Plasmid pPCP1-derived sRNA HmsA promotes biofilm formation of Yersinia pestis
© The Author(s). 2016
Received: 24 March 2016
Accepted: 29 July 2016
Published: 4 August 2016
The ability of Yersinia pestis to form a biofilm is an important characteristic in flea transmission of this pathogen. Y. pestis laterally acquired two plasmids (pPCP1and pMT1) and the ability to form biofilms when it evolved from Yersinia pseudotuberculosis. Small regulatory RNAs (sRNAs) are thought to play a crucial role in the processes of biofilm formation and pathogenesis.
A pPCP1-derived sRNA HmsA (also known as sR084) was found to contribute to the enhanced biofilm formation phenotype of Y. pestis. The concentration of c-di-GMP was significantly reduced upon deletion of the hmsA gene in Y. pestis. The abundance of mRNA transcripts determining exopolysaccharide production, crucial for biofilm formation, was measured by primer extension, RT-PCR and lacZ transcriptional fusion assays in the wild-type and hmsA mutant strains. HmsA positively regulated biofilm synthesis-associated genes (hmsHFRS, hmsT and hmsCDE), but had no regulatory effect on the biofilm degradation-associated gene hmsP. Interestingly, the recently identified biofilm activator sRNA, HmsB, was rapidly degraded in the hmsA deletion mutant. Two genes (rovM and rovA) functioning as biofilm regulators were also found to be regulated by HmsA, whose regulatory effects were consistent with the HmsA-mediated biofilm phenotype.
HmsA potentially functions as an activator of biofilm formation in Y. pestis, implying that sRNAs encoded on the laterally acquired plasmids might be involved in the chromosome-based regulatory networks implicated in Y. pestis-specific physiological processes.
The genus Yersinia is composed of 11 species, including three human pathogenic species: Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica. Y. pestis is thought to have evolved from Y. pseudotuberculosis 5021–7022 years ago . Despite >90 % genome identity between Y. pestis and Y. pseudotuberculosis, the disease caused by these two species differs dramatically. Y. pseudotuberculosis is a self-limiting gastroenteric pathogen that does not usually form biofilms [2, 3]. By contrast, Y. pestis is a deadly pathogen responsible for three human plague pandemics. It is transmitted to mammals and/or humans by infected flea bites or by direct contact with infected animals . Y. pestis must survive and adapt to the complex microenvironments of multiple hosts during its infectious process [4, 5]. During its evolution from Y. pseudotuberculosis, Y. pestis acquired two unique plasmids, pPCP1 and pMT1, which are crucial for the processes of pathogenesis and flea transmission [6–8]. Plasmid pPCP1 is a 9.5 kb plasmid that encodes the plasminogen activator Pla, a surface protease that is essential for mediating primary pneumonic plague [7, 9].
The formation of biofilm within the flea digestive tract is important for natural transmission of Y. pestis because complete blockage of the proventriculus promotes frequent biting by fleas and thus increases the opportunities for transmission [4, 10]. A dense bacterial aggregate embedded in a self-produced exopolysaccharide (EPS) matrix facilitates the adaptation to complex microenvironments [3, 10, 11]. The hmsHFRS locus encodes the structural proteins required for the synthesis and transport of EPS, a major component of the Y. pestis biofilm [12, 13]. EPS expression is controlled at the post-transcriptional level by the intracellular concentration of the c-di-GMP second messenger , which is synthesized by diguanylate cyclases HmsD/HmsT and degraded by the phosphodiesterase HmsP in Y. pestis [15–17]. Several transcriptional regulators have been discovered that are involved in biofilm formation in Y. pestis. For example, Fur, a regulator of iron metabolism, can repress biofilm formation by negatively regulating the hmsT gene . RcsA, a negative regulator of biofilms, is reported to be functionally defective in Y. pestis . RovM, which is directly induced under specific microenvironments and represses the expression of the rovA gene, also regulates biofilm formation . We recently reported the role of RovA in biofilm formation of Y. pestis . The PhoPQ two-component system, a LysR-type transcriptional regulator YfbA and the carbon storage regulator CsrA have recently been shown to contribute to biofilm formation of Y. pestis [22–24].
Y. pestis has to adapt to diverse environmental conditions during its complex life cycle by modulating the expression of metabolic, cell surface and virulence factors. In bacteria there are different levels at which gene expression can be regulated. Small regulatory RNAs (sRNAs) play important regulatory roles at the post-transcriptional level in bacterial physiology and pathogenesis, including biofilm formation . They are reported to exert their regulatory functions by interacting with specific mRNAs or proteins and thus influence translation and mRNA stability upon sensing environmental cues [26, 27]. The identification of more than 100 sRNAs, identified by RNomics and deep sequencing, facilitates the study of post-transcriptional mechanisms of gene regulation in Y. pestis [28–32]. Post-transcriptional regulation and the underlying role of certain novel sRNAs in virulence and host adaptation have begun to be addressed in the genus Yersinia in recent years [32–34].
HmsB, a chromosome-encoded sRNA (also known as sR035), was identified by our previous study  and subsequently shown to promote biofilm formation by increasing EPS production in Y. pestis . The plasmid pPCP1-deriving sRNA HmsA (also known as sR084) was initially found to be highly abundant in Y. pestis grown in vitro and positively regulated by the CRP protein, a global regulator of catabolite repression . Here we observed an altered biofilm phenotype in the hmsA mutant of Y. pestis. Based on results measuring the abundance of mRNA transcripts determining EPS production, HmsA was found to potentially function as an activator of biofilm formation in Y. pestis by modulating the intracellular level of c-di-GMP molecules. Interestingly, the recently identified biofilm-associated sRNA, HmsB, was significantly downregulated in the hmsA deletion mutant. Furthermore, transcription of the biofilm regulators, RovA and RovM, also seemed to be affected by HmsA, which might partially account for the biofilm phenotype.
Bacterial strains, plasmids and oligonucleotides used in this study
Y. pestis wild-type strain 201
Y. pestis WT strain carrying plasmid pBAD-TF
Y. pestis WT strain lacking plasmid pPCP1
hmsA deletion mutant derived from Y. pestis WT strain 201
∆hmsA strain carrying plasmid pBAD-TF
∆hmsA strain carrying plasmid pBAD-HmsA
hmsS deletion mutant derived from Y. pestis WT strain
fur deletion mutant derived from Y. pestis WT strain
hmsB deletion mutant derived from Y. pestis WT strain
A low-copy lacZ fusion vector
Transcriptional fusion vector modified from pBAD/HisA
HmsA overexpressing plasmid by inserting a DNA fragment amplified by primer HmsA-pBAD-F/R into pBAD-TF
Biofilm formation assays
Three methods were used to evaluate biofilm formation by Y. pestis, as described previously . For the rugose colony morphology assay, 5 μL of bacterial glycerol stocks were spotted onto LB plates, which were dried for 2 d at 37 °C and cooled for at least 2 h at room temperature, these plates were incubated at 26 °C for 1 week. The surface morphology of each bacterial colony was photographed.
A crystal violet staining assay was used to detect biofilm formation in vitro. One hundred microliters of bacterial glycerol stock were added to 18 mL of LB medium for cultivation to stationary phase at 26 °C. The bacterial cultures were diluted 1:20 into 18 mL of LB medium for cultivation to an optical density (OD620) of about 1.0 and stored at 4 °C for 8 h. The bacterial cultures were diluted 1:20 and 1 mL of the diluted cultures was transferred into the 24-well tissue culture plates; these plates were incubated with shaking at 230 rpm for 24 h at 26 °C. The OD620 values of the cultures containing planktonic cells were determined and used for normalization. The pellicle was gently washed three times with H2O, and incubated at 80 °C for 15 min. The biofilms were stained with 2 mL of 0.1 % crystal violet for 15 min. The wells were washed three times with 2 mL of H2O, the biofilms were dissolved with 2 mL of dimethylsulfoxide for 1 h. Crystal violet staining was determined by the OD570 values and the relative amount of biofilm formation was indicated by the OD570/OD620 values. Three biological replicates and two technical replicates were performed for each strain.
For Caenorhabditis elegans killing assays, bacterial biofilm formation in vivo was determined by the percentage of fourth-stage larvae and adults (L4/adult) of C. elegans after incubation of nematode eggs on Y. pestis lawns. The nematode eggs were collected from lysates of adult C. elegans grown on Escherichia coli OP50 lawns on NGM agar plates. About 200 to 300 nematode eggs were placed on each bacterial lawn, followed by incubation at 20 °C for 72 h, the number of nematodes, fourth-stage larvae and adults of C. elegans were counted. Three biological replicates were performed for each strain.
Measurement of the intracellular c-di-GMP levels
The intracellular c-di-GMP levels were determined by a chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) method. One hundred microliters of bacterial glycerol stocks were added to 18 mL of LB medium for cultivation to stationary phase at 26 °C. The bacterial cultures were diluted 1:20 into 18 mL of LB medium for cultivation to an OD620 of about 1.0, and were then stored at 4 °C for 8 h. The bacterial cultures were diluted 1:20 into 18 mL LB medium for cultivation to middle exponential phase (OD620 ≈ 1.0) at 26 °C. Bacterial cultures were harvested for the extraction of c-di-GMP. The samples were loaded onto an API 4000-QTRAP mass spectrometer equipped with an electrospray ionization source (Applied Biosystems). One microliter of bacterial culture was harvested and the protein concentration was determined using a Micro BCA Protein Assay Kit (Thermo Scientific). The c-di-GMP levels were expressed as pmol/mg of bacterial protein.
RNA extraction and northern blotting
Y. pestis strains were grown in LB medium at 26 °C to an OD620 of about 1.0 and stored at 4 °C for 8 h. The bacterial cultures were diluted 1:20 into 18 mL LB medium for cultivation to middle exponential phase (OD620 ≈ 1.0) at 26 °C. Before harvesting, bacterial cultures were mixed with double-volume RNAprotect Regent (Qiagen) to minimize RNA degradation. Total RNA was isolated using TRIzol Reagent (Invitrogen). The quantity and quality of RNA were determined by NANODROP spectrophotometry (Thermo Scientific). Northern blotting was carried out using a DIG Northern Starter Kit (Roche) following the manufacturer’s protocol as described by Beckmann et al. . Total RNA samples (5–10 μg) were denatured at 95 °C for 3 min, separated on 6 % polyacrylamide-7 M urea gels, and RNA samples were transferred onto Hybond N+ membranes (GE Healthcare) by electroblotting. The membranes were UV-crosslinked and pre-hybridized for 1 h, and DIG-labeled RNA probes generated using the primers HmsA/HmsB-NB-F/R were added. The membranes were then hybridized overnight at 68 °C in DIG Easy Hyb buffer (Roche) according to the manufacturer’s protocols. Multiple exposures to X-ray film were taken to achieve the desired signal strength. RNA probes were synthesized by in vitro transcription using T7 RNA polymerase, then RNA was immunologically detected and scanned.
Primer extension assay
Primer extension assays were performed using a primer extension system-AMV reverse transcriptase kit (Promega) as previously reported by our group  with slight modifications. Total RNA (10 μg) was reverse-transcribed using the 32P-labelled primer HmsA-PE-R. The cDNA products were subjected to electrophoresis in a 6 % polyacrylamide-8 M urea gel. The gel was then analyzed by autoradiography (Kodak film). To serve as sequence ladders, sequencing reactions were also performed with the same primers used for primer extension, using the AccuPower & Top DNA Sequencing Kit (Bioneer).
Genes with a promoter-proximal DNA region were cloned into the low-copy-number transcriptional fusion vector pRW50 , which harbors a promoterless lacZ reporter gene. The recombinant plasmid or the empty pRW50 (negative control) was transformed into Y. pestis strains and the β-Galactosidase Enzyme Assay System (Promega) was used to measure β-galactosidase activity in three independent cellular extracts .
Quantitative RT-PCR (qRT-PCR)
The cDNA was synthesized from 5 μg of RNA using the ThermoScrip RT-PCR System (Invitrogen). Real-time PCR was performed in duplicate for each RNA sample using the TransStart™ Green qPCR SuperMix UDG (TransGen Biotech) with an appropriate cDNA dilution as a template. Three biological replicates were performed for each strain. Control reactions were carried out in parallel in the absence of the reverse transcriptase, 16S rRNA was used as an internal standard to normalize the expression levels of the tested sRNA candidates. Relative quantitative analysis was performed across different cDNA templates using the LightCycler 480 software (Bio-Rad).
Enhanced role of HmsA in biofilm formation
The role of HmsA as a regulator of biofilm formation was subsequently confirmed by crystal violet staining and nematode development assays. Quantitative determination by crystal violet staining revealed an approximately 50 % decrease in bacterial biofilm of the Y. pestis ΔhmsA mutant at the air-liquid interface compared with the WT strain and the ΔhmsA::HmsA strain (Fig. 1b). Incubation of nematode eggs on bacterial lawns of the WT and ΔhmsA::HmsA strain, revealed that only a small percentage (below 20 %) of eggs grew to L4/adult nematodes due to abundant attachment of biofilms to nematode heads. By contrast, bacterial lawns of the ∆hmsA and ∆hmsS mutant strains allowed the growth of about 40 % and 95 % of eggs into L4/adult nematodes, respectively (Fig. 1c). These results indicated that HmsA enhances biofilm formation in Y. pestis.
Impact of HmsA on the biosynthesis of c-di-GMP molecules
Biofilm formation in Y. pestis is regulated by the intracellular concentration of the second messenger c-di-GMP. As HmsA influences the extent of biofilm formation by Y. pestis, we hypothesized that deletion of hmsA would result in decreased c-di-GMP levels. To test the effect of HmsA on c-di-GMP synthesis in Y. pestis cells, the concentration of c-di-GMP was determined in the WT, ∆hmsA and ΔhmsA::HmsA strains by HPLC-MS/MS. The production of c-di-GMP was clearly reduced in the ∆hmsA mutant compared with the WT and ΔhmsA::HmsA mutant (Fig. 1d). The c-di-GMP levels were comparable between the WT and ΔhmsA::HmsA strain grown under the same conditions. However, absence of the hmsA gene resulted in a reduction in c-di-GMP levels by about 50 %. These data indicated that HmsA plays a role in modulation of c-di-GMP levels, and that c-di-GMP levels correlate with the amount of biofilm produced.
HmsA affects the transcriptional regulation of structural genes for EPS synthesis
Most sRNAs derived from intergenic regions act as trans-acting sRNAs repressing translation and destroying mRNA stability via base pairing with their target mRNAs. Translational repression of a target mRNA results in active or passive mRNA degradation. We used a primer extension assay and RT-PCR to determine the HmsA-mediated regulatory effects on biofilm-related genes at the transcriptional and post-transcriptional level in Y. pestis. A lacZ transcriptional fusion assay was also performed to explore the possibility that abundance changes occur at the transcriptional level.
HmsA affects the regulation of genes for c-di-GMP synthesis and degradation
HmsA affects the regulation of the biofilm-associated sRNA HmsB
Regulatory effects of HmsA on biofilm-related regulators (RovA and RovM) and the ferric uptake regulator (Fur)
Y. pestis evolved from Y. pseudotuberculosis by acquiring novel pathogenic traits, which conferred the ability to cause fatal disease in mammals, and the capacity to form a biofilm in fleas [8, 41, 47]. Two laterally acquired plasmids, pPCP1 and pMT1, have been shown to be essential for Y. pestis pathogenesis and flea transmission, respectively [6–8]. Here we present evidence that plasmid pPCP1 contributes to biofilm formation in Y. pestis. Our study showed that HmsA was implicated in chromosome-encoded regulatory networks of biofilm formation by modulating the concentration of c-di-GMP and EPS production. Until now, two regulatory sRNAs were reported to affect biofilm formation in Y. pestis, which constituted a new class of regulators of biofilm matrix production. Strikingly, the stability of HmsB appeared to be impaired by HmsA. A consecutive 13-nt base pairing region was found at the 5ʹ-terminal region of HmsA and within HmsB using the IntaRNA program (Additional file 2: Figure S2a). Whether the potentially direct interaction between HmsA and HmsB accounts for blocking of the accessibility to ribonuclease remains to be experimentally determined.
Notably, most of the tested genes were transcriptionally regulated, suggesting the indirect role of HmsA. Only hmsT and hmsCDE appeared to be post-transcriptionally regulated. In general, sRNAs promote translational activation by either increasing accessibility of the ribosomal binding site on mRNA or adjusting mRNA susceptibility to ribonucleolytic degradation . We predicted the secondary structure of the 5' UTR of HmsT/HmsC and potential interactions between HmsA and HmsT/HmsC using the Mfold (http://mfold.rit.albany.edu) algorithm and the IntaRNA program (http://rna.informatik.uni-freiburg.de/IntaRNA), respectively. The stem-loop structure was found to be located close to the ribosomal binding site or translational start site of both HmsT and HmsC (Additional file 2: Figure S2b and S2c). Interestingly, the interactive sites between HmsA and HmsT/HmsC partially overlap with the stem-loop-forming sites of HmsT/HmsC, which is consistent with the traditional view of sRNA-mediated target activation. However, the precise mechanism by which the hmsT and hmsD transcripts, as well as other mRNAs, are directly targeted by HmsA remains to be further investigated.
In this study the plasmid pPCP1-deriving sRNA HmsA was characterized as an activator of biofilm formation in Y. pestis. The biofilm-associated genes were found regulated by HmsA, implying that sRNAs encoded on the laterally acquired plasmids might involve in the chromosome-based regulatory networks. It provides a further insight into importance of sRNAs in Y. pestis-specific physiology and evolution.
This study was funded by the National Basic Research Program of China (2014CB744405), the National Natural Science Foundation of China (31430006) and the State Key of Pathogen and Biosecurity (Academy of Military Medical Science, SKLPBS1418).
Availability of data and materials
All data generated or analysized during this study are included in this published article and its supplementary information files.
YH, RY and DZ conceived the study and designed the experiments. ZL performed all the experiments. XG and HW contributed to RNA extraction and northern blotting assay. HF and LL participated in primer extension assay and qRT-PCR. YY and RC performed computational analysis and figure construction. The manuscript was written by YH and ZL, and was revised by RY. All the authors read and approved the final manuscript, The authors declare that they have no competing interests.
The authors declare that they have no competing interests.
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