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The Type VI Secretion System (T6SS) is a widespread and highly effective mechanism of microbial warfare; it confers the ability to efficiently kill susceptible cells within close proximity. Due to its large physical size, complexity, and ballistic basis for intoxication, it has widely been assumed to incur significant growth costs in the absence of improved competitive outcomes. In this study, we precisely examine the fitness costs of constitutive T6SS firing in the bacteriumVibrio cholerae. We find that, contrary to expectations, constitutive expression of the T6SS has a negligible impact on growth, reducing growth fitness by 0.025 ± 0.5% (95% CI) relative to a T6SS− control. Mathematical modeling of microbial populations demonstrates that, due to clonal interference, constitutive expression of the T6SS will often be neutral, with little impact on evolutionary outcomes. Our findings underscore the importance of precisely measuring the fitness costs of microbial social behaviors and help explain the prevalence of the T6SS across Gram‐negative bacteria.

 

Chronic, highly antibiotic-resistant infections in cystic fibrosis (CF) lungs contribute to increasing morbidity and mortality.Pseudomonas aeruginosa, a common CF pathogen, exhibits resistance to multiple antibiotics, contributing to antimicrobial resistance (AMR). These bacterial populations display genetic and phenotypic diversity, but it is unclear how this diversity affects susceptibility to bacteriocins. R-pyocins, i.e., bacteriocins produced byP. aeruginosa, are phage-tail-like antimicrobials. R-pyocins have potential as antimicrobials, however, recent research suggests the diversity ofP. aeruginosavariants within CF lung infections leads to varying susceptibility to R-pyocins. This variation may be linked to changes in lipopolysaccharide (LPS), acting as the R-pyocin receptor. Currently, it is unknown how frequently R-pyocin-susceptible strains are in chronic CF lung infection, particularly when considering the heterogeneity within these strains. In this study, we tested the R2-pyocin susceptibility of 139P.aeruginosavariants from 17 sputum samples of 7 CF patients and analyzed LPS phenotypes. We found that 83% of sputum samples did not have R2-pyocin-resistant variants, while nearly all samples contained susceptible variants. There was no correlation between LPS phenotype and R2-pyocin susceptibility, though we estimate that about 76% of sputum-derived variants lack an O-specific antigen, 40% lack a common antigen, and 24% have altered LPS cores. The absence of a correlation between LPS phenotype and R-pyocin susceptibility suggests that LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Our research supports the potential of R-pyocins as therapeutic agents, as many infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations.

Cystic fibrosis (CF) patients often experience chronic, debilitating lung infections caused by antibiotic-resistantPseudomonas aeruginosa, contributing to antimicrobial resistance (AMR). The genetic and phenotypic diversity ofP. aeruginosapopulations in CF lungs raises questions about their susceptibility to non-traditional antimicrobials, like bacteriocins. In this study, we focused on R-pyocins, a type of bacteriocin with high potency and a narrow killing spectrum. Our findings indicate that a large number of infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations, supporting their potential use as therapeutic agents. The absence of a clear correlation between lipopolysaccharide (LPS) phenotypes and R-pyocin susceptibility suggests that LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Understanding the relationship between LPS phenotypes and R-pyocin susceptibility is crucial for developing effective treatments for these chronic infections.

 

Surface attached communities of microbes grow in a wide variety of environments. Often, the size of these microbial community is constrained by their physical surroundings. However, little is known about how size constraints of a colony impact the outcome of microbial competitions. Here, we use individual-based models to simulate contact killing between two bacterial strains with different killing rates in a wide range of community sizes. We found that community size has a substantial impact on outcomes; in fact, in some competitions the identity of the most fit strain differs in large and small environments. Specifically, when at a numerical disadvantage, the strain with the slow killing rate is more successful in smaller environments than in large environments. The improved performance in small spaces comes from finite size effects; stochastic fluctuations in the initial relative abundance of each strain in small environments lead to dramatically different outcomes. However, when the slow killing strain has a numerical advantage, it performs better in large spaces than in small spaces, where stochastic fluctuations now aid the fast killing strain in small communities. Finally, we experimentally validate these results by confining contact killing strains ofVibrio choleraein transmission electron microscopy grids. The outcomes of these experiments are consistent with our simulations. When rare, the slow killing strain does better in small environments; when common, the slow killing strain does better in large environments. Together, this work demonstrates that finite size effects can substantially modify antagonistic competitions, suggesting that colony size may, at least in part, subvert the microbial arms race.

 

Quorum sensing is a term describing bacterial cell-to-cell communication systems for monitoring and responding to changes in population density. This primer serves as an introduction to the canonical LuxR-LuxI-type quorum sensing circuits common to many species of Gram-negative bacteria. Quorum sensing can synchronize behaviours across a community. Different species employ quorum sensing strategies to control specific behaviours such as bioluminescence, virulence factor production, secondary metabolite production, and biofilm formation.

 

During the biofilm life cycle, bacteria attach to a surface and then reproduce, forming crowded, growing communities. Many theoretical models of biofilm growth dynamics have been proposed; however, difficulties in accurately measuring biofilm height across relevant time and length scales have prevented testing these models, or their biophysical underpinnings, empirically. Using white light interferometry, we measure the heights of microbial colonies with nanometer precision from inoculation to their final equilibrium height, producing a detailed empirical characterization of vertical growth dynamics. We propose a heuristic model for vertical growth dynamics based on basic biophysical processes inside a biofilm: diffusion and consumption of nutrients and growth and decay of the colony. This model captures the vertical growth dynamics from short to long time scales (10 min to 14 d) of diverse microorganisms, including bacteria and fungi. 

The diversity of multicellular organisms is, in large part, due to the fact that multicellularity has evolved many times independently. Nonetheless, multicellular organisms all share a universal biophysical trait: cells are attached to each other. All mechanisms of cellular attachment belong to one of two broad classes; intercellular bonds are either re-formable, or they are not. Both classes of multicellular assembly are common in nature, having evolved dozens of times independently. In this review, we detail these varied mechanisms as they exist in multicellular organisms. We also discuss the evolutionary implications of different intercellular attachment mechanisms on nascent multicellular organisms. The type of intercellular bond present during early steps in the transition to multicellularity constrains future evolutionary and biophysical dynamics for the lineage, affecting the origin of multicellular life cycles, cell-cell communication, cellular differentiation, and multicellular morphogenesis. The types of intercellular bonds used by multicellular organisms may thus result in some of the most impactful historical constraints on the evolution of multicellularity. 

ABSTRACT Mutations in regulatory mechanisms that control gene expression contribute to phenotypic diversity and thus facilitate the adaptation of microbes and other organisms to new niches. Comparative genomics can be used to infer rewiring of regulatory architecture based on large effect mutations like loss or acquisition of transcription factors but may be insufficient to identify small changes in noncoding, intergenic DNA sequence of regulatory elements that drive phenotypic divergence. In human-derived Vibrio cholerae , the response to distinct chemical cues triggers production of multiple transcription factors that can regulate the type VI secretion system (T6), a broadly distributed weapon for interbacterial competition. However, to date, the signaling network remains poorly understood because no regulatory element has been identified for the major T6 locus. Here we identify a conserved cis -acting single nucleotide polymorphism (SNP) controlling T6 transcription and activity. Sequence alignment of the T6 regulatory region from diverse V. cholerae strains revealed conservation of the SNP that we rewired to interconvert V. cholerae T6 activity between chitin-inducible and constitutive states. This study supports a model of pathogen evolution through a noncoding cis -regulatory mutation and preexisting, active transcription factors that confers a different fitness advantage to tightly regulated strains inside a human host and unfettered strains adapted to environmental niches. IMPORTANCE Organisms sense external cues with regulatory circuits that trigger the production of transcription factors, which bind specific DNA sequences at promoters (“ cis ” regulatory elements) to activate target genes. Mutations of transcription factors or their regulatory elements create phenotypic diversity, allowing exploitation of new niches. Waterborne pathogen Vibrio cholerae encodes the type VI secretion system “nanoweapon” to kill competitor cells when activated. Despite identification of several transcription factors, no regulatory element has been identified in the promoter of the major type VI locus, to date. Combining phenotypic, genetic, and genomic analysis of diverse V. cholerae strains, we discovered a single nucleotide polymorphism in the type VI promoter that switches its killing activity between a constitutive state beneficial outside hosts and an inducible state for constraint in a host. Our results support a role for noncoding DNA in adaptation of this pathogen. 

Abstract Evolutionary arms races are broadly prevalent among organisms including bacteria, which have evolved defensive strategies against various attackers. A common microbial aggression mechanism is the type VI secretion system (T6SS), a contact-dependent bacterial weapon used to deliver toxic effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins that neutralize effectors. However, less is known about factors that protect non-sibling bacteria from T6SS attacks independently of cognate immunity proteins. In this study, we observe that human Escherichia coli commensal strains sensitive to T6SS attacks from Vibrio cholerae are protected when co-cultured with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find that cells lacking the cAMP receptor protein (CRP), which regulates expression of hundreds of genes in response to glucose, survive significantly better against V. cholerae T6SS attacks even in the absence of glucose. Finally, we show that the glucose-mediated T6SS protection varies with different targets and killers. Our findings highlight the first example of an extracellular small molecule modulating a genetically controlled response for protection against T6SS attacks. This discovery may have major implications for microbial interactions during pathogen-host colonization and survival of bacteria in environmental communities.