You might have heard of antibiotic resistance, which is one of the biggest public health issues of our time. In addition to the measures that should be taken at all levels of society to combat this issue, research to identify new antimicrobial targets to alleviate the rapid emergence of antibiotic resistance has become crucial. Cell division proteins in bacteria are now being investigated as potential new antibiotic targets. Cell division is like a dance, where cell division proteins have to follow a tight choreography, but what are bacterial cell division proteins? Think of a bacterium as a simplified version of yourself. We are made up of hundreds of different types of cells, each type adapted... (More)
A Tale of Two Proteins: SepF2 and SepF3
You might have heard of antibiotic resistance, which is one of the biggest public health issues of our time. In addition to the measures that should be taken at all levels of society to combat this issue, research to identify new antimicrobial targets to alleviate the rapid emergence of antibiotic resistance has become crucial. Cell division proteins in bacteria are now being investigated as potential new antibiotic targets. Cell division is like a dance, where cell division proteins have to follow a tight choreography, but what are bacterial cell division proteins? Think of a bacterium as a simplified version of yourself. We are made up of hundreds of different types of cells, each type adapted to do a different job. For our cells to do their job, they rely on instruction manuals, the genes, which tell our cells to make proteins. Proteins contribute to our growth and maintenance and perform different functions, and so do the bacterial proteins. Central to the cell division machinery in all bacteria is the protein FtsZ. Bacteria typically reproduce by binary fission, where genetic material is duplicated, septum is formed and the bacterium divides into daughter cells, each with a complete copy of the DNA. Streptomycetes, those bacterial superheroes who save hundreds of thousands of lives each year by the antibiotics they produce, are unique in their growth and cell division. They have a very interesting complex life cycle and two types of cell division: the formation of cross walls in vegetative hyphae and the sporulation septa during sporulation, both of which rely on FtsZ. Cell division in bacteria is accomplished through the formation of a Z-ring (FtsZ ring) at the site of division. A Z-ring needs an anchor, the protein FtsA, to become tethered to the membrane. Streptomycetes lack FtsA but have SepF for FtsZ membrane tethering. In our model organism, Streptomyces venezuelae, as well as in other streptomycetes, there are genes for two additional homologues of SepF, SepF2 and SepF3 proteins, which seem to have an interesting story to tell!
The story of sepF2 and sepF3 began when Sen et al., (2020), members of our lab team, isolated and investigated mutations of these two genes. The authors had very interesting findings, but there were still missing pieces of the puzzle. The aim of our project was to clarify the roles of sepF2 and sepF3 in cell division. We investigated many aspects of cell division in connection with single deletion mutations of these genes. We also generated a sepF2 sepF3 double mutant to clarify how the functions of these two genes might intersect. The experiments carried out concentrated on sporulation-specific cell division, although we anticipated an effect of the double mutation during vegetative growth. Using Fluorescent Protein Labelling techniques and different microscopic techniques, we were able to follow the expression of different genes and the behavior of the cell division machinery, visualize different cellular structures, and study the effect of mutations on different processes.
Our results show that the deletion of sepF2 doesn’t show an effect, whereas the deletion of sepF3 leads to many defects: spores of the mutant cannot detach from each other and stick in long chains, the typical regular spacing of sporulation septa is missing in the sepF3 mutant, and nucleoid partitioning among the spore compartments, the mechanism which ensure that each spore has a copy of the DNA, also becomes defective. We also observed by live-cell imaging that nucleoids seem to be moving between the spore compartments which suggested that the septa were not completely closed. We confirmed by applying a microscopy method called Fluorescence Recovery After Photobleaching that sporulation septa are improperly closed in the sepF3 mutant. The mutant also appeared to lack the compartmentalization of the wild-type strains and seemed to form spores in regions where spores should not be formed, i.e., in the main hyphal branches. We investigated this defect and revealed that the sepF3 mutant has such compartmentalization, so it’s still unclear why the deletion of this gene can lead to sporulation in the main hyphal branches.
After the isolation of the sepF2 sepF3 double mutant, we conducted many tests to see how the inactivation of both genes affected the phenotype. By visualizing the nucleoids, we found differences between the sepF3 mutant and the double mutant strains: nucleoid partitioning in the sepF3 mutant is defective, whereas the double mutant has a defect in nucleoid compaction, the mechanism of packaging the genetic material in the nucleoid region. We examined the colony appearance of the double mutant strain and found that the mutant has whiter and fluffier colonies. The colonies also look smaller and bald when they are separated from each other, a phenotype which suggested an effect of the mutant during vegetative growth. Staining of hyphal cross-walls in vegetative hyphae showed, in addition to regular cross-walls, large irregular stained spots which appeared larger and more abundant in the double mutant strain. Although there are still gaps in our understanding of the broad roles of SepF2 and SepF3 in cell division, we managed to answer vital parts of the research question and provided insights on the tasks these two proteins evolved to fulfil. The results of our project also shed light on the importance of studying genetic interactions and synthetic phenotypes since although the inactivation of sepF2 alone doesn’t show an effect, the inactivation of both genes has an effect that is different from what was seen in the single mutant strains.
Targeting proteins involved in cell division for antibiotic therapies requires extensive research on how the division machinery works. We also believe that by studying this unique nonpathogenic organism, we can provide answers which might help in fighting diseases caused by pathogenic organisms. For example, studies in cell morphogenesis, development and genetics of Streptomyces and Mycobacterium discovered remarkable analogies between these two organisms and uncovering these similarities can make Streptomyces research more valuable in terms of fighting pathogenic mycobacterial species. Other aspects that make Streptomyces research valuable include the ability of these organisms to produce a variety of important compounds, like antibiotics, anticancer agents, and immunosuppressants. This makes research in Streptomyces very significant in the pharmaceutical industry. Finally, these organisms provide a valuable source of natural products that can be developed into novel antibiotics needed to fight many infections that are currently resistant to existing antibiotics.
Master’s Degree Project in Molecular Biology, 60 credits, 2021
Department of Biology, Lund University
Supervisor: Klas Flärdh
Professor of Microbiology, Dept. of Biology, Faculty of Science, Lund University (Less)