SaxA-Mediated Isothiocyanate Metabolism in Phytopathogenic Pectobacteria

Abstract

Pectobacteria are devastating plant pathogens that infect a large variety of crops, including members of the family Brassicaceae. To infect cabbage crops, these plant pathogens need to overcome the plant's antibacterial defense mechanisms, where isothiocyanates are liberated by hydrolysis of glucosinolates. Here, we found that a Pectobacterium isolate from the gut of cabbage root fly larvae was particularly resistant to isothiocyanate and even seemed to benefit from the abundant Brassica root metabolite 2-phenylethyl isothiocyanate as a nitrogen source in an ecosystem where nitrogen is scarce. The Pectobacterium isolate harbored a naturally occurring mobile plasmid that contained a sax operon. We hypothesized that SaxA was the enzyme responsible for the breakdown of 2-phenylethyl isothiocyanate. Subsequently, we heterologously produced and purified the SaxA protein and characterized the recombinant enzyme. It hydrolyzed 2-phenylethyl isothiocyanate to yield the products carbonyl sulfide and phenylethylamine. It was also active toward another aromatic isothiocyanate but hardly toward aliphatic isothiocyanates. It belongs to the class B metal-dependent beta-lactamase fold protein family but was not, however, able to hydrolyze beta-lactam antibiotics. We discovered that several copies of the saxA gene are widespread in full and draft Pectobacterium genomes and therefore hypothesize that SaxA might be a new pathogenicity factor of the genus Pectobacterium, possibly compromising food preservation strategies using isothiocyanates. P ectobacteria are phytopathogens that cause tuber soft rot and blackleg (stem rot) in many horticulturally and economically important crops during cultivation, transport, or storage. Most Pectobacterium spp., e.g., Pectobacterium carotovorum or P. wasabiae, can be detrimental to many different plants, such as potato, sugar beet, cabbage, wasabi, chicory, and Ornithogalum plants, whereas other Pectobacterium spp., e.g., P. atrosepticum and P. betavasulorum, have a more narrow host range of potato and sugar beet plants, respectively (1).Several pathogenicity factors of soft-rot-causing Pectobacterium spp. have been found during the last decades (2-4). Of these, plant cell wall-degrading enzymes produced by Pectobacterium spp. have a large impact on the progress of the disease, as their production leads to the degradation of invaded plant tissue (2, 3). The production of these enzymes is dependent on cell density and regulated by quorum sensing through N-acetylhomoserine lactone (5) and intracellular regulators (6, 7). Motility and nutrient uptake are also factors influencing phytopathogenicity (4,(8)(9)(10). Besides these general pathogenicity factors, phytopathogens have to overcome the toxicity of plant allelochemicals. Important allelochemicals of members of the family Brassicaceae (cabbages and mustards) are isothiocyanates that are liberated by the glucosinolate-myrosinase defense system (11-13). The antimicrobial effect of isothiocyanates has been mainly attributed to their reactivity w...

Root and shoot glucosinolate allocation patterns follow optimal defence allocation theory

“…; Welte et al . ). This would allow this species to feed on plant organs with high levels of 2PE‐GSL and other ITC forming GSLs.…”

Reviews and Syntheses: Carbonyl Sulfide as a Multi-scale Tracer for Carbon and Water Cycles

“…Biotic OCS production from cryptogamic covers is a possibility: In bacteria, novel enzymatic pathways 10 have been described that degrade thiocyanate and isothiocyanate and render OCS as a byproduct (Bezsudnova et al, 2007;Hussain et al, 2013;Katayama et al, 1992;Welte et al, 2016). Evidence for OCS emissions following SCN -degradation has been observed from a range of environmental samples from aquatic and terrestrial origins, indicating a wide distribution of OCS-emitting microorganisms in nature (Yamasaki et al, 2002).…”

“…Hydrolysis of isothiocyanate, another breakdown product of 15 glucosinolates (Hanschen et al, 2014), by the SaxA protein also yields OCS, as shown in phytopathogenic Pectobacterium sp. (Welte et al, 2016). Some Actinomycetales bacteria and Mucoromycotina fungi, both commonly found in soils, are also known to emit OCS, but the origin and pathway remains to be elucidated (Masaki et al, 2016;Ogawa et al, 2016).…”

Single gene enables plant pathogenic Pectobacterium to overcome host‐specific chemical defence

“…Brassicales plants employ the GSN–myrosinase defence system which, on tissue damage, releases a blend of toxic volatiles, of which ITCs are particularly toxic to insects and bacteria (Wittstock et al ., ; Dufour et al ., ). We have shown previously that the bacterial saxA gene encodes a hydrolase that can detoxify ITCs in vitro and that the gene is widespread among Enterobacterales (Welte et al ., ; van den Bosch et al ., ). In this study, we demonstrated that deletion of two different saxA gene variants results in greatly reduced virulence of Pectobacterium spp.…”

“…These observations may be the effect of convergent evolution, although differential effectiveness against particular variations of GSN/ITC profiles of hosts cannot be excluded. The presence of two saxA genes in the genome of P. polaris NCPPB3395 and the presence of three saxA genes with unique origins in strains of P. wasabiae (Welte et al ., ) suggest that there may be added benefit to diversifying genomic saxA gene content when infecting specific hosts. The analysis of the distribution of saxA genes in the Pectobacterium genus showed that most Pectobacterium strains isolated from a Brassica host contain one or more saxA genes in their genome, with the exception of one group of P. odoriferum strains (S6, S6–2, and BCS7).…”