PHA granule formation and degradation by Cupriavidus necator under different nutritional conditions.

Polyhydroxyalkanoates (PHA) are polymers produced by microorganisms with increasing commercialization potential; Cupriavidus necator has been the model microorganism to research PHA production. Despite many contributions concerning the formation and degradation of PHA granules, as well as the morphological changes in cells, these phenomena have not been univocally explained yet. Thus, this study aims to integrate the microscopic and analytical analysis to characterize changes in bacterial cell/PHA granules morphology, PHA content, and yield coefficients under different cultivation strategies of C. necator ATCC 17697. The cell size and morphology, granule size and amount, residual biomass, and PHA concentration along the fermentation and degradation depend greatly on nutritional conditions and cultivation time of C. necator. It was proposed to calculate a yield coefficient for the residual biomass production in the PHA utilization stage, related to the bacteria's ability to survive without a carbon source in the culture medium by utilizing the accumulated PHA previously. Maximum granule length reached 1.07 µm after 72 h of PHA accumulation stage under optimum nutritional conditions. This value is twice the values previously reported for C. necator. It is important since the larger PHA granules facilitate the recovery of PHA and different application development.

Biosynthesis of polyhydroxyalkanoates from vegetable oil under the co-expression of fadE and phaJ genes in Cupriavidus necator.

The acyl-CoA dehydrogenase (FadE) and (R)-specific enoyl-CoA hydratase (PhaJ) are functionally related to the degradation of fatty acids and the synthesis of polyhydroxyalkanoates (PHAs). To verify this, a recombinant Cupriavidus necator H16 harboring the plasmid -pMPJAS03- with fadE from Escherichia coli strain K12 and phaJ1 from Pseudomonas putida strain KT2440 under the arabinose promoter (araC-PBAD) was constructed. The impact of co-expressing fadE and phaJ genes on C. necator H16/pMPJAS03 maintaining the wild-type synthase on short-chain-length/medium-chain-length PHA formation from canola or avocado oil at different arabinose concentrations was investigated. The functional activity of fadEE.c led to obtaining higher biomass and PHA concentrations compared to the cultures without expressing the gene. While high transcriptional levels of phaJ1P.p, at 0.1% of arabinose, aid the wild-type synthase to polymerize larger-side chain monomers, such as 3-Hydroxyoctanoate (3HO) and 3-Hydroxydecanoate (3HD). The presence of even small amounts of 3HO and 3HD in the co-polymers significantly depresses the melting temperature of the polymers, compared to those composed of pure 3-hydroxybutyrate (3HB). Our data presents supporting evidence that the synthesis of larger-side chain monomers by the recombinant strain relies not only upon the affinity of the wild-type synthase but also on the functionality of the intermediate supplying enzymes.

Hydrogen and polyhydroxybutyrate production from wheat straw hydrolysate using Caldicellulosiruptor species and Ralstonia eutropha in a coupled process.

This report presents an integrated biorefinery concept in which wheat straw hydrolysate was treated with co-cultures of osmotolerant thermophilic bacterial strains, Caldicellulosiruptor saccharolyticus and C. owensensis to obtain hydrogen, while the liquid effluent containing acetate and residual glucose was used as feed for polyhydroxybutyrate (PHB) production by Ralstonia eutropha. The Caldicellulosiruptor spp. co-culture consumed 10.8 g/L of pretreated straw sugars, glucose and xylose, producing 134 mmol H2/L. PHB accumulation by R. eutropha was first studied in minimal salts medium using acetate with/without glucose as carbon source. Addition of salts promoted cell growth and PHB production in the effluent. Fed-batch cultivation in a nitrogen limited medium with 40% (v/v) aeration resulted in a cell density of 15.1 g/L with PHB content of 80.1% w/w and PHB concentration of 12.1 g/L, while 20% aeration gave a cell density of 11.3 g/L with 83.4% w/w PHB content and 9.4 g/L PHB concentration.

Genome characteristics dictate poly-R-(3)-hydroxyalkanoate production in Cupriavidus necator H16.

Cupriavidus necator H16 is a well-recognized enterprise with efficient manufacturing machineries to produce diverse polymers belonging to polyhydroxyalkanoates (PHAs) family. The genome fingerprints, including PHA machinery proteins and fatty acid metabolism, had educated engineering strategies to enhance PHAs production. This outstanding progress has enlightened us to present an exhaustive examination of the ongoing research, addressing the great potential design of genome features towards PHA production and furthermore, we show how those acquired knowledge have been explored in other biotechnological applications. This updated-review concludes that the combination of an optimal strain selection, suitable metabolic engineering and a large-scale fermentation on oil substrates is critical to endow the ability of incorporating mcl-PHAs monomers in this organism.

Heterologous expression of phaC2 gene and poly-3-hydroxyalkanoate production by recombinant Cupriavidus necator strains using canola oil as carbon source.

Many heterologous transformation studies have been carried out using the Cupriavidus necator PHB-4 strain to investigate the expression characteristics of various polyhydroxyalkanoate (PHA) synthase enzymes. In this study, we generated a recombinant C. necator PHB-4 strain by transforming a plasmid (pMRC03) harbouring the synthetic phaC2 gene of Pseudomonas putida CA-3. Under conditions favourable for expression of the phaC2 P.putCA-3 gene, canola oil was used as carbon source for the synthesis of PHAs. The expressed synthase polymerised monomers of 3-hydroxybutyrate (3-HB), 3-hydroxyvalerate (3-HV) and 3-hydroxyhexanoate (3-HHx) in the recombinant C. necator PHB-4 (pMRC03) strain. We then co-expressed the phaC2P.putCA-3 gene with the native phaC1C.ne gene in wild type Cupriavidus necator H16 (C. necator H16 (pMRC03)). This co-expression produced a PHA blend of 3-HB, 3-HV, 3-HHx and 3-hydroxyoctanoate (3-HO) monomers in the presence of canola oil. Gas chromatography analysis revealed the presence of 94mol% 3-HB, 1mol% 3-HV, 4mol% 3-HHx and 1mol% 3-HO in a tetra-polymer. Thus, we confirmed that a synthetic phaC2 gene encoding the synthase enzyme is functionally active with substrates ranging from short to medium chain length PHAs.

Poly(3-Hydroxybutyrate) Production in Repeated fed-Batch with Cell Recycle Using a Medium with low Carbon Source Concentration.

Among approaches applied to obtain high productivity and low production costs in bioprocesses are high cell density and the use of low cost substrates. Usually low cost substrates, as waste/agroindustrial residues, have low carbon concentration, which leads to a difficulty in operating bioprocesses. Real time control of process for intracellular products is also difficult. The present study proposes a strategy of repeated fed-batch with cell recycle to attain high cell density of Cupriavidus necator and high poly(3-hydroxybutyrate) (P(3HB)) productivity, using a substrate with low carbon source concentration (90 g l(-1)). Also, the use of the oxygen uptake rate data was pointed out as an on line solution for process control, once P(3HB) is an intracellular product. The results showed that total biomass (X), residual biomass (Xr) and P(3HB) values at the end of the culture were 61.6 g l(-1), 19.3 g l(-1) and 42.4 g l(-1) respectively, equivalent to 68.8 % of P(3HB) in the cells, and P(3HB) productivity of 1.0 g l(-1) h(-1). Therefore, the strategy proposed was efficient to achieve high productivity and high polymer content from a medium with low carbon source concentration.

Use of enzymes in extraction of polyhydroxyalkanoates produced by Cupriavidus necator.

Poly(3-hydroxybutyrate) (P(3HB)) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV), are biodegradable thermoplastic polymers. They are members of the polyhydroxyalkanoate (PHA) family, synthesized and accumulated as a carbon and energy reserve by a variety of microorganisms. The aim of this study was to evaluate the use of the proteases Corolase® L10, Alcalase® 2.4L, Corolase® 7089 and Protemax® FC and glycosidases Celumax® BC, Rohament® CL and Rohalase® Barley for the recovery of P(3HB) and P(3HB-co-3HV) synthesized by Cupriavidus necator. The enzyme Celumax® BC provided better lysis of the bacterial cell membrane and the results for the optimization of the operating conditions showed that this enzyme is most stable in acetate buffer at pH 4.0, bath at 60°C, hydrolysis time of 1 h and concentration of 0.02% (w/w). The optimization of the operating conditions showed that the enzyme Celumax® BC provided better lysis of the bacterial cell in acetate buffer at pH 4.0, bath at 60°C, hydrolysis time of 1 h and concentration of 0.02% (w/w). These conditions resulted in lysis of the membrane of the bacteria with a recovery of 93.2% P(3HB-co-3HV) with 94% purity. The results showed that the use of enzymes for the polymer extraction is an efficient process that assists in the cell disruption of Cupriavidus necator.

Cupriavidus necator isolates are able to fix nitrogen in symbiosis with different legume species.

The aim of the present study was to identify a collection of 35 Cupriavidus isolates at the species level and to examine their capacity to nodulate and fix N(2). These isolates were previously obtained from the root nodules of two promiscuous trap species, Phaseolus vulgaris and Leucaena leucocephala, inoculated with soil samples collected near Sesbania virgata plants growing in Minas Gerais (Brazil) pastures. Phenotypic and genotypic methods applied for this study were SDS-PAGE of whole-cell proteins, and 16S rRNA and gyrB gene sequencing. To confirm the ability to nodulate and fix N(2), the presence of the nodC and nifH genes was also determined, and an experiment was carried out with two representative isolates in order to authenticate them as legume nodule symbionts. All 35 isolates belonged to the betaproteobacterium Cupriavidus necator, they possessed the nodC and nifH genes, and two representative isolates were able to nodulate five different promiscuous legume species: Mimosa caesalpiniaefolia, L. leucocephala, Macroptilium atropurpureum, P. vulgaris and Vigna unguiculata. This is the first study to demonstrate that C. necator can nodulate legume species.

Carbon source pulsed feeding to attain high yield and high productivity in poly(3-hydroxybutyrate) (PHB) production from soybean oil using Cupriavidus necator.

Poly(3-hydroxybutyrate) (PHB) biosynthesis from soybean oil by Cupriavidus necator was studied using a bench scale bioreactor. The highest cell concentration (83 g l(-1)) was achieved using soybean oil at 40 g l(-1) and a pulse of the same concentration. The PHB content was 81% (w/w), PHB productivity was 2.5 g l(-1) h(-1), and the calculated Y(p/s) value was 0.85 g g(-1). Growth limitation and the onset of PHB biosynthesis took place due to exhaustion of P, and probably also Cu, Ca, and Fe.

Strict and direct transcriptional repression of the pobA gene by benzoate avoids 4-hydroxybenzoate degradation in the pollutant degrader bacterium Cupriavidus necator JMP134.

As other environmental bacteria, Cupriavidus necator JMP134 uses benzoate as preferred substrate in mixtures with 4-hydroxybenzoate, strongly inhibiting its degradation. The mechanism underlying this hierarchical use was studied. A C. necator benA mutant, defective in the first step of benzoate degradation, is unable to metabolize 4-hydroxybenzoate when benzoate is also included in the medium, indicating that this substrate and not one of its catabolic intermediates is directly triggering repression. Reverse transcription polymerase chain reaction analysis revealed that 4-hydroxybenzoate 3-hydroxylase-encoding pobA transcripts are nearly absent in presence of benzoate and a fusion of pobA promoter to lacZ reporter confirmed that benzoate drastically decreases the transcription of this gene. Expression of pobA driven by a heterologous promoter in C. necator benA mutant, allows growth on 4-hydroxybenzoate in presence of benzoate, overcoming its repressive effect. In contrast with other bacteria, regulators of benzoate catabolism do not participate in repression of 4-hydroxybenzoate degradation. Moreover, the effect of benzoate on pobA promoter can be observed in heterologous strains with the sole presence of PobR, the transcriptional activator of pobA gene, indicating that PobR is enough to fully reproduce the phenomenon. This novel mechanism for benzoate repression is probably mediated by direct action of benzoate over PobR.

Production of polyhydroxyalkanoates (PHAs) with canola oil as carbon source.

Wautersia eutropha was able to synthesize medium chain length polyhydroxyalkanoates (PHAs) when canola oil was used as carbon source. W. eutropha was cultivated using fructose and ammonium sulphate as carbon and nitrogen sources, respectively, for growth and inoculum development. The experiments were done in a laboratory scale bioreactor in three stages. Initially, the biomass was adapted in a batch culture. Secondly, a fed-batch was used to increase the cell dry weight and PHA concentration to 4.36 g L(-1) and 0.36 g L(-1), respectively. Finally, after the addition of canola oil as carbon source a final concentration of 18.27 g L(-1) PHA was obtained after 40 h of fermentation. With canola oil as carbon source, the polymer content of the cell dry matter was 90%. The polymer was purified from dried cells and analyzed by FTIR, NMR and DSC using PHB as reference. The polymer produced by W. eutropha from canola oil had four carbon monomers in the structure of the PHA and identified by 1H and 13C NMR analysis as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxyoctanoate (3HO), and 3-hydroxydodecanoate (3HDD).

Mineralization of PCBs by the genetically modified strain Cupriavidus necator JMS34 and its application for bioremediation of PCBs in soil.

Polychlorobiphenyls (PCBs) are classified as "high-priority pollutants." Diverse microorganisms are able to degrade PCBs. However, bacterial degradation of PCBs is generally incomplete, leading to the accumulation of chlorobenzoates (CBAs) as dead-end metabolites. To obtain a microorganism able to mineralize PCB congeners, the bph locus of Burkholderia xenovorans LB400, which encodes one of the most effective PCB degradation pathways, was incorporated into the genome of the CBA-degrading bacterium Cupriavidus necator JMP134-X3. The bph genes were transferred into strain JMP134-X3, using the mini-Tn5 transposon system and biparental mating. The genetically modified derivative, C. necator strain JMS34, had only one chromosomal insertion of bph locus, which was stable under nonselective conditions. This modified bacterium was able to grow on biphenyl, 3-CBA and 4-CBA, and degraded 3,5-CBA in the presence of m-toluate. The strain JMS34 mineralized 3-CB, 4-CB, 2,4'-CB, and 3,5-CB, without accumulation of CBAs. Bioaugmentation of PCB-polluted soils with C. necator strain JMS34 and with the native B. xenovorans LB400 was monitored. It is noteworthy that strain JMS34 degraded, in 1 week, 99% of 3-CB and 4-CB and approximately 80% of 2,4'-CB in nonsterile soil, as well as in sterile soil. Additionally, the bacterial count of strain JMS34 increased by almost two orders of magnitude in PCB-polluted nonsterile soil. In contrast, the presence of native microflora reduced the degradation of these PCBs by strain LB400 from 73% (sterile soil) to approximately 50% (nonsterile soil). This study contributes to the development of improved biocatalysts for remediation of PCB-contaminated environments.

3-Chlorobenzoate is taken up by a chromosomally encoded transport system in Cupriavidus necator JMP134.

Cupriavidus necator JMP134(pJP4) is able to grow on 3-chlorobenzoate (3-CB), a model chloroaromatic pollutant. Catabolism of 3-CB is achieved via the expression of the chromosomally encoded benABCD genes and the tfd genes from plasmid pJP4. Since passive diffusion of benzoic acid derivatives at physiological pH is negligible, the uptake of this compound should be facilitated by a transport system. However, no transporter has so far been described to perform this function, and identification of chloroaromatic compound transporters has been limited. In this work, uptake experiments using 3-[ring-UL-(14)C]CB showed an inducible transport system in strain JMP134, whose expression is activated by 3-CB and benzoate. A similar level of 3-CB uptake was found for a mutant strain of JMP134, defective in chlorobenzoate degradation, indicating that metabolic drag is not an important component of the measured uptake rate. Competitive inhibitor assays showed that uptake of 3-CB was inhibited by benzoate and, to a lesser degree, by 3-CB and 3,5-dichlorobenzoate, but not by any of 12 other substituted benzoates tested. The expression of several gene candidates for this transport function was analysed by RT-PCR, including both permease-type and ABC-type ATP-dependent transporters. Induction of a chromosomally encoded putative permease transporter (benP gene) was found specifically in the presence of 3-CB or benzoate. A benP knockout mutant of strain JMP134 displayed an almost complete loss of 3-CB transport activity. This is to our knowledge the first report of a 3-CB transporter.

Metabolic reconstruction of aromatic compounds degradation from the genome of the amazing pollutant-degrading bacterium Cupriavidus necator JMP134.

Cupriavidus necator JMP134 is a model for chloroaromatics biodegradation, capable of mineralizing 2,4-D, halobenzoates, chlorophenols and nitrophenols, among other aromatic compounds. We performed the metabolic reconstruction of aromatics degradation, linking the catabolic abilities predicted in silico from the complete genome sequence with the range of compounds that support growth of this bacterium. Of the 140 aromatic compounds tested, 60 serve as a sole carbon and energy source for this strain, strongly correlating with those catabolic abilities predicted from genomic data. Almost all the main ring-cleavage pathways for aromatic compounds are found in C. necator: the beta-ketoadipate pathway, with its catechol, chlorocatechol, methylcatechol and protocatechuate ortho ring-cleavage branches; the (methyl)catechol meta ring-cleavage pathway; the gentisate pathway; the homogentisate pathway; the 2,3-dihydroxyphenylpropionate pathway; the (chloro)hydroxyquinol pathway; the (amino)hydroquinone pathway; the phenylacetyl-CoA pathway; the 2-aminobenzoyl-CoA pathway; the benzoyl-CoA pathway and the 3-hydroxyanthranilate pathway. A broad spectrum of peripheral reactions channel substituted aromatics into these ring cleavage pathways. Gene redundancy seems to play a significant role in the catabolic potential of this bacterium. The literature on the biochemistry and genetics of aromatic compounds degradation is reviewed based on the genomic data. The findings on aromatic compounds biodegradation in C. necator reviewed here can easily be extrapolated to other environmentally relevant bacteria, whose genomes also possess a significant proportion of catabolic genes.

Role of eukaryotic microbiota in soil survival and catabolic performance of the 2,4-D herbicide degrading bacteria Cupriavidus necator JMP134.

Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C. necator JMP134 could be isolated after soil enrichment. In native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5alpha (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.

Enzymatic recovery and purification of polyhydroxybutyrate produced by Ralstonia eutropha.

Polyhydroxybutyrate (PHB) is the most studied among a wide variety of polyhydroxyalkanoates, bacterial biodegradable polymers known as potential substitutes for conventional plastics. This work aimed at evaluating the use of enzymes to recover and purify the PHB produced by Ralstonia eutropha DSM545. Screening experiments allowed the selection of trypsin, bromelain and lysozyme among six enzymes, based on their efficiency in lysing cells of a non-PHB producing R. eutropha strain. Then, process conditions for high efficiency in PHB purification from the DSM545 cells were searched for the enzymes previously selected. The best result was achieved with 2.0% of bromelain (enzyme mass per biomass), equivalent to 14.1 U ml(-1), at 50 degrees C and pH 9.0, resulting in 88.8% PHB purity. Aiming at improving the process efficiency and reducing the enzyme cost, experiments were carried out with pancreatin, leading to 90.0% polymer purity and an enzyme cost three times lower than the one obtained with bromelain. The molecular mass analysis of PHB showed no polymer degradation. Therefore, this work demonstrates the potential of using enzymes in order to recover and purify PHB and bacterial biopolymers in general.

Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways.

Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 plasmid was able to capture chromosomal genes and form hybrid plasmids with the IncP-1 alpha plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants.

Production of poly(3-hydroxybutyrate) by solid-state fermentation with Ralstonia eutropha.

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g(-1) medium on soy cake in 36 h, and 0.7 mg g(-1) medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g(-1) medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.

Phosphate feeding strategy during production phase improves poly(3-hydroxybutyrate-co-3-hydroxyvalerate) storage by Ralstonia eutropha.

The effect of a phosphate feeding strategy and the optimal rate of biomass production ( r(x)) during the production phase of P(3HB-co-3HV) in a 6-l fermentor were determined in cultures of Ralstonia eutropha with the goal of enhancing polymer productivity. Rates of biomass production ( r(x)) between 0.00 and 0.20 gx r l(-1) h(-1) were monitored during the production phase. When a low rate of cell growth was maintained ( r(x) of 0.02 gx r l(-1) h(-1)), polymer production improved, resulting in a final cell mass, P(3HB-co-3HV) mass, and P(3HB-co-3HV) content of 98.2 g, 62.0 g and 63.1 wt%, respectively, after 27.3 h. The maximum polymer productivity obtained during the production phase was 1.36 g l(-1 )h(-1).

Efficient turnover of chlorocatechols is essential for growth of Ralstonia eutropha JMP134(pJP4) in 3-chlorobenzoic acid.

Ralstonia eutropha JMP134(pJP4) degrades 3-chlorobenzoate (3-CB) by using two not completely isofunctional, pJP4-encoded chlorocatechol degradation gene clusters, tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II). Introduction of several copies of each gene cluster into R. eutropha JMP222, which lacks pJP4 and thus accumulates chlorocatechols from 3-CB, allows the derivatives to grow in this substrate. However, JMP222 derivatives containing one chromosomal copy of each cluster did not grow in 3-CB. The failure to grow in 3-CB was the result of accumulation of chlorocatechols due to the limiting activity of chlorocatechol 1,2-dioxygenase (TfdC), the first enzyme in the chlorocatechol degradation pathway. Micromolar concentrations of 3- and 4-chlorocatechol inhibited the growth of strains JMP134 and JMP222 in benzoate, and cells of strain JMP222 exposed to 3 mM 3-CB exhibited a 2-order-of-magnitude decrease in viability. This toxicity effect was not observed with strain JMP222 harboring multiple copies of the tfdC(I) gene, and the derivative of strain JMP222 containing tfdC(I)D(I)E(I)F(I) plus multiple copies of the tfdC(I) gene could efficiently grow in 3-CB. In addition, tfdC(I) and tfdC(II) gene mutants of strain JMP134 exhibited no growth and impaired growth in 3-CB, respectively. The introduction into strain JMP134 of the xylS-xylXYZL genes, encoding a broad-substrate-range benzoate 1,2-dioxygenase system and thus increasing the transformation of 3-CB into chlorocatechols, resulted in derivatives that exhibited a sharp decrease in the ability to grow in 3-CB. These observations indicate that the dosage of chlorocatechol-transforming genes is critical for growth in 3-CB. This effect depends on a delicate balance between chlorocatechol-producing and chlorocatechol-consuming reactions.