Case Studies in the Assessment of Microbial Fitness: Seemingly Subtle Changes Can Have Major Effects on Phenotypic Outcomes.

Following the completion of an adaptive evolution experiment, fitness evaluations are routinely conducted to assess the magnitude of adaptation. In doing so, proper consideration should be given when determining the appropriate methods as trade-offs may exist between accuracy and throughput. Here, we present three instances in which small changes in the framework or execution of fitness evaluations significantly impacted the outcomes. The first case illustrates that discrepancies in fitness conclusions can arise depending on the approach to evaluating fitness, the culture vessel used, and the sampling method. The second case reveals that variations in environmental conditions can occur associated with culture vessel material. Specifically, these subtle changes can greatly affect microbial physiology leading to changes in the culture pH and distorting fitness measurements. Finally, the last case reports that heterogeneity in CFU formation time can result in inaccurate fitness conclusions. Based on each case, considerations and recommendations are presented for future adaptive evolution experiments.

Trade-offs, trade-ups, and high mutational parallelism underlie microbial adaptation during extreme cycles of feast and famine.

Microbes are evolutionarily robust organisms capable of rapid adaptation to complex stress, which enables them to colonize harsh environments. In nature, microbes are regularly challenged by starvation, which is a particularly complex stress because resource limitation often co-occurs with changes in pH, osmolarity, and toxin accumulation created by metabolic waste. Often overlooked are the additional complications introduced by eventual resource replenishment, as successful microbes must withstand rapid environmental shifts before swiftly capitalizing on replenished resources to avoid invasion by competing species. To understand how microbes navigate trade-offs between growth and survival, ultimately adapting to thrive in environments with extreme fluctuations, we experimentally evolved 16 Escherichia coli populations for 900 days in repeated feast/famine conditions with cycles of 100-day starvation before resource replenishment. Using longitudinal population-genomic analysis, we found that evolution in response to extreme feast/famine is characterized by narrow adaptive trajectories with high mutational parallelism and notable mutational order. Genetic reconstructions reveal that early mutations result in trade-offs for biofilm and motility but trade-ups for growth and survival, as these mutations conferred positively correlated advantages during both short-term and long-term culture. Our results demonstrate how microbes can navigate the adaptive landscapes of regularly fluctuating conditions and ultimately follow mutational trajectories that confer benefits across diverse environments.

Evolution of pH-sensitive transcription termination during adaptation to repeated long-term starvation.

Fluctuating environments that consist of regular cycles of co-occurring stress are a common challenge faced by cellular populations. For a population to thrive in constantly changing conditions, an ability to coordinate a rapid cellular response is essential. Here, we identify a mutation conferring an arginine-to-histidine (Arg to His) substitution in the transcription terminator Rho. The rho R109H mutation frequently arose in E. coli populations experimentally evolved under repeated long-term starvation conditions, during which feast and famine result in drastic environmental pH fluctuations. Metagenomic sequencing revealed that populations containing the rho mutation also possess putative loss-of-function mutations in ydcI, which encodes a recently characterized transcription factor associated with pH homeostasis. Genetic reconstructions of these mutations show that the rho allele confers a plastic alkaline-induced reduction of Rho function that, when found in tandem with a ΔydcI allele, leads to intracellular alkalinization and genetic assimilation of Rho mutant function. We further identify Arg to His substitutions at analogous sites in rho alleles from species originating from fluctuating alkaline environments. Our results suggest that Arg to His substitutions in global regulators of gene expression can serve to rapidly coordinate complex responses through pH sensing and shed light on how cellular populations across the tree of life use environmental cues to coordinate rapid responses to complex, fluctuating environments.

The Identity of the Constriction Region of the Ribosomal Exit Tunnel Is Important to Maintain Gene Expression in Escherichia coli.

Mutational changes in bacterial ribosomes often affect gene expression and consequently cellular fitness. Understanding how mutant ribosomes disrupt global gene expression is critical to determining key genetic factors that affect bacterial survival. Here, we describe gene expression and phenotypic changes presented in Escherichia coli cells carrying an uL22(K90D) mutant ribosomal protein, which displayed alterations during growth. Ribosome profiling analyses revealed reduced expression of operons involved in catabolism, indole production, and lysine-dependent acid resistance. In general, translation initiation of proximal genes in several of these affected operons was substantially reduced. These reductions in expression were accompanied by increases in the expression of acid-induced membrane proteins and chaperones, the glutamate-decarboxylase regulon, and the autoinducer-2 metabolic regulon. In agreement with these changes, uL22(K90D) mutant cells had higher glutamate decarboxylase activity, survived better in extremely acidic conditions, and generated more biofilm in static cultures compared to their parental strain. Our work demonstrates that a single mutation in a non-conserved residue of a ribosomal protein affects a substantial number of genes to alter pH resistance and the formation of biofilms. IMPORTANCE All newly synthesized proteins must pass through a channel in the ribosome named the exit tunnel before emerging into the cytoplasm, membrane, and other compartments. The structural characteristics of the tunnel could govern protein folding and gene expression in a species-specific manner but how the identity of tunnel elements influences gene expression is less well-understood. Our global transcriptomics and translatome profiling demonstrate that a single substitution in a non-conserved amino acid of the E. coli tunnel protein uL22 has a profound impact on catabolism, cellular signaling, and acid resistance systems. Consequently, cells bearing the uL22 mutant ribosomes had an increased ability to survive acidic conditions and form biofilms. This work reveals a previously unrecognized link between tunnel identity and bacterial stress adaptation involving pH response and biofilm formation.