Cloning and nucleotide base sequence analysis of a spectinomycin adenyltransferase AAD(9) determinant from Enterococcus faecalis

LeBlanc, Donald J.; Lee, L N; Inamine, Julia M.

doi:10.1128/aac.35.9.1804

Cited by 154 publications

(107 citation statements)

References 27 publications

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“…Insertion-duplication mutagenesis using the pJDC9-derivative pEGR91 containing an internal fragment of the ciaR gene has been described (14). Alternatively, the gene was disrupted by insertion of the spectinomycin resistance gene aad9 from pDL278 (31), and no differences in phenotypes from the pJDC9 derivative was noted. The R6ciaR null (Spc r ) mutant (R6ciaR::aad9) has been described (36).…”

Section: Methodsmentioning

confidence: 99%

The CiaRH System ofStreptococcus pneumoniaePrevents Lysis during Stress Induced by Treatment with Cell Wall Inhibitors and by Mutations inpbp2xInvolved in β-Lactam Resistance

et al. 2006

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The two-component signal-transducing system CiaRH of Streptococcus pneumoniae plays an important role during the development of beta-lactam resistance in laboratory mutants. We show here that a functional CiaRH system is required for survival under many different lysis-inducing conditions. Mutants with an activated CiaRH system were highly resistant to lysis induced by a wide variety of early and late cell wall inhibitors, such as cycloserine, bacitracin, and vancomycin, and were also less susceptible to these drugs. In contrast, loss-of-function CiaRH mutants were hypersusceptible to these drugs and were apparently unable to maintain a stationary growth phase in normal growth medium and under choline deprivation as well. Moreover, disruption of CiaR in penicillin-resistant mutants with an altered pbp2x gene encoding low-affinity PBP2x resulted in severe growth defects and rapid lysis. This phenotype was observed with pbp2x genes containing point mutations selected in the laboratory and with highly altered mosaic pbp2x genes from penicillin-resistant clinical isolates as well. This documents for the first time that PBP2x mutations required for development of beta-lactam resistance are functionally not neutral and are tolerated only in the presence of the CiaRH system. This might explain why cia mutations have not been observed in penicillin-resistant clinical isolates. The results document that the CiaRH system is required for maintenance of the stationary growth phase and for prevention of autolysis triggered under many different conditions, suggesting a major role for this system in ensuring cell wall integrity.

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Section: Methodsmentioning

confidence: 99%

The CiaRH System ofStreptococcus pneumoniaePrevents Lysis during Stress Induced by Treatment with Cell Wall Inhibitors and by Mutations inpbp2xInvolved in β-Lactam Resistance

et al. 2006

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“…S. pneumoniae D39 derivatives with replacement mutations in the psa genes were constructed by using plasmid pCZA342 (Hoskins et al, 1999), which encodes apramycin (for selection in E. coli) and erythromycin (for selection in S. pneumoniae) resistance markers. The strategy for constructing null mutants requires generation of a construct containing a spectinomycinresistance marker (LeBlanc et al, 1991) flanked by PCRgenerated fragments upstream and downstream of the gene to be mutated cloned into pCZA342. Plasmid pCZA342 can replicate in E. coli but not in S. pneumoniae so transformants are screened for an erythromycin-sensitive, spectinomycinresistant phenotype.…”

Section: Methodsmentioning

confidence: 99%

In vivo characterization of the psa genes from Streptococcus pneumoniae in multiple models of infection

et al. 2002

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Differential fluorescence induction technology was used to identify promoters of Streptococcus pneumoniae genes that are expressed during lung infection of the mouse. Among the promoter clones that were identified multiple times was the psa promoter, which drives expression of the psaBCA operon. These genes have been identified previously and shown to encode a manganese permease system as well as play a role in the virulence of this organism. Mutations in psaB, psaC or psaA result in growth limitation in low manganese. The expression of the psa operon was examined in vivo and the virulence of deletion mutants of psaB, psaC, psaA and psaBCA was assessed in four different animal models of infection. The psa promoter was induced more than ten-fold in vivo using an intraperitoneal chamber implant model. The psaB, psaC and psaA mutants were completely attenuated in systemic, respiratory tract and otitis media infections. In addition, these mutants were unable to grow in an implanted peritoneal chamber, but growth was restored by the addition of manganese to the chambers.

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“…In the vicR mutant, there was no transcription termination sequence between the aad gene, which included its native promoter (LeBlanc et al, 1991), and the 39 fragment of the interrupted vicR gene. The transcript levels of vicK and vicX in vicR : : aad were down-regulated compared with the wt strain (Fig.…”

Section: Genes Potentially Regulated By Vicrkmentioning

confidence: 99%

Defects in ex vivo and in vivo growth and sensitivity to osmotic stress of group A Streptococcus caused by interruption of response regulator gene vicR

et al. 2006

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The regulator VicR of the two-component regulatory system VicRK is essential in several Gram-positive bacteria. However, the authors were able to generate an unconditional vicR insertional mutant of group A Streptococcus. This mutant grew well in rich media but not in non-immune human blood and serum, had attenuated virulence, and was unstable in mice. Complementation of the mutant with vicR expressed in trans restored its phenotype to wild-type. A vicK deletion mutant had a phenotype similar to that of the vicR mutant. Phagocytosis and killing of the vicR mutant were normal, suggesting that VicRK does not regulate processes involved in evasion of host defence. Microarray analysis showed that vicR inactivation down-regulated the transcription of 13 genes, including putative cell wall hydrolase gene pcsB and spy1058–1060, which encode a putative phosphotransferase system enzyme II for carbohydrate transport, and upregulated the expression of five genes, including spy0183 and spy0184, which encode an osmoprotectant transporter OpuA. Consistent with microarray analysis, the vicR mutant took up more of the osmoprotectants betaine and proline and was sensitive to osmotic stress, indicating that vicR inactivation induced osmotic stress and increased susceptibility to osmotic pressure. Additionally, a spy1060 deletion mutant also displayed attenuated virulence. These results suggest that VicRK regulates processes involved in cell wall metabolism, nutrient uptake, and osmotic protection.

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Cloning and nucleotide base sequence analysis of a spectinomycin adenyltransferase AAD(9) determinant from Enterococcus faecalis

Cited by 154 publications

References 27 publications

The CiaRH System ofStreptococcus pneumoniaePrevents Lysis during Stress Induced by Treatment with Cell Wall Inhibitors and by Mutations inpbp2xInvolved in β-Lactam Resistance

The CiaRH System ofStreptococcus pneumoniaePrevents Lysis during Stress Induced by Treatment with Cell Wall Inhibitors and by Mutations inpbp2xInvolved in β-Lactam Resistance

In vivo characterization of the psa genes from Streptococcus pneumoniae in multiple models of infection

Defects in ex vivo and in vivo growth and sensitivity to osmotic stress of group A Streptococcus caused by interruption of response regulator gene vicR

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