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HmsB enhances biofilm formation in Yersinia pestis.

Fang N, Qu S, Yang H, Fang H, Liu L, Zhang Y, Wang L, Han Y, Zhou D, Yang R - Front Microbiol (2014)

Bottom Line: The hmsHFRS operon is responsible for biosynthesis and translocation of biofilm matrix exopolysaccharide.Further gene regulation experiments disclose that HmsB stimulates the expression of hmsB, hmsCDE, hmsT, and hmsHFRS but represses that of hmsP.HmsB most likely acts as a major activator of biofilm formation in Y. pestis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology Beijing, China.

ABSTRACT
The hmsHFRS operon is responsible for biosynthesis and translocation of biofilm matrix exopolysaccharide. Yersinia pestis expresses the two sole diguanylate cyclases HmsT and HmsD and the sole phosphodiesterase HmsP, which are specific for biosynthesis and degradation, respectively, of 3',5'-cyclic diguanosine monophosphate (c-di-GMP), a second messenger promoting exopolysaccharide production. In this work, the phenotypic assays indicates that Y. pestis sRNA HmsB enhances the production of c-di-GMP, exopolysaccharide, and biofilm. Further gene regulation experiments disclose that HmsB stimulates the expression of hmsB, hmsCDE, hmsT, and hmsHFRS but represses that of hmsP. HmsB most likely acts as a major activator of biofilm formation in Y. pestis. This is the first report of regulation of Yersinia biofilm formation by a sRNA. Data presented here will promote us to gain a deeper understanding of the complex regulatory circuits controlling Yersinia biofilm formation.

No MeSH data available.


Related in: MedlinePlus

HmsB-dependent expression of hmsB. (A) Primer extension. The mRNA levels of hmsB in WT or ΔhmsB were determined by primer extension. The Sanger sequence ladders (lanes G, C, A, and T) and the primer extension products of hmsB were analyzed with an 8 M urea-6% acrylamide sequencing gel. The transcription start site of hmsB was indicated by underlined nucleotide A. (B) LacZ fusion. The PhmsB:lacZ transcriptional fusion vector was transformed into WT or ΔhmsB, and then the hmsB promoter activities (miller units of β-galactosidase activity) were determined in bacterial cellular extracts.
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Figure 3: HmsB-dependent expression of hmsB. (A) Primer extension. The mRNA levels of hmsB in WT or ΔhmsB were determined by primer extension. The Sanger sequence ladders (lanes G, C, A, and T) and the primer extension products of hmsB were analyzed with an 8 M urea-6% acrylamide sequencing gel. The transcription start site of hmsB was indicated by underlined nucleotide A. (B) LacZ fusion. The PhmsB:lacZ transcriptional fusion vector was transformed into WT or ΔhmsB, and then the hmsB promoter activities (miller units of β-galactosidase activity) were determined in bacterial cellular extracts.

Mentions: The relative mRNA level of each of hmsB (Figure 3A), hmsC(Figure 4A), hmsT (Figure 5A), and hmsH (Figure 6A) was measured in WT or ΔhmsB by primer extension assay, and the results showed that the mRNA level of each of the above four genes decreased considerably in ΔhmsB relative to WT. Notably, this assay detected a single transcription start site (nucleotide A) located at nucleotide position 472430 on CO92 genome, which confirmed the above 5′-RACE result. The promoter-proximal region of each of hmsB (Figure 3B), hmsC (Figure 4B), hmsT (Figure 5B), and hmsH (Figure 6B) was cloned into the transcriptional lacZ fusion reporter vector pRW50, and the corresponding recombinant vector was introduced into WT or ΔhmsB to determine the target promoter activity; it was shown that the promoter activity of each of the above four genes was significantly reduced in ΔhmsB relative to WT. Further Western blot assay confirmed that biosynthesis of each of HmsD (Figure 4C), HmsT (Figure 5C), and HmsF (Figure 6C) decreased in ΔhmsB relative to WT. Notably, observations from transcriptional lacZ fusion experiments denoted that HmsB-dependent expression of hmsB, hmsCDE, hmsT, and hmsHFRS most likely involved mechanisms of gene transcriptional regulation.


HmsB enhances biofilm formation in Yersinia pestis.

Fang N, Qu S, Yang H, Fang H, Liu L, Zhang Y, Wang L, Han Y, Zhou D, Yang R - Front Microbiol (2014)

HmsB-dependent expression of hmsB. (A) Primer extension. The mRNA levels of hmsB in WT or ΔhmsB were determined by primer extension. The Sanger sequence ladders (lanes G, C, A, and T) and the primer extension products of hmsB were analyzed with an 8 M urea-6% acrylamide sequencing gel. The transcription start site of hmsB was indicated by underlined nucleotide A. (B) LacZ fusion. The PhmsB:lacZ transcriptional fusion vector was transformed into WT or ΔhmsB, and then the hmsB promoter activities (miller units of β-galactosidase activity) were determined in bacterial cellular extracts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264472&req=5

Figure 3: HmsB-dependent expression of hmsB. (A) Primer extension. The mRNA levels of hmsB in WT or ΔhmsB were determined by primer extension. The Sanger sequence ladders (lanes G, C, A, and T) and the primer extension products of hmsB were analyzed with an 8 M urea-6% acrylamide sequencing gel. The transcription start site of hmsB was indicated by underlined nucleotide A. (B) LacZ fusion. The PhmsB:lacZ transcriptional fusion vector was transformed into WT or ΔhmsB, and then the hmsB promoter activities (miller units of β-galactosidase activity) were determined in bacterial cellular extracts.
Mentions: The relative mRNA level of each of hmsB (Figure 3A), hmsC(Figure 4A), hmsT (Figure 5A), and hmsH (Figure 6A) was measured in WT or ΔhmsB by primer extension assay, and the results showed that the mRNA level of each of the above four genes decreased considerably in ΔhmsB relative to WT. Notably, this assay detected a single transcription start site (nucleotide A) located at nucleotide position 472430 on CO92 genome, which confirmed the above 5′-RACE result. The promoter-proximal region of each of hmsB (Figure 3B), hmsC (Figure 4B), hmsT (Figure 5B), and hmsH (Figure 6B) was cloned into the transcriptional lacZ fusion reporter vector pRW50, and the corresponding recombinant vector was introduced into WT or ΔhmsB to determine the target promoter activity; it was shown that the promoter activity of each of the above four genes was significantly reduced in ΔhmsB relative to WT. Further Western blot assay confirmed that biosynthesis of each of HmsD (Figure 4C), HmsT (Figure 5C), and HmsF (Figure 6C) decreased in ΔhmsB relative to WT. Notably, observations from transcriptional lacZ fusion experiments denoted that HmsB-dependent expression of hmsB, hmsCDE, hmsT, and hmsHFRS most likely involved mechanisms of gene transcriptional regulation.

Bottom Line: The hmsHFRS operon is responsible for biosynthesis and translocation of biofilm matrix exopolysaccharide.Further gene regulation experiments disclose that HmsB stimulates the expression of hmsB, hmsCDE, hmsT, and hmsHFRS but represses that of hmsP.HmsB most likely acts as a major activator of biofilm formation in Y. pestis.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology Beijing, China.

ABSTRACT
The hmsHFRS operon is responsible for biosynthesis and translocation of biofilm matrix exopolysaccharide. Yersinia pestis expresses the two sole diguanylate cyclases HmsT and HmsD and the sole phosphodiesterase HmsP, which are specific for biosynthesis and degradation, respectively, of 3',5'-cyclic diguanosine monophosphate (c-di-GMP), a second messenger promoting exopolysaccharide production. In this work, the phenotypic assays indicates that Y. pestis sRNA HmsB enhances the production of c-di-GMP, exopolysaccharide, and biofilm. Further gene regulation experiments disclose that HmsB stimulates the expression of hmsB, hmsCDE, hmsT, and hmsHFRS but represses that of hmsP. HmsB most likely acts as a major activator of biofilm formation in Y. pestis. This is the first report of regulation of Yersinia biofilm formation by a sRNA. Data presented here will promote us to gain a deeper understanding of the complex regulatory circuits controlling Yersinia biofilm formation.

No MeSH data available.


Related in: MedlinePlus