Studies on the Utilization of Hydrocarbons by Microorganisms

In the course of study on the utilization of methyl-substituents of mono-cyclic aromatic hydrocarbons by Pseudomonas aeruginosa S668B2, some organic acids and phenolic compounds were found to be produced in culture broth.

Strain S668B2 was capable of producing ultraviolet absorbing and fluorescent substances from m-xylene. These substances were isolated in the form of crystal and identified as 3-methyl salicylic acid and m-toluic acid.

Strain S668B2 also produced ultraviolet absorbing and fluorescent substances from pseudocumene (1,2,4-trimethyl benzene). These substances were isolated in the crystalline form and identified as 3,4-dimethyl benzoic acid and 3,4-dimethyl phenol.

Strain S668B″ did not attack o-xylene. Under the similar conditions Pseudomonas desmolytica S449B3, which produced a large amount of cumic acid from p-cymene, did not oxidize o-xylene, but grew on p-xylene, m-xylene and 1,2,4-trimethyl benzene.

None out of 364 soil samples gave microorganisms which utilize o-xylene as a sole carbon source.     

The Degradation of Isopropylbenzene and Isobutylbenzene by Pseudomonas sp.

To clarify biodegradation pathways of isoalkyl substituted aromatic hydrocarbons, oxidation products of isopropylbenzene and isobutylbenzene by Ps. desmolytica S449B1 and Ps. convexa S107B1 were examined.

Oxidation products from isopropylbenzene were determined to be 3-isopropylcatechol and (+)-2-hydroxy-7-methyI-6-oxooctanoic acid. Isobutylbenzene was also oxidized to 3-isobutylcatechol and (+)-2-hydroxy-8-methyl-6-oxononanoic acid by the same strains.

From these results, the existence of an unknown reductive step in the degradation of these isoalkyl substituted aromatic hydrocarbons and the initial oxidation of these aromatic hydrocarbons by the strains were made clear. The degradation pathways of isopropylbenzene and isobutylbenzene by these strains were discussed.     

Anaerobic oxidation of the aromatic plant hydrocarbon p-cymene by newly isolated denitrifying bacteria

The capability of nitrate-reducing bacteria to degrade alkyltoluenes in the absence of molecular oxygen was investigated with the three isomers of xylene, ethyltoluene, and isopropyltoluene (cymene) in enrichment cultures inoculated with freshwater mud. Denitrifying enrichment cultures developed most readily (within 4 weeks) with p-cymene, a natural aromatic hydrocarbon occurring in plants, and with m-xylene (within 6 weeks). Enrichment of denitrifiers that utilized m-ethyltoluene and p-ethyltoluene was slow (within 8 and 12 weeks, respectively); no enrichment cultures were obtained with the other alkylbenzenes within 6 months. Anaerobic degradation of p-cymene, which has not been reported before, was studied in more detail. Two new types of denitrifying bacteria with oval cells, strains pCyN1 and pCyN2, were isolated; they grew on p-cymene (diluted in an inert carrier phase) and nitrate with doubling times of 12 and 16 h, respectively. Strain pCyN1, but not strain pCyN2, also utilized p-ethyltoluene and toluene. Both strains grew with some alkenoic monoterpenes structurally related to p-cymene, e.g., α-terpinene. In addition, the isolates utilized p-isopropylbenzoate, and mono- and dicarboxylic aliphatic acids. Determination of the degradation balance of p-cymene and growth with acetate and nitrate indicated the capacity for complete oxidation of organic substrates under anoxic conditions. Adaptation studies with cells of strain pCyN1 suggest the existence of at least two enzyme systems for anaerobic alkylbenzene utilization, one metabolizing p-cymene and p-ethyltoluene, and the other metabolizing toluene. Excretion of p-isopropylbenzoate during growth on p-cymene indicated that the methyl group is the site of initial enzymatic attack. Although both strains were facultatively aerobic, as revealed by growth on acetate under air, growth on p-cymene under oxic conditions was observed only with strain pCyN1. Strains pCyN1 and pCyN2 are closely related to members of the Azoarcus-Thauera cluster within the β-subclass of the Proteobacteria, as revealed by 16S rRNA gene sequence analysis. This cluster encompasses several described denitrifiers that oxidize toluene and other alkylbenzenes. Received: 15 July 1998 / Revision received: 29 July 1999 / Accepted: 2 August 1999     

Studies on the Utilization of Hydrocarbons by Microorganisms

In the course of investigation of alicyclic hydrocarbon-utilizing microorganisms, five strains of ethylcyclohexane-utilizing bacteria were isolated from soil samples.

Among those bacteria, the strain S6B1 that was identified as Alcaligenes faecalis, showed the best growth in shaking culture.

The strain S6B1 was found to produce 4-ethylcyclohexanol from ethylcyclohexane.

This substance separated from culture broth was purified and identified to be trans-4-ethylcyclohexanol by the use of NMR.     

A novel n-alkane-degrading bacterium as a minor member of p-xylene-degrading sulfate-reducing consortium

A p-xylene-degrading, sulfate-reducing enrichment culture was characterized by analyzing the response of its members to changes in the available substrate. The culture was inoculated into media containing other substrates, resulting in the establishment of benzoate-, acetate-, and lactate-utilizing enrichment cultures. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the enriched cultures targeting 16S rRNA genes showed quite simple band patterns. The predominant band from the benzoate-utilizing enrichment culture was identical to that from the original enrichment culture utilizing p-xylene. A single, dominant DGGE band was observed in common from the acetate- and lactate-utilizing enrichment cultures. A novel sulfate-reducing bacterium, strain PL12, was isolated from the lactate-utilizing enrichment culture. The 16S rRNA gene sequence of strain PL12 was identical to that of the dominant DGGE band in the acetate- and lactate-utilizing enrichment cultures and distinct from the dominant sequences in the original p-xylene-degrading and benzoate-utilizing enrichment cultures. Phylogenetic analysis of the 16S rRNA gene sequences showed that the isolate belonged to the family Desulfobacteraceae in the class Deltaproteobacteria. The isolated strain PL12 could utilize n-hexane and n-decane as substrates, but could not utilize benzoate, p-xylene and other aromatic hydrocarbons. These results suggest that the p-xylene degradation observed in the original enrichment culture was performed by the dominant bacterium corresponding to DGGE band pXy-K-13 (Nakagawa et al. 2008). The novel strain PL12 might have been utilizing metabolites of p-xylene.     

Preferential utilization of petroleum oil hydrocarbon components by microbial consortia reflects degradation pattern in aliphatic–aromatic hydrocarbon binary mixtures

In this study, the abilities of two microbial consortia (Y and F) to degrade aliphatic–aromatic hydrocarbon mixtures were investigated. Y consortium preferentially degraded the aromatic hydrocarbon fractions in kerosene, while F consortium preferentially degraded the aliphatic hydrocarbon fractions. Degradation experiments were performed under aerobic conditions in sealed bottles containing liquid medium and n-octane or n-decane as representative aliphatic hydrocarbons or toluene, ethylbenzene or p-xylene as representative aromatic hydrocarbons (all at 100 mg/l). Results demonstrated that the Y consortium degraded p-xylene more rapidly than n-octane. It degraded toluene, ethylbenzene and p-xylene more rapidly than decane. In comparison, the F consortium degraded n-octane more rapidly than toluene, ethylbenzene or p-xylene, and n-decane more rapidly than toluene, ethylbenzene or p-xylene. 16S rRNA gene sequencing revealed that the Y consortium was dominated by Betaproteobacteria and the F consortium by Gammaproteobacteria, and in particular Pseudomonas. This could account for their metabolic differences. The substrate preferences of the two consortia showed that the aliphatic–aromatic hydrocarbon binary mixtures, especially the n-decane–toluene/ethylbenzene/p-xylene pairs, reflected their degradation ability of complex hydrocarbon compounds such as kerosene. This suggests that aliphatic–aromatic binary systems could be used as a tool to rapidly determine the degradation preferences of a microbial consortium.     

Microbial Oxidation of α-Methylstyrene and β-Methylstyrene

An unidentified bacterial strain S107B1, isolated from soil by use of isopropylbenzene as a carbon source, was shown to bring about oxidation of α-methylstyrene and β-methylstyrene,

One of the oxidation products produced from α-methylstyrene was identified as the new compound, (—)-cis-23-dihydroxy-1-isopropenyl-6-cyclohexene.

The same strain S107B1 also oxidized β-methylstyrene and produced 3-phenylpropionaldehyde and benzoic acid.

From these results, the existence of reductive step for the aerobic degradation of these aromatic hydrocarbons by this strain was made clear. The initial attack on these aromatic hydrocarbons and a cyclohexenediol compound formed from α-methylstyrene were discussed.     

Physiological and Genetic Comparison of Two Aromatic Hydrocarbon-degrading Sphingomonas Strains

Sphingomonas yanoikuyae strain B1 is able to degrade a wider range of aromatic hydrocarbons than S. paucimobilis strain TNE12 can degrade. Various culture techniques were used to corroborate that B1 used m-xylene, biphenyl, toluene, naphthalene, and phenanthrene as sole carbon and energy sources. In contrast, TNE12 could not mineralize m-xylene, biphenyl, toluene, or naphthalene. However, fluoranthene served as carbon and energy source for TNE12 but not B1. Southern blots were performed using the cloned genomic region (approximately 23 kb) containing the degradative genes for the upstream pathways for biphenyl and m-xylene and a TOL plasmid-type meta operon from B1 as a probe against the Kpn I restriction-digested total DNA of TNE12. This 23 kb probe hybridized to three Kpn I-digested fragments of TNE12 DNA; thus significant homology existed between the aromatic hydrocarbon-degrading genes of B1 and TNE12. Further work with smaller probes revealed, however, that TNE12 DNA fragments did not hybridize with the probe containing the genes encoding for xylene monooxygenase and part of an aromatic dioxygenase. A recombinant plasmid, which contains only the genes for xylene monooxygenase, is able to complement TNE12 on m-xylene. These genes are, therefore, probably missing from TNE12. Hence, TNE12 cannot use monocylclic aromatics whereas B1 can. Pulsed field gel electrophoresis coupled with Southern blotting revealed that the aromatic degradative genes were on an approximately 240 kb plasmid of TNE12; the same genes in B1 are known to be chromosomal.     

Bioluminescent bioreporter integrated-circuit sensing of microbial volatile organic compounds

A bioluminescent bioreporter for the detection of the microbial volatile organic compound p-cymene was constructed as a model sensor for the detection of metabolic by-products indicative of microbial growth. The bioreporter, designated Pseudomonas putida UT93, contains a Vibrio fischeri luxCDABE gene fused to a p-cymene/p-cumate-inducible promoter derived from the P. putida F1 cym operon. Exposure of strain UT93 to 0.02–850 ppm p-cymene produced self-generated bioluminescence in less than 1.5 h. Signals in response to specific volatile organic compounds (VOCs) such as m- and p-xylene and styrene, also occurred, but at two-fold lower bioluminescent levels. The bioreporter was interfaced with an integrated-circuit microluminometer to create a miniaturized hybrid sensor for remote monitoring of p-cymene signatures. This bioluminescent bioreporter integrated-circuit device was capable of detecting fungal presence within approximately 3.5 h of initial exposure to a culture of p-cymene-producing Penicillium roqueforti.     

Degradation of <Emphasis Type="Italic">o</Emphasis>-xylene and <Emphasis Type="Italic">m</Emphasis>-xylene by a novel sulfate-reducer belonging to the genus <Emphasis Type="Italic">Desulfotomaculum</Emphasis>

A strictly anaerobic bacterium, strain OX39, was isolated with o-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. Apart from o-xylene, strain OX39 grew on m-xylene and toluene and all three substrates were oxidized completely to CO₂. Induction experiments indicated that o-xylene, m-xylene, and toluene degradation were initiated by different specific enzymes. Methylbenzylsuccinate was identified in supernatants of cultures grown on o-xylene and m-xylene, and benzylsuccinate was detected in supernatants of toluene-grown cells, thus indicating that degradation was initiated in all three cases by fumarate addition to the methyl group. Strain OX39 was sensitive towards sulfide and depended on Fe(II) in the medium as a scavenger of the produced sulfide. Analysis of the PCR-amplified 16S rRNA gene revealed that strain OX39 affiliates with the gram-positive endospore-forming sulfate reducers of the genus Desulfotomaculum and is the first hydrocarbon-oxidizing bacterium in this genus.     

Anaerobic Degradation of p-Xylene by a Sulfate-Reducing Enrichment Culture

A strictly anaerobic enrichment culture was obtained with p-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. p-Xylene was completely oxidized to CO₂. The enrichment culture depended on Fe(II) in the medium as a scavenger of the produced sulfide. 4-Methylbenzylsuccinic acid and 4-methylphenylitaconic acid were identified in supernatants of cultures indicating that degradation of p-xylene was initiated by fumarate addition to one of the methyl groups. Therefore, p-xylene degradation probably proceeds analogously to toluene degradation by Thauera aromatica or anaerobic degradation pathways for o- and m-xylene.     

Isolation,Identification and Substrate Assimilation Specificity of Some Aromatic Hydrocarbon Utilizing Bacteria

As a result of screening isoalkyl or isoalkenyl substituted aromatic hydrocarbon assimilating microorganisms, 19 strains of isopropylbenzene assimilating bacteria were isolated. Thirteen of these strains were found to grow on α-methylstyrene and all 4 strains tested were also found to grow on isobutylbenzene.

Among them, 2 strains (S107B1 and S182BI) were selected for further study and were identified as Ps. convexa and Ps. ovalis, respectively.

Furthermore, some examined aromatic hydrocarbon utilizing bacteria were classified into two groups by differences in substrate assimilation specificity.     

Specific biodegradation of polychlorinated biphenyls (PCBs) facilitated by plant terpenoids

The aim of this study was to examine how plant terpenoids, as natural growth substrates or inducers, would affect the biodegradation of PCB congeners. Various PCB degraders that could grow on biphenyl and several terpenoids were tested for their PCB degradation capabilities. Degradation activities of the PCB congeners, 4,4′-dichlorobiphenyl (4,4′-DCBp) and 2,2′-dichlorobiphenyl (2,2′-DCBp), were initially monitored through a resting cell assay technique that could detect their degradation products. The PCB degraders,Pseudomonas sp. P166 andRhodococcus sp. T104, were found to grow on both biphenyl and terpenoids ((S)-(−) limonene,p-cymene and α-terpinene) whereasArthrobacter sp. B1B could not grow on the terpenoids as a sole carbon source. The B1B strain grown on biphenyl exhibited good degradation activity for 4,4′-DCBp and 2,2′-DCBp, while the activity of strains P166 and T104 was about 25% that of the B1B strain, respectively. Concomitant GC analysis, however, demonstrated that strain T104, grown on (S)-(−) limonene,p-cymene and α-terpinene, could degrade 4,4′-DCBp up to 30%, equivalent to 50% of the biphenyl induction level. Moreover, strain T104 grown on (S)-(−) limonene, could also degrade 2,2′-DCBp up to 30%. This indicates that terpenoids, widely distributed in nature, could be utilized as both growth and/or inducer substrate(s) for PCB biodegradation in the environment.     

Identification,cloning, and characterization of a multicomponent biphenyl dioxygenase from <Emphasis Type="Italic">Sphingobium yanoikuyae</Emphasis> B1

Sphingobium yanoikuyae B1 utilizes both polycyclic aromatic hydrocarbons (biphenyl, naphthalene, and phenanthrene) and monocyclic aromatic hydrocarbons (toluene, m- and p-xylene) as its sole source of carbon and energy for growth. The majority of the genes for these intertwined monocyclic and polycyclic aromatic pathways are grouped together on a 39 kb fragment of chromosomal DNA. However, this gene cluster is missing several genes encoding essential enzymatic steps in the aromatic degradation pathway, most notably the genes encoding the oxygenase component of the initial polycyclic aromatic hydrocarbon (PAH) dioxygenase. Transposon mutagenesis of strain B1 yielded a mutant blocked in the initial oxidation of PAHs. The transposon insertion point was sequenced and a partial gene sequence encoding an oxygenase component of a putative PAH dioxygenase identified. A cosmid clone from a genomic library of S. yanoikuyae B1 was identified which contains the complete putative PAH oxygenase gene sequence. Separate clones expressing the genes encoding the electron transport components (ferredoxin and reductase) and the PAH dioxygenase were constructed. Incubation of cells expressing the dioxygenase enzyme system with biphenyl or naphthalene resulted in production of the corresponding cis-dihydrodiol confirming PAH dioxygenase activity. This demonstrates that a single multicomponent dioxygenase enzyme is involved in the initial oxidation of both biphenyl and naphthalene in S. yanoikuyae B1.     

Physicochemical and immunochemical characterization of salicylate 5-hydroxylase,m-hydroxybenzoate 6-hydroxylase and p-hydroxybenzoate 3-hydroxylase from Rhodococcus erythropolis

Salicylate 5-hydroxylase (SAL5H), m-hydroxybenzoate 6-hydroxylase (MHB6H), and p-hydroxybenzoate 3-hydroxylase (PHB3H) from Gram-positive Rhodococcus erythropolis strain S1 were characterized physicochemically and immunochemically. The subunit size and amino acid composition of SAL5H, MHB6H, and PHB3H from strain S1 showed properties similar to those of other flavin-containing aromatic compound monooxygenases such as p-hydroxybenzoate hydroxylase and salicylate 1-hydroxylase (SAL1H), belonging to p-hydroxybenzoate hydroxylase-class, except for homotetrameric structure and cofactor specficity. The N-terminal amino acid sequence of MHB6H from strain S1 indicated significant similarity of ADP-binding region in the N-terminal portion of the enzyme with that known for SAL1H from Pseudomonas putida. Immunochemical properties, determined while conducting serological experiments, showed SAL5H and MHB6H from strain S1 to be immunologically different from PHB3H from strain S1, while SAL5H and MHB6H to apparently share partial antigenic determinants.     

Anaerobic degradation of ethylbenzene and other aromatic hydrocarbons by new denitrifying bacteria

Anaerobic degradation of alkylbenzenes with side chains longer than that of toluene was studied in freshwater mud samples in the presence of nitrate. Two new denitrifying strains, EbN1 and PbN1, were isolated on ethylbenzene and n-propylbenzene, respectively. For comparison, two further denitrifying strains, ToN1 and mXyN1, were isolated from the same mud with toluene and m-xylene, respectively. Sequencing of 16SrDNA revealed a close relationship of the new isolates to Thauera selenatis. The strains exhibited different specific capacities for degradation of alkylbenzenes. In addition to ethylbenzene, strain EbN1 utilized toluence, but not propylbenzene. In contrast, propylbenzene-degrading strain PbN1 did not grow on toluene, but was able to utilize ethylbenzene. Strain ToN1 used toluene as the only hydrocarbon substrate, whereas strain mXyN1 utilized both toluene and m-xylene. Measurement of the degradation balance demonstrated complete oxidation of ethylbenzene to CO₂ by strain EbN1. Further characteristic substrates of strains EbN1 and PbN1 were 1-phenylethanol and acetophenone. In contrast to the other isolates, strain mXyN1 did not grow on benzyl alcohol. Benzyl alcohol (also m-methylbenzyl alcohol) was even a specific inhibitor of toluene and m-xylene utilization by strain mXyN1. None of the strains was able to grow on any of the alkylbenzenes with oxygen as electron acceptor. However, polar aromatic compounds such as benzoate were utilized under both oxic and anoxic conditions. All four isolates grew anaerobically on crude oil. Gas chromatographic analysis of crude oil after growth of strain ToN1 revealed specific depletion of toluene.     

Anaerobic degradation of p-xylene in sediment-free sulfate-reducing enrichment culture

Anaerobic degradation of p-xylene was studied with sulfate-reducing enrichment culture. The enrichment culture was established with sediment-free sulfate-reducing consortium on crude oil. The crude oil-degrading consortium prepared with marine sediment revealed that toluene, and xylenes among the fraction of alkylbenzene in the crude oil were consumed during the incubation. The PCR-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene for the p-xylene degrading sulfate-reducing enrichment culture showed the presence of the single dominant DGGE band pXy-K-13 coupled with p-xylene consumption and sulfide production. Sequence analysis of the DGGE band revealed a close relationship between DGGE band pXy-K-13 and the previously described marine sulfate-reducing strain oXyS1 (similarity value, 99%), which grow anaerobically with o-xylene. These results suggest that microorganism corresponding to pXy-K-13 is an important sulfate-reducing bacterium to degrade p-xylene in the enrichment culture.     

Aromatic hydrocarbon degradation by Sphingomonas yanoikuyae B1

Sphingomonas yanoikuyae B1 is able to grow on a wide variety of aromatic compounds including biphenyl, naphthalene, phenanthrene, toluene, m-, and p-xylene. In addition, the initial enzymes for degradation of biphenyl have the ability to metabolize a wide variety of different polycyclic aromatic hydrocarbons. The catabolic pathways for the degradation of both the monocyclic and polycyclic aromatic hydrocarbons are intertwined, joining together at the level of (methyl)benzoate and catechol. Both upper branches of the catabolic pathways are induced when S. yanoikuyae B1 is grown on either class of compound. An analysis of the genes involved in the degradation of these aromatic compounds reveals that at least six operons are involved. The genes are not arranged in discrete pathway units but are combined in groups with genes for the degradation of both classes of compounds in the same operon. Genes for multiple dioxygenases are present perhaps explaining the ability of S. yanoikuyae B1 to grow on a wide variety of aromatic compounds. Received 10 August 1997/ Accepted in revised form 15 August 1997     

Anaerobic Activation of p-Cymene in Denitrifying Betaproteobacteria: Methyl Group Hydroxylation versus Addition to Fumarate

The betaproteobacteria “Aromatoleum aromaticum” pCyN1 and “Thauera” sp. strain pCyN2 anaerobically degrade the plant-derived aromatic hydrocarbon p-cymene (4-isopropyltoluene) under nitrate-reducing conditions. Metabolite analysis of p-cymene-adapted “A. aromaticum” pCyN1 cells demonstrated the specific formation of 4-isopropylbenzyl alcohol and 4-isopropylbenzaldehyde, whereas with “Thauera” sp. pCyN2, exclusively 4-isopropylbenzylsuccinate and tentatively identified (4-isopropylphenyl)itaconate were observed. 4-Isopropylbenzoate in contrast was detected with both strains. Proteogenomic investigation of p-cymene- versus succinate-adapted cells of the two strains revealed distinct protein profiles agreeing with the different metabolites formed from p-cymene. “A. aromaticum” pCyN1 specifically produced (i) a putative p-cymene dehydrogenase (CmdABC) expected to hydroxylate the benzylic methyl group of p-cymene, (ii) two dehydrogenases putatively oxidizing 4-isopropylbenzyl alcohol (Iod) and 4-isopropylbenzaldehyde (Iad), and (iii) the putative 4-isopropylbenzoate-coenzyme A (CoA) ligase (Ibl). The p-cymene-specific protein profile of “Thauera” sp. pCyN2, on the other hand, encompassed proteins homologous to subunits of toluene-activating benzylsuccinate synthase (termed [4-isopropylbenzyl]succinate synthase IbsABCDEF; identified subunits, IbsAE) and protein homologs of the benzylsuccinate β-oxidation (Bbs) pathway (termed BisABCDEFGH; all identified except for BisEF). This study reveals that two related denitrifying bacteria employ fundamentally different peripheral degradation routes for one and the same substrate, p-cymene, with the two pathways apparently converging at the level of 4-isopropylbenzoyl-CoA.     

Characterization of a Spontaneously Occurring Mutant of the TOL20 Plasmid in Pseudomonas putida MT20: Possible Regulatory Implications

Pseudomonas putida MT20 carries a plasmid (TOL20) that codes for the enzymes responsible for the catabolism of toluene, m- and p-xylene to benzoate, and m- and p-toluate, respectively, followed by meta cleavage of the aromatic ring. Growth on 5 mM benzoate selects very strongly for (i) strains that have been cured of the plasmid and (ii) strains with an intermediate growth pattern (the B3 phenotype) that retain the ability to grow on toluene, m-xylene, and benzoate but are unable to grow on m-toluate. Both types of strains were selected because they are no longer able to oxidize benzoate by the plasmid pathway but instead use an alternative route, the ortho or β-ketoadipate pathway, which is chromosomally coded and supports faster growth. Evidence that one strain with the B3 phenotype, MT20-B3, has a regulatory mutation that prevents induction of the meta-pathway enzymes by benzoate and m-toluate, but which enables them to be induced by toluene and m-xylene, is presented. The plasmid in this strain, as in most of the others with the same phenotype, is nonconjugative. Analysis of MT20-B3, together with revertants of it and other noninducible mutants, has led to a model for the regulation of the plasmid-coded enzymes in MT20, in which it is proposed that the early enzymes for degradation of m-toluate and benzoate are positively controlled by two regulator molecules, one of which interacts with toluene and m-xylene as inducers and the other of which interacts with benzoate and m-toluate. It is argued that MT20-B3 and strains with a similar phenotype arose as a result of a deletion of the gene coding for the second regulator molecule.