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Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity.

ABSTRACT: Streptococcus pneumoniae (pneumococcus) infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs) are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallographic and functional studies, we report that the catalytic activity of adenylate kinase from S . pneumoniae (SpAdK) serotype 2 D39 is essential for growth. We determined the crystal structure of SpAdK in two conformations: ligand-free open form and closed in complex with a two-substrate mimic inhibitor adenosine pentaphosphate (Ap5A). Crystallographic analysis of SpAdK reveals Arg-89 as a key active site residue. We generated a conditional expression mutant of pneumococcus in which the expression of the adk gene is tightly regulated by fucose. The expression level of adk correlates with growth rate. Expression of the wild-type adk gene in fucose-inducible strains rescued a growth defect, but expression of the Arg-89 mutation did not. SpAdK increased total cellular ATP levels. Furthermore, lack of functional SpAdK caused a growth defect in vivo. Taken together, our results demonstrate that SpAdK is essential for pneumococcal growth in vitro and in vivo.

SUBMITTER: Thach TT

PROVIDER: S-EPMC4141199 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity.

Thach Trung Thanh TT Luong Truc Thanh TT Lee Sangho S Rhee Dong-Kwon DK

FEBS open bio 20140708

Streptococcus pneumoniae (pneumococcus) infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs) are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallogr ...[more]

PMID: 25180151

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Project description:Streptococcus pneumoniae is an ovoid-shaped Gram-positive bacterium that grows by carrying out peripheral and septal peptidoglycan (PG) synthesis, analogous to model bacilli, such as Escherichia coli and Bacillus subtilis In the model bacilli, FtsZ and FtsA proteins assemble into a ring at midcell and are dedicated to septal PG synthesis but not peripheral PG synthesis; hence, inactivation of FtsZ or FtsA results in long filamentous cells unable to divide. Here, we demonstrate that FtsA and FtsZ colocalize at midcell in S. pneumoniae and that partial depletion of FtsA perturbs septum synthesis, resulting in elongated cells with multiple FtsZ rings that fail to complete septation. Unexpectedly, complete depletion of FtsA resulted in the delocalization of FtsZ rings and ultimately cell ballooning and lysis. In contrast, depletion or deletion of gpsB and sepF, which in B. subtilis are synthetically lethal with ftsA, resulted in enlarged and elongated cells with multiple FtsZ rings, with deletion of sepF mimicking partial depletion of FtsA. Notably, cell ballooning was not observed, consistent with later recruitment of these proteins to midcell after Z-ring assembly. The overproduction of FtsA stimulates septation and suppresses the cell division defects caused by the deletion of sepF and gpsB under some conditions, supporting the notion that FtsA shares overlapping functions with GpsB and SepF at later steps in the division process. Our results indicate that, in S. pneumoniae, both GpsB and SepF are involved in septal PG synthesis, whereas FtsA and FtsZ coordinate both peripheral and septal PG synthesis and are codependent for localization at midcell.IMPORTANCEStreptococcus pneumoniae (pneumococcus) is a clinically important human pathogen for which more therapies against unexploited essential targets, like cell growth and division proteins, are needed. Pneumococcus is an ovoid-shaped Gram-positive bacterium with cell growth and division properties that have important distinctions from those of rod-shaped bacteria. Gaining insights into these processes can thus provide valuable information to develop novel antimicrobials. Whereas rods use distinctly localized protein machines at different cellular locations to synthesize peripheral and septal peptidoglycans, we present evidence that S. pneumoniae organizes these two machines at a single location in the middle of dividing cells. Here, we focus on the properties of the actin-like protein FtsA as an essential orchestrator of peripheral and septal growth in this bacterium.

Project description:The VicRK two-component system of S. pneumoniae (Pneumococcus) is a member of the essential WalRK (YycFG) family found in low G+C gram positive bacteria. Previously, we showed that phosphorylated VicR response regulator (RR) is essential, because of its strong positive regulation of the pcsB gene, which mediates cell division. Little is known about the signals sensed by the VicK or WalK histidine kinases (HK), and it is unknown why pneumococcal VicK is not essential under aerobic growth conditions, whereas other the WalK HKs in other species are essential. VicK contains the only PAS domain in the Pneumococcal genome. PAS domains in other HKs have been shown to sense oxygen or other metabolic signals, such as NAD+, and it has been speculated that VicK may be involved in sensing oxygen in S. pneumoniae. Contrary to previous reports, we found that delta vicK mutants do not grow anaerobically, implying a conditional requirement for the VicK HK. Point mutations and domain deletions in vicK indicated that the autokinase activity of VicK is required for anaerobic growth, whereas the PAS domain and VicK phosphatase activity are not required. These combined results are consistent with the idea that maintenance of VicR~P level is required for anaerobic growth. The inability of delta vicK mutants to grow anaerobically was suppressed by spontaneous high- and low-growth yield mutants. Spontaneous suppressor mutants were still VicR dependent and did not result in bypass of the VicR signaling pathway. The genome of a high yield suppressor was sequenced using the Illumina method, which identified three mutations in the suppressor, two of which are predicted to play roles in phosphate transport (pnpR RR and phosphate ABC transporter). Manual sequencing of regions in additional independently isolated ΔvicK suppressor mutants identified similar mutations, and deletion of the pnpR RR gene in the ΔvicK background fully suppressed the anaerobic growth requirement for the VicK HK. Ongoing experiments will determine whether this suppression requires the PnpS HK or is mediated through changes in signaling. Finally, Western blotting showed that the level of VicK during anaerobic growth is about 3-fold less than that of aerobic cultures. This result suggests that the level of VicRKX operon expression is lower anaerobically, which may prevent crosstalk that occurs during aerobic culture to phosphorylate VicR.

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Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity.

Publications

Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity.

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