Genetic Dissection of Specificity Determinants in the Interaction of HPr with Enzymes II of the Bacterial Phosphoenolpyruvate:Sugar Phosphotransferase System in<i>Escherichia coli</i>

Reichenbach, Birte; Breustedt, Daniel A.; Stülke, Jörg; Rak, Bodo; Görke, Boris

doi:10.1128/jb.00236-07

Cited by 12 publications

(11 citation statements)

References 47 publications

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“…However, this result is consistent with reports for other bacterial ptsH mutants (40). One explanation for this observation is that A. actinomycetemcomitans possesses HPr-like proteins, such as diphosphoryltransfer protein, that can partially substitute for HPr when grown in certain media (44). In addition, the bacteria may be utilizing other carbon sources in the complex medium.…”

Section: Discussionsupporting

confidence: 81%

Screen for Leukotoxin Mutants in Aggregatibacter actinomycetemcomitans : Genes of the Phosphotransferase System Are Required for Leukotoxin Biosynthesis

et al. 2008

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Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans is a pathogen that causes localized aggressive periodontitis and extraoral infections including infective endocarditis. Recently, we reported that A. actinomycetemcomitans is beta-hemolytic on certain growth media due to the production of leukotoxin (LtxA). Based on this observation and our ability to generate random transposon insertions in A. actinomycetemcomitans, we developed and carried out a rapid screen for LtxA mutants. Using PCR, we mapped several of the mutations to genes that are known or predicted to be required for LtxA production, including ltxA, ltxB, ltxD, and tdeA. In addition, we identified an insertion in a gene previously not recognized to be involved in LtxA biosynthesis, ptsH. ptsH encodes the protein HPr, a phosphocarrier protein that is part of the sugar phosphotransferase system. HPr results in the phosphorylation of other proteins and ultimately in the activation of adenylate cyclase and cyclic AMP (cAMP) production. The ptsH mutant showed only partial hemolysis on blood agar and did not produce LtxA. The phenotype was complemented by supplying wild-type ptsH in trans, and real-time PCR analysis showed that the ptsH mutant produced approximately 10-fold less ltxA mRNA than the wild-type strain. The levels of cAMP in the ptsH mutant were significantly lower than in the wild-type strain, and LtxA production could be restored by adding exogenous cAMP to the culture.

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Section: Discussionsupporting

confidence: 81%

Screen for Leukotoxin Mutants in Aggregatibacter actinomycetemcomitans : Genes of the Phosphotransferase System Are Required for Leukotoxin Biosynthesis

et al. 2008

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“…1A). Without IPTG, only background levels of enzyme activity were obtained, indicating the known, weak leakiness of the terminator (22,52). However, when licT was expressed to the same extent as bglG (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The strains also carried a deletion of the bgl operon to avoid interference with the naturally encoded BglG protein and its negative regulator, BglF, which is a homolog of B. subtilis BglP. To assess antitermination activity, a reporter plasmid was used that carries the bgl terminator t 2 between a constitutive promoter (P 16 ) and the lacZ reporter gene, as has been described previously (22,23,52) (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…For a better understanding of these interactions, we started to swap PTS components of the Gram-negative organism E. coli with their homologous counterparts from the Grampositive bacterium B. subtilis. Application of this approach to HPr revealed a signature motif in its interacting surface that determines the specificity of HPr for either Gram-negative or -positive EIIs (52). In the present study, we extended this approach to the antitermination proteins and transplanted the B. subtilis licT gene to E. coli.…”

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Insight into Bacterial Phosphotransferase System-Mediated Signaling by Interspecies Transplantation of a Transcriptional Regulator

et al. 2011

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The bacterial sugar:phosphotransferase system (PTS) delivers phosphoryl groups via proteins EI and HPr to the EII sugar transporters. The antitermination protein LicT controls ␤-glucoside utilization in Bacillus subtilis and belongs to a family of bacterial transcriptional regulators that are antagonistically controlled by PTS-catalyzed phosphorylations at two homologous PTS regulation domains (PRDs). LicT is inhibited by phosphorylation of PRD1, which is mediated by the ␤-glucoside transporter EII Bgl . Phosphorylation of PRD2 is catalyzed by HPr and stimulates LicT activity. Here, we report that LicT, when artificially expressed in the nonrelated bacterium Escherichia coli, is likewise phosphorylated at both PRDs, but the phosphoryl group donors differ. Surprisingly, E. coli HPr phosphorylates PRD1 rather than PRD2, while the stimulatory phosphorylation of PRD2 is carried out by the HPr homolog NPr. This demonstrates that subtle differences in the interaction surface of HPr can switch its affinities toward the PRDs. NPr transfers phosphoryl groups from EI Ntr to EIIA Ntr . Together these proteins form the paralogous PTS Ntr , which controls the activity of K ؉ transporters in response to unknown signals. This is achieved by binding of dephosphorylated EIIA Ntr to other proteins. We generated LicT mutants that were controlled either negatively by HPr or positively by NPr and were suitable bio-bricks, in order to monitor or to couple gene expression to the phosphorylation states of these two proteins. With the aid of these tools, we identified the stringent starvation protein SspA as a regulator of EIIA Ntr phosphorylation, indicating that PTS Ntr represents a stress-related system in E. coli.

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“…The EIIs are grouped into seven families, which are not related to each other and have evolved from different sources [6]. A signature motif important for carrying out all these interactions has recently been identified for E. coli HPr [8]. Nevertheless, HPr uses the same interaction surface to interact with a large number of unrelated proteins in its host.…”

Section: Unique Features Of the Bacterial Ptsmentioning

confidence: 99%

Regulation of Carbohydrate Utilization by Phosphotransferase System‐Mediated Protein Phosphorylation

Görke¹,

Reichenbach

2009

Bacterial Signaling

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Genetic Dissection of Specificity Determinants in the Interaction of HPr with Enzymes II of the Bacterial Phosphoenolpyruvate:Sugar Phosphotransferase System inEscherichia coli

Cited by 12 publications

References 47 publications

Screen for Leukotoxin Mutants in Aggregatibacter actinomycetemcomitans : Genes of the Phosphotransferase System Are Required for Leukotoxin Biosynthesis

Screen for Leukotoxin Mutants in Aggregatibacter actinomycetemcomitans : Genes of the Phosphotransferase System Are Required for Leukotoxin Biosynthesis

Insight into Bacterial Phosphotransferase System-Mediated Signaling by Interspecies Transplantation of a Transcriptional Regulator

Regulation of Carbohydrate Utilization by Phosphotransferase System‐Mediated Protein Phosphorylation

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