A direct link between DNA replication regulation and central carbon metabolism (CCM) has been previously demonstrated in Bacillus subtilis and Escherichia coli, as effects of certain mutations in genes coding for replication proteins could be specifically suppressed by particular mutations in genes encoding CCM enzymes. However, specific molecular mechanism(s) of this link remained unknown. In this report, we demonstrate that various CCM metabolites can suppress the effects of mutations in different replication genes of E. coli on bacterial growth, cell morphology, and nucleoid localization. This provides evidence that the CCM-replication link is mediated by metabolites rather than direct protein-protein interactions. On the other hand, action of metabolites on DNA replication appears indirect rather than based on direct influence on the replication machinery, as rate of DNA synthesis could not be corrected by metabolites in short-term experiments. This corroborates the recent discovery that in B. subtilis, there are multiple links connecting CCM to DNA replication initiation and elongation. Therefore, one may suggest that although different in detail, the molecular mechanisms of CCM-dependent regulation of DNA replication are similar in E. coli and B. subtilis, making this regulation an important and common constituent of the control of cell physiology in bacteria.

Marine bacteria display significant versatility in adaptation to variations in the environment and stress conditions, including temperature shifts. Shewanella baltica plays a major role in denitrification and bioremediation in the marine environment, but is also identified to be responsible for spoilage of ice-stored seafood. We aimed to characterize transcriptional response of S. baltica to cold stress in order to achieve a better insight into mechanisms governing its adaptation. We exposed bacterial cells to 8 °C for 90 and 180 min, and assessed changes in the bacterial transcriptome with RNA sequencing validated with the RT-qPCR method. We found that S. baltica general response to cold stress is associated with massive downregulation of gene expression, which covered about 70% of differentially expressed genes. Enrichment analysis revealed upregulation of only few pathways, including aminoacyl-tRNA biosynthesis, sulfur metabolism and the flagellar assembly process. Downregulation was observed for fatty acid degradation, amino acid metabolism and a bacterial secretion system. We found that the entire type II secretion system was transcriptionally shut down at low temperatures. We also observed transcriptional reprogramming through the induction of RpoE and repression of RpoD sigma factors to mediate the cold stress response. Our study revealed how diverse and complex the cold stress response in S. baltica is.

Bacterial resistance to known antibiotics comprises a serious threat to public health. Propagation of multidrug-resistant pathogenic strains is a reason for undertaking a search for new therapeutic strategies, based on newly developed chemical compounds and the agents present in nature. Moreover, antibiotic treatment of infections caused by enterotoxin toxin-bearing strain—enterohemorrhagic Escherichia coli (EHEC) is considered hazardous and controversial due to the possibility of induction of bacteriophage-encoded toxin production by the antibiotic-mediated stress. The important source of potentially beneficial compounds are secondary plant metabolites, isothiocyanates (ITC), and phytoncides from the Brassicaceae family. We reported previously that sulforaphane and phenethyl isothiocyanate, already known for their chemopreventive and anticancer features, exhibit significant antibacterial effects against various pathogenic bacteria. The mechanism of their action is based on the induction of the stringent response and accumulation of its alarmones, the guanosine penta- and tetraphosphate. In this process, the amino acid starvation path is employed via the RelA protein, however, the precise mechanism of amino acid limitation in the presence of ITCs is yet unknown. In this work, we asked whether ITCs could act synergistically with each other to increase the antibacterial effect. A set of aliphatic ITCs, such as iberin, iberverin, alyssin, erucin, sulforaphen, erysolin, and cheirolin was tested in combination with sulforaphane against E. coli. Our experiments show that all tested ITCs exhibit strong antimicrobial effect individually, and this effect involves the stringent response caused by induction of the amino acid starvation. Interestingly, excess of specific amino acids reversed the antimicrobial effects of ITCs, where the common amino acid for all tested compounds was glycine. The synergistic action observed for iberin, iberverin, and alyssin also led to accumulation of (p)ppGpp, and the minimal inhibitory concentration necessary for the antibacterial effect was four- to eightfold lower than for individual ITCs. Moreover, the unique mode of ITC action is responsible for inhibition of prophage induction and toxin production, in addition to growth inhibition of EHEC strains. Thus, the antimicrobial effect of plant secondary metabolites by the stringent response induction could be employed in potential therapeutic strategies.

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Vibrio cholerae represents a constant threat to public health, causing widespread infections, especially in developing countries with a significant number of fatalities and serious complications every year. The standard treatment by oral rehydration does not eliminate the source of infection, while increasing antibiotic resistance among pathogenic V. cholerae strains makes the therapy difficult. Thus, we assessed the antibacterial potential of plant-derived phytoncides, isothiocyanates (ITC), against V. cholerae O365 strain. Sulforaphane (SFN) and 2-phenethyl isothiocyanate (PEITC) ability to inhibit bacterial growth was assessed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values indicate that these compounds possess antibacterial activity and are also effective against cells growing in a biofilm. Tested ITC caused accumulation of stringent response alarmone, ppGpp, which indicates induction of the global stress response. It was accompanied by bacterial cytoplasm shrinkage, the inhibition of the DNA, and RNA synthesis as well as downregulation of the expression of virulence factors. Most importantly, ITC reduced the toxicity of V. cholerae in the in vitro assays (against Vero and HeLa cells) and in vivo, using Galleria mellonella larvae as an infection model. In conclusion, our data indicate that ITCs might be considered promising antibacterial agents in V. cholerae infections.