Ellis L. Kline

Convergent and divergent points in catabolic pathways involved in utilization of fluoranthene, naphthalene, anthracene, and phenanthrene by Sphingomonas paucimobilis var. EPA505

Catabolic pathways for utilization of naphthalene (NAP), anthracene (ANT), phenanthrene (PHE), and fluoranthene (FLA) by Sphingomonas paucimobilis EPA505 were identified. Accumulation of catabolic intermediates was investigated with three classes of Tn5 mutants with the following polycyclic aromatic hydrocarbon (PAH)-negative phenotypes; (class I NAP− PHE− FLA−, class II NAP− PHE−, and class III FLA−). Class I mutant 200pbhA had a Tn5 insertion within a meta ring fission dioxygenase (pbhA), and a ferredoxin subunit gene (pbhB) resided directly downstream. Mutant 200pbhA and other class I mutants lost the ability to catalyze the initial dihydroxylation step and did not transform NAP, ANT, PHE, or FLA. Class I mutant 401 accumulated salicylic acid, 2-hydroxy-3-naphthoic acid, 1-hydroxy-2-naphthoic acid, and hydroxyacenaphthoic acid during incubation with NAP, ANT, PHE, or FLA, respectively. Class II mutant 132pbhC contained the Tn5 insertion in an aldolase hydratase (pbhC) and accumulated what appeared to be meta ring fission products: trans-o-hydroxybenzylidene pyruvate, trans-o-hydroxynaphylidene pyruvate, and trans-o-hydroxynaphthyl-oxobutenoic acid when incubated with NAP, ANT, and PHE, respectively. When mutant 132pbhC was incubated with 1-hydroxy-2-naphthoic acid, it accumulated trans-o-hydroxybenzylidene pyruvate. Class III mutant 104ppdk had a Tn5 insertion in a pyruvate phosphate dikinase gene that affected expression of a FLA-specific gene and accumulated a proposed meta ring fission product; trans-o-hydroxyacenaphyl-oxobutenoic acid during incubation with FLA. Trans-o-hydroxyacenaphyl-oxobutenoic acid was degraded to acenaphthenone that accumulated with class III mutant 611. Acenaphthenone was oxidized via incorporation of one molecule of dioxygen by another oxygenase. 2,3-Dihydroxybenzoic acid was the final FLA-derived catabolic intermediate detected. Analysis of PAH utilization mutants revealed that there are convergent and divergent points involved in NAP, ANT, PHE, and FLA utilization by S. paucimobilis EPA505. Journal of Industrial Microbiology & Biotechnology (2001) 26, 369–382.

Identification of four structural genes and two putative promoters necessary for utilization of phenanthrene naphthalene, fluoranthene, and by Sphingomonas paucimobilis var. EPA505.

Abstract Sphingomonas paucimobilis var. EPA505 utilizes fluoranthene (FLA), naphthalene (NAP), and phenanthrene (PHE) as sole carbon sources for energy and growth. A genetic library of EPA505 was constructed using mini-Tn5 promoter reporter genes encoding for tetracycline resistance (tcp−) or luminescence (luxABp−). Out of 2250 Tn5 mutants, ten were deficient in utilization of FLA, NAP, and/or PHE as sole carbon sources. Three classes of Tn5 mutants were defined: classI (nap−phe−fla−), classII (nap−phe−), and classIII (fla−). Four of five mutants in classI did not express dioxygenase function, whereas one classI mutant and all classII and classIII mutants retained dioxygenase activity. In Tn5 tcp− classI mutants 200 and 394 (dioxygenase negative) and classII mutant 132 (dioxygenase positive), promoter reporter was expressed when induced with FLA, NAP, PHE, other polycyclic aromatic hydrocarbons (PAHs), and several proposed PAH-derived catabolites. The Tn5 tcp− derived classIII mutant 104 was induced only with PAHs and not with PAH-derived catabolites. DNA sequence analysis of cloned regions of classI mutant 200 revealed that Tn5 inserted into a gene that shared (96%) DNA sequence homology with 2,3-dihydroxybiphenyl 1,2-dioxygenase that is designated pbhA. Nucleotide sequences downstream of pbhA shared (84%) homology to a Rieske-type ferredoxin subunit gene of a multicomponent dioxygenase designated pbhB. The Tn5 tcp− in classII mutant 132 occurred within sequences that shared (74%) homology with a trans-o-hydroxybenzylidene-pyruvate hydratase-aldolase gene (pbhC). Sequence analysis of the region proximal to this gene revealed a putative promoter that contained a binding site for a LysR transcriptional activator. In classIII mutant 104, the Tn5 tcp− resided within a region that shared 94% nucleotide homology to that of a pyruvate phosphate dikinase gene known to be involved in cellular uptake of glucose. The FLA-specific catabolic gene disrupted in mutant 104 was designated phbD. Functional and sequence analyses of promoter probe mutants allowed identification of four genes necessary for the utilization of PAHs that are controlled by at least two promoters that are affected by a wide range of aromatic compounds.

Imidazole acetic acid as a substitute for cAMP

Abstract The ability of imidazole acetic acid (IA) to substitute for cAMP was demonstrated by use of a series of strains carrying a lesion in the cya structural gene. The substitution of IA for cAMP was specific for the L-arabinose operon in that this compound was ineffective in substituting for cAMP in the lactose or maltose catabolic systems. The cAMP receptor protein (CRP) and the araC gene product were necessary for the IA mediated induction of the L-arabinose system.

Loss of butylate-utilizing ability by a Flavobacterium

Abstract Flavobacterium sp. strain VI.15 (But + ) was isolated from a vernolate-history soil employing an indicator medium (MSBT). This organism was capable of utilizing butylate (But + ), EPTC (Ept + ), and vernolate (Ver + ) as a primary carbon source. Butylate-minus derivatives of the herbicide-utilizing wild-type were isolated on MSBTE differential indicator composed of minimal salts + 200 μg ml −1 butylate + 70 μg ml −1 yeast extract + 25 μg ml −1 2,3,5-triphenyltetrazolium chloride. VI.15 (But + ) was plated on the differential indicator and plasmid moieties present within this bacterium were induced to segregate. Loss of pSMB2 as a function of physical (temperature) and acridine orange-curing corresponded with a loss of butylate-utilizing ability. Moreover, spontaneous loss of carbamothioate-utilizing abilites upon cold storage (4°C) on minimal media further suggested plasmid involvement in carbamothioate biodegradation.

Benzyl derivative facilitation of transcription in Escherichia coli at the ara and lac operon promoters: Metabolite gene regulation (MGR)

SummaryA number of benzyl derivatives have been tested for their ability to induce the expression of the araBAD operon in an Escherichia coli K-12 strain. Those derivatives shown to be stimulatory include: benzoic acid (BA), paramino benzoic acid (PABA), para-hydroxy benzoic acid (PHBA), ortho-amino benzoic acid (OABA), 3-hydroxy-4-methoxy phenylethylamine (MTA), and 4-hydroxy-3-methoxyphenol acetic acid (HVA). The araC gene product was necessary to facilitate the induction. To further characterize if the inductive effect was mediated at the level of transcription, an araBAD-tetracycline resistant (Tcr) operon fusion plasmid (pAP-B) was employed. Benzyl derivatives which induce expression of the araBAD operon in situ also induced a Tcr phenotype with pAP-B. Both indole acetic acid (IAA) and imidazole (IM), which were previously shown to circumvent the necessity for cAMP in the induction of the araBAD operon, also induced a Tcr phenotype with pAP-B. Induction of lac or othe cAMP responding operons with the inducing molecules at the chromosomal level was not detectable when assessed by carbon utilization. However, a lacZYA-Tcr operon fusion plasmid (pLPI) did respond to IAA and several of the inducing benzyl derivatives. Catabolite repression of chromosomal araBAD expression was reversed when the exogenous concentration of OABA was elevated. Similar effects on the Tcr phenotypes conferred by pAP-B and pLP1 were observed when OABA or several other inducing benzyl derivatives were present exogenously.

An amylase gene from Drosophila pseudoobscura is expressed in Escherichia coli: Functional selection and biochemical comparisons of the fly- and clone-produced amylases

An amylase gene from Drosophila pseudoobscura was isolated from a genomic library constructed in pBR322 and cloned in Escherichia coli by selecting for the ability of its product to hydrolyze starch, a carbon source not normally utilized by E. coli. Hybridization of pAMY17F to D. pseudoobscura polytene chromosomes shows a positive signal at the amylase pseudogene locus (bank 78, chromosome 3). The chimeric plasmid pAMY17F, has been altered in such a way as to increase amylase expression. Southern and Northern hybridizations to the cloned amylase DNA indicate that the source of the gene is from D. pseudoobscura. Biochemical properties such as pH optima, substrate specificities, electrophoretic analyses, inhibitor sensitivities, heat stabilities, temperature responsiveness and molecular weights indicate that the amylases produced by the fly and bacterial clone are similar and have similar properties. It appears that E. coli/pAMY17F is producing an amylase like that found in D. pseudoobscura.

The effect of an ilvA Mu phage insertion on ilv gene expression in Escherichia coli K-12

Abstract Physiological analysis of an E. coli K-12 strain carrying a Mu phage integrated into the ilvA structural gene shows that there is a polar affect on ilvD gene expression, whereas, the ilvE gene maintains a normal multivalent regulation response. It was also demonstrated that the ilvC and ilvB genes can be derepressed and repressed in response to ilv multivalent control. These experiments demonstrate that the ilvE structural gene can be regulated independently from the ilvA and ilvD structural genes and that the ilvC structural gene does not require the complete ilvA gene product (threonine deaminase) for its induction.

Effects of the flrA regulatory locus on biosynthesis and excretion of amino acids in EscherichiacoliBr

We have partially characterized phenotypic effects of an unusual amino acid regulatory locus, flrA, in E. coli B/r that alters the expression of the ilv and leu operons [Kline, E.L (1972) J. Bacteriol. 110:1127-1134]. This study demonstrated that a primary effect of the flrA7 mutation in haploid strains was overproduction of valine. In diploid strains (FflrA+/flrA7) this mutation resulted in excretion of valine, isoleucine, leucine, aspartate, threonine, glutamate, histidine and lysine. Increased excretion of amino acids by mutant strains might be explained by a membrane alteration or by flrA encoding a positive regulatory factor that affects the ilv operon and has pleiotropic effects on other amino acid operons.