Community quorum sensing signalling and quenching: microbial granular biofilm assembly.

BACKGROUND: Recent reports exploring the role of gradients of quorum sensing (QS) signals in functional activated sludge have raised the question of whether shared systems of signalling synthesis and degradation, or quorum quenching (QQ), across the community inform of the means by which QS biology regulate floccular and granular biofilm assembly. AIMS: In this study, we aimed to explore the species origin and interactive role of QS and QQ activities in such highly diverse microbial biofilm communities. METHODS: Here, such aims were addressed systematically by a comprehensive multi-pronged RNA-sequencing, microbiological and analytical chemistry experimental approach, using two related but independently evolved floccular and granular sludge communities. RESULTS: Our data revealed a distinct difference between the QS and QQ potentials of the two communities, with different species largely displaying either QS or QQ functions. The floccular sludge community showed a high rate of QQ activity, and this rate was dependent on the acyl chain length demonstrating specificity of degradation. When the floccular biomass was transformed into the granular sludge, the QQ activity of the community was reduced by 30%. N-acyl homoserine lactones with four to eight carbons on the acyl chain accumulated at the granular stage, and their concentrations were at least threefold higher than those of the floccular stage. These findings corroborated meta-community analysis where a major shift in the dominant species from potential signal quenchers to producers was observed during the transition from flocs to granules, indicating the role of species composition and associated signalling activities in coordinating community behaviours. CONCLUSIONS: This study suggests that QQ has an important function in regulating community level QS signalling, and provides a mechanistic insight into the role of QS biology in complex community assembly.

The role of quorum sensing signalling in EPS production and the assembly of a sludge community into aerobic granules.

Quorum sensing (QS) signalling has been extensively studied in single species populations. However, the ecological role of QS in complex, multi-species communities, particularly in the context of community assembly, has neither been experimentally explored nor theoretically addressed. Here, we performed a long-term bioreactor ecology study to address the links between QS, organization and composition of complex microbial communities. The conversion of floccular biomass to highly structured granules was found to be non-random, but strongly and positively correlated with N-acyl-homoserine-lactone (AHL)-mediated QS. Specific AHLs were elevated up to 100-fold and were strongly associated with the initiation of granulation. Similarly, the levels of particular AHLs decreased markedly during the granular disintegration phase. Metadata analysis indicated that granulation was accompanied by changes in extracellular polymeric substance (EPS) production and AHL add-back studies also resulted in increased EPS synthesis. In contrast to the commonly reported nanomolar to micromolar signal concentrations in pure culture laboratory systems, QS signalling in the granulation ecosystem occurred at picomolar to nanomolar concentrations of AHLs. Given that low concentrations of AHLs quantified in this study were sufficient to activate AHL bioreporters in situ in complex granular communities, AHL mediated QS may be a common feature in many natural and engineered ecosystems, where it coordinates community behaviour.

Phenotypic diversification and adaptation of Serratia marcescens MG1 biofilm-derived morphotypes.

We report here the characterization of dispersal variants from microcolony-type biofilms of Serratia marcescens MG1. Biofilm formation proceeds through a reproducible process of attachment, aggregation, microcolony development, hollow colony formation, and dispersal. From the time when hollow colonies were observed in flow cell biofilms after 3 to 4 days, at least six different morphological colony variants were consistently isolated from the biofilm effluent. The timing and pattern of variant formation were found to follow a predictable sequence, where some variants, such as a smooth variant with a sticky colony texture (SSV), could be consistently isolated at the time when mature hollow colonies were observed, whereas a variant that produced copious amounts of capsular polysaccharide (SUMV) was always isolated at late stages of biofilm development and coincided with cell death and biofilm dispersal or sloughing. The morphological variants differed extensively from the wild type in attachment, biofilm formation, and cell ultrastructure properties. For example, SSV formed two- to threefold more biofilm biomass than the wild type in batch biofilm assays, despite having a similar growth rate and attachment capacity. Interestingly, the SUMV, and no other variants, was readily isolated from an established SSV biofilm, indicating that the SUMV is a second-generation genetic variant derived from SSV. Planktonic cultures showed significantly lower frequencies of variant formation than the biofilms (5.05 x 10(-8) versus 4.83 x 10(-6), respectively), suggesting that there is strong, diversifying selection occurring within biofilms and that biofilm dispersal involves phenotypic radiation with divergent phenotypes.

Quorum sensing-controlled biofilm development in Serratia liquefaciens MG1.

Serratia liquefaciens MG1 contains an N-acylhomoserine lactone-mediated quorum-sensing system that is known to regulate swarming motility colonization. In this study, we describe for S. liquefaciens MG1 the development of a novel biofilm consisting of cell aggregates and differentiated cell types, such as cell chains and long filamentous cells. Furthermore, quorum sensing is shown to be crucial for normal biofilm development and for elaborate differentiation. A mutant of S. liquefaciens MG1 that was incapable of synthesizing extracellular signal formed a thin and nonmature biofilm lacking cell aggregates and differentiated cell chains. Signal-based complementation of this mutant resulted in a biofilm with the wild-type architecture. Two quorum-sensing-regulated genes (bsmA and bsmB) involved in biofilm development were identified, and we propose that these genes are engaged in fine-tuning the formation of cell aggregates at a specific point in biofilm development.