Biology subjects

Noda-Garcia, L.

Publications and source records attributed to Noda-Garcia, L..

6 recordsLinked to original sources

bioRxiv2026

Species-specific metabolic networks shape evolutionary routes to functional rescue

Metabolic networks are highly interconnected. Still, it remains unclear whether organism- and environment-specific factors shape their capacity to evolve in response to metabolic stress or whether it follows general principles. Here, we studied metabolic evolvability in Bacillus subtilis using auxotrophic mutants lacking central biosynthetic enzymes. Across two genetic backgrounds with contrasting biofilm-forming capacities and under direct or gradual selection through nutrient gradients, B. subtilis bypassed 9 of 17 essential functions. Rescue was more frequent in the biofilm-proficient background and under gradual selection, which was also associated with more mutations in coding regions, particularly nonsynonymous. Adaptation proceeded through loss-of-function mutations that relieved regulatory or enzymatic constraints and redirected metabolic flux. Comparison with Escherichia coli revealed substantial differences in bypassability and genetic routes that persisted under matched conditions, although two cross-species solutions converged at the pathway level. Our data show species-specific metabolic networks shape available rescue routes, while ecological context influences their evolutionary accessibility during adaptation.

evolutionary biology↗

bioRxiv2026

Nutrient environments shape amino acid auxotrophy and cross-feeding

Auxotrophy, the absence of biosynthetic capacity for essential metabolites, is widespread in microbes and is thought to shape interactions within communities. Auxotrophies are often treated as fixed properties of organisms; however, evidence indicates that auxotrophic phenotypes can depend on environmental context, thereby affecting community assembly by cross-feeding. Here, we systematically quantify how nutrient environments shape both auxotrophy and cross-feeding. Using matched sets of six amino acid auxotrophs in Escherichia coli and Bacillus subtilis, we measured monoculture and pairwise coculture growth across 40 carbon and nitrogen environments. We find that auxotrophy itself is highly environment dependent, with strains growing in a substantial fraction of amino acid-free conditions despite lacking key biosynthetic enzymes. Cross-feeding likewise varies widely across species, environments, and auxotroph pairs. Despite this variability, cross-feeding outcomes exhibit consistent patterns within and across species. In particular, B. subtilis cross-feeds better overall and cross-feeding growth is better predicted by auxotroph type (i.e., which amino acids the strain cannot produce) than by environmental context. A machine-learning model recapitulates this pattern, identifying auxotroph type as the strongest predictor of cross-feeding growth, similar to species identity, exceeding the contributions of nutrient environment and prototroph growth. Together, these results show that environmental context reshapes both metabolic need and exchange, yet cross-feeding follows emergent patterns linked to the species and auxotrophy type, with implications for predicting community assemblies and the evolution of gene loss.

ecology↗

bioRxiv2026

Adaptive laboratory evolution unlocks membrane permeability as a key limitation in long-chain alcohol metabolism by Pseudomonas putida KT2440

Pseudomonas putida KT2440, renowned for its diverse metabolic capabilities, is a promising platform for downstream processing and revalorization of recalcitrant molecules. In this study, we examined and optimized P. putida KT2440s ability to utilize long-chain alcohols. These molecules are byproducts of the degradation of polyethylene (PE), the most widely used plastic. Using them as feedstock for microbial growth would close the plastic-derived carbon cycle, reducing environmental pollution. First, we discovered that P. putida KT2440 can use long-chain alcohols as the sole carbon and energy source. Using adaptive laboratory evolution (ALE), we generated variants with improved growth rates on long-chain alcohols, specifically 1-hexadecanol and 1-eicosanol. Mutations that became fixed during ALE provided insights into the mechanism, highlighting the importance of cell-substrate interaction. By heterologously expressing a hydrocarbon transporter-encoding gene, we successfully reproduced the ALE-derived phenotype, demonstrating that the bottleneck in long-chain alcohol utilization is not substrate transformation but uptake. These findings lay the groundwork for the potential application of P. putida KT2440 for the degradation of PE.

bioengineering↗

bioRxiv2025

A synthetic bacterium that degrades and assimilates poly(ethylene terephthalate)

Polyethylene terephthalate (PET) is the fourth most commonly used plastic worldwide. Like all plastics, post-consumer PET is poorly managed and accumulates in the environment, posing significant ecological threats. After 70 years of accumulation, microorganisms capable of degrading and assimilating PET have been isolated, demonstrating that PET can be broken down and converted into valuable cellular biomass or metabolic products. These natural isolates, however, are poorly characterized and challenging to genetically manipulate, which limits their further optimization and applicability. Here, we engineer a well-established synthetic biology chassis for the biodegradation and assimilation of PET. We modified the bacterium Pseudomonas putida KT2440 to heterologously express an active PET-hydrolytic enzyme extracellularly and to metabolize PET biodegradation products. The resulting strain, named PETBuster, was capable of growing on PET as the sole carbon source on solid and liquid media. We achieved 91% PET degradation after 21 days of culture, with a doubling time of 3.6 days, under mesophilic conditions. In this way, we demonstrate that PET fermentation is feasible, opening the door to the production of valuable chemicals from waste.

synthetic biology↗

bioRxiv2021

Directed evolution of the rRNA methylating enzyme Cfr reveals molecular basis of antibiotic resistance

Alteration of antibiotic binding sites through modification of ribosomal RNA (rRNA) is a common form of resistance to ribosome-targeting antibiotics. The rRNA-modifying enzyme Cfr methylates an adenosine nucleotide within the peptidyl transferase center, resulting in the C-8 methylation of A2503 (m8A2503). Acquisition of cfr results in resistance to eight classes of ribosome-targeting antibiotics. Despite the prevalence of this resistance mechanism, it is poorly understood whether and how bacteria modulate Cfr methylation to adapt to antibiotic pressure. Moreover, direct evidence for how m8A2503 alters antibiotic binding sites within the ribosome is lacking. In this study, we performed directed evolution of Cfr under antibiotic selection to generate Cfr variants that confer increased resistance by enhancing methylation of A2503 in cells. Increased rRNA methylation is achieved by improved expression and stability of Cfr through transcriptional and post-transcriptional mechanisms, which may be exploited by pathogens under antibiotic stress as suggested by natural isolates. Using a variant which achieves near-stoichiometric methylation of rRNA, we determined a 2.2 [A] cryo-EM structure of the Cfr-modified ribosome. Our structure reveals the molecular basis for broad resistance to antibiotics and will inform the design of new antibiotics that overcome resistance mediated by Cfr.

biochemistry↗

bioRxiv2018

Chance and pleiotropy dominate genetic diversity in complex bacterial environments

How does environmental complexity affect the evolution of single genes? Here, we measured the effects of a set of mutants of Bacillus subtilis glutamate dehydrogenase across 19 different environments - from homogenous single cell populations in liquid media to heterogeneous biofilms, plant roots and soil communities. The effects of individual gene mutations on organismal fitness were highly reproducible in liquid cultures. Strikingly, however, 84% of the tested alleles showed opposing fitness effects under different growth conditions (environmental pleiotropy). In biofilms and soil samples, different alleles dominated in parallel replica experiments. Accordingly, we found that in these heterogeneous cell communities the fate of mutations was dictated by a combination of selection and drift. The latter relates to programmed prophage excisions that occurred along biofilm development. Overall, per individual condition, by the combined action of selection, pleiotropy and chance, a wide range of glutamate dehydrogenase mutations persisted and sometimes fixated. However, across longer periods and multiple environments, nearly all this diversity would be lost - indeed, considering all environments and conditions we have tested, wild-type is the fittest allele.

microbiology↗