Screening of Metagenomic and Genomic Libraries Reveals Three Classes of Bacterial Enzymes That Overcome the Toxicity of Acrylate

Abstract

Acrylate is produced in significant quantities through the microbial cleavage of the highly abundant marine osmoprotectant dimethylsulfoniopropionate, an important process in the marine sulfur cycle. Acrylate can inhibit bacterial growth, likely through its conversion to the highly toxic molecule acrylyl-CoA. Previous work identified an acrylyl-CoA reductase, encoded by the gene acuI, as being important for conferring on bacteria the ability to grow in the presence of acrylate. However, some bacteria lack acuI, and, conversely, many bacteria that may not encounter acrylate in their regular environments do contain this gene. We therefore sought to identify new genes that might confer tolerance to acrylate. To do this, we used functional screening of metagenomic and genomic libraries to identify novel genes that corrected an E. coli mutant that was defective in acuI, and was therefore hyper-sensitive to acrylate. The metagenomic libraries yielded two types of genes that overcame this toxicity. The majority encoded enzymes resembling AcuI, but with significant sequence divergence among each other and previously ratified AcuI enzymes. One other metagenomic gene, arkA, had very close relatives in Bacillus and related bacteria, and is predicted to encode an enoyl-acyl carrier protein reductase, in the same family as FabK, which catalyses the final step in fatty-acid biosynthesis in some pathogenic Firmicute bacteria. A genomic library of Novosphingobium, a metabolically versatile alphaproteobacterium that lacks both acuI and arkA, yielded vutD and vutE, two genes that, together, conferred acrylate resistance. These encode sequential steps in the oxidative catabolism of valine in a pathway in which, significantly, methacrylyl-CoA is a toxic intermediate. These findings expand the range of bacteria for which the acuI gene encodes a functional acrylyl-CoA reductase, and also identify novel enzymes that can similarly function in conferring acrylate resistance, likely, again, through the removal of the toxic product acrylyl-CoA.

Mechanistic insight into acrylate metabolism and detoxification in marine dimethylsulfoniopropionate‐catabolizing bacteria

“…Acrylate, which can represent 10–15% of the total carbon in coral exudates (Tapiolas et al ., ), is also an important carbon source in marine environment. Many marine bacteria that cleave DMSP subsequently metabolise the product acrylate as a carbon source for growth (Yoch, ; Todd et al ., ; Curson et al ., , Curson et al ., ). However, acrylate is toxic and is further converted into other intracellular toxic compounds, such as the electrophile acryloyl‐CoA that attacks sulfhydryl groups, which are detrimental to bacteria (Clayden et al ., ; Todd et al ., ).…”

“…Recently, SAR11 α‐proteobacteria (order Pelagibacterales), which are the most abundant heterotrophic bacteria in the oceans, were shown to both cleave and demethylate DMSP (Sun et al ., ). There are many studies of microbial DMSP cleavage and demethylation, but only two detailed reports of acrylate utilization in DMSP‐catabolizing bacteria, those being Halomonas HTNK1 (an oceanospirillales) and Ruegeria pomeroyi DSS‐3 (a Roseobacter) (Todd et al ., ; Reisch et al ., ; Curson et al ., ). Two different pathways for acrylate utilization and detoxification were proposed, the AcuN‐AcuK pathway and the PrpE‐AcuI pathway, which use the acryloyl‐CoA transferase (AcuN) and acryloyl‐CoA hydratase (AcuK) or the propionate‐CoA ligase (PrpE) and acryloyl‐CoA reductase (AcuI) enzymes, respectively, as the key enzymes (Todd et al ., ; Reisch et al ., ).…”

“…B) (Reisch et al ., ). AcuI is believed to be responsible for the detoxification of acrylate (Herrmann et al ., ; Curson et al ., ). Consistent with this, an R. pomeroyi acuI ‐ mutant loses the ability to use acrylate as a sole carbon source and becomes hypersensitive to this metabolite (Todd et al ., ).…”

DddY is a bacterial dimethylsulfoniopropionate lyase representing a new cupin enzyme superfamily with unknown primary function

“…Thus, genome context can provide important hints regarding the enzymatic function of ddd+ genes. Acrylate, the verified product of all cupin Ddd+ enzymes ( Table 1), is toxic, 33,34 and previous studies indicated proximal genes that relate to acrylate catabolism. These include: acuI, a zinc/iron dependent alcohol reductase that converts acrylyl-CoA into propanoyl-CoA; 17,33 acuK (a dehydratase) and acuN (a CoA transferase) that can jointly convert acrylate into 3-hydroxypropionate; 35 or cytochrome dependence oxi-reductases that could also catabolize acrylate.…”

A putative 3‐hydroxyisobutyryl‐CoA hydrolase is required for efficient symbiotic nitrogen fixation in Sinorhizobium meliloti and Sinorhizobium fredii NGR234

“…HIB‐CoA hydrolases are required for l ‐valine catabolism and appear to prevent the accumulation of toxic metabolic intermediates, particularly methacrylyl‐CoA. Evidence presented here and elsewhere (Curson et al ., , PLoS ONE 9:e97660) demonstrated that Smb20752 and NGR_b20860 can also prevent metabolic toxicity, are required for l ‐valine metabolism, and play an undefined role in 3‐hydroxybutyrate catabolism. We present evidence that the symbiotic defect of the HIB‐CoA hydrolase mutants is independent of the inability to catabolize l ‐valine and suggest it relates to the toxicity resulting from metabolism of other compounds possibly related to 3‐hydroxybutyric acid.…”