Norspermidine is not a self-produced trigger for biofilm disassembly.
ABSTRACT: Formation of Bacillus subtilis biofilms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the polyamine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 ?M prevents B. subtilis biofilm formation, (3) endogenous norspermidine is present in biofilms at 50-80 ?M, and (4) norspermidine prevents biofilm formation by condensing biofilm exopolysaccharide. In contrast, we find that, at concentrations up to 200 ?M, exogenous norspermidine promotes biofilm formation. We find that norspermidine is absent in wild-type B. subtilis biofilms at all stages, and higher concentrations of exogenous norspermidine eventually inhibit planktonic growth and biofilm formation in an exopolysaccharide-independent manner. Moreover, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, confirming that norspermidine is not physiologically relevant to biofilm function in this species.
Project description:The polyamine norspermidine is one of the major polyamines synthesized by Vibrionales and has also been found in various aquatic organisms. Norspermidine is among the environmental signals that positively regulate Vibrio cholerae biofilm formation. The NspS/MbaA signaling complex detects extracellular norspermidine and mediates the response to this polyamine. Norspermidine binding to the NspS periplasmic binding protein is thought to inhibit the phosphodiesterase activity of MbaA, increasing levels of the biofilm-promoting second messenger cyclic diguanylate monophosphate, thus enhancing biofilm formation. V. cholerae can also synthesize norspermidine using the enzyme NspC as well as import it from the environment. Deletion of the nspC gene was shown to reduce accumulation of bacteria in biofilms, leading to the conclusion that intracellular norspermidine is also a positive regulator of biofilm formation. Because V. cholerae uses norspermidine to synthesize the siderophore vibriobactin it is possible that intracellular norspermidine is required to obtain sufficient amounts of iron, which is also necessary for robust biofilm formation. The objective of this study was to assess the relative contributions of intracellular and extracellular norspermidine to the regulation of biofilm formation in V. cholerae. We show the biofilm defect of norspermidine synthesis mutants does not result from an inability to produce vibriobactin as vibriobactin synthesis mutants do not have diminished biofilm forming abilities. Furthermore, our work shows that extracellular, but not intracellular norspermidine, is mainly responsible for promoting biofilm formation. We establish that the NspS/MbaA signaling complex is the dominant mediator of biofilm formation in response to extracellular norspermidine, rather than norspermidine synthesized by NspC or imported into the cell.
Project description:Biofilm formation in Vibrio cholerae is in part regulated by norspermidine, a polyamine synthesized by the enzyme carboxynorspermidine decarboxylase (NspC). The absence of norspermidine in the cell leads to a marked reduction in V. cholerae biofilm formation by an unknown mechanism. In this work, we show that overexpression of nspC results in large increases in biofilm formation and vps gene expression as well as a significant decrease in motility. Interestingly, increased NspC levels do not lead to increased concentrations of norspermidine in the cell. Our results show that NspC levels inversely regulate biofilm and motility and implicate the presence of an effective feedback mechanism maintaining norspermidine homeostasis in V. cholerae. Moreover, we provide evidence that NspC and the norspermidine sensor protein, NspS, provide independent and distinct inputs into the biofilm regulatory network.
Project description:Biofilms are structured communities of bacteria that are held together by an extracellular matrix consisting of protein and exopolysaccharide. Biofilms often have a limited lifespan, disassembling as nutrients become exhausted and waste products accumulate. D-amino acids were previously identified as a self-produced factor that mediates biofilm disassembly by causing the release of the protein component of the matrix in Bacillus subtilis. Here we report that B. subtilis produces an additional biofilm-disassembly factor, norspermidine. Dynamic light scattering and scanning electron microscopy experiments indicated that norspermidine interacts directly and specifically with exopolysaccharide. D-amino acids and norspermidine acted together to break down existing biofilms and mutants blocked in the production of both factors formed long-lived biofilms. Norspermidine, but not closely related polyamines, prevented biofilm formation by B. subtilis, Escherichia coli, and Staphylococcus aureus.
Project description:Biofilms are defined as aggregation of single cell microorganisms and associated with over 80% of all the microbial infections. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen capable of leading to various infections in immunocompromised people. Recent studies showed that norspermidine, a kind of polyamine, prevented and disrupted biofilm formation by some Gram-negative bacterium. In this study, the effects of norspermidine on P. aeruginosa biofilm formation and eradication were tested. Microtiter plate combined with crystal violet staining was used to study the effects of norspermidine on P. aeruginosa initial attachment, then we employed SEM (scanning electron microscope), qRT-PCR, and QS-related virulence factor assays to investigate how norspermidine prevent biofilm formation by P. aeruginosa. We reported that high-dose norspermidine had bactericide effect on P. aeruginosa, and norspermidine began to inhibit biofilm formation and eradicate 24-h mature biofilm at concentration of 0.1 and 1 mmol/L, respectively, probably by preventing cell-surface attachment, inhibiting swimming motility, and downregulating QS-related genes expression. To investigate the potential utility of norspermidine in preventing device-related infections, we found that catheters immersed with norspermidine were effective in eradicating mature biofilm. These results suggest that norspermidine could be a potent antibiofilm agent for formulating strategies against P. aeruginosa biofilm.
Project description:The present study aimed at investigating the influence of norspermidine on the formation of dual-species biofilms composed of Streptococcus mutans (S. mutans) and Streptococcus sanguinis (S. sanguinis). Crystal violet assay was conducted to assess the formation of single-species biofilms of S. mutans and S. sanguinis, and the growth curve was carefully observed to monitor the growth of these two species of bacteria. Fluorescence in situ hybridization (FISH) and MTT array were used to analyze the composition and metabolic activity of the dual-species biofilms, respectively. Extracellular polysaccharides (EPS)/bacteria staining, anthrone method, and scanning electron microscopy (SEM) imaging were conducted to study the synthesis of EPS by dual-species biofilms. Lactic acid assay and pH were measured to detect dual-species biofilm acid production. We found that norspermidine had different effects on S. mutans and S. sanguinis including their growth and biofilm formation. Norspermidine regulated the composition of the dual-species biofilms, decreased the ratio of S. mutans in dual-species biofilms, and reduced the metabolic activity, EPS synthesis, and acid production of dual-species biofilms. Norspermidine regulated dual-species biofilms in an ecological way, suggesting that it may be a potent reagent for controlling dental biofilms and managing dental caries.
Project description:Ubiquitous polyamine spermidine is not required for normal planktonic growth of Bacillus subtilis but is essential for robust biofilm formation. However, the structural features of spermidine required for B. subtilis biofilm formation are unknown and so are the molecular mechanisms of spermidine-stimulated biofilm development. We report here that in a spermidine-deficient B. subtilis mutant, the structural analogue norspermidine, but not homospermidine, restored biofilm formation. Intracellular biosynthesis of another spermidine analogue, aminopropylcadaverine, from exogenously supplied homoagmatine also restored biofilm formation. The differential ability of C-methylated spermidine analogues to functionally replace spermidine in biofilm formation indicated that the aminopropyl moiety of spermidine is more sensitive to C-methylation, which it is essential for biofilm formation, but that the length and symmetry of the molecule is not critical. Transcriptomic analysis of a spermidine-depleted B. subtilis speD mutant uncovered a nitrogen-, methionine-, and S-adenosylmethionine-sufficiency response, resulting in repression of gene expression related to purine catabolism, methionine and S-adenosylmethionine biosynthesis and methionine salvage, and signs of altered membrane status. Consistent with the spermidine requirement in biofilm formation, single-cell analysis of this mutant indicated reduced expression of the operons for production of the exopolysaccharide and TasA protein biofilm matrix components and SinR antagonist slrR Deletion of sinR or ectopic expression of slrR in the spermidine-deficient ?speD background restored biofilm formation, indicating that spermidine is required for expression of the biofilm regulator slrR Our results indicate that spermidine functions in biofilm development by activating transcription of the biofilm matrix exopolysaccharide and TasA operons through the regulator slrR.
Project description:The aquatic bacterium and human intestinal pathogen, Vibrio cholerae, senses and responds to a variety of environment-specific cues to regulate biofilm formation. Specifically, the polyamines norspermidine and spermidine enhance and repress V. cholerae biofilm formation, respectively. These effects are relevant for understanding V. cholerae pathogenicity and are mediated through the periplasmic binding protein NspS and the transmembrane bis-(3'-5') cyclic diguanosine monophosphate (c-di-GMP) phosphodiesterase MbaA. However, the levels of spermidine required to inhibit biofilm formation through this pathway are unlikely to be encountered by V. cholerae in aquatic reservoirs or within the human host during infection. We therefore hypothesized that other polyamines in the gastrointestinal tract may control V. cholerae biofilm formation at physiological levels. The tetramine spermine has been reported to be present at nearly 50 ?m concentrations in the intestinal lumen. Here, we report that spermine acts as an exogenous cue that inhibits V. cholerae biofilm formation through the NspS-MbaA signaling system. We found that this effect probably occurs through a direct interaction of spermine with NspS, as purified NspS protein could bind spermine in vitro Spermine also inhibited biofilm formation by altering the transcription of the vps genes involved in biofilm matrix production. Global c-di-GMP levels were unaffected by spermine supplementation, suggesting that biofilm formation may be regulated by variations in local rather than global c-di-GMP pools. Finally, we propose a model illustrating how the NspS-MbaA signaling system may communicate exogenous polyamine content to the cell to control biofilm formation in the aquatic environment and within the human intestine.
Project description:Polyamines are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including biofilm formation, thus indicating that polyamines may have potential in the control of unwanted biofilm. In the present study, the effects of the polyamines norspermidine and spermidine on biofilms of 10 potentially pathogenic wild-type strains of Escherichia coli serotype O103:H2, Salmonella enterica subsp. enterica serovar Typhimurium, and S. enterica serovar Agona were investigated. We found that exogenously supplied norspermidine and spermidine did not mediate disassembly of preformed biofilm of any of the E. coli and S. enterica strains. However, the polyamines did affect biofilm production. Interestingly, the two species reacted differently to the polyamines. Both polyamines reduced the amount of biofilm formed by E. coli but tended to increase biofilm formation by S. enterica. Whether the effects observed were due to the polyamines specifically targeting biofilm formation, being toxic for the cells, or maybe a combination of the two, is not known. However, there were no indications that the effect was mediated through binding to exopolysaccharides, as earlier suggested for E. coli. Our results indicate that norspermidine and spermidine do not have potential as inhibitors of S. enterica biofilm. Furthermore, we found that the commercial polyamines used contributed to the higher pH of the test medium. Failure to acknowledge and control this important phenomenon may lead to misinterpretation of the results.
Project description:<h4>Unlabelled</h4>In bacteria, the functions of polyamines, small linear polycations, are poorly defined, but these metabolites can influence biofilm formation in several systems. Transposon insertions in an ornithine decarboxylase (odc) gene in Agrobacterium tumefaciens, predicted to direct synthesis of the polyamine putrescine from ornithine, resulted in elevated cellulose. Null mutants for odc grew somewhat slowly in a polyamine-free medium but exhibited increased biofilm formation that was dependent on cellulose production. Spermidine is an essential metabolite in A. tumefaciens and is synthesized from putrescine in A. tumefaciens via the stepwise actions of carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase (CASDC). Exogenous addition of either putrescine or spermidine to the odc mutant returned biofilm formation to wild-type levels. Low levels of exogenous spermidine restored growth to CASDH and CASDC mutants, facilitating weak biofilm formation, but this was dampened with increasing concentrations. Norspermidine rescued growth for the odc, CASDH, and CASDC mutants but did not significantly affect their biofilm phenotypes, whereas in the wild type, it stimulated biofilm formation and depressed spermidine levels. The odc mutant produced elevated levels of cyclic diguanylate monophosphate (c-di-GMP), exogenous polyamines modulated these levels, and expression of a c-di-GMP phosphodiesterase reversed the enhanced biofilm formation. Prior work revealed accumulation of the precursors putrescine and carboxyspermidine in the CASDH and CASDC mutants, respectively, but unexpectedly, both mutants accumulated homospermidine; here, we show that this requires a homospermidine synthase (hss) homologue.<h4>Importance</h4>Polyamines are small, positively charged metabolites that are nearly ubiquitous in cellular life. They are often essential in eukaryotes and more variably in bacteria. Polyamines have been reported to influence the surface-attached biofilm formation of several bacteria. In Agrobacterium tumefaciens, mutants with diminished levels of the polyamine spermidine are stimulated for biofilm formation, and exogenous provision of spermidine decreases biofilm formation. Spermidine is also essential for A. tumefaciens growth, but the related polyamine norspermidine exogenously rescues growth and does not diminish biofilm formation, revealing that the growth requirement and biofilm control are separable. Polyamine control of biofilm formation appears to function via effects on the cellular second messenger cyclic diguanylate monophosphate, regulating the transition from a free-living to a surface-attached lifestyle.
Project description:Polyamines are small organic cations found in all cells, and the biosynthetic pathway is well described in eukaryotes and Escherichia coli. The characterized pathway uses decarboxylated S-adenosylmethionine as the aminopropyl group donor to form spermidine from putrescine by the key enzymes S-adenosylmethionine decarboxylase and spermidine synthase. We report here the in vivo characterization of an alternative polyamine biosynthetic pathway from Vibrio cholerae, the causative agent of human cholera. The pathway uses aspartate beta-semialdehyde as the aminopropyl group donor and consists of a fused protein containing l-2,4-diaminobutyrate aminotransferase and l-2,4-diaminobutyrate decarboxylase, a carboxynorspermidine dehydrogenase (CANSDH), and a carboxynorspermidine decarboxylase (CANSDC). We show that in V. cholerae, this pathway is required for synthesis of both sym-norspermidine and spermidine. Heterologous expression of the V. cholerae pathway in E. coli results in accumulation of the nonnative polyamines diaminopropane and sym-norspermidine. Genetic deletion of the V. cholerae CANSDC led to accumulation of carboxynorspermidine, whereas deletion of either CANSDC or the putative CANSDH led to loss of sym-norspermidine and spermidine. These results allowed unambiguous identification of the gene encoding CANSDH. Furthermore, deletion of either CANSDH or CANSDC led to a 50-60% reduction in growth rate of planktonic cells and severely reduced biofilm formation, which could be rescued by exogenously supplied sym-norspermidine but not spermidine. The pathway was not required for infectivity in a mouse model of V. cholerae infection. Notably, the alternative polyamine biosynthetic pathway is widespread in bacteria and is likely to play a previously unrecognized role in the biology of these organisms.