J Mol Biol, 1985 Jun 5, 183(3), 517 - 8 Preliminary X-ray study of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida N.C.I.B . 9869; Shamala N et al.; Single crystals of p-cresol methylhydroxylase, a flavocytochrome c from Pseudomonas putida, have been prepared . The crystals are orthorhombic, space group P212121 with unit cell parameters; a = 140.3 A, b = 130.6 A and c = 74.1 A . They contain a single non-symmetric dimer per asymmetric unit and diffract to at least 2.5 A resolution. Appl Environ Microbiol, 1985 Jun, 49(6), 1494 - 501 Toxic effects of chlorinated and brominated alkanoic acids on Pseudomonas putida PP3: selection at high frequencies of mutations in genes encoding dehalogenases; Weightman AJ et al.; Mutant strains of Pseudomonas putida PP3 capable of utilizing monochloroacetate (MCA) and dichloroacetate (DCA) as the sole sources of carbon and energy were isolated from chemostat cultures . The mutants differed from the parent strain in that they could grow on products of MCA and DCA dehalogenation (catalyzed by inducible dehalogenases I and II) and were resistant to growth inhibition by the two substrates . The growth inhibition of strain PP3 by MCA, DCA, and other halogenated alkanoic acids was studied . Sensitivity to dehalogenase substrates was related to the expression of the dehalogenase genes . For example, mutants producing elevated levels of one or both of the dehalogenases were sensitive to 2-monochloropropionate and 2-monochlorobutanoate at concentrations which did not affect the growth of strain PP3 . P . putida PP1, the parent of strain PP3, was resistant to the inhibitory effects of MCA and DCA . Spontaneous mutants of strain PP3, also resistant to MCA and DCA, were selected at high frequency, and four different classes of these strains were distinguished on the basis of dehalogenase phenotype . All dehalogenase-producing mutants were inducible; no constitutive mutant has yet been isolated . Most of the resistant mutants examined did not produce one or both of the dehalogenase, and over half of those tested failed to revert back to the parental (strain PP3) phenotype, indicating that the observed mutations involved high-frequency deletion of DNA base sequences affecting expression of genes encoding dehalogenases and associated permease(s). J Bacteriol, 1985 Jun, 162(3), 865 - 71 Cloning of genes specifying carbohydrate catabolism in Pseudomonas aeruginosa and Pseudomonas putida; Cuskey SM et al.; A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769 . The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance . Mutants of P . aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase . Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase . However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer . Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells . Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes . The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant . Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida. Biochem J, 1985 Jun 1, 228(2), 325 - 35 p-Cresol methylhydroxylase . Assay and general properties; McIntire W et al.; p-Cresol methylhydroxylase from Pseudomonas putida, an anaerobic dehydrogenase that catalyses the oxidation of p-cresol to p-hydroxybenzyl alcohol and then to p-hydroxybenzaldehyde, is an enzyme of great interest in several respects . One of these is the fact that its flavoprotein and cytochrome c subunits may be reversibly dissociated with ease, with full regeneration of the activity and its native properties on recombining the components . Bisubstrate kinetic analysis of the unresolved enzyme gives parallel-line kinetics in double-reciprocal plots, whereas the reaction of the separated flavoprotein subunit with substrates is described by converging lines . The mechanistic implications of these behaviours are discussed . Reductive titration with dithionite results in the uptake of 3 electrons by the enzyme, with the intermediate formation of the anionic flavin radical {McIntire, Edmondson, Hopper & Singer (1981) Biochemistry 20, 3068-3075} . Reductive titration with substrates resulted initially only in reduction of the cytochrome subunit, followed by formation of the anionic radical and finally the fully reduced enzyme . These observations suggest rapid intermolecular electron transfer between p-cresol methylhydroxylase molecules . This paper also examines the effect of pH and ionic strength on the activity and specificity of the enzyme with respect to substrates and natural, as well as artificial, electron acceptors . The absorption coefficients of the enzyme and of its subunits in various oxidation states are also presented. J Bacteriol, 1985 Jun, 162(3), 1325 - 8 SAL-TOL in vivo recombinant plasmid pKF439; Furukawa K et al.; SAL-TOL in vivo recombinant plasmid pKF439 was characterized in a strain from a mixed culture of bacteria harboring various degradative plasmids . Analysis of the gene organization of pKF439 revealed that the 57-kilobase TOL fragment, including the 40-kilobase TOL metabolic region, was inserted into the complete SAL replicon at the position of SmaI-C within XhoI-B of SAL . The molecular size of pKF439 was calculated to be 138 kilobases . pKF439 could be transferred to Pseudomonas putida and Pseudomonas aeruginosa at high frequency, and the transconjugants gained the ability to grow with m-xylene, m-toluate, and salicylate as the sole carbon source. Biochem J, 1985 May 15, 228(1), 257 - 62 The active transport of 2-keto-D-gluconate in vesicles prepared from Pseudomonas purida; Agbanyo F et al.; The transport of 2-keto-D-gluconate (alpha-D-arabino-2-hexulopyranosonic acid; 2KGA) in vesicles prepared from glucose-grown Pseudomonas putida occurs by a saturable process with a Km of 110.0 +/- 2.9 microM and a Vmax . of 0.55 +/- 0.04 nmol X min-1 X (mg of protein)-1 . The provision of phenazine methosulphate/ascorbate or L-malate leads to an accumulation of intravescular 2KGA, a decrease in the Km value to 50 +/- 2.1 microM and 35 +/- 2.9 microM respectively and no change in the Vmax . In the presence of electron donors the transport of 2KGA is inhibited by the respiratory poisons antimycin A, rotenone and the uncoupler 2,4-dinitrophenol . 2KGA transport is also competitively inhibited by 4-deoxy-4-fluoro-2-keto- or 3-deoxy-3-fluoro-2-keto-D-gluconate with Ki values of 50 microM and 160 microM respectively . The carrier system for 2KGA is repressed in vesicles from cells grown on succinate . Such vesicles transport 2KGA by non-specific physical diffusion with a Km value of infinity in the absence or presence of electron donors . Vesicles from glucose or succinate grown cells, in the presence of phenazine methosulphate/ascorbate at pH 6.6, generate a proton-motive force (delta p) of approx . 140 mV . The delta p, composed of proton gradient (delta pH) and a membrane potential (delta psi), is collapsed in the presence of dinitrophenol . Based on the results obtained with valinomycin, nigericin and carbonyl cyanide m-chlorophenylhydrazone, the active transport of 2KGA at pH 6.6 is coupled predominately to the delta pH component of delta p. Genetika, 1985 May, 21(5), 872 - 4 {Effective method of transduction with virulent phage pf16 using specific mutants of Pseudomonas putida PpG1}; Gorbunova SA et al.; A procedure of simple selection of Pseudomonas putida PpG1 mutants is described . The mutants can be used for transduction with virulent pf16 phage, giving reliable results . The frequency of transduction of chromosomal markers ilv and trp was 10(-6) . Also, transduction of plasmid RP4 with phage pf16 was shown with the frequency of 10(-7). J Appl Bacteriol, 1985 Apr, 58(4), 425 - 9 A simple most probable number method for the enumeration of sulphate-reducing bacteria in biocide containing waters; Battersby NS et al.; A simple most probable number (MPN) method has been developed for the enumeration of sulphate-reducing bacteria (SRB) in biocide-containing waters . The medium used is based on source water, it contains no toxic thioglycollate and is resistant to oxidation through mishandling . Reduction is by a suspension of Pseudomonas putida which acts as a powerful adsorbent of biguanide, phenolic, quaternary ammonium compound, glutaraldehyde and isothiazolone biocides . Good recoveries of SRB type strains were obtained using this method and were comparable to other published techniques . Recovery of SRB in mixed culture was comparable to that using a standard laboratory technique. J Bacteriol, 1985 Apr, 162(1), 203 - 8 Conjugative mapping of pyruvate, 2-ketoglutarate, and branched-chain keto acid dehydrogenase genes in Pseudomonas putida mutants; Sykes PJ et al.; Branched-chain keto acid dehydrogenase, an enzyme in the common pathway of branched-chain amino acid catabolism of Pseudomonas putida, is a multienzyme complex which catalyzes the oxidative decarboxylation of branched-chain keto acids . The objective of the present study was to isolate strains with mutations of this and other keto acid dehydrogenases and to map the location of the mutations on the chromosome of P . putida . Several strains with mutations of branched-chain keto acid dehydrogenase, two pyruvate and two 2-ketoglutarate dehydrogenase, were isolated, and the defective subunits were identified by biochemical analysis . By using a recombinant XYL-K plasmid to mediate conjugation, these mutations were mapped in relation to a series of auxotrophic and other catabolic mutations . The last time of entry recorded was at approximately 35 min, and the data were consistent with a single point of entry . Branched-chain keto acid dehydrogenase mutations affecting E1, E1 plus E2, and E3 subunits mapped at approximately 35 min . One other strain affected in the common pathway was deficient in branched-chain amino acid transaminase, and the mutation was mapped at 16 min . The mutations in the two pyruvate dehydrogenase mutants, one deficient in E1 and the other deficient in E1 plus E2, mapped at 22 minutes . The 2-ketoglutarate dehydrogenase mutation affecting the E1 subunit mapped at 12 minutes . A 2-ketoglutarate dehydrogenase mutant deficient in E3 was isolated, but the mutation proved too leaky to map. J Gen Microbiol, 1985 Apr, 131 ( Pt 4), 885 - 96 Chromosomal map of Pseudomonas putida PPN, and a comparison of gene order with the Pseudomonas aeruginosa PAO chromosomal map; Morgan AF et al.; The generalized transducing phage Pf16h2 has been used to confirm linkage relationships of chromosomal markers of Pseudomonas putida previously determined from their time-of-entry in Hfr crosses, and to map new auxotrophic mutations . By means of spot matings using Hfr donors of known origin of transfer, catabolic markers forming part of a closely linked group of operons referred to as a superoperonic cluster have been shown to be chromosomally located and their map positions determined . R-prime-mediated interspecific complementation has been used to equate functionally 21 auxotrophic loci in P . putida and P . aeruginosa, and the distribution of these loci on the two genetic maps has been compared . While both maps reveal that auxotrophic markers are largely restricted to about 40% of the chromosome and that auxotrophic markers of similar phenotype are not clustered, there is evidence of at least seven chromosomal rearrangements since divergence from a presumed common ancestor. J Bacteriol, 1985 Apr, 162(1), 138 - 46 Cloning and expression in Escherichia coli of histidine utilization genes from Pseudomonas putida; Consevage MW et al.; A library of the Pseudomonas putida chromosome, prepared through the use of the cosmid pJB8 ligated to a partial Sau3A digest of bacterial DNA, followed by in vitro packaging into bacteriophage lambda particles, was used to construct a strain of Escherichia coli which contained the genes for histidine utilization . This isolate produced a repressor product and all five enzymes required in Pseudomonas spp . for histidine dissimilation, whereas none of these could be detected in the nontransduced parent E . coli strain . When this transductant was grown on various media containing histidine or urocanate as the inducer, it was observed that production of the cloned histidine degradative enzymes was influenced somewhat by the choice of nitrogen source used but not by the carbon source . The recombinant cosmid was isolated and found to consist of 21.1 kilobase pairs of DNA, with approximately 16 kilobase pairs derived from Pseudomonas DNA and the remainder being from the pJB8 vector . Digestion of this insert DNA with EcoRI provided a 6.1-kilobase-pair fragment which, upon ligation in pUC8 and transformation into an E . coli host, was found to encode histidine ammonia-lyase and urocanase . The inducible nature of this production indicated that the hut repressor gene also was present on this fragment . Insertional inactivation of the histidine ammonia-lyase and urocanase genes by the gamma-delta transposon has permitted location of these structural genes and has provided evidence that transcription proceeds from urocanase through histidine ammonia-lyase . Mapping of the 16-kilobase-pair Pseudomonas DNA segment with restriction enzymes and subcloning of additional portions, one of which contained the gene for formiminoglutamate hydrolase and another that could constitutively express activities for both imidazolone propionate hydrolase and formylglutamate hydrolase, has provided evidence for the organization of all hut genes. Can J Microbiol, 1985 Apr, 31(4), 381 - 6 Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa; Mutharia LM et al.; A rapid colony immunoblot screening procedure was used to demonstrate the surface localization of porin protein F on bacterial colonies of Pseudomonas aeruginosa . By this method, we demonstrated that protein F was accessible to four different specific monoclonal antibodies in a wide variety of both mucoid and nonmucoid P . aeruginosa strains . Controls were performed to demonstrate that, using this procedure, only surface-exposed epitopes bound monoclonal antibodies and that nonspecific binding of monoclonal antibodies either to cells lacking protein F or to mucoid exopolysaccharide did not occur . Monoclonal antibodies MA4-4, MA2-10, and MA4-10, specific for protein F, also interacted with colonies of Pseudomonas putida and Pseudomonas syringae, whereas the protein F specific monoclonal antibody MA5-8 interacted only with P . aeruginosa strains . Using the above-named monoclonal antibodies, we investigated the antigenic structure of protein F . Monoclonal antibodies MA4-4, MA2-10, and MA4-10 bound to 29-31 kilodalton proteolytic fragments produced after papain or trypsin digestion of purified protein F or of protein F in outer membranes or intact cells . Antibody MA5-8 did not interact with proteolytically digested protein F but did interact with two of the six fragments produced after partial cyanogen bromide cleavage of protein F . Antibodies MA4-4, MA2-10, and MA4-10 did not interact with protein F after reduction of its internal disulphide bonds with 2-mercaptoethanol; in contrast, the reactivity of MA5-8 was unaffected . This data suggests that there are at least two distinct highly conserved surface epitopes on porin protein F. FEBS Lett, 1985 Mar 25, 182(2), 485 - 8 Periplasmic location of p-cresol methylhydroxylase in Pseudomonas putida; Hopper DJ et al.; The cellular location of the flavocytochrome c, p-cresol methylhydroxylase was investigated in two strains of Pseudomonas putida . In both cases the enzymes were shown to be located in the periplasmic fraction by their release during treatment of the bacteria with EDTA and lysozyme in a solution containing a high concentration of sucrose . For strain NCIB 9869 the finding is in accord with the suggestion that the physiological acceptor for the enzyme is azurin as this too was shown to be located mostly in the periplasm. J Mol Biol, 1985 Mar 20, 182(2), 353 - 5 Crystal structure of muconate lactonizing enzyme at 6.5 A resolution; Goldman A et al.; We have obtained crystals of Pseudomonas putida muconate lactonizing enzyme . They diffract to better than 2.4 A resolution and have two monomers in the asymmetric unit, related by a non-crystallographic 2-fold axis . The cell dimensions are 139.3 A X 139.3 A X 84.1 A, and the space group is I4 . The electron density map at 6.5 A resolution shows that the enzyme is an octamer with D4 symmetry. J Biol Chem, 1985 Feb 25, 260(4), 2355 - 63 Purification and properties of ferredoxinTOL . A component of toluene dioxygenase from Pseudomonas putida F1; Subramanian V et al.; Toluene dioxygenase oxidizes toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene . This reaction is catalyzed by a multienzyme system that is induced in cells of Pseudomonas putida F1 during growth on toluene . One of the components of toluene dioxygenase has been purified to homogeneity and shown to be an iron-sulfur protein that has been designated ferredoxinTOL . The molecular weight of ferredoxinTOL was calculated to be 15,300, and the purified protein was shown to contain 2 g of atoms each of iron- and acid-labile sulfur which appear to be organized as a single {2Fe-2S}cluster . Solutions of ferredoxinTOL were brown in color and showed absorption maxima at 277, 327, and 460 nm . A shoulder in the spectrum of the oxidized protein was discernible at 575 nm . Reduction with sodium dithionite or NADH and ferredoxinTOL reductase resulted in a decrease in visible absorbance at 460 and 575 nm, with a concomitant shift in absorption maxima to 382 and 438 nm . The redox potential of ferredoxinTOL was estimated to be -109 mV . In the oxidized state, the protein is diamagnetic . However, upon reduction it exhibited prominent electron paramagnetic resonance signals with anisotropy in g values (gx = 1.81, gy = 1.86, and gz = 2.01) . Anaerobic reductive titrations revealed that ferredoxinTOL is a one-electron carrier that accepts electrons from NADH in a reaction that is mediated by a flavoprotein (ferredoxinTOL reductase) . The latter is the first component in the toluene dioxygenase system . Reduced ferredoxinTOL can transfer electrons to cytochrome c or to a terminal iron-sulfur dioxygenase (ISP-TOL) which catalyzes the incorporation of molecular oxygen into toluene and related aromatic substrates. Biochemistry, 1985 Jan 15, 24(2), 301 - 8 Presence and quantity of dehydroalanine in histidine ammonia-lyase from Pseudomonas putida; Consevage MW et al.; Dehydroalanine is present in the histidine ammonia-lyase (histidase) from Pseudomonas putida ATCC 12633 as shown by reaction of purified enzyme with K14CN or NaB3H4 and subsequent identification of {14C}aspartate or {3H}alanine, respectively, following acid hydrolysis of the labeled protein . When labeling with cyanide was conducted under denaturing conditions, 4 mol of {14C}cyanide was incorporated per mol of enzyme (Mr 220 000), equivalent to one dehydroalanine residue being modified per subunit in this protein composed of four essentially identical subunits . In native enzyme, inactivation of catalytic activity by cyanide was complete when 1 mol of {14C}cyanide had reacted per mol of histidase, suggesting that modification of any one of the four dehydroalanine residues in the tetrameric enzyme was sufficient to prevent catalysis at all sites . Loss of activity on treatment with cyanide could be blocked by the addition of the competitive inhibitor cysteine or substrate if Mn2+ was also present . Cross-linking of native enzyme with dimethyl suberimidate produced no species larger than tetramer, thereby eliminating the possibility that an aggregation phenomenon might explain why only one-fourth of the dehydroalanyl residues was modified by cyanide during inactivation . A labeled tryptic peptide was isolated from enzyme inactivated with {14C}cyanide . Its composition was different from that of a tryptic peptide previously isolated from other histidases and shown to contain a highly reactive and catalytically important cysteine residue . Such a finding indicates the dehydroalanine group is distinct from the active site cysteine . Treatment of crude extracts with {14C}cyanide and purification of the inactive enzyme yielded labeled protein that release {14C}aspartate on acid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS) Gene, 1985, 38(1-3), 73 - 84 Development of broad-host-range vectors for expression of cloned genes in Pseudomonas; Werneke JM et al.; The cloning and expression of genes in Pseudomonas have been difficult, until now, due to the absence of vector systems that contain multiple restriction sites downstream from promoter sequences that are functional in Pseudomonas . We report here the construction of several broad-host-range vectors that can be utilized in either Pseudomonas or Escherichia coli and that rely on easily selectable antibiotic resistance markers with multiple cloning sites . These vectors were constructed by inserting the entire pUC13 sequence into derivatives of the RSF1010 wide-host-range plasmid . From this construction, other derivatives were obtained, specifically a lacZ::KmR fusion gene which provides an easily selectable marker in both E . coli and Pseudomonas . These vectors have been used to express the Pseudomonas putida cytochrome P450 monoxygenase gene in a P450-deficient P . putida strain . Thus, these vectors allow for the cloning, expression and selection of Pseudomonas genes in Pseudomonas by complementation. Biochemistry, 1985 Jan 1, 24(1), 204 - 10 Biodehalogenation: reactions of cytochrome P-450 with polyhalomethanes; Castro CE et al.; The products, stoichiometry, and kinetics of the oxidation of the enzyme cytochrome P-450 cam by five polyhalomethanes and chloronitromethane are described . The reactivity of the enzyme is compared with that of deuteroheme and with the enzyme in its native cell, Pseudomonas putida (PpG-786) . In all cases, the reaction entails hydrogenolysis of the carbon-halogen bond: 2FeIIP + RCXn----2FeIIIP + RCHXn-1 (P = porphyrin or P-450 cam in vitro and in vivo) . Trichloronitromethane was the fastest reacting substrate, and chloroform was the slowest . The results establish that P . putida is a valid whole cell model for the reductase activity of the P-450 complement in these reactions . The reactions of cytochrome P-450 with polyhaloalkanes proceed in a manner quite analogous to other iron(II) proteins in the G conformation . The chemistry observed for the enzyme parallels that of its iron(II) porphyrin active site . Iron-bonded carbenes are not intermediates, and hydrolytically stable iron alkyls are not products of these reactions. Gene, 1985, 36(3), 301 - 9 Transcriptional control of the nah and sal hydrocarbon-degradation operons by the nahR gene product; Schell MA; The positively regulated nah and sal operons of the NAH7 plasmid from Pseudomonas putida encode the enzymes for metabolism of naphthalene via salicylate . To study their coordinate regulation, a 6-kb DNA fragment containing the entire nahA gene (encoding naphthalene dioxygenase), the gene of the nah operon, was cloned into a RSF1010 plasmid derivative . Analysis of expression of nahA from the nah promoter in either Escherichia coli or Pseudomonas putida showed that a 1.6-kb DNA fragment from the nahR (nah operon regulatory locus) region was required in trans for (i) induction by salicylate; (ii) high-level expression of nahA, and (iii) complementation of nahR- mutants . Measurement of transcription in induced and uninduced P . putida showed that induction of the nah and sal operons occurred at the transcriptional level . The trans-acting positive regulatory gene, nahR, however, was constitutively transcribed. Avian Dis, 1985 Jan-Mar, 29(1), 246 - 9 Cyclocoelum mutabile infection and aortic rupture in an American coot (Fulica americana); Branton SL et al.; An American coot (Fulica americana) was found dead within the enclosed research compound of the South Central Poultry Research Laboratory at Mississippi State, Mississippi . Gross and microscopic examinations revealed the bird to be in good body condition; however, blood from the beak cavity and external nares was present . Biliary congestion, hemopericardium, blood-filled air sacs, and a ruptured, ascending aorta were also noted . Nineteen trematodes (Cyclocoelum mutabile) were found within the body cavity at necropsy . Bacteriological examination revealed the presence of Escherichia coli in both the heart and liver and Pseudomonas putida in the liver only . No virus was isolated. J Mol Appl Genet, 1985, 3(1), 26 - 35 Identification of the promoter of the Pseudomonas gene coding for carboxypeptidase G2; Minton NP et al.; A 213-bp region of noncoding DNA upstream of the ATG start codon of the Pseudomonas carboxypeptidase G2 (CPG2) structural gene has been shown to contain the CPG2 promoter . The mRNA start point (+1) on the DNA sequence has been identified by mapping the 5' end of the CPG2 transcript . The identified promoter region contains a -10 region (TATAAG) that closely resembles the Escherichia coli consensus sequence (TATAAT), but has no easily recognisable -35 region . The lack of homology in the -35 region explains why this particular pseudomonad gene is poorly expressed in E . coli . Similar sequence differences may turn out to be the cause of the generally observed inefficient expression of Pseudomonas genes in E . coli . The promoter region also carries a sequence (CTGGCACTCGAATTGCT) that closely matches the consensus nif promoter sequence (CTGGPyAPyPuNNNNTTGCA) of Klebsiella pneumoniae and Rhizobium, and a similar sequence (ATGGCATGGCGGTTGCT) found in the promoter region of the xylABC operon of the TOL plasmid of Pseudomonas putida. Appl Environ Microbiol, 1985 Jan, 49(1), 19 - 23 Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems; Wiggins BA et al.; Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication . The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml . The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml . In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E . coli population exceeded 10(4) CFU/ml . The mineralization of glucose was not affected in E . coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage . The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage . We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml. Gene, 1985, 36(1-2), 143 - 50 Omega mutagenesis in gram-negative bacteria: a selectable interposon which is strongly polar in a wide range of bacterial species; Frey J et al.; We have used the 2.0-kb DNA fragment omega {Prentki and Krisch, Gene 29 (1984) 303-313} to mutagenize in vitro a broad-host-range plasmid carrying the entire meta-cleavage pathway of the Pseudomonas putida TOL plasmid pWW0 . The mutant plasmids were subsequently introduced by conjugal mobilization into a variety of Gram-negative bacteria . The omega fragment carries a selectable marker (aadA+; SpcR/SmR), which is expressed in all species tested, as well as flanking transcription and translation termination signals and synthetic polylinkers . Expression of the plasmid-borne catechol 2,3-dioxygenase (C23O) gene, situated downstream from the site of omega insertion, was substantially reduced in all strains tested . The transcription terminators originally cloned from bacteriophage T4 gene 32, are apparently functional in a wide range of hosts . Insertional mutagenesis with the omega 'interposon' can thus be used in a wide variety of species, with the advantages of a positive selection for the presence of the fragment, the termination of RNA and protein synthesis beyond the site of insertion, and genetic stability of the resulting mutation. Mol Gen Genet, 1985, 200(1), 65 - 7 Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other gram negative bacteria; Boulnois GJ et al.; The properties of pLG221, a derivative of the ColIb plasmid carrying the transposon Tn5 are described . This plasmid can be used to introduce Tn5 by conjugation from Escherichia coli into a variety of Gram negative bacteria outside the host range for maintenance of ColIb . Plasmid pLG221, and a similar plasmid pLG223 carrying Tn10 may be of general utility as vectors for transposon-mediated mutagenesis in a variety of Gram negative bacteria. Basic Life Sci, 1985, 30, 657 - 62 Shuttle vector for Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa; Lushnikov AA et al.; A hybrid plasmid capable of replication in 2 different genera, Escherichia and Pseudomonas, was constructed . This plasmid DNA can be used as a cloning vector in E . coli and pseudomonades cells . The described hybrid plasmid pLD411 has been constructed on the basis of 2 small E . coli vector R-plasmids used in our laboratory and cryptic plasmid pWW2 or P . putida MT1 . Plasmid pLD411 DNA was mapped with restrictases; its biological activity in transformations of different bacterial strains was studied, and the characteristics of transformed cells were also described. J Biol Chem, 1984 Dec 10, 259(23), 14389 - 93 Novel reactivity of cytochrome P-450-CAM . Methyl hydroxylation of 5,5-difluorocamphor; Eble KS et al.; The interaction of the camphor hydroxylating P-450 isolated from Pseudomonas putida grown on camphor (P-450-CAM) with 5,5-difluorocamphor, a substrate analog in which the two methylene hydrogens at the normal site of hydroxylation have been replaced with fluorine, has been examined . This compound binds tightly to the enzyme with a dissociation constant and UV-visible absorption spectrum identical to that observed with d-camphor . In the presence of the reconstituted P-450-CAM system, 5,5-difluorocamphor is metabolized at a rate approximately one-third the rate of the physiological substrate, d-camphor, resulting in the formation of a hydroxylated product with a molecular weight of 204 as well as a minor (less than 3%) hydroxylated product of molecular weight 184 . Isotopically labeled molecular oxygen (18O2) is incorporated into the major product while labeled oxygen from water (H218O) is not incorporated, clearly indicating that the hydroxyl oxygen originates from dioxygen . Proton NMR characterization (400 MHz) of the major product has led to its assignment as 5,5-difluoro-9-hydroxy-camphor, with supporting structural evidence provided by the mass spectral fragmentation pattern . The formation of 9-hydroxylated product represents the first example of methyl hydroxylation catalyzed by cytochrome P-450-CAM, indicates a change in regio-selectivity when the normal site of reaction is blocked, and supports the hypothesis that the delivery of the oxygen atom occurs from the exo side of the camphor molecule. J Bacteriol, 1984 Dec, 160(3), 1003 - 9 Isolation and characterization of Pseudomonas putida PpF1 mutants defective in the toluene dioxygenase enzyme system; Finette BA et al.; Pseudomonas putida PpF1 degraded toluene via a dihydrodiol pathway to tricarboxylic acid cycle intermediates . The initial reaction was catalyzed by a multicomponent enzyme, toluene dioxygenase, which oxidized toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol) . The enzyme consisted of three protein components: NADH-ferredoxintol oxidoreductase (reductasetol), ferredoxintol, and a terminal oxygenase which is an iron-sulfur protein (ISPtol) . Mutants blocked in each of these components were isolated after mutagenesis with nitrosoguanidine . Mutants occurred as colony morphology variants when grown in the presence of toluene on indicator plates containing agar, mineral salts, a growth-supporting nutrient (arginine), 2,3,5-triphenyltetrazolium chloride (TTC), and Nitro Blue Tetrazolium (NBT) . Under these conditions, wild-type colonies appeared large and red as a result of TTC reduction . Colonies of reductasetol mutants were white or white with a light blue center, ferredoxintol strains were light blue with a dark blue center, and strains that lacked ISPtol gave dark blue colonies . Blue color differences in the mutant colonies were due to variations in the extent of NBT reduction . Strains lacking all three components appeared white . Toluene dioxygenase mutants were characterized by assaying toluene dioxygenase activity in crude cell extracts which were complemented with purified preparations of each protein component . Between 40 and 60% of the putative mutants selected from the NBT-TTC indicator plates were unable to grow with toluene as the sole source of carbon and energy . This method should prove extremely useful in isolating mutants in other multicomponent oxygenase enzyme systems. Biochem J, 1984 Dec 1, 224(2), 617 - 21 Stereochemistry of 1-(4'-hydroxyphenyl)ethanol produced by hydroxylation of 4-ethylphenol by p-cresol methylhydroxylase; McIntire W et al.; Enzymic hydroxylation of 4-ethylphenol by (a) Pseudomonas putida and (b) highly purified p-cresol methylhydroxylase gave optically active 1-(4'-hydroxyphenyl)-ethanol . The products were transformed into the phenolic methyl ethers and shown to contain 69.5% and 65.6%, respectively, of the (S)-(-)-isomer . The stereochemistry of the reaction is discussed in terms of three distinct steps occurring at the active site of the enzyme. J Biochem (Tokyo), 1984 Nov, 96(5), 1587 - 91 Formaldehyde dehydrogenase from Pseudomonas putida: a zinc metalloenzyme; Ogushi S et al.; The NAD+-dependent formaldehyde dehydrogenase from Pseudomonas putida C-83 was found to contain 4 gram atoms of zinc per mol, corresponding to 2 gram atoms of zinc per subunit monomer . Treatment of the enzyme with o-phenanthroline resulted in removal of 1 gram atom of zinc per subunit and caused a complete inactivation of the enzyme . The activity lost was restored by the addition of zinc ions, by which the zinc content was also reversed to almost the same level as that of the native enzyme . Another zinc atom that was resistant to metal chelator-treatment was liberated from the enzyme only after the irreversible denaturation of the enzyme . These results indicate that the formaldehyde dehydrogenase of P . putida is a zinc metalloenzyme and one of two zinc atoms per subunit participates in the catalytic activity of the enzyme, another zinc being presumably involved in maintaining the native conformation of the enzyme . Treatment of the enzyme with bipyridine also caused a reversible inactivation of the enzyme, but the zinc content remained unchanged . The spectrophotometric analysis indicated that the formation of a enzyme-Zn-bipyridine complex took place . Incubation of the enzyme with p-chloromercuribenzoate also resulted in a complete loss of the activity . These results suggest that an intrinsic zinc and sulfhydryl group together with NAD+ participate in the dehydrogenation reaction of substrate by the enzyme. J Bacteriol, 1984 Nov, 160(2), 797 - 800 TOL plasmid can prevent induction of chemotactic responses to aromatic acids; Harwood CS et al.; Growth conditions that elicited positive chemotaxis to benzoate and m-toluate in TOL- Pseudomonas putida cells failed to elicit taxis to these compounds in TOL+ cells . The inability of TOL+ cells to respond to these aromatic acids appears to be due to the preferential expression of TOL-encoded genes for aromatic degradation over chromosomally encoded genes . Expression of chromosomal genes for aromatic degradation is required for cells to form beta-ketoadipate, the inducer of benzoate and m-toluate taxis. J Bacteriol, 1984 Nov, 160(2), 622 - 8 Aromatic acids are chemoattractants for Pseudomonas putida; Harwood CS et al.; A quantitative capillary assay was used to show that aromatic acids, compounds that are chemorepellents for Escherichia coli and Salmonella sp., are chemoattractants for Pseudomonas putida PRS2000 . The most effective attractants were benzoate; p-hydroxybenzoate; the methylbenzoates; m-, p-, and o-toluate; salicylate; DL-mandelate; beta-phenylpyruvate; and benzoylformate . The chemotactic responses to these compounds were inducible . Taxis to benzoate and m-toluate was induced by beta-ketoadipate, a metabolic intermediate formed when benzoate is dissimilated via enzymes specified by chromosomal genes . Benzoylformate taxis was induced by benzoylformate and L(+)-mandelate . Taxis to mandelate, benzoylformate, and beta-phenylpyruvate was exhibited by cells grown on mandelate, but not by cells grown on benzoate . Cells grown on benzoate were chemotactic to benzoate, the toluates, p-hydroxybenzoate, and salicylate . These results show that P . putida synthesizes at least two distinct chemoreceptors for aromatic acids . Although DL-mandelate was an effective attractant in capillary assays, additional experiments indicated that the cells were actually responding to benzoylformate, a metabolite formed from mandelate . With the exception of mandelate taxis, chemotaxis to aromatic acids was not dependent on the expression of pathways for aromatic degradation . Therefore, the tactic responses exhibited by cells cannot be attributed to an effect of the oxidation of aromatic acids on the energy metabolism of cells. EMBO J, 1984 Nov, 3(11), 2461 - 6 Transcription of the TOL plasmid toluate catabolic pathway operon of Pseudomonas putida is determined by a pair of co-ordinately and positively regulated overlapping promoters; Mermod N et al.; Expression of the meta-cleavage pathway operon of TOL plasmid pWW0 of Pseudomonas putida is positively regulated by the xylS gene product . We have sequenced the promoter region of this operon and localized the transcription initiation sites . Two overlapping promoters, designated Pm1 and Pm2, are responsible for the positively regulated expression of the meta-pathway operon . Mutants of P . putida were isolated that expressed the meta-cleavage pathway operon constitutively . Several plasmid-located mutations that led to constitutivity were characterized by sequencing and the transcription initiation sites on mutant plasmids localized . This resulted in the identification of newly created promoters whose functioning did not require the xylS product . Comparison of the promoter sequences obtained suggests a tentative consensus sequence for promoters of P . putida which is significantly different from that of E . coli. J Bacteriol, 1984 Oct, 160(1), 279 - 87 Genetic and physical analyses of Caulobacter crescentus trp genes; Winkler ME et al.; Caulobacter crescentus trp mutants were identified from a collection of auxotrophs . Precursor feeding experiments, accumulation studies, and complementation experiments resulted in the identification of six genes corresponding to trpA, trpB, trpC, trpD, trpE, and trpF . Genetic mapping experiments demonstrated that the trp genes were in two clusters, trpCDE and trpFBA, and a 5.4-kilobase restriction fragment from the C . crescentus chromosome was isolated that contained the trpFBA gene cluster . Complementation experiments with clones containing the 5.4-kilobase fragment indicated that trpF was expressed in Escherichia coli and that all three genes were expressed in Pseudomonas putida . This expression was lost in both organisms when the pBR322 tet gene promoter was inactivated, indicating that all three genes were transcribed in the same orientation from the tet promoter . Thus, the C . crescentus promoters do not seem to be expressed in E . coli or P . putida . Complementation of the C . crescentus trp mutants indicated that the tet promoter was not necessary for expression in C . crescentus and suggested that at least two native promoters were present for expression of the trpF, trpB, and trpA genes . Taken together, these results indicate that C . crescentus promoters may have structures that are significantly different from the promoters of other gram-negative species. J Bacteriol, 1984 Oct, 160(1), 251 - 5 Transposon mutagenesis analysis of meta-cleavage pathway operon genes of the TOL plasmid of Pseudomonas putida mt-2; Harayama S et al.; Hybrid plasmids containing the regulated meta-cleavage pathway operon of TOL plasmid pWWO were mutagenized with transposon Tn1000 or Tn5 . The resulting insertion mutant plasmids were examined for their ability to express eight of the catabolic enzymes in Escherichia coli . The physical locations of the insertions in each of 28 Tn1000 and 5 Tn5 derivative plasmids were determined by restriction endonuclease cleavage analysis . This information permitted the construction of a precise physical and genetic map of the meta-cleavage pathway operon . The gene order xylD (toluate dioxygenase), L (dihydroxycyclohexidiene carboxylate dehydrogenase), E (catechol 2,3-dioxygenase), G (hydroxymuconic semialdehyde dehydrogenase), F (hydroxymuconic semialdehyde hydrolase), J (2-oxopent-4-enoate hydratase), I (4-oxalocrotonate decarboxylase), and H (4-oxalocrotonate tautomerase) was established, and gene sizes were estimated . Tn1000 insertions within catabolic genes exerted polar effects on distal structural genes of the operon, but not on an adjacent regulatory gene xylS. Mikrobiologiia, 1984 Sep-Oct, 53(5), 822 - 5 {Phosphate and glucose accumulation by Pseudomonas cultures in relation to their arsenic resistance}; Mynbaeva BN et al.; The effect of arsenite and arsenate on 14C-glucose and 32-P-phosphate transport was studied in the cells of Pseudomonas aeruginosa 561 sensitive to arsenite and in the cells of Pseudomonas putida 18 oxidizing arsenite and resistant to arsenic . Transport and accumulation of phosphate and glucose were inhibited in the presence of arsenite in the cells of P . aeruginosa 561 whereas arsenate inhibited only phosphate accumulation . Arsenite and arsenate had hardly any effect at the initial transport rate and on the overall accumulation of phosphate and glucose in the cells of P . putida 18 . The resistance to arsenite is supposed to be caused by selective impermeability of the cellular membranes to arsenite and arsenate. Gene, 1984 Sep, 29(3), 323 - 30 Nucleotide sequence of the promoter region of the xylDEGF operon on TOL plasmid of Pseudomonas putida; Inouye S et al.; The transcription initiation site of the xylDEGF operon on the TOL plasmid of Pseudomonas putida mt-2 was determined in P . putida and in Escherichia coli by S1 nuclease and reverse transcriptase mapping . The induced synthesis of mRNA started at the same start point in both P . putida and E . coli, although the amount of mRNA in E . coli cells was less than that in P . putida . The nucleotide sequence of the region surrounding the start point was also determined . The ribosome-binding site (RBS) complementary to the 3' end of the 16S rRNA of Pseudomonas aeruginosa and E . coli preceded the predicted start codon for the xylD gene . The consensus nucleotide sequence for E . coli promoters was not found in the region preceding the transcription start point . On the other hand, the sequences of the "-10" and the "-35" regions of the xylDEGF operon revealed some homology with the respective, previously determined sequences of the xylABC operon of the TOL plasmid. J Biol Chem, 1984 Aug 10, 259(15), 9790 - 8 Sequence of the small subunit of yeast carbamyl phosphate synthetase and identification of its catalytic domain; Nyunoya H et al.; The yeast gene CPA1 coding for the small subunit of arginine-specific carbamyl phosphate synthetase has been cloned by complementation of a cpa 1 mutant with a plasmid library of total yeast chromosomal DNA . Two of the plasmids, pJL113/ST4 and pJL113/ST15, contain DNA inserts in opposite orientations with overlapping sequences of 2.6 kilobases . The nucleotide sequence of a 2.2-kilobase region of the DNA insert carrying the CPA1 gene has been determined . The CPA1 gene has been identified to be 1233 nucleotides long and to code for a polypeptide of 411 amino acids with a calculated molecular weight of 45,358 . The amino acid sequence encoded in CPA1 is homologous to the recently determined sequence of the small subunit of Escherichia coli carbamyl phosphate synthetase (Piette, J., Nyunoya, H., Lusty, C.J., Cunin, R., Weyens, G., Crabeel, M., Charlier, D., Glandsdorff, N., and Pierard, A . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 4134-4138) over the entire length of the polypeptide chain . Comparison of the amino acid sequences of the small subunits of yeast and E . coli carbamyl phosphate synthetases to the sequences of Component II of anthranilate and p-aminobenzoate synthases suggests that these amidotransferases are evolutionarily related . The most highly conserved region of the yeast and E . coli enzymes includes a cysteine residue previously found to be at the active site of Pseudomonas putida anthranilate synthase Component II (Kawamura, M., Keim, P.S., Goto, Y., Zalkin, H., and Heinrikson, R.L . (1978) J . Biol . Chem . 253, 4659-4668) . Based on the observed homologies in the primary sequences of the other amidotransferases examined, we propose a 13-amino acid long sequence to be part of the catalytic domain of this class of enzymes. Steroids, 1984 Aug, 44(2), 175 - 93 Phenolic 9,10-secosteroids as products of the catabolism of bile acids by a Pseudomonas sp; Park RJ; The obligate aerobe, Pseudomonas putida ATCC 31752, efficiently utilises bile acids as a source of carbon and energy for growth and maintenance . When aeration is considerably restricted, a consequence to the catabolism of the bile acids in a fermentor is an accumulation of certain steroidal catabolites . Evidence is presented to show that among these are hydroxy-9,10-seco-1,3,5 (10)-androstratriene-9, 17-diones and those from four of the common bile acids, cholic, chenodeoxycholic, hyodeoxycholic and deoxycholic acids have been isolated and their structures determined . The product of catabolism of hyodeoxycholic acid appears to exist in a hemi-acetal form which readily forms an acetal during isolation procedures . All but one of these are described for the first time. J Bacteriol, 1984 Aug, 159(2), 678 - 82 Novel system for recognizing and eliminating foreign DNA in Pseudomonas putida; Kim K et al.; Derivatives of pQSR49 (R1162::Tn1) containing cloned fragments of Escherichia coli chromosomal DNA are stable in E . coli but unstable in Pseudomonas putida . Similar derivatives containing P . putida chromosomal DNA are stable in both species . Instability is a consequence of plasmid loss during growth and is not due to death or inhibition of growth of plasmid-containing cells . Average copy numbers per cell of the unstable hybrid plasmids are similar to that of pQSR49, indicating that instability is not the result of reduced replication of plasmid DNA . A plasmid unrelated to pQSR49, RK2, becomes unstable in the presence of the unstable hybrids but only when it also contains foreign DNA . The data suggest an inducible mechanism in P . putida active in the elimination of plasmid-borne foreign DNA from the cell. J Biol Chem, 1984 Jul 10, 259(13), 8091 - 4 Resolution of 5-oxo-L-prolinase into a 5-oxo-L-proline-dependent ATPase and a coupling protein; Seddon AP et al.; 5-Oxo-L-prolinase catalyzes a reaction in which the endergonic cleavage of 5-oxo-L-proline to form L-glutamate is coupled to the exergonic cleavage of ATP to ADP and Pi . In the present research, the enzyme present in a strain of Pseudomonas putida isolated from soil by enrichment culture was found to be composed of two protein components . Neither component alone could catalyze the 5-oxoprolinase reaction, but the reaction was effectively catalyzed when they were mixed . One component (A) exhibited 5-oxo-L-proline-dependent ATPase activity indicating that Component A can interact with both ATP and 5-oxo-L-proline . The other component (coupling protein; B) does not exhibit ATPase activity nor is there evidence that it binds 5-oxo-L-proline . The findings are consistent with (but do not prove) the hypothesis that the Component A catalyzes an initial step in the reaction which involves 5-oxoproline and ATP, such as phosphorylation of 5-oxoproline . The coupling protein (B) may function as a catalyst that converts a phosphorylated form of 5-oxoproline to glutamate, or it might alter the conformation of Component A so as to facilitate the reaction. Appl Environ Microbiol, 1984 Jul, 48(1), 108 - 13 Effect of restricted aeration on catabolism of cholic acid by two Pseudomonas species; Smith MG et al.; Examination of some previously isolated bile acid-utilizing Pseudomonas strains showed that Pseudomonas sp . ATCC 31752, together with other fluorescent strains, can be assigned to Pseudomonas putida biotype B, whereas Pseudomonas sp . ATCC 31753, like most other nonfluorescent strains, is an unrecognized phenotype . A study was made of the growth of these two species at 25 degrees C and pH 7.0 in a fermentor with 2.5 g of sodium cholate liter-1 as sole carbon source, and the catabolism of the cholate and its products was followed by high-pressure liquid chromatographic and thin-layer chromatographic examination . At aeration rates of either 150 or 5 ml min-1 liter-1, growth of each species followed the same catabolic pathway . 7 alpha, 12 beta-Dihydroxy-1,4-androstadiene-3,17-dione was the major catabolite formed, with 0.3 g liter-1 being the maximum concentration that accumulated at the higher aeration rate, whereas 1.4 g liter-1 accumulated at the lower aeration rate, irrespective of the species used . The latter yield is sufficiently high to be of potential commercial value if such a catabolite were found to be economically useful for steroid drug manufacture . It is postulated that the rate-limiting step in cholic acid catabolism by these species at the lower aeration rate is 9 alpha-hydroxylation, a step requiring molecular oxygen, hence, the marked effect of oxygen limitation on catabolite accumulation . Another consequence of oxygen limitation is the production of a red pigment in the culture medium, which, however, does not affect catabolite recovery. J Bacteriol, 1984 Jun, 158(3), 920 - 7 Isolation and expression of Rhizobium japonicum cloned DNA encoding an early soybean nodulation function; Sutton BC et al.; A first visible step in the nodulation of legumes by Rhizobium spp . is the deformation and curling of root hairs . We have identified and cloned DNA sequences encoding this function from two strains of Rhizobium japonicum (USDA 122 and USDA 110) with a weakly homologous probe from Rhizobium meliloti . Root hair curling encoded by the cloned DNA fragments was examined on soybeans (Glycine soja ) after conjugative transfer of these sequences in broad-host-range vectors to various bacterial genera . Pseudomonas putida gave unambiguous expression of the root hair curling genes . This enabled us to identify the 8.7-kilobase EcoRI fragments encoding root hair curling from each strain . The phenotypes encoded by the plasmids pBS1 (derived from strain USDA 122) and pBS2 (derived from strain USDA 110) are distinct and represent a phenotype characteristic of their parent R . japonicum strains . Subclones of pBS1 and pBS2 were generated in single and multicopy vectors, and their expression was analyzed in P . putida . We established that a 4.2-kilobase internal Sa/I fragment of pBS1 and a 3.5-kilobase SstI -EcoRI fragment of pBS2 are sufficient to confer root hair curling on soybeans. J Bacteriol, 1984 Jun, 158(3), 1025 - 32 Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp . strain B13; Lehrbach PR et al.; DNA fragments containing the xylD and xylL genes of TOL plasmid pWW0 -161 of Pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahG gene of the NAH plasmid NAH7 , which codes for salicylate hydroxylase, were cloned in pBR322 vector plasmid . Deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites . The pBR322-based plasmids were ligated to the broad host range cloning vector pKT231 , or derivatives of it, and the hybrid plasmids were introduced into Pseudomonas sp . B13( WR1 ), a bacterium able to degrade 3-chlorobenzoate but not 4-chlorobenzoate, 3,5- dichlorobenzoate , salicylate, or chlorosalicylates . The cloned xylD gene expanded the catabolic range of WR1 to include 4-chlorobenzoate, whereas the cloned xylD - xylL genes enabled the isolation of derivatives of WR1 that degraded 3-chlorobenzoate, 4-chlorobenzoate, and 3,5- dichlorobenzoate . The cloned nahG gene extended the catabolic range of WR1 to include salicylate and 3-, 4-, and 5- chlorosalicylate . Genetika, 1984 Jun, 20(6), 907 - 14 {Introduction of the hybrid plasmid RP4::D3112 into Pseudomonas putida cells requires the presence of specific mutation in the phage genome}; Gorbunova SA et al.; The wild type of D3112, a transposable phage of Pseudomonas aeruginosa can not be introduced as a portion of the hybrid plasmid RP4::D3112 into Pseudomonas putida cells . It is only possible when phage D3112 carries mutations designated lpc (lethal for P . putida and Escherichia coli) . Analysis of heteroduplex molecules between DNAs of phages D3112w+ and D3112lpc demonstrated the absence of nonhomology regions, which suggests that lpc is a point mutation . The lpc2 mutation was located within the interval 20-29.9 kb of the phage genome. Biochemistry, 1984 May 22, 23(11), 2478 - 82 Structural analysis of the cysteine-containing peptides from the major 3-methylcholanthrene-induced isozyme of cytochrome P-450 (P-450c) in rat liver microsomes; Haniu M et al.; Cytochrome P-450c, the major 3-methylcholanthrene-inducible isozyme of cytochrome P-450 in rat liver microsomes, was subjected to proteolytic digestion after S-carboxymethylation of the protein, and the peptides were resolved by high-pressure liquid chromatography . Since it is now recognized that cytochromes P-450 contain a thiolate as the axial fifth ligand of the heme, seven peptides containing eight cysteines were subjected to microsequence analysis . One cysteine-containing peptide (Tsa-56) was shown to possess 46-69% homology with a common peptide found in five other cytochromes P-450 but is not anticipated to be the heme-binding segment on the basis of X-ray crystallographic results obtained with Pseudomonas putida cytochrome P-450cam . Analysis of the other cysteine-containing peptides in cytochrome P-450c revealed two peptides (Tsa-54 and T-46) of only limited homology with the highly conserved region of cytochromes P-450cam, P-450LM2, P-450b, and P-450e that are all presumed to contain the heme-binding cysteine . Another peptide that contained two cysteines and a stretch of hydrophobic residues (Tsa-47) had limited sequence homology with a similar peptide found in several other cytochromes P-450 . This domain is located a short distance from the proposed heme-binding cysteine in other cytochromes P-450 . Sequence analysis of a cysteine-containing peptide (T-30) from another 3-methylcholanthrene-inducible rat liver cytochrome P-450 (cytochrome P-450d) revealed 91% homology with peptide T-46 from cytochrome P-450c, but this peptide shows no significant homology with any of the cysteine-containing peptides from other cytochromes P-450.(ABSTRACT TRUNCATED AT 250 WORDS) Anal Biochem, 1984 May 1, 138(2), 421 - 4 Purification of bacterial L-methionine gamma-lyase; Nakayama T et al.; A rapid procedure for the purification of L-methionine gamma-lyase from Pseudomonas putida ICR 3460 by DEAE- TOYOPEARL 650M and DEAE-Sephadex A-50 column chromatography is presented . The enzyme was purified with an average yield of 75% and showed about 10-fold higher specific activity than the enzyme from P . putida (= P . ovalis) IFO 3738 reported previously (H . Tanaka, N . Esaki , and K . Soda (1976) FEBS Lett . 66, 307-311) . The present enzyme has a molecular weight of about 172,000 and consists of four subunits with identical molecular weights (43,000) . It shows the typical absorption spectrum of pyridoxal enzyme with maxima at 278 and 420 nm, and contains 4 mol of pyridoxal 5'-phosphate per mole of enzyme . The enzyme has a multicatalytic function similar to the enzyme of P . putida IFO 3738 (K . Soda, H . Tanaka, and N . Esaki (1983) Trends Biochem . Sci . 8, 214-217). Mikrobiologiia, 1984 May-Jun, 53(3), 471 - 5 {Effect of plasmids from various incompatibility groups on the development of bacteriophages specific to Pseudomonas aeruginosa and Pseudomonas putida}; Kulakov LA et al.; The aim of this work was to study the effect of plasmids belonging to different incompatibility groups on the growth of bacteriophages in Pseudomonas aeruginosa and Pseudomonas putida strains . The growth of bacteriophages was shown to be limited most often due to the presence in cells of plasmids belonging to the P-2 incompatibility group . Plasmids of the Inc P-2 group differed from one another in the spectrum of bacteriophages whose growth they limited . Phages whose growth was suppressed in strains containing plasmids of the P-5, P-9 or P-10 incompatibility groups were found . Some plasmids showed no specific interaction with bacteriophages . The plasmids investigated differed in the studied trait in P . aeruginosa and P . putida cells . In contrast to P . aeruginosa PAO, P . putida PpGI plasmid containing cells did not maintain the growth of donor-specific bacteriophages and, to a lesser degree, limited the growth of phages specific for P . putida PpGI. J Gen Microbiol, 1984 May, 130 ( Pt 5), 1169 - 81 The use of Mudlac transposons as tools for vital staining to visualize clonal and non-clonal patterns of organization in bacterial growth on agar surfaces; Shapiro JA; When a histochemical stain for beta-galactosidase activity is applied to growth of Gram-negative bacteria on agar medium, the pigmentation is non-uniform and capable of revealing internal colony organization into different cell types . Use of an Escherichia coli strain with a thermosensitive lac repressor indicates that colonies expand by addition of new cells at the periphery and that older cells which have synthesized beta-galactosidase early in development remain in the centre . Mixed inocula of different strains show clonal exclusiveness as they proliferate outwards . Mudlac transposons can create genetic fusions that place beta-galactosidase expression under a variety of regulatory systems . Stained surface cultures of E . coli and Pseudomonas putida strains carrying Mudlac insertions in plasmids reveal a variety of flower-like staining patterns . These patterns display both clonal (i.e . sectorial) and non-clonal (circular and radial) features which are heritable within a given strain . The non-clonal aspects of the patterns reflect phenotypic differentiation without genetic change . These observations indicate that bacterial growth on agar surfaces is a highly regulated process similar, in many respects, to the development of specific multicellular tissues and organisms. J Bacteriol, 1984 May, 158(2), 597 - 602 Attachment of diaminopimelic acid to bdelloplast peptidoglycan during intraperiplasmic growth of Bdellovibrio bacteriovorus 109J; Ruby EG et al.; An early event in the predatory lifestyle of Bdellovibrio bacteriovorus 109J is the attachment of diaminopimelic acid (DAP) to the peptidoglycan of its prey . Attachment occurs over the first 60 min of the growth cycle and is mediated by an extracellular activity(s) produced by the bdellovibrio . Some 40,000 DAP residues are incorporated into the Escherichia coli bdelloplast wall, amounting to ca . 2 to 3% of the total initial DAP content of its prey cells . Incorporation of DAP occurs when E . coli, Pseudomonas putida, or Spirillum serpens are the prey organisms . The structurally similar compounds lysine, ornithine, citrulline, and 2,4-diaminobutyric acid are not attached . The attachment process is not affected by heat-killing the prey nor by the addition of inhibitors of either energy generation (cyanide, azide, or arsenate), protein or RNA synthesis (chloramphenicol and rifamycin), or de novo synthesis of cell wall (penicillin or vancomycin) . Approximately one-third of the incorporated DAP is exchangeable with exogenously added unlabeled DAP, whereas the remaining incorporated DPA is solubilized only during the lysis of the bdelloplast wall . Examination of DAP incorporation at low prey cell densities suggests that bdellovibrios closely couple the incorporation to an independent, enzymatic solubilization of DAP by a peptidase . The data indicate that DAP incorporation is a novel process, representing the second example of the ability of the bdellovibrio to biosynthetically modify the wall of its prey. J Bacteriol, 1984 Apr, 158(1), 389 - 92 Plasmid-determined silver resistance in Pseudomonas stutzeri isolated from a silver mine; Haefeli C et al.; A silver-resistant strain of Pseudomonas stutzeri was isolated from a silver mine . It harbored three plasmids, the largest of which (pKK1; molecular weight, 49.4 X 10(6)) specified silver resistance . Plasmid pKK1 was apparently nonconjugative but could be transferred to Pseudomonas putida by mobilization with plasmid R68.45. FEBS Lett, 1984 Mar 26, 168(2), 265 - 70 Relationship of lipoamide dehydrogenases from Pseudomonas putida to other FAD-linked dehydrogenases; Delaney R et al.; Pseudomonas putida produces two lipoamide dehydrogenases, LPD-glc and LPD-val . LPD-val is specifically required as the lipoamide dehydrogenase of branched-chain keto acid dehydrogenase and LPD-glc fulfills all other requirements for lipoamide dehydrogenase . Both proteins are dimers with one FAD per subunit . LPD-glc has an absorption maximum at 455 nm, but LPD-val has a maximum at 460 nm . Comparison of amino acid compositions revealed that LPD-glc was more closely related to Escherichia coli and pig heart lipoamide dehydrogenase than to LPD-val . LPD-val did not appear to be closely related to any of the proteins compared with the possible exception of mercuric reductase. Proc Natl Acad Sci U S A, 1984 Mar, 81(6), 1664 - 8 Camphor hydroxylase of Pseudomonas putida: vestiges of sequence homology in cytochrome P-450CAM, putidaredoxin, and related proteins; Dus KM; The amino acid sequences of cytochrome P-450CAM and putidaredoxin of the camphor hydroxylase {camphor, reduced-putida-ferredoxin:oxygen oxidoreductase (5-hydroxylating), EC 1.14.15.1} of Pseudomonas putida are compared to each other and then to the sequences of bovine adrenodoxin and cytochrome b5 . The comparisons reveal areas of homology indicating that these four proteins may share a common evolutionary origin . Moreover, homologous segments can be recognized by proper alignment of the sequence of cytochrome P-450CAM to recently determined sequences of other P-450 hemeproteins . Further scrutiny indicates vestiges of internal sequence duplications that have been conserved in limited segments by the constraints of selection over a long time period, while other segments of the sequence diverged . It is predicted that the corresponding reductases will show a similar sequence pattern . The conserved regions of internal sequence repetition may serve a special purpose in protein-protein interactions within the multienzyme systems. Proc Natl Acad Sci U S A, 1984 Mar, 81(6), 1649 - 53 Coding nucleotide sequence of 3-methylcholanthrene-inducible cytochrome P-450d cDNA from rat liver; Kawajiri K et al.; We determined the coding nucleotide sequence of the mRNA for a 3-methylcholanthrene-inducible cytochrome P-450, P-450d, of rat liver by sequence analysis of cloned cDNAs . The predicted amino acid sequence of the cytochrome is composed of 513 amino acids, and its NH2-terminal sequence of 30 amino acids completely coincides with that reported from analysis of the purified cytochrome P-450d . The amino acid composition of the deduced sequence also agrees well with that determined from the purified protein . Computer-aided analysis was carried out to compare the complete primary structures of five species of cytochrome P-450, two molecular species of phenobarbital-inducible rat liver cytochrome P-450 (P-450b and P-450e), phenobarbital-inducible rabbit liver cytochrome P-450LM2, 3-methylcholanthrene-inducible rat liver cytochrome P-450d, and camphor-hydroxylating P-450 of Pseudomonas putida (P-450CAM) . It is concluded therefrom that the time of divergence between cytochrome P-450b (P-450e) and P-450d is much earlier than that of branching between phenobarbital-inducible cytochromes P-450 of rat and rabbit . One highly conserved cysteine-containing region that is close to the COOH terminus is found in all of these cytochrome P-450 sequences, indicating that the heme-binding site is the cysteine residue in this region. Proc Natl Acad Sci U S A, 1984 Mar, 81(6), 1688 - 91 Nucleotide sequence surrounding transcription initiation site of xylABC operon on TOL plasmid of Pseudomonas putida; Inouye S et al.; The xylABC operon on the TOL plasmid directs the synthesis of enzymes for conversion of toluene to benzoate and is positively controlled by the regulatory gene xylR . In the study here the nucleotide sequence was determined for the regulatory region of this operon . The in vivo transcription initiation site of the operon was determined by S1 nuclease and reverse transcriptase mapping . RNA was prepared from m-methylbenzyl alcohol-induced cells of Pseudomonas putida and Escherichia coli carrying pTN2, a derivative of the TOL plasmid containing the structural and regulatory genes of the entire toluene-degrading pathway . The amount of E . coli mRNA was estimated to be only 10% of that of P . putida mRNA . Consensus sequences of the -10 region (Pribnow box) and the -35 region (RNA polymerase recognition site) in E . coli genes were not found in the region preceding the transcription initiation site, whereas a sequence complementary to the 3' end of the 16S rRNA of Pseudomonas aeruginosa and E . coli existed in front of the predicted start codon of the xylA gene . These results explain the inefficient expression of TOL genes in E . coli. J Bacteriol, 1984 Mar, 157(3), 937 - 9 Use of ureidopenicillins for selection of plasmid vector transformants in Pseudomonas aeruginosa and Pseudomonas putida; Day DL et al.; Broad-host-range plasmids coding for beta-lactamase were successfully selected after transformation of Pseudomonas strains . Transformants of both Pseudomonas aeruginosa and Pseudomonas putida containing plasmid pRO1614 were isolated in media containing low concentrations of piperacillin . These strains were also susceptible to other ureidopenicillins . Similar selections of transformants with carbenicillin, ampicillin, or ticarcillin required high concentrations of antibiotics and yielded backgrounds of spontaneous resistant mutants. Arch Biochem Biophys, 1984 Feb 1, 228(2), 660 - 6 Oxidation of glycine by Pseudomonas putida requires a specific lipoamide dehydrogenase; Sokatch JR et al.; Pseudomonas putida produces two lipoamide dehydrogenases with molecular weights of 49,000 and 56,000 designated LPD-val and LPD-glc, respectively . LPD-val is required for oxidation of valine, since it is specifically utilized as the E3 component of branched-chain keto acid dehydrogenase . Since glycine oxidation by bacteria and mammals also requires lipoamide dehydrogenase, we desired to determine which lipoamide dehydrogenase would be used by the P . putida glycine oxidation system . When grown in a medium with glycine as the sole nitrogen source, P . putida produced a single lipoamide dehydrogenase with a molecular weight of 56,000 and which reacted with antiserum to LPD-glc . The partially purified glycine oxidation system from P . putida was stimulated by LPD-glc but not by LPD-val and was inhibited by anti-LPD-glc, but not by anti-LPD-val . It was not possible to detect LPD-val in extracts of cells grown in glucose-glycine medium by the use of anti-LPD-val . LPD-glc was five times as active as LPD-val in catalyzing the oxidation of purified protein H, the heat-stable, lipoic acid-containing protein of the glycine oxidation system . These results indicate that LPD-glc is specifically utilized for glycine oxidation in P . putida. J Bacteriol, 1984 Feb, 157(2), 385 - 90 Permeability of the boundary layers of Bdellovibrio bacteriovorus 109J and its bdelloplasts to small hydrophilic molecules; Cover WH et al.; Measurements of the sucrose-permeable and -impermeable volumes during Bdellovibrio bacteriovorus attack on Escherichia coli or Pseudomonas putida showed that the volume of the bdelloplast increased over that of the substrate cell . Although the pattern of the increase differed with the two organisms, the volumes reached maximum at about 60 min into the bdellovibrio growth cycle . By this time, the cytoplasmic membranes of the attacked cells were completely permeable to sucrose . The kinetics of increase in sucrosepermeable volumes were similar to the kinetics of attachment and penetration (Varon and Shilo, J . Bacteriol . 95:744-753, 1968) . These data show that the original cytoplasmic and periplasmic compartmentalization of the substrate cell ceases to exist with respect to small hydrophilic molecules during bdellovibrio attack . In contrast, the effective pore size of the outer membrane of the substrate cell to small oligosaccharides remains unaltered during bdelloplast formation as was shown by direct measurements of its exclusion limits . The major porin protein of E . coli, OmpF, was recoverable from the bdelloplast outer membrane fraction until the onset of lysis . The Braun lipoprotein was removed from the bdelloplast wall early, and OmpA was lost in the terminal part of the bdellovibrio growth cycle. Biochem J, 1984 Feb 1, 217(3), 667 - 73 An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy; Geary PJ et al.; Benzene dioxygenase from Pseudomonas putida comprises three components, namely a flavoprotein (NADH:ferredoxin oxidoreductase; Mr 81000), an intermediate electron-transfer protein, or ferredoxin (Mr 12000) with a {2Fe-2S} cluster, and a terminal dioxygenase containing two {2Fe-2S} iron-sulphur clusters (Mr 215000), which requires two additional Fe2+ atoms/molecule for oxygenase activity . The ferredoxin and the dioxygenase give e.s.r . signals in the reduced state with rhombic symmetry and average g values of 1.92 and 1.896 respectively . The mid-point redox potentials were determined by e.s.r . titration at pH 7.0 to be -155 mV and -112 mV respectively . The signal from the dioxygenase shows pronounced g anisotropy and most closely resembles those of 4-methoxybenzoate mono-oxygenase from Pseudomonas putida and the {2Fe-2S} 'Rieske' proteins of the quinone-cytochrome c region of electron-transport chains of respiration and photosynthesis. J Biol Chem, 1984 Jan 10, 259(1), 249 - 54 Hydroxyproline 2-epimerase of Pseudomonas . Subunit structure and active site studies; Ramaswamy SG; Hydroxyproline 2-epimerase of Pseudomonas putida was purified to homogeneity by an improved procedure . The native enzyme consists of two probably identical subunits . Alkylation of the active site with labeled reagents resulted in the loss of 80-85% of the activity but the incorporation of only one alkyl group even though the active site contains a Cys residue from each of the two subunits . This result suggests that the enzyme shows half-site reactivity . The labeled enzyme was further subjected to exhaustive alkylation with unlabeled iodoacetate, permitting tryptic hydrolysis and isolation of an active site peptide in 30% yield . The specific radioactivity of the peptide was consistent with the first result, that only 1 mol of alkyl group was initially incorporated into active site . The active site peptide (14 residues) was sequenced and found to possess homology with the clostridial proline racemase. Folia Microbiol (Praha), 1984, 29(3), 242 - 7 Degradation of 3-chlorobenzoate in soil by pseudomonads carrying biodegradative plasmids; Pertsova RN et al.; Degradation of continuously added 3-chlorobenzoate (3-CB) was studied in samples of chernozem soil . Soil columns were inoculated with Pseudomonas putida growing on 3-CB and carrying the biodegradation plasmid and with Pseudomonas aeruginosa incapable of growth on 3-CB and carrying the inserted biodegradation plasmid pBS 2 determining ortho-cleavage of the aromatic ring . While the 3-CB degradation was observed in both inoculated variants, the native microflora of the soil under study was incapable to degrade 3-CB . Among pseudomonads isolated from inoculated soil at different stages of cultivation and growth on 3-CB, some had the taxonomic features of P . putida as well as those differing in 1-5 characteristics . The study of the activities of the enzymes cleaving the aromatic ring revealed the presence of pyrocatechol 1,2-dioxygenase in the isolated strains only, as estimated by means of benzoate and 3-CB as substrates. Mol Gen Genet, 1984, 197(3), 384 - 91 Reversal by DNA amplifications of an unusual mutation blocking alkane and alcohol utilization in Pseudomonas putida; McBeth DL et al.; We analyzed the reversion of strains carrying alk208, a mutation in the alkBAC (alkane utilization) region of the Pseudomonas CAM-OCT plasmid . Reversion of alk208 was stimulated 25 to 75-fold by small doses of UV-irradiation . All alkane hydroxylase-positive (AlkB+) revertants proved to be aliphatic alcohol dehydrogenase-positive (AlkC+) as well, whereas AlkC+ revertants could be either AlkB+ or AlkB- . Most of the AlkB- AlkC+ partial revertants produced AlkC- segregants at measurable frequencies . UV-irradiation substantially increased the rate of AlkC- segregation . Most segregants reverted to AlkB+ or AlkC+ at frequencies similar to the original alk208 strain . Dot blot hybridization analyses using cloned probes from various regions of CAM-OCT revealed that the partial revertants contained specific amplications of alk DNA . The endpoints of these amplifications mapped in at least two regions . AlkC- segregants had lost the DNA amplifications. Biomed Biochim Acta, 1984, 43(11), K17 - 24 Chemical modification of tyrosine residues at the active centre of cytochrome P-450 CAM; Janig GR et al.; Soluble cytochrome P-450 CAM from Pseudomonas putida (EC 1.14.14.1) was chemically modified with tetranitromethane . At least five out of totally nine tyrosine residues are accessible to nitration as shown by tryptic peptide mapping using HPLC . Modification in the presence of the inhibitor metyrapone and subsequent peptide mapping indicate the location of one tyrosine residue at the active centre of cytochrome P-450 CAM. Prog Clin Biol Res, 1984, 144A, 355 - 63 Purification and properties of amino acid racemase from Aeromonas punctata subsp . caviae; Inagaki K et al.; An amino acid racemase, which occurs in the cytoplasmic fraction of Aeromonas punctata subsp . caviae, has been purified to homogeneity by the criteria of electrophoresis and ultracentrifugation . The enzyme has a molecular weight of about 80,000 and consists of two subunits identical in molecular weight (about 40,000) . The enzyme contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme, and exhibits absorption maxima at 280 nm and 420 nm . The holoenzyme is resolved by dialysis against hydroxylamine to yield the inactive apoenzyme, which is reconstituted by the addition of pyridoxal 5'-phosphate to recover the full activity . The enzyme catalyzes racemization of a number of amino acids, e.g . lysine, ornithine, ethionine, arginine, glutamine, and methionine . The Michaelis constants were determined: 1 mM for L-lysine; 0.9 mM for D-lysine; 0.9 mM for L-ornithine; 1 mM for L-arginine; and 2.6 microM for pyridoxal 5'-phosphate . This enzyme is similar in enzymological properties to the racemase of Pseudomonas putida, but is distinct from it in immunochemical properties. J Gen Microbiol, 1984 Jan, 130 ( Pt 1), 69 - 76 L-arginine utilization by Pseudomonas species; Stalon V et al.; The utilization of arginine was studied in several different Pseudomonas species . The arginine decarboxylase and agmatine deiminase pathways were found to be characteristic of Pseudomonas species of group I as defined by Palleroni et al . (1974) . Pseudomonas putida strains had three distinct arginine catabolic pathways initiated by arginine decarboxylase, arginine deiminase and arginine oxidase, respectively . The two former routes were also present in P . fluorescens and P . mendocina and in P . aeruginosa which also used arginine by a further unknown pathway . None of these pathways occurred in P . cepacia strains; agmatine catabolism seemed to follow an unusual route involving guanidinobutyrate as intermediate. Mol Gen Genet, 1984, 195(3), 511 - 5 Escherichia coli and Pseudomonas putida RNA polymerases display identical contacts with promoters; Gragerov AI et al.; Methylation protection experiments with four promoters (P1 and P2 of the pBR322 plasmid, lacUV5 and lambda P0) have shown that the RNA polymerases from Escherichia coli and Pseudomonas putida, while differing in the primary structure of the subunits involved in DNA binding, display identical patterns of DNA contacts . Nor do these enzymes differ in covalent cross-linking patterns with a partially apurinized promoter . We conclude that the two RNA polymerases have very similar structures of DNA binding centers . The evolutionary conservation of this structure may account for the fact that diverse RNA polymerases often recognize and efficiently use promoters of distant bacterial species. J Biol Chem, 1983 Dec 10, 258(23), 14219 - 32 Ligand binding to heme proteins . An evaluation of distal effects; Mims MP et al.; The O2, CO, and alkyl isocyanide-binding properties of a variety of vertebrate and invertebrate heme proteins have been compared in detail to those of protoheme mono-3-(1-imidazoyl)-propylamide monomethyl ester in aqueous suspensions of soap micelles . The proteins examined include: cytochrome P-450cam from Pseudomonas putida, beef heart cytochrome c oxidase, yeast cytochrome c peroxidase, alpha and beta subunits of human hemoglobin, sheep hemoglobin, carp hemoglobin, sperm whale myoglobin, horse heart myoglobin, a monomeric hemoglobin from Glycera dibranchiata, erythrocruorin from Chironomusthummii, soybean leghemoglobin, and several hemoglobins that lack distal histidines . The smallest bimolecular rates were observed for cytochrome P-450 containing bound camphor, cytochrome c oxidase, and cytochrome c peroxidase . In the case of P-450, the extremely low isonitrile binding rates (approximately 1 M-1 S-1 at 20 degrees C) are due to steric exclusion by bound camphor molecules . For the oxidase and peroxidase, inhibition of CO and isonitrile binding appears to be due to the polar nature of the active sites . In the cases of animal hemoglobins and myoglobins, the sixth coordination positions appear to be designed to accommodate diatomic molecules with no steric hindrance by distal protein residues . Protein resistance to the diffusion of CO and O2 does not limit the observed association rate constants . In contrast, ligands containing three or more atoms are sterically hindered both in their final bound positions and during diffusion to the active site . The magnitude of this hindrance (greater than or equal to 2 kcal/mol) exhibits a complex dependence on ligand size and shape . The most important protein residue appears to be His E7 . In addition to restricting the size of the sixth coordination position, the distal histidine is also capable of forming a hydrogen bond with bound oxygen molecules . The strength of this hydrogen bond was estimated to be -2 and -1 kcal/mol for mammalian myoglobins and hemoglobins, respectively, and accounts for the smaller CO/O2 partition constants (M values) observed for these proteins in comparison to the constants observed for pentacoordinate model heme compounds. Appl Biochem Biotechnol, 1983 Dec, 8(6), 481 - 9 Synthesis of S-(carboxymethyl)-D-cysteine by 3-chloro-D-alanine chloride-lyase of pseudomonas putida CR 1-1; Nagasawa T et al.; S-(Carboxymethyl)-D-cysteine, which is an important component of semisynthetic cephalosporin, MT-141, was enzymatically synthesized . S-(Ethoxy-carbonyl-methyl)-D-cystein was synthesized from 3-chloro- D-alanine and ethyl thioglycolate by the beta-replacement reaction of 3-chloro-D-alanine chloride-lyase from Pseudomonas putida CR 1-1 and subsequently hydrolyzed by alkali . The synthesized S-(carboxymethyl)-D-cysteine was isolated from a large scale reaction mixture and identified physicochemically . The reaction conditions for the synthesis of S-(ethoxycarbonylmethyl)-D-cysteine were optimized using resting cells of P . putida CR 1-1. J Bacteriol, 1983 Dec, 156(3), 1222 - 7 Molecular cloning of the Pseudomonas carboxypeptidase G2 gene and its expression in Escherichia coli and Pseudomonas putida; Minton NP et al.; The gene coding for carboxypeptidase G2 was cloned from Pseudomonas sp . strain RS-16 into Escherichia coli W5445 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322 . The plasmid isolated, pNM1, was restriction mapped, and the position of the gene on the 5.8-megadalton insert was pinpointed by subcloning . The expression of carboxypeptidase in E . coli was 100-fold lower than in the Pseudomonas sp . strain . When the cloned gene was subcloned into the Pseudomonas vector pKT230 and introduced into Pseudomonas putida 2440, a 30-fold increase in expression over that obtained in E . coli was observed . High expression (up to 5% soluble protein) was obtained in E . coli by subcloning a 3.1-megadalton Bg/II fragment into the BamHI site of pAT153 . The increased expression was orientation dependent and is presumed to be due to transcriptional readthrough from the Tc promoter of the vector . Production of carboxypeptidase was shown to be induced (two-fold) by the presence of folic acid, and the mature protein was shown to be located in the periplasmic space of E . coli. Gene, 1983 Dec, 26(2-3), 273 - 82 Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida . Construction of broad-host-range, controlled-expression vectors; Bagdasarian MM et al.; A broad-host-range vector, pKT240, containing the structural gene (aph) for aminoglycoside phosphotransferase (APH), without promoter, has been constructed . Insertion of DNA fragments carrying promoters upstream of aph gene into the unique EcoRI site of this vector results in the expression of the aph gene and consequently the resistance of the host cells to streptomycin . The new vector has been used to show that the hybrid trp-lac (tac) promoter and the promoter of the lacIQ gene of Escherichia coli are active in Pseudomonas putida . Derivatives of pKT240 containing tac and lacIQ sequences may be used as wide-host-range expression vectors . Regulated overproduction of APH and catechol 2,3-oxygenase can be obtained with the aid of the new vectors in both E . coli and P . putida. Experientia, 1983 Nov 15, 39(11), 1273 - 5 The microbial oxygenation of the benzylisoquinoline alkaloid laudanosine; Canonica L et al.; The microbial transformation of the benzylisoquinoline alkaloid laudanosine by a strain of Pseudomonas putida gives a metabolite in which O-demethylation of 1 methoxyl group of ring C, and introduction of 1 ketonic oxygen at C9 and 1 phenolic oxygen at ring C have occurred . Also, O-methylcoripalline is formed in this transformation. Biochimie, 1983 Nov-Dec, 65(11-12), 629 - 35 Properties and function of malate enzyme from Pseudomonas putida; Garrido-Pertierra A et al.; Malate enzyme (L-malate: NADP+ oxidoreductase (oxalacetate-decarboxylating, EC 1.1.1.40)) has been purified from Pseudomonas putida to 99 per cent homogeneity by heat, ammonium suphate fractionation, gel filtration and anion exchange chromatography . Sodium dodecylsulphate-(SDS)-polyacrylamide disc gel electrophoresis analysis showed an approximate tetrameric subunit with a molecular weight of 52,000 . The purified enzyme showed a pH optimum between 8.0 and 8.5 (for Tris-HCl buffer) and required bivalent cations for catalysis; monovalent ions like K+ and NH4+ acted as very effective activators . The temperature-activity relationship for the malate enzyme from 35-80 degrees C showed broken Arrhenius plots with an inflexion at 65 degrees C . The enzyme halflife was 30s at 85 degrees C . The enzyme showed hyperbolic kinetics for both substrates with apparent Km values of 4.0 X 10(-4) M and 2.3 X 10(-5) M for L-malate and NADP+ respectively . From the study of the effects of some compounds on the enzyme, the physiological significance of those produced by fumarate, succinate and oxalacetate can be emphasized. J Bacteriol, 1983 Nov, 156(2), 487 - 92 Plasmid-encoded regulation of colicin E1 gene expression; Ebina Y et al.; A plasmid-encoded factor that regulates the expression of the colicin E1 gene was found in molecular cloning experiments . The 2,294-base-pair AvaII fragment of the colicin E1 plasmid (ColE1) carrying the colicin E1 structural gene and the promoter-operator region had the same information with respect to the repressibility and inducibility of colicin E1 synthesis as the original ColE1 plasmid . An operon fusion was constructed between the 204-bp fragment containing the colicin E1 promoter-operator and xylE, the structural gene for catechol 2,3-dioxygenase encoded on the TOL plasmid of Pseudomonas putida . The synthesis of the dioxygenase from the resulting plasmid occurred in recA+, but not in recA- cells and was derepressed in the recA lexA(Def) double mutant . These results indicate that the ColE1 plasmid has no repressor gene for colicin E1 synthesis and that the lexA protein functions as a repressor . Colicin E1 gene expression was adenosine 3',5'-phosphate (cAMP) dependent . Upon the removal of two PvuII fragments (2,000 bp in length) from the ColE1 plasmid, the induced synthesis of colicin E1 occurred in the adenylate-cyclase mutant even without cAMP . The 3,100-bp Tth111I fragment of the ColE1 plasmid cloned on pACYC177 restored the cAMP dependency of the deleted ColE1 plasmid . Since the deleted fragments correspond to the mobility region of ColE1, the cAMP dependency of the gene expression should be somehow related to the plasmid mobilization function. Biochemistry, 1983 Oct 25, 22(22), 5231 - 6 Enzymes of the beta-ketoadipate pathway in Pseudomonas putida: primary and secondary kinetic and equilibrium deuterium isotope effects upon the interconversion of (+)-muconolactone to cis,cis-muconate catalyzed by cis,cis-muconate cycloisomerase; Ngai KL et al.; Primary and secondary kinetic and secondary equilibrium deuterium isotope effect studies on the cis,cis-muconate cycloisomerase catalyzed interconversion of cis,-cis-muconate (CCM) and (+)-muconolactone (ML) have been performed . The primary and solvent kinetic deuterium isotope effects upon Vmax for the reactions of (+)-{5R-2H}muconolactone in water (HOH) and (+)-muconolactone in deuterium oxide (DOD) to form cis,cis-muconate are about 2.5-2.6 with the heavier isotopic reactions being the slower ones . The secondary equilibrium isotope effect for the formation cis,-cis-{2,3,4,5-2H4} muconate from (+)-{2,3,4,5S-2H4}muconolactone is 1.32 for KH/KD = {( cis,cis-muconate}/{(+)-muconolactone})/ {(+)-{2,3,4,5S-2H4}muconolactone}) and agrees well with the measured value of 1.45 on the basis of the fumarase reaction {Cook, P . F., Blanchard, J . S., & Cleland, W . W . (1980) Biochemistry 19, 4853-4858} . The secondary kinetic deuterium isotope effect determined by the equilibrium perturbation method {Cleland, W . W . (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W . W., O'Leary, M . H., & Northrop, D . B., Eds.) pp 153-175, University Park Press, Baltimore, MD} for the conversion of cis,cis-{2,3,4,5-2H4} muconate to (+)-{2,3,4,5S-2H4}muconolactone is 0.66, expressed as (VmaxCCM(H)/KmCCM(H}/(VmaxCCM(D)/KmCCM(D} . From the equilibrium and kinetic secondary deuterium isotope effects, the calculated value for the kinetic secondary deuterium isotope effect for the reverse reaction, (VmaxML(H)/KmML(H}/(VmaxML(D)/KmML(D}, is about 0.96.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1983 Oct 25, 22(22), 5223 - 30 Enzymes of the beta-ketoadipate pathway in Pseudomonas putida: kinetic and magnetic resonance studies of the cis,cis-muconate cycloisomerase catalyzed reaction; Ngai KL et al.; Steady-state kinetic analysis of the divalent metal ion requiring cis,cis-muconate cycloisomerase catalyzed interconversion of cis,cis-muconate and (+)-muconolactone obeys Michaelis-Menten kinetics and the Haldane relationship from pH 6.2 to 8.3 . The pH vs . kcat/Km profiles suggest free-enzyme apparent pKa values of 6.2 and 7.4: the reciprocal behavior of the data with respect to the latter pKa value is consistent with base-acid catalysis by the enzyme involving proton removal from the lactone and protonation of cis,cis-muconate, respectively . This catalysis by the enzyme of proton transfer is consistent with the stereospecific incorporation of solvent deuterium into the pro-5R position of (+)-muconolactone in the enzyme-catalyzed reaction: in reverse, the departure of the carboxylic oxygen atom and proton from the C(4) and C(5) carbon atoms follows a syn (cis) route {Avigad, G., & Englard, S . (1969) Fed . Proc., Fed . Am . Soc . Exp . Biol . 28, 345, Abstr . 486} . The titration of enzyme freed of divalent metal ion with manganous ion, monitored by electron paramagnetic resonance spectroscopy and steady-state kinetic measurements, indicates a single binding site per subunit characterized by KdissE X Mn = {E} {Mn2+}/{E X Mn2+} = 4.5 and 3.0 microM, respectively, the latter value analyzed via a rapid equilibrium mechanism . The paramagnetic effects of Mn2+ on the 1/T1 and 1/T2 values for the H-5S proton of (+)-muconolactone in the E X ML X Mn ternary complex provide an estimate of the correlation time, tau c, at 5 X 10(-9) s from the T1/T2 ratio, indicating that the condition of rapid exchange of (+)-muconolactone in solution with the ternary complex obtains.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Biophys Res Commun, 1983 Oct 14, 116(1), 30 - 8 Heme binding and substrate-protected cysteine residues in P-450cam; Haniu M et al.; Reactions of the Pseudomonas putida cytochrome P-450-substrate complex or enzyme alone with 14C-labeled iodoacetic acid have been investigated at pH 7.0 . After subsequent conversion of all of the cysteine residues to S-beta-carboxymethylcysteinyl residues, tryptic peptides of the derivative were separated by either high performance liquid chromatography or two dimensional electrophoresis, and their amino acid compositions and partial sequences were determined . All but cysteine residue-134 reacted to some extent . This result implicated residue-134 as the thiol group which is the axial heme ligand since the heme was intact in all of the derivatives made . Reaction of the enzyme-substrate complex with "cold" iodoacetic acid followed by substrate removal and reaction with 14C-labeled iodoacetic acid resulted in radiolabeling of mainly cysteine-240 . This suggested cysteine-240 to be an active site cysteine residue. Science, 1983 Oct 14, 222(4620), 167 - 9 Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo; Ensley BD et al.; A fragment of plasmid NAH7 from Pseudomonas putida PpG7 has been cloned and expressed in Escherichia coli HB101 . Growth of the recombinant Escherichia coli in nutrient medium results in the formation of indigo . The production of this dye is increased in the presence of tryptophan or indole . Several bacteria that oxidize aromatic hydrocarbons to cis-dihydrodiols also oxidize indole to indigo . The results suggest that indigo formation is due to the combined activities of tryptophanase and naphthalene dioxygenase. J Bacteriol, 1983 Oct, 156(1), 89 - 94 Cloning of genes for naphthalene metabolism in Pseudomonas putida; Grund AD et al.; Plasmid pIG7 DNA cloned in Pseudomonas putida with the broad-host-range vectors pRK290 and pKT240 expresses the genes encoding nephthalene oxidation in the presence of the intermediate substrate, salicylate, or the gratuitous inducer, anthranilate . Two operons, nahAF and nahGK, cloned from the EcoRI fragment A (25 kilobases) are under wild-type regulation by the nahR locus . Deletion plasmids provide a restriction map of both operons . Double transformants containing structural and regulatory cistron nahR in trans are used to demonstrate positive control of expression. FEBS Lett, 1983 Sep 5, 161(1), 100 - 2 Redox potential of the cytochrome c in the flavocytochrome p-cresol methylhydroxylase; Hopper DJ; The redox potential of the cytochrome c in 5 flavocytochrome c proteins, all p-cresol methylhydroxylases purified from species of Pseudomonas, was measured . All gave similar values ranging from 226-250 mV . Two of the enzymes, from Pseudomonas putida NC1B 9866 and NC1B 9869, were resolved into their flavoprotein and cytochrome subunits and the redox potentials of the isolated cytochrome c subunits measured . The values for these were 60-70 mV below those for the whole enzymes but, in both cases, reconstitution of active enzyme by addition of the flavoprotein subunit restored the original potential. Mikrobiologiia, 1983 Sep-Oct, 52(5), 771 - 6 {Degradation of 3-chlorobenzoic acid by a Pseudomonas putida strain}; Grishchenkov VG et al.; A Pseudomonas putida strain 87 capable of assimilating 3-chlorobenzoic acid as a sole source of carbon and energy (3Cba+) was isolated . Treatment with mitomycin C eliminated the 3Cba+ phenotype in 1% of cells in the population . The 3Cba+ phenotype was transferred at a low frequency in the process of conjugation to other bacteria belonging to the genus Pseudomonas . Determinants localized on the plasmid are presumed to be responsible for the capability to assimilate 3-chlorobenzoic acid . A scheme is proposed for the oxidation of 3-chlorobenzoic acid on the basis of studying the products of its degradation . Two catechol 1,2-dioxygenases are present in strains with the 3Cba+ phenotype as was shown by analysing the activity of enzymes catalysing cleavage of the aromatic cycle and by studying their induction . One of the two seems to be encoded by chromosomal genes while the other is encoded by plasmid genes and determines the capability of the culture to cleave the chlorinated pyrocatechol. Vopr Med Khim, 1983 Jul-Aug, 29(4), 131 - 5 {Improved procedure for isolation and purification of methionine gamma-lyase from Pseudomonas putida}; Berezov TT et al.; An improved, simplified and relatively rapid procedure is developed for isolation and purification of a new antitumor enzyme--methionine gamma-lyase from Pseudomonas putida . The method includes five steps instead of seven steps in previous procedure with a good yield of the enzyme . The purified enzyme was shown to be homogeneous by the criteria of disc gel electrophoresis . The highly homogeneous preparations of the enzyme exhibited the absorption maxima at 280 and 420 nm . The detailed studies on antileukemic activity of the methionine gamma-lyase are currently in progress. Biull Eksp Biol Med, 1983 May, 95(5), 87 - 8 {Cytotoxic effect of methionine-gamma-lyase on neoplastic cells in culture}; Pekhov AA et al.; The influence of methionine-gamma-lyase from Pseudomonas putida on DNA synthesis by CaOv and L-8 cell lines has been studied . The agent has been demonstrated to inhibit the incorporation of 3H-thymidine into L-8 cell line and to have no effect on CaOv cells. Plasmid, 1983 May, 9(3), 325 - 30 Replication of derivatives of the broad host range plasmid RK2 in two distantly related bacteria; Schmidhauser TJ et al.; A 0.7-kb segment of the broad host range plasmid RK2 containing the replication origin of this plasmid will replicate in Escherichia coli and Pseudomonas putida when this segment is joined to a 1.8-kb region of RK2 designated traA* . The presence of another region of RK2, designated trfB, that previously was implicated in RK2 replication had no effect on the maintenance of the RK2 trfA*-oriV replicon in these two organisms . These observations indicate a requirement for a minimal account of information for replication of this broad host range plasmid in two distantly related bacteria. J Bacteriol, 1983 Apr, 154(1), 508 - 12 Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli; Clarke PH et al.; R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains . Expression in P . putida was similar to that in P . aeruginosa and was repressed by exogenous arginine . Expression in E . coli was 2 to 4% of that in P . aeruginosa . Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E . coli in this respect. Arch Biochem Biophys, 1983 Apr 1, 222(1), 207 - 15 Proton coupling in the ligand-binding reaction of ferric cytochrome P-450 from Pseudomonas putida; Totani K et al.; Effects of pH on the ligand-binding reactions of ferric heme in cytochrome P-450 from Pseudomonas putida (camphor 5-monooxygenase, EC 1.14.15.1) were studied by using cyanide, N-methylimidazole, pyridine, and ethylisocyanide as ligands . In all cases, affinity of the ferric heme for the ligand was found to increase as pH of the medium was raised from around 6 to 9 . Depending on the ligand, the increase was 10- to 1000-fold and the shapes of their pH-affinity curves were remarkably different . Analyses such pH profiles disclosed the presence of a dissociable group in the enzyme with a pK value of approximately 9.5 and that its ionization greatly enhanced the affinity of the heme for ligands . When a dissociable ligand such as hydrogen cyanide and N-methylimidazole was used, the dissociated form of the ligand had a higher affinity toward the heme than the undissociated form . The shapes of the pH-affinity curves were successfully simulated as overlapping curves of ionization reactions of the ligand and the dissociable group . In addition, size of the ligand molecule was shown to be also important in the binding reaction: relatively large molecules such as pyridine, ethylisocyanide, and N-methylimidazole bound to the enzyme in a competitive manner against d-camphor concentration, whereas the binding of a smaller molecule such as cyanide was inhibited by the substrate in a noncompetitive manner . On the basis of these findings, control mechanisms for the ligand-binding reactions of the cytochrome P-450 from P . putida are discussed. Biochem Biophys Res Commun, 1983 Mar 29, 111(3), 809 - 16 Synthesis of D-cysteine-related amino acids by 3-chloro-D-alanine chloride-lyase of Pseudomonas putida CR 1-1; Nagasawa T et al.; Using the beta-replacement reaction of 3-chloro-D-alanine chloride-lyase from Pseudomonas putida CR 1-1, S-methyl-, S-ethyl-, S-n-propyl-, S-n-butyl-, S-phenyl-, S-benzyl-, S-(2,3-dihydroxypropyl)- and S-(2-hydroxyethyl)-D-cysteine were synthesized from 3-chloro-D-alanine and methyl-, ethyl-, n-propyl-, n-butyl-, phenyl-, benzyl-mercaptans, alpha-thioglycerol and 2-mercaptoethanol, respectively . The enzymatically synthesized D-cysteine-related amino acids were isolated from the large scale reaction mixture and identified physicochemically. J Biol Chem, 1983 Mar 10, 258(5), 2923 - 8 Complete nucleotide sequence of the metapyrocatechase gene on the TOI plasmid of Pseudomonas putida mt-2; Nakai C et al.; Metapyrocatechase which catalyzes the oxygenative ring cleavage of catechol to form alpha-hydroxymuconic epsilon-semialdehyde is encoded by the xylE gene on the TOL plasmid of Pseudomonas putida mt-2 . We have cloned the xylE region in Escherichia coli and determined the nucleotide sequence of the DNA fragment of 985 base pairs around the gene . The fragment included only one open translational frame of sufficient length to accommodate the enzyme . The predicted amino acid sequence consisted of 307 residues, and its NH2- and COOH-terminal sequences were in perfect agreement with those of the enzyme recently determined (Nakai, C., Hori, K., Kagamiyama, H., Nakazawa, T., and Nozaki, M . (1983) J . Biol . Chem . 258, 2916-2922) . A mutant plasmid was isolated which did not direct the synthesis of the active enzyme . This plasmid had a DNA region corresponding to the NH2-terminal two-thirds of the polypeptide . From the deduced amino acid sequence, the secondary structure was predicted . Around 10 base pairs upstream from the initiator codon for metapyrocatechase, there was a base sequence which was complementary to the 3'-end of 16 S rRNAs from both E.coli and Pseudomonas aeruginosa . A preferential usage of C- and G-terminated codons was found in the coding region xylE, which contributed to the relatively high G + C content (57%) of this gene. J Biochem (Tokyo), 1983 Mar, 93(3), 807 - 17 Structural characteristics of cytochrome P-450 . Possible location of the heme-binding cysteine in determined amino-acid sequences; Gotoh O et al.; Computer-aided analyses were made of the complete amino-acid sequences of two P-450 species, the phenobarbital-inducible major P-450 of rat liver microsomes (P-450PB) and camphor-hydroxylating P-450 of Pseudomonas putida (P-450cam) . Statistically significant homology was recognized between the two P-450 sequences, but these sequences were not related to those of other groups of hemoproteins, such as hemoglobins, peroxidases, and cytochrome c's and b's . Two highly homologous regions, HR1 and HR2, and two other weakly homologous regions were found on optimally matched alignment of the P-450 sequences . The secondary structures of the two P-450's predicted by current prediction methods bear strong resemblance at these homologous regions . Both HR1 and HR2 contain a cysteine residue near the center of the homologous regions, and they are the only regions that show significant homology among all 48 combinations of local sequences around the cysteine residues (six on P-450PB and eight on P-450cam) . HR1 is located in the N-proximal half of the molecule, is rich in hydrophilic residues, and is predicted to be helical . On the other hand, HR2 is close to the C-terminus, has intermediate hydrophobicity, and may take a complex secondary structure of a turn-sheet-helix . The amino-acid sequences around the HR1 and HR2 regions are also well conserved in another P-450 species, rabbit P-450LM2. Proc Natl Acad Sci U S A, 1983 Feb, 80(4), 1101 - 5 Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene; Zukowski MM et al.; A method to isolate fragments of DNA that promote gene expression in Bacillus subtilis is described . The system is based on production of catechol 2,3-dioxygenase {CatO2ase; catechol:oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.2} encoded by the Pseudomonas putida TOL plasmid gene xylE . The gene was transferred to aB . subtilis/Escherichia coli plasmid vector to construct pTG402 . Although xylE is functionally expressed in E . coli, CatO2ase is not detected in B . subtilis unless a fragment of DNA capable of promoting gene expression is ligated into a cleavage site on pTG402 upstream from xylE . Fragments of chromosomal DNA from B . subtilis, Bacillus licheniformis, Bacillus pumilus, and E . coli are shown to promote xylE gene expression in B . subtilis . The special feature of the system is the method of detection: colonies of cells that express xylE become yellow within seconds after selection plates are sprayed with catechol, a colorless substrate that is converted by CatO2ase to the yellow product, 2-hydroxymuconic semialdehyde . The complete nucleotide sequence of xylE is presented . Strong complementarity between the ribosome binding site and 16S rRNA suggests that xylE mRNA translation in B . subtilis may commence at the same site as that recognized by P . putida . Identity of CatO2ase produced in B . subtilis, E . coli, and P . putida support the hypothesis . Our sensitive color assay offers an approach to develop plasmid gene expression vectors for a wide variety of host organisms. J Bacteriol, 1983 Feb, 153(2), 822 - 9 Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7; Schell MA; The genes encoding the enzymes responsible for conversion of naphthalene to 2-hydroxymuconic acid (nahA through nahI) are contained on a 25-kilobase EcoRI fragment of an 85-kilobase NAH plasmid of Pseudomonas putida . These genes were cloned into the plasmid vectors pBR322 and RSF1010 to obtain the recombinant plasmids pKGX505 and pKGX511, respectively . To facilitate cloning and analysis, an NAH7 plasmid containing a Tn5 transposon in the salicylate hydroxylase gene (nahG) was used to derive the EcoRI fragment . The genes for naphthalene degradation were expressed at a low level in Escherichia coli strains containing the fragment on the recombinant plasmids pKGX505 or pKGX511 . This was shown by the ability of whole cells to convert naphthalene to salicylic acid and by in vitro enzyme assays . The expression of at least two of these genes in E . coli appeared to be regulated by the presence of the inducer salicylic acid . In addition, high-level expression and induction appear to be mediated by an NAH plasmid promoter and a regulatory gene located on the fragment . A restriction endonuclease cleavage map of the cloned fragment was generated, and the map positions of several nah genes were determined by analysis of various subcloned DNA fragments. J Bacteriol, 1983 Feb, 153(2), 969 - 75 Mutations affecting lipoamide dehydrogenases of Pseudomonas putida; Sokatch JR et al.; Pseudomonas putida grown on valine produces two lipoamide dehydrogenases, LPD-glu (Mr, 56,000 and LPD-val (Mr, 49,000) . The 49,000-dalton protein is used by P . putida for branched-chain keto acid dehydrogenase, whereas the 56,000-dalton protein is presumably used for pyruvate and 2-ketoglutarate dehydrogenases . The objective of this study was to isolate and characterize mutants of P . putida with mutations affecting lipoamide dehydrogenases in order to study the relationship of these two proteins . Mutant JS287 lacked LPD-val, the lipoamide dehydrogenase which is induced by growth on valine and is specific for branched-chain keto acid dehydrogenase, and had normal amounts of LPD-glu, the lipoamide dehydrogenase which is formed during growth on glucose and which is probably used by both pyruvate and 2-ketoglutarate dehydrogenases . Mutant JS94 was a pleiotropic mutant with defects in 2-ketoglutarate, branched-chain, and lipoamide dehydrogenases . Proteolysis of LPD-glu and LPD-val produced completely different digestion products, suggesting that these two proteins are products of separate structural genes . Antisera prepared against LPD-glu reacted only with LPD-glu, whereas antisera prepared against LPD-val reacted with LPD-val and cross-reacted with LPD-glu . Although mutant JS94 did not produce active lipoamide dehydrogenase, cell-free extracts of this mutant contained a protein which cross-reacted with anti-LPD-val. Biochemistry, 1983 Jan 4, 22(1), 143 - 9 Steroid 21-hydroxylase (cytochrome P-450) from porcine adrenocortical microsomes: microsequence analysis of cysteine-containing peptides; Yuan PM et al.; The steroid 21-hydroxylase cytochrome P-450 from porcine adrenocortical microsomes was purified to homogeneity . The protein exhibited two NH2-terminal sequences, one of which was identical with the first but lacking the NH2-terminal methionine . The sequence was extremely hydrophobic but had little homology to the 17 alpha-hydroxylase/C17,20-lyase isolated from neonatal porcine testes or to rat or rabbit liver microsomal cytochromes P-450 . The cysteine-containing fragments of the S-carboxymethylated protein were purified by high-performance liquid chromatography and sequenced . Three of the cysteine-containing peptides exhibited significant sequence homology with peptides from the major phenobarbital-induced rat liver cytochrome P-450 (P-450b) and two with peptides from cytochrome P-450cam (camphor methylene hydroxylase from Pseudomonas putida) . The presence of conserved regions in the primary sequences of these proteins appears likely to provide clues to the nature of their heme-binding domains. Int J Biochem, 1983, 15(6), 867 - 70 Immunological relatedness of histidine ammonia-lyases from some species of Pseudomonas: taxonomic implication; Rokosu AA; 1 . Histidine ammonia-lyases (histidase EC 4.3.1.3) from Pseudomonas testosteroni NCIB 10808 and Pseudomonas putida NCIB 10807 were purified and specific antibody was raised to each separately in a rabbit . 2 . Immunological cross-reactions of each antibody to histidine ammonia-lyases from various species of Pseudomonas were examined by the enzyme inhibition test . 3 . The immunological data obtained suggest that these Pseudomonas species can be classi