Los narcos: Genios de la fun morality

In the second part of the thesis, I look at the difference between toxin and antitoxin gene pairs that create a PSK phenotype and those that do not. As a model system, I used the toxin and antitoxin gene pair barnase and barstar from Bacillus amy- loliquefaciens, which can be found unlinked on the chromosome. Barnase is a small (12.4kDA) RNase (Hartley and Smeaton 1973), bound with high affinity (kd 10−14_M)

by barstar, its intracellular inhibitor (Wang et al. 2004; Hartley 1993). Together, they are not known to exhibit PSK: I am interested in what conditions are necessary for them to do so.

Functions of barnase and barstar

The physiological or ecological functions of barstar and barnase are unknown, though they are often assumed to help the cell to acquire nutrients from the environment. In support of this, Bacillus spp. are adapted to low nutrient habitats (soil), and secrete a host of extracellular degradative enzymes for nutrient uptake prior to sporulation (Kharitonova and Vershinina 2009). Bacillus spp. have pathways for importing extra-

cellular ribonucleic acids (Saxild and Nygaard 1991; Beaman et al. 1983) and either re-incorporating them into their RNA (Bodmer and Grether 1965) or catabolising them as a source of nutrients (Schuch et al. 1999). The biosynthesis of a variety of related guanyl-specific RNases can be induced by phosphorous (Znamenskaya et al. 1995; Ulyanova et al. 2011) and nitrogen limitation (Kharitonova and Vershinina 2009). But the evidence for barnase to act similarly is inconstistent. The barnase gene is flanked by genes involved in nitrogen, phosphorous and carbon metabolism (Ulyanova et al. 2011). Yet the expression of barnase does not appear to be regulated by low levels of phosphorous and nitrogen in the environment (Znamenskaya et al. 1995; Ulyanova et al. 2011). This would be expected if its primary role was to raise nutrient levels in a manner similar to related RNases.

1.3 Exploring the parameter space of PSK 31 Other theories on the role of barnase have considered the possibility of barnase acting as a toxin on neighboring cells, instead of or in addition to a role in nutrient scavenging. For example, Ramos et al. (2006) created a heterologous expression system in E. coli and found that barnase expression could lead to zones of inhibition in surrounding lawns composed of other E. coli or other soil bacteria. The presence of barstar in the test strain used for the lawn eliminated the zone of inhibition. The authors suggested that barnase could act similarly to bacteriocins (Kleanthous 2010).

The two views on the role of barnase are not necessarily exclusive. It has also been speculated, for example, that barnase could aid in nutrient uptake at the onset of sporulation by killing neighboring cells (Ulyanova et al. 2011), a process known as cannibalism. Two such systems have been described in B. subtilis, skf and sdp, under control of the Spo0A transcription factor (González-Pastor 2011; Claverys et al. 2006). As B. amyloliquefaciens lacks known cannibal operons, barnase could fulfill this function. Similar to cannibal operons, barnase is not expressed in null-Spo0A mutants and has been reported as stationary phase dependent (Paddon et al. 1989). Barstar may have a binding site for the SigW transcription factor, which regulates cell immunity to toxins, and is inactive in null-Spo0A bacteria (Ulyanova et al. 2011). As such barstar expression may also be SpoA regulated. Most work done on barnase expression and function has been preliminary and isolated. Further research will be necessary before strong statements can be made about the role of this protein in B. amyloliquefaciens in its natural environment.

Biochemistry and secretion of barstar and barnase

Biochemically, barnase is guanyl-specific, cleaving single stranded RNA downstream of guanine nucleotides with a preference for downstream purines (Mossakowska et al. 1989; Day et al. 1992; Bastyns et al. 1994). The RNA is cleaved via a two-step process (Figure 1.3), as diagrammed below. The activity of barnase inside the cell is inhibited by a one to one binding to barstar (Hartley and Smeaton 1973). The negatively charged amino acids on barstar sterically block the positively charged active site on barnase through strong electrostatic binding (Buckle and Fersht 1994) (Figure 1.4). This interaction is extremely strong, with a dissociation constant between the two proteins at 10-14_{M. The dissociation constant is the concentration of protein at which}

half is unbound to the other protein, such that the more tightly the two interact the lower the dissociation constant will be.

Figure 1.3: Hydrolysis of RNA by barnase.Barnase is a guanyl-specific RNase. Single stranded RNA is cleaved downstream of guanine nucleotides in a two-step process, with a cyclic phosphate intermediate formed (the transesterification step) followed by hydrolysis of the intermediate,

yielding a new guanosine 30 _{phosphate and 5}0_{-OH end (Rushizky et al.}

1963). The general base is the amino acid glu73, while the general acid is his102 (Schreiber et al. 1997; Mossakowska et al. 1989).

Figure 1.4: Interactions between barnase and barstar. Barstar (pink) has negatively charged amino acids that interact with and sterically block the active site of barnase (blue). Conformation from (Buckle and Fersht 1994), as viewed in Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/

1.3 Exploring the parameter space of PSK 33 In its native host, B. amyloliquefaciens, barnase is translated as a pre-proprotein (Paddon et al. 1989). The 26 amino acid pre sequence is an export signal that is cleaved during translocation of the cell membrane (Chen and Nagarajan 1993). Ex- port of barnase on its native signal sequence is slow relative to other signal sequences: barnase has been shown in the cytoplasm up to 90 minutes after translation in B. subtilis. This may account for the presence of barstar (Chen and Nagarajan 1993), as the analogous but rapidly exported RNase binase, from Bacillus intermedius, apparently lacks an intracellular inhibitor (Ulyanova et al. 2011). Barnase is ex- ported independently of barstar, allowing it to be active extracellularly (Paddon

et al. 1989).

The pro-sequence (13 amino acids) is cleaved by extracellular serine proteases after secretion, to make the mature barnase protein (Paddon et al. 1989). Its presence does not appear to affect overall refolding rates or catalytic activity of barnase (Gray

et al. 1993). It does increase protein binding to chaperon GroEL, potentially aiding

in transport of the protein (Gray et al. 1993; Chen and Nagarajan 1993). Barnase is also bound by the E. coli chaperon SecB (Stenberg and Fersht 1997). SecB transfers proteins to a translocase that secretes proteins through the cell membranes and is relatively conserved between E. coli and B. subtilis (Harwood and Cranenburgh 2008).

Barstar and barnase as a model system

The biochemical activity of barnase and barstar is similar to that seen in type II TA systems. The type II toxins MazF, HigB and VapC are all ribonucleases (Van Melderen and De Bast 2009), also tightly bound by intracellular inhibitors (Brzozowska and Zielenkiewicz 2013). As a secreted toxin, barnase also has some similarities to bacteriocins. These similarities, plus the well-studied biochemistry of the proteins, prompted their selection to test the necessary conditions for a given toxin and antitoxin to become a PSK.

There is precedent for genes to become addictive in new environmental and genetic contexts. As genes move from locations between and within replicons and between different cells and external environments, both their function and the selective pres- sures acting on them can change (Heinemann and Silby 2003). Context, for example,

affects the spread of antibiotic resistance via PSK-like mechanisms.

Genetic context changes as genes move from replicon to replicon. Many antibiotic resistance genes were originally chromosomal genes, being selected for as resistance mechanisms on plasmids (Martinez 2012; Baquero et al. 2008). Environmental context changes as genes move into new cells in new environments. Some resistance genes now seen on plasmids originated from producer strains, to protect the bacterium from its own antibiotics, or strains that co-exist with producer strains (Laskaris

et al.2010; Benveniste and Davies 1973; Davies and Davies 2010). But some genes

that function as antibiotic resistance mechanisms have alternate functions in their original host (Davies and Davies 2010; Forsberg et al. 2012; Martinez 2012; Laskaris

et al.2010). The gene qnrA, which encodes resistance to quinolines, comes from the

non-antibiotic producing aquatic bacterium Shewanella algae (Poirel et al. 2005). Now it has become an important pathway for resistance to synthetic antibiotics such as quinolines.

Once the genes become mobilized, they can potentially spread to new hosts. Lacking their original biochemical and genetic context, they can still be maintained and selected for if they confer antibiotic resistance, a form of exaptation (Baquero et

al. 2008). In the presence of lethal concentrations of antibiotics in the environments,

these resistance genes become addictive and spread quickly through populations (Cooper and Heinemann 2000).

I am interested in using barstar and barnase to test the importance of cellular and genetic context for expression of the PSK phenotype. Within the cell, I consider the relative expression levels and stability of the different components necessary for barstar and barnase to exhibit PSK in a manner similar to type II TA systems. The role of genetic context is explored by testing the hypothesis that a given secreted toxin can induce PSK when expressed from a plasmid, in a manner analogous to antibiotics.

Ultimately, I am interested in the evolution of PSK systems in bacteria. PSKs have successfully colonized large portions of the bacterial gene pool, proliferating on both mobile elements and chromosomes. The movement and interactions of genes in new contexts has the potential both to drive evolution of PSKs and select for their maintenance. The ability of genes to become addictive in new contexts, and selection for such phenotypes on mobile elements through competition, has implica- tions for selection of genes onto plasmids and subsequent spread through bacterial populations.

Chapter 2

Materials and Methods