At its most fundamental level, life is a dance between a shifting array of microscopic partners. To unlock the cellular choreography, John D. Helmann, Microbiology, has devoted his career to studying a single-celled bacterium known as Bacillus subtilis. The organism is well-known in scientific research as the preferred model for bacteria generally, as well as specifically for gram-positive bacteria (those with a thick peptidoglycan layer making up their cell wall). And although B. subtilis is not a human pathogen, it is closely related, genetically and functionally, to many infectious microbes, such as Staphylococcus aureus, Listeria monocytogenes, and bacteria in the Streptococcus family, which means research findings about B. subtilis are generally applicable to a host of disease-causing agents.
“It’s a soil bacteria that’s found all over the planet, a simple organism with about 4000 genes, but it has a lot of complexity,” Helmann says. “It’s also naturally transformable, so the genetic tools are just head and shoulders above other organisms of interest, especially in the past before we had genomics and whole genome sequencing.”
The complexity and malleability of B. subtilis cell processes mean that Helmann can use the organism both as an example of how bacteria grow, survive, and infect, and as a stepping stone to understanding the biology of multicelled organisms, such as humans. “Most of our cells have virtually identical genomes, but we have cells of many, many different types that all come together to form the various tissues and organs that make up our bodies,” he says. “The differences between these cell types come down to which genes are turned on and which are turned off in each cell. Very early in my career, I was drawn to the question of what causes those genes to switch on and off.”
Sigma Factors: Making the Hop to Antibiotic Resistance
The answer to what causes gene expression has a lot to do with regulation of the enzyme RNA polymerase (RNAP), Helmann explains. During transcription, RNAP copies DNA, which contains an organism’s genetic code, into messenger RNA (mRNA), which then carries the genetic instructions to ribosomes for translation into proteins.
With B. subtilis as his model, Helmann has spent a large portion of his time investigating the bacteria’s sigma factors, which are the transcription factors that allow RNAP to recognize the start points of genes so the enzyme can decide where to begin transcribing DNA into mRNA. In particular, the Helmann lab explores how sigma factors control regulatory proteins that help cells adapt to three main types of stressors: compounds that interfere with the function of the cell’s outer layer, or envelope; changes in the availability of metal ions; and oxidative stress.
In one research path, Helmann and his colleagues have focused on the actions of a group of B. subtilis transcription factors—known as extracytoplasmic function (ECF) sigma factors—which tend to be activated under conditions of stress to the cell envelope. For example, one or more ECF sigma factors are triggered when the bacterium is exposed to antibiotics. “This often leads to resistance against the antibiotic,” Helmann says.
“We want to understand what sort of stress responses cells mount when they are first exposed to an antibiotic...[and whether that allows them] to persist long enough to acquire a resistance mutation.”
The Helmann lab is contributing to the scientific understanding of antibiotic resistance by exploring the role of sigma factors in more detail. “We want to understand what sort of stress responses cells mount when they are first exposed to an antibiotic at low levels and whether these responses then allow them to tolerate the presence of the antibiotic without dying,” Helmann says. “The cells may then be able to persist long enough to acquire a resistance mutation that gives them the ability to ignore the antibiotic and proliferate.”
The Metal Ion Tango
Helmann and his colleagues also have investigated the effects of metal ion limitation on B. subtilis. While a scarcity of metal ions can happen for a number of reasons, limiting access to metals is also one of the ways the human body’s innate immune system fights a bacterial infection. “Bacteria need metal ions for growth,” Helmann explains. “So the body may restrict the bacteria’s access to iron, manganese, and zinc. This can hinder bacteria, but it’s not always obvious why they stop growing. What processes fail?”
In the case of zinc, the researchers recently showed that limiting access to the metal limits the ability of B. subtilis to make folate, which it needs as a cofactor for some enzymes. They also found that the bacterium has mechanisms to prioritize the distribution of limited metal ions within the cell. “Under zinc-limiting conditions, where the cell is barely able to get enough zinc to sustain life, we found that certain proteins very specifically capture whatever zinc is available and hand it off to another protein that contributes to the biosynthesis of folate,” Helmann says.
Recently Helmann and his colleagues shifted their focus to the converse situation, asking how cells deal with an overload of metals. There are various reasons why this can happen. Often, cells with limited access to a certain metal respond by making high-affinity transporters that rapidly import a lot of the missing nutrient into the cell once it becomes available again. As a result, the cell can become overloaded. “Cells have transporters to pump excess metal ions back out,” Helmann says. “And we’ve identified those efflux transporters for iron and manganese just within the last few years.”
The researchers have also identified the major mechanism by which metal ions intoxicate cells—a process called mismetalation, meaning an enzyme that would normally bind one type of metal ion at a very specific binding site binds a different metal ion there instead. In the case of B. subtilis, they found that excess zinc will mismetalate a protein called PerR. “This leads to dysregulation of the genes normally regulated by PerR, which results in the cell making too much heme (an iron-containing compound), which then triggers an oxidative stress response,” Helmann says.
Following the Lead of Forward Genetics
Over almost four decades of research, Helmann has seen important changes in his field—in particular, the shift from using a classic reverse-genetics approach, for which researchers identify a gene or protein of interest and then mutate the gene to see what happens, to the rewards of forward genetics. “With forward genetics, we expose the bacterium to an antibiotic, for example, without trying to preconceive which genes are important,” Helmann says. “We let the bacterium evolve to become more resistant to antibiotics, and then we ask, which genes have changed? The change could have happened anywhere on the entire genome. It’s tremendously satisfying and also very stimulating because you don’t know what you’re going to find. You’re really letting the cell solve the problem for you.”