Biomedical subjects

D R Helinski

Publications and source records attributed to D R Helinski.

At least 37 records · Page 2Linked to original sources

PubMed1996

Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein.

The parDE operon, located within the 3.2-kb stabilization region of plasmid RK2, encodes antitoxin (ParD) and toxin (ParE) proteins that stabilize the maintenance of this broad-host-range plasmid via a postsegregational killing mechanism. A ParE protein derivative, designated ParE', was purified by construction of a fusion protein, GST-ParE, followed by glutathione-agarose binding and cleavage of the fusion protein. ParE' has three additional amino acids on the N terminus and a methionine residue in place of the native leucine residue. The results of glutathione-agarose affinity binding and glutaraldehyde cross-linking indicate that ParE' exists as a dimer in solution and that it binds to the dimeric form of ParD to form a tetrameric complex. The formation of this complex is presumably responsible for the ability of ParD to neutralize ParE toxin activity. Previous studies demonstrated that the parDE operon is autoregulated as a result of the binding of the ParD protein to the parDE promoter. ParE' also binds to the parDE promoter but only in the presence of the autoregulatory ParD protein. ParE', in the presence or absence of the ParD protein, does not bind to any other part of the 3.2-kb stabilization region. The binding of the ParE' protein to ParD did not alter the DNase I footprint pattern obtained as a result of ParD binding to the parDE promoter. The role of ParE in binding along with ParD to the promoter, if any, remains unclear.

Bacterial Proteins↗

PubMed1996

Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2.

A 3.2-kb fragment encoding five genes, parCBA/DE, in two divergently transcribed operons promotes stable maintenance of the replicon of the broad-host-range plasmid RK2 in a vector-independent manner in Escherichia coli. The parDE operon has been shown to contribute to stabilization through the postsegregational killing of plasmid-free daughter cells, while the parCBA operon encodes a resolvase, ParA, that mediates the resolution of plasmid multimers through site-specific recombination. To date, evidence indicates that multimer resolution alone does not play a significant role in RK2 stable maintenance by the parCBA operon in E. coli. It has been proposed, instead, that the parCBA region encodes an additional stability mechanism, a partition system, that ensures that each daughter cell receives a plasmid copy at cell division. However, studies carried out to date have not directly determined the plasmid stabilization activity of the parCBA operon alone. An assessment was made of the relative contributions of postsegregational killing (parDE) and the putative partitioning system (parCBA) to the stabilization of mini-RK2 replicons in E. coli. Mini-RK2 replicons carrying either the entire 3.2-kb (parCBA/DE) fragment or the 2.3-kb parCBA region alone were found to be stably maintained in two E. coli strains tested. The stabilization found is not due to resolution of multimers. The stabilizing effectiveness of parCBA was substantially reduced when the plasmid copy number was lowered, as in the case of E. coli cells carrying a temperature-sensitive mini-RK2 replicon grown at a nonpermissive temperature. The presence of the entire 3.2-kb region effectively stabilized the replicon, however, under both low- and high-copy-number-conditions. In those instances of decreased plasmid copy number, the postsegregational killing activity, encoded by parDE, either as part of the 3.2-kb fragment or alone played the major role in the stabilization of mini-RK2 replicons within the growing bacterial population. Our findings indicate that the parCBA operon functions to stabilize by a mechanism other than cell killing and resolution of plasmid multimers, while the parDE operon functions solely to stabilize plasmids by cell killing. The relative contribution of each system to stabilization depends on plasmid copy number and the particular E. coli host.

Bacterial Proteins↗

PubMed1995

The oxygen sensor protein, FixL, of Rhizobium meliloti. Role of histidine residues in heme binding, phosphorylation, and signal transduction.

The two-component system sensor/response regulator pair, FixL/FixJ, controls the expression of Rhizobium meliloti nitrogen fixation (nif and fix) genes in response to changes in oxygen concentration. A truncated version of FixL, FixL*, is an oxygen-binding hemoprotein kinase that phosphorylates and dephosphorylates the nif and fix gene transcriptional activator, FixJ. Phosphorylation of FixJ is required for optimal transcriptional activation, and anaerobic conditions in vitro result in a substantial increase in the level of FixJ-phosphate. In this study, site-directed mutagenesis was carried out at histidine residues in FixL*. Mutant FixL* derivatives were purified and analyzed in vitro for their heme/oxygen binding properties and phosphorylation/dephosphorylation activities. Mutation of histidine 285, the putative autophosphorylation site, to glutamine results in the loss of FixL* phosphorylation activities. However, this mutant protein retains a substantial level of FixJ-phosphate dephosphorylation activity. Mutation of histidine 194 to asparagine results in the loss of heme binding and in the failure of FixL* to regulate its phosphorylation/dephosphorylation activities in response to changes in oxygen concentration. The FixL*H194N mutant protein also exhibits an increased FixJ phosphorylation activity under aerobic conditions. This study provides further evidence for the importance of the heme binding domain of FixL* in regulating FixJ phosphorylation and dephosphorylation activities in response to oxygen.

Bacterial Proteins↗

PubMed1995

Involvement of the host initiator function dnaA in the replication of coliphage lambda.

We demonstrate that the initiation of coliphage lambda DNA replication is dependent on the host initiator function dnaA, provided that the lambdoid prophage Rac is absent. Presence of Rac compensated the absence of dnaA function, causing initiation of replication. In dnaAts rac+ cells at 43 degrees, most of parental phage DNA molecules, after one round of theta replication, switched to a replication with features of the sigma mode and produced progeny at high yield. Initiation of replication of the lambda Pts1 mutant at 43 degrees was blocked by dnaA function; however, under dnaA-rac+ conditions all parental phage DNA molecules, after one round of theta replication, switched to the sigma mode and produced progeny at high yield. Taking into account our recent finding that transcriptional activation of ori lambda seems to be dnaA-regulated (to be published elsewhere), we suggest that the DnaA-lambda Pts1 incompatibility occurs at the insertion of the ori lambda-bound lambda O-lambda P-DnaB preprimosome between the complementary lambda DNA strands. The role of Rac and the mechanism of the switch from theta to sigma mode of lambda phage DNA replication are discussed.

Bacterial Proteins↗

PubMed1995

Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2.

The par region of the broad-host-range, IncP alpha plasmid RK2 has been implicated as a stability determinant by its ability to enhance the maintenance of mini-RK2 plasmids or heterologous replicons in a growing population of host cells. The region consists of two operons: parCBA, which encodes a multimer resolution system, and parDE, which specifies a postsegregational response mechanism that is toxic to plasmidless segregants. To assess the importance of this region to the stable maintenance of the complete RK2 plasmid in different hosts, we used the vector-mediated excision (VEX) deletion system to specifically remove the entire par region or each operon separately from an otherwise intact RK2 plasmid carrying a lacZ marker. The par region was found to be important to stable maintenance of RK2lac (pRK2526) in Escherichia coli and five other gram-negative hosts (Agrobacterium tumefaciens, Azotobacter vinelandii, Acinetobacter calcoaceticus, Caulobacter crescentus, and Pseudomonas aeruginosa). However, the relative importance of the parCBA and parDE operons varied from host to host. Deletion of parDE had no effect on the maintenance of pRK2526 in A. calcoaceticus, but it severely reduced pRK2526 maintenance in A. vinelandii and resulted in significant instability in the other hosts. Deletion of parCBA did not alter pRK2526 stability in E. coli, A. tumefaciens, or A. vinelandii but severely reduced plasmid maintenance in A. calcoaceticus and P. aeruginosa. In the latter two hosts and C. crescentus, the delta parCBA mutant caused a notable reduction in growth rate in the absence of selection for the plasmid, indicating that instability resulting from the absence of parCBA may trigger the postsegregational response mediated by parDE. We also examined the effect of the conjugal transfer system on RK2 maintenance in E. coli. Transfer-defective traJ and traG mutants of pRK2526 were stably maintained in rapidly growing broth cultures. On solid medium, which should be optimal for IncP-mediated conjugation, colonies from cells containing the pRK2526 tra mutants displayed significant numbers of white (Lac-) sectors on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates, whereas sectors appeared rarely in colonies from tra+ plasmid-containing cells. Both the traJ and traG mutations further reduced the maintenance of the already unstable deltapar derivative. Thus, these experiments with defined mutations in an intact RK2 plasmid have revealed (i) that the par region allows RK2 to adapt to the different requirements for stable maintenance in various hosts and (ii) that conjugal transfer can contribute to the maintenance of RK2 in a growing population, particularly under conditions that are favorable to RK2 transfer.

Bacterial Proteins↗

PubMed1994

Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis.

The IncP alpha promiscuous plasmid (R18, R68, RK2, RP1 and RP4) comprises 60,099 bp of nucleotide sequence, encoding at least 74 genes. About 40 kb of the genome, designated the IncP core and including all essential replication and transfer functions, can be aligned with equivalent sequences in the IncP beta plasmid R751. The compiled IncP alpha sequence revealed several previously unidentified reading frames that are potential genes. IncP alpha plasmids carry genetic information very efficiently: the coding sequences of the genes are closely packed but rarely overlap, and occupy almost 86% of the genome's nucleotide sequence. All of the 74 genes should be expressed, although there is as yet experimental evidence for expression of only 60 of them. Six examples of tandem-in-frame initiation sites specifying two gene products each are known. Two overlapping gene arrangements occupy different reading frames of the same region. Intergenic regions include most of the 25 promoters; transcripts are usually polycistronic. Translation of most of the open reading frames seems to be initiated independently, each from its own ribosomal binding and initiation site, although, a few cases of coupled translation have been reported. The most frequently used initiation codon is AUG but translation for a few open reading frames begins at GUG or UUG. The most common stop-codon is UGA followed by UAA and then UAG. Regulatory circuits are complex and largely dependent on two components of the central control operon. KorA and KorB are transcriptional repressors controlling at least seven operons. KorA and KorB act synergistically in several cases by recognizing and binding to conserved nucleotide sequences. Twelve KorB binding sites were found around the IncP alpha sequence and these are conserved in R751 (IncP beta) with respect to both sequence and location. Replication of IncP alpha plasmids requires oriV and the plasmid-encoded initiator protein TrfA in combination with the host-encoded replication machinery. Conjugative plasmid transfer depends on two separate regions occupying about half of the genome. The primary segregational stability system designated Par/Mrs consists of a putative site-specific recombinase, a possible partitioning apparatus and a post-segregational lethality mechanism, all encoded in two divergent operons. Proteins related to the products of F sop and P1 par partitioning genes are separately encoded in the central control operon.

Amino Acid Sequence↗

PubMed1994

The parDE operon of the broad-host-range plasmid RK2 specifies growth inhibition associated with plasmid loss.

Recently, a 0.8 kb region of the broad-host-range plasmid RK2 has been shown to be sufficient to stabilize plasmids in a vector-independent, broad-host-range manner under some but not all growth conditions (Roberts, R. C. & Helinski, D. R. (1992). J. Bacteriol. 174, 8119-8132). This region encompasses the parDE operon, which encodes the small proteins ParD and ParE, both of which are required for the plasmid stabilization. This paper demonstrates that the 0.8 kb region encodes the capacity to inhibit cell growth of Escherichia coli, presumably of those bacteria that have lost plasmids carrying this stabilization region, and this inhibition appears to be associated with cell killing and bacterial cell filamentation. A good correlation was observed between the capacity of wild-type and mutated 0.8 kb regions to promote stable maintenance of a temperature-sensitive RK2 replicon plasmid and to inhibit bacterial cell division under specified medium conditions. The properties of the wild-type and mutant 0.8 kb regions further indicate that the ParE protein is responsible for the growth inhibition and the ParD protein neutralizes the toxic activity of the ParE protein. This is consistent with the finding that the presence of the parD gene in trans destabilizes a temperature-sensitive RK2 replicon carrying a copy of the functional 0.8 kb region. This destabilization appears to be the result of ParD protein-mediated suppression of growth inhibition, thus allowing survival of cells that have lost the temperature-sensitive plasmid. These observations indicate that the 0.8 kb sequence of RK2 encodes a growth inhibition function that is likely to play a role in the plasmid stabilization.

Bacterial Proteins↗

PubMed1994

Isolation and characterization of DNA-binding mutants of a plasmid replication initiation protein utilizing an in vivo binding assay.

An in vivo screen was developed for the identification of mutants of the RK2 replication initiation protein, TrfA, that were altered in their binding to the iterons within the plasmid RK2 origin of replication. This assay is based on an antibiotic selection system originally described by Elledge, Sugiono, Guarente, and Davis (Proc. Natl. Acad. Sci. USA86, 3689-3693, 1989) for the isolation in vivo of genes encoding sequence-specific DNA-binding proteins. A TrfA-specific binding site consisting of two 17-bp iterons separated by a nonrandom 6-bp spacer was placed 3' to a strong constitutive promoter. This promoter-iteron fragment was then inserted into the assay vector convergent to the aadA gene such that an increased level of spectinomycin resistance by the Escherichia coli host was dependent on the binding of wild-type TrfA protein to the binding site. The in vivo system was used to specifically isolate TrfA mutants which were either defective in binding or capable of effecting increased levels of spectinomycin resistance as compared to wild-type TrfA. The defective TrfA mutants isolated by this screen were purified and found to be considerably less effective in DNA binding by in vitro gel mobility shift assays. The map location was determined for these six defective TrfA mutants. Each of the mutations consisted of a single base change and mapped within codons extending over a 162 amino acid sequence. All of the mutants which were capable of effecting increased levels of spectinomycin resistance in the in vivo DNA-binding assay also showed some alteration in RK2 replication in vivo with most of the mutants having a copy-up phenotype similar to previously isolated TrfA mutants able to maintain an eight-iteron RK2 origin plasmid at a higher copy number.

Amino Acid Sequence↗

PubMed1993

Characteristics and significance of DNA binding activity of plasmid stabilization protein ParD from the broad host-range plasmid RK2.

A region of the plasmid RK2 has been shown to stabilize plasmid replicons in a broad host-range manner. This region encodes two divergently transcribed operons: parCBA and parDE. The parCBA operon specifies a multimer resolution system, while the parDE operon alone is capable of stabilizing an RK2-derived minireplicon under defined growth conditions in several different Gram-negative bacteria. The observed autoregulation of the parDE operon is most likely the result of ParD protein binding within the PparDE region. The characteristics of ParD binding to this region and the role of such binding in plasmid stabilization were examined with purified ParD protein. The results indicate that the binding of a single dimer of ParD protein to the promoter region most likely blocks interaction of RNA polymerase holoenzyme with the promoter. DNase I protection experiments indicate that ParD binds to a discrete sequence of 48 base pairs in length. While the binding of ParD to PparDE is essential for proper regulation of expression of the ParD and ParE proteins in vivo, the analyses of binding properties of mutant ParD proteins suggest that binding to this region does not play a direct role in plasmid stabilization.

Amino Acid Sequence↗

PubMed1993

Mutants of the two-component regulatory protein FixJ of Rhizobium meliloti that have increased activity at the nifA promoter.

FixL and FixJ belong to a two-component regulatory system in Rhizobium meliloti that induces the expression of numerous nitrogen-fixation genes during symbiosis with alfalfa. FixJ is a positive activator required for transcription of the regulatory genes nifA and fixK, while FixL is an oxygen-binding hemoprotein capable of regulating the phosphorylation status of both itself and FixJ, in response to oxygen availability. In this study, we isolated four FixJ mutants that display increased activity at the nifA promoter (PnifA) in Escherichia coli. All four mutants possess amino acid changes in a domain of FixJ that is conserved in other response regulator proteins, and all exhibit increased activity at PnifA in R. meliloti that is dependent on the presence of FixL. One of the mutant proteins, while less efficient at accepting phosphate from a truncated derivative of FixL (FixL*), nevertheless has a phosphorylated form that is more stable than the phosphorylated form of wild-type (wt) FixJ and is more resistant to the phosphatase activity of FixL*. The wt FixJ-phosphate was found to have a half-life of approximately 4 h, which makes it an unusually long-lived response regulator protein. The exceptional stability of wt FixJ-phosphate and the altered phosphorylation properties observed for the mutant are discussed in relation to signal transduction in the FixLJ system.

Bacterial Proteins↗

PubMed1993

Essentiality of the three carboxyl-terminal amino acids of the plasmid RK2 replication initiation protein TrfA for DNA binding and replication activity in gram-negative bacteria.

In a previous study of mutations in trfA, the gene encoding the replication initiation protein of the broad host-range plasmid RK2, a carboxyl-terminal deletion of 3 amino acids of the TrfA protein was found to be completely nonfunctional for RK2 replication in Escherichia coli and other Gram-negative bacteria. In this work site-directed mutagenesis of the trfA gene was carried out to construct TrfA proteins altered in the three carboxyl-terminal positions. Specifically, TrfA proteins with deletions or substitutions of the terminal cysteine, lysine, and arginine (codons 380-382, respectively) were constructed and characterized for their ability to initiate replication from an RK2 origin in vivo in E. coli, Azotobacter vinelandii, Pseudomonas putida, and Agrobacterium tumefaciens and for binding activity to the iterons at the replication origin. Substitutions of the cysteine at position 380 with a glycine or an arginine resulted in a TrfA protein defective in binding to the RK2 origin and, therefore, defective in replication initiation activity in all four Gram-negative bacteria. Substitution of a serine at that position preserved limited function in replication and DNA binding. The lysine at position 381 could be changed to a glutamine without any obvious change in TrfA function. Deletion of the terminal arginine at position 382 did not affect the ability of TrfA to bind to origin iterons but caused a complete loss of replication activity in all four bacteria. Substitution of this terminal arginine with alanine, serine, or glutamic acid also produced replication-defective TrfA protein in all four bacterial hosts while not affecting iteron binding activity. However, substitution of this arginine with a lysine resulted in a loss of replication activity in E. coli and A. vinelandii but had no effect in P. putida and A. tumefaciens. These observations suggest that the terminal arginine plays an essential role in the activity of the TrfA protein, possibly interaction with host proteins, which can be separated from its iteron binding activity.

Amino Acids↗

PubMed1993

Mutations in the gene encoding the replication-initiation protein of plasmid RK2 produce elevated copy numbers of RK2 derivatives in Escherichia coli and distantly related bacteria.

Mini-replicons of the broad-host-range plasmid RK2 with increased copy number (cn) due to mutations in the gene encoding the essential replication initiation protein TrfA are described. The cn of these derivatives have been determined in Escherichia coli, Pseudomonas aeruginosa and Agrobacterium tumefaciens and were found to be elevated in all three bacterial hosts. One of the cn mutations was introduced into the intact 60-kb RK2 plasmid by homologous recombination in vivo, resulting in an approximately twofold cn increase. The expression of trfA from this mutant RK2 plasmid did not respond to the cn change as predicted by a simple transcription rate-limitation, replication control model. Implications for the model of RK2 replication control and the potential use of mutant RK2 mini-replicons as high-copy broad-host-range gene cloning vectors are discussed.

Bacterial Proteins↗

PubMed1993

Oxygen regulation of nifA transcription in vitro.

In Rhizobium meliloti, transcription of the key nitrogen-fixation regulatory genes nifA and fixK is induced in response to microaerobiosis through the action of the FixL and FixJ proteins. These two proteins are sensor and regulator homologues, respectively, of a large family of bacterial two-component systems involved in sensing and responding to environmental changes. A soluble, truncated form of the membrane protein FixL, FixL*, has been shown to be a hemoprotein that phosphorylates and dephosphorylates FixJ in response to oxygen tension. Here we use an in vitro transcription system to prove that FixJ is a transcriptional activator of both nifA and fixK and that phosphorylation of FixJ markedly increases its activity. Phosphorylation was achieved either by preincubating FixJ with FixL* and ATP or by exposing FixJ to the inorganic phospho donor ammonium hydrogen phosphoramidate. Both FixJ and FixJ-phosphate formed heparin-resistant complexes under the assay conditions used. Lastly, we were able to show that anaerobiosis, in the presence of FixL* and ATP, greatly stimulates FixJ activity at the nifA promoter with either Escherichia coli or R. meliloti RNA polymerase. This use of atmospheric oxygen to control nifA transcription in vitro represents a reconstitution of a bacterial two-component signal transduction system in its entirety, from effector to ultimate target, by the use of purified components.

Adenosine Triphosphate↗

PubMed1993

Autophosphorylation and phosphatase activities of the oxygen-sensing protein FixL of Rhizobium meliloti are coordinately regulated by oxygen.

The FixL and FixJ proteins of Rhizobium meliloti control the expression of other nif and fix genes in response to oxygen levels. FixL is a hemoprotein kinase that senses oxygen availability and responds to the absence of oxygen by activation of its autophosphorylating activity followed by transfer of the phosphate to FixJ. FixJ in turn activates the nifA and fixK promoters. In vitro studies reported here with a soluble truncated version of FixL (FixL*) indicate that, while low oxygen tension specifically increases the autophosphorylating activity of FixL*, the ability of phospho-FixL* to act as a phosphate donor to FixJ is not affected by the presence or absence of oxygen. FixL* is also shown to possess a phosphatase activity that is repressed under anaerobic conditions only when the protein is in the phosphorylated form. A fixL mutant that induces a higher level of nifA promoter activity in the presence of fixJ in vivo displayed both an increased autophosphorylating activity and a decreased phosphatase activity in vitro. These data provide evidence for a role for both autophosphorylation and phosphatase activities of FixL in the mechanism by which oxygen tension within the alfalfa nodule induces expression of bacterial nitrogen fixation genes during symbiosis.

Adenosine Triphosphate↗

PubMed1993

DNA sequence requirements for interaction of the RK2 replication initiation protein with plasmid origin repeats.

Replication of plasmid RK2 in a variety of Gram-negative bacteria requires its origin of replication and the plasmid-encoded TrfA proteins (TrfA-33 and TrfA-44). The initiation of replication requires that the TrfA proteins bind to a series of 17-base pair (bp) direct repeats located within the RK2 origin. The conserved 17-bp repeats are arranged in tandem and are separated by less conserved spacer sequences of 4-6 bp in length. A series of plasmids containing one or two iterons, with or without the less conserved spacer sequences, were constructed to analyze the DNA sequence requirements for binding of TrfA-33 to the iterons. In addition to the analysis of TrfA binding in vitro, the plasmid constructs were examined for their ability to exert incompatibility toward an RK2 replicon in Escherichia coli. These analyses revealed that the conserved 17-bp iteron sequence itself is not sufficient for TrfA binding; the adjacent less conserved spacer sequences are also required. Site-specific mutagenesis was carried out to determine the importance of specific bases within the spacer sequence for binding activity and a consensus sequence for a TrfA-specific binding unit was determined. DNase I and methylation interference footprinting procedures were also carried out to characterize the TrfA-binding unit complex. Finally, it was shown that the binding of the TrfA-33 protein to two adjacent TrfA binding units on a DNA fragment is not substantially affected by the relative orientation or spacing between the two units.

Bacterial Proteins↗

PubMed1993

The oxygen sensor FixL of Rhizobium meliloti is a membrane protein containing four possible transmembrane segments.

Regulation of nitrogen fixation genes in Rhizobium meliloti is mediated by two proteins, FixL and FixJ, in response to oxygen availability. FixL is an oxygen-binding hemoprotein with kinase and phosphatase activities that is thought to sense oxygen levels directly and to transmit this signal to FixJ via phosphorylation-dephosphorylation reactions. FixJ controls the expression of other regulatory genes, including nifA, that regulate the transcription of genes required for symbiotic nitrogen fixation. We have been studying the structural and functional features of FixL that are required for oxygen sensing. We constructed mutant derivatives and confirmed that FixL consists of 505 amino acids instead of 464, as originally reported. Hydropathy plots of the full-length protein, together with TnphoA insertional analysis, lead us to propose that FixL is likely to be a polytopic integral membrane protein containing four membrane-spanning segments. We have also constructed an N-terminal deletion of the FixL protein whose in vivo activity indicates that the hydrophobic membrane-spanning regions are not absolutely required for oxygen sensing in vivo. We also report that FixL shares homology in its N terminus with other sensor proteins, including KinA from Bacillus subtilis and NtrB from Bradyrhizobium parasponia. The region of homology comprises a 70-amino-acid residue stretch that is also conserved in two oxygenases, P-450 and isopenicillin synthase.

Amino Acid Sequence↗

PubMed1992

The FixL protein of Rhizobium meliloti can be separated into a heme-binding oxygen-sensing domain and a functional C-terminal kinase domain.

Transcription of nitrogen fixation (nif and fix) genes in Rhizobium meliloti is induced by a decrease in oxygen concentration. The products of two genes, fixL and fixJ, are responsible for sensing and transmitting the low-oxygen signal. The proteins encoded by fixL and fixJ (FixL and FixJ, respectively) are homologous to a family of bacterial proteins that transduce environmental signals through a common phosphotransfer mechanism [David, M., Daveran, M., Batut, J., Dedieu, A., Domergue, O., Ghai, J., Hertig, C., Boistard, P. & Khan, D. (1988) Cell 54, 671-683]. FixL, the oxygen sensor, is a membrane protein. It has previously been shown that a soluble derivative of FixL, FixL*, is an oxygen-binding hemoprotein and a kinase that autophosphorylates and also phosphorylates FixJ [Gilles-Gonzalez, M. A., Ditta, G. S. & Helinski, D. R. (1991) Nature (London) 350, 170-172]. In this work, deletion derivatives of fixL* were constructed and overexpressed in Escherichia coli, and the truncated proteins were purified. We show that a fragment of FixL from amino acid residue 127 to residue 260 binds heme, retains the ability to bind oxygen, and has no detectable kinase activity. A C-terminal fragment of FixL, beginning at residue 260, fails to bind heme but is active as a kinase. We also demonstrate that anaerobiosis results in an enhancement of FixL* autophosphorylation and FixJ phosphorylation activities in vitro. Finally, we show that the heme-binding region of FixL is required in vitro for oxygen regulation of its kinase activities.

Bacterial Proteins↗

PubMed1992

Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

Base Sequence↗