Lipopeptides constitute a structurally diverse group of metabolites produced by various bacterial and fungal genera. In the past decades, research on lipopeptides has been fueled by their antimicrobial, antitumour, immunosuppressant and surfactant activities. However, the natural functions of lipopeptides in the lifestyles of the producing microorganisms have received considerably less attention. The substantial structural diversity of lipopeptides suggests that these metabolites have different natural roles, some of which may be unique to the biology of the producing organism. This review gives a detailed overview of the versatile functions of lipopeptides in the biology of Pseudomonas and Bacillus species, and highlights their role in competitive interactions with coexisting organisms, including bacteria, fungi, oomycetes, protozoa, nematodes and plants. Their functions in cell motility, leading to colonization of novel habitats, and in the formation and development of highly structured biofilms are discussed in detail. Finally, this review provides an update on lipopeptide detection and discovery as well as on novel regulatory mechanisms and genes involved in lipopeptide biosynthesis in these two bacterial genera.

Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics

The interaction of bacteria with surfaces has important implications in a range of areas, including bioenergy, biofouling, biofilm formation, and the infection of plants and animals. Many of the interactions of bacteria with surfaces produce changes in the expression of genes that influence cell morphology and behavior, including genes essential for motility and surface attachment. Despite the attention that these phenotypes have garnered, the bacterial systems used for sensing and responding to surfaces are still not well understood. An understanding of these mechanisms will guide the development of new classes of materials that inhibit and promote cell growth, and complement studies of the physiology of bacteria in contact with surfaces. Recent studies from a range of fields in science and engineering are poised to guide future investigations in this area. This review summarizes recent studies on bacteria–surface interactions, discusses mechanisms of surface sensing and consequences of cell attachment, provides an overview of surfaces that have been used in bacterial studies, and highlights unanswered questions in this field.

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Bacteria-surface interactions.

Bacteria grow in either planktonic form or as biofilms, which are attached to either inert or biological surfaces. Both growth forms are highly relevant states in nature and of paramount scientific focus. However, interchanges between bacteria in these two states have been little explored. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we show that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances in nature. We hypothesized and proved that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. These findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations.

https://www.pnas.org/content/pnas/109/32/13088.full.pdf

Bacterial swimmers that infiltrate and take over the biofilm matrix

The dense aggregation of cells on a surface, as seen in biofilms, inevitably results in both environmental and cellular heterogeneity. For example, nutrient gradients can trigger cells to differentiate into various phenotypic states. Not only do cells adapt physiologically to the local environmental conditions, but they also differentiate into cell types that interact with each other. This allows for task differentiation and, hence, the division of labor. In this article, we focus on cell differentiation and the division of labor in three bacterial species: Myxococcus xanthus, Bacillus subtilis, and Pseudomonas aeruginosa. During biofilm formation each of these species differentiates into distinct cell types, in some cases leading to cooperative interactions. The division of labor and the cooperative interactions between cell types are assumed to yield an emergent ecological benefit. Yet in most cases the ecological benefits have yet to be elucidated. A notable exception is M. xanthus, in which cell differentiation within fruiting bodies facilitates the dispersal of spores. We argue that the ecological benefits of the division of labor might best be understood when we consider the dynamic nature of both biofilm formation and degradation.

Division of Labor in Biofilms: the Ecology of Cell Differentiation.

Some plant-associated Bacillus strains produce induced systemic resistance (ISR) in the host, which contributes to their protective effect against phytopathogens. Little is known about the variety of elicitors responsible for ISR that are produced by Bacillus strains. Working with a particular strain, we have previously identified the surfactin lipopeptide as a main compound stimulating plant immune-related responses. However, with the perspective of developing Bacillus strains as biocontrol agents, it is important to establish whether a central role of surfactin is generally true for isolates belonging to the B. subtilis/amyloliquefaciens complex. To that end, we set up a comparative study involving a range of natural strains. Their secretomes were first tested for triggering early defense events in cultured tobacco cells. Six isolates with contrasting activities were further evaluated for ISR in plants, based both on macroscopic disease reduction and on stimulation of the oxylipin pathway as defense mechanism. A strong correlation was found between defense-inducing activity and the amount of surfactin produced by the isolates. These results support the idea of a widespread role for surfactin as a nonvolatile elicitor formed by B. subtilis/amyloliquefaciens, and screening for strong surfactin producers among strains naturally secreting multiple antibiotics could be an efficient approach to select good candidates as biopesticides.

https://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-09-13-0262-R

Plant Defense Stimulation by Natural Isolates of Bacillus Depends on Efficient Surfactin Production

Highly branched dendritic swarming of B. subtilis on synthetic B-medium involves a developmental-like process that is absolutely dependent on flagella and surfactin secretion. In order to identify new swarming genes, we targeted the two-component ComPA signalling pathway and associated global regulators. In liquid cultures, the histidine kinase ComP, and the response regulator ComA, respond to secreted pheromones ComX and CSF (encoded by phrC) in order to control production of surfactin synthases and ComS (competence regulator). In this study, for what is believed to be the first time, we established that distinct early stages of dendritic swarming can be clearly defined, and that they are amenable to genetic analysis. In a mutational analysis producing several mutants with distinctive phenotypes, we were able to assign the genes sfp (activation of surfactin synthases), comA, abrB and codY (global regulators), hag (flagellin), mecA and yvzB (hag-like), and swrB (motility), to the different swarming stages. Surprisingly, mutations in genes comPX, comQ, comS, rapC and oppD, which are normally indispensable for import of CSF, had only modest effects, if any, on swarming and surfactin production. Therefore, during dendritic swarming, surfactin synthesis is apparently subject to novel regulation that is largely independent of the ComXP pathway; we discuss possible alternative mechanisms for driving srfABCD transcription. We showed that the phrC mutant, largely independent of any effect on surfactin production, was also, nevertheless, blocked early in swarming, forming stunted dendrites, with abnormal dendrite initiation morphology. In a mixed swarm co-inoculated with phrC sfp+ and phrC+ sfp (GFP), an apparently normal swarm was produced. In fact, while initiation of all dendrites was of the abnormal phrC type, these were predominantly populated by sfp cells, which migrated faster than the phrC cells. This and other results indicated a specific migration defect in the phrC mutant that could not be trans-complemented by CSF in a mixed swarm. CSF is the C-terminal pentapeptide of the surface-exposed PhrC pre-peptide and we propose that the residual PhrC 35 aa residue peptide anchored in the exterior of the cytoplasmic membrane has an apparently novel extracellular role in swarming.

Identification of genes required for different stages of dendritic swarming in Bacillus subtilis, with a novel role for phrC.

The non-domesticated Bacillus subtilis strain 3610 displays, over a wide range of humidity, hyper-branched, dendritic, swarming-like migration on a minimal agar medium. At high (70 %) humidity, the laboratory strain 168 sfp
+ (producing surfactin) behaves very similarly, although this strain carries a frameshift mutation in swrA, which another group has shown under their conditions (which include low humidity) is essential for swarming. We reconcile these different results by demonstrating that, while swrA is essential for dendritic migration at low humidity (30–40 %), it is dispensable at high humidity. Dendritic migration (flagella- and surfactin-dependent) of strains 168 sfp
+
swrA and 3610 involves elongation of dendrites for several hours as a monolayer of cells in a thin fluid film. This enabled us to determine in situ the spatiotemporal pattern of expression of some key players in migration as dendrites develop, using gfp transcriptional fusions for hag (encoding flagellin), comA (regulation of surfactin synthesis) as well as eps (exopolysaccharide synthesis). Quantitative (single-cell) analysis of hag expression in situ revealed three spatially separated subpopulations or cell types: (i) networks of chains arising early in the mother colony (MC), expressing eps but not hag; (ii) largely immobile cells in dendrite stems expressing intermediate levels of hag; and (iii) a subpopulation of cells with several distinctive features, including very low comA expression but hyper-expression of hag (and flagella). These specialized cells emerge from the MC to spearhead the terminal 1 mm of dendrite tips as swirling and streaming packs, a major characteristic of swarming migration. We discuss a model for this swarming process, emphasizing the importance of population density and of the complementary roles of packs of swarmers driving dendrite extension, while non-mobile cells in the stems extend dendrites by multiplication.

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers.

Krzysztof Hinc

Papers

Identification of genes required for different stages of dendritic swarming in Bacillus subtilis, with a novel role for phrC.

Single-cell analysis in situ in a Bacillus subtilis swarming community identifies distinct spatially separated subpopulations differentially expressing hag (flagellin), including specialized swarmers.