Discovery and Characterization of a Gram-Positive Pel Polysaccharide Biosynthetic Gene Cluster

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Discovery and Characterization of a Gram-Positive Pel Polysaccharide Biosynthetic Gene Cluster bioRxiv preprint doi: https://doi.org/10.1101/768473; this version posted September 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster 2 3 Gregory B. Whitfield1,2, Lindsey S. Marmont1,2,#a, Cedoljub Bundalovic-Torma1,2,#b, Erum 4 Razvi1,2, Elyse J. Roach3, Cezar M. Khursigara3, John Parkinson1,2,4, and P. Lynne Howell1,2,* 5 6 7 1Program in Molecular Medicine, The Hospital for Sick Children, Toronto, ON, CANADA 8 2Department of Biochemistry, University of Toronto, Toronto, ON, CANADA 9 3Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, CANADA 10 4Department of Molecular Genetics, University of Toronto, Toronto, ON, CANADA 11 #aCurrent Address: Department of Microbiology, Harvard Medical School, Boston, MA, USA. 12 #bCurrent Address: Department of Cell Systems Biology, University of Toronto, Toronto, ON, 13 CANADA. 14 15 *Corresponding author 16 E-mail: [email protected] (PLH) 1 bioRxiv preprint doi: https://doi.org/10.1101/768473; this version posted September 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 17 Abstract 18 Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, 19 and the signalling pathways that regulate their production are largely unknown. In a companion 20 study, we developed a computational pipeline for the unbiased identification of homologous 21 bacterial operons and applied this algorithm to the analysis of synthase-dependent 22 exopolysaccharide biosynthetic systems (https://doi.org/10.1101/769745). Here, we explore the 23 finding that many species of Gram-positive bacteria have operons with similarity to the 24 Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from 25 Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for 26 biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the 27 degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3’,5’-dimeric guanosine 28 monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we 29 identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally 30 regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit 31 significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive 32 organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of 33 Bacilli, Clostridia, Streptococci, and Actinobacteria suggests that Pel may be a common biofilm 34 matrix component in many Gram-positive bacteria. 35 36 Author summary 37 The Pel polysaccharide is required for biofilm formation in P. aeruginosa and we have 38 previously found that the genes necessary for biosynthesis of this polymer are broadly distributed 39 across Gram-negative bacteria. Herein, we show that many species of Gram-positive bacteria 2 bioRxiv preprint doi: https://doi.org/10.1101/768473; this version posted September 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 40 also possess Pel biosynthetic genes and demonstrate that these genes are used Bacillus cereus for 41 biofilm formation. We show that Pel production in B. cereus is regulated by c-di-GMP and have 42 identified two enzymes, a diguanylate cyclase, CdgF, and a phosphodiesterase, CdgE, that 43 control the levels of this bacterial signalling molecule. While Pel production in B. cereus also 44 requires the binding of c-di-GMP to the receptor PelD, the divergence of this protein in 45 Streptococci suggests a c-di-GMP independent mechanism of regulation is used in this species. 46 The discovery of a Pel biosynthetic gene cluster in Gram-positive bacteria and our 47 characterization of the pel operon in B. cereus suggests that Pel is a widespread biofilm 48 component across all bacteria. 3 bioRxiv preprint doi: https://doi.org/10.1101/768473; this version posted September 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 49 Introduction 50 Bacteria are routinely challenged by a variety of environmental conditions that test their 51 ability to survive. One mechanism employed by a wide range of microorganisms to withstand 52 these challenges is to form a structured multicellular community, or biofilm. Bacteria growing as 53 a biofilm are more tolerant of the presence of toxic compounds [1,2] and predation by protists 54 [3,4], and exhibit increased persistence during infection due to antimicrobial tolerance [5,6] and 55 the ability to evade the host immune response [7,8]. As a result, biofilm formation is linked not 56 only to chronic bacterial infection in humans [9] [10], and plants and animals of economic 57 importance [11-13], but also to the contamination of industrial facilities involved in processing 58 food [14,15], and pulp and paper [16]. 59 Biofilm formation requires the production of extracellular material, or matrix, that allows 60 bacteria to adhere to each other and/or to a surface [17]. While this matrix can be composed of 61 protein adhesins, functional amyloid, extracellular DNA, and polysaccharides, the relative 62 importance and abundance of each of these components varies greatly between bacterial species 63 [17]. The complexity of the biofilm matrix is further amplified by the unique structural and 64 functional characteristics of the matrix components produced by different bacteria, such as the 65 cepacian polysaccharide produced by Burkholderia species [18], the CdrA adhesin of 66 Pseudomonads [19], or TasA fibres from Bacilli [20]. Indeed, there is even variation in the 67 utilization of matrix components for biofilm formation between different strains of the same 68 species [21]. As a result of this complexity, identification of the biofilm matrix composition of 69 many clinically and industrially relevant biofilm-forming organisms remains unresolved [22]. 70 Despite the complexity of biofilm matrix structure, there are components that are utilized 71 by a wide range of organisms. In particular, cellulose [23] and poly-β-(1,6)-N-acetyl-D- 4 bioRxiv preprint doi: https://doi.org/10.1101/768473; this version posted September 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 72 glucosamine (PNAG; [24]) have been identified as biofilm components across many bacterial 73 genera. In a companion paper [25], we developed a computational pipeline that enables the 74 unbiased identification of functionally related gene clusters from genome sequence data. 75 Applying this pipeline to all bacterial phyla, we performed a systematic search for synthase- 76 dependent polysaccharide biosynthetic operons involved in cellulose, PNAG, alginate and Pel 77 polysaccharide production. From this search we identified pel gene clusters not only in Gram- 78 negative species where it was originally identified but, unexpectantly, a range of Gram-positive 79 species. Herein, we show that one of these pel gene clusters, pelDEADAFG, from Bacillus cereus 80 ATCC 10987, is involved in the biosynthesis of a Pel-like polysaccharide which is essential for 81 biofilm formation by this organism. We identified a diguanylate cyclase, CdgF, and a cyclic- 82 3’5’-dimeric guanosine monophosphate (c-di-GMP) phosphodiesterase, CdgE, that regulate 83 biofilm formation and demonstrate that production of the polysaccharide is regulated post- 84 translationally through binding of c-di-GMP to the inhibitory site of a degenerate GGDEF 85 domain-containing receptor similar to PelD from Pseudomonas aeruginosa [26]. Our data not 86 only expand the range of organisms in which Pel loci are found, but also identify novel, 87 functionally distinct relatives of the Pel locus in Gram-positive bacteria that were missed in prior 88 analyses [27]. This study establishes Pel as contributing to the arsenal of biofilm formation 89 mechanisms acquired by Gram positives, as well as providing a rare example of post- 90 translational c-di-GMP-mediated regulation of biofilm formation in this group of organisms. 91 92 Results 93 Identification of Pel biosynthetic loci in Gram-positive bacteria 5 bioRxiv preprint doi: https://doi.org/10.1101/768473; this version posted September 18, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 94 The Pel polysaccharide was first identified in the Gram-negative opportunistic pathogen 95 P. aeruginosa, where its biosynthesis has been linked to the pelABCDEFG operon [28]. 96 Bioinformatic and biochemical analysis of the protein products of the pel operon has revealed a 97 complex membrane-spanning molecular machine (Fig. S1). Previous work from our lab 98 identified pel operons in bacterial genomes through bioinformatic searches using the outer 99 membrane lipoprotein PelC as a query sequence. We used this small outer membrane lipoprotein 100 as it is unique to Pel biosynthesis [27]. While this work expanded our knowledge of bacteria with 101 pel operons considerably, the use of PelC as a search sequence limits this analysis to Gram- 102 negative bacteria. To overcome this, we developed a computational pipeline that allows for the 103 unbiased identification of homologous bacterial operons [25]. A search for pel loci using the 104 protein coding sequences of the P. aeruginosa PAO1 pel operon as a reference set identified 105 pelF and pelG loci in 43 Gram-positive phyla, including commonly studied species of Bacilli, 106 Clostridia, and Streptococci [25].
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