A single-amino-acid substitution in the TvbS1 receptor results in decreased susceptibility to infection by avian sarcoma and leukosis virus subgroups B and D and resistance to infection by subgroup E in vitro and in vivo

The avian sarcoma and leukosis virus (ASLV) family of retroviruses contains five highly related envelope subgroups (A to E) thought to have evolved from a common viral ancestor in the chicken population. Three genetic loci in chickens determine the susceptibility or resistance of cells to infection by the subgroup A to E ASLVs. Some inbred lines of chickens display phenotypes that are somewhere in between either efficiently susceptible or resistant to infection by specific subgroups of ASLV. The tvb gene encodes the receptor for subgroups B, D, and E ASLVs. The wild-type Tvb^S1 receptor confers susceptibility to subgroups B, D, and E ASLVs. In this study, the genetic defect that accounts for the altered susceptibility of an inbred chicken line, line M, to infection by ASLV(B), ASLV(D), and ASLV(E) was identified. The tvb gene in line M, tvb^r2, encodes a mutant Tvb^S1 receptor protein with a substitution of a serine for a cysteine at position 125 (C125S). Here, we show that the C125S substitution in Tvb^S1 significantly reduces the susceptibility of line M cells to infection by ASLV(B) and ASLV(D) and virtually eliminates susceptibility to ASLV(E) infection both in cultured cells and in the incidence and growth of avian sarcoma virus-induced sarcomas in chickens. The C125S substitution significantly reduces the binding affinity of the Tvb^S1 receptor for the subgroup B, D, and E ASLV envelope glycoproteins. These are the first results that demonstrate a possible role of the cysteine-rich domain 3 in the function of the Tvb receptors.     

Nuclear Receptor CAR Represses TNFα-Induced Cell Death by Interacting with the Anti-Apoptotic GADD45B

Background

Phenobarbital (PB) is the most well-known among numerous non-genotoxic carcinogens that cause the development of hepatocellular carcinoma (HCC). PB activates nuclear xenobiotic receptor Constitutive Active/Androstane Receptor (CAR; NR1I3) and this activation is shown to determine PB promotion of HCC in mice. The molecular mechanism of CAR-mediated tumor promotion, however, remains elusive at the present time. Here we have identified Growth Arrest and DNA Damage-inducible 45β (GADD45B) as a novel CAR target, through which CAR represses cell death.

Methodology/Principal Findings

PB activation of nuclear xenobiotic receptor CAR is found to induce the Gadd45b gene in mouse liver throughout the development of HCC as well as in liver tumors. Given the known function of GADD45B as a factor that represses Mitogen-activated protein Kinase Kinase 7 - c-Jun N-terminal Kinase (MKK7-JNK) pathway-mediated apoptosis, we have now demonstrated that CAR interacts with GADD45B to repress Tumor Necrosis Factor α ( TNFα)-induced JNK1 phosphorylation as well as cell death. Primary hepatocytes, prepared from Car^+/+, Car^−/−, Gadd45b^+/+ and Gadd45b^−/− mice, were treated with TNFα and Actinomycin D to induce phosphorylation of JNK1 and cell death. Co-treatment with the CAR activating ligand TCPOBOP (1,4 bis[2-(3,5-dichloropyridyloxy)]benzene) has resulted in repression of both phosphorylation and cell death in the primary hepatocytes from Car^+/+ but not Car^−/−mice. Repression by TCPOBOP was not observed in those prepared from Gadd45b^−/− mice. In vitro protein-protein interaction and phosphorylation assays have revealed that CAR interacts with MKK7 and represses the MKK7-mediated phosphorylation of JNK1.

Conclusions/Significance

CAR can form a protein complex with GADD45B, through which CAR represses MKK7-mediated phosphorylation of JNK1. In addition to activating the Gadd45b gene, CAR may repress death of mouse primary hepatocytes by forming a GADD45B complex and repressing MKK7-mediated phosphorylation of JNK1. The present finding that CAR can repress cell death via its interaction with GADD45B provides an insight for further investigations into the CAR-regulated molecular mechanism by which PB promotes development of HCC.     

The Unique Envelope Gene of the Subgroup J Avian Leukosis Virus Derives from ev/J Proviruses, a Novel Family of Avian Endogenous Viruses

A new subgroup of avian leukosis virus (ALV), designated subgroup J, was identified recently. Viruses of this subgroup do not cross-interfere with viruses of the avian A, B, C, D, and E subgroups, are not neutralized by antisera raised against the other virus subgroups, and have a broader host range than the A to E subgroups. Sequence comparisons reveal that while the subgroup J envelope gene includes some regions that are related to those found in env genes of the A to E subgroups, the majority of the subgroup J gene is composed of sequences either that are more similar to those of a member (E51) of the ancient endogenous avian virus (EAV) family of proviruses or that appear unique to subgroup J viruses. These data led to the suggestion that the ALV-J env gene might have arisen by multiple recombination events between one or more endogenous and exogenous viruses. We initiated studies to investigate the origin of the subgroup J envelope gene and in particular to determine the identity of endogenous sequences that may have contributed to its generation. Here we report the identification of a novel family of avian endogenous viruses that include env coding sequences that are over 95% identical to both the gp85 and gp37 coding regions of subgroup J viruses. We call these viruses the ev/J family. We also report the isolation of ev/J-encoded cDNAs, indicating that at least some members of this family are expressed. These data support the hypothesis that the subgroup J envelope gene was acquired by recombination with expressed endogenous sequences and are consistent with acquisition of this gene by only one recombination event.     

Genetic Mapping of the Cloned Subgroup A Avian Sarcoma and Leukosis Virus Receptor Gene to the TVA Locus

A chicken gene conferring susceptibility to subgroup A avian sarcoma and leukosis viruses (ASLV-A) was recently identified by a gene transfer strategy. Classical genetic approaches had previously identified a locus, TVA, that controls susceptibility to ASLV-A. Using restriction fragment length polymorphism (RFLP) mapping in inbred susceptible (TVA*S) and resistant (TVA*R) chicken lines, we demonstrate that in 93 F₂ progeny an RFLP for the cloned receptor gene segregates with TVA. From these analyses we calculate that the cloned receptor gene lies within 5 centimorgans of TVA, making it highly probable that the cloned gene is the previously identified locus TVA. The polymorphism that distinguishes the two alleles of TVA in these inbred lines affects the encoded amino acid sequence of the region of Tva that encompasses the viral binding domain. However, analysis of the genomic sequence encoding this region of Tva in randomly bred chickens suggests that the altered virus binding domain is not the basis for genetic resistance in the chicken lines analyzed.Avian sarcoma and leukosis viruses (ASLV) may be classified into several subgroups based on properties of the viral envelope proteins. Five major subgroups of ASLV (A to E) have been defined based on immunological reactivity of the viral envelope proteins, host range, and infection interference patterns (reviewed in reference 18). The subgroup specificity of the virus maps to the env gene, and distinct hypervariable regions within env have been shown to determine the viral subgroup (2, 3, 7, 8).Patterns of susceptibility of inbred chicken lines to infection with the various ASLV subgroups suggest that three loci control viral entry for the five major ASLV subgroups (11, 12, 15, 17). Dominant alleles at the TVA and TVC (TVA*S and TVC*S) loci confer susceptibility to subgroups A and C viruses, respectively. The TVB locus is more complex, with various alleles determining infection by subgroup B, D, and E viruses. TVA, TVB, and TVC appear to act at the level of viral entry into the cell; therefore, it has been assumed that the TV loci encode or control expression of the cellular receptors for ASLV (5, 13).Recently, a gene that confers susceptibility to subgroup A ASLV (ASLV-A) was cloned by a gene transfer strategy (1, 19). Molecular clones containing coding sequences for this gene relieve the block to viral entry when expressed in a number of mammalian cell lines (1, 19). The gene encodes a surface glycoprotein that has been shown to bind directly and specifically to the ASLV-A envelope protein (4, 9). In addition, binding of the receptor protein to ASLV-A envelope protein induces conformational changes in the viral glycoproteins that appear to be associated with viral entry (10). Finally, an antibody to the receptor protein specifically blocks infection of chicken cells by subgroup A viruses, suggesting that this gene encodes the normal ASLV-A receptor on chicken cells (1). Taken together, these results demonstrate that the cloned gene encodes a subgroup A virus receptor.Since it is clear that the cloned gene encodes an ASLV-A receptor, the protein supposed to be encoded by the classical TVA locus, we asked whether the identified receptor gene mapped to the TVA locus. In this paper we show that a restriction fragment length polymorphism (RFLP) of DNA from inbred chickens can be used to demonstrate that the cloned ASLV-A receptor gene maps to within 5 centimorgans (cM) of the TVA locus as defined by susceptibility to ASLV-A, making it highly probable that the cloned gene is TVA. In addition, analysis of the genomic sequences encoding the viral interaction domain of the receptor in both inbred and randomly bred chickens demonstrates that alterations in this region of the receptor are not responsible for the resistance phenotype.

TVA segregation analysis.

Inbred chicken lines homozygous for TVA alleles encoding either sensitivity (TVA*S) or resistance (TVA*R) were used to generate birds in which the TVA segregation pattern could be studied. Regional Poultry Research Lab lines 6₃ (TVA*S/*S TVB*S/*S) and 7₂ (TVA*R/*R TVB*R/*R) (6) were crossed, and the resulting heterozygous F₁ progeny were mated to generate 93 F₂ chicks. The susceptibility phenotype of the F₂ chicks was determined on the basis of tumor formation following subcutaneous injection of 500 focus-forming units (FFU) of subgroup A Bryan high-titer Rous sarcoma virus (RSV) (RAV-1) and 500 FFU of subgroup B Bryan high-titer RSV (RAV-2) into the right and left wing webs, respectively, of 4-week-old chicks. At 2, 3, and 4 weeks postinjection the wings were palpated to check for tumor development. Chicks were scored as positive for susceptibility (e.g., TVA*S/*S or TVA*S/*R) if a tumor of any size formed in the appropriate wing web.Table ​Table11 summarizes the distribution of susceptibility to subgroups A and B RSV in the F₂ chicks. Segregation of TVB yielded roughly the predicted frequency of susceptible (67 observed versus 70 expected) and resistant (26 observed versus 23 expected) progeny for a dominant locus (P > 0.05). In contrast, the distribution of subgroup A-susceptible and -resistant birds deviated significantly from the expected 3:1 ratio, with an excessive number of subgroup A-resistant chicks observed and a ratio of 2:1 (χ² = 3.4) (Table ​(Table1).1). Previous analysis of the segregation of TVA using the same chicken lines did not reveal an altered distribution of sensitive and resistant birds (6), suggesting that the bias seen here may be a chance deviation from the expected ratio. Segregation of two endogenous virus loci, ALVE2 (previously designated ev2) (carried by line 7₂) and ALVE3 (previously designated ev3) (carried by line 6₃), also gave roughly the expected 3:1 ratio in these F₂ chicks (data not shown). When the segregation bias in the ASLV-A-susceptible birds was accounted for, then susceptibility to subgroup A and B RSV segregated independently, as was expected since the TVA and TVB genes have been reported to be unlinked (6). Recent mapping studies also confirmed that TVA and TVB are found on different linkage groups (3a).

TABLE 1

Segregation of resistance and susceptibility to subgroup A and B viruses in F₂ progeny obtained by crossing lines 6₃ and 7₂

High-Resolution Linkage Map of Mouse Chromosome 13 in the Vicinity of the Host Resistance LocusLgn1

Natural resistance of inbred mouse strains to infection withLegionella pneumophilais controlled by the expression of a single dominant gene on chromosome 13, designatedLgn1.The genetic difference atLgn1is phenotypically expressed as the presence or absence of intracellular replication ofL. pneumophilain host macrophages. In our effort to identify theLgn1gene by positional cloning, we have generated a high-resolution linkage map of theLgn1chromosomal region. For this, we have carried out extensive segregation analysis in a total of 1270 (A/J × C57BL/6J) × A/J informative backcross mice segregating the resistance allele of C57BL/6J and the susceptibility allele of A/J. Additional segregation analyses were carried out in three preexisting panels of C57BL/6J ×Mus spretusinterspecific backcross mice. A total of 39 DNA markers were mapped within an interval of approximately 30 cM overlapping theLgn1region. Combined pedigree analyses for the 5.4-cM segment overlappingLgn1indicated the locus order and the interlocus distances (in cM):D13Mit128–(1.4)–D13Mit194–(0.1)–D13Mit147–(0.9)–D13Mit36–(0.9)–D13Mit146–(0.2)–Lgn1/D13Mit37–(1.0)–D13Mit70.Additional genetic linkage studies of markers not informative in the A/J × C57BL/6J cross positionedD13Mit30, -72, -195,and-203, D13Gor4, D13Hun35,andMtap5in the immediate vicinity of theLgn1locus. The marker density and resolution of this genetic linkage map should allow the construction of a physical map of the region and the isolation of YAC clones overlapping the gene.     

The Coxsackievirus-Adenovirus Receptor Protein Can Function as a Cellular Attachment Protein for Adenovirus Serotypes from Subgroups A, C, D, E, and F

Attachment of an adenovirus (Ad) to a cell is mediated by the capsid fiber protein. To date, only the cellular fiber receptor for subgroup C serotypes 2 and 5, the so-called coxsackievirus-adenovirus receptor (CAR) protein, has been identified and cloned. Previous data suggested that the fiber of the subgroup D serotype Ad9 also recognizes CAR, since Ad9 and Ad2 fiber knobs cross-blocked each other’s cellular binding. Recombinant fiber knobs and ³H-labeled Ad virions from serotypes representing all six subgroups (A to F) were used to determine whether the knobs cross-blocked the binding of virions from different subgroups. With the exception of subgroup B, all subgroup representatives cross-competed, suggesting that they use CAR as a cellular fiber receptor as well. This result was confirmed by showing that CAR, produced in a soluble recombinant form (sCAR), bound to nitrocellulose-immobilized virions from the different subgroups except subgroup B. Similar results were found for blotted fiber knob proteins. The subgroup F virus Ad41 has both short and long fibers, but only the long fiber bound sCAR. The sCAR protein blocked the attachment of all virus serotypes that bound CAR. Moreover, CHO cells expressing human CAR, in contrast to untransformed CHO cells, all specifically bound the sCAR-binding serotypes. We conclude therefore that Ad serotypes from subgroups A, C, D, E, and F all use CAR as a cellular fiber receptor.     

Artificial insertion of a dominant gene for resistance to avian leukosis virus into the germ line of the chicken

Summary This report describes the unique biological properties of a transgenic chicken line that contains a defective avian leukosis virus (ALV) proviral insert that we call alv6. Chick embryo fibroblasts (CEF) containing this insert express subgroup A envelope glycoprotein since they yield focus-forming pseudotype virus when co-cultivated with transformed quail cells expressing envelope-defective Bryan high-liter Rous sarcoma virus (RSV). In addition, these cells display high interference to subgroup A RSV but not to subgroup B RSV infection. Chickens containing this insert are highly resistant to pathogenic subgroup A ALV infection, but show little immunological tolerance to subgroup B ALV infection. Thus we have artificially inserted a dominant gene for resistance to avian leukosis infection into the chicken germ line.     

High-resolution linkage map in the vicinity of the Lp locus

Looptail (Lp) is a mutation that profoundly affects neurulation in mouse and is characterized by craniorachischisis, an open neural tube extending from the midbrain to the tail in embryos homozygous for the mutation. Lp maps to the distal portion of mouse chromosome 1, and as part of a positional cloning approach, we have generated a high-resolution linkage map of the Lp chromosomal region. For this, we have carried out extensive segregation analysis in a total of 706 backcross mice informative for Lp and derived from two crosses, (Lp/ + X SJL/J)F1 X SJL/J and (Lp/ + X SWR/J)F1 X SWR/J. In addition, 269 mice from a (Mus spretus X C57BL/6J)F1 X C57BL/6J interspecific backcross were also used to order marker loci and calculate intergene distances for this region. With these mice, a total of 28 DNA markers corresponding to either cloned genes or anonymous markers of the SSLP or SSCP-types were mapped within a 5-cM interval overlapping the Lp region, with the following locus order and interlocus distances (in cM): centromere-D1Mit110 / Atp1β1 / Cd3ζ / Cd3η / D1Mit145 — D1Hun14 / D1Mit15 — D1Mit111 / D1Mit112 — D1Mit114 — D1Mit148 / D1Mit205/ D1Mit36 / D1Mit146 / D1Mit147 / D1Mit270 / D1Hun13 — Fcgr2 — Mpp — Apoa2/Fcer1γ - Lp - D1Mit149 / Spna1/Fcer1α-Eph1-Hlx1/D1Mit62. These studies have allowed the delineation of a maximum genetic interval for Lp of 0.5 cM, a size amenable to physical mapping techniques.     

Heterologous Expression of the Desulfovibrio gigas [NiFe] Hydrogenase in Desulfovibrio fructosovorans MR400

The ability of Desulfovibrio fructosovorans MR400 ΔhynABC to express the heterologous cloned [NiFe] hydrogenase of Desulfovibrio gigas was investigated. The [NiFe] hydrogenase operon from D. gigas, hynABCD, was cloned, sequenced, and introduced into D. fructosovorans MR400. A portion of the recombinant heterologous [NiFe] hydrogenase was totally matured, exhibiting catalytic and spectroscopic properties identical to those of the native D. gigas protein. A chimeric operon containing hynAB from D. gigas and hynC from D. fructosovorans placed under the control of the D. fructosovorans hynAp promoter was constructed and expressed in D. fructosovorans MR400. Under these conditions, the same level of activity was obtained as with the D. gigas hydrogenase operon.     

Genetic Analysis of Audiogenic Seizure Susceptibility in C57bl/6j x Dba/2j Recombinant Inbred Strains of Mice

The inheritance of susceptibility to audiogenic seizures (ASs) was studied in the C57BL/6J (B6) and DBA/2J (D2) progenitor strains, their reciprocal F₁ hybrids, backcross generations and in 21 B6 x D2 recombinant inbred (RI) strains of mice at 21 days of age. All of the D2 mice tested experienced ASs, whereas none of the B6 mice responded to the sound. Although 23% of the F₁ mice experienced wild running, they were generally as resistant to ASs as their B6 parents. Mice of the F₁ x B6 backcross generation were also resistant to ASs. In the F₁ x D2 backcross generation, however, a significant preponderance (72%) of AS-susceptible mice was found. No significant association was observed between any of the four coat-color phenotypes that were segregating in this generation and susceptibility to ASs. A continuous distribution of mean seizure severity scores and several new audiogenic response phenotypes, distinctly different from the phenotypes of either progenitor strain, were found among the 21 RI strains. These and the results from the F₁ x D2 backcross generation suggest that the difference in AS susceptibility between 21-day-old B6 and D2 mice cannot be under the control of a single locus. In addition, no association was found between AS susceptibility and the chromosome 4 markers Lyb-2, Mup-1 and b among the 21 RI strains. An association was observed, however, between AS susceptibility and the Ah locus. Several of the RI strains that were AS resistant at 21 days of age became AS susceptible as adults. One RI strain was susceptible to ASs at both young and adult ages. The B6, D2 and F₁ mice were completely resistant to ASs at adult ages. Genetic differences were found among the RI strains for the incidence, onset, duration, and type of severity of ASs. A remarkable amount of phenotypic variability in the audiogenic response, which can be attributed only to the influence of environmental factors, occurred within several of the RI strains. A multiple-factor mode of inheritance involving a physiological threshold can account for our observations.     

mmr, a Mycobacterium tuberculosis Gene Conferring Resistance to Small Cationic Dyes and Inhibitors

The mmr gene, cloned from Mycobacterium tuberculosis, was shown to confer to Mycobacterium smegmatis resistance to tetraphenylphosphonium (TPP), erythromycin, ethidium bromide, acriflavine, safranin O, and pyronin Y. The gene appears to code for a protein containing four transmembrane domains. Studies of [³H]TPP intracellular accumulation strongly suggest that the resistance mediated by the Mmr protein involves active extrusion of TPP.     

Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus

In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-¹⁴C]18:0)-CoA was synthesized from [1-¹⁴C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-¹⁴C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-¹⁴C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [¹⁴C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.     

Identification and characterization of a shared TNFR-related receptor for subgroup B, D, and E avian leukosis viruses reveal cysteine residues required specifically for subgroup E viral entry

Genetic and receptor interference data have indicated the presence of one or more cellular receptors for subgroup B, D, and E avian leukosis viruses (ALV) encoded by the s1 allele of the chicken tvb locus. Despite the prediction that these viruses use the same receptor, they exhibit a nonreciprocal receptor interference pattern: ALV-B and ALV-D can interfere with infection by all three viral subgroups, but ALV-E only interferes with infection by subgroup E viruses. We identified a tvb(s1) cDNA clone which encodes a tumor necrosis factor receptor-related receptor for ALV-B, -D, and -E. The nonreciprocal receptor interference pattern was reconstituted in transfected human 293 cells by coexpressing the cloned receptor with the envelope (Env) proteins of either ALV-B or ALV-E. This pattern of interference was also observed when soluble ALV surface (SU)-immunoglobulin fusion proteins were bound to this cellular receptor before viral challenge. These data demonstrate that viral Env-receptor interactions can account for the nonreciprocal interference between ALV subgroups B, D, and E. Furthermore, they indicate that a single chicken gene located at tvb(s1) encodes receptors for these three viral subgroups. The TVB(S1) protein differs exclusively at residue 62 from the published subgroup B- and D-specific receptor, encoded by the s3 allele of tvb. Residue 62 is a cysteine in TVB(S1) but is a serine in TVB(S3), giving TVB(S1) an even number of cysteines in the extracellular domain. We present evidence for a disulfide bond requirement in TVB(S1) for ALV-E infection but not for ALV-B infection. Thus, ALV-B and ALV-E interact in fundamentally different ways with this shared receptor, a finding that may account for the observed biological differences between these two ALV subgroups.     

Lysophosphatidic Acid Acyltransferase from Coconut Endosperm Mediates the Insertion of Laurate at the sn-2 Position of Triacylglycerols in Lauric Rapeseed Oil and Can Increase Total Laurate Levels

Expression of a California bay laurel (Umbellularia californica) 12:0-acyl-carrier protein thioesterase, bay thioesterase (BTE), in developing seeds of oilseed rape (Brassica napus) led to the production of oils containing up to 50% laurate. In these BTE oils, laurate is found almost exclusively at the sn-1 and sn-3 positions of the triacylglycerols (T.A. Voelker, T.R. Hayes, A.C. Cranmer, H.M. Davies [1996] Plant J 9: 229–241). Coexpression of a coconut (Cocos nucifera) 12:0-coenzyme A-preferring lysophosphatitic acid acyltransferase (D.S. Knutzon, K.D. Lardizabal, J.S. Nelsen, J.L. Bleibaum, H.M. Davies, J.G. Metz [1995] Plant Physiol 109: 999–1006) in BTE oilseed rape seeds facilitates efficient laurate deposition at the sn-2 position, resulting in the acccumulation of trilaurin. The introduction of the coconut protein into BTE oilseed rape lines with laurate above 50 mol % further increases total laurate levels.     

Application of a Propionyl Coenzyme A Synthetase for Poly(3-Hydroxypropionate-co-3-Hydroxybutyrate) Accumulation in Recombinant Escherichia coli

The genetic operon for propionic acid degradation in Salmonella enterica serovar Typhimurium contains an open reading frame designated prpE which encodes a propionyl coenzyme A (propionyl-CoA) synthetase (A. R. Horswill and J. C. Escalante-Semerena, Microbiology 145:1381–1388, 1999). In this paper we report the cloning of prpE by PCR, its overexpression in Escherichia coli, and the substrate specificity of the enzyme. When propionate was utilized as the substrate for PrpE, a K_m of 50 μM and a specific activity of 120 μmol · min⁻¹ · mg⁻¹ were found at the saturating substrate concentration. PrpE also activated acetate, 3-hydroxypropionate (3HP), and butyrate to their corresponding coenzyme A esters but did so much less efficiently than propionate. When prpE was coexpressed with the polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha in recombinant E. coli, a PHA copolymer containing 3HP units accumulated when 3HP was supplied with the growth medium. To compare the utility of acyl-CoA synthetases to that of an acyl-CoA transferase for PHA production, PHA-producing recombinant strains were constructed to coexpress the PHA biosynthetic genes with prpE, with acoE (an acetyl-CoA synthetase gene from R. eutropha [H. Priefert and A. Steinbüchel, J. Bacteriol. 174:6590–6599, 1992]), or with orfZ (an acetyl-CoA:4-hydroxybutyrate-CoA transferase gene from Clostridium propionicum [H. E. Valentin, S. Reiser, and K. J. Gruys, Biotechnol. Bioeng. 67:291–299, 2000]). Of the three enzymes, PrpE and OrfZ enabled similar levels of 3HP incorporation into PHA, whereas AcoE was significantly less effective in this capacity.     

Biodegradation of Atrazine by Agrobacterium radiobacter J14a and Use of This Strain in Bioremediation of Contaminated Soil

We examined the ability of a soil bacterium, Agrobacterium radiobacter J14a, to degrade the herbicide atrazine under a variety of cultural conditions, and we used this bacterium to increase the biodegradation of atrazine in soils from agricultural chemical distribution sites. J14a cells grown in nitrogen-free medium with citrate and sucrose as carbon sources mineralized 94% of 50 μg of [¹⁴C-U-ring]atrazine ml⁻¹ in 72 h with a concurrent increase in the population size from 7.9 × 10⁵ to 5.0 × 10⁷ cells ml⁻¹. Under these conditions cells mineralized the [ethyl-¹⁴C]atrazine and incorporated approximately 30% of the ¹⁴C into the J14a biomass. Cells grown in medium without additional carbon and nitrogen sources degraded atrazine, but the cell numbers did not increase. Metabolites produced by J14a during atrazine degradation include hydroxyatrazine, deethylatrazine, and deethyl-hydroxyatrazine. The addition of 10⁵ J14a cells g⁻¹ into soil with a low indigenous population of atrazine degraders treated with 50 and 200 μg of atrazine g⁻¹ soil resulted in two to five times higher mineralization than in the noninoculated soil. Sucrose addition did not result in significantly faster mineralization rates or shorten degradation lag times. However, J14a introduction (10⁵ cells g⁻¹) into another soil with a larger indigenous atrazine-mineralizing population reduced the atrazine degradation lag times below those in noninoculated treatments but did not generally increase total atrazine mineralization.     

Sporadic Distribution of tRNACCUArg Introns among α-Purple Bacteria: Evidence for Horizontal Transmission and Transposition of a Group I Intron

A group I intron interrupts the tRNA_CCU^Arg gene of the α-purple bacterium Agrobacterium tumefaciens (B. Reinhold-Hurek and D. A. Shub, Nature [London] 357:173–176, 1992). In this study, we assess the distribution of the corresponding intron among 12 additional species of α-purple bacteria. Of 10 newly identified tRNA_CCU^Arg genes, we found only two that contained an intron homologous to that of the Agrobacterium tRNA_CCU^Arg intron. This restricted and scattered distribution of the tRNA_CCU^Arg intron among α-purple bacteria is consistent with a recent origin and horizontal transmission. Primary and secondary structural similarities between tRNA_UAA^Leu introns found in strains of the cyanobacterium Microcystis aeruginosa (K. Rudi and K. S. Jacobsen, FEMS Microbiol. Lett. 156:293–298, 1997) and α-purple tRNA_CCU^Arg introns suggest that these introns share a more recent common ancestor than either does with other known cyanobacterial tRNA_UAA^Leu introns.     

The receptor for the subgroup C avian sarcoma and leukosis viruses, Tvc, is related to mammalian butyrophilins, members of the immunoglobulin superfamily

The five highly related envelope subgroups of the avian sarcoma and leukosis viruses (ASLVs), subgroup A [ASLV(A)] to ASLV(E), are thought to have evolved from an ancestral envelope glycoprotein yet utilize different cellular proteins as receptors. Alleles encoding the subgroup A ASLV receptors (Tva), members of the low-density lipoprotein receptor family, and the subgroup B, D, and E ASLV receptors (Tvb), members of the tumor necrosis factor receptor family, have been identified and cloned. However, alleles encoding the subgroup C ASLV receptors (Tvc) have not been cloned. Previously, we established a genetic linkage between tvc and several other nearby genetic markers on chicken chromosome 28, including tva. In this study, we used this information to clone the tvc gene and identify the Tvc receptor. A bacterial artificial chromosome containing a portion of chicken chromosome 28 that conferred susceptibility to ASLV(C) infection was identified. The tvc gene was identified on this genomic DNA fragment and encodes a 488-amino-acid protein most closely related to mammalian butyrophilins, members of the immunoglobulin protein family. We subsequently cloned cDNAs encoding Tvc that confer susceptibility to infection by subgroup C viruses in chicken cells resistant to ASLV(C) infection and in mammalian cells that do not normally express functional ASLV receptors. In addition, normally susceptible chicken DT40 cells were resistant to ASLV(C) infection after both tvc alleles were disrupted by homologous recombination. Tvc binds the ASLV(C) envelope glycoproteins with low-nanomolar affinity, an affinity similar to that of binding of Tva and Tvb with their respective envelope glycoproteins. We have also identified a mutation in the tvc gene in line L15 chickens that explains why this line is resistant to ASLV(C) infection.     

Curing of Yeast [URE3] Prion by the Hsp40 Cochaperone Ydj1p Is Mediated by Hsp70

[URE3] is a prion of the yeast Ure2 protein. Hsp40 is a cochaperone that regulates Hsp70 chaperone activity. When overexpressed, the Hsp40 Ydj1p cures yeast of [URE3], but the Hsp40 Sis1p does not. On the basis of biochemical data Ydj1p has been proposed to cure [URE3] by binding soluble Ure2p and preventing it from joining prion aggregates. Here, we mutagenized Ydj1p and find that disrupting substrate binding, dimerization, membrane association, or ability to transfer substrate to Hsp70 had little or no effect on curing. J-domain point mutations that disrupt functional interactions of Ydj1p with Hsp70 abolished curing, and the J domain alone cured [URE3]. Consistent with heterologous J domains possessing similar Hsp70 regulatory activity, the Sis1p J domain also cured [URE3]. We further show that Ydj1p is not essential for [URE3] propagation and that depletion of Ure2p is lethal in cells lacking Ydj1p. Our data imply that curing of [URE3] by overproduced Ydj1p does not involve direct interaction of Ydj1p with Ure2p but rather works through regulation of Hsp70 through a specific J-protein/Hsp70 interaction.     

Identification of Genes Encoding Conjugated Bile Salt Hydrolase and Transport in Lactobacillus johnsonii 100-100

Cytosolic extracts of Lactobacillus johnsonii 100-100 (previously reported as Lactobacillus sp. strain 100-100) contain four heterotrimeric isozymes composed of two peptides, α and β, with conjugated bile salt hydrolase (BSH) activity. We now report cloning, from the genome of strain 100-100, a 2,977-bp DNA segment that expresses BSH activity in Escherichia coli. The sequencing of this segment showed that it contained one complete and two partial open reading frames (ORFs). The 3′ partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5′ and 3′ deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used to extend and complete the 948-nucleotide sequence of the BSH gene 3′ of the cloned segment. The predicted amino acid sequence of the 5′ partial ORF (651 nucleotides) was about 80% similar to the C-terminal half of the largest, complete ORF (1,353 nucleotides), and these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily. E. coli DH5α cells transformed with a construct containing these ORFs, in concert with an extracellular factor produced by strain 100-100, demonstrated levels of uptake of [¹⁴C]taurocholic acid that were increased as much as threefold over control levels. [¹⁴C]Cholic acid was taken up in similar amounts by strain DH5α pSportI (control) and DH5α p2000 (transport clones). These findings support a hypothesis that the ORFs are conjugated bile salt transport genes which may be arranged in an operon with BSH genes.