Plasmid pPCP1-derived sRNA HmsA promotes biofilm formation of Yersinia pestis

Liu et al. BMC Microbiology (2016) 16:176 DOI 10.1186/s12866-016-0793-5 RESEARCH ARTICLE Open Access Plasmid pPCP1-derived sRNA HmsA promotes biofilm formation of Yersinia pestis Zizhong Liu1,2, Xiaofang Gao1,3, Hongduo Wang1,4, Haihong Fang1, Yanfeng Yan1, Lei Liu1, Rong Chen1,5, Dongsheng Zhou1, Ruifu Yang1* and Yanping Han1* Abstract Background: 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. Results: 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. Conclusion: 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. Background 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 [1]. 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 * Correspondence: ; 1 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, No. 20, Dongdajie, Fengtai, Beijing 100071, China Full list of author information is available at the end of the article humans by infected flea bites or by direct contact with infected animals [4]. 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 © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. BMC Microbiology (2016) 16:176 [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 [14], 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 [18]. RcsA, a negative regulator of biofilms, is reported to be functionally defective in Y. pestis [19]. RovM, which is directly induced under specific microenvironments and represses the expression of the rovA gene, also regulates biofilm formation [20]. We recently reported the role of RovA in biofilm formation of Y. pestis [21]. 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 posttranscriptional level in bacterial physiology and pathogenesis, including biofilm formation [25]. 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 [28] and subsequently shown to promote biofilm formation by increasing EPS production in Y. pestis [35]. 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 repr (...truncated)