Structure of Desulfitobacterium hafniense PylSc, a pyrrolysyl-tRNA synthetase

Pyrrolysine, the 22nd genetically-encoded amino acid, is charged onto its specific tRNA by PylS, a pyrrolysyl-tRNA synthetase. While PylS is found as a single protein in certain archaeal methanogens, in the Gram-positive bacterium Desulfitobacterium hafniense, PylS is divided into two separate proteins, PylSn and PylSc, corresponding to the N-terminal and C-terminal domains of the single PylS protein found in methanogens. Previous crystallographic studies have provided the structure of a truncated C-terminal portion of the archaeal Methanosarcina mazei PylS associated with catalysis. Here, we report the apo 2.1 Å resolution structure of the intact D. hafniense PylSc protein and compare it to structures of the C-terminal truncated PylS from methanogenic species. In PylSc, the hydrophobic pocket binding the ring of pyrrolysine is more constrained than in the archaeal enzyme; other structural differences are also apparent.     

Desulfitobacterium hafniense is present in a high proportion within the biofilms of a high-performance pentachlorophenol-degrading, methanogenic fixed-film reactor

We developed a pentachlorophenol (PCP)-degrading, methanogenic fixed-film reactor by using broken granular sludge from an upflow anaerobic sludge blanket reactor. This methanogenic consortium was acclimated with increasing concentrations of PCP. After 225 days of acclimation, the reactor was performing at a high level, with a PCP removal rate of 1,173 muM day(-1), a PCP removal efficiency of up to 99%, a degradation efficiency of approximately 60%, and 3-chlorophenol as the main chlorophenol residual intermediate. Analyses by PCR-denaturing gradient gel electrophoresis (DGGE) showed that Bacteria and Archaea in the reactor stabilized in the biofilms after 56 days of operation. Important modifications in the profiles of Bacteria between the original granular sludge and the reactor occurred, as less than one-third of the sludge DGGE bands were still present in the reactor. Fluorescence in situ hybridization experiments with probes for Archaea or Bacteria revealed that the biofilms were composed mostly of Bacteria, which accounted for 70% of the cells. With PCR species-specific primers, the presence of the halorespiring bacterium Desulfitobacterium hafniense in the biofilm was detected very early during the reactor acclimation period. D. hafniense cells were scattered in the biofilm and accounted for 19% of the community. These results suggest that the presence of PCP-dehalogenating D. hafniense in the biofilm was crucial for the performance of the reactor.     

Recognition of pyrrolysine tRNA by the Desulfitobacterium hafniense pyrrolysyl-tRNA synthetase

Pyrrolysine (Pyl), the 22nd co-translationally inserted amino acid, is incorporated in response to a UAG amber stop codon. Pyrrolysyl-tRNA synthetase (PylRS) attaches Pyl to its cognate tRNA, the special amber suppressor tRNA(Pyl). The genes for tRNA(Pyl) (pylT) and PylRS (pylS) are found in all members of the archaeal family Methanosarcinaceae, and in Desulfitobacterium hafniense. The activation and aminoacylation properties of D. hafniense PylRS and the nature of the tRNA(Pyl) identity elements were determined by measuring the ability of 24 mutant tRNA(Pyl) species to be aminoacylated with the pyrrolysine analog N-epsilon-cyclopentyloxycarbonyl-l-lysine. The discriminator base G73 and the first base pair (G1.C72) in the acceptor stem were found to be major identity elements. Footprinting analysis showed that PylRS binds tRNA(Pyl) predominantly along the phosphate backbone of the T-loop, the D-stem and the acceptor stem. Significant contacts with the anticodon arm were not observed. The tRNA(Pyl) structure contains the highly conserved T-loop contact U54.A58 and position 57 is conserved as a purine, but the canonical T- to D-loop contact between positions 18 and 56 was not present. Unlike most tRNAs, the tRNA(Pyl) anticodon was shown not to be important for recognition by bacterial PylRS.     

Genome sequence of Desulfitobacterium hafniense DCB-2, a Gram-positive anaerobe capable of dehalogenation and metal reduction

Background

Enterococci are among the leading causes of hospital-acquired infections in the United States and Europe, with Enterococcus faecalis and Enterococcus faecium being the two most common species isolated from enterococcal infections. In the last decade, the proportion of enterococcal infections caused by E. faecium has steadily increased compared to other Enterococcus species. Although the underlying mechanism for the gradual replacement of E. faecalis by E. faecium in the hospital environment is not yet understood, many studies using genotyping and phylogenetic analysis have shown the emergence of a globally dispersed polyclonal subcluster of E. faecium strains in clinical environments. Systematic study of the molecular epidemiology and pathogenesis of E. faecium has been hindered by the lack of closed, complete E. faecium genomes that can be used as references.

Results

In this study, we report the complete genome sequence of the E. faecium strain TX16, also known as DO, which belongs to multilocus sequence type (ST) 18, and was the first E. faecium strain ever sequenced. Whole genome comparison of the TX16 genome with 21 E. faecium draft genomes confirmed that most clinical, outbreak, and hospital-associated (HA) strains (including STs 16, 17, 18, and 78), in addition to strains of non-hospital origin, group in the same clade (referred to as the HA clade) and are evolutionally considerably more closely related to each other by phylogenetic and gene content similarity analyses than to isolates in the community-associated (CA) clade with approximately a 3?C4% average nucleotide sequence difference between the two clades at the core genome level. Our study also revealed that many genomic loci in the TX16 genome are unique to the HA clade. 380 ORFs in TX16 are HA-clade specific and antibiotic resistance genes are enriched in HA-clade strains. Mobile elements such as IS16 and transposons were also found almost exclusively in HA strains, as previously reported.

Conclusions

Our findings along with other studies show that HA clonal lineages harbor specific genetic elements as well as sequence differences in the core genome which may confer selection advantages over the more heterogeneous CA E. faecium isolates. Which of these differences are important for the success of specific E. faecium lineages in the hospital environment remain(s) to be determined.     

Isolation and characterization of Tn-Dha1, a transposon containing the tetrachloroethene reductive dehalogenase of Desulfitobacterium hafniense strain TCE1

A new 9.9 kb catabolic transposon, Tn-Dha1, containing the gene responsible for tetrachloroethene (PCE) reductive dechlorination activity, was isolated from Desulfitobacterium hafniense strain TCE1. Two fully identical copies of the insertion sequence ISDha1, a new member of the IS256 family, surround the gene cluster pceABCT, a truncated gene for another transposase and a short open reading frame with homology to a member of the twin-arginine transport system (tatA). Evidence was obtained by Southern blot for an alternative form of the transposon element as a circular molecule containing only one copy of ISDha1. This latter structure most probably represents a dead-end product of the transposition of Tn-Dha1. Strong indications for the transposition activity of ISDha1 were given by polymerase chain reaction (PCR) amplification and sequencing of the intervening sequence located between both inverted repeats (IR) of ISDha1 (IR junction). A stable genomic ISDha1 tandem was excluded by quantitative real-time PCR. Promoter mapping of the pceA gene, encoding the reductive dehalogenase, revealed the presence of a strong promoter partially encoded in the right inverted repeat of ISDha1. A sequence comparison with pce gene clusters from Desulfitobacterium sp. strains PCE-S and Y51 and from Dehalobacter restrictus, all of which show 100% identity for the pceAB genes, indicated that both Desulfitobacterium strains seem to possess the same transposon structure, whereas only the pceABCT gene cluster is conserved in D. restrictus.     

Degradation of Chlorophenols Using Pentachlorophenol-Degrading Bacteria Sphingomonas chlorophenolica in a Batch Reactor

Chlorophenols are common environmental contaminants that have been used as the major component in wide-spectrum biocides in industry and agriculture. Many chlorophenols tend to persist in the environment and may become public health hazards. This research studied the ability of the pentachlorophenol (PCP)-degrading bacterium Sphingomonas chlorophenolica to degrade and dechlorinate other chlorophenols. In addition, the characteristics of S. chlorophenolica were also investigated. When S. chlorophenolica cells were preincubated with PCP, the lag phase PCP degradation periods became shorter and the PCP concentrations that could be removed became higher. S. chlorophenolica was able to completely degrade 2,3,6-trichlorophenol (2,3,6-TCP), 2,4,6-trichlorophenol (2,4,6-TCP), 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP), and PCP within 38.1, 15.1, 11.8, and 11.8 h, and to release concentrations of 50.1, 60.9, 63.7, and 58.5 mg/L chloride at the same period of time. In the presence of supplementary carbon sources, the PCP removal efficiency increased with the presence of glucose or pyruvate. However, the removal efficiency of 75 mg/L 2,4-dichlorophenol did not increase with supplemental carbon sources.     

Functional characterization of the trigger factor protein PceT of tetrachloroethene-dechlorinating Desulfitobacterium hafniense Y51

Desulfitobacterium hafniense strain Y51 dechlorinates tetrachloroethene to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by the action of the PceA reductive dehalogenase encoded by pceA. The pceA gene constitutes a gene cluster with pceB, pceC, and pceT. However, the gene components, except for pceA, still remained to be characterized. In the present study, we characterized the function of PceT. PceT of strain Y51 showed a sequence homology with trigger factor proteins, although it is evolutionally distant from the well-characterized trigger factor protein of Escherichia coli. The PceT protein tagged with 6x histidine was expressed as a soluble form in E. coli. The recombinant PceT fusion protein exhibited peptidyl-proryl cis–trans isomerase activity toward the chromogenic peptide N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The PceT fusion protein also exhibited chaperon activity towards the chemically denatured citrate synthase. Immunoprecipitation analysis using antibodies raised against PceA and PceT demonstrated that PceT specifically binds to the precursor form of PceA with an N-terminal twin-arginine translocation (TAT) signal sequence. On the other hand, PceT failed to bind the mature form of PceA that lost the TAT signal sequence. This is the first report in dehalorespiring bacteria, indicating that PceT is responsible for the correct folding of the precursor PceA.     

Cyanophycin synthetase-like enzymes of non-cyanobacterial eubacteria: characterization of the polymer produced by a recombinant synthetase of Desulfitobacterium hafniense

Some bacterial genomes were found to contain genes encoding putative proteins with considerable sequence homology to cyanophycin synthetase CphA of cyanobacteria. Such a gene from the Gram-positive, spore-forming anaerobe Desulfitobacterium hafniense was cloned. Expression in Escherichia coli resulted in the formation of a polydispers copolymer of aspartic acid and arginine, with a minor amount of lysine, of about 30 kDa molecular mass. In contrast to cyanophycin, this polymer was water-soluble. The structure of the polymer formed by the synthetase from Desulfitobacterium hafniense was studied by enzymatic degradation with the cyanophycin-specific hydrolase cyanophycinase, and by chemical and mass-spectroscopic analyses. Despite of the differences in solubility, indicating that both polymers cannot be completely identical, the chemical structure was found to be very similar to that of cyanophycin. The results suggest that the use of cyanophycin-like polymers as a nitrogen-rich reserve material is not restricted to cyanobacteria, and that such polymers may not necessarily be stored in granules.     

Impact of Vitamin B12 on Formation of the Tetrachloroethene Reductive Dehalogenase in Desulfitobacterium hafniense Strain Y51

Corrinoids are essential cofactors of reductive dehalogenases in anaerobic bacteria. Microorganisms mediating reductive dechlorination as part of their energy metabolism are either capable of de novo corrinoid biosynthesis (e.g., Desulfitobacterium spp.) or dependent on exogenous vitamin B₁₂ (e.g., Dehalococcoides spp.). In this study, the impact of exogenous vitamin B₁₂ (cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the subcellular localization of the reductive PCE dehalogenase was investigated in the Gram-positive Desulfitobacterium hafniense strain Y51, a bacterium able to synthesize corrinoids de novo. PCE-depleted cells grown for several subcultivation steps on fumarate as an alternative electron acceptor lost the tetrachloroethene-reductive dehalogenase (PceA) activity by the transposition of the pce gene cluster. In the absence of vitamin B₁₂, a gradual decrease of the PceA activity and protein amount was observed; after 5 subcultivation steps with 10% inoculum, more than 90% of the enzyme activity and of the PceA protein was lost. In the presence of vitamin B₁₂, a significant delay in the decrease of the PceA activity with an ∼90% loss after 20 subcultivation steps was observed. This corresponded to the decrease in the pceA gene level, indicating that exogenous vitamin B₁₂ hampered the transposition of the pce gene cluster. In the absence or presence of exogenous vitamin B₁₂, the intracellular corrinoid level decreased in fumarate-grown cells and the PceA precursor formed catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that exogenous vitamin B₁₂ is not incorporated into the PceA precursor, even though it affects the transposition of the pce gene cluster.     

Effects of 2-Bromoethanesulfonic Acid and 2- Chloroethanesulfonic Acid on Acetate Utilization in a Continuous-Flow Methanogenic Fixed-Film Column

2-Bromoethanesulfonic acid (BESA) and 2-chloroethanesulfonic acid (CESA) have been reported to be potent inhibitors of methane formation during methanogenic decomposition in batch cultures. However, in a laboratory-scale continuous-flow methanogenic fixed-film column containing a predominance of acetate-decarboxylating methanogens, BESA at 6 × 10⁻⁴ M produced only a 41% inhibition of acetate utilization, and CESA at 5.4 × 10⁻⁴ M produced a 37% inhibition of acetate utilization. BESA and CESA concentrations were not monitored in the effluent, so their fate is unknown. The organisms in the column were capable of degrading trace halogenated aliphatic compounds (~30 μg/liter) with acetate (100 mg/liter) as the primary substrate. Previous exposure of the cells to halogenated organic compounds may have conferred resistance to BESA and CESA. Degradation of the inhibitor compounds is another possible explanation for the observed effects.     

Effects of Chloromethanes on Growth of and Deletion of the pce Gene Cluster in Dehalorespiring Desulfitobacterium hafniense Strain Y51

The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 μM, but not carbon dichloride even at 1 mM.     

Resolution of culture Clostridium bifermentans DPH-1 into two populations, a Clostridium sp. and tetrachloroethene-dechlorinating Desulfitobacterium hafniense strain JH1

Clostridium bifermentans strain DPH-1 reportedly dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene. Cultivation-based approaches resolved the DPH-1 culture into two populations: a nondechlorinating Clostridium sp. and PCE-dechlorinating Desulfitobacterium hafniense strain JH1. Strain JH1 carries pceA, encoding a PCE reductive dehalogenase, and shares other characteristics with Desulfitobacterium hafniense strain Y51.     

Effects of chloromethanes on growth of and deletion of the pce gene cluster in dehalorespiring Desulfitobacterium hafniense strain Y51

The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 microM, but not carbon dichloride even at 1 mM.     

Biodegradation of Pentachlorophenol in a Continuous Anaerobic Reactor Augmented with Desulfitobacterium frappieri PCP-1

In this work, a strain of anaerobic pentachlorophenol (PCP) degrader, Desulfitobacterium frappieri PCP-1, was used to augment a mixed bacterial community of an anaerobic upflow sludge bed reactor degrading PCP. To estimate the efficiency of augmentation, the population of PCP-1 in the reactor was enumerated by a competitive PCR technique. The PCP-1 strain appeared to compete well with other microorganisms of the mixed bacterial community, with its population increasing from 10⁶ to 10¹⁰ cells/g of volatile suspended solids within a period of 70 days. Proliferation of strain PCP-1 allowed for a substantial increase of the volumetric PCP load from 5 to 80 mg/liter of reaction volume/day. A PCP removal efficiency of 99% and a dechlorination efficiency of not less than 90.5% were observed throughout the experiment, with 3-Cl-phenol and phenol being observable dechlorination intermediates.     

Degradation of Methanethiol by Methylotrophic Methanogenic Archaea in a Lab-Scale Upflow Anaerobic Sludge Blanket Reactor

In a lab-scale upflow anaerobic sludge blanket reactor inoculated with granular sludge from a full-scale wastewater treatment plant treating paper mill wastewater, methanethiol (MT) was degraded at 30°C to H₂S, CO₂, and CH₄. At a hydraulic retention time of 9 h, a maximum influent concentration of 6 mM MT was applied, corresponding to a volumetric loading rate of 16.5 mmol liter⁻¹ day⁻¹. The archaeal community within the reactor was characterized by anaerobic culturing and denaturing gradient gel electrophoresis analysis, cloning, and sequencing of 16S rRNA genes and quantitative PCR. Initially, MT-fermenting methanogenic archaea related to members of the genus Methanolobus were enriched in the reactor. Later, they were outcompeted by Methanomethylovorans hollandica, which was detected in aggregates but not inside the granules that originated from the inoculum, the microbial composition of which remained fairly unchanged. Possibly other species within the Methanosarcinacaea also contributed to the fermentation of MT, but they were not enriched by serial dilution in liquid media. The archaeal community within the granules, which was dominated by Methanobacterium beijingense, did not change substantially during the reactor operation. Some of the species related to Methanomethylovorans hollandica were enriched by serial dilutions, but their growth rates were very low. Interestingly, the enrichments could be sustained only in the presence of MT and did not utilize any of the other typical substrates for methylotrophic methanogens, such as methanol, methyl amine, or dimethylsulfide.