Summary: The translation process, central to life, is tightly connected to the one‐carbon (1‐C) metabolism via a plethora of macromolecule modifications and specific effectors. Using manual genome annotations and putting together a variety of experimental studies, we explore here the possible reasons of this critical interaction, likely to have originated during the earliest steps of the birth of the first cells. Methionine, S‐adenosylmethionine and tetrahydrofolate dominate this interaction. Yet, 1‐C metabolism is unlikely to be a simple frozen accident of primaeval conditions. Reactive 1‐C species (ROCS) are buffered by the translation machinery in a way tightly associated with the metabolism of iron–sulfur clusters, zinc and potassium availability, possibly coupling carbon metabolism to nitrogen metabolism. In this process, the highly modified position 34 of tRNA molecules plays a critical role. Overall, this metabolic integration may serve both as a protection against the deleterious formation of excess carbon under various growth transitions or environmental unbalanced conditions and as a regulator of zinc homeostasis, while regulating input of prosthetic groups into nascent proteins. This knowledge should be taken into account in metabolic engineering.

Summary: Despite decades of studies meant to analyse the bacterial response to carbon limitation, we still miss a high‐resolution overview of the situation. All gene expression changes observed in such conditions cannot solely be accounted for by the global regulator Crp either free or bound to its effector, cyclic AMP . Here, for the first time, we evaluated the response of both CDS (protein‐coding sequence) and nc RNA (non‐coding RNA ) genes to carbon limitation, revealed cellular functions of differentially expressed genes systematically, quantified the contribution of Crp‐ cAMP and other factors to regulation and deciphered regulation strategies at a genomewide scale. Approximately one‐third of the differentially expressed genes we identified responded to Crp‐ cAMP via its direct or indirect control, while the remaining genes were subject to growth rate‐dependent control or were controlled by other regulators, especially RpoS. Importantly, gene regulation mechanisms can be established by expression pattern studies. Here, we propose a comprehensive picture of how cells respond to carbon scarcity. The global regulation strategies thus exposed illustrate that the response of cell to carbon scarcity is not limited to maintaining sufficient carbon metabolism via cAMP signalling while the main response is to adjust metabolism to cope with a slow growth rate.

Small non-translated regulatory RNAs control plenty of bacterial vital activities. The small RNA GcvB has been extensively studied, indicating the multifaceted roles of GcvB beyond amino acid metabolism. However, few reported GcvB-dependent regulation in minimal medium. Here, by applying a high-resolution RNA-seq assay, we compared the transcriptomes of a wild-type Escherichia coli K-12 strain and its gcvB deletion derivative grown in minimal medium and identified putative targets responding to GcvB, including flu, a determinant gene of auto-aggregation. The following molecular studies and the enhanced auto-aggregation ability of the gcvB knockout strain further substantiated the induced expression of these genes. Intriguingly, the reduced expression of OxyR (the oxidative stress regulator) in the gcvB knockout strain was identified to account for the increased expression of flu. Additionally, GcvB was characterized to up-regulate the expression of OxyR at the translational level. Accordingly, compared to the wild type, the GcvB deletion strain was more sensitive to oxidative stress and lost some its ability to eliminate endogenous reactive oxygen species. Taken together, we reveal that GcvB regulates oxidative stress response by up-regulating OxyR expression. Our findings provide an insight into the diversity of GcvB regulation and add an additional layer to the regulation of OxyR.

Iron is an essential element because it functions as a cofactor of many enzymes, but excess iron causes cell damage. Iron hemostasis in Escherichia coli was transcriptionally maintained by the ferric uptake regulator (Fur). Despite having been studied extensively, the comprehensive physiological roles and mechanisms of Fur-coordinated iron metabolism still remain obscure. In this work, by integrating a high-resolution transcriptomic study of the Fur wild-type and knockout Escherichia coli K-12 strains in the presence or absence of iron with high-throughput ChIP-seq assay and physiological studies, we revisited the regulatory roles of iron and Fur systematically and discovered several intriguing features of Fur regulation. The size of the Fur regulon was expanded greatly, and significant discrepancies were observed to exist between the regulations of Fur on the genes under its direct repression and activation. Fur showed stronger binding strength to the genes under its repression, and genes that were repressed by Fur were more sensitive to Fur and iron regulation as compared to the genes that were activated by Fur. Finally, we found that Fur linked iron metabolism to many essential processes, and the systemic regulations of Fur on carbon metabolism, respiration, and motility were further validated or discussed. These results highlight how Fur and Fur-controlled iron metabolism affect many cellular processes in a systematic way.

Summary: How bacteria adjust gene expression to cope with variable environments remains open to question. Here, we investigated the way global gene expression changes in E. coli correlated with the metabolism of seven carbon substrates chosen to trigger a large panel of metabolic pathways. Coarse‐grained analysis of gene co‐expression identified a novel regulation pattern: we established that the gene expression trend following immediately the reduction of growth rate (GR) was correlated to its initial expression level. Subsequent fine‐grained analysis of co‐expression demonstrated that the Crp regulator, coupled with a change in GR, governed the response of most GR‐dependent genes. By contrast, the Cra, Mlc and Fur regulators governed the expression of genes responding to non‐glycolytic substrates, glycolytic substrates or phosphotransferase system transported sugars following an idiosyncratic way. This work allowed us to expand additional genes in the panel of gene complement regulated by each regulator and to elucidate the regulatory functions of each regulator comprehensively. Interestingly, the bulk of genes controlled by Cra and Mlc were, respectively, co‐regulated by Crp‐ or GR‐related effect and our quantitative analysis showed that each factor took turns to work as the primary one or contributed equally depending on the conditions.

Motility is finely regulated and is crucial to bacterial processes including colonization and biofilm formation. There is a trade-off between motility and growth in bacteria with molecular mechanisms not fully understood. Hypermotile Escherichia coli could be isolated by evolving non-motile cells on soft agar plates. Most of the isolates carried mutations located upstream of the flhDC promoter region, which upregulate the transcriptional expression of the master regulator of the flagellum biosynthesis, FlhDC. Here, we identified that spontaneous mutations in clpX boosted the motility of E. coli largely, inducing several folds of changes in swimming speed. Among the mutations identified, we further elucidated the molecular mechanism underlying the ClpX^V78F mutation on the regulation of E. coli motility. We found that the V78F mutation affected ATP binding to ClpX, resulting in the inability of the mutated ClpXP protease to degrade FlhD as indicated by both structure modeling and in vitro protein degradation assays. Moreover, our proteomic data indicated that the ClpX^V78F mutation elevated the stability of known ClpXP targets to various degrees with FlhD as one of the most affected. In addition, the specific tag at the C-terminus of FlhD being recognized for ClpXP degradation was identified. Finally, our transcriptome data characterized that the enhanced expression of the motility genes in the ClpX^V78F mutations was intrinsically accompanied by the reduced expression of stress resistance genes relating to the reduced fitness of the hypermotile strains. A similar pattern was observed for previously isolated hypermotile E. coli strains showing high expression of flhDC at the transcriptional level. Hence, clpX appears to be a hot locus comparable to the upstream of the flhDC promoter region evolved to boost bacterial motility, and our finding provides insight into the reduced fitness of the hypermotile bacteria.