Genetic linkage and cotransfer of a novel, vanB-containing transposon (Tn5382) and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate

Mechanisms for the intercellular transfer of VanB-type vancomycin resistance determinants and for the almost universal association of these determinants with those for high-level ampicillin resistance remain poorly defined. We report the discovery of Tn5382, a ca. 27-kb putative transposon encoding VanB-type glycopeptide resistance in Enterococcus faecium. Open reading frames internal to the right end of Tn5382 and downstream of the vanXB dipeptidase gene exhibit significant homology to genes encoding the excisase and integrase of conjugative transposon Tn916. The ends of Tn5382 are also homologous to the ends of Tn916, especially in regions bound by the integrase enzyme. PCR amplification experiments indicate that Tn5382 excises to form a circular intermediate in E. faecium. Integration of Tn5382 in the chromosome of E. faecium C68 has occurred 113 bp downstream of the stop codon for the pbp5 gene, which encodes high-level ampicillin resistance in this clinical isolate. Transfer of vancomycin, ampicillin, and tetracycline resistance from C68 to an E. faecium recipient strain occurs at low frequency in vitro and is associated with acquisition of a 130- to 160-kb segment of DNA that contains Tn5382, the pbp5 gene, and its putative repressor gene, psr. The interenterococcal transfer of this large chromosomal element appears to be the primary mechanism for vanB operon spread in northeast Ohio. These results expand the known family of Tn916-related transposons, suggest a mechanism for vanB operon entry into and dissemination among enterococci, and provide an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance in clinical E. faecium strains.     

Conjugal transfer of transposon Tn916 from Enterococcus faecalis to Bacillus licheniformis

Transposon Tn916 was conjugally transferred from Enterococcus faecalis to Bacillus licheniformis bacterial strains ATCC 10716 and ATCC 9945A by filter and broth matings. The Tn916 transfer frequencies to B. licheniformis ranged from 10(-7) to 10(-5) selecting for tetracycline-resistant (Tcr) transconjugants depending on broth or filter mating. Movement of Tn916 was demonstrated when Tcr B. licheniformis transconjugants were mated with Bacillus subtilis strain W23. Tn916 insertion caused several auxotrophic and bacitracin deficient mutants. Southern blot analyses of HindIII chromosomal digests extracted from Tcr transconjugants showed that the transposon inserted at different sites in the B. licheniformis chromosome and the copy number of Tn916 varied. This approach should be useful for genetic studies of B. licheniformis.     

pDUAL: a transposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo

We describe a transposon gamma delta-containing cosmid cloning vector, pDUAL (previously called pJANUS), and demonstrate an efficient strategy for isolating nested deletions in both large-scale and small-scale DNA sequencing efforts. This "deletion factory" strategy takes advantage of the ability of gamma delta (Tn1000) to generate deletions that extend from an end of the transposon into adjacent DNA when gamma delta transposes to new sites in the same DNA molecule. pDUAL contains the contraselectable (conditional lethal) sacB+ (sucrose sensitivity) and strA+ (streptomycin sensitivity) genes just outside each end of an engineered gamma delta and selectable kan+ (Kanr) and tet+ (Tetr) genes between the cloning site and sacB and strA, respectively. Selection on sucrose tetracycline medium yields deletions that extend from one gamma delta end for various distances into the cloned DNA, while selection on streptomycin kanamycin medium yields comparable deletions in the other direction. Both types of deletions are recoverable because the essential plasmid replication origin is embedded in the gamma delta component and is thereby retained in each deletion product. Pilot experiments with pDUAL clones showed that deletion end points can be mapped or selected by plasmid size and that both DNA strands of any single clone can be accessed for sequencing by using a pair of universal primers specific for sequences that are just interior to the gamma delta ends.     

Enterococcus faecalis gene transfer under natural conditions in municipal sewage water treatment plants

The ability of Enterococcus faecalis to transfer various genetic elements under natural conditions was tested in two municipal sewage water treatment plants. Experiments in activated sludge basins of the plants were performed in a microcosm which allowed us to work under sterile conditions; experiments in anoxic sludge digestors were performed in dialysis bags. We used the following naturally occurring genetic elements: pAD1 and pIP1017 (two so-called sex pheromone plasmids with restricted host ranges, which are transferred at high rates under laboratory conditions); pIP501 (a resistance plasmid possessing a broad host range for gram-positive bacteria, which is transferred at low rates under laboratory conditions); and Tn916 (a conjugative transposon which is transferred under laboratory conditions at low rates to gram-positive bacteria and at very low rates to gram-negative bacteria). The transfer rate between different strains of E. faecalis under natural conditions was, compared to that under laboratory conditions, at least 10(5)-fold lower for the sex pheromone plasmids, at least 100-fold lower for pIP501, and at least 10-fold lower for Tn916. In no case was transfer from E. faecalis to another bacterial species detected. By determining the dependence of transfer rates for pIP1017 on bacterial concentration and extrapolating to actual concentrations in the sewage water treatment plant, we calculated that the maximum number of transfer events for the sex pheromone plasmids between different strains of E. faecalis in the municipal sewage water treatment plant of the city of Regensburg ranged from 10(5) to 10(8) events per 4 h, indicating that gene transfer should take place under natural conditions.     

Cloning and expression of a plasmid pBS195 gene, determining oxygenase activity, in Escherichia coli cells

Plasmid pBS195, detected in a strain of Lactobacillus sp. isolated from long-living persons, has a broad host range, including Gram-positive and Gram-negative microorganisms [1]. Plasmid-harboring colonies of the strain Escherichia coli HB101 give a color reaction with catechol. This indicates that genes mediating the activity of oxygenase are present in this plasmid. The high activity level of this enzyme, mediated by pBS195, and substrate specificity, which has not bee detected in any known metapyrocatechases, were found in cells of E. coli. Hybridization with a 32P-labeled fragment containing the NahC gene revealed a region of homology with a 1.6-kb EcoR I- BamH I fragment of plasmid pBS195. Deletion variants of this plasmid that lost oxygenase activity confirmed the location of the oxygenase gene in this region. The gene responsible for oxygenase activity in the plasmid was cloned on the pUC19 vector in E. coli cells. The expression of the cloned gene is controlled by the lac promoter of this vector. Physical, hybridization, and deletion analyses as well as analysis of polypeptides, which are synthesized in E. coli mini-cells, showed that this activity requires the participation of a polypeptide with molecular mass of 34 kDa.     

New genetic techniques for group B streptococci: high-efficiency transformation, maintenance of temperature-sensitive pWV01 plasmids, and mutagenesis with Tn917

Three techniques were developed to improve the genetic manipulation of group B streptococci (GBS). We first optimized a protocol for transformation of GBS by electroporation, which provided transformation efficiencies of 10(5) CFU/microgram. Variables that influenced the transformation efficiency were the glycine content of the competent cell growth media, the electric field strength during electroporation, the electroporation buffer composition, the host origin of the transforming plasmid, and the concentration of selective antibiotic at the final plating. Our transformation protocol provides an efficiency sufficient for cloning from ligation reactions directly into GBS, obviating an intermediate host such as Escherichia coli. Second, temperature-sensitive plasmids of the pWV01 lineage were shown to transform GBS, and their temperature-sensitive replication was confirmed. Lastly, the temperature-sensitive pWV01 plasmid pTV1OK, which contains Tn917, was used as a transposon delivery vector for the construction of genomic Tn917 mutant libraries. We have shown, for the first time, that Tn917 transposes to the GBS chromosome and at a frequency of 10(-3)/CFU. Furthermore, representative clones from a Tn917 library contained single transposon insertions that were randomly located throughout the chromosome. These techniques should provide useful methods for cloning, mutagenesis, and characterization of genes from GBS.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Tn4451 from Clostridium perfringens is a mobilizable transposon that encodes the functional Mob protein, TnpZ

The 6.3 kb Clostridium perfringens transposon Tn4451 encodes a 50 kDa protein, TnpZ, which has amino acid sequence similarity to a group of plasmid mobilization and recombination proteins that comprise the Mob/Pre family. Members of this family interact with an upstream palindromic sequence called an RSA site, and an RSA-like sequence has been identified upstream of the tnpZ gene. In Escherichia coli, in the presence of a chromosomally integrated derivative of the broad-host-range IncP plasmid, RP4, TnpZ was able to promote plasmid mobilization in cis and was able to function in trans to enable the mobilization of a co-resident plasmid carrying an RSA site. It was also able to mediate the conjugative transfer of plasmids from E. coli to C. perfringens. Site-directed mutagenesis of two bases within the RSA site resulted in a significant reduction in mobilization frequency, demonstrating that the RSA site is required for efficient plasmid mobilization. TnpZ is the only Mob/Pre protein known to be associated with a transposable genetic element, and Tn4451 is the first mobilizable but non-self-transmissible transposon to be identified from a gram-positive bacterium.     

Effect of duodenum-preserving resection of the head of the pancreas on endocrine and exocrine pancreatic function in patients with chronic pancreatitis

Two mobilizable cloning vectors, designated pABW1 and pAWB2, were constructed basing on the E. coli vector pBGS18 and oriT originating from RK2. In pABW2 the kanamycin resistance gene was replaced by a novel tetracycline resistance cassette derived from Tn1721. Both vectors, specific for E. coli, allow to perform the cloning steps in E. coli and then to efficiently transfer the constructs by conjugation to the host of choice. A vector which cannot propagate in the given host can be applied for identification of the host specific plasmid replicator regions. With the use of pABW2 we defined the minimal replicator region of pTAV202-a mini-derivative of the large pTAV1 plasmid of P. versutus. We also proved that RepC' encoded on this fragment is the principal initiator replication protein and that oriV is located along its coding sequence.     

Relationship of p53 molecular abnormalities with flow cytometry and growth factor receptor content in lung cancer

The nucleotide sequence of the 3.5-kb right junction fragment of the streptococcal conjugative transposon Tn5252 was obtained. The DNA fragment was found to carry four putative genes one of which displayed a high degree of similarity to prokaryotic 5C-cytosine methyltransferases carrying multiple sequence specificities. No cognate endonuclease gene was detected in the sequenced DNA. Purified methylase polypeptide synthesized in a T7 promoter-controlled overexpression system was found to lack methylase activity while the cell extracts of host cells containing the recombinant plasmid carrying the methylase gene were active. In vivo mutations in the methylase gene did not seem to affect the transferability of the element.     

Virulence and arsenic resistance in Yersiniae

The genus Yersinia contains three pathogenic species: Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica. Only a few biotypes and serotypes of Y. enterocolitica are pathogenic, and these form two distinct groups: some are of low virulence, and they are encountered worldwide; others, mainly encountered in North America, are markedly more virulent. All pathogenic yersiniae possess a 70-kb virulence plasmid called pYV which encodes secreted antihost proteins called Yops as well as a type III secretion machinery that is required for Yop secretion. Genes encoding Yop synthesis and secretion are tightly clustered in three quadrants of the pYV plasmid. We show here that in the low-virulence strains of Y. enterocolitica, the fourth quadrant of the plasmid contains a new class II transposon, Tn2502. This transposon encodes a defective transposase, but transposition can be complemented in trans by Tn2501, another class II transposon. Tn2502 was not detected in the pYV plasmids of the more virulent American strains of Y. enterocolitica, of Y. pseudotuberculosis, and of Y. pestis. Tn2502 confers arsenite and arsenate resistance. This resistance involves four genes; three are homologous to the arsRBC genes present on the Escherichia coli chromosome, but no homolog of the fourth one, arsH, has been found. The systematic presence of such a resistance operon on a virulence plasmid is unusual and could be related to the recent spread of low-virulence Y. enterocolitica strains. The presence of this ars operon also constitutes the first significant difference between the pYV plasmids from different Yersinia species.     

An avian pathogenic Escherichia coli strain produces a hemolysin, the expression of which is dependent on cyclic AMP receptor protein gene function

An avian pathogenic Escherichia coli strain M1000 showed a clear zone of erythrocyte lysis on sheep blood agar plates. The hemolytic activity was not detected in the culture supernatant nor was any DNA sequence homologous to the E. coli alpha-hemolysin gene detected in the chromosome or plasmid DNA of the strain, indicating that the observed hemolysis was different from alpha-type. To identify the genetic determinant responsible for the hemolysis, we performed random Tn5 insertional mutagenesis and obtained one mutant, named M5005, that totally lacked the hemolytic activity. Cloning and nucleotide sequencing of the region flanking the transposon insertion site in the M5005 chromosome revealed that the transposon was inserted within an open reading frame of the cyclic AMP receptor protein (CRP) gene, which is involved in one of the global regulatory networks of gene expression in E. coli. Nucleotide sequence analysis of the intact crp gene of the strain M1000 showed that the CRP protein of M1000 is 99% identical to that of K-12. Introduction of the intact crp gene on a low copy plasmid into the mutant M5005 restored the hemolytic phenotype, confirming that the mutation site in M5005 was in the crp gene. CRP plays a central role in catabolite repression, the phenomenon by which the synthesis of many enzymes required to metabolize various sugars is repressed in the presence of glucose. When the hemolytic activity of E. coli M1000 grown in the presence of glucose was examined, the hemolysis was totally impaired. These results indicate that the avian pathogenic E. coli strain M1000 produces a hemolysin the expression of which is dependent on crp gene function.     

Cloning and characterization of the lactococcal plasmid-encoded type II restriction/modification system, LlaDII

The LlaDII restriction/modification (R/M) system was found on the naturally occurring 8.9-kb plasmid pHW393 in Lactococcus lactis subsp. cremoris W39. A 2.4-kb PstI-EcoRI fragment inserted into the Escherichia coli-L. lactis shuttle vector pCI3340 conferred to L. lactis LM2301 and L. lactis SMQ86 resistance against representatives of the three most common lactococcal phage species: 936, P335, and c2. The LlaDII endonuclease was partially purified and found to recognize and cleave the sequence 5'-GC decreases NGC-3', where the arrow indicates the cleavage site. It is thus an isoschizomer of the commercially available restriction endonuclease Fnu4HI. Sequencing of the 2.4-kb PstI-EcoRI fragment revealed two open reading frames arranged tandemly and separated by a 105-bp intergenic region. The endonuclease gene of 543 bp preceded the methylase gene of 954 bp. The deduced amino acid sequence of the LlaDII R/M system showed high homology to that of its only sequenced isoschizomer, Bsp6I from Bacillus sp. strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases. The genetic organizations of the LlaDII and Bsp6I R/M systems are identical. Both methylases have two recognition sites (5'-GCGGC-3' and 5'-GCCGC-3') forming a putative stemloop structure spanning part of the presumed -35 sequence and part of the intervening region between the -35 and -10 sequences. Alignment of the LlaDII and Bsp6I methylases with other m5C methylases showed that the protein primary structures possessed the same organization.     

Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method

An efficient method for constructing a recombinant adenovirus (Ad) vector, based on an in vitro ligation, has been developed. To insert the foreign gene into an adenoviral DNA, we introduced three unique restriction sites, I-CeuI, SwaI, and PI-SceI, into the E1 deletion site of the vector plasmid, which contains a complete E1, E3-deleted adenovirus type 5 genome. I-CeuI and PI-SceI are intron-encoded endonucleases with a sequence specificity of at least 9-10 and 11 bp, respectively. A shuttle plasmid, pHM3, containing multiple cloning sites between the I-CeuI and PI-SceI sites, was constructed. After the gene of interest was inserted into this shuttle plasmid, the plasmid for E1-deleted adenovirus vector could be easily prepared by in vitro ligation using the I-CeuI and PI-SceI sites. SwaI digestion of the ligation products prevented the production of a plasmid containing a parental adenovirus genome (null vector). After transformation into E. coli, more than 90% of the transformants had the correct insert. To make the vector, a PacI-digested, linearized plasmid was transfected into 293 cells, resulting in a homogeneous population of recombinant virus. The large number and strategic location of the unique restriction sites will not only increase the rapidity of production of new first-generation vectors for gene transfer but will allow for rapid further improvements in the vector DNA backbone.     

Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate

The Clostridium perfringens enterotoxin gene is on a transposon-like element, Tn5565, integrated in the chromosome in human food poisoning strains. The flanking IS elements, IS1470 A and B, are related to IS30. The IS element found in the transposon, IS1469, is related to IS200 and has been found upstream of cpe in all Type A strains. PCR and sequencing studies from cell extracts and plasmid isolations of C. perfringens indicate that Tn5565 can form a circular form with the tandem repeat (IS1470)2, similar to the transposition intermediates described for a number of IS elements.     

Applications of transposition mutagenesis in antibiotic producing streptomycetes

Several transposons have been developed from the streptomycete insertion sequence IS493. They have broad host specificity in Streptomyces species and insert relatively randomly into a consensus target sequence of gNCaNTgNNy. Collectively, they have specialized features that facilitate the following: cloning of DNA flanking insertions; physical mapping of insertions; construction of highly stable mutants; and efficient construction of mutant libraries. All of the transposons can be introduced into streptomycetes by conjugation from E. coli, and can be delivered by curing the temperature sensitive delivery plasmid. Tn5099 was used to physically map genes involved in daptomycin and red pigment production in Streptomyces roseosporus, and to clone daptomycin biosynthetic genes. Tn5099 was also used in Streptomyces fradiae to identify and clone a neutral genomic site for the insertion of a second copy of the tylF gene. Recombinants containing two copies of the tylF gene carried out the normally rate limiting conversion of macrocin to tylosin very efficiently, thus causing substantial increases in tylosin yield.     

Complete nucleotide sequence of pSK41: evolution of staphylococcal conjugative multiresistance plasmids

The 46.4-kb nucleotide sequence of pSK41, a prototypical multiresistance plasmid from Staphylococcus aureus, has been determined, representing the first completely sequenced conjugative plasmid from a gram-positive organism. Analysis of the sequence has enabled the identification of the probable replication, maintenance, and transfer functions of the plasmid and has provided insights into the evolution of a clinically significant group of plasmids. The basis of deletions commonly associated with pSK41 family plasmids has been investigated, as has the observed insertion site specificity of Tn552-like beta-lactamase transposons within them. Several of the resistance determinants carried by pSK41-like plasmids were found to be located on up to four smaller cointegrated plasmids. pSK41 and related plasmids appear to represent a consolidation of antimicrobial resistance functions, collected by a preexisting conjugative plasmid via transposon insertion and IS257-mediated cointegrative capture of other plasmids.     

Formation of deletional derivatives of the Ti-plasmid pGV3850 in a conjugative transfer from Agrobacterium tumefaciens to Escherichia coli

The process of the transfer of the Ti-plasmid vector pGV3850 with the plasmid pBR322 inserted into the T-DNA region from Agrobacterium tumefaciens to a non-plasmid strain of Escherichia coli was studied. The transferred Ti-plasmid was found to be exposed to deletions and formed a wide range of derivatives with a size ranging from 3-4 kb to 50 kb, maintained in E. coli due to ColE1-replicon. The Ti-plasmid is also inserted into the chromosome of the recipient bacterium with at least a 100-fold lower frequency than the formation of deletional derivatives. It was shown that the induction of vir genes controlling the transfer of T-DNA into plants has no appreciable effect on the efficiency of obtaining transconjugates in mating with E. coli. The deletion of the genetic material of megaplasmids with the inserted functional site OriV ColE1, as a result of the conjugative transfer from cells of different bacteria to the cells of E. coli, was proposed for molecular cloning.     

Molecular cloning and nucleotide sequence of an endo-1,5-alpha-L-arabinase gene from Bacillus subtilis

The nucleotide sequence of the gene encoding an endo-1,5-alpha-L-arabinase (protopectinase C) of Bacillus subtilis was determined by sequencing fragments amplified by the cassette-ligation-mediated PCR (CLM-PCR). The gene covering the start and stop codon was amplified by PCR with two specific primers, which were designed from the sequence data determined by CLM-PCR. An approximately 1.5-kb amplification product was cloned into the vector pUC119, forming a plasmid termed pPPC. An ORF that encodes the arabinase composed of 324 amino acids including a 33-amino-acid signal peptide was assigned. Comparison of the deduced amino acid sequence of the enzyme with that of an Aspergillus niger endoarabinase showed 37% identity in a 207-amino-acid overlap. The optimal nucleotide sequence for catabolite repression of B. subtilis was found upstream of the structural gene. In a culture of Escherichia coli DH5alpha cells harboring pPPC, no arabinase activity was detected, either intracellularly or extracellularly, suggesting that the B. subtilis promotor is not functional in this transformant. In B. subtilis IFO 3134 strain, production of protopectinase C was repressed by readily metabolizable carbohydrates. In contrast, productivity (total enzyme activity/bacterial growth) of the enzyme was increased about fourfold in the presence of 0.75 M potassium phosphate in the culture medium. The phosphate anion seemed to be involved in the stimulation of protopectinase C production in this stain.