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

The Role of rpoE in stationary phase mutagenesis in Bacillus

Stationary phase mutagenesis is a phenomenon whereby random mutations are generated in non-dividing cells. In order to understand how these mutations arise, we use Bacillus subtilis, a gram positive rod-shaped model organism. It is hypothesize that increased transcription promotes stationary phase mutagenesis in this organism. We therefore examined the role of rpoE, a gene that encodes RNA polymerase ! subunit and proposed to influence efficiency of transcription. To this end, we will first generate a strain bearing a deletion in the rpoE gene. In order to determine if this gene is important for mutagenesis, we will examine the accumulation of mutations in this strain compared to the wild type by scoring for reversion to auxotrophy. If rpoE is significant in this process, we will expect a difference between the accumulation of mutations in the mutant strain and wild type. This project is a step towards understanding stationary phase mutagenesis, a process that has implications in evolution, drug resistance and cancer formation

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

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

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

The Bacillus Subtilis K-State Promotes Stationary-Phase Mutagenesis via Oxidative Damage

Bacterial cells develop mutations in the absence of cellular division through a process known as stationary-phase or stress-induced mutagenesis. This phenomenon has been studied in a few bacterial models, including Escherichia coli and Bacillus subtilis; however, the underlying mechanisms between these systems differ. For instance, RecA is not required for stationary-phase mutagenesis in B. subtilis like it is in E. coli. In B. subtilis, RecA is essential to the process of genetic transformation in the subpopulation of cells that become naturally competent in conditions of stress. Interestingly, the transcriptional regulator ComK, which controls the development of competence, does influence the accumulation of mutations in stationary phase in B. subtilis. Since recombination is not involved in this process even though ComK is, we investigated if the development of a subpopulation (K-cells) could be involved in stationary-phase mutagenesis. Using genetic knockout strains and a point-mutation reversion system, we investigated the effects of ComK, ComEA (a protein involved in DNA transport during transformation), and oxidative damage on stationary-phase mutagenesis. We found that stationary-phase revertants were more likely to have undergone the development of competence than the background of non-revertant cells, mutations accumulated independently of DNA uptake, and the presence of exogenous oxidants potentiated mutagenesis in K-cells. Therefore, the development of the K-state creates conditions favorable to an increase in the genetic diversity of the population not only through exogenous DNA uptake but also through stationary-phase mutagenesis

Mfd Affects Global Transcription and the Physiology of Stressed Bacillus subtilis Cells

© Copyright © 2021 Martin, Sundararajan, Ermi, Heron, Gonzales, Lee, Anguiano-Mendez, Schilkey, Pedraza-Reyes and Robleto. For several decades, Mfd has been studied as the bacterial transcription-coupled repair factor. However, recent observations indicate that this factor influences cell functions beyond DNA repair. Our lab recently described a role for Mfd in disulfide stress that was independent of its function in nucleotide excision repair and base excision repair. Because reports showed that Mfd influenced transcription of single genes, we investigated the global differences in transcription in wild-type and mfd mutant growth-limited cells in the presence and absence of diamide. Surprisingly, we found 1,997 genes differentially expressed in Mfd– cells in the absence of diamide. Using gene knockouts, we investigated the effect of genetic interactions between Mfd and the genes in its regulon on the response to disulfide stress. Interestingly, we found that Mfd interactions were complex and identified additive, epistatic, and suppressor effects in the response to disulfide stress. Pathway enrichment analysis of our RNASeq assay indicated that major biological functions, including translation, endospore formation, pyrimidine metabolism, and motility, were affected by the loss of Mfd. Further, our RNASeq findings correlated with phenotypic changes in growth in minimal media, motility, and sensitivity to antibiotics that target the cell envelope, transcription, and DNA replication. Our results suggest that Mfd has profound effects on the modulation of the transcriptome and on bacterial physiology, particularly in cells experiencing nutritional and oxidative stress

Efecto de la fertilización con calcio, fósforo y molibdeno en la fijación de nitrógeno y rendimiento en el frijól comun

58 p.El cultivo del frijol común (Phaseolus vulgaris L.) es de mucha importancia en Centroamérica. Es un componente básico de la dieta diaria de los habitantes de esta región, con un consumo percapita anual es 14.6 kg, y una fuente importante de proteínas, ya que el grano posee entre 18 y 24% de proteína (FAO, 1978). En honduras, el frijol es cultivado mayormente por pequeños agricultores agricultores; el 70% de la producción de frijol proviene de fincas de 3 ha o menos, y el cultivo ocupa una extensión total aproximada de 100,000 ha de tierra cultivable. A pesar de esto honduras sigue siendo importador de frijol para poder abastecer su demanda interna (Adams, 1984)