Horizontal Transfer of Tetracycline Resistance among Chlamydia spp. In Vitro

ABSTRACT There are no examples of stable tetracycline resistance in clinical strains of Chlamydia trachomatis. However, the swine pathogen Chlamydia suis is commonly tetracycline resistant, both in America and in Europe. In tested U.S. strains, this resistance is mediated by a genomic island carrying a tet(C) allele. In the present study, the ability of C. suis to mobilize tet(C) into other chlamydial species was examined. Differently antibiotic resistant strains of C. suis, C. trachomatis, and Chlamydia muridarum were used in coculture experiments to select for multiply antibiotic resistant progeny. Coinfection of mammalian cells with a naturally occurring tetracycline-resistant strain of C. suis and a C. muridarum or C. trachomatis strain containing selected mutations encoding rifampin (rifampicin) or ofloxacin resistance readily produced doubly resistant recombinant clones that demonstrated the acquisition of tetracycline resistance. The resistance phenotype in the progeny from a C. trachomatis L2/oflR-C. suis R19/tetR cross resulted from integration of a 40-kb fragment into a single ribosomal operon of a recipient, leading to a merodiploid structure containing three rRNA operons. In contrast, a cross between C. suis R19/tetR and C. muridarum MoPn/oflR led to a classical double-crossover event transferring 99 kb of DNA from C. suis R19/tetR into C. muridarum MoPn/oflR. Tetracycline resistance was also transferred to recent clinical strains of C. trachomatis. Successful crosses were not obtained when a rifampin-resistant Chlamydophila caviae strain was used as a recipient for crosses with C. suis or C. trachomatis. These findings provide a platform for further exploration of the biology of horizontal gene transfer in Chlamydia while bringing to light potential public health concerns generated by the possibility of acquisition of tetracycline resistance by human chlamydial pathogens.

thyA as a Selection Marker in Construction of Food-Grade Host-Vector and Integration Systems for Streptococcus thermophilus

ABSTRACT We constructed food-grade host-vector and integration systems for Streptococcus thermophilus by using a thymidylate synthase gene (thyA) as the selection marker. Two thyA genes, thyASt and thyALb , were cloned from S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively. Thymidine-requiring mutants of S. thermophilus were obtained after successive cultures in the presence of trimethoprim, and one of them, TM1-1, was used as the host. Food-grade vectors were constructed by using either thyASt or thyALb as the selection marker. Transformants of TM1-1 created by using these vectors were selected for thymidine autotrophy as efficiently as for erythromycin resistance. By using the host-vector system developed in this way, a foreign amylase gene (amyA) was expressed in TM1-1 and was also integrated into the chromosome by use of a temperature-sensitive integration vector constructed with thyALb as the selection marker via a double-crossover event. The results obtained show that thyA is an efficient and safe selection marker for S. thermophilus that is suitable for food applications.

Site-Directed Disruption of the fimA and fimF Fimbrial Genes of Xylella fastidiosa

Xylella fastidiosa causes Pierce's disease, a serious disease of grape, citrus variegated chlorosis, almond and oleander leaf scorches, and many other similar diseases. Although the complete genome sequences of several strains of this organism are now available, the function of most genes in this organism, especially those conferring virulence, is lacking. Attachment of X. fastidiosa to xylem vessels and insect vectors may be required for virulence and transmission; therefore, we disrupted fimA and fimF, genes encoding the major fimbrial protein FimA and a homolog of the fimbrial adhesin MrkD, to determine their role in the attachment process. Disruption of the fimA and fimF genes in Temecula1 and STL grape strains of X. fastidiosa was obtained by homologous recombination using plasmids pFAK and pFFK, respectively. These vectors contained a kanamycin resistance gene cloned into either the fimA or fimF genes of X. fastidiosa grape strains Temecula1 or STL. Efficiency of transformation was sufficiently high (≈600 transformants per μg of pFFK DNA) to enable selection of rare recombination events. Polymerase chain reaction and Southern blot analyses of the mutants indicated that a double crossover event had occurred exclusively within the fimA and fimF genes, replacing the chromosomal gene with the disrupted gene and abolishing production of the corresponding proteins, FimA or FimF. Scanning electron microscopy revealed that fimbriae size and number, cell aggregation, and cell size were reduced for the FimA¯ or FimF¯ mutants of X. fastidiosa when compared with the parental strain. FimA¯ or FimF¯ mutants of X. fastidiosa remained pathogenic to grapevines, with bacterial populations slightly reduced compared with those of the wild-type X. fastidiosa cells. These mutants maintained their resistance to kanamycin in planta for at least 6 months in the greenhouse.

Molecular Characterization of a Stress-Inducible Gene from Lactobacillus helveticus

ABSTRACT A gene (htrA) coding for a stress-inducible HtrA-like protein from Lactobacillus helveticus CNRZ32 was cloned, sequenced, and characterized. The deduced amino acid sequence of the gene exhibited 30% identity with the HtrA protein fromEscherichia coli; the putative catalytic triad and a PDZ domain that characterize the HtrA family of known bacterial serine proteases were also found in the sequence. Expression of the L. helveticus htrA gene in a variety of stress conditions was analyzed at the transcriptional level. The strongest induction, resulting in over an eightfold increase in the htrAtranscription level, was found in growing CNRZ32 cells exposed to 4% (wt/vol) NaCl. Enhanced htrA mRNA expression was also seen in CNRZ32 cells after exposure to puromycin, ethanol, or heat. The reporter gene gusA was integrated in theLactobacillus chromosome downstream of the htrApromoter by a double-crossover event which also interrupted the wild-type gene. The expression of gusA in the stress conditions tested was similar to that of htrA itself. In addition, the presence of an intact htrA gene facilitated growth under heat stress but not under salt stress.

Structure and biological activity of human homologs of the raf/mil oncogene.

Two human genes homologous to the raf/mil oncogene have been cloned and sequenced. One, c-raf-2, is a processed pseudogene; the other, c-raf-1, contains nine exons homologous to both raf and mil and two additional exons homologous to mil. A 3' portion of c-raf-1 containing six of the seven amino acid differences relative to murine v-raf can substitute for the 3' portion of v-raf in a transformation assay. Sequence homologies between c-raf-1 and Moloney leukemia virus at both ends of v-raf indicate that the viral gene was acquired by homologous recombination. Although the data are consistent with the traditional model of retroviral transduction, they also raise the possibility that the transduction occurred in a double crossover event between proviral DNA and the murine gene.

Structure and biological activity of human homologs of the raf/mil oncogene

Two human genes homologous to the raf/mil oncogene have been cloned and sequenced. One, c-raf-2, is a processed pseudogene; the other, c-raf-1, contains nine exons homologous to both raf and mil and two additional exons homologous to mil. A 3' portion of c-raf-1 containing six of the seven amino acid differences relative to murine v-raf can substitute for the 3' portion of v-raf in a transformation assay. Sequence homologies between c-raf-1 and Moloney leukemia virus at both ends of v-raf indicate that the viral gene was acquired by homologous recombination. Although the data are consistent with the traditional model of retroviral transduction, they also raise the possibility that the transduction occurred in a double crossover event between proviral DNA and the murine gene.