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Biochemical characterization of three putative ATPases from a new type IV secretion system of Aeromonas veronii plasmid pAC3249A.

ABSTRACT: BACKGROUND: Type four secretion systems (TFSS) are bacterial macromolecular transport systems responsible for transfer of various substrates such as proteins, DNA or protein-DNA complexes. TFSSs encode two or three ATPases generating energy for the secretion process. These enzymes exhibit highest sequence conservation among type four secretion components. RESULTS: Here, we report the biochemical characterization of three ATPases namely TraE, TraJ and TraK (VirB4, VirB11 and VirD4 homologs of the Agrobacterium tumefaciens transfer system, respectively) from the transfer system of Aeromonas veronii plasmid pAC3249A. ATPases were expressed as His-tag fusion proteins in E. coli and purified by affinity chromatography. ATP binding and ATP hydrolysis experiments were performed with the purified ATPases. TraE and TraK showed strong binding to TNP-ATP and TNP-CTP (fluorescent analogs of ATP and CTP respectively) whereas TraJ showed weak binding. The optimum temperature range for the three ATPases was between 42 degrees C and 50 degrees C. Highest ATP hydrolysis activity for all the ATPases was observed in the presence of Mg2+ and Mn2+. However, TraJ and TraK also showed activity in the presence of Co2+. TraJ exhibited the highest specific activity of all the three ATPases with vmax 118 +/- 5.68 nmol/min/mg protein and KM 0.58 +/- 0.10 mM. CONCLUSIONS: This is the first biochemical characterization of conjugative transport ATPases encoded by a conjugative plasmid from Aeromonas. Our study demonstrated that the three ATPases of a newly reported TFSS of A. veronii plasmid pAc3249A are functional in both ATP hydrolysis and ATP binding.

SUBMITTER: Rangrez AY

PROVIDER: S-EPMC2831815 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Biochemical characterization of three putative ATPases from a new type IV secretion system of Aeromonas veronii plasmid pAC3249A.

Rangrez Ashraf Y AY Abajy Mohammad Y MY Keller Walter W Shouche Yogesh Y Grohmann Elisabeth E

BMC biochemistry 20100209

<h4>Background</h4>Type four secretion systems (TFSS) are bacterial macromolecular transport systems responsible for transfer of various substrates such as proteins, DNA or protein-DNA complexes. TFSSs encode two or three ATPases generating energy for the secretion process. These enzymes exhibit highest sequence conservation among type four secretion components.<h4>Results</h4>Here, we report the biochemical characterization of three ATPases namely TraE, TraJ and TraK (VirB4, VirB11 and VirD4 ho ...[more]

PMID: 20144229

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Project description:Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.

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Dataset Information

Biochemical characterization of three putative ATPases from a new type IV secretion system of Aeromonas veronii plasmid pAC3249A.

Publications

Biochemical characterization of three putative ATPases from a new type IV secretion system of Aeromonas veronii plasmid pAC3249A.

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