Introduction of the emm6 gene into an emm-deleted strain of Streptococcus pyogenes restores its ability to resist phagocytosis.

To complete the formal proof that the M protein functions to protect the group A streptococcus from phagocytosis and to lay the groundwork for the molecular genetic dissection of this major virulence factor, we have reintroduced a wild-type allele of the M protein gene (emm) into the chromosome of a strain from which it had previously been deleted. For this purpose, we used homologous recombination with an engineered plasmid that was introduced into the Streptococcus pyogenes chromosome by an electroporation technique that we developed. The plasmid pJRS120E was constructed in Escherichia coli and contains the structural gene for the antiphagocytic M6 protein (emm6) of S. pyogenes JRS4 and a selectable marker (ermAM) between the chromosomal regions that flank emm6 in S. pyogenes. This plasmid was introduced into S. pyogenes strain JRS75, a strain that is not resistant to phagocytosis as a result of the allelic replacement of the emm6 gene by a kanamycin resistance gene (aphA3). DNA hybridization analysis indicated that in erythromycin-resistant strain JRS115, homologous recombination resulted in the replacement of the aphA3 gene of JRS75 with the introduced emm6 allele. Strain JRS115 produces approximately the same amount of M6 as does the parental emm6+ strain JRS4, as assayed by Western blot analysis, and is resistant to phagocytosis.     

Nucleotide sequence of the gene for heat-stable enterotoxin II of Escherichia coli.

Previously, the gene for heat-stable enterotoxin II (STII) has been mapped by transposon Tn5 insertion mutagenesis in the chimeric R-Ent plasmid pCG86 (Mazaitis, A. J., R. Maas, and W. K. Maas, J. Bacteriol. 145:97-105, 1981). DNA segments containing this gene were cloned from the wild-type and STII-insertion-mutant plasmid. The position of the Tn5 insertion was determined, and a 530-base-pair-long segment of the wild-type plasmid corresponding to the Tn5 insertion site was sequenced. An open reading frame for the STII gene was identified and is characterized by typical promoter and ribosome binding site sequences. The deduced STII structural gene codes for a protein 71 amino acids long, including a typical signal peptide of 23 amino acids and a mature protein of 48 amino acids. The size and overall structure of STII are similar to those of STI, but the amino acid compositions of the two heat-stable enterotoxins are completely different.     

Molecular mechanisms of tetracycline and macrolide resistance and emm characterization of Streptococcus pyogenes isolates in Tunisia

Streptococcus pyogenes or group A Streptococcus, a major human pathogen, remains susceptible to beta-lactams, but resistance to other antibiotics is becoming more common. The purpose of this study was to characterize both phenotypic and genotypic epidemiological markers of group A Streptococcus and to identify the mechanisms of resistance to macrolides and tetracyclines. A total of 103 strains, isolated at Charles Nicolle University Hospital of Tunis, were investigated. The rate of resistance to erythromycin was low (5%), whereas a high rate of tetracycline resistance was found (70%). All the macrolide-resistant isolates expressed the constitutive macrolide, lincosamide, and streptograminB phenotype and harbored the erm(B) gene. Resistance to tetracycline was mainly due to the tet(M) gene, which is commonly associated with the conjugative transposon Tn916. No significant association was found between erm(B) and tet(M) genes. The tetracycline-resistant strains belonged to 28 distinct emm types. Among them, emm118 was the most prevalent type, followed by emm42, std432, emm76, and emm18. However, emm1, emm4, emm6, emm28, and emm3 were the most frequent types among tetracycline susceptible isolates. Only emm118 and emm42 types (p ≤ 0.05) were significantly associated with resistance to tetracycline.     

Disease symptoms in transgenic tobacco induced by integrated gene VI of cauliflower mosaic virus

A chimeric vector (pKR 612B1) containing the neomycin phosphotransferase (APH) gene from the Tn5 transposon under the control of the gene VI promoter of cauliflower mosaic virus (CaMV) and the cloned gene VI region (SalI-BstEII) of the same virus were used to cotransform tobacco protoplasts. Using the polyethylene glycol transformation procedure, a large number of protoplasts were transformed and proved to be resistant to kanamycin (Km). Whole Km-resistant plants were regenerated and shown to contain the integrated foreign genes. DNA from transformed clones was analyzed by Southern blot hybridization, showing the presence of the Tn5-derived gene and the viral gene. Transgenic plants containing the viral gene show mild mosaic patterns and fasciation. The expression of the gene VI product was detected by immunoblots.     

Tn916 insertional inactivation of multiple genes on the chromosome of Streptococcus mutans GS-5.

Streptococcus mutans GS-5 was transformed with the Escherichia coli plasmid pAM150 containing the cloned streptococcal transposon Tn916. Southern blot analyses with the tetracycline-resistant determinant of Tn916 showed that Tn916 was inserted into the chromosome of S. mutans at a variety of different sites. Tn916 insertions resulted in the inactivation of genes that code for various steps in the biosynthesis of several different amino acids. Two auxotrophs which contained a single copy of Tn916 were shown to revert to prototrophy at frequencies of about 10(-8). All of the revertant prototrophs were susceptible to tetracycline, indicating regeneration of the functional gene by excision of Tn916.     

Effects on virulence of mutations in a locus essential for hyaluronic acid capsule expression in group A streptococci.

Mucoid or highly encapsulated strains of group A streptococci have been associated both with unusually severe infections and with acute rheumatic fever. Previously, we described an acapsular mutant, TX4, derived from a mucoid M-type 18 strain of a group A streptococcus by transposon mutagenesis (M. R. Wessels, A. E. Moses, J. B. Goldberg, and T. J. DiCesare, Proc. Natl. Acad. Sci. USA 88:8317-8321, 1991). We now report studies further characterizing strain TX4 as well as an additional acapsular mutant, TX72. Strain TX4 was found to contain a 9.5-kb deletion of chromosomal DNA adjacent to the site of transposon Tn916 insertion. Cloned chromosomal DNA from TX4 flanking the transposon insertion site was used as a probe to demonstrate the presence of homologous regions in 11 of 11 wild-type group A streptococcal strains of various M protein types. A second acapsular mutant, TX72, had a single transposon insertion and had no apparent deletion of chromosomal DNA. The Tn916 insertion in TX72 was mapped to the hasA locus (encoding hyaluronate synthase), which lies within the chromosomal region deleted in TX4. Strain TX72 was avirulent in mice and sensitive to phagocytic killing in vitro. Transduction of either the insertion-deletion mutation from TX4 or the simple insertion mutation from TX72 to a type 24 group A streptococcus strain also resulted in loss of capsule expression, demonstrating that a homologous region of the chromosome controls capsule expression in another serotype of group A streptococci. We conclude that the hyaluronic acid capsule plays an important role in virulence and that a region of the chromosome essential for capsular polysaccharide expression is conserved among diverse group A streptococcal strains.     

A system for generalized mutagenesis of Haemophilus ducreyi.

The lack of a generalized mutagenesis system for Haemophilus ducreyi has hampered efforts to identify virulence factors expressed by this sexually transmitted pathogen. To address this issue, the transposable element Tn1545-delta 3, which encodes resistance to kanamycin, was evaluated for its ability to insert randomly into the H. ducreyi chromosome and produce stable, isogenic mutants. Electroporation of H. ducreyi with 1 microgram of plasmid pMS1 carrying Tn1545-delta 3 resulted in the production of 10(4) kanamycin-resistant transformants; Southern blot analysis of a number of these transformants indicated that insertion of the transposon into the chromosome occurred at a number of different sites. This pMS1-based transposon delivery system was used to produce an H. ducreyi mutant that expressed an altered lipooligosaccharide (LOS). Passage of this mutant in vitro in the presence or absence of kanamycin did not affect the stability of the transposon insertion. To confirm that the observed mutant phenotype was the result of the transposon insertion, a chromosomal fragment containing Tn1545-delta 3 was cloned from this H. ducreyi LOS mutant. Electroporation of the wild-type H. ducreyi strain with this DNA fragment yielded numerous kanamycin-resistant transformants, the majority of which had the same altered LOS phenotype as the original mutant. Southern blot analysis confirmed the occurrence of proper allelic exchange in the LOS-deficient transformants obtained in this backcross experiment. The ability of Tn1545-delta 3 to produce insertion mutations in H. ducreyi should facilitate genetic analysis of this pathogen.     

Characterization of vanA-containing Enterococcus faecium isolates carrying Tn5397-like and Tn916/Tn1545-like transposons in wild boars (Sus Scrofa)

The presence of vanA-containing Enterococcus faecium isolates was demonstrated in two of 67 fecal samples (3%) of healthy wild boars recovered in Portugal. One isolate per sample was studied further (E. faecium PG5V and PG48V). The mechanisms of resistance for other antibiotics were analyzed in these two vancomycin-resistant isolates, as well as the presence of genes related with virulence factors. The tet(M), tet(L), and erm(B) genes, associated with tetracycline or erythromycin resistance, were identified in both isolates, and the presence of aph(3')-IIIa gene (associated with kanamycin resistance) was detected in one of them. E. faecium PG48V exhibited an ampicillin minimum inhibitory concentration (MIC) of 64 microg/ml and showed eight amino acid substitutions in the carboxy-terminal end of the PBP5 protein (461Q --> K, 470H --> Q, 485M --> A, 496N --> K, 499A --> T, 525E --> D, 586V --> L, and 629E --> V) with respect to the reference sequence (GenBank accession number X84860). The purK-6 allele was identified in isolate PG48V, which also harbored the cylL(L)L(S), entA, and entB genes, encoding cytolysin, enterocin A, and enterocin B, respectively. The purK-3 allele was found in the PG5V isolate. The two vanA-containing isolates harbored the Tn916/Tn1545-like mobile element, and the Tn5397-like transposon was also found in isolate PG48V. The intestinal tract of wild boars could be a reservoir of vanA-containing enterococci.     

Detection of a genetic linkage between genes coding for resistance to tetracycline and erythromycin in Clostridium difficile

Elements carrying more than one antibiotic resistance gene have never been found in Clostridium difficile, one of the major causes of nosocomial diarrheic diseases. In this study, C. difficile isolates were investigated for a possible genetic linkage between tet(M) and erm(B), the most frequent genes found in strains resistant to tetracycline and erythromycin. In the majority of C. difficile strains, tet(M) is carried by Tn5397. However, tet(M) genes carried by Tn916-like elements have been found in recent clinical isolates. As far as erythromycin resistance is concerned, the only completely characterized transposon harboring an erm(B) gene in C. difficile is Tn5398, even if ErmB determinants probably carried by other elements have been identified. Among the 100 C. difficile isolates screened in this study, 27 were positive for tet(M) and erm(B). Twenty five of these strains were positive for tndX, used as marker for Tn5397, whereas two were positive for int, used as marker for Tn916-like elements. The latter isolates showed two tet(M) genes: one was carried by a Tn916-like element, able to transfer to a recipient C. difficile strain, whereas the second was genetically linked to an erm(B) in a composite element probably unable to conjugate. Molecular analysis of C. difficile cd1911 tet(M)-erm(B) DNA sequence demonstrated that this region has arisen by recombination of DNA fragments from different plasmids and transposons. This is the first demonstration that C. difficile is able to accumulate and maintain antibiotic resistance genes, as observed in other pathogens.     

Transposon Tn916 mutagenesis in Bacillus anthracis.

Mutagenesis of Bacillus anthracis by the streptococcal tetracycline resistance transposon Tn916 is described. Tn916 was transferred from Streptococcus faecalis DS16C1 to B. anthracis VNR-1 by conjugation in a standard filter mating procedure. Tetracycline-resistant (Tcr) transconjugants were obtained at a frequency of 1.6 X 10(-8) per donor CFU. When donor and recipient cells were treated with nafcillin before conjugation, the frequency was increased nearly 10-fold. Nafcillin pretreatment of donor and recipient strains was used in all subsequent conjugation experiments. S.faecalis CG110, containing multiple chromosomal insertions of Tn916, transferred the transposon to B. anthracis VNR-1 at a frequency of 9.3 x 10(-5). A Tcr B. anthracis transconjugant, strain VNR-1-tet-1, transferred Tn916 to B. anthracis UM23-1 and Bacillus subtilis BST1 at frequencies of 2.1 x 10(-4) and 5.8 X 10(-6), respectively. The transfer of Tn916 occurred only on membrane filters, since no Tcr transconjugants were obtained when strains VNR-1-tet-1 and UM23-1 were mixed and incubated in broth culture. The presence of the Tn916-associated tetM gene in Tcr B. anthracis and B. subtilis transconjugants was confirmed in hybridization experiments by using a 5-kilobase-pair DNA fragment containing the tetM gene as a probe. Of 3,000 B. anthracis UM23-1 Tcr transconjugants tested, 21 were phenylalanine auxotrophs and 2 were auxotrophic for phenylalanine, tyrosine, and tryptophan.     

Insertion of Tn916 in Neisseria meningitidis resulting in loss of group B capsular polysaccharide.

We recently found that the 16.4-kb conjugative transposon Tn916 could be introduced into Neisseria meningitidis by transformation and that it appeared to transpose to many different sites in the chromosome of recipient meningococci. In order to identify transposon-induced alterations of specific meningococcal virulence determinants, a library of meningococcal Tetr transformants containing Tn916 was made and screened for those altered in the production of group B capsular polysaccharide. A capsule-defective mutant, M7, was identified by using monoclonal and polyclonal antisera to group B polysaccharide in immunoblot and agar antiserum procedures. Growth of M7 was similar to that of the parent strain. M7 produced no group B capsular polysaccharide by rocket immunoelectrophoresis, and the mutation was stable during laboratory passage. The capsule-defective phenotype was linked to Tetr, as demonstrated by immunoblot and Southern blot analysis of progeny Tetr transformants (transformants of the parent strain obtained with DNA from M7). A capsule-deficient mutant, O8, was identified by using a similar approach. Analysis of the Tn916 insertions in M7 and O8 indicated that a significant portion of the transposon on either side of the tetM determinant had been lost. The ability of Tn916 to generate defined, stable mutations in meningococcal virulence determinants is demonstrated by our study.     

Electroporation of a suicide plasmid bearing a transposon into Brucella abortus

The creation of bacterial mutants by transposon mutagenesis has facilitated the identification of regulatory and structural genes. In the case of B. abortus the number of reported transposon mutants created by mating or P1 infection has been relatively small. We studied the conditions necessary to introduce Tn5 bearing a kanamycin resistance gene (KnR) into B. abortus by electroporation. The highest frequency of Tn5 transposition was obtained using B. abortus 2308 harvested at a density of 5.2 x 10(8) cells/ml; 0.5 microgram of plasmid was electroporated for 10 ms at 625 V (equivalent to 12.5 kV/cm). The frequency of Tn5 transposition obtained under optimum conditions was estimated to be around 18-20 insertions per 10(10) Brucella. The phase of growth (or the number of generations) had a strong influence on the frequency of transposition. Dot blot analysis confirmed that all KnR clones appearing after 4 days of incubation at 37 degrees C carried Tn5 in their genomes. Furthermore, the randomnes of Tn5 insertion was verified by Southern analysis of chromosomal DNA extracted from knR clones and hybridized with labeled Tn5.     

Expression of K99 adhesion antigen controlled by the Escherichia coli tryptophan operon promoter.

The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [O101:K99] has been cloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J. D. A. van Embden, F. K. de Graaf, L. M. Schouls, and J. S. Teppma, Infect. Immun. 29:1125-1133, 1980). Cells harboring one such construction, known as pK99-64, are capable of expressing K99 antigen on the cell surface. We replaced the natural promoter sequence for the gene encoding the K99 pilus subunit with a strong, inducible exogenous promoter, the E. coli tryptophan (trp) operon promoter, to construct the plasmid pBR-TrpK99. E. coli cells harboring pBR-TrpK99 or a similar construction in the plasmid pDR540, known as pKO-TrpK99, upon induction with 3-beta-indoleacrylic acid, produced about fourfold more K99 antigen than did cells bearing pK99-64 with the natural promoter. Expression of the pilus antigen was found to be under control of the tryptophan promoter. Plasmid instability was encountered, however, in cells bearing pKO-TrpK99 when the trp promoter was derepressed. Introduction of the aminoglycoside 3'-phosphotransferase gene of transposable element Tn5 into pKO-TrpK99 to generate pKON-TrpK99 effectively stabilized the plasmid in cells grown under identical conditions in medium containing kanamycin.     

Cloning of the filamentous hemagglutinin of Bordetella pertussis and its expression in Escherichia coli.

Bordetella pertussis UT25 DNA was cloned into the kanamycin resistance gene of cosmid pCP13 to construct a genomic library in Escherichia coli LE392. One clone containing plasmid pDB441 expressed the filamentous hemagglutinin (FHA) as identified by protein immunoblots with the use of rabbit anti-B. pertussis antiserum, rabbit anti-FHA antiserum, and a monoclonal antibody to FHA. FHA is a protein of 220 to 210 kilodaltons, but the immunoreactive FHA, as expressed in E. coli, was larger than that expressed in B. pertussis, suggesting that there was a difference in the processing of this protein between these two bacteria. The fha gene was mapped to a 6.5-kilobase pair DNA fragment by the use of various restriction endonucleases. The kanamycin resistance gene of pCP13 was found to provide the promoter function but probably not the translation start signal for the fha gene. Conjugative transfer of pDB441 to B. pertussis BP353, a transposon Tn5-induced FHA mutant, increased the expression of the FHA over that seen with wild-type B. pertussis.     

Chromosomal map location of the alpha-hemolysin structural gene in Staphylococcus aureus NCTC 8325.

The alpha-hemolysin structural gene, hly+, previously cloned, insertionally inactivated, and introduced into the chromosome by allele replacement (M.O. O'Reilly, J.C.S. De Azavedo, S. Kennedy, and T.J. Foster, Microb. Pathog. 1:125-138, 1986), was shown by protoplast fusion and transformation to be in the gene order purC-hly-uraB-omega[chr::Tn916]1101-thrB on the chromosome of Staphylococcus aureus NCTC 8325. This location is clearly distinct from that of the agr determinant, a regulatory gene affecting several extracellular proteins, including alpha-hemolysin, located between tmn and ilv.     

Distribution of the erm (B) gene, tetracycline resistance genes, and Tn1545-like transposons in macrolide- and lincosamide-resistant enterococci from pigs and humans

The distribution of the erm (B) and the tetracycline resistance genes tet(K), tet(L), tet(M), tet(O), and tet(S) was investigated among macrolide- and lincosamide-resistant enterococci originating from humans, pigs, and pork carcasses. The presence of transposons of the Tn916/Tn1545 family was also traced in these isolates. Furthermore, the porcine strains were tested for the presence of glycopeptide resistance genes vanA and vanB. The erm(B) gene was found in 85% of the porcine and in all human isolates. Ninety-eight percent of the porcine and 89% of the human erm(B)-positive enterococci carried the tet(M) gene. Seventy-seven percent and 70%, respectively, of these strains harbored a Tn1545-like element. Tet(L) was observed in 68% of the porcine and in 65% of the human enterococci. The other tetracycline resistance genes were very rare and the glycopeptide resistance genes vanA and vanB were not detected among the porcine isolates. The similar frequencies of resistance genes and the highly mobile Tn1545-like transposon among porcine and human enterococci might indicate exchange of resistant strains or their resistance genes between humans and pigs or the existence of a common reservoir.     

GAA trinucleotide repeat region regulates M9/pMGA gene expression in Mycoplasma gallisepticum

Mycoplasma gallisepticum, the cause of chronic respiratory infections in the avian host, possesses a family of M9/pMGA genes encoding an adhesin(s) associated with hemagglutination. Nucleotide sequences of M9/pMGA gene family members indicate extensive sequence similarity in the promoter regions of both the transcribed and silent genes. The mechanism that regulates M9/pMGA gene expression is unknown, but studies have revealed an apparent correlation between gene expression and the number of tandem GAA repeat motifs located upstream of the putative promoter. In this study, transposon Tn4001 was used as a vector with the Escherichia coli lacZ gene as the reporter system to examine the role of the GAA repeats in M9/pMGA gene expression in M. gallisepticum. A 336-bp M9 gene fragment (containing the GAA repeat region, the promoter, and the translation start codon) was amplified by PCR, ligated with a lacZ gene from E. coli, and inserted into the Tn4001-containing plasmid pISM2062. This construct was transformed into M. gallisepticum PG31. Transformants were filter cloned on agar supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to monitor lacZ gene expression on the basis of blue/white color selection. Several cycles of filter cloning resulted in cell lineages in which lacZ gene expression alternated between the On and Off states in successive generations of progeny clones. The promoter regions of the M9-lacZ hybrid genes of individual progeny clones were amplified by PCR and sequenced. The only differences between the promoter regions of the blue and white colonies were in the number of GAA repeats. Clones that expressed lacZ had exactly 12 tandem copies of the GAA repeat. Clones that did not express lacZ invariably had either more than 12 (14 to 16) or fewer than 12 (5 to 11) GAA repeats. Southern analysis of M. gallisepticum chromosomal DNA confirmed that the phase-variable expression of the lacZ reporter gene was not caused by Tn4001 transposition. These data strongly indicate that changes in the length of the GAA repeat region are responsible for regulating M9/pMGA gene expression.     

Presence and mechanism of antimicrobial resistance among enterococci from cats and dogs

The presence and mechanism of acquired resistance to erythromycin, tylosin, lincomycin, quinupristin/dalfopristin, tetracycline, chloramphenicol, gentamicin, kanamycin, and vancomycin were determined in 97 and 104 enterococci isolated from rectal swabs of cats and dogs, respectively. Eleven feline and three canine enterococcal isolates contained the aac(6')-Ie-aph(2')-Ia gene encoding high-level resistance to gentamicin, an antibiotic often used for treating enterococcal infections in humans. The combination of erm(B) and vat(E) genes encoding resistance to streptogramins was detected in one canine quinupristin/dalfopristin-resistant Enterococcus faecium isolate. Four quinupristin/dalfopristin-resistant enterococci only contained the erm(B) gene. Cross resistance against macrolides and lincosamides (30%) and resistance against tetracyclines (55%) was found to be widely distributed among enterococci from pets. In all of the feline and in 93% of the canine macrolide and lincosamide-resistant isolates, this resistance was encoded by the erm(B) gene. tet(M) was the most prevalent tetracycline resistance gene. It was detected in 91% of the feline and 86% of the canine tetracycline- resistant enterococci. A high occurrence of the Tn916/Tn1545 transposon family was found among these tet(M)-positive isolates. Enterococci from pet animals with resistance against vancomycin were not found. This study shows that enterococci from the intestinal microbiota of cats and dogs may act as a reservoir of resistance genes for animal or human pathogens.