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Complete sequences of KPC-2-encoding plasmid p628-KPC and CTX-M-55-encoding p628-CTXM coexisted in Klebsiella pneumoniae.

Wang L, Fang H, Feng J, Yin Z, Xie X, Zhu X, Wang J, Chen W, Yang R, Du H, Zhou D - Front Microbiol (2015)

Bottom Line: A carbapenem-resistant Klebsiella pneumoniae strain 628 was isolated from a human case of intracranial infection in a Chinese teaching hospital.ΔTn3-ISKpn27-bla KPC-2-ΔISKpn6-ΔTn1722 and this transposon together with a mercury resistance (mer) gene locus constitutes a 34 kb acquired drug-resistance region. bla KPC-2 has two transcription starts (nucleotides G and C located at 39 and 250 bp upstream of its coding region respectively) which correspond to two promoters, i.e., the intrinsic P1 and the upstream ISKpn27/Tn3-provided P2 with the core -35/-10 elements TAATCC/TTACAT and TTGACA/AATAAT respectively. bla CTX-M-55 is mobilized in an ISEcp1-bla CTX-M-55-Δorf477 transposition unit and appears to be the sole drug-resistant determinant in p628-CTXM. bla CTX-M-55 possesses a single transcription start (nucleotides G located at 116 bp upstream of its coding region) corresponding to the ISEcp1-provided P1 promoter with the core -35/-10 element TTGAAA/TACAAT.All the above detected promoters display a characteristic of constitutive expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Laboratory, The First Hospital Affiliated to Henan University Kaifeng, China.

ABSTRACT
A carbapenem-resistant Klebsiella pneumoniae strain 628 was isolated from a human case of intracranial infection in a Chinese teaching hospital. Strain 628 produces KPC-2 and CTX-M-55 encoded by two different conjugative plasmids, i.e., the IncFIIK plasmid p628-KPC and the IncI1 plasmid p628-CTXM respectively. bla KPC-2 is captured by a Tn1722-based unit transposon with a linear structure. ΔTn3-ISKpn27-bla KPC-2-ΔISKpn6-ΔTn1722 and this transposon together with a mercury resistance (mer) gene locus constitutes a 34 kb acquired drug-resistance region. bla KPC-2 has two transcription starts (nucleotides G and C located at 39 and 250 bp upstream of its coding region respectively) which correspond to two promoters, i.e., the intrinsic P1 and the upstream ISKpn27/Tn3-provided P2 with the core -35/-10 elements TAATCC/TTACAT and TTGACA/AATAAT respectively. bla CTX-M-55 is mobilized in an ISEcp1-bla CTX-M-55-Δorf477 transposition unit and appears to be the sole drug-resistant determinant in p628-CTXM. bla CTX-M-55 possesses a single transcription start (nucleotides G located at 116 bp upstream of its coding region) corresponding to the ISEcp1-provided P1 promoter with the core -35/-10 element TTGAAA/TACAAT. All the above detected promoters display a characteristic of constitutive expression. Coexistence of bla KPC and bla CTX-M in K. pneumoniae has been reported many times but this is the first report to gain deep insights into genetic platforms, promoters, and expression of the two coexisting bla genes with determination of entire nucleotide sequences of the two corresponding plasmids.

No MeSH data available.


Related in: MedlinePlus

Organization and expression of bla genes. (A) Gene organization. Boxed arrows stand for length/direction of indicated genes. Broken-line arrows represent transcription starts for indicated bla genes. (B) Primer extension. Primer extension assay of the RNA transcript of each bla gene was performed for strain 628 cultured with addition of increasing amounts of imipenem or ampicillin. Lanes C, T, A, and G represent Sanger sequencing reactions. Lanes 0, 3, and 6 stand for 0, 3, and 6 mg/L imipenem. Lanes 0, 200, and 400 stand for 0, 200, and 400 mg/L ampicillin. Transcription start of each bla gene is indicated by arrow with nucleotide, and minus number under arrow indicates nucleotide position upstream of indicated bla gene. Representative data from at least two independent biological replicates are shown.
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Figure 5: Organization and expression of bla genes. (A) Gene organization. Boxed arrows stand for length/direction of indicated genes. Broken-line arrows represent transcription starts for indicated bla genes. (B) Primer extension. Primer extension assay of the RNA transcript of each bla gene was performed for strain 628 cultured with addition of increasing amounts of imipenem or ampicillin. Lanes C, T, A, and G represent Sanger sequencing reactions. Lanes 0, 3, and 6 stand for 0, 3, and 6 mg/L imipenem. Lanes 0, 200, and 400 stand for 0, 200, and 400 mg/L ampicillin. Transcription start of each bla gene is indicated by arrow with nucleotide, and minus number under arrow indicates nucleotide position upstream of indicated bla gene. Representative data from at least two independent biological replicates are shown.

Mentions: In this work, the primer extension assay detected two transcription starts, i.e., nucleotides G and C located at 39 and 250 bp upstream of blaTn1722−basedKPC−2 from p628-KPC respectively; the corresponding two promoters were designated P1 and P2ISKpn27∕Tn3 with the core −35/−10 elements TAATCC/TTACAT and TTGACA/AATAAT respectively (Figures 5, 6). The first 74 bp fragments upstream of blaTn4401bKPC−2 and blaTn1722−basedKPC−2 are essentially identical; the P1 promoter is located within this 74 bp region and thereby shared by blaTn4401bKPC−2 and blaTn1722−basedKPC−2 (Figure 6). The next 280 bp region upstream of the above 74 bp fragment for blaTn4401bKPC−2 is dramatically divergent at nucleotide level from the counterpart for blaTn1722−basedKPC−2; these two distinct 280 bp regions contain P2ISKpn7 and P2ISKpn27∕Tn3 respectively. The −35 element of P2ISKpn7 is provided by ISKpn7 inserted at 319 bp upstream of blaTn4401bKPC−2, while the −35 and −10 elements of P2ISKpn27∕Tn3 are provided by ISKpn27 and Tn3 inserted at 281 and 75 bp upstream of blaTn1722−basedKPC−2 respectively (Figure 6).


Complete sequences of KPC-2-encoding plasmid p628-KPC and CTX-M-55-encoding p628-CTXM coexisted in Klebsiella pneumoniae.

Wang L, Fang H, Feng J, Yin Z, Xie X, Zhu X, Wang J, Chen W, Yang R, Du H, Zhou D - Front Microbiol (2015)

Organization and expression of bla genes. (A) Gene organization. Boxed arrows stand for length/direction of indicated genes. Broken-line arrows represent transcription starts for indicated bla genes. (B) Primer extension. Primer extension assay of the RNA transcript of each bla gene was performed for strain 628 cultured with addition of increasing amounts of imipenem or ampicillin. Lanes C, T, A, and G represent Sanger sequencing reactions. Lanes 0, 3, and 6 stand for 0, 3, and 6 mg/L imipenem. Lanes 0, 200, and 400 stand for 0, 200, and 400 mg/L ampicillin. Transcription start of each bla gene is indicated by arrow with nucleotide, and minus number under arrow indicates nucleotide position upstream of indicated bla gene. Representative data from at least two independent biological replicates are shown.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4541600&req=5

Figure 5: Organization and expression of bla genes. (A) Gene organization. Boxed arrows stand for length/direction of indicated genes. Broken-line arrows represent transcription starts for indicated bla genes. (B) Primer extension. Primer extension assay of the RNA transcript of each bla gene was performed for strain 628 cultured with addition of increasing amounts of imipenem or ampicillin. Lanes C, T, A, and G represent Sanger sequencing reactions. Lanes 0, 3, and 6 stand for 0, 3, and 6 mg/L imipenem. Lanes 0, 200, and 400 stand for 0, 200, and 400 mg/L ampicillin. Transcription start of each bla gene is indicated by arrow with nucleotide, and minus number under arrow indicates nucleotide position upstream of indicated bla gene. Representative data from at least two independent biological replicates are shown.
Mentions: In this work, the primer extension assay detected two transcription starts, i.e., nucleotides G and C located at 39 and 250 bp upstream of blaTn1722−basedKPC−2 from p628-KPC respectively; the corresponding two promoters were designated P1 and P2ISKpn27∕Tn3 with the core −35/−10 elements TAATCC/TTACAT and TTGACA/AATAAT respectively (Figures 5, 6). The first 74 bp fragments upstream of blaTn4401bKPC−2 and blaTn1722−basedKPC−2 are essentially identical; the P1 promoter is located within this 74 bp region and thereby shared by blaTn4401bKPC−2 and blaTn1722−basedKPC−2 (Figure 6). The next 280 bp region upstream of the above 74 bp fragment for blaTn4401bKPC−2 is dramatically divergent at nucleotide level from the counterpart for blaTn1722−basedKPC−2; these two distinct 280 bp regions contain P2ISKpn7 and P2ISKpn27∕Tn3 respectively. The −35 element of P2ISKpn7 is provided by ISKpn7 inserted at 319 bp upstream of blaTn4401bKPC−2, while the −35 and −10 elements of P2ISKpn27∕Tn3 are provided by ISKpn27 and Tn3 inserted at 281 and 75 bp upstream of blaTn1722−basedKPC−2 respectively (Figure 6).

Bottom Line: A carbapenem-resistant Klebsiella pneumoniae strain 628 was isolated from a human case of intracranial infection in a Chinese teaching hospital.ΔTn3-ISKpn27-bla KPC-2-ΔISKpn6-ΔTn1722 and this transposon together with a mercury resistance (mer) gene locus constitutes a 34 kb acquired drug-resistance region. bla KPC-2 has two transcription starts (nucleotides G and C located at 39 and 250 bp upstream of its coding region respectively) which correspond to two promoters, i.e., the intrinsic P1 and the upstream ISKpn27/Tn3-provided P2 with the core -35/-10 elements TAATCC/TTACAT and TTGACA/AATAAT respectively. bla CTX-M-55 is mobilized in an ISEcp1-bla CTX-M-55-Δorf477 transposition unit and appears to be the sole drug-resistant determinant in p628-CTXM. bla CTX-M-55 possesses a single transcription start (nucleotides G located at 116 bp upstream of its coding region) corresponding to the ISEcp1-provided P1 promoter with the core -35/-10 element TTGAAA/TACAAT.All the above detected promoters display a characteristic of constitutive expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Laboratory, The First Hospital Affiliated to Henan University Kaifeng, China.

ABSTRACT
A carbapenem-resistant Klebsiella pneumoniae strain 628 was isolated from a human case of intracranial infection in a Chinese teaching hospital. Strain 628 produces KPC-2 and CTX-M-55 encoded by two different conjugative plasmids, i.e., the IncFIIK plasmid p628-KPC and the IncI1 plasmid p628-CTXM respectively. bla KPC-2 is captured by a Tn1722-based unit transposon with a linear structure. ΔTn3-ISKpn27-bla KPC-2-ΔISKpn6-ΔTn1722 and this transposon together with a mercury resistance (mer) gene locus constitutes a 34 kb acquired drug-resistance region. bla KPC-2 has two transcription starts (nucleotides G and C located at 39 and 250 bp upstream of its coding region respectively) which correspond to two promoters, i.e., the intrinsic P1 and the upstream ISKpn27/Tn3-provided P2 with the core -35/-10 elements TAATCC/TTACAT and TTGACA/AATAAT respectively. bla CTX-M-55 is mobilized in an ISEcp1-bla CTX-M-55-Δorf477 transposition unit and appears to be the sole drug-resistant determinant in p628-CTXM. bla CTX-M-55 possesses a single transcription start (nucleotides G located at 116 bp upstream of its coding region) corresponding to the ISEcp1-provided P1 promoter with the core -35/-10 element TTGAAA/TACAAT. All the above detected promoters display a characteristic of constitutive expression. Coexistence of bla KPC and bla CTX-M in K. pneumoniae has been reported many times but this is the first report to gain deep insights into genetic platforms, promoters, and expression of the two coexisting bla genes with determination of entire nucleotide sequences of the two corresponding plasmids.

No MeSH data available.


Related in: MedlinePlus