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 stringent response regulates the poly-ß-hydroxybutyrate (PHB) synthesis in Azotobacter vinelandii.

The stringent response exerted by (p)ppGpp and RNA-polymerase binding protein DksA regulates gene expression in diverse bacterial species. To control gene expression (p)ppGpp, synthesized by enzymes RelA and SpoT, interacts with two sites within the RNA polymerase; site 1, located in the interphase between subunits ß' and ω (rpoZ), and site 2 located in the secondary channel that is dependent on DksA protein. In Escherichia coli, inactivation of dksA results in a reduced sigma factor RpoS expression. In Azotobacter vinelandii the synthesis of polyhydroxybutyrate (PHB) is under RpoS regulation. In this study, we found that the inactivation of relA or dksA, but not rpoZ, resulted in a negative effect on PHB synthesis. We also found that the dksA, but not the relA mutation reduced both rpoS transcription and RpoS protein levels, implying that (p)ppGpp and DksA control PHB synthesis through different mechanisms. Interestingly, despite expressing rpoS from a constitutive promoter in the dksA mutant, PHB synthesis was not restored to wild type levels. A transcriptomic analysis in the dksA mutant, revealed downregulation of genes encoding enzymes needed for the synthesis of acetyl-CoA, the precursor substrate for PHB synthesis. Together, these data indicate that DksA is required for optimal expression of RpoS which in turn activates transcription of genes for PHB synthesis. Additionally, DksA is required for optimal transcription of genes responsible for the synthesis of precursors for PHB synthesis.

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.

Agricultural land use among mestizo colonist and indigenous populations: Contrasting patterns in the Amazon.

This paper compares land use patterns of mestizo colonists and indigenous populations in the central Ecuadorian Amazon, based on data from a household survey covering mestizo colonist, Kichwa and Shuar households. As expected, colonists mostly engage in commercial agriculture and cattle ranching, but there are substantial differences in land use patterns between the Shuar and the Kichwa. The Shuar engage in cash cropping and cattle ranching, and on average, devote even more land to agricultural uses than mestizo colonists in this sample. In contrast, the Kichwa engage more in subsistence crop production and less in commercial agriculture. Such different patterns appear related to local conditions, earlier migratory and settlement patterns, and the level of exposure to markets. The implications of this for policy are explored in the conclusions.