Usnic acid, a compound produced by various lichen species, has been demonstrated previously to inhibit growth of different bacteria and fungi; however, mechanism of its antimicrobial activity remained unknown. In this report, we demonstrate that usnic acid causes rapid and strong inhibition of RNA and DNA synthesis in Gram-positive bacteria, represented by Bacillus subtilis and Staphylococcus aureus, while it does not inhibit production of macromolecules (DNA, RNA, and proteins) in Escherichia coli, which is resistant to even high doses of this compound. However, we also observed slight inhibition of RNA synthesis in a Gram-negative bacterium, Vibrio harveyi. Inhibition of protein synthesis in B. subtilis and S. aureus was delayed, which suggest indirect action (possibly through impairment of transcription) of usnic acid on translation. Interestingly, DNA synthesis was halted rapidly in B. subtilis and S. aureus, suggesting interference of usnic acid with elongation of DNA replication. We propose that inhibition of RNA synthesis may be a general mechanism of antibacterial action of usnic acid, with additional direct mechanisms, such as impairment of DNA replication in B. subtilis and S. aureus.

bThe pathogenicity of enterohemorrhagic Escherichia coli (EHEC) depends on production of Shiga toxins, which are encoded by stx genes located in the genomes of lambdoid prophages. Efficient expression of these genes requires prophage induction and lytic development of phages. Treatment of EHEC infections is problematic due to not only the resistance of various strains to antibiotics but also the fact that many antibiotics cause prophage induction, thus resulting in high-level expression of stx genes. Here we report that E. coli growth, Shiga toxin-converting phage development, and production of the toxin by EHEC are strongly inhibited by phenethyl isothiocyanate (PEITC). We demonstrate that PEITC induces the stringent response in E. coli that is mediated by massive production of a global regulator, guanosine tetraphosphate (ppGpp). The stringent response induction arises most probably from interactions of PEITC with amino acids and from amino acid deprivation-mediated activation of ppGpp synthesis. In mutants unable to synthesize ppGpp, development of Shiga toxin-converting phages and production of Shiga toxin are significantly enhanced. Therefore, ppGpp, which appears at high levels in bacterial cells after stimulation of its production by PEITC, is a negative regulator of EHEC virulence and at the same time efficiently inhibits bacterial growth. This is in contrast to stimulation of virulence of different bacteria by this nucleotide reported previously by others.

DNA replication is coupled to growth by an unknown mechanism. Here, we investigated this coupling by analyzing growth and replication in 15 mutants of central carbon metabolism (CCM) cultivated in three rich media. In about one-fourth of the condition tested, defects in replication resulting from changes in initiation or elongation were detected. This uncovered 11 CCM genes important for replication and showed that some of these genes have an effect in one, two or three media. Additional results presented here and elsewhere (Jannière, L., Canceill, D., Suski, C., et al. (2007), PLoS One, 2, e447.) showed that, in the LB medium, the CCM genes important for DNA elongation (gapA and ackA) are genetically linked to the lagging strand polymerase DnaE while those important for initiation (pgk and pykA) are genetically linked to the replication enzymes DnaC (helicase), DnaG (primase) and DnaE. Our work thus shows that the coupling between growth and replication involves multiple, medium-dependent links between CCM and replication. They also suggest that changes in CCM may affect initiation by altering the functional recruitment of DnaC, DnaG and DnaE at the chromosomal origin, and may affect elongation by altering the activity of DnaE at the replication fork. The underlying mechanism is discussed.

The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp0] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase. IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.

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