Emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168

Abstract The emergence of resistance to glutaraldehyde in spores of Bacillus subtilis 168 was examined. Resistance to an organic solvent (toluene), heat and lysozyme were included for comparison. A sequential development of resistance was observed, with toluene resistance occuring early on in sporulation (stages III and IV), thermal resistance at early stage V, lysozyme resistance at middle stage V and glutaraldehyde resistance arising late in stage V. Studies with sporulation mutants also indicate that glutaraldehyde resistance is acquired even later than lysozyme resistance and may therefore possibly be considered as a very late marker event for sporulation, characterizing late stages of B. subtilis 168 spore formation.     

Emergence and development of chlorhexidine resistance during sporulation of Bacillus subtilis 168

Abstract During sporulation of Bacillus subtilis strain 168 initiated by step-down conditions, resistance to chlorhexidine diacetate (CHA) developed at about t _3.5, before heat but after toluene resistance. Mutants blocked at stage IV of sporulation were sensitive to all three treatments. Stage V mutants were toluene resistant but moderately sensitive to heat and CHA. A stage VI mutant was resistant to all three treatments. Thus, chlorhexidine resistance is likely to be a result of spore coat, rather than of cortex, development.     

Adaptation of Rhodococcus erythropolis cells to high concentrations of toluene

Cells of Rhodococcus erythropolis DCL14 were adapted to increasing toluene concentrations in a mechanically stirred reactor. When the initial non-adapted cells were placed in contact with toluene, only 10.5% of cells remained viable after 1 h in the presence of 20% (v/v) toluene, while 8.6% of cells were viable after 28 h in the presence of an organic phase containing 80% (v/v) toluene in n-dodecane. Cell adaptation was studied by following the toluene consumption rate, the viability of the cell population, and the composition of the bacteria cellular membrane in the presence of increasing concentrations of toluene in the reactor. A maximum toluene concentration of 4.9 M, which corresponds to 52.4% (v/v) toluene in the organic phase, was achieved, toluene being consumed at 10.7 mg/(h mg protein). The adapted cells showed a substantially increased resistance to 50% ethanol and to concentrations of Betadine and Micropur tablets currently used in water purification, when compared to non-adapted cells.     

pKJ1, a naturally occurring conjugative plasmid coding for toluene degradation and resistance to streptomycin and sulfonamides.

Pseudomonas sp. TA8 isolated by m-toluate enrichment from an aqueous sample metabolized toluene and m- and p-xylenes via the meta cleavage pathway, and manifested specific resistance to streptomycin and sulfonamides. A variety of experiments revealed that the pKJ1 plasmid of about 150 megadaltons carried by TA8 specified both the toluene and xylene degradative function (the Tol function) and streptomycin/sulfonamide resistance. The deletion of a segment of pKJ1 (about 22 megadaltons) resulted in the loss of the Tol function. pKJ1 was not assigned to Pseudomonas incompatibility group P-1, P-2, P-3, or P-9.     

Improving the catabolic functions in the toluene-resistant strain Pseudomonas putidaS12

The metabolic range of the solvent-resistant bacterium, Pseudomonas putida S12, was successfully expanded by transferring into it catabolic plasmids for the degradation of toluene, biphenyl and naphthalene. Enhancement of the catabolic possibilities of P. putida S12 strain does not interfere with its toluene resistance properties. In this way we obtained derivatives which are able to use the heterologous genes in a two-phase system.     

Production of L-DOPA(3,4-dihydroxyphenyl-L-alanine) from benzene by using a hybrid pathway

As an alternative approach to the production of L-DOPA from a cheap raw material, we constructed a hybrid pathway consisting of toluene dioxygenase, toluene cis-glycol dehydrogenase, and tyrosine phenol-lyase. In this pathway, catechol is formed from benzene through the sequential action of toluene dioxygenase and toluene cis-glycol dehydrogenase, and L-DOPA is synthesized from the resulting catechol in the presence of pyruvate and ammonia by tyrosine phenol-lyase cloned from Citrobacter freundii. When the hybrid pathway was expressed in E. coli, production of L-DOPA was as low as 3 mM in 4 h due to the toxic effect of benzene on the cells. In order to reduce lysis of cells, Pseudomonas aeruginosa was employed as an alternative, which resulted in accumulation of about 14 mM L-DOPA in 9 h, showing a stronger resistance to benzene.     

Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E

In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The ttgGHI genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single ttgH mutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.     

A set of genes encoding a second toluene efflux system in Pseudomonas putida DOT-T1E is linked to the tod genes for toluene metabolism

Sequence analysis in Pseudomonas putida DOT-T1E revealed a second toluene efflux system for toluene metabolism encoded by the ttgDEF genes, which are adjacent to the tod genes. The ttgDEF genes were expressed in response to the presence of aromatic hydrocarbons such as toluene and styrene in the culture medium. To characterize the contribution of the TtgDEF system to toluene tolerance in P. putida, site-directed mutagenesis was used to knock out the gene in the wild-type DOT-T1E strain and in a mutant derivative, DOT-T1E-18. This mutant carried a Tn5 insertion in the ttgABC gene cluster, which encodes a toluene efflux pump that is synthesized constitutively. For site-directed mutagenesis, a cassette to knock out the ttgD gene and encoding resistance to tellurite was constructed in vitro and transferred to the corresponding host chromosome via the suicide plasmid pKNG101. Successful replacement of the wild-type sequences with the mutant cassette was confirmed by Southern hybridization. A single ttgD mutant, DOT-T1E-1, and a double mutant with knock outs in the ttgD and ttgA genes, DOT-T1E-82, were obtained and characterized for toluene tolerance. This was assayed by the sudden addition of toluene (0.3% [vol/vol]) to the liquid culture medium of cells growing on Luria-Bertani (LB) medium (noninduced) or on LB medium with toluene supplied via the gas phase (induced). Induced cells of the single ttgD mutant were more sensitive to sudden toluene shock than were the wild-type cells; however, noninduced wild-type and ttgD mutant cells were equally tolerant to toluene shock. Noninduced cells of the double DOT-T1E-82 mutant did not survive upon sudden toluene shock; however, they still remained viable upon sudden toluene shock if they had been previously induced. These results are discussed in the context of the use of multiple efflux pumps involved in solvent tolerance in P. putida DOT-T1E.     

Endophytes and their potential to deal with co-contamination of organic contaminants (toluene) and toxic metals (nickel) during phytoremediation

The aim was to investigate if engineered endophytes that are capable of degrading organic contaminants, and deal with or ideally improve uptake and translocation of toxic metals, can improve phytoremediation of mixed organic-metal pollution. As a model system, yellow lupine was inoculated with the endophyte Burkholderia cepacia VM1468 possessing (a) the pTOM-Bu61 plasmid, coding for constitutive toluene/TCE degradation, and (b) the chromosomally inserted ncc-nre Ni resistance/sequestration system. As controls, plants were inoculated with B. vietnamiensis BU61 (pTOM-Bu61) and B. cepacia BU72 (containing the ncc-nre Ni resistance/sequestration system). Plants were exposed to mixes of toluene and Ni. Only inoculation with B. cepacia VM1468 resulted in decreased Ni and toluene phytotoxicity, as measured by a protective effect on plant growth and decreased activities of enzymes involved in antioxidative defence (catalase, guaiacol peroxidase, superoxide dismutase) in the roots. Besides, plants inoculated with B. cepacia VM1468 and B. vietnamiensis BU61 released less toluene through the leaves than non-inoculated plants and those inoculated with B. cepacia BU72. Ni-uptake in roots was slightly increased for B. cepacia BU72 inoculated plants. These results indicate that engineered endophytes have the potential to assist their host plant to deal with co-contamination of toxic metals and organic contaminants during phytoremediation.     

Isolation and Characterization of Toluene-Sensitive Mutants from the Toluene-Resistant Bacterium Pseudomonas putida GM73

To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, Pseudomonas putida GM73, we carried out Tn5 mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10⁻⁶). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn5-inserted genes, and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by ttg genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from ttg8 did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.     

The pGRT1 plasmid of Pseudomonas putida DOT-T1E encodes functions relevant for survival under harsh conditions in the environment

Pseudomonas putida DOT-T1E has the capacity to grow in the presence of high concentrations of toluene. This ability is mainly conferred by an efflux pump encoded in a self-transmissible 133 kb plasmid named pGRT1. Sequence analysis of the pGRT1 plasmid revealed several key features. Most of the genes related to the plasmid maintenance functions show similarity with those encoded on pBVIE04 from Burkholderia vietnamensis G4, and knock-out mutants in several of these genes confirmed their roles. Two additional plasmid DNA fragments were incorporated into the plasmid backbone by recombination and/or transposition; in these DNA regions, apart from multiple recombinases and transposases, several stress-related and environmentally relevant functions are encoded. We report that plasmid pGRT1 not only confers the cells with tolerance to toluene but also resistance to ultraviolet light. We show here the implication of a new protein in solvent tolerance which controls the level of expression of the TtgGHI efflux pump, as well as the implication of a protein with homology to the universal stress protein in solvent tolerance and ultraviolet light resistance. Furthermore, this plasmid encodes functions that allow the cells to chemotactically respond to toluene and participate in iron scavenging.     

Effect of solvent adaptation on the antibiotic resistance in Pseudomonas putida S12

The effect of the adaptation to toluene on the␣resistance to different antibiotics was investigated in the␣solvent-resistant strain Pseudomonas putida S12. We␣followed the process of the solvent adaptation of P.␣putida S12 by cultivating the strain in the presence␣of␣increasing concentrations of toluene and studied␣the correlation of this gradual adaptation to the resistance towards antibiotics. It was shown that the tolerance to various chemically and structurally unrelated antibiotics, with different targets in the cell, increased during this gradual adaptation. The survival of P. putida S12 in the presence of antibiotics like tetracycline, nigericin, polymyxin B, piperacillin or chloramphenicol increased 30- to and 1000-fold after adaptation to 600 mg/l toluene. However, cells grown in the absence of any solvents lost their adaptation to toluene even when grown in the presence of antibiotics. Results are discussed in terms of the physico-chemical properties of membranes as affected by the observed cis/trans isomerization of unsaturated fatty acids, as well as in terms of the active efflux of molecules from the cytoplasmic membrane. Received: 9 May 1997 / Received revision: 4 July 1997 / Accepted: 4 July 1997     

Temporal dissociation of late events in Bacillus subtilis sporulation from expression of genes that determine them

During sporulation in replacement medium, resistance to toluene to heating at 65 degrees C, to lysozyme, and to heating at 80 degrees C appeared in sequence between 4 and 8 h after the induction of sporulation (i.e., between t4 and t8). The addition of sufficient chloramphenicol at t4.5 to prevent protein synthesis nevertheless allowed the emergence of all of these types of resistance except lysozyme resistance. The numbers of spores with these types of resistance (lysozyme resistance again excepted) increased about fourfold when phenylmethylsulfonyl fluoride (an inhibitor of serine protease activity) was also present. Thus, the observed increases in resistance in the 2 h after the addition of chloramphenicol resulted from the utilization of preformed protein elements. Dipicolinate did not seem to be a determining factor in the development of any of these forms of resistance. Electron micrographs showed that inhibition of protein synthesis did not prevent deposition of the outer layers of the spores. Lysozyme resistance developed differently; synthesis of the relevant proteins began later (t5), and continued synthesis was necessary up to t8. Some processing of proteins made earlier was a prerequisite for lysozyme resistance. Therefore, it appears that from the viewpoint of regulation, the expression of the genes and the production of the proteins for resistance to toluene, heating at 65 degrees C, and heating at 80 degrees C are all stage IV sporulation events, although the resistance properties themselves appear only during stages V and VI. Lysozyme resistance is the only real late event among those examined. The germination characteristics of the spores, which are also late events, are discussed in this context, as they too are dependent on proteins that are synthesized much earlier.     

Transgenic plants of Petunia hybrida harboring the CYP2E1 gene efficiently remove benzene and toluene pollutants and improve resistance to formaldehyde

The CYP2E1 protein belongs to the P450 enzymes family and plays an important role in the metabolism of small molecular and organic pollutants. In this study we generated CYP2E1 transgenic plants of Petunia using Agrobacterium rhizogenes K599. PCR analysis confirmed that the regenerated plants contained the CYP2E1 transgene and the rolB gene of the Ri plasmid. Southern blotting revealed the presence of multiple copies of CYP2E1 in the genome of transgenic plants. Fluorescent quantitative PCR revealed exogenous CYP2E1 gene expression in CYP2E1 transgenic plants at various levels, whereas no like expression was detected in either GUS transgenic plants or wild-types. The absorption of benzene and toluene by transgenic plants was analyzed through quantitative gas chromatography. Transgenic plants with high CYP2E1 expression showed a significant increase in absorption capacity of environmental benzene and toluene, compared to control GUS transgenic and wild type plants. Furthermore, these plants also presented obvious improved resistance to formaldehyde. This study, besides being the first to reveal that the CYP2E1 gene enhances plant resistance to formaldehyde, also furnishes a new method for reducing pollutants, such as benzene, toluene and formaldehyde, by using transgenic flowering horticultural plants.     

Mutations in Genes Involved in the Flagellar Export Apparatus of the Solvent-Tolerant Pseudomonas putida DOT-T1E Strain Impair Motility and Lead to Hypersensitivity to Toluene Shocks

Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of 1% (vol/vol) toluene in the culture medium. Random mutagenesis with mini-Tn5-'phoA-Km allowed us to isolate a mutant strain (DOT-T1E-42) that formed blue colonies on Luria-Bertani medium supplemented with 5-bromo-4-chloro-3-indolylphosphate and that, in contrast to the wild-type strain, was unable to tolerate toluene shocks (0.3%, vol/vol). The mutant strain exhibited patterns of tolerance or sensitivity to a number of antibiotics, detergents, and chelating agents similar to those of the wild-type strain. The mutation in this strain therefore seemed to specifically affect toluene tolerance. Cloning and sequencing of the mutation revealed that the mini-Tn5-'phoA-Km was inserted within the fliP gene, which is part of the fliLMNOPQRflhBA cluster, a set of genes that encode flagellar structure components. FliP is involved in the export of flagellar proteins, and in fact, the P. putida fliP mutant was nonmotile. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of toluene tolerance and motility unequivocally assigned FliP a function in solvent resistance. An flhB knockout mutant, another gene component of the flagellar export apparatus, was also nonmotile and hypersensitive to toluene. In contrast, a nonpolar mutation at the fliL gene, which encodes a cytoplasmic membrane protein associated with the flagellar basal body, yielded a nonmotile yet toluene-resistant strain. The results are discussed regarding a possible role of the flagellar export apparatus in the transport of one or more proteins necessary for toluene tolerance in P. putida DOT-T1E to the periplasm.     

Isolation and transposon mutagenesis of a Pseudomonas putida KT2442 toluene-resistant variant: involvement of an efflux system in solvent resistance

A toluene-resistant variant of Pseudomonas putida KT2442, strain TOL, was isolated after liquid cultivation under xylene followed by toluene for 1 month in each condition. Almost all the populations of the variant strain formed small but readily visible colonies under toluene within 24 h at 30°C. The toluene-resistant strain also showed an increase in resistance to some unrelated antibiotics. Several toluene-sensitive Tn5 mutants have been isolated from the toluene-resistant strain and showed various levels of sensitivity. Most of these mutations did not cause significant changes in antibiotic resistance; however, one of the mutants (TOL-4) was highly susceptible to both organic solvents and various antibiotics, especially β-lactams. Sequencing analysis revealed that the mutation in TOL-4 had been introduced into a gene that may encode a transporter protein of an efflux system. This efflux system is very similar to one of the multidrug efflux systems of Pseudomonas aeruginosa. These observations indicate that a multidrug efflux system plays a major role in the organic solvent resistance of P. putida TOL. However, several other genes may also be involved. Received: December 18, 1997 / Accepted: March 16, 1998     

Identification and characterization of a transmissible linear plasmid from Rhodococcus erythropolis BD2 that encodes isopropylbenzene and trichloroethene catabolism.

Rhodococcus erythropolis BD2, which is able to utilize isopropylbenzene as a sole carbon and energy source, was shown to contain a conjugative linear plasmid, pBD2. The estimated size of pBD2 is 208 to 212 kb. Linear plasmid-deficient strains had lost both the isopropylbenzene degradation and trichloroethene degradation characteristics, as well as the arsenite resistance and mercury resistance phenotypes. Reintroduction of pBD2 restored all four characteristics. Conjugational transfer of pBD2 to a plasmidless mutant of strain BD2 and other R. erythropolis strains occurred at frequencies between 3.5 x 10(-5) and 2.6 x 10(-3) transconjugants per recipient. R. erythropolis BD2 degrades isopropylbenzene via 3-isopropylcatechol and 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate. Both isopropylbenzene-oxidizing and meta-cleavage activities were shown to correspond with the presence of pBD2. Southern hybridizations with DNA encoding the toluene dioxygenase structural genes (todC1C2BA) from Pseudomonas putida F1 revealed homology to linear plasmid DNA. These results indicate that the isopropylbenzene degradation pathway encoded by linear plasmid pBD2 is initiated by an isopropylbenzene dioxygenase analogous to toluene dioxygenase.