Biology subjects

Bruna, R. E.

Publications and source records attributed to Bruna, R. E..

3 recordsLinked to original sources

bioRxiv2020

DNA transduction in Sodalis species: implications for the genetic modification of uncultured endosymbionts of insects

Bacteriophages (phages) are ubiquitous in nature. These viruses play a number of central roles in microbial ecology and evolution by, for instance, promoting horizontal gene transfer (HGT) among bacterial species. The ability of phages to mediate HGT through transduction has been widely exploited as an experimental tool for the genetic study of bacteria. As such, bacteriophage P1 represents a prototypical generalized transducing phage with a broad host range that has been extensively employed in the genetic manipulation of Escherichia coli and a number of other model bacterial species. Here we demonstrate that P1 is capable of infecting, lysogenizing and promoting transduction in members of the bacterial genus Sodalis, including the maternally inherited insect endosymbiont Sodalis glossinidius. While establishing new tools for the genetic study of these bacterial species, our results suggest that P1 may be used to deliver DNA to many Gram negative endosymbionts in their insect host, thereby circumventing a culturing requirement to genetically manipulate these organisms. SummaryA large number of economically important insects maintain intimate associations with maternally inherited endosymbiotic bacteria. Due to the inherit nature of these associations, insect endosymbionts cannot be usually isolated in pure culture nor genetically manipulated. Here we use a broad-host range bacteriophage to deliver exogenous DNA to an insect endosymbiont and a closely related free-living species. Our results suggest that broad host range bacteriophages can be used to genetically alter insect endosymbionts in their insect host and, as a result, bypass a culturing requirement to genetically alter these bacteria.

microbiology↗

bioRxiv2020

Limitation of phosphate assimilation maintains cytoplasmic magnesium homeostasis

Phosphorus (P) is an essential component of several core biological molecules. In bacteria, P is mainly acquired as inorganic orthophosphate (Pi). Once in the cytoplasm, Pi is incorporated into adenosine triphosphate (ATP), which exists primarily as a Mg2+ salt. Notably, whereas P is essential, excess of cytosolic Pi hinders growth. Here we demonstrate that cytotoxic effects of excessive Pi uptake result from its assimilation into ATP and subsequent disruption of Mg2+ dependent processes. We show that Salmonella enterica cells experiencing cytoplasmic Mg2+ starvation restrict Pi uptake, thereby limiting the availability of an ATP precursor. This response prevents excessive ATP synthesis, overproduction of ribosomal RNA, chelation of free cytoplasmic Mg2+ and the destabilization of Mg2+-dependent core processes that ultimately hinder bacterial growth and leads to loss of cellular viability. We demonstrate that, even when cytoplasmic Mg2+ is not limiting, excessive Pi uptake leads to increased ATP synthesis, depletion of free cytoplasmic Mg2+, inhibition of translation and growth. Our results establish that bacteria must restrict Pi uptake to prevent the depletion of cytoplasmic Mg2+. Furthermore, they provide a framework to understand the molecular basis of Pi cytotoxicity and reveal a regulatory logic employed by bacterial cells to control P assimilation. ImportancePhosphorus (P) is essential for life. As the fifth most abundant element in living cells, P is required for the synthesis of an array of biological molecules including (d)NTPs, nucleic acids and membranes. Organisms typically acquire environmental P as inorganic phosphate. While essential for growth and viability, excessive intracellular Pi is toxic for both bacteria and eukaryotes. Using the bacterium Salmonella enterica as a model, we demonstrate that Pi cytotoxicity is manifested following its assimilation into ATP, which acts as a chelating agent for intracellular cations, most notably, Mg2+. These results identify physiological processes disrupted by excessive Pi and elucidate a regulatory logic employed by bacteria to prevent uncontrollable P assimilation.

microbiology↗

bioRxiv2020

Conjugal DNA transfer in the maternally inherited symbiont of tsetse flies Sodalis glossinidius

Stable associations between insects and bacterial species are widespread in nature. This is the case for many economically important insects, such as tsetse flies. Tsetse flies are the vectors of Trypanosoma brucei, the etiological agent of African trypanosomiasis--a zoonotic disease that incurs a high socioeconomic cost in endemic regions. Populations of tsetse flies are often infected with the bacterium Sodalis glossinidius. Following infection, S. glossinidius establishes a chronic, stable association characterized by vertical (maternal) and horizontal (paternal) modes of transmission. Due to the stable nature of this association, S. glossinidius has been long sought as a means for the implementation of anti-Trypanosoma paratransgenesis in tsetse flies. However, the lack of tools for the genetic modification of S. glossinidius has hindered progress in this area. Here we establish that S. glossinidius is amenable to DNA uptake by conjugation. We show that conjugation can be used as a DNA delivery method to conduct forward and reverse genetic experiments in this bacterium. This study serves as an important step in the development of genetic tools for S. glossinidius. The methods highlighted here should guide the implementation of genetics for the study of the tsetse-Sodalis association and the evaluation of S. glossinidius-based tsetse fly paratransgenesis strategies. ImportanceTsetse flies are the insect vectors of T. brucei, the causative agent of African sleeping sickness--a zoonotic disease that inflicts a substantial economic cost to a broad region of sub-Saharan Africa. Notably, tsetse flies can be infected with the bacterium S. glossinidius to establish an asymptomatic chronic infection. This infection can be inherited by future generations of tsetse flies allowing S. glossinidius to spread and persist within populations. To this effect, S. glossinidius has been considered as a potential expression platform to create flies which reduce T. brucei stasis and lower overall parasite transmission to humans and animals. However, the efficient genetic manipulation of S. glossinidius has remained a technical challenge due to its complex growth requirements and uncharacterized physiology. Here we exploit a natural mechanism of DNA transfer among bacteria and develop an efficient technique to genetically manipulate S. glossinidius for future studies in reducing trypanosome transmission.

microbiology↗