repA expression per gene copy were reduced under these conditions, and the data strongly suggest that there is an inverse proportionality between the specific rate of plasmid replication viz repA expression and the copy number/gene dosage of the plasmid.

Relationship between pNG2, an Emr plasmid in Corynebacterium diphtheriae, and plasmids in aerobic skin coryneforms.

Erythromycin-resistant (Emr) coryneforms from cutaneous lesions and erythromycin-susceptible (Ems) coryneforms from normal skin sites were screened for plasmids. Approximately one-third of the 40 isolates carried one or more plasmids ranging in mass from 2.5 to 36 megadaltons, all exhibiting different restriction enzyme digest patterns. In contrast, only Corynebacterium diphtheriae strains comprising a single cohort of apparently identical Emr, pNG2-carrying isolates have been identified as plasmid carriers. Homology was demonstrated between pNG2 and a number of fragments in restriction enzyme digests of plasmids from both Emr and Ems skin coryneforms under high-stringency conditions. However, none was detected between pNG2 and the genomic or plasmid DNAs of Emr staphylococci or streptococci isolated concurrently with the Emr coryneforms. One coryneform plasmid, pNG34, exhibited extensive homology with pNG2, and many comigrating fragments were observed. Very little relationship was observed between C. diphtheriae and the skin coryneforms when their genomic DNAs were hybridized. The origin and presence of pNG2 in Emr C. diphtheriae is discussed in relation to these findings.

Viability of palindromic DNA is restored by deletions occurring at low but variable frequency in plasmids of Escherichia coli

Palindromic arrangements of 2 × 400 to 2 × 1517 bp of nucleotide sequences were created by in vitro manipulation of Escherichia coli plasmids. As a consequence of its method of formation, each palindrome possessed at its center the recognition site for a particular restriction endonuclease. Eight out of eight palindromes, having at their centers the recognition sites for BamHI, BglI, BglI, HindIII, PstI, SalI, XhoI, and XmaIII, were shown to be inviable when transformed into E. coli. The smallest of these palindromes had a half-length of approx. 400 bp. The lethality of palindromic sequences for their carrier plasmids was circumvented at low frequencies by spontaneous in vivo deletion events which removed the centers of symmetry of the palindromes. The frequencies of such deletions were less than 1% in most cases, but varied significantly both with the palindromic sequence in question and with the surrounding nonpalindromic sequences of the carrier plasmid. We confirmed the viability of a plasmid with a 147-bp palindrome [Bergsma et al., Gene 20 (1982) 157–167] and found that this palindrome (derived from SV40) does not confer viability on the plasmids with long palindromes.

Selection methods in bacilli for recombinants and transformants of intra- and interspecific fused protoplasts.

The intraspecific fusion frequencies obtained with the direct selection method on a semi-synthetic regeneration medium between strains of B. subtilis and B. licheniformis were distributed from 9.9 X 10(-2) to 4.5 X 10(-3), which was one or two orders higher than those of interspecific recombinations between B. subtilis and B. licheniformis. The regeneration media were also useful for selecting interspecific transformants from plasmid carrier to noncarrier. This selection could be used as a primary selection method for inter- and intraspecific recombinants obtained by protoplast fusion.

New runaway-replication-plasmid cloning vectors and suppression of runaway replication by novobiocin

Two new cloning vectors (pBEU28 and pBEU50) with temperature-controlled runaway-replication properties are described. pBEU28 is similar to aphA + (Kan R) plasmid pBEU2 but lacks a 1.8-kb duplication which is responsible for plasmid instability. pBEU50 is an analog of pBR313 and pBR322 in that it carries bla +(Amp R), which can be used for selection, and tet +(Tet R) which can be inactivated by cloning at HindIII and BamHI restriction sites. Sublethal concentrations of novobiocin were exploited to suppress runaway replication and to restore the viability of the plasmid carriers. By this method copB deletion mutants of two temperature-controlled, conditional runaway-replication plasmids were detected and isolated. The unconditional runaway-replication property of these plasmids leads us to hypothesize that there are at least two controls of plasmid R1 copy number and that the copB-dependent control is temperature-sensitive in the conditional runaway replication mutants. The novobiocin suppression of the runaway replication permitted us to clone dnaN + on pBEU28 and to identify its presence at 42°C with a dnaN59 transformation recipient which was temperature-sensitive due to a defect in the dnaN gene.

Tn554: Isolation and characterization of plasmid insertions

Tn554, a transposon in Staphylococcus aureus that carries determinants of spectinomycin resistance and inducible macrolide-lincosamide resistance, is characterized by a highly efficient transposition, exceptional site specificity for insertion, and inhibition of transposition by a copy of the transposon inserted at its preferred chromosomal site. In this communication we describe the characteristics of a number of rare, secondary-site insertions of Tn554 into several related penicillinase plasmids. These plasmid insertions display considerable variation in the frequencies with which they can act as transposon donors, as well as in the frequencies at which they undergo apparently precise excision. Transposition from the plasmid transposon donors is ordinarily a duplicative process and these subsequent transposition events always return Tn554 to its preferred site in the S. aureus chromosome; such derivatives are indistinguishable from the primary chromosomal insertion from which they were originally derived. We also report an unusual relationship between Tn554 and the transducing phage, φ11, in which Tn554 is frequently transferred independently of its plasmid carrier. We suggest that the bacteriophage may play an important role in the mobility of Tn554, in addition to the usual transduction mechanism, in a process that we have referred to as “hitchhiking.”