Corrinoid auxotrophy in the obligate organohalide-respiring Dehalobacter restrictus strain PER-K23

Background Corrinoids are an essential cofactor of reductive dehalogenases, the key enzymes of organohalide respiration (OHR). Dehalobacter restrictus is an obligate OHR bacterium (OHRB) able to conserve energy with tetrachloroethene, but is unable to de novo synthesize corrinoids. Genome analysis of D. restrictus strain PER-K23 however revealed the presence of the complete corrinoid biosynthesis pathway. Objectives The aim of the present study is to understand the corrinoid metabolism of D. restrictus strain PER-K23 at the level of biosynthesis, regulation and transport and to compare it to contrasting situations in other Dehalobacter strains. Methods Genome analysis was performed with standard bioinformatic tools. Both transcriptomic and proteomic approaches were applied on D. restrictus strain PER-K23 cells cultivated in media with alternative corrinoid conditions. Gene expression was further addressed using targeted reverse transcription and quantitative PCR. Growth and dechlorination of D. restrictus strain PER-K23 was followed by measuring cell numbers and chloride release, respectively. Results and Conclusion Annotation and analysis of genes involved in corrinoid metabolism revealed a 101-bp deletion in the cbiH gene resulting in a shift of the reading frame and leading to a non-functional enzyme. This mutation, which is not present in the genome of other Dehalobacter strains, indicates that cbiH represents a possible checkpoint behind corrinoid auxotrophy. The expression of most corrinoid biosynthetic genes is controlled by cobalamin riboswitches. Experimental evidence of this latter mechanism was obtained. Comparative ’omics’ analyses of corrinoid-starved cells revealed an increased production of corrinoid transporters and proteins involved in corrinoid salvaging. Taken together, these data suggest that D. restrictus strain PER-K23 has recently lost its capacity of de novo corrinoid synthesis. References : Holliger et al., 1998, Kruse et al., 2013; Rupakula et al., 2013, Tang et al., 2012

Corrinoid auxotrophy in the obligate organohalide respiring Dehalobacter restrictus

Christof Holliger, Julien Maillard, Aamani Rupakula Boyanapalli, Kiyan Shabestary

Background Corrinoids are an essential cofactor of reductive dehalogenases, the key enzymes of the environmental-friendly process of organohalide respiration (OHR). Dehalobacter restrictus strain PER-K23 is an obligate OHR bacterium (OHRB) able to conserve energy with tetrachloroethene, but is unable to de novo synthesize corrinoids (1). Genome analysis of D. restrictus however revealed the presence of the complete corrinoid biosynthesis pathway (2,3). Objectives The aim of the present study is to understand the corrinoid metabolism of D. restrictus at the level of biosynthesis, regulation and transport and to compare it to contrasting situations in other OHR bacteria. Methods Genome analysis was performed with standard bioinformatic tools. Both transcriptomic and proteomic approaches were applied on D. restrictus cells cultivated in media with alternative corrinoid conditions. Gene expression was further addressed using targeted reverse transcription and quantitative PCR. Conclusions Annotation and analysis of genes involved in corrinoid metabolism revealed a 101-bp deletion in the cbiH gene resulting in a shift of the reading frame and leading to a non-functional enzyme. This mutation, which is not present in the genome of other Dehalobacter spp., indicates that cbiH represents a possible checkpoint behind corrinoid auxotrophy. The expression of most corrinoid biosynthetic genes is likely to be controlled by cobalamin riboswitches. Experimental evidence of this latter mechanism is under scrutiny. Comparative ’omics’ analyses of corrinoid-starved cells revealed an increased production of corrinoid transporters and proteins involved in corrinoid salvaging. Taken together, these data suggest that D. restrictus has recently lost its capacity of de novo corrinoid synthesis. References (1) Holliger et al. (1998), Arch Microbiol 169, 313. (2) Kruse et al. (2013), Stand Genomic Sci, submitted. (3) Rupakula et al. (2013), R Soc Phil Trans B, in press.

2013

Corrinoid Auxotrophy in the Obligate Organohalide-Respiring Dehalobacter restrictus strain PER-K23

Christof Holliger, Yue Lu, Julien Maillard, Aamani Rupakula Boyanapalli, Hauke Smidt

Introduction: Corrinoids are an essential cofactor of reductive dehalogenases, the key enzymes of organohalide respiration (OHR). Dehalobacter restrictus strain PER-K23 is an obligate OHR bacterium able to conserve energy with tetrachloroethene, but is unable to de novo synthesize corrinoids. In contrast, genome analysis of D. restrictus revealed the presence of the complete corrinoid biosynthesis pathway. Objectives: To understand the corrinoid metabolism of D. restrictus at the level of biosynthesis, regulation and transport and compare it to other emerging Dehalobacter genomes Methods: Genomes of Dehalobacter spp. was obtained from the JGI. Proteomics were applied on D. restrictus cells cultivated in media supplemented with corrinoids spanning a range from 0 to 250 µg/l. Gene expression analysed using targeted reverse transcription and quantitative PCR. Results and Discussion: A non-functional cbiH gene All the genes required for corrinoid biosynthesis & transport were identified in D. restrictus. However, there was a 101-bp deletion in the precorrin-3B C17-methyltransferase encoding cbiH. This mutation is not present in the genome of other Dehalobacter strains indicating that a non-functional cbiH could be the possible cause of corrinoid auxotrophy in D. restrictus. Active regulation of corrinoid metabolism The expression of corrinoid biosynthetic genes in D. restrictus was verified experimentally to be regulated by cobalamin riboswitches. Analysis of corrinoid-starved D. restrictus cells revealed an increased transcription of the genes encoded directly downstream of the riboswitches. After corrinoid addition all operons were repressed, with most pronounced reduction for two operons, termed operon 1 and 2, encoding enzymes involved in corrinoid transport and salvaging only found in the genome of D. restrictus (73- and 346-fold, respectively). Overproduction of corrinoid-salvaging proteins (CbiZ) and transporters Proteomics of corrinoid-starved cells revealed on average a 45-fold over-production of operon 2 thereby reinforcing the trend from transcriptomics. Taken together, these data suggest that D. restrictus has lost its ability of de novo corrinoid synthesis and instead evolved a strategy for augmented corrinoid uptake and modification to fulfill its corrinoid requirement.

2014

The genome of Dehalobacter restrictus - high gene redundancy for a restricted metabolism

Christof Holliger, Julien Maillard, Aamani Rupakula Boyanapalli, Hauke Smidt

Organohalide respiration (OHR) is a bacterial anaerobic respiratory metabolism dedicated to use halogenated compounds as terminal electron acceptors. OHR bacteria are strictly anaerobic microorganisms which use reductive dehalogenases (RdhA) as key enzymes in this respiration process (1,2). Dehalobacter restrictus, an obligate OHR bacterium has been shown to use exclusively tetra- (PCE) and trichloroethene (TCE) as electron acceptors. The PCE RdhA (so called PceA) has been characterized previously (3). D. restrictus genome is currently being sequenced (in collaboration with JGI) revealing an unexpected high number of rdhA genes. Aim: The overall aim of this study is to characterize the functional diversity of rdhA genes in D. restrictus. Specific attention will be given to the use made by this bacterium of such redundant genetic information, and to the possible evolution mechanisms by which these numerous gene copies have emerged. Methods: The rdh gene clusters will be analyzed using bioinformatics comparing their diversity and gene composition to the well-characterized cpr (2) and pce (4) gene clusters in OHR isolates. Although D. restrictus is characterized by a restricted metabolism, different culture conditions will be tested either by replacing PCE with other organohalides, or by spiking organohalides in a culture growing on PCE. The level of transcription of the rdhA genes in D. restrictus will be measured by a targeted approach using optimized RNA extraction, RT, PCR and qPCR protocols. A special focus will also be given to the operon structure of the active pceABCT gene cluster of D. restrictus. Results: So far the analysis of the 2.9 Mb draft genome of D. restrictus revealed the presence of 20 different rdh gene clusters. While most of them are scattered in the available genome contigs, an array of 4 consecutive rdhAB operons is interspersed with regulatory genes suggesting a tight transcription regulation. When compared to the vast family of RdhA sequences in databases, D. restrictus RdhAs reflect the diversity observed in Firmicutes and are fairly distant to Dehalococcoides-type RdhAs. Preliminary transcription analysis of the 20 rdhA genes in standard growth conditions suggests that only the known pceA gene is significantly used in PCE/TCE dechlorination. Proteomic data will be obtained to confirm this view. More transcriptional analysis from cells subjected to other organohalides will be also presented. Conclusion: Based on genome analysis, D. restrictus seems to adopt an intermediate position between the facultative OHR bacteria such as Desulfitobacteria, and the obligate ones like Dehalococcoides members. The high rdhA gene redundancy appears as an evolutionary strategy to grow on many organohalides. This hypothesis still needs to be proven experimentally. References: (1) Holliger et al., Eds. Häggblom & Bossert, Kluwer Academic, 2003, p115. (2) Smidt & de Vos, Annu. Rev. Microbiol., 2004, 58:43. (3) Maillard et al., Appl. Environ. Microbiol., 2003, 69:4628. (4) Maillard et al., Environ. Microbiol., 2005, 7:107.

2012