Valiya Kalladi, W. B. | Search bioRxiv

bioRxiv2026

TCA cycle entry point, growth variability and amino acid utilization in Alteromonas macleodii ATCC 27126

Amino acid catabolism is a vital metabolic process in bacteria, providing energy, carbon and potentially nitrogen as resources, and affecting global cycles of these elements. The ability of a bacterium to catabolize an amino acid is often inferred from the presence of the relevant catabolic pathways in its genome, yet the "gene=function" inference is not straightforward. Here, we use growth assays in 96 well plates on individual amino acids and their combinations to directly measure the ability of a model marine bacterium, Alteromonas macleodii ATCC 27126, to utilize these resources for growth. With the exception of aspartate and glutamate, which did not support growth in any of our experiments, ATCC 27126 grew on all other amino acids. However, the probability of growth, together with growth yield and rate, differed depending on the entry point of the catabolic pathway to central carbon metabolism, with robust growth occurring only on amino acids catabolized into pyruvate or acetyl CoA. Growth on combinations of two amino acids revealed reproducible patterns, the clearest being inhibition of growth on other amino acids by asparagine, aspartate and their degradation product, oxaloacetate. Finally, growth was different in test tubes compared with 96 well plates. Our results reveal hidden complexity in amino acid utilization and suggest a "TCA-centric" viewpoint for amino acid utilization, perhaps reflecting the high metabolic flexibility of pyruvate and specific regulatory aspects of the TCA cycle in Alteromonas.

ecology↗

bioRxiv2025

Biological insights and methodological challenges learned from working with a diverse heterotrophic marine bacterial library

BackgroundOrganized collections of bacterial strains can help bridge the gap between studying model organisms and communities, through comparative experiments between genetically and phenotypically diverse strains. We describe the establishment and initial characterization of a library of 62 marine heterotrophic bacteria, selected to represent a significant fraction of the genome-encoded functional diversity and a wide range of known phytoplankton-bacteria interactions. We focus on important but often undiscussed aspects of collecting and maintaining such a library, verifying strain identity, and applying classical microbiological methods across diverse strains. ResultsCultured strains contain up to hundreds of mutations compared with the reference genomes, with non-synonymous mutations in rpoB and/or rpoC genes observed in [~]15% of the cultures. Most strains grow well at 25{degrees}C, but the dependence of growth rate on temperature and the width of the temperature niche vary between strains in a systematic manner. We describe steps towards designing a universal, defined, minimal media for marine bacteria, revealing that growth inhibition on amino acids and peptides by carbohydrates is widespread. Cell counts obtained from flow cytometry and colony plating differ systematically, as do different methods to assess motility. Finally, we discuss traits potentially related to microbial interactions such as hemolysis, biofilm formation, and antibiotic resistance. Gammaproteobacteria such as Alteromonas, Pseudoalteromonas, and Vibrio reveal consistently robust growth, and activity, perhaps explaining why these clades are well-explored. ConclusionExplicitly discussing the insights and challenges of working with strain libraries will pave the way to robust, reproducible, and generalizable mapping of bacterial traits across diversity.

microbiology↗