The Bacillus phage SPß and its relatives: a temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components.

The Bacillus phage SPß has been known for about 50 years, but only a few strains are available. We isolated four new wild-type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SPß-like phages. Sequence data revealed the genome replication strategy and the genome packaging mode of SPß-like phages. We extracted 55 SPß-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resemble four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SPß-like prophages consists of 38 proteins. The integration cassette proved to be not conserved, even though, present in all strains. It consists of distinct integrases. Analysis of SPß transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.

Laboratory Evolution of Microbial Interactions in Bacterial Biofilms.

Microbial adaptation is conspicuous in essentially every environment, but the mechanisms of adaptive evolution are poorly understood. Studying evolution in the laboratory under controlled conditions can be a tractable approach, particularly when new, discernible phenotypes evolve rapidly. This is especially the case in the spatially structured environments of biofilms, which promote the occurrence and stability of new, heritable phenotypes. Further, diversity in biofilms can give rise to nascent social interactions among coexisting mutants and enable the study of the emerging field of sociomicrobiology. Here, we review findings from laboratory evolution experiments with either Pseudomonas fluorescens or Burkholderia cenocepacia in spatially structured environments that promote biofilm formation. In both systems, ecotypes with overlapping niches evolve and produce competitive or facilitative interactions that lead to novel community attributes, demonstrating the parallelism of adaptive processes captured in the lab.

synphage: a pipeline for phage genome synteny graphics focused on gene conservation.

Motivation: Visualization and comparison of genome maps of bacteriophages can be very effective, but none of the tools available on the market allow visualization of gene conservation between multiple sequences at a glance. In addition, most bioinformatic tools running locally are command line only, making them hard to setup, debug, and monitor. Results: To address these motivations, we developed synphage, an easy-to-use and intuitive tool to generate synteny diagrams from GenBank files. This software has a user-friendly interface and uses metadata to monitor the progress and success of the data transformation process. The output plot features colour-coded genes according to their degree of conservation among the group of displayed sequences. The strength of synphage lies also in its modularity and the ability to generate multiple plots with different configurations without having to re-process all the data. In conclusion, synphage reduces the bioinformatic workload of users and allows them to focus on analysis, the most impactful area of their work. Availability and implementation: The synphage tool is implemented in the Python language and is available from the GitHub repository at https://github.com/vestalisvirginis/synphage. This software is released under an Apache-2.0 licence. A PyPI synphage package is available at https://pypi.org/project/synphage/ and a containerized version is available at https://hub.docker.com/r/vestalisvirginis/synphage. Contributions to the software are welcome whether it is reporting a bug or proposing new features and the contribution guidelines are available at https://github.com/vestalisvirginis/synphage/blob/main/CONTRIBUTING.md.

Unique relationships between phages and endospore-forming hosts.

As part of their survival strategy under harsh environmental conditions, endospore-forming bacteria can trigger a sporulation developmental program. Although the regulatory cascades that precisely control the transformation of vegetative bacteria into mother cells and resilient spores have been described in detail, less is known about how bacteriophages that prey on endospore-formers exploit sporulation. Herein, we argue that phages infecting these bacteria have evolved several specific molecular mechanisms, not yet known in other bacteria, that manifest from the phage-driven alliance to negative effects on the host. We anticipate that the relationships between phages and endospore-formers outlined here will inspire studies on phage ecology and evolution, and could facilitate important advances in the development of phage therapies against pathogenic spore-formers.

Host control by SPß phage regulatory switch as potential manipulation strategy.

The interaction between temperate phages and their bacterial hosts has always been one of the most controversial in nature. As genetic parasites, phages need their hosts to propagate, while the host may take advantage of the genetic arsenal carried in the phage genome. This intriguing host-parasite interplay with an evident mutualistic implication could be challenged by recent discoveries of alternative phage lifestyles and regulatory systems that seem to support a manipulative strategy pursued by the phage. Through two fascinating novel mechanisms concerning the active lysogeny and a phage-encoded quorum sensing system, referred as 'Arbitrium', employed by SPß-like phages of Bacilli, we propose the parasite manipulation as ecological relationship between certain temperate phages and bacteria.

DegQ is an important policing link between quorum sensing and regulated adaptative traits in Bacillus subtilis.

Quorum sensing (QS) is a widespread bacterial communication system that controls important adaptive traits in a cell density-dependent manner. However, mechanisms by which QS-regulated traits are linked within the cell and mechanisms by which these links affect adaptation are not well understood. In this study, Bacillus subtilis was used as a model bacterium to investigate the link between the ComQXPA QS system, DegQ, surfactin and protease production in planktonic and biofilm cultures. The work tests two alternative hypotheses predicting that hypersensitivity of the QS signal-deficient mutant (comQ::kan) to exogenously added ComX, resulting in increased surfactin production, is linked to an additional genetic locus, or alternatively, to overexpression of the ComX receptor ComP. Results are in agreement with the first hypothesis and show that the P srfAA hypersensitivity of the comQ::kan mutant is linked to a 168 strain-specific mutation in the P degQ region. Hence, the markerless ΔcomQ mutant lacking this mutation is not overresponsive to ComX. Such hyper-responsiveness is specific for the P srfAA and not detected in another ComX-regulated promoter, the P aprE , which is under the positive control by DegQ. Our results suggest that DegQ by exerting differential effect on P srfAA and P aprE acts as a policing mechanism and the intracellular link, which guards the cell from an overinvestment into surfactin production. IMPORTANCE DegQ levels are known to regulate surfactin synthesis and extracellular protease production, and DegQ is under the control of the ComX-dependent QS. DegQ also serves as an important policing link between these QS-regulated processes, preventing overinvestment in these costly processes. This work highlights the importance of DegQ, which acts as the intracellular link between ComX production and the response by regulating extracellular degradative enzyme synthesis and surfactin production.

New Phage-Derived Antibacterial Enzyme PolaR Targeting Rothia spp.

Rothia is an opportunistic pathogen, particularly life-threatening for the immunocompromised. It is associated with pneumonia, endocarditis, peritonitis and many other serious infections, including septicemia. Of note, Rothia mucilaginousa produces metabolites that support and increase overgrowth of Pseudomonas aeruginosa, one of the ESKAPE bacteria. Endolysins are considered as antibacterial enzymes derived from bacteriophages that selectively and efficiently kill susceptible bacteria without harming human cells or the normal microbiome. Here, we applied a computational analysis of metagenomic sequencing data of the gastric mucosa phageome extracted from human patients' stomach biopsies. A selected candidate anti-Rothia sequence was produced in an expression system, purified and confirmed as a Rothia mucilaginosa- and Rothia dentocariosa-specific endolysin PolaR, able to destroy bacterial cells even when aggregated, as in a biofilm. PolaR had no cytotoxic or antiproliferative effects on mammalian cells. PolaR is the first described endolysin selectively targeting Rothia species, with a high potential to combat infections caused by Rothia mucilaginosa and Rothia dentocariosa, and possibly other bacterial groups. PolaR is the first antibacterial enzyme selected from the gastric mucosa phageome, which underlines the biological complexity and probably underestimated biological role of the phageome in the human gastric mucosa.

Bacillus velezensis stimulates resident rhizosphere Pseudomonas stutzeri for plant health through metabolic interactions.

Trophic interactions play a central role in driving microbial community assembly and function. In gut or soil ecosystems, successful inoculants are always facilitated by efficient colonization; however, the metabolite exchanges between inoculants and resident bacteria are rarely studied, particularly in the rhizosphere. Here, we used bioinformatic, genetic, transcriptomic, and metabonomic analyses to uncover syntrophic cooperation between inoculant (Bacillus velezensis SQR9) and plant-beneficial indigenous Pseudomonas stutzeri in the cucumber rhizosphere. We found that the synergistic interaction of these two species is highly environmental dependent, the emergence of syntrophic cooperation was only evident in a static nutrient-rich niche, such as pellicle biofilm in addition to the rhizosphere. Our results identified branched-chain amino acids (BCAAs) biosynthesis pathways are involved in syntrophic cooperation. Genome-scale metabolic modeling and metabolic profiling also demonstrated metabolic facilitation among the bacterial strains. In addition, biofilm matrix components from Bacillus were essential for the interaction. Importantly, the two-species consortium promoted plant growth and helped plants alleviate salt stress. In summary, we propose a mechanism in which synergic interactions between a biocontrol bacterium and a partner species promote plant health.

Peptide signaling without feedback in signal production operates as a true quorum sensing communication system in Bacillus subtilis.

Bacterial quorum sensing (QS) is based on signal molecules (SM), which increase in concentration with cell density. At critical SM concentration, a variety of adaptive genes sharply change their expression from basic level to maximum level. In general, this sharp transition, a hallmark of true QS, requires an SM dependent positive feedback loop, where SM enhances its own production. Some communication systems, like the peptide SM-based ComQXPA communication system of Bacillus subtilis, do not have this feedback loop and we do not understand how and if the sharp transition in gene expression is achieved. Based on experiments and mathematical modeling, we observed that the SM peptide ComX encodes the information about cell density, specific cell growth rate, and even oxygen concentration, which ensure power-law increase in SM production. This enables together with the cooperative response to SM (ComX) a sharp transition in gene expression level and this without the SM dependent feedback loop. Due to its ultra-sensitive nature, the ComQXPA can operate at SM concentrations that are 100-1000 times lower than typically found in other QS systems, thereby substantially reducing the total metabolic cost of otherwise expensive ComX peptide.

Surfactin Facilitates Horizontal Gene Transfer in Bacillus subtilis.

Genetic competence for the uptake and integration of extracellular DNA is a key process in horizontal gene transfer (HGT), one of the most powerful forces driving the evolution of bacteria. In several species, development of genetic competence is coupled with cell lysis. Using Bacillus subtilis as a model bacterium, we studied the role of surfactin, a powerful biosurfactant and antimicrobial lipopeptide, in genetic transformation. We showed that surfactin itself promotes cell lysis and DNA release, thereby promoting HGT. These results, therefore, provide evidence for a fundamental mechanism involved in HGT and significantly increase our understanding of the spreading of antibiotic resistance genes and diversification of microbial communities in the environment.

Phages carry interbacterial weapons encoded by biosynthetic gene clusters.

Bacteria produce diverse specialized metabolites that mediate ecological interactions and serve as a rich source of industrially relevant natural products. Biosynthetic pathways for these metabolites are encoded by organized groups of genes called biosynthetic gene clusters (BGCs). Understanding the natural function and distribution of BGCs provides insight into the mechanisms through which microorganisms interact and compete. Further, understanding BGCs is extremely important for biocontrol and the mining of new bioactivities. Here, we investigated phage-encoded BGCs (pBGCs), challenging the relationship between phage origin and BGC structure and function. The results demonstrated that pBGCs are rare, and they predominantly reside within temperate phages infecting commensal or pathogenic bacterial hosts. Further, the vast majority of pBGCs were found to encode for bacteriocins. Using the soil- and gut-associated bacterium Bacillus subtilis, we experimentally demonstrated how a temperate phage equips a bacterium with a fully functional BGC, providing a clear competitive fitness advantage over the ancestor. Moreover, we demonstrated a similar transfer of the same phage in prophage form. Finally, using genetic and genomic comparisons, a strong association between pBGC type and phage host range was revealed. These findings suggest that bacteriocins are encoded in temperate phages of a few commensal bacterial genera. In these cases, lysogenic conversion provides an evolutionary benefit to the infected host and, hence, to the phage itself. This study is an important step toward understanding the natural role of bacterial compounds encoded by BGCs, the mechanisms driving their horizontal transfer, and the sometimes mutualistic relationship between bacteria and temperate phages.

Impact of Rap-Phr system abundance on adaptation of Bacillus subtilis.

Microbes commonly display great genetic plasticity, which has allowed them to colonize all ecological niches on Earth. Bacillus subtilis is a soil-dwelling organism that can be isolated from a wide variety of environments. An interesting characteristic of this bacterium is its ability to form biofilms that display complex heterogeneity: individual, clonal cells develop diverse phenotypes in response to different environmental conditions within the biofilm. Here, we scrutinized the impact that the number and variety of the Rap-Phr family of regulators and cell-cell communication modules of B. subtilis has on genetic adaptation and evolution. We examine how the Rap family of phosphatase regulators impacts sporulation in diverse niches using a library of single and double rap-phr mutants in competition under 4 distinct growth conditions. Using specific DNA barcodes and whole-genome sequencing, population dynamics were followed, revealing the impact of individual Rap phosphatases and arising mutations on the adaptability of B. subtilis.

Pervasive prophage recombination occurs during evolution of spore-forming Bacilli.

Phages are the main source of within-species bacterial diversity and drivers of horizontal gene transfer, but we know little about the mechanisms that drive genetic diversity of these mobile genetic elements (MGEs). Recently, we showed that a sporulation selection regime promotes evolutionary changes within SPß prophage of Bacillus subtilis, leading to direct antagonistic interactions within the population. Herein, we reveal that under a sporulation selection regime, SPß recombines with low copy number phi3Ts phage DNA present within the B. subtilis population. Recombination results in a new prophage occupying a different integration site, as well as the spontaneous release of virulent phage hybrids. Analysis of Bacillus sp. strains suggests that SPß and phi3T belong to a distinct cluster of unusually large phages inserted into sporulation-related genes that are equipped with a spore-related genetic arsenal. Comparison of Bacillus sp. genomes indicates that similar diversification of SPß-like phages takes place in nature. Our work is a stepping stone toward empirical studies on phage evolution, and understanding the eco-evolutionary relationships between bacteria and their phages. By capturing the first steps of new phage evolution, we reveal striking relationship between survival strategy of bacteria and evolution of their phages.

Quantitative image analysis of microbial communities with BiofilmQ.

Biofilms are microbial communities that represent a highly abundant form of microbial life on Earth. Inside biofilms, phenotypic and genotypic variations occur in three-dimensional space and time; microscopy and quantitative image analysis are therefore crucial for elucidating their functions. Here, we present BiofilmQ-a comprehensive image cytometry software tool for the automated and high-throughput quantification, analysis and visualization of numerous biofilm-internal and whole-biofilm properties in three-dimensional space and time.

Metal ions weaken the hydrophobicity and antibiotic resistance of Bacillus subtilis NCIB 3610 biofilms.

Surface superhydrophobicity makes bacterial biofilms very difficult to fight, and it is a combination of their matrix composition and complex surface roughness which synergistically protects these biomaterials from wetting. Although trying to eradicate biofilms with aqueous (antibiotic) solutions is common practice, this can be a futile approach if the biofilms have superhydrophobic properties. To date, there are not many options available to reduce the liquid repellency of biofilms or to prevent this material property from developing. Here, we present a solution to this challenge. We demonstrate how the addition of metal ions such as copper and zinc during or after biofilm formation can render the surface of otherwise superhydrophobic B. subtilis NCIB 3610 biofilms completely wettable. As a result of this procedure, these smoother, hydrophilic biofilms are more susceptible to aqueous antibiotics solutions. Our strategy proposes a scalable and widely applicable step in a multi-faceted approach to eradicate biofilms.

Cheaters shape the evolution of phenotypic heterogeneity in Bacillus subtilis biofilms.

Biofilms are closely packed cells held and shielded by extracellular matrix composed of structural proteins and exopolysaccharides (EPS). As matrix components are costly to produce and shared within the population, EPS-deficient cells can act as cheaters by gaining benefits from the cooperative nature of EPS producers. Remarkably, genetically programmed EPS producers can also exhibit phenotypic heterogeneity at single-cell level. Previous studies have shown that spatial structure of biofilms limits the spread of cheaters, but the long-term influence of cheating on biofilm evolution is not well understood. Here, we examine the influence of EPS nonproducers on evolution of matrix production within the populations of EPS producers in a model biofilm-forming bacterium, Bacillus subtilis. We discovered that general adaptation to biofilm lifestyle leads to an increase in phenotypical heterogeneity of eps expression. However, prolonged exposure to EPS-deficient cheaters may result in different adaptive strategy, where eps expression increases uniformly within the population. We propose a molecular mechanism behind such adaptive strategy and demonstrate how it can benefit the EPS producers in the presence of cheaters. This study provides additional insights on how biofilms adapt and respond to stress caused by exploitation in long-term scenario.

Privatization of Biofilm Matrix in Structurally Heterogeneous Biofilms.

The self-produced biofilm provides beneficial protection for the enclosed cells, but the costly production of matrix components makes producer cells susceptible to cheating by nonproducing individuals. Despite detrimental effects of nonproducers, biofilms can be heterogeneous, with isogenic nonproducers being a natural consequence of phenotypic differentiation processes. For instance, in Bacillus subtilis biofilm cells differ in production of the two major matrix components, the amyloid fiber protein TasA and exopolysaccharides (EPS), demonstrating different expression levels of corresponding matrix genes. This raises questions regarding matrix gene expression dynamics during biofilm development and the impact of phenotypic nonproducers on biofilm robustness. Here, we show that biofilms are structurally heterogeneous and can be separated into strongly and weakly associated clusters. We reveal that spatiotemporal changes in structural heterogeneity correlate with matrix gene expression, with TasA playing a key role in biofilm integrity and timing of development. We show that the matrix remains partially privatized by the producer subpopulation, where cells tightly stick together even when exposed to shear stress. Our results support previous findings on the existence of "weak points" in seemingly robust biofilms as well as on the key role of linkage proteins in biofilm formation. Furthermore, we provide a starting point for investigating the privatization of common goods within isogenic populations.IMPORTANCE Biofilms are communities of bacteria protected by a self-produced extracellular matrix. The detrimental effects of nonproducing individuals on biofilm development raise questions about the dynamics between community members, especially when isogenic nonproducers exist within wild-type populations. We asked ourselves whether phenotypic nonproducers impact biofilm robustness, and where and when this heterogeneity of matrix gene expression occurs. Based on our results, we propose that the matrix remains partly privatized by the producing subpopulation, since producing cells stick together when exposed to shear stress. The important role of linkage proteins in robustness and development of the structurally heterogeneous biofilm provides an entry into studying the privatization of common goods within isogenic populations.

Evolved Biofilm: Review on the Experimental Evolution Studies of Bacillus subtilis Pellicles.

For several decades, laboratory evolution has served as a powerful method to manipulate microorganisms and to explore long-term dynamics in microbial populations. Next to canonical Escherichia coli planktonic cultures, experimental evolution has expanded into alternative cultivation methods and species, opening the doors to new research questions. Bacillus subtilis, the spore-forming and root-colonizing bacterium, can easily develop in the laboratory as a liquid-air interface colonizing pellicle biofilm. Here, we summarize recent findings derived from this tractable experimental model. Clonal pellicle biofilms of B. subtilis can rapidly undergo morphological and genetic diversification creating new ecological interactions, for example, exploitation by biofilm non-producers. Moreover, long-term exposure to such matrix non-producers can modulate cooperation in biofilms, leading to different phenotypic heterogeneity pattern of matrix production with larger subpopulation of "ON" cells. Alternatively, complementary variants of biofilm non-producers, each lacking a distinct matrix component, can engage in a genetic division of labor, resulting in superior biofilm productivity compared to the "generalist" wild type. Nevertheless, inter-genetic cooperation appears to be evanescent and rapidly vanquished by individual biofilm formation strategies altering the amount or the properties of the remaining matrix component. Finally, fast-evolving mobile genetic elements can unpredictably shift intra-species interactions in B. subtilis biofilms. Understanding evolution in clonal biofilm populations will facilitate future studies in complex multispecies biofilms that are more representative of nature.

Publisher Correction: Collapse of genetic division of labour and evolution of autonomy in pellicle biofilms.

In the version of this Article originally published, author Carolina Falcón Garcia's name was coded wrongly, resulting in it being incorrect when exported to citation databases. This has now been corrected, though no visible changes will be apparent.

Impaired competence in flagellar mutants of Bacillus subtilis is connected to the regulatory network governed by DegU.

The competent state is a developmentally distinct phase, in which bacteria are able to take up and integrate exogenous DNA into their genome. Bacillus subtilis is one of the naturally competent bacterial species and the domesticated laboratory strain 168 is easily transformable. In this study, we report a reduced transformation frequency of B. subtilis mutants lacking functional and structural flagellar components. This includes hag, the gene encoding the flagellin protein forming the filament of the flagellum. We confirm that the observed decrease of the transformation frequency is due to reduced expression of competence genes, particularly of the main competence regulator gene comK. The impaired competence is due to an increase in the phosphorylated form of the response regulator DegU, which is involved in regulation of both flagellar motility and competence. Altogether, our study identified a close link between motility and natural competence in B. subtilis suggesting that hindrance in motility has great impact on differentiation of this bacterium not restricted only to the transition towards sessile growth stage.