Colony morphotype diversification as a signature of bacterial evolution

Abstract The appearance of colony morphotypes is a signature of genetic diversification in evolving bacterial populations. Colony structure highly depends on the cell–cell interactions and polymer production that are adjusted during evolution in an environment that allows the development of spatial structures. Nucci and colleagues describe the emergence of a rough and dry morphotype of a noncapsulated Klebsiella variicola strain during a laboratory evolution study, resembling genetic changes observed in clinical isolates.

Bacterial evolution in the laboratory is studied to understand the underl ying genetic c hanges selected under specific experimental conditions.Intriguingl y, certain m utations detected in the laboratory can also be observed in bacterial isolates from clinical or environmental settings, highlighting the r ele v ance of laboratory evolution experiments .T he first experimental evolution studies that exploited biofilm as laboratory model have immediately recognized the rapid emergence of colony morphotypes (Rainey and Tra visano 1998, P oltak and Cooper 2011, Martin et al. 2016 ).Colon y mor phology div ersification has been since also observ ed in host-microbe interactions (Pestrak et al. 2018, Blake et al. 2021, Nor dgaar d et al. 2022 ) and various in-vitro biofilm experiments (K o vács and Dr a goš 2019 , Xu et al. 2022 ).The colony morphotypes fr equentl y differ in the ability to pr oduce extr acellular pol ymeric substances that constitute a matrix connecting the cells in a population.When bacteria are evolving in a spatially structured envir onment, the div ersity in matrix pr oduction benefits the population, the mixture of evolved clones with variable levels of matrix secretion has higher population productivity or cell amount than the homogenous ancestor population.The difference in matrix production is due to mutations that either alter the regulation of the biosynthetic gene clusters, the function of metabolic pathways contributing to the synthesis of extracellular polymeric substances, or dir ectl y the synthesis machinery.These phenotypic changes influence cell-cell interactions.In addition to identifying the genetic changes , i.e .mechanistic understanding of affected pr ocesses, r esearc hers ar e also intrigued by the underl ying selection pr essur e and the impact of genetic differentiation on sociomicrobiology.
In their recent MicroLife publication, Nucci and colleagues identify the diversification of noncapsulated Klebsiella variicola strains e volv ed for 675 generations in environments with differing nutrient le v els (Nucci et al. 2023a ).After plating the independent populations from their evolution experiments, the researchers observ ed the emer gence of a unique colon y type r esembling the rough and dry morphotype , i.e .rdar-like , observed in other Enterobacteria (Fig. 1 ).While the rdar-like morphotypes rely on the expression of curli or exopolysaccharides in Enterobacteria , K. variicola rdar-like colonies carry mutations in either the mrkH gene that encodes a transcriptional regulator controlling genes related to type 3 fimbriae production or the nac gene that codes a regulator for genes expressed in nitrogen-limited condition.Interestingl y, mr kH is disrupted by an insertion sequence (IS) element in certain clones that display the rdar-like morphology.IS elements driv e r a pid loss of K. pneumoniae ca psule pr oduction in experimentall y e volv ed population (Nucci et al. 2022 ).IS elements have been previously implicated in the evolution of fuzzy spreader colon y mor photypes of Bacillus thuringiensis , where an IS4-like element disrupts a gene encoding a guan yl yltr ansfer ase, causing incr eased hydr ophobicity and a ggr egation (Lin et al. 2022 ).In contrast to the enhanced aggregation of B. thuringiensis fuzzy morphotypes, K. variicola rdar-like deriv ativ es display diminished a ggr egation (Nucci et al. 2023a ).Ne v ertheless, the observ ed par allelism in the role of IS elements during the evolution of novel morphotypes highlights the impact of mobile genetic elements in r a pid adaptation of bacteria, although it might be notable in a speciesor strain-specific manner (Nucci et al. 2022, Hu et al. 2023 ).
The rdar-like clones of K. variicola display increased growth rate and fitness advantage compared with the ancestor (Nucci et al. 2023a ).Ho w e v er, the fitness adv anta ge is mostl y pr ominent when the morphotype is in minority, demonstrating a negative frequency selection.Indeed, the morphotype frequency seems to increase during the experimental evolution, achieving up to 66% abundance in certain lineages, but displaying ∼16% frequency at the end of the study.
The rdar-like morphotype was detected after plating by Nucci and colleagues ( 2023a ) when a noncapsulated K. variicola background was used, while colonies with distinct morphology were less a ppar ent in ca psulated str ains that follo w ed a differ ent e volutionary path (Nucci et al. 2022 ).Subsequent experiments with strains carrying the mutant mrkH or nac alleles demonstrated that these m utations conv ey lo w er influence on cell-to-cell a ggr egation in the capsulated background and the fitness effects were onl y mar ginall y lar ger in ca psulated str ains compar ed to nonca psulated strains.Historical contingency, where the existing mutations influence the subsequent adaptation path might explain the lar ger rdar-like mor photype fr equency in nonca psulated K. v ariicola .This highlights the opportunity to disco ver no v el e volutionary paths in strains lacking the most frequently mutated targets.Deleting the three most frequently mutated pathways in Pseudomonas fluorescens and subsequent experimental evolution in a static micr ocosm r e v ealed 13 ne w m utational pathways that all r esult in wrinkl y spr eader colon y mor photype (Lind et al. 2015 ).Inter estingl y, the fitness benefits of these novel mutations are also present in the ancestor background, although not as elevated as the common targeted paths, suggesting a hierarchical appearance of mutations driven by the superior fitness benefit (Lind et al. 2015 ).Furthermor e, r emov al of exopol ysacc haride synthesis in B. subtilis permits the evolution of clones with enhanced biofilm formation, explained by the production of novel, cysteine containing amyloid fibre variants (Dragoš et al. 2018 ).In contrast to the P. fluorescens example, the reconstituted B. subtilis strains with cysteineencompassing amyloid fibres convey a disadv anta ge in the presence of the exopol ysacc haride (Dr a goš et al. 2018 ).
Finally, the w ork b y Nucci et al. highlights the r ele v ance of labor atory e volution for r eal-life scenarios.Detailed anal ysis of K. pneumoniae genomes r e v ealed compar able IS element insertion in the mrkH gene of numerous clinical isolates.Isolates with interrupted mr kH genes wer e mostl y originated fr om human host samples, including urine and blood as the main source (Nucci et al. 2023a ).Yet, the frequency of these morphotypes is low in Klebsiella isolates, potentially explained by the negative frequency selection of these mutations .T he detection of specific mutations or gene disruptions in natural populations further validate the relevance of experimental evolution studies in the laboratory settings as pr e viousl y r eported in Klebsiella (Nucci et al. 2023b ) and in other species (Tr av erse et al. 2013, Lin et al. 2022 ).
The study of Nucci et al. ( 2023a ) highlights the po w er of experimental evolution to understand genetic adaptation behind colon y mor phology div ersification and connects the m utational landscape of laboratory-based experimental settings to natural bacterial populations.