Transposon Tn5 Insertion Research Articles

The Vi antigen of Salmonella typhi: molecular analysis of the viaB locus.

Strains of Salmonella typhi isolated from the blood of patients with typhoid fever invariably express a capsular polysaccharide, termed the Vi antigen. Vi antigen expression is controlled by two separate chromosomal loci, viaA and viaB. The viaA locus is commonly found in enteric bacteria. In contrast, the viaB locus appears to be specific to Vi-expressing strains of Salmonella and Citrobacter. Here the cloning, expression and analysis of viaB determinants from S. typhi Ty2 is described. Whole-cell DNA from strain Ty2 was size-fractionated and cloned into the pLA2917 cosmid vector. A recombinant cosmid, pVT1, conferring a Vi-positive phenotype upon Escherichia coli and upon the Vi-non-expressing strain Ty21a of S. typhi, was characterized and used for further studies. Transposon Tn5 insertion mutagenesis demonstrated that the Vi-antigen-encoding region on pVT1 consisted of a 15 kb fragment. A subclone, designated pVT3, which contained an 18 kb insert, was sufficient to confer Vi antigen expression upon E. coli and S. typhi Ty21a. Results of recombination experiments indicated that this DNA sequence was the viaB locus of S. typhi Ty2. In E. coli SE5000 maxicells, the viaB determinants encoded at least eight polypeptides, with molecular masses of 80, 65, 59, 48, 44, 39, 35 and 28 kDa. Functional characterization of viaB mutations in S. typhi Ty2 suggested that the 80 and 65 kDa proteins were required for cell-surface localization of the Vi antigen.

Transposon Tn5 as an identifiable marker in rhizobia: Survival and genetic stability of Tn5 mutant bean rhizobia under temperature stressed conditions in desert soils

Five transposon Tn5 insertion mutants of a beanRhizobium strain (Rhizobium leguminosarum b. v.phaseoli) were used in an ecological study to evaluate the extent to which transposon Tn5 was stable to serve as an identifiable marker in rhizobia under a high temperature stress condition in two Sonoran Desert soils. All the mutants possessed single chromosomal insertions of the transposon. In both soils, under the temperature stress conditions that were employed (40°C), both wild type and mutant populations possessing functional transposable elements declined rapidly. After 12 days, mutant cells, when screened using the Tn5 coded antibiotic resistance markers, were significantly less in number than when they were screened using only their intrinsic antibiotic resistance markers. There were no significant differences in numbers between the mutant cell population and the wild type when the mutant cells were screened using only the intrinsic antibiotic resistance markers. DNA-DNA hybridizations using a probe indicated neither deletion nor transposition of the transposable element. The results indicate that transposon DNA sequences are present within cells under high temperature stress conditions, but kanamycin/neomycin resistance is not expressed by some of these cells, suggesting that Tn5 undergoes a possible functional inactivation under these conditions. The possible implications of these findings are discussed.

MarA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli

Stable chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, derived by exposing susceptible cells to low concentrations of tetracycline or chloramphenicol, express cross-resistance to structurally unrelated antibiotics. The entire resistance phenotype is reversed to susceptibility by insertion of transposon Tn5 into a locus, designated marA, near 34 min on the chromosome (A. M. George and S. B. Levy, J. Bacteriol. 155:541-548, 1983). Strains in which 39 kbp of chromosomal DNA, including marA, had been deleted were unable to produce Mar mutants. The deletion strain could be complemented in trans by introduction of intact marA+ on plasmid F'506. Junction fragments from a strain containing marA::Tn5 were cloned, exploiting kanamycin resistance on Tn5 for selection. They were used as probes to search a phasmid library of E. coli K-12 for recombinants containing the marA+ region. Two phasmids which contained regions hybridizing to this probe were identified and shown to complement delta marA in a deletion strain. From one phasmid, several marA-containing fragments were cloned: those of greater than or equal to 7.8 kbp restored the ability to form Mar mutants in a deletion strain. These Mar mutants were shown to be dependent on the cloned marA fragment. Chromosomal as well as recombinant Mar mutants showed increased expression of a marA-specific mRNA species of about 1.4 kb, which was barely or not detectable in wild-type strains. Exposure of mutants and, to a lesser extent, parental strains to tetracycline or chloramphenicol resulted in elevated levels of mRNA which hybridized to the marA probe. These results indicate that the marA locus is needed for production of Mar mutants and is regulated, responding to at least two antibiotics to which it controls resistance.

Survival of genetically engineeredPseudomonas fluorescensin soil in competition with the parent strain

The population dynamics of two genetically engineered Pseudomonas fluorescens strains, D5 and C5t, introduced into a loamy sand soil, in competition with a spontaneous antibiotic-resistant mutant of the corresponding wildtype strain was studied. Strain D5 contained an insertion of transposon Tn5 in its genome, whereas strain C5t was obtained by insertion of Tn5::tox, a Tn5-derivative containing a Bacillus thuringiensis var. morrisoniδ-endotoxin gene, into the chromosome using a suicide vector system. Southern hybridization analysis demonstrated the absence of vector sequences, and the presence of single copies of either Tn5 or Tn5::tox in the respective strains. Western blotting and a bio-assay on larvae of Anopheles stephensi suggested the tox gene was functional in clone C5t. Both D5 and C5t were prototrophic and their generation times in minimal medium were slightly below that of the corresponding wild-type strain. Tn5 and Tn5::tox were stable in both clones during growth in minimal medium for 16 generations. During growth in competition with the wild-type strain, D5 competed well, however C5t was outcompeted from 50 to below 3% of the population in 40 generations. During growth in competition in the sterile loamy sand, both strains were outcompeted by the parent strain; strain C5t was less competitive than D5. In non-sterile loamy sand, the introduced mixed populations showed a slow decline; both C5t and D5 were outcompeted by the parent strain. The decreased fitness of both modified strains, although significant, was considered to be small in ecological terms. Further, the addition of 10% bentonite clay to the loamy sand resulted in a significant enhancement of survival of the mixed populations, and a stabilization of the proportions between the modified strains and the parent. Finally, there was a trend towards a decrease in the proportion modified strain/parent strain in both mixes in the rhizosphere of wheat.

Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification

The structural gene, nirS, for the respiratory nitrite reductase (cytochrome cd1) from Pseudomonas stutzeri was identified by (i) sequencing of the N-terminus of the purified protein and partial sequencing of the cloned gene, (ii) immunoscreening of clones from a lambda gt11 expression library, (iii) mapping of the transposon Tn5 insertion site in the nirS mutant strain MK202, and (iv) complementation of strain MK202 with a plasmid carrying the insert from an immunopositive lambda clone. A mutation causing overproduction of cytochrome c552 mapped on the same 8.6 kb EcoRI fragment within 1.7 kb of the mutation affecting nirS. Two mutations affecting nirD, which cause the synthesis of an inactive cytochrome cd1 lacking heme d1, mapped 1.1 kb apart within a 10.5 kb EcoRI fragment contiguous with the fragment carrying nirS. Nir- mutants of another type that had low level synthesis of cytochrome cd1, had Tn5 insertions within an 11 kb EcoRI fragment unlinked to the nirS+ and nirD+ fragments. Cosmid mapping provided evidence that nirS and nirD, and the previously identified gene cluster for nitrous oxide respiration are closely linked. The nirS gene and the structural gene for nitrous oxide reductase, nosZ, are transcribed in the same direction and are separated by approximately 14 kb. Several genes for copper processing are located within the intervening region.

Adenylate cyclase toxin from Bordetella pertussis. The relationship between induction of cAMP and hemolysis.

Bordetella pertussis produces a calmodulin-activated adenylate cyclase (AC) that exists in several forms. Only one form of AC, of apparent 200 kDa, is a toxin that penetrates eukaryotic cells and generates uncontrolled levels of intracellular cAMP. Recombination studies in transposon Tn5-insertion mutants of B. pertussis and amino acid sequence homology with alpha-hemolysin of Escherichia coli suggested that AC toxin may also have a hemolytic activity. Here, we demonstrate that only the toxic form of B. pertussis AC possesses hemolytic activity. Immunoblotting of membranes from sheep erythrocytes throughout the process of cell lysis detects the presence and accumulation of only the 200-kDa form of B. pertussis AC. cAMP generation induced by AC toxin in sheep erythrocytes is immediate whereas appearance of hemolysis is delayed by about 1 h and requires a higher level of AC toxin activity. Addition of exogenous calmodulin to sheep erythrocyte incubation medium potentiates the hemolytic activity of AC toxin but blocks cAMP generation. Extracellular Ca2+ at mM concentrations is absolutely required for cAMP generation but not for hemolysis. However, binding of AC toxin to sheep erythrocytes in the absence of exogenous Ca2+ followed by reincubation of cells in a toxin-free buffer containing Ca2+ leads to an immediate rise in intracellular cAMP. Human erythrocytes bind AC toxin and generate cAMP but are resistant to lysis. These results show that binding of AC toxin to erythrocytes can cause both cAMP generation and hemolysis or only one of these depending on conditions applied and cell type used.

Mutations that affect Tn5 insertion into pBR322: importance of local DNA supercoiling

The major hot spot of transposon Tn5 insertion in plasmid pBR322 (hot spot I) is in the promoter for the tetracycline resistance gene (tet). We made a series of pBR322 derivatives with mutations in and around this promoter and assayed their effects on insertion of Tn5 into hot spot I. Those mutations which reduced transcription from the tet promoter also reduced the frequency of insertion into hot spot I. Transcription and translation of tet are thought to cause the formation of paired domains of negative and positive supercoiling in pBR322. An amber codon in tet, 345 base pairs from hot spot I, decreases the negative supercoiling of the DNA segment containing hot spot I because it terminates translation of tet prematurely. We report here that this amber mutation also reduces insertion into hot spot I. These results suggest that the ability of Tn5 to insert into its major hot spot in pBR322 depends directly on negative supercoiling of the target DNA.

Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules

Bacteroid differentiation was examined in developing and mature alfalfa nodules elicited by wild-type or Fix- mutant strains of Rhizobium meliloti. Ultrastructural studies of wild-type nodules distinguished five steps in bacteroid differentiation (types 1 to 5), each being restricted to a well-defined histological region of the nodule. Correlative studies between nodule development, bacteroid differentiation, and acetylene reduction showed that nitrogenase activity was always associated with the differentiation of the distal zone III of the nodule. In this region, the invaded cells were filled with heterogeneous type 4 bacteroids, the cytoplasm of which displayed an alternation of areas enriched with ribosomes or with DNA fibrils. Cytological studies of complementary halves of transversally sectioned mature nodules confirmed that type 4 bacteroids were always observed in the half of the nodule expressing nitrogenase activity, while the presence of type 5 bacteroids could never be correlated with acetylene reduction. Bacteria with a transposon Tn5 insertion in pSym fix genes elicited the development of Fix- nodules in which bacteroids could not develop into the last two ultrastructural types. The use of mutant strains deleted of DNA fragments bearing functional reiterated pSym fix genes and complemented with recombinant plasmids, each carrying one of these fragments, strengthened the correlation between the occurrence of type 4 bacteroids and acetylene reduction. A new nomenclature is proposed to distinguish the histological areas in alfalfa nodules which account for and are correlated with the multiple stages of bacteroid development.

Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium

Transposon Tn5 insertions causing anaerobic cysteine auxotrophy were isolated from a Salmonella typhimurium cysI parent (auxotrophic under aerobic but not anaerobic conditions). Insertions in one mutant group appeared to be in cysG. A second group of insertions, designated asr (anaerobic sulfite reduction), were located near map unit 53 on the S. typhimurium chromosome. They did not cause aerobic or anaerobic auxotrophy in a cys1+ background but did prevent dissimilatory sulfite reduction. Plasmids containing asr DNA cloned from wild-type S. typhimurium conferred anaerobic prototrophy and the ability to produce hydrogen sulfide from sulfite on an Escherichia coli cys1 mutant.

Growth of Genetically Altered Pseudomonas solanacearum in Soil and Rhizosphere

AbstractThe extent to which a bacterium can grow in soil and in the rhizosphere is influenced by the presence or absence of various genes. The effect of genetic alteration of the egl gene, encoding a β‐1,4‐endoglucanase, on the growth of Pseudomonas solanacearum E.F. Smith (the causal agent of bacterial wilt) in soil and in the rhizosphere of tomato (Lycopersicon esculentum Mill.), common purslane (Portulaca oleracea L.), and pearl millet (Pennisetum glaucum (L.) R. Br.) was determined. Endoglucanase production by P. solanacearum was either inactivated by transposon (Tn5) insertion mutagenesis or enhanced twofold by increasing the egl copy number with a recombinant plasmid (pHE3). In a test of the stability of the genetic alterations in two different soils, the transposon in the chromosome was detectable for >120 d, but the recombinant plasmid was apparently lost after 14 d in one soil and after 120 d in the other. In comparison to the wild‐type strain, the generation times of the strains with the transposon and the recombinant plasmid increased 16 and 25%, respectively, in one soil solution and 35 and 53% in the other soil solution; this correlated well with the ability of the strains to grow in the same freshly moistened soils. Under conditions of soil inoculation, the genetically altered strains did not wilt tomato seedings significantly faster or slower than the wild‐type strain. Although all of the P. solanacearum strains, including the wild type, grew in the rhizosphere of gnotobiotically grown tomato plants, none of the strains grew in the rhizosphere of tomato, common purslane, or pearl millet under nonsterile conditions. The data suggest that genetic alterations slow the growth of P. solanacearum in freshly moistened soil, and that endoglucanase is of minor importance to the growth of P. solanacearum under these conditions.

Genetic analysis of tag mutants of Myxococcus xanthus provides evidence for two developmental aggregation systems

Temperature-dependent aggregation mutants (tag) of the myxobacterium Myxococcus xanthus aggregated into mounds and developed into fruiting bodies normally at 28 degrees C; however, they failed to form mounds at 34 degrees C. The timing of sporulation was unaffected by the mutations, and normal numbers of spores were produced at both permissive and restrictive temperatures. This class of mutations was originally identified through screening of ethyl methanesulfonate (EMS)-generated mutations. Subsequent work identified a linked insertion of transposon Tn5, which was used to map the EMS-generated mutations to four loci. In this paper, we describe the cloning of the tag loci and the use of transposon mutagenesis to further analyze the tag loci. Nine tag complementation groups spanning 8.5 kilobase pairs of DNA were identified through mapping of 28 independent Tn5 insertions. All insertion and deletion mutants had the same phenotype as the EMS mutants: they were temperature sensitive for mound formation. This result suggests that M. xanthus has at least two sets of genes for developmental aggregation. The tag genes constitute one set of these genes; they are required for normal development at 34 degrees C but are not required for normal development at 28 degrees C.

Cloning and Characterization ofRhizobium melilotiLoci Required for Symbiotic Root Nodule Invasion

An immunological assay of root nodule polypeptides was used to analyze the nodules induced by 25 symbiotically defective Rhizobium meliloti mutants. Differences in polypeptide accumulation in these nodules were used to divide the mutants into three subsets. One subset, containing two mutant strains, was further analyzed. Nodules induced by these mutant strains lack both infection threads and bacteria. The kinetics of nodule formation by these mutant strains, by an exoB mutant, and by mixed mutant inocula suggest that the gene products required for nodule invasion may also influence nodule meristem induction. One of the two mutants characterized in this study contains a transposon Tn5 insertion in the ndvB locus, which probably results in the loss of beta-glucan synthesis. The second mutant contains a transposon in a previously uncharacterized locus. RNA analysis suggests that the newly identified locus is transcribed in free-living cultures of ndvB and exoB strains, as well as in the parental R. meliloti strain. Southern blot analysis suggests that at least a portion of this locus is duplicated. This duplication may explain the apparently leaky phenotype of the mutant strain.

A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction

We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.

Chemotactic control of the two flagellar systems of Vibrio parahaemolyticus

Vibrio parahaemolyticus synthesizes two distinct flagellar organelles, the polar flagellum (Fla), which propels the bacterium in a liquid environment (swimming), and the lateral flagella (Laf), which are responsible for movement over surfaces (swarming). Chemotactic control of each of these flagellar systems was evaluated separately by analyzing the behavioral responses of strains defective in either motility system, i.e., Fla+ Laf- (swimming only) or Fla- Laf+ (swarming only) mutants. Capillary assays, modified by using viscous solutions to measure swarming motility, were used to quantitate chemotaxis by the Fla+ Laf- or Fla- Laf+ mutants. The behavior of the mutants was very similar with respect to the attractant compounds and the concentrations which elicited responses. The effect of chemotaxis gene defects on the operation of the two flagellar systems was also examined. A locus previously shown to encode functions required for chemotactic control of the polar flagellum was cloned and mutated by transposon Tn5 insertion in Escherichia coli, and the defects in this locus, che-4 and che-5, were then transferred to the Fla+ Laf- or Fla- Laf+ strains of V. parahaemolyticus. Introduction of the che mutations into these strains prevented chemotaxis into capillary tubes and greatly diminished movement of bacteria over the surface of agar media or through semisolid media. We conclude that the two flagellar organelles, which consist of independent motor-propeller structures, are directed by a common chemosensory control system.

Development of an Adhesion Assay and Characterization of an Adhesion-Deficient Mutant of Pseudomonas fluorescens

A sand column adhesion assay was developed which distinguishes the adhesion abilities of a number of pseudomonads isolated from fine sandy loam. Pseudomonas fluorescens Pf0-1 which adhered at >90% of the total cells added was subjected to transposon Tn5 insertion mutagenesis. From 2,500 Pf0-1::Tn5 mutants examined in the sand column assay, two adhesion-deficient Pf0-1 mutants showing <50% attachment were isolated. Marker exchange analysis of one of these mutants, Pf0-5, confirmed that the decreased adhesion was linked to the Tn5 insertion in the chromosome. The growth rate of Pf0-5 in enriched media and sterile soil was similar to that of the wild type; in minimal medium, however, Pf0-5 grew faster. In a soil column assay, less Pf0-5 than wild-type bacteria were recovered, suggesting a decreased ability to persist in soil. A 34-kilodalton major outer membrane protein present in the wild type was missing in Pf0-5. Transmission electron microscopy of the cell surface revealed that the wild-type possessed polar flagella which were absent in the mutant.

Altered imino diacid synthesis and transcription in crown gall tumors with transposon Tn5 insertions in the 3' end of the octopine synthase gene

Octopine synthase encoded by the T-DNA (transferred DNA) locus ocs synthesizes N2-(D-1-carboxyethyl)-L-amino acids in octopine-type crown gall tumors. So far, derivatives of only basic amino acids have been isolated. We have detected a glutamine derivative and called it heliopine. Tumors induced by several Ti plasmids with transposon Tn5 insertions in the 3' end of ocs still synthesized small quantities of N2-(1-carboxyethyl)-arginine and N2-(1-carboxyethyl)-glutamine. In addition, N2-(1,3-dicarboxypropyl)-asparagine, which is absent in wild-type octopine tumors, was detected in these tumors. These three imino diacids (octopine, heliopine, and asparaginopine, respectively, or their isomers) were undetectable in tumors induced by Ti plasmids harboring deletions of the ocs gene. Poly(A)+ RNAs which hybridize to the ocs sequence can also be detected in the ocs::Tn5 tumors; these RNAs, however, were heterogeneous in size and shorter in length than the normal ocs mRNA. These results indicate that mutant ocs products synthesize imino diacids in these ocs::Tn5 tumors.

Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.

Dsg, a gene required for cell-cell interaction early in Myxococcus development

dsg mutants of Myxococcus xanthus are conditionally defective in fruiting body development, including sporulation. Unable to develop on their own, these mutants can assemble fruiting bodies with spores if they are mixed with wild-type cells. To elucidate the developmental defect in dsg mutants by close comparison with wild type, such mutants have been backcrossed by transduction, using a closely linked insertion of transposon Tn5 for selection. Backcrossed dsg mutants form aggregates that are larger, less compact, and less symmetrical than dsg+ fruiting bodies. Also, the starvation-induced sporulation in dsg aggregates is delayed and reduced. However, dsg mutants can be induced by glycerol or dimethyl sulfoxide to sporulate at levels approaching those of wild type. dsg mutants may thus have a primary defect early in development which diminishes their capacity to aggregate and which indirectly decreases the number of fruiting body spores. The linked insertion of Tn5 also facilitated cloning the dsg gene. The cloned dsg+ allele was shown to be dominant to both the dsg-429 and dsg-439 alleles, and both mutant alleles were shown to belong to the same genetic complementation group. Subcloning of restriction fragments, deletions, and insertions of transposon Tn5 agree in locating the dsg gene to an 850-base-pair segment of the cloned region.

General method for site-directed mutagenesis in Escherichia coli O18ac:K1:H7: deletion of the inducible superoxide dismutase gene, sodA, does not diminish bacteremia in neonatal rats

A defined deletion in the Escherichia coli K-12 sodA gene (encoding manganese-superoxide dismutase) linked to a nontransposable selectable marker was generated by transposon Tn5 insertion in combination with in vitro mutagenesis. This mutant allele was used to replace the wild-type sodA gene in an E. coli clinical isolate of serotype O18ac:K1:H7 by bacteriophage P1 transduction. The O18ac:K1:H7 sodA mutant contained no manganese-superoxide dismutase and no hybrid manganese-iron-superoxide dismutase. The sodA mutant was more sensitive to paraquat toxicity than were the parental strain and an isogenic mutant bearing an analogously constructed sodA+ Tn5 insertion allele. In a suckling rat model for bacteremia following oral inoculation of E. coli K1, the sodA mutant was undiminished in its capabilities both to colonize the gastrointestinal tract and, surprisingly, to cause bacteremia. In conjunction with the rat model for E. coli K1 pathogenesis, the method for site-directed mutagenesis described in this paper permits determination of the role played in colonization and bacteremia by any K1 gene which either has a homolog in E. coli K-12 or can be cloned and manipulated therein.

Assembly of Escherichia coli fumarate reductase holoenzyme.

The production and assembly of the four fumarate reductase polypeptides into holoenzyme was studied in vivo in a T7-promoter-conditional expression system. No posttranslational modification of any of the subunits was detected, although the ratio of polypeptides produced varied with the temperature at which expression occurred. FrdC and FrdD, the membrane anchor polypeptides, assembled rapidly into the membrane and then were capped with FrdA and FrdB in separate events. Truncation of the C-terminal domain of FrdD by insertion of transposon Tn5 into the frdD cistron interfered with membrane insertion of the anchor polypeptides and assembly of the holoenzyme. Proteolytic degradation of truncated FrdD was implicated in the production of a soluble FrdABC trimer.