GTPase-dependent signaling in bacteria: characterization of a membrane-binding site for era in Escherichia coli

Lin, Yi-Ping; Sharer, J. Daniel; March, Paul E.

doi:10.1128/jb.176.1.44-49.1994

Cited by 33 publications

(41 citation statements)

References 40 publications

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“…coli and S. mutans Era proteins appear to partition between the cytoplasm and the cytoplasmic membrane [15,22 -24]. The binding of Era to the membrane can be disrupted by salt treatment [15,24]. In this study, we show that a deletion of 45 residues, i.e.…”

Section: Discussionmentioning

confidence: 79%

Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C-terminal part of the Streptococcus pneumoniae Era GTPase

Hang¹,

Meier²,

Zhao³

2001

Eur J Biochem

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Era, an essential GTPase, plays a regulatory role in several cellular processes. The Era protein of Streptococcus pneumoniae has recently been shown to bind to 16S rRNA and the cytoplasmic membrane. However, exact locations of Era responsible for RNA-and membrane-binding were unknown. To identify the regions in Era that interact with the RNA and membrane, the C-terminal part of S. pneumoniae Era was systematically deleted while the N-terminal part, responsible for the GTPase activity of the protein, was kept intact. The resulting truncated Era proteins were purified and characterized. The C-terminal deletion of 9 or 19 amino-acid residues did not affect 16S rRNA-binding activity while further deletions of the C-terminus (29-114 amino-acid residues) abolished the activity. These results indicate that the integrity of the putative KH domain of Era, spanning the amino-acid residues between < 22-83 from the C-terminus, is required for 16S rRNA-binding. Furthermore, the Era proteins with a deletion up to 45 residues from the C-terminus retained membrane-binding activity, but longer deletions significantly reduced the activity. These results indicate that part of the putative KH domain is also required for membrane-binding. Thus, these results indicate for the first time that the regions critical for the membrane-and 16S rRNA-binding activities of Era overlap. The era gene with a deletion of 9 or 19 codons from its 3 0 terminus complemented an Escherishia coli mutant strain deficient in Era production whereas the genes with longer deletions failed to do so, thereby indicating that the KH domain is essential for Era function. Taken together, the results of this study indicate that the putative KH domain is required for 16S rRNA-binding activity and that part of the KH domain is also required for membrane-binding activity. The results also suggest that the interaction between Era and 16S rRNA is essential for bacterial growth.

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Section: Discussionmentioning

confidence: 79%

Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C-terminal part of the Streptococcus pneumoniae Era GTPase

Hang¹,

Meier²,

Zhao³

2001

Eur J Biochem

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“…Immunoelectron microscopic studies localized the E. coli Era protein on the internal side of the cytoplasmic membrane and also revealed that the Era protein exists in patches on the membrane (Gollop & March, 1991a). Later, biochemical studies confirmed that the E. coli Era protein is associated with the cytoplasmic membrane (Lin et al, 1994). This association was GTP-or GDPdependent and could be disrupted by salt treatment (Lin et al, 1994).…”

Section: Introductionmentioning

confidence: 82%

“…Later, biochemical studies confirmed that the E. coli Era protein is associated with the cytoplasmic membrane (Lin et al, 1994). This association was GTP-or GDPdependent and could be disrupted by salt treatment (Lin et al, 1994). These studies led to the suggestions that potential binding sites exist on the membrane for the Era protein (Lin et al, 1994).…”

Section: Introductionmentioning

confidence: 83%

“…This association was GTP-or GDPdependent and could be disrupted by salt treatment (Lin et al, 1994). These studies led to the suggestions that potential binding sites exist on the membrane for the Era protein (Lin et al, 1994). However, the significance of this membrane binding activity of Era and the part of Era that is responsible for this association remain unknown.…”

Section: Introductionmentioning

confidence: 99%

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Biochemical and molecular analyses of the C-terminal domain of Era GTPase from Streptococcus pneumoniae

Zhao¹,

Meier²,

Peery³

et al. 1999

Microbiology

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Era, an essential GTPase, is present in many bacteria and Mycoplasma spp. and appears to play a major role in the cell cycle and other cellular processes. To further understand its function, an era gene from Streptococcus pneumoniae was identified and cloned, and a mutant era gene with a deletion of 68 codons from its 3'4erminus was constructed. The truncated Era protein was then purified and characterized, and the ability of the truncated era gene to complement an Escherichia coli mutant strain defective in Era production was examined. Like the full-length Era protein, the truncated Era protein was able to bind and hydrolyse GTP, but its binding activity was increased twofold and its hydrolytic activity was reduced sevenfold when compared with those of the full-length Era protein. Unlike the full-length Era protein, the truncated Era protein lost its ability to bind to the E. coli cytoplasmic membrane. The fulllength era gene was able to complement the €. coli mutant deficient in Era production when carried on pACYCl84, while the truncated era gene failed to do so when carried on pACYCl84, pBR322 or pUC18. The cellular amounts of the truncated Era and the full-length Era proteins in €. coli and S. pneumoniae, respectively, were then determined by Western blot analysis. In addition, the minimal amount of the S. pneumoniae Era protein needed for complementation of the E. coli mutant was also measured. Taken together, these results suggest that the C-terminus of the Era protein might be responsible for the binding of the protein to the cytoplasmic membrane and be essential for function.

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“…This situation might be artifactual and due to the histidine (and acidic residue)-rich region or might result from modifications of the protein. Possible modifications include (i) metal ions bound to the histidine-rich region, (ii) nucleotides bound to the G domains (23), and (iii) unknown groups modifying the three cysteinyl residues at the amino terminus (see above). These possibilities are currently under investigation.…”

Section: (X)-t-v-(x)-g-(x)-g-t-s-a-i-g Correspond To the Sequence Exmentioning

confidence: 99%

Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

et al. 1994

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The products of the Rhizobium leguminosarum hyp gene cluster are necessary for synthesis of a functional uptake [NiFe] hydrogenase system in symbiosis with pea plants, and at least for HypB and HypF, a role in hydrogenase-specific nickel metabolism has been postulated (L. Rey, J. Murillo, Y. Hernando, E. Hidalgo, E. Cabrera, J. Imperial, and T. Ruiz-Argiieso, Mol. Microbiol. 8:471-481, 1993). The R. leguminosarum hypB gene product has been overexpressed in Escherichia coli and purified by immobilized nickel chelate affinity chromatography in a single step. The purified recombinant HypB protein was able to bind 3.9 + 0.1 Ni2+ ions per HypB monomer in solution. Co2+, Cu2+, and Zn2+ ions competed with Ni2+ with increasing efficiency.Monospecific HypB antibodies were raised and used to show that HypB is synthesized in R. eguminosarum microaerobic vegetative cells and pea bacteroids but not in R. leguminosarum aerobic cells. HypB protein synthesized by R. kguminosarum microaerobic vegetative cells could also be isolated by immobilized nickel chelate affinity chromatography. A histidine-rich region at the amino terminus of the protein (23-HGHHHH DGHHDHDHDHDHHRGDHEHDDHHH-54) is proposed to play a role in nickel binding, both in solution and in chelated form.Rhizobium leguminosarum bv. viciae possesses an H2 uptake system that is able to oxidize H2 generated by the nitrogenase complex as a byproduct of the N2 reduction reaction (8,40). This system consists of an uptake [NiFe] hydrogenase and accessory proteins, and it is only expressed in the plant symbiotic state. The main features of the system have been studied by our laboratory in the Pisum-pea bacteroid symbiosis. The genetic determinants for the H2 uptake system are clustered in a 15-kb DNA region (hup region) in the symbiotic plasmid (21,22). This region has been sequenced, and 17 potential genes have been identified. The first six genes constitute the hydrogenase structural operon and include the genes hupS and hupL, encoding the hydrogenase polypeptides (13), and four additional genes, hupCDEF (14). A five-gene cluster containing hupGHIJK has been identified downstream the hydrogenase structural operon (38 Purification of the HypB protein by Ni(II)-NTA-agarose chromatography. The Ni(II)-nitrilotriacetic acid (NTA)-agarose matrix was obtained from Diagen (Dusseldorf, Germany), and the manufacturer's recommendations for its use (12) were followed, with minor modifications as follows.(i) Denaturing conditions. Frozen cells from a 100-ml induced culture were lysed in the presence of 6 M guanidineHCl (3.5 ml), and cell extracts were applied to an Ni(II)-NTAagarose column (2 by 0.8 cm). Proteins were stepwise eluted by means of buffers of decreasing pH, 8.0, 6.3, 5.9, and 4.5, all of which contained 8 M urea, at a flow rate of 0.5 ml min-1. Fractions (1 ml) were collected, and portions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining (18).(ii) Nondenaturing conditions. All the manipulations were carri...

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GTPase-dependent signaling in bacteria: characterization of a membrane-binding site for era in Escherichia coli

Cited by 33 publications

References 40 publications

Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C-terminal part of the Streptococcus pneumoniae Era GTPase

Analysis of the interaction of 16S rRNA and cytoplasmic membrane with the C-terminal part of the Streptococcus pneumoniae Era GTPase

Biochemical and molecular analyses of the C-terminal domain of Era GTPase from Streptococcus pneumoniae

Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis

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