This website does readability filtering of other pages. All styles, scripts, forms and ads are stripped. If you want your website excluded or have other feedback, use this form.

Identification of the Mutation Responsible for the Temperature-Sensitive Lipopolysaccharide O-antigen Defect in the Pseudomonas Aeruginosa Cystic Fibrosis Isolate 2192 - PubMed

Clipboard, Search History, and several other advanced features are temporarily unavailable. Skip to main page content

National Institutes of Health National Library of Medicine National Center for Biotechnology Information NCBI homepage Log in

Account

Logged in as:
username
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Actions . 2013 Apr;195(7):1504-14. doi: 10.1128/JB.01999-12. Epub 2013 Jan 25.

Identification of the Mutation Responsible for the Temperature-Sensitive Lipopolysaccharide O-antigen Defect in the Pseudomonas Aeruginosa Cystic Fibrosis Isolate 2192

Michael R Davis Jr  1 Artur MuszynskiIvonne V LollettChristopher L PritchettRussell W CarlsonJoanna B Goldberg Affiliations

Affiliation

  • 1 Department of Microbiology, Immunology, and Cancer Biology, University of Virginia Health Sciences Center, Charlottesville, VA, USA.
Free PMC article Item in Clipboard

Identification of the Mutation Responsible for the Temperature-Sensitive Lipopolysaccharide O-antigen Defect in the Pseudomonas Aeruginosa Cystic Fibrosis Isolate 2192

Michael R Davis Jr et al. J Bacteriol. 2013 Apr. Free PMC article Actions . 2013 Apr;195(7):1504-14. doi: 10.1128/JB.01999-12. Epub 2013 Jan 25.

Authors

Michael R Davis Jr  1 Artur MuszynskiIvonne V LollettChristopher L PritchettRussell W CarlsonJoanna B Goldberg

Affiliation

  • 1 Department of Microbiology, Immunology, and Cancer Biology, University of Virginia Health Sciences Center, Charlottesville, VA, USA.
Item in Clipboard
Favorites

Abstract

Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients is characterized by a series of genotypic and phenotypic changes that reflect the transition from acute to chronic infection. These include the overproduction of the exopolysaccharide alginate and the loss of complete lipopolysaccharide (LPS). LPS is a major component of the Gram-negative outer membrane and is composed of lipid A, core oligosaccharide, and O antigen. In this report, we show that the LPS defect of the P. aeruginosa chronic infection isolate 2192 is temperature sensitive. When grown at 25°C, 2192 expresses serotype O1 LPS with a moderate chain length and in reduced amounts relative to those of a wild-type serotype O1 laboratory strain (stO1). In contrast, 2192 expresses no LPS O antigen when grown at 37°C. This is the first time that a temperature-sensitive defect in O-antigen production has been reported. Using complementation analyses with a constructed wbpM deletion mutant of stO1, we demonstrate that the temperature-sensitive O-antigen production defect in 2192 is due to a mutation in wbpM, which encodes a UDP-4,6-GlcNAc dehydratase involved in O-antigen synthesis. The mutation, a deletion of a single amino acid (V636) from the extreme C terminus of WbpM, renders the protein less stable than its wild-type counterpart. This residue of WbpM, which is critical for stability and function, is located outside of the recognized domains of the protein and may provide insight into the structure-function relationship of this enzyme, which is found in all 20 serotypes of P. aeruginosa. We also identify a promoter of wbpM, map a transcriptional start site of wbpM, and show that mucoidy plays a role in the loss of expression of high-molecular-weight LPS in this CF isolate.

Figures

Fig 1

5

2192 exhibits temperature-dependent LPS expression...

Fig 1

13

2192 exhibits temperature-dependent LPS expression. P. aeruginosa strain stO1 was grown at 37...

Fig 1 2192 exhibits temperature-dependent LPS expression. P. aeruginosa strain stO1 was grown at 37°C overnight, and strain 2192 was grown at either 25°C or 37°C. Cultures were adjusted to an OD600 of 0.5, and LPS was prepared by the method of Davis and Goldberg (21), run on an 8% polyacrylamide gel, and subjected to Western blotting using rabbit polyclonal antisera against serotype O1.

Fig 2

5

IATS serotype O1 O-antigen locus...

Fig 2

13

IATS serotype O1 O-antigen locus. Genes are represented by arrows. Where a gene...

Fig 2 IATS serotype O1 O-antigen locus. Genes are represented by arrows. Where a gene name is given, it is based on that of its highest-scoring homolog in other P. aeruginosa genomes. (Adapted from reference with permission.)

Fig 3

5

Complementation of 2192 with wild-type...

Fig 3

13

Complementation of 2192 with wild-type stO1 genes. The coding regions of wild-type stO1...

Fig 3 Complementation of 2192 with wild-type stO1 genes. The coding regions of wild-type stO1 O-antigen genes that are mutated in P. aeruginosa 2192 were provided on plasmid pUCP18Ap to strain 2192. Strains were grown overnight at either 25°C (A) or 37°C (B). LPS was isolated, and Western blot analysis was performed as described for Fig. 1.

Fig 4

5

Transcription of wbpM and characterization...

Fig 4

13

Transcription of wbpM and characterization of promoter P1. (A) RNA was isolated from...

Fig 4 Transcription of wbpM and characterization of promoter P1. (A) RNA was isolated from stO1 or 2192 and was converted to cDNA. The amount of wbpM is reported as the number of target transcripts per 10 omlA transcripts ± standard error and was determined by RT-qPCR. Data represent the results of three independent experiments with triplicate technical replicates. ***, P < 0.001. (B) Primer extension analysis was used to identify the transcriptional start site. GATC represents the sequencing ladder generated using oligonucleotide wbpMPE1. −C represents a negative control consisting of end-labeled wbpMPE1 incubated with ΔwbpM total RNA in a reverse transcription reaction. PE represents total RNA from stO1 incubated with wbpmPE1 in a reverse transcription reaction. The arrow indicates the start site of transcription. The sequence upstream of wbpM is delineated, and +1 indicates the first nucleotide in the mRNA. (C) Promoter P1 of wbpM in serotype O1, as predicted by BPROM. The predicted −35 and −10 boxes and a putative Shine-Dalgarno (S-D) sequence are underlined; the + 1 site is marked. The sequence of the forward primer used to generate an ∼500-bp fragment containing P1 for lacZ fusions is shown in boldface italics. The reverse primer (not shown) (5′-CCAGCTCTCCCTACTCCTTTC-3′) is located ∼500 bp downstream of the indicated wbpM start site. (D and E) P1-lacZ fusion strains were grown to exponential phase, and cells were collected. Reverse promoter constructs were used as a control. β-Galactosidase activity was determined using cleared whole-cell lysates and is reported in Miller units. Data represent the results of 3 independent experiments carried out in triplicate. A significant difference (***, P < 0.001) was found between P1-For and P1-Rev at both growth phases. P1-For, P1 in the forward orientation relative to lacZ; P1-Rev, P1 in the reverse orientation.

Fig 5

5

LPS in wbpM O1 and...

Fig 5

13

LPS in wbpM O1 and wbpM 2192 complemented the Δ wbpM mutant. P...

Fig 5 LPS in wbpMO1 and wbpM2192 complemented the ΔwbpM mutant. P. aeruginosa strain stO1, its ΔwbpM mutant, and 2192 were provided with pHERD20T and were grown overnight at 25°C or 37°C in the presence of 2% arabinose. In addition, the ΔwbpM mutant was provided with either pHERD20T-wbpMO1 or pHERD20T-wbpM2192 and was grown overnight at 25°C or 37°C in the presence of 2% arabinose. HMW LPS is indicated by an arrow. LPS was isolated, and Western blot analysis was performed as described for Fig. 1.

Fig 6

5

Induction of expression of wbpM...

Fig 6

13

Induction of expression of wbpM O1 -Flag from pHERD20T in the Δ wbpM...

Fig 6 Induction of expression of wbpMO1-Flag from pHERD20T in the ΔwbpM mutant and 2192. P. aeruginosa strain stO1, its ΔwbpM mutant, and 2192 were grown overnight at 37°C. In addition, the ΔwbpM mutant and 2192 were provided with pHERD20T-wbpMO1-Flag and were grown in LB supplemented with arabinose at 0%, 0.2%, 0.50%, 1.0%, or 2.0%. LPS was isolated, and Western blot analysis was performed as described for Fig. 1. Whole-cell lysates were also prepared. LPS preparations and whole-cell lysates were run on 8% polyacrylamide gels and were subjected to Western blotting using rabbit polyclonal antisera against serotype O1 (A) and mouse anti-Flag antisera (B), respectively.

Fig 7

5

Monitoring the stability of WbpM...

Fig 7

13

Monitoring the stability of WbpM O1 -Flag and WbpM 2192 -Flag in P...

Fig 7 Monitoring the stability of WbpMO1-Flag and WbpM2192-Flag in P. aeruginosa. Cultures of either 2192 with pHERD20T-wbpMO1-Flag (A) or pHERD20T-wbpM2192-Flag (B) or the ΔwbpM mutant with pHERD20T-wbpMO1-Flag (C) or pHERD20T-wbpM2192-Flag (D) were grown in the presence of 2% (wt/vol) arabinose for 2 h to induce transcription from the PBAD promoter on pHERD20T (I), washed, and then grown in the presence of 1% (wt/vol) glucose to inhibit transcription. Uninduced (UI) cultures were used as controls. Samples were taken for 4 h at hourly intervals following glucose inhibition, and whole-cell lysates were prepared. These were run on 8% polyacrylamide gels and were subjected to Western blotting with monoclonal anti-Flag sera. Monoclonal anti-RpoA serum was used as a loading control. See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Grant support

LinkOut - more resources

Full-text links HighWire Free PMC article Citation text Send To Feedback