Current lab projects
Promiscuity, Serendipity and Metabolic Innovation
The proteomes of most bacteria contain 1000-2000 enzymes, and each enzyme has an unknown number of promiscuous activities. Consequently, bacteria can access many thousands of catalytic activities when a new enzyme is needed to enhance fitness or even to enable survival.聽We are interested in the potential for evolution of novel metabolic pathways patched together from multiple promiscuous enzymes.
Figure 1. A serendipitous pathway that can restore synthesis of PLP when pdxB is deleted.
Promiscuous enzymes can serve as the starting point for evolution of new enzyme activities. However, their evolutionary potential goes further; multiple promiscuous activities can be patched together to generate novel metabolic pathways. We call these pathways 鈥渟erendipitous鈥 because their assembly relies upon the fortuitous combination of promiscuous activities available within the proteome, which can differ substantially in different organisms, or in the same organism under different environmental conditions.
We are studying the emergence of serendipitous pathways in a model system in which we have deleted a gene (pdxB) that is essential for synthesis of the essential cofactor pyridoxal 5-phosphate (PLP).聽PLP is needed in relatively small quantities because it is a cofactor (i.e. it is regenerated after every catalytic cycle). Thus, even an inefficient low-flux pathway can enable cells lacking pdxB to grow.聽We have succeeded in restoring robust growth of 鈭pdxB E. coli within as few as 150 generations. Figure 1 shows one serendipitous pathway that can restore PLP synthesis. We are currently characterizing another.
There is a deep reservoir of catalytic potential lurking in the proteome that can be drawn upon to meet a new environmental challenge.
Evolution in Action: An Evolving Metabolic Pathway for Degradation of a Toxic Pesticide
In the early 20th century, numerous anthropogenic chlorinated chemicals such as DDT, lindane, PCP, atrazine, tetrachloroethylene, and PCBs were introduced with little concern over their ultimate fate in the environment. Many of these compounds persist in the environment, posing risks to the health of humans and wildlife. We are interested in the assembly of new metabolic pathways that allow microbes to degrade recalcitrant and toxic pesticides.
Figure 2. The pathway for conversion of PCP to 脽-ketoadipate, which can be further degraded via the TCA cycle. PcpB, PCP hydroxylase; PcpD, tetrachlorobenzoquinone (TCBQ) reductase; PcpC, tetrachlorohydroquinone (TCHQ) dehalogenase; PcpA, 2,6-dichlorohydroquinone (DCHQ) dioxygenase; PcpE, maleylacetate reductase. Comparison of the genome of S. chlorophenolicum with that of the closely related S. japonicum suggest that the enzymes in the cyan and yellow segments are encoded by genes acquired by horizontal gene transfer (HGT) after divergence of the two species.
Biodegradation of anthropogenic compounds is often inefficient because microbes have not yet evolved enzymes that efficiently catalyze the steps needed to convert such compounds into metabolites in central carbon metabolism.聽Further, the ability of microbes to degrade novel compounds can be impaired when the compound or its degradation products are toxic.聽
Pentachlorophenol (PCP) is a toxic anthropogenic pesticide that was first introduced in the 1930s. Sphingobium chlorophenolicum can mineralize PCP to CO2, HCl and H2O (Figure 2).聽The ability of S. chlorophenolicum to degrade PCP is remarkable because PCP and the first four downstream metabolites are toxic.聽PCP dissipates the proton-motive force. Tetrachlorobenzoquinone is a highly reactive alkylating agent. Tetrachlorohydroquinone, 2,5,6-trichlorohydroquinone, and 2,6-dichlorohydroquinone react with O2 to generate reactive oxygen species. We are currently using high-throughput sequencing methods (RNA-Seq and Tn-Seq) as well as physiological approaches to discover how S. chlorophenolicum is able to withstand the stresses caused by PCP and its metabolites, and how its resistance to these myriad toxic effects can be improved by mutations.
Evolution of a New Enzyme by Gene Duplication and Divergence
We are interested in the trajectories by which microbes evolve improved fitness when a newly recruited promiscuous enzyme limits growth rate. New enzymes often evolve from promiscuous activities of previously existing enzymes by a process of gene duplication and divergence. This process is complicated by numerous factors, including the need to maintain the original function of the enzyme and the occurrence of mutations elsewhere in the genome that improve fitness by other mechanisms.
ArgC (N-acetyl-L-g-glutamylphosphate reductase) is essential for growth of E. coli on glucose because it is required for synthesis of arginine.聽However, a point mutation in proA that changes Glu383 to Ala allows ProA (glutamylphosphate reductase) to serve both functions, albeit poorly (Figure 3). We refer to E383A ProA as ProA*.聽We have evolved several lineages of a strain lacking argC and carrying proA* under selection for improved growth on glucose in the presence of proline to select for improved synthesis of arginine (Figure 4).聽Growth is improved by a mutation in the promoter of the proBA* operon and massive amplification of the region surrounding proA*. We have also detected a聽mutation in proA* and several additional genomic mutations. We are in the processing of dissecting the mechanisms behind their effects. This work will help us understand the interplay between mutations in the gene encoding the weak-link enzyme that lead toward a newly needed enzyme and genomic mutations that compensate for growth impairment by other mechanisms.
Figure 3. The reactions catalyzed by ProA and ArgC are chemically identical. The substrates differ only in the absence or presence of an acetyl moiety on the amino group.
Figure 4. Changes in growth rate and proA* copy number during evolution of 鈭argCproA* E. coli containing a promoter mutation on glucose + proline.
Synonymous 鈮 Silent
Synonymous mutations have traditionally been considered to be silent with respect to fitness because they do not change the encoded amino acid. However, synonymous mutations can alter the mRNA structures in ways that alter translation initiation, mRNA stability, or even protein folding due to changes in the tempo of translation. We are interested in how synonymous mutations improve fitness when bacteria are subjected to strong selective pressures.
Figure 5. Growth rates and levels of ProA* in strains carrying a promoter mutation and other mutations relative to those of the strain carrying just M1. Black, no other mutations in head region; blue, intergenic mutation; red, synonymous mutations in codon 2; white, synonymous mutation in codon 3; magenta, synonymous mutation in codon 4; cyan, synonymous mutations in codon 6; yellow, non-synonymous mutation M5. Data points representing strains with the promoter mutation and two additional mutations are shown with colored stripes corresponding to the colors used for individual mutations. Error bars represent 1 SD.
We deleted argC in S. enterica and introduced a point mutation that changes Glu382 to Ala in order to examine how the process of gene duplication and divergence proceeds in Salmonella聽rather than E. coli.聽In contrast to the results in E. coli, gene amplification did not occur in S. enterica. After evolution of this strain for up to 250 generations, we found a promoter mutation and two different synonymous mutations in the first six codons of proA* itself.聽The synonymous mutations, far from being silent, increase growth rate 30-80%.聽Other synonymous mutations in this region have similarly strong effects, ranging from a 2-fold increase in growth rate to total prevention of growth (Figure 5). These effects seem to be due to alterations in the efficiency of translation initiation caused by changes in secondary structure around the AUG start codon.