The ability of minor groove binding agents to inhibit

The ability of minor groove binding agents to inhibit the activity of DNA helicases is not without precedence. Similar effective inhibition of the unwinding of DNA by the BLM and Werner\'s DNA helicases by the minor groove binder distamycin A has been observed. Further, and similar to RecBCD, intercalating agents such as ethidium bromide and actinomycin D had little effect on any of these proteins. These results argue that for several DNA helicases, interaction of the protein with the minor groove ahead of the translocating enzyme as observed for RecBCD, plays a critical role in the mechanism of DNA unwinding. This, however, is not a general rule applicable to all DNA helicases, as the E. coli UvrD enzyme is inhibited by intercalating agents and not by minor groove binding agents., During translocation and DNA unwinding, RecBCD hydrolyzes two to three ATP molecules per base-pair unwound., Each agent decreased both the rate of DNA unwinding, and concomitantly, the rate of ATP hydrolysis (Table 3). For each inhibitor, the reduction in DNA unwinding paralleled that of the rate of ATP hydrolysis. For Et743, the most potent inhibitor, only a twofold decrease in the ATP utilization efficiency was observed. Thus, these inhibitors slow the progress of the translocating enzyme without perturbing significantly the allosteric interaction that activates the enzyme for ATP hydrolysis.
Materials and Methods
Introduction Living species are continuously subjected to alkylating stress by both endogenous and exogenous species that can covalently modify metabolites and biological macromolecules. Particularly, DNA can be alkylated by reactive species leading to the generation of miscoding bases possibly providing lethal modifications in genetic information (1). Methylating agents, like methyl methane sulphonate (MMS), consist in a large category of reactive chemical compounds that can attack nucleophilic sites on DNA bases causing covalent modifications 2, 3, 4. DNA modification seriously impairs transcription and replication or disorganizes the cell-cycle checkpoints driving eukaryotic retinoic acid receptor to apoptosis. Since these molecules are ubiquitous and hence unavoidable, all living organisms have developed several repair or defence mechanisms to overcome their effects and to protect the DNA molecule. Many bacteria build up inducible response pathways that enhance cellular resistance to defend against unpredictable levels of environmental alkylating agents 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. Alkylation response mechanisms also occur in Mycobacterium tuberculosis (MTB), a non sporogenous aerobic pathogenic bacterium, responsible for tuberculosis disease (TB), still one of the most important cause of death due to a unique pathogen. A distinguishing feature of MTB is the presence of a complex cell wall much thicker than in other bacteria, characterized by a high concentration of extremely long chain lipids. This cellular architecture, which confers resistance to environmental factors and antibiotic substances, is important in the pathogenicity of tuberculosis infection because it can affect immune response and granuloma formation 15, 16. Current therapeutic treatments of TB disease require the use of multiple anti-mycobacterial drugs such as rifampicin and isoniazid (17). Recently we demonstrated that treatment of E. coli with small amounts of MMS caused a strong decrease in biofilm formation due to the reduced expression of the enzyme NanA (18). A global investigation of M. smegmatis response to alkylating agents was then pursued by differential proteomics to evaluate the protein expression profiles under alkylating stress conditions and to identify the most affected cellular pathways. Surprisingly, contrary to E. coli, when we incubated M. smegmatis with a sublethal amount of MMS a strong increase in biofilm formation was detected. Quantitative analysis of proteomic profiles demonstrated that most of the M. smegmatis proteins upregulated following MMS treatment are involved in biofilm formation and/or cell wall biosynthesis. Tailored experiments confirmed that under stress conditions M. smegmatis elicits physical defence mechanisms by increasing biofilm formation. Among the upregulated proteins, we focused our attention on the bifunctional enzyme GlmU whose expression was largely increased under biofilm inducing conditions and that was reported to be involved in biofilm production in E. coli, S. epidermidis and S. aureus 19, 20, 21. Experiments with both conditional deletion and overexpressing glmU mutants suggested that down regulation of GlmU decreased M. smegmatis capabilities to produce biofilm whereas overexpression of the enzyme increased biofilm formation. These data were further supported by inhibition of GlmU acetyltransferase activity with two different inhibitors resulting in a clear decrease of biofilm formation, thus confirming the role of GlmU in the process of biofilm production in M. smegmatis.