Defining the regulatory mechanisms of sigma factor RpoS degradation in Azotobacter vinelandii and Pseudomonas aeruginosa.

In several Gram-negative bacteria, the general stress response is mediated by the alternative sigma factor RpoS, a subunit of RNA polymerase that confers promoter specificity. In Escherichia coli, regulation of protein levels of RpoS involves the adaptor protein RssB, which binds RpoS for presenting it to the ClpXP protease for its degradation. However, in species from the Pseudomonadaceae family, RpoS is also degraded by ClpXP, but an adaptor has not been experimentally demonstrated. Here, we investigated the role of an E. coli RssB-like protein in two representative Pseudomonadaceae species such as Azotobacter vinelandii and Pseudomonas aeruginosa. In these bacteria, inactivation of the rssB gene increased the levels and stability of RpoS during exponential growth. Downstream of rssB lies a gene that encodes a protein annotated as an anti-sigma factor antagonist (rssC). However, inactivation of rssC in both A. vinelandii and P. aeruginosa also increased the RpoS protein levels, suggesting that RssB and RssC work together to control RpoS degradation. Furthermore, we identified an in vivo interaction between RssB and RpoS only in the presence of RssC using a bacterial three-hybrid system. We propose that both RssB and RssC are necessary for the ClpXP-dependent RpoS degradation during exponential growth in two species of the Pseudomonadaceae family.

The ribosome rescue pathways SsrA-SmpB, ArfA, and ArfB mediate tolerance to heat and antibiotic stresses in Azotobacter vinelandii.

Bacteria have a mechanism to rescue stalled ribosomes known as trans-translation consisting of SsrA, a transfer-messenger RNA (tmRNA), and the small protein SmpB. Other alternative rescue mechanisms mediated by ArfA and ArfB proteins are present only in some species. Ribosome rescue mechanisms also play a role in tolerance to antibiotics and various stresses such as heat. This study shows that the genome of the soil bacterium A. vinelandii harbours genes encoding for tmRNA, SmpB, two paralogs of ArfA (arfA1 and arfA2), and ArfB. A number of mutant strains carrying mutations in the ssrA, arfA1, arfA2, and arfB genes were constructed and tested for their growth and susceptibility to heat and the antibiotic tetracycline. We found that the inactivation of both ssrA and one or the two arfA genes was detrimental to growth and caused a higher susceptibility to heat and to the antibiotic tetracycline. Interestingly, the arfB mutant strain was unable to grow after 2 h of incubation at 45°C. Inactivation of arfB in the ssrA-arfA1-arfA2 strain caused a lethal phenotype since the quadruple mutant could not be isolated. Taken together, our data suggest that both arfA1 and arfA2, as well as arfB, are functional as back up mechanisms, and that the ArfB pathway has an essential role that confers A. vinelandii resistance to high temperatures.

Genotoxic Effects of Aluminum Chloride and Their Relationship with N-Nitroso-N-Methylurea (NMU)-Induced Breast Cancer in Sprague Dawley Rats.

Recently, soluble forms of aluminum for human use or consumption have been determined to be potentially toxic due to their association with hepatic, neurological, hematological, neoplastic, and bone conditions. This study aims to assess the genotoxic effect of aluminum chloride on genomic instability associated with the onset of N-nitroso-N-methylurea (NMU)-induced breast cancer in Sprague Dawley rats. The dietary behavior of the rats was assessed, and the concentration of aluminum in the mammary glands was determined using atomic absorption spectroscopy. Genomic instability was determined in the histological sections of mammary glands stained with hematoxylin and eosin. Moreover, micronucleus in peripheral blood and comet assays were performed. The results of dietary behavior evaluation indicated no significant differences between the experimental treatments. However, aluminum concentration in breast tissues was high in the +2000Al/-NMU treatment. This experimental treatment caused moderate intraductal cell proliferation, lymph node hyperplasia, and serous gland adenoma. Furthermore, micronucleus and comet test results revealed that +2000Al/-NMU led to a genotoxic effect after a 10-day exposure and the damage was more evident after a 15-day exposure. Therefore, in conclusion, genomic instability is present and the experimental conditions assessed are not associated with breast cancer.