A transposon mutant library of <i>Bacillus cereus</i> ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle‐biofilm formation

Okshevsky, Mira; Louw, Matilde Greve; Lamela, Elena Otero; Nilsson, Marie; Tolker‐Nielsen, Tim

doi:10.1002/mbo3.552

Cited by 33 publications

(41 citation statements)

References 73 publications

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“…Indeed some bacteria were reported to form freefloating pellicles (Robertson et al, 2013). Another specificity is that flagellar motility seems to be mandatory for pellicle formation, which is not a rule for SSA-biofilm (Armitano et al, 2013;Guttenplan and Kearns, 2013;Okshevsky et al, 2017). This is coherent with the fact that bacteria need to actively locate the air-liquid interface in order to form a pellicle.…”

Section: Introductionmentioning

confidence: 99%

Control of pellicle biogenesis involves the diguanylate cyclases PdgA and PdgB, the c‐di‐GMP binding protein MxdA and the chemotaxis response regulator CheY3 in Shewanella oneidensis

Gambari

Boyeldieu

Armitano

et al. 2018

Environmental Microbiology

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Summary Shewanella oneidensis is an aquatic proteobacterium with remarkable respiratory and chemotactic abilities. It is also capable of forming biofilms either associated to surfaces (SSA‐biofilm) or at the air–liquid interface (pellicle). We have previously shown that pellicle biogenesis in S. oneidensis requires the flagellum and the chemotaxis regulatory system including CheA3 kinase and CheY3 response regulator. Here we searched for additional factors involved in pellicle development. Using a multicopy library of S. oneidensis chromosomal fragments, we identified two genes encoding putative diguanylate cyclases (pdgA and pdgB) and allowing pellicle formation in the non‐pellicle‐forming cheY3‐deleted mutant. A mutant deleted of both pdgA and pdgB is affected during pellicle development. By overexpressing phosphodiesterase encoding genes, we confirmed the key role of c‐di‐GMP in pellicle biogenesis. The mxd operon, previously proposed to encode proteins involved in exopolysaccharide biosynthesis, is also essential for pellicle formation. In addition, we showed that the MxdA protein, containing a degenerate GGDEF motif, binds c‐di‐GMP and interacts with both CheY3 and PdgA. Therefore, we propose that pellicle biogenesis in S. oneidensis is controlled by a complex pathway that involves the chemotaxis response regulator CheY3, the two putative diguanylate cyclases PdgA and PdgB, and the c‐di‐GMP binding protein MxdA.

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Section: Introductionmentioning

confidence: 99%

Control of pellicle biogenesis involves the diguanylate cyclases PdgA and PdgB, the c‐di‐GMP binding protein MxdA and the chemotaxis response regulator CheY3 in Shewanella oneidensis

Gambari

Boyeldieu

Armitano

et al. 2018

Environmental Microbiology

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“…S3). BCE_5588 is homologous to the UDP-glucose-4-epimerase, GalE, and a transposon screen published during the course of this study has already implicated this gene in biofilm formation [42]. As BCE_5582 was predicted by the Phyre 2 algorithm [30] to adopt a structure similar to the endo-α-1,4- N -acetylgalactosaminidase domain of P. aeruginosa PelA (PelA H Pa ; [43]), we refer to BCE_5582 as PelA H Bc herein.…”

Section: Resultsmentioning

confidence: 99%

“…Transposon insertions in BCE_5585 , BCE_5586 , and BCE_5587, which we have termed pelA DA , pelF , and pelG herein, led to an impairment in biofilm formation and based on these results, Okshevsky et al hypothesized that the BCE_5583 – BCE_5587 operon (herein pelDEA DA FG ) may be involved in the production of an extracellular polysaccharide [42]. Two distinct transposon insertions were also observed in cdgF , which was the only DGC identified in the screen, as well as an insertion in BCE_5588 that completely abrogated biofilm formation in ATCC 10987 [42]. BCE_5588, downstream of the pel operon, is similar to the UDP-glucose-4-epimerase GalE [34] and we hypothesized that BCE_5588 may be involved in the generation of the precursor nucleotide-sugars required for polysaccharide biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, eDNA, exopolysaccharide, and protein components have all been described as matrix components in various B. cereus strains [52–54]. As is the case amongst a diverse array of biofilm forming bacteria, eDNA has been shown to be crucial for biofilm formation in ATCC 14579 [52], the environmental B. cereus isolate AR156 [55], and ATCC 10987 [42]. First characterized in Bacillus subtilis [56], the exopolysaccharide producing epsA-O operon is also present in B. cereus , although its contribution to biofilm formation appears to be strain dependent.…”

Section: Discussionmentioning

confidence: 99%

“…First characterized in Bacillus subtilis [56], the exopolysaccharide producing epsA-O operon is also present in B. cereus , although its contribution to biofilm formation appears to be strain dependent. In the plant associated B. cereus strain 905, deletion of the eps locus does not affect biofilm formation [57], while in ATCC 10987 eps inactivation completely abrogated biofilm formation [42]. Strain-dependent utilization of different polysaccharide biosynthetic loci for biofilm formation has been observed in P. aeruginosa , where Pel and/or the Psl polysaccharide are variably required [21].…”

Section: Discussionmentioning

confidence: 99%

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Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

Whitfield

Marmont

Bundalovic-Torma

et al. 2019

Preprint

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AbstractOur understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems (https://doi.org/10.1101/769745). Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3’,5’-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of Bacilli, Clostridia, Streptococci, and Actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.Author summaryThe Pel polysaccharide is required for biofilm formation in P. aeruginosa and we have previously found that the genes necessary for biosynthesis of this polymer are broadly distributed across Gram-negative bacteria. Herein, we show that many species of Gram-positive bacteria also possess Pel biosynthetic genes and demonstrate that these genes are used Bacillus cereus for biofilm formation. We show that Pel production in B. cereus is regulated by c-di-GMP and have identified two enzymes, a diguanylate cyclase, CdgF, and a phosphodiesterase, CdgE, that control the levels of this bacterial signalling molecule. While Pel production in B. cereus also requires the binding of c-di-GMP to the receptor PelD, the divergence of this protein in Streptococci suggests a c-di-GMP independent mechanism of regulation is used in this species. The discovery of a Pel biosynthetic gene cluster in Gram-positive bacteria and our characterization of the pel operon in B. cereus suggests that Pel is a widespread biofilm component across all bacteria.

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Novel Genes Required for Surface-Associated Motility in Acinetobacter baumannii

2021

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Acinetobacter baumannii is an opportunistic and increasingly multi-drug resistant human pathogen rated as a critical priority one pathogen for the development of new antibiotics by the WHO in 2017. Despite the lack of flagella, A. baumannii can move along wet surfaces in two different ways: via twitching motility and surface-associated motility. While twitching motility is known to depend on type IV pili, the mechanism of surface-associated motility is poorly understood. In this study, we established a library of 30 A. baumannii ATCC® 17978™ mutants that displayed deficiency in surface-associated motility. By making use of natural competence, we also introduced these mutations into strain 29D2 to differentiate strain-specific versus species-specific effects of mutations. Mutated genes were associated with purine/pyrimidine/folate biosynthesis (e.g. purH, purF, purM, purE), alarmone/stress metabolism (e.g. Ap4A hydrolase), RNA modification/regulation (e.g. methionyl-tRNA synthetase), outer membrane proteins (e.g. ompA), and genes involved in natural competence (comEC). All tested mutants originally identified as motility-deficient in strain ATCC® 17978™ also displayed a motility-deficient phenotype in 29D2. By contrast, further comparative characterization of the mutant sets of both strains regarding pellicle biofilm formation, antibiotic resistance, and virulence in the Galleria mellonella infection model revealed numerous strain-specific mutant phenotypes. Our studies highlight the need for comparative analyses to characterize gene functions in A. baumannii and for further studies on the mechanisms underlying surface-associated motility.

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A transposon mutant library of Bacillus cereus ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle‐biofilm formation

Cited by 33 publications

References 73 publications

Control of pellicle biogenesis involves the diguanylate cyclases PdgA and PdgB, the c‐di‐GMP binding protein MxdA and the chemotaxis response regulator CheY3 in Shewanella oneidensis

Control of pellicle biogenesis involves the diguanylate cyclases PdgA and PdgB, the c‐di‐GMP binding protein MxdA and the chemotaxis response regulator CheY3 in Shewanella oneidensis

Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

Novel Genes Required for Surface-Associated Motility in Acinetobacter baumannii

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