Emergence of Macrolide Resistance Gene mph(B) in Streptococcus uberis and Cooperative Effects with rdmC-Like Gene

ABSTRACT Streptococcus uberis UCN60 was resistant to spiramycin (MIC = 8 μg/ml) but susceptible to erythromycin (MIC = 0.06 μg/ml), azithromycin (MIC = 0.12 μg/ml), josamycin (MIC = 0.25 μg/ml), and tylosin (MIC = 0.5 μg/ml). A 2.5-kb HindIII fragment was cloned from S. uberis UCN60 DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to spiramycin by inactivation. The sequence analysis of the fragment showed the presence of an rdmC-like gene that putatively encoded a protein belonging to the alpha/beta hydrolase family and of the first 196 nucleotides of the mph(B) gene putatively encoding a phosphotransferase known to inactivate 14-, 15-, and 16-membered macrolides in E. coli. The entire mph(B) gene was then identified in S. uberis UCN60. The two genes were expressed alone or in combination in E. coli, Staphylococcus aureus, and Enterococcus faecalis. Analysis of MICs revealed that rdmC-like alone did not confer resistance to erythromycin, tylosin, and josamycin in those three hosts. It conferred resistance to spiramycin in E. coli and E. faecalis but not in S. aureus. mph(B) conferred resistance in E. coli to erythromycin, tylosin, josamycin, and spiramycin but only low levels of resistance in E. faecalis and S. aureus to spiramycin (MIC = 8 μg/ml). The combination of mph(B) and rdmC-like genes resulted in a resistance to spiramycin and tylosin in the three hosts that significantly exceeded the mere addition of the resistance levels conferred by each resistance mechanism alone.

Study of Abnormal NHEJ Function in Human Myeloid Leukemia.

Non-homologous end joining (NHEJ) is a major mechanism by which eukaryote cells can repair the DNA double-strand break (DSB) and protect the cell from further damages. Evidences have suggested that the genomic instability caused by deficient DNA repair function may contribute to the development of solid tumor, while its role in leukemogenesis has not been adequately studied. To study the NHEJ function in myeloid leukemia, an in vitro system was developed for clinical samples by using the linear plasmid pUC18 DNA digested with EcoR I as assay system.. Nuclear protein extracted from leukemic cells and mononuclear cells (MNCs) from normal BM or PB, was incubated with linear plasmid pUC18 DNA under certain conditions. Plasmid DNA was separated by agarose gel electrophoresis and imaged by SYBR greenIstaining. End-ligation efficiency was assessed by dividing the densitometry readings for the sum of all converted plasmid products by the sum of all products. This assay was performed on 7 myeloid leukemia cell lines, 16 samples of normal BM or PB cells, 20 cases of CML cells and 19 cases of de novo AML cells. E. coli strain DH5α was electrotransformed with pUC18 DNA end-joined by nuclear proteins from normal BM or leukemia cells, and was plated on LB agar, containing X-gal and IPTG. Correct ligation of cut plasmid DNA resulted in blue colonies while faulty repair would result in white colonies. The percentage of white colonies over total colonies gave the frequency of misrepair. Primers around the EcoR I site were designed and colony PCR was performed on blue and white colonies. Sequencing of PCR products was performed. Antibodies against the nuclear repair proteins Ku70, Ku86 and DNA-PKcs were used for antibody abrogation studies. We have found that the mean repair efficiency of normal MNCs was 18.6±13.1% (0~46.6%),. The ligation efficiencies in myeloid leukemic cell lines ranged 10.6%~31.0% (mean 22.4%, P>0.05). The mean ligation efficiency was significantly higher in CML cells as compared with normal MNCs (24.8±14.9% v.s 18.6%, p=0.024). The DNA repair capacity of de novo acute myeloid leukemia cells(7.2%~76.9%)was markedly increased as compared with normal MNCs (mean 41.1±15.4% v.s 18.6%, p<0.0001 ). The mean frequency of misrepair from AML and CML cells were considerable higher (AML, 8.17% and CML, 2.10%) than that of normal BM cells (0.91%). Most misrepair were small deletions near EcoR I site. Large deletions (>100bp) were found from assays with AML cells. Three abnormalities of sequence deletion, inversion and insertion were found in a rare misrepair white colony with AML cells. The DNA end-ligation efficiencies of AML cells could be dramatically inhibited by antibodies against proteins Ku70, Ku86 and DNA-PK. We concluded that the Cell-free NHEJ assay system can be used to examine the clinical leukemic samples. NHEJ efficiencies were slightly enhanced in most myeloid leukemia cell lines compared with normal MNCs. The end-ligation efficiency was significantly higher in primary myeloid leukemia cells. The overactive but more error-prone NHEJ function relied on the activity of Ku and DNA-PK proteins for DSB repair may contribute to genomic instability in AML cells.

New lnu(C) Gene Conferring Resistance to Lincomycin by Nucleotidylation in Streptococcus agalactiae UCN36

ABSTRACT Streptococcus agalactiae UCN36 was resistant to lincomycin (MIC = 16 μg/ml) but susceptible to clindamycin (MIC = 0.12 μg/ml) and erythromycin (MIC = 0.06 μg/ml). A 4-kb HindIII fragment was cloned from S. agalactiae UCN36 total DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to lincomycin. The sequence analysis of the fragment showed the presence of a 1,724-bp element delineated by imperfect inverted repeats (22 of 25 bp) and inserted in the operon for capsular synthesis of S. agalactiae UCN36. This element carried two open reading frames (ORF). The deduced amino acid sequence of the upstream ORF displayed similarity with transposases from anaerobes and IS1. The downstream ORF, lnu(C), encoded a 164-amino-acid protein with 26% to 27% identity with the LnuAN2, LnuA, and LnuA′ lincosamide nucleotidyltransferases reported for Bacteroides and Staphylococcus, respectively. Crude lysates of E. coli AG100A containing the cloned lnu(C) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl2. Mass spectrometry experiments demonstrated that the LnuC enzyme catalyzed adenylylation of lincomycin.

Cloning of the rDNA repeat unit from a British entomopathogenic nematode (Steinernematidae) and its potential for species identification

SUMMARYThe entire ribosomal DNA repeat unit of a steinernematid species (Nashes isolate) was cloned as three separate EcoR I fragments in the plasmid pUC18. An equimolar cocktail of these three clones was used to identify Steinernema species on Southern blots as each species displays its own unique restriction fragment length polymorphisms. The clones also identified two new species isolated in a soil survey of coastal regions of Britain. One of the clones (pSn4.0) can detect length heterogeneities in the rDNA repeat unit of various isolates of some of the species, particularly the most common in the United Kingdom, S. feltiae. These differences in the rDNA repeat unit length remained constant over several years for one isolate of S. feltiae, but were different for each of the geographical isolates studied to date.

Plasmids in bacteria exposed to activated neutrophils mediate mutagenesis when transferred to new hosts

The plasmid pUC18 contains a lacZ alpha-complementation gene that codes for a small peptide that can complement the delta M15 mutation of the Escherichia coli lacZ (beta-galactosidase) gene, converting bacteria carrying that mutated gene from the lacZ- to the lacZ+ phenotype. This plasmid was used in experiments designed to study mutagenesis by human neutrophils. E coli carrying pUC18 were incubated with neutrophils under conditions in which little ingestion of the bacteria took place; the plasmid was then isolated and transformed into an E coli strain (BOZO) that carries the lacZ delta M15 mutation. Of these transformants, 11 of 205,000 were lacZ, suggesting that in these 11, alpha-complementation had been lost through a mutation. No lac- colonies were detected among several hundred thousand BOZO transformed with plasmid isolated from incubations in which phagocytosis could take place, nor from incubations from which neutrophils were omitted. Despite the lac- phenotype of these 11 transformants, plasmids reisolated from nine of them showed normal alpha-complementing ability when transformed into fresh BOZO. These findings indicated that in these nine, the mutations were located in the chromosomes of the transformed BOZO. It thus appears that on exposure to activated neutrophils, a plasmid may acquire a lesion (? mutation) that can somehow be transferred to the genome of a recipient microorganism, resulting in repair of the damaged plasmid accompanied by mutation of the recipient's chromosome. Restriction mapping of the DNA from four of these nine chromosomal mutants suggested that the mutations did not represent major insertions or deletions in the portion of the bacterial chromosome corresponding to the pUC18 lac operon insert, nor in the remainder of the lacZ delta M15 gene. These results confirm previous work showing that exposure to activated neutrophils can induce mutations in biological systems, and provides an experimental model in which the mechanism of neutrophil-mediated mutagenesis may be examined.

Plasmids in bacteria exposed to activated neutrophils mediate mutagenesis when transferred to new hosts

The plasmid pUC18 contains a lacZ alpha-complementation gene that codes for a small peptide that can complement the delta M15 mutation of the Escherichia coli lacZ (beta-galactosidase) gene, converting bacteria carrying that mutated gene from the lacZ- to the lacZ+ phenotype. This plasmid was used in experiments designed to study mutagenesis by human neutrophils. E coli carrying pUC18 were incubated with neutrophils under conditions in which little ingestion of the bacteria took place; the plasmid was then isolated and transformed into an E coli strain (BOZO) that carries the lacZ delta M15 mutation. Of these transformants, 11 of 205,000 were lacZ, suggesting that in these 11, alpha-complementation had been lost through a mutation. No lac- colonies were detected among several hundred thousand BOZO transformed with plasmid isolated from incubations in which phagocytosis could take place, nor from incubations from which neutrophils were omitted. Despite the lac- phenotype of these 11 transformants, plasmids reisolated from nine of them showed normal alpha-complementing ability when transformed into fresh BOZO. These findings indicated that in these nine, the mutations were located in the chromosomes of the transformed BOZO. It thus appears that on exposure to activated neutrophils, a plasmid may acquire a lesion (? mutation) that can somehow be transferred to the genome of a recipient microorganism, resulting in repair of the damaged plasmid accompanied by mutation of the recipient's chromosome. Restriction mapping of the DNA from four of these nine chromosomal mutants suggested that the mutations did not represent major insertions or deletions in the portion of the bacterial chromosome corresponding to the pUC18 lac operon insert, nor in the remainder of the lacZ delta M15 gene. These results confirm previous work showing that exposure to activated neutrophils can induce mutations in biological systems, and provides an experimental model in which the mechanism of neutrophil-mediated mutagenesis may be examined.

Natural Transformation of Plasmid DNA in Escherichia coli in Sterilized Soil Column

The present study was carried out to assess transformability of natural and laboratory strains of Escherichia coli by plasmid DNA under different transformation conditions in sterilized soil column. Transformation experiments were carried out in laboratory conditions and in sterile soil columns with CaCl2-treated competent cells and non-competent cells at log phase and stationary phase of growth using the broad host range plasmid pUC18. In soil column experiments, transformants were obtained after CaCl2 induced competence in both E. coli K12 DH5α and strain BM09 in the frequency of 10-8 to 10-9. In natural transformation assays, transformants appeared only in log phase cells of strain DH5α at a lower frequency (5.0 x 10-9), and in CaCl2-competent BM09 cells, but not in fresh cells. Thus the major limiting factor for natural transformation in environmental E. coli in soil column is probably the absence of a competent state. The significance of this finding has been discussed with respect to generally observed lower antibiotic resistance in environmental E. coli isolates from aquatic sources. Keywords: Natural transformation; Plasmid DNA; Escherichia coli; Competent stateDOI: http://dx.doi.org/10.3329/bjm.v25i1.4856 Bangladesh J Microbiol, Volume 25, Number 1, June 2008, pp 49-52