Quantitative Proteomic Analysis of Biological Processes and Responses of the Bacterium<i>Desulfovibrio desulfuricans</i>ND132 upon Deletion of Its Mercury Methylation Genes

Chen, Qian; Chen, Hongmei; Johs, Alexander; Xia, Lu; An, Jiachun; Pierce, Eric M.; Parks, Jerry M.; Elias, Dwayne A.; Hettich, Robert L.; Gu, Baohua

doi:10.1002/pmic.201700479

Cited by 27 publications

(30 citation statements)

References 52 publications

(107 reference statements)

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“…We speculate that the fraction of HgcA is equivalent to approximately 0.0004% of the total protein concentration in cell lysates (see supplemental material), which would correspond to approximately 1 in 220,000 proteins in the cell lysate. Such low abundance would explain difficulties in identifying HgcA protein fragments and low transcript levels in previously published studies (20,27,28). The calculated kinetic parameters resulting from this HgcA abundance estimate are k cat ϭ 3 ϫ 10 Ϫ3 s Ϫ1 , and k cat /K m ϭ 9 ϫ 10 5 M Ϫ1 · s Ϫ1 .…”

Section: Discussionmentioning

confidence: 92%

“…Reverse transcription-PCR (RT-PCR) data from ND132 (20) and proteomics studies in ND132 (27) and G. sulfurreducens PCA (28) indicated that the abundance of HgcA and HgcB in Hg methylators is extremely low. Owing to the low abundance and resultant difficulties in determining accurate concentrations of HgcA and HgcB in the cell lysates, we used the obtained kinetic parameters to estimate turnover numbers (k cat ), catalytic efficiencies (k cat /K m ), and free energies of activation based on transition state theory (29,30) for a range of enzyme concentrations expressed as a fraction of total cellular protein concentration (Table 1, Fig.…”

Section: Resultsmentioning

confidence: 99%

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Kinetics of Enzymatic Mercury Methylation at Nanomolar Concentrations Catalyzed by HgcAB

Date

Parks

Rush

et al. 2019

Appl Environ Microbiol

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Methylmercury (MeHg) is a potent bioaccumulative neurotoxin that is produced by certain anaerobic bacteria and archaea. Mercury (Hg) methylation has been linked to the gene pair hgcAB, which encodes a membrane-associated corrinoid protein and a ferredoxin. Although microbial Hg methylation has been characterized in vivo, the cellular biochemistry and the specific roles of the gene products HgcA and HgcB in Hg methylation are not well understood. Here, we report the kinetics of Hg methylation in cell lysates of Desulfovibrio desulfuricans ND132 at nanomolar Hg concentrations. The enzymatic Hg methylation mediated by HgcAB is highly oxygen sensitive, irreversible, and follows Michaelis-Menten kinetics, with an apparent K m of 3.2 nM and V max of 19.7 fmol · min Ϫ1 · mg Ϫ1 total protein for the substrate Hg(II). Although the abundance of HgcAB in the cell lysates is extremely low, Hg(II) was quantitatively converted to MeHg at subnanomolar substrate concentrations. Interestingly, increasing thiol/Hg(II) ratios did not impact Hg methylation rates, which suggests that HgcAB-mediated Hg methylation effectively competes with cellular thiols for Hg(II), consistent with the low apparent K m . Supplementation of 5-methyltetrahydrofolate or pyruvate did not enhance MeHg production, while both ATP and a nonhydrolyzable ATP analog decreased Hg methylation rates in cell lysates under the experimental conditions. These studies provide insights into the biomolecular processes associated with Hg methylation in anaerobic bacteria. IMPORTANCE The concentration of Hg in the biosphere has increased dramatically over the last century as a result of industrial activities. The microbial conversion of inorganic Hg to MeHg is a global public health concern due to bioaccumulation and biomagnification of MeHg in food webs. Exposure to neurotoxic MeHg through the consumption of fish represents a significant risk to human health and can result in neuropathies and developmental disorders. Anaerobic microbial communities in sediments and periphyton biofilms have been identified as sources of MeHg in aquatic systems, but the associated biomolecular mechanisms are not fully understood. In the present study, we investigate the biochemical mechanisms and kinetics of MeHg formation by HgcAB in sulfate-reducing bacteria. These findings advance our understanding of microbial MeHg production and may help inform strategies to limit the formation of MeHg in the environment.

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Section: Discussionmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Kinetics of Enzymatic Mercury Methylation at Nanomolar Concentrations Catalyzed by HgcAB

Date

Parks

Rush

et al. 2019

Appl Environ Microbiol

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“…We speculate that the fraction of HgcA is equivalent to approximately 0.0004% of the total protein concentration in cell lysates (see Supplementary Materials), which would correspond to approximately 1 in 220,000 proteins in the cell lysate. Such low abundance would explain difficulties in identifying HgcA protein fragments and low transcript levels in previously published studies (20,27,28). The calculated kinetic parameters resulting from this HgcA abundance estimate are k cat = 3·10 -3 s -1 , and kcat/KM = 9·10 5 M -1 · s -1 .…”

Section: Discussionmentioning

confidence: 89%

“…RT-PCR data from ND132 (20) and proteomics studies in ND132 (27) and G. sulfurreducens PCA (28) indicated that the abundance of HgcA and HgcB in Hg methylators is extremely low. Owing to the low abundance and resultant difficulties in determining accurate concentrations of HgcA and HgcB in the cell lysates, we used the obtained kinetic parameters to estimate turnover numbers (kcat), catalytic efficiencies (kcat/KM), and free energies of activation based on transition state theory (29,30) for a range of enzyme concentrations expressed as a fraction of total cellular protein concentration (Table 1, Fig.…”

Section: Enzyme Kinetics Of Hgcab-mediated Hg Methylationmentioning

confidence: 99%

Kinetics of enzymatic mercury methylation at nanomolar concentrations catalyzed by HgcAB

Date

Parks

Rush

et al. 2019

Preprint

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Methylmercury (MeHg) is a potent neurotoxin that bioaccumulates in fish. MeHg is generated by anaerobic bacteria and archaea possessing the gene pair hgcAB. Although bacterial mercury (Hg) methylation has been characterized in vivo, the specific role of HgcAB in catalyzing Hg methylation is not well understood. Here we report the kinetics of HgcAB-mediated Hg methylation in cell lysates of Desulfovibrio desulfuricans ND132 at nanomolar Hg concentrations. The enzymatic Hg methylation mediated by HgcAB is highly oxygensensitive, irreversible, and follows Michaelis-Menten kinetics with an apparent KM of 3.2 nM and Vmax of 19.7 fmol·min -1 ·mg -1 total protein for the substrate Hg(II). Although the abundance of HgcAB in the cell lysates is extremely low, Hg(II) was quantitatively converted to MeHg at subnanomolar substrate concentrations. Supplementation with ATP, methyltetrahydrofolate, or pyruvate did not enhance MeHg production under the experimental conditions. Insight into the kinetics of Hg methylation catalyzed by HgcAB advances our understanding of the complex global Hg cycle.

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“…In terms of coverage this is in with good keeping with other proteomic studies with metal trails in bacteria, including those of Desulfovibrio sp. (28, 29).…”

Section: Resultsmentioning

confidence: 99%

Shotgun proteomics analysis of nanoparticle-synthesisingDesulfovibrio alaskensisin response to platinum and palladium

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Imrie²,

Mühlbauer

et al. 2019

Preprint

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Platinum and palladium are much sought-after metals of global critical importance in terms of abundance and availability. At the nano-scale these metals are of even higher value due to their catalytic abilities for industrial applications. Desulfovibrio alaskensis is able to capture ionic forms of both of these metals, reduce them, and synthesize elemental nanoparticles. Despite this ability very little is known about the biological pathways involved in the formation of these nanoparticles. Proteomic analysis of D. alaskensis in response to platinum and palladium has highlighted those proteins involved in both the reductive pathways and the wider stress-response system. A core set of 13 proteins was found in both treatments and consisted of proteins involved in metal transport and reduction. There were also 7 proteins specific to either platinum or palladium. Over-expression of one of these platinum-specific genes, a NiFe hydrogenase small subunit (Dde_2137), resulted in the formation of larger nanoparticles. This study improves our understanding of the pathways involved in the metal resistance mechanism of Desulfovibrio and informs how we can tailor the bacterium for nanoparticle production, enhancing its application as a bioremediation tool and as way to capture contaminant metals from the environment.ImportanceBacteria, in particularly D. alaskensis, represent a biological and greener way to capture high value metals such as platinum group metals from environmental and industrial waste streams. The recovery of these metals in nanoparticle forms adds extra value to this process as they can be used in a variety of different industrial applications as they have exceptional catalytic capabilities. D. alaskensis ability to do this, has been widely reported, though very little is understood about the underlying protein and genetic components. It is by understanding the biological basis of this capability that we can further improve and adapt this bacterium to be better at bioremediation and to control its ability to do so.

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Quantitative Proteomic Analysis of Biological Processes and Responses of the BacteriumDesulfovibrio desulfuricansND132 upon Deletion of Its Mercury Methylation Genes

Cited by 27 publications

References 52 publications

Kinetics of Enzymatic Mercury Methylation at Nanomolar Concentrations Catalyzed by HgcAB

Kinetics of Enzymatic Mercury Methylation at Nanomolar Concentrations Catalyzed by HgcAB

Kinetics of enzymatic mercury methylation at nanomolar concentrations catalyzed by HgcAB

Shotgun proteomics analysis of nanoparticle-synthesisingDesulfovibrio alaskensisin response to platinum and palladium

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