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, 184 (5), 1253-61

PduA Is a Shell Protein of Polyhedral Organelles Involved in Coenzyme B(12)-dependent Degradation of 1,2-propanediol in Salmonella Enterica Serovar Typhimurium LT2

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PduA Is a Shell Protein of Polyhedral Organelles Involved in Coenzyme B(12)-dependent Degradation of 1,2-propanediol in Salmonella Enterica Serovar Typhimurium LT2

Gregory D Havemann et al. J Bacteriol.

Abstract

Salmonella enterica forms polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. These organelles are thought to consist of a proteinaceous shell that encases coenzyme B(12)-dependent diol dehydratase and perhaps other enzymes involved in 1,2-propanediol degradation. The function of these organelles is unknown, and no detailed studies of their structure have been reported. Genes needed for organelle formation and for 1,2-propanediol degradation are located at the 1,2-propanediol utilization (pdu) locus, but the specific genes involved in organelle formation have not been identified. Here, we show that the pduA gene encodes a shell protein required for the formation of polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. A His(6)-PduA fusion protein was purified from a recombinant Escherichia coli strain and used for the preparation of polyclonal antibodies. The anti-PduA antibodies obtained were partially purified by a subtraction procedure and used to demonstrate that the PduA protein localized to the shell of the polyhedral organelles. In addition, electron microscopy studies established that strains with nonpolar pduA mutations were unable to form organelles. These results show that the pduA gene is essential for organelle formation and indicate that the PduA protein is a structural component of the shell of these organelles. Physiological studies of nonpolar pduA mutants were also conducted. Such mutants grew similarly to the wild-type strain at low concentrations of 1,2-propanediol but exhibited a period of interrupted growth in the presence of higher concentrations of this growth substrate. Growth tests also showed that a nonpolar pduA deletion mutant grew faster than the wild-type strain at low vitamin B(12) concentrations. These results suggest that the polyhedral organelles formed by S. enterica during growth on 1,2-propanediol are not involved in the concentration of 1,2-propanediol or coenzyme B(12), but are consistent with the hypothesis that these organelles moderate aldehyde production to minimize toxicity.

Figures

FIG. 1.
FIG. 1.
Overexpression and purification of the His6-PduA protein. Lane 1, molecular mass markers; lane 2, uninduced boiled cell lysate; lane 3, induced boiled cell lysate; lane 4, soluble fraction; lane 5, inclusion body preparation; lane 6, combined fractions 12 to 15 from Ni2+ column elution. Molecular masses in kilodaltons are shown at the left.
FIG. 2.
FIG. 2.
Western analysis with unsubtracted (A) and subtracted (B) anti-PduA polyclonal antibody preparations. For both panels: lane 1, S. enterica serovar Typhimurium LT2; lane 2, BE182 (ΔpduA mutant); lane 3, BE232 (isogenic to BE233, except that the expression plasmid lacks an insert); lane 4, BE233 (PduA expression strain). Molecular masses in kilodaltons are shown at the left of each blot. Total protein loaded in each lane was equivalent to that from cells at an OD600 of 0.02.
FIG. 3.
FIG. 3.
Localization of diol dehydratase and the PduA protein by immunoelectron microscopy. Cells were grown on succinate minimal medium supplemented with 1,2-propanediol to induce expression of the pdu operon. For each panel the arrows point to gold particles that indicate the location of either diol dehydratase or the PduA protein. The strain and protein labeled in each panel are as follows. (A) S. enterica, diol dehydratase. (B) BE182 (ΔpduA), diol dehydratase. (C) S. enterica, PduA. (D) BE182 (ΔpduA), PduA. Bars at lower right-hand corners are 100 nm in length.
FIG. 4.
FIG. 4.
Complementation of a ΔpduA mutation for formation of polyhedral organelles. Strain BE228 (ΔpduA/pTA749-PduA expression plasmid) was grown on minimal succinate medium supplemented with 1,2-propanediol and IPTG and then examined by transmission electron microscopy. Strain BE228 was grown with 0.01 (A) and 0.1 (B) mM IPTG, respectively. (A) The numbered arrows indicate the locations of the following structures: 1, polar inclusion body; 2, polyhedral organelles; 3, abnormally shaped organelles. (B) The arrows indicate the aberrant rod-like structures observed in some cells. Bars at the lower right-hand corners are 100 nm in length.
FIG. 5.
FIG. 5.
Growth of the wild-type strain and a pduA mutant on 1,2-propanediol-CN-B12 minimal medium. Results for the wild type, S. enterica serovar Typhimurium LT2 (▪), and BE182 (nonpolar ΔpduA) (•) are shown. Cells were cultured as described in Materials and Methods.
FIG. 6.
FIG. 6.
Effects of various 1,2-propanediol concentrations on the growth of strain BE182 (ΔpduA) and the wild-type strain. Cells were cultured on 1,2-propanediol-CN-B12 minimal media having the following 1,2-propanediol concentrations: 0.4% (▪, □), 0.2% (▴, ▵), 0.1% (•, ○), and 0.05% (⧫, ◊). Closed symbols, wild type, S. enterica serovar Typhimurium LT2. Open symbols, BE182 (ΔpduA).
FIG. 7.
FIG. 7.
Effects of various CN-B12 concentrations on the growth of strain BE182 (ΔpduA) and the wild-type strain (S. enterica). Cells were cultured on 1,2-propanediol minimal medium supplemented with CN-B12 at the concentrations indicated. ▪, wild-type; •, BE182 (ΔpduA). Error bars represent one standard deviation.

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