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.

Genetic and Enzymatic Properties of Metallo-β-Lactamase VIM-5 from a Clinical Isolate of Enterobacter cloacae

ABSTRACT A VIM-5-producing Enterobacter cloacae isolate (EDV/1) was identified in a collection of clinical strains stored before 2002. The gene, bla VIM-5, was located on a 2,712-bp BamHI-HindIII fragment of a 23-kbp (approximately) nonconjugative plasmid (pEDV5) in a class 1 integron as a single gene cassette.

Characterization of Transcriptional Regulatory Genes for Biphenyl Degradation in Rhodococcus sp. Strain RHA1

ABSTRACT Transcription of the bphA1A2A3A4C1B genes, which are responsible for the conversion of biphenyl and polychlorinated biphenyl to the meta-cleavage products in Rhodococcus sp. strain RHA1, was examined. The bphA1 promoter (P bphA1 ) was identified and was shown to promote transcription induction by biphenyl and ethylbenzene. An 8.8-kb HindIII fragment that promotes transcription induction of P bphA1 in Rhodococcus erythropolis IAM1399 was isolated from the region downstream of bphB by using a reporter plasmid containing P bphA1 . Analysis of the nucleotide sequence of this fragment revealed a set of putative two-component regulatory system genes, which were designated bphS and bphT. Deletion analysis of the 8.8-kb HindIII fragment indicated that bphT is responsible for the basal activation of P bphA1 and that both bphS and bphT are required for the elevated basal activation of and transcriptional induction by biphenyl of P bphA1 . These results support the notion that bphS and bphT encode a sensor kinase and a response regulator, respectively, of a two-component regulatory system. The bphS and bphT genes promote transcriptional induction by a variety of aromatic compounds, including biphenyl, benzene, alkylbenzenes, and chlorinated benzenes. A promoter activity assay and reverse transcription (RT)-PCR analysis revealed a weak constitutive promoter in the adjacent region upstream of bphS. RT-PCR analysis indicated that there is induced transcription of bphA1 through bphT, in which P bphA1 is thought to take part. An insertionally inactivated bphS mutant, SDR1, did not grow on biphenyl. Growth was restored by introduction of an intact bphS gene into SDR1. These results indicate that at least bphS is indispensably responsible for the growth of RHA1 on biphenyl.

Mutation in GDP-Fucose Synthesis Genes of Sinorhizobium fredii Alters Nod Factors and Significantly Decreases Competitiveness to Nodulate Soybeans

We mutagenized Sinorhizobium fredii HH103-1 with Tn5- B20 and screened about 2,000 colonies for increased β-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3′ end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5′ end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-Lfucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into noeL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.

A 150-megadalton plasmid in Rhizobium etli strain TAL182 contains genes for nodulation competitiveness on Phaseolus vulgaris L.

Rhizobium etli TAL182, a competitive strain for the nodulation of Phaseolus beans, occupied more than 99% of the nodules when co-inoculated in various proportions with Rhizobium TAL1145 or Rhizobium tropici CIAT899. Two overlapping cosmid clones, pUHR68 and pUHR69, containing genes for nodulation competitiveness from TAL182, were isolated by functional complementation of strain TAL1145. Using one of these cosmid clones, we constructed two Tn5-insertion mutants of TAL182 defective in nodulation competitiveness. The Tn5 insertions in both mutants were localized in identical positions within a 4.6-kb HindIII fragment. One mutant, RUH120, was complemented for nodulation competitiveness by this HindIII fragment. The cloned DNA in pUHR68 is a part of a plasmid, 150 MDa in size, in TAL182 and does not show homology with TAL1145 genomic DNA. The 4.6-kb HindIII fragment contains a gene(s) required for nodulation competitiveness on beans, which is present only in some R. etli strains and absent in other Rhizobium spp.Key words: nitrogen fixation, competition, nodule, common bean.

Conservation of a satellite DNA sequence (SATB) in the tilapiine and haplochromine genome (Pisces: Cichlidae)

We have cloned and sequenced a 1900-bp EcoRI fragment (SATB) from the tilapiine fish Oreochromis niloticus. The SATB sequence is highly reiterated in the tilapiine genome and organized in long tandem arrays. A 760-bp HindIII fragment, an internal component of SATB, has also been cloned and sequenced from the related tilapiine species Oreochromis hornorum. Hybridization of the radiolabeled 760-bp HindIII repeat detected the presence of the SATB repeat in the genomes of several tilapiine species as well as the haplochromine species Haplochromis (Protomelas) similis. The 760-bp HindIII fragment did not hybridize to genomic DNA of Etroplus maculatus (an Asian cichlid) or to that of Cichlasoma meeki (a South American cichlid). The SATB repeat sequence is 56% AT and constitutes 0.2–5% of the tilapiine genome depending on the species examined. Four imperfect 21-bp direct repeat sequences are present within the cloned 1900-bp EcoRI repeat. Alignment of the four direct repeats from the O. niloticus cloned 1900-bp DNA and the two homologous direct repeats from the O. hornorum 760-bp HindIII repeat revealed a core motif of 11 bp that exhibits 100% sequence identity between all of the direct repeats. The conservation of this motif in the SATB repeat suggests that this sequence may be under selective constraint.Key words: satellite DNA, Cichlidae, direct repeats.

Frequent changes in the number of reiterated ribosomal RNA genes throughout the life cycle of the basidiomycete Coprinus cinereus.

We have examined the stability of the tandemly repeated genes that encode the ribosomal RNA in Coprinus cinereus. These genes are contained within two linked HindIII fragments in a 3.0-Mb chromosome. We monitored the size of these fragments in both mitotic and meiotic segregants using the contour-clamped homogeneous electric field (CHEF) method. No length changes were observed in the smaller HindIII fragment (100 kb; 10 repeats) among the DNAs prepared from 46 asexual spore derivatives (oidia) or 128 meiotic segregants (basidiospores from 32 tetrads). However, the larger HindIII fragment (1100 kb; 120 repeats) did exhibit variability. Substantial changes, involving up to 40% of the larger HindIII fragment were recorded in 7 of 46 oidial isolates (including 4 of 22 transformed derivatives). To learn if the changes were confined to the vegetative portion of the life cycle, we examined transmission of HindIII variants through three crosses. In the first two crosses (16 tetrads total), no changes were observed in the large HindIII fragment. However, in the third cross (16 tetrads), each tetrad showed at least one alteration. In half of the tetrads from the third cross, the altered patterns segregated 2:2, suggesting that the changes occurred after mating but prior to premeiotic DNA replication. We conclude that breakage and rejoining reactions within the rDNA are frequent and are not confined to any particular stage of the life cycle. It also appears that certain repeats are sheltered from these events. Finally, marked differences in rDNA stability were observed in the cross analyzed.