Antibiotic resistance in Bacillus subtilis as affected by transcriptional derepression and the stringent response

Bacterial cells under conditions of starvation or prolonged non-lethal selective pressures accumulate mutations in highly transcribed genes. This process is part of cellular programs to increase genetic diversity in conditions of stress, also known as stationary phase or stress-induced mutagenesis. This experiment investigated mutation frequencies for antibiotic resistance as affected by the stringent response. The stringent response is a global cellular process that initiates at the cessation of growth and mediates changes in gene expression that repress synthesis of ribosome components. We used Bacillus subtilis strains that differ in RelA proficiency. The relA gene controls the synthesis of (p)ppGpp, the signaling molecule which mediates the stringent response. Since genes involved in protein synthesis are repressed during the stringent response, we hypothesize that relaxed mutants express a higher accumulation of mutations that confer resistance to tetracycline than cells that become stringent. Resistance to tetracycline may be acquired by altering components of the small subunit of bacterial ribosomes. Utilizing an overlay procedure and increasing times of incubation under nutritional stress, stationary cells were prompted for resistance to tetracycline. Our results showed that relA- cells expressed a higher accumulation of Tcr mutations than the one observed in wild type cells. These results provide evidence that transcriptional derepression in cells under non-lethal stress mediates mutagenic events. Implications in antibiotic resistance are further discussed

The Effect of CodY on stationary phase mutagenesis in Bacillus subtilis

We examine the notion that cells in conditions of stress accumulate mutation is in genes under selection via transcription processes. CodY is a global transcriptional regulator in many Gram positives, including soil and pathogenic microbes. In conditions of exponential growth and when branch chain amino acids and GTP are in abundance CodY acts as a transcriptional repressor of many metabolic operons. This transitional repression saves the cell energy and allows efficient use of resources. In conditions of starvation, CodY relieves repression of genes involved in acquisition of nutrients and degradation of carbon sources (genes under selection). Here, we compare the accumulation of mutations in genes under selection in wild type and CodY

DNA secondary structures and their contribution to mutagenesis in B. subtilis stationary phase cells

It is widely known and accepted that the cause of many mutations in cells are generated during the replication process of actively dividing cells, however more recent research has shown that mutations also arise in non growing conditions, a phenomenon known stationary phase mutagenesis. Much of what is known come from studies in eukaryotic and bacterial models. It is proposed that in nongrowing cells, the process of transcription plays an important role in mutagenesis. I will test the hypothesis that secondary structures formed of DNA generated transcription promote mutagenesis. The sequences transcriptiongenerated structures are speculated to be prone to mutations by exposing regions of single stranded DNA to lesions. To test this hypothesis, I examined the Bacillus subtilis gene thiF, predicted by in silico analysis to be prone to mutations at particular locations during transcription. By altering the base sequence of this gene, the stability of its stem-loop structures is affected, thereby allowing us to test whether transcription of the altered sequence influences accumulation of in thiF. Our assay for detection of mutations is based on reversion to thiamine auxotrophy in cells under conditions of starvation. Ultimately, these experiments will increase our understanding of how mutations occur in cells of all domains of life

Mutations to antibiotic resistance during stringent response in B. subtilis

The relA gene in Bacillus subtilis controls a variety of factors during the stringent response which is a response to starvation of amino acids. The stringent response inhibits DNA synthesis and transcription of genes of tRNA, rRNA, and ribosomal proteins and promotes synthesis of the required amino acids. The objective of my project is to determine if a strain of B. subtilis that has a knockout mutation for the relA gene will accumulate a higher number of mutations that confer resistance to antibiotics that inhibit translation. It is proposed that because the relA gene inhibits transcription of ribosomal proteins, a strain lacking this gene will transcribe more rRNA and ribosomal proteins and promote the generation of mutations that target the translation process

The Role of recN in stationary phase mutagenesis in bacillus subtilis

Here, we examine mutagenic programs that are independent of growth, such aspects of the evolutionary process are novel and have been implicated in the formation of cancers in animal cells and the acquisition of antibiotic resistance in animal pathogens. Adaptive or stationary phase mutagenesis is a genetic program to in increase diversity in cells under conditions of stress whereby cells escape non-dividing conditions. Previous research has shown that recombination functions are required to generate mutations that promote growth in Escherichia coli cells starved for carbon. This project tests the hypothesis that recombination functions are required for the generation of mutations that promote growth in response to amino acid starvation stresses in Bacillus subtilis cells. In B. subtilis cells, recN, in addition to recA, mediates recombination events and may influence the formation of adaptive mutations. A RecN- strain will be generated by standard molecular techniques and compared to a RecN+ strain for its ability to accumulate mutations that affect amino acid biosynthesis. We speculate that recN does not affect stationary phase mutagenesis in B. subtilis and discussed other novel mechanisms mediating the generation of mutations in non-dividing cells

Constructing an ArgF- strain of Bacillus subtilis

The goal of our research is to determine whether the level of transcription of a gene is correlated with the level of mutation in that gene. One factor involved in the mutability of a transcribed gene is the ability of the single stranded DNA to form secondary stem loop structures (SLS), in the wake of the transcription bubble, that contain unpaired mutable bases. We are interested in correlating the levels of mutation with transcription in the argF gene, which is predicted by bioinformatic analysis to be highly mutable. To achieve this goal, Allison will first construct a non-polar argF genetic knockout using a kanamycin cassette. Then, she will test the phenotype of the ArgF- strain. If a biochemical suppressor is present, she will disrupt the next possible genetic candidate. She will also build an IPTG-inducible construct containing argF with a stop codon in the loop of a putative SLS. This will be introduced into ArgF- Bacillus subtilis and assayed for the accumulation of mutations under starvation conditions, in the presence and absence of IPTG

MUSE reveals extended circumnuclear outflows in the seyfert 1 NGC 7469

NGC 7469 is a well-known luminous infrared galaxy, with a circumnuclear star formation ring ∼830 pc radius) surrounding a Seyfert 1 active galactic nucleus (AGN). Nuclear unresolved winds were previously detected in X-rays and ultraviolet, as well as an extended biconical outflow in infrared coronal lines. We search for extended outflows by measuring the kinematics of the Hβ and [O III] λ5007 optical emission lines, in data of the Very Large Telescope/Multi-unit Spectroscopic Explorer integral field spectrograph. We find evidence of two outflow kinematic regimes: One slower regime extending across most of the star formation (SF) ring-possibly driven by the massive SF-and a faster regime (with a maximum velocity of -715 km s-1), only observed in [O III], in the western region between the AGN and the massive star-forming regions of the ring, likely AGN-driven. This work shows a case where combined AGN/SF feedback can be effectively spatially resolved, opening up a promising path toward a deeper understanding of feedback processes in the central kiloparsec of AGNSupport from CONACyT (Mexico) grant CB-2016-01-286316 is acknowledged. J.P.T.P. acknowledges DAIP-UGto (Mexico) for granted support (0173/2019). Y.A. acknowledges support from project PID2019-107408GB-C42 (Ministerio de Ciencia e Innovación, Spain). S.F.S. thanks the support of CONACYT grants CB-285080 and FC-2016-01- 1916, and funding from the PAPIIT-DGAPA-IN100519 (UNAM) project. L.G. was funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 839090. This work has been partially supported by the Spanish grant PGC2018-095317-B-C21 within the European Funds for Regional Development (FEDER). E.B acknowledges the support from Comunidad de Madrid through the Atracción de Talento grant 2017-T1/TIC-5213. This research has been partially funded by the Spanish State Research Agency (AEI) Project MDM-2017-0737 Unidad de Excelencia “María de Maeztu”- Centro de Astrobiología (INTA-CSIC

Policing the legume-Rhizobium symbiosis: a critical test of partner choice

In legume-Rhizobium symbioses, specialised soil bacteria fix atmospheric nitrogen in return for carbon. However, ineffective strains can arise, making discrimination essential. Discrimination can occur via partner choice, where legumes prevent ineffective strains from entering, or via sanctioning, where plants provide fewer resources. Several studies have inferred that legumes exercise partner choice, but the rhizobia compared were not otherwise isogenic. To test when and how plants discriminate ineffective strains we developed sets of fixing and non-fixing strains that differed only in the expression of nifH - essential for nitrogen fixation - and could be visualised using marker genes. We show that the plant is unable to select against the non-fixing strain at the point of entry, but that non-fixing nodules are sanctioned. We also used the technique to characterise mixed nodules (containing both a fixing and a non-fixing strain), whose frequency could be predicted using a simple diffusion model. We discuss that sanctioning is likely to evolve in preference to partner choice in any symbiosis where partner quality cannot be adequately assessed until goods or services are actively exchanged

Bacteria-inducing legume nodules involved in the improvement of plant growth, health and nutrition

Bacteria-inducing legume nodules are known as rhizobia and belong to the class Alphaproteobacteria and Betaproteobacteria. They promote the growth and nutrition of their respective legume hosts through atmospheric nitrogen fixation which takes place in the nodules induced in their roots or stems. In addition, rhizobia have other plant growth-promoting mechanisms, mainly solubilization of phosphate and production of indoleacetic acid, ACC deaminase and siderophores. Some of these mechanisms have been reported for strains of rhizobia which are also able to promote the growth of several nonlegumes, such as cereals, oilseeds and vegetables. Less studied are the mechanisms that have the rhizobia to promote the plant health; however, these bacteria are able to exert biocontrol of some phytopathogens and to induce the plant resistance. In this chapter, we revised the available data about the ability of the legume nodule-inducing bacteria for improving the plant growth, health and nutrition of both legumes and nonlegumes. These data showed that rhizobia meet all the requirements of sustainable agriculture to be used as bio-inoculants allowing the total or partial replacement of chemicals used for fertilization or protection of crops