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.     

Novel toluene elimination system in a toluene-tolerant microorganism

In studies of Pseudomonas putida IH-2000, a toluene-tolerant microorganism, membrane vesicles (MVs) were found to be released from the outer membrane when toluene was added to the culture. These MVs were found to be composed of phospholipids, lipopolysaccharides (LPS), and very low amounts of outer membrane proteins. The MVs also contained a higher concentration of toluene molecules (0.172 +/- 0. 012 mol/mol of lipid) than that found in the cell membrane. In contrast to the wild-type strain, the toluene-sensitive mutant strain 32, which differs from the parent strain in LPS and outer membrane proteins, did not release MVs from the outer membrane. The toluene molecules adhering to the outer membrane are eliminated by the shedding of MVs, and this system appears to serve as an important part of the toluene tolerance system of IH-2000.     

Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system

Pseudomonas putida T-57 was isolated from an activated sludge sample after enrichment on mineral salts basal medium with toluene as a sole source of carbon. P. putida T-57 utilizes n-butanol, toluene, styrene, m-xylene, ethylbenzene, n-hexane, and propylbenzene as growth substrates. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10-90% (v/v), and was tolerant to organic solvents whose log P(ow) (1-octanol/water partition coefficient) was higher than 2.5. Enzymatic and genetic analysis revealed that P. putida T-57 used the toluene dioxygenase pathway to catabolize toluene. A cis-toluene dihydrodiol dehydrogenase gene (todD) mutant of T-57 was constructed using a gene replacement technique. The todD mutant accumulated o-cresol (maximum 1.7 g/L in the aqueous phase) when cultivated in minimal salts basal medium supplemented with 3% (v/v) toluene and 7% (v/v) 1-octanol. Thus, T-57 is thought to be a good candidate host strain for bioconversion of hydrophobic substrates in two-phase (organic-aqueous) systems.     

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.     

Global and cognate regulators control the expression of the organic solvent efflux pumps TtgABC and TtgDEF of Pseudomonas putida

Pseudomonas putida DOT-T1E grows on a water-toluene double liquid phase. Toluene tolerance in this microorganism is mainly achieved by at least two efflux pumps that belong to the RND family. The TtgDEF efflux pump is induced by toluene, whereas the other efflux pump, called TtgABC, is expressed at a high level in cells not exposed to toluene and at a lower level in cells grown with toluene. The ttgR gene is adjacent to the ttgABC operon and is transcribed divergently from ttgA. The expression level of ttgR was fourfold higher in cells growing in the presence of toluene than in its absence. In a TtgR-deficient background, expression from the ttgA promoter increased about 20-fold, suggesting that TtgR represses expression from the ttgA promoter. In this mutant, background expression of the ttgR gene was also much higher than in the wild-type background; however, its level of expression increased in the presence of toluene. In a ttgR mutant background, expression from the ttgD promoter followed the same pattern of expression as in the wild type. Analysis of a P. putida pTn5cat mutant that exhibited increased sensitivity to a sudden toluene shock, regardless of whether or not it was previously exposed to low toluene concentrations, revealed that pTn5cat had interrupted an lrp-like gene. The ttgR gene was expressed at very high levels in this mutant, with concomitant repression of expression of the ttgABC operon. The second ttgDEF efflux pump was expressed at low levels in this mutant strain, suggesting that the Lrp-like protein is a global regulatory protein involved in the solvent-tolerant response of this strain.     

A Pseudomonas putida cardiolipin synthesis mutant exhibits increased sensitivity to drugs related to transport functionality

Biological membranes have evolved different mechanisms to modify their composition in response to chemical stimuli in a process called 'homeoviscous adaptation'. Among these mechanisms, modifications in the ratio of saturated/unsaturated fatty acids and in cis/trans fatty acid isomers, cyclopropanation and changes in the phospholipids head group composition have been observed. To further understand the role of phospholipid head groups in solvent stress adaptation, we knocked out the cls (cardiolipin synthase) gene in Pseudomonas putida DOT-T1E. As expected, cls mutant membranes contained less cardiolipin than those of the wild-type strain. Although no significant growth rate defect was observed in the cls mutant compared with the wild-type strain, mutant cells were significantly smaller than the wild-type cells. The cls mutant was more sensitive to toluene shocks and to several antibiotics than the parental strain, suggesting either that the RND efflux pumps involved in the extrusion of these drugs were not working efficiently or that membrane permeability was altered in the mutant. Membranes of the cls mutant strain seemed to be more rigid than those of the parental strain, as observed by measurements of fluorescence polarization using the DPH probe, which intercalates into the membranes. Ethidium bromide is pumped out in Pseudomonas putida by at least one RND efflux pump involved in antibiotic and solvent resistance, and the higher rate of accumulation of ethidium bromide inside mutant cells indicated that functioning of the efflux pumps was compromised as a consequence of the alteration in phospholipid head group composition.     

A mutant of Candida albicans deficient in beta-N-acetylglucosaminidase (chitobiase)

A mutant of Candida albicans ATCC 10261 was isolated that was defective in the production of beta-N-acetylglucosaminidase (chitobiase). The mutant grew normally in minimal medium supplemented with either glucose or N-acetyl-D-glucosamine (GlcNAc) as carbon and energy source, and the cells formed germ-tubes at 37 degrees C when induced to do so with GlcNAc. However, unlike the wild-type parent strain, the mutant strain did not utilize N,N'-diacetylchitobiose for growth. The mutant and parent strains had similar growth rates on glucose or GlcNAc, similar rates of uptake of these sugars and similar rates of 14C-labelled amino acid incorporation. The chitobiase mutant did, however, contain 53-85% more chitin than the wild-type strain. No reversion of the mutant phenotype was observed following induction of mitotic recombination with UV light, suggesting that the mutant allele (chi) was carried homozygously in the chitobiase-deficient mutant. Although the chitobiase-deficient mutant was pathogenic, it was not as virulent as the wild-type strain.     

Fatty acid biosynthesis is involved in solvent tolerance in Pseudomonas putida DOT-T1E

The unusual tolerance of Pseudomonas putida DOT-T1E to toluene is based on the extrusion of this solvent by constitutive and inducible efflux pumps and rigidification of its membranes via phospholipid alterations. Pseudomonas putida DOT-T1E-109 is a solvent-sensitive mutant. Mutant cells were less efficient in solvent extrusion than the wild-type cells, as shown by the limited efflux of 14C-1,2,4-trichlorobenzene from the cell membranes, despite the fact that the efflux pumps are overexpressed as a result of increased expression of the ttgDEF and ttgGHI efflux pump operons. This limitation could be the result of alterations in the outer membrane because the mutant cells released more beta-lactamase to the external medium than the wild-type cells. The mutant P. putida DOT-T1E-109 showed negligible synthesis of fatty acids in the presence of sublethal concentrations of toluene as revealed by analysis of 13CH3-13COOH incorporation into fatty acids. In contrast, the mutant strain in the absence of solvents, and the wild-type strain, both in the presence and in the absence of toluene, incorporated 13CH3-13COOH at a high rate into de novo synthesized lipids. The mutation in P. putida DOT-T1E-109 increases sensitivity to the solvent because of a limited efflux of the solvent from the cell membranes with the concomitant inhibition of fatty acid biosynthesis.     

Involvement of the cis/trans Isomerase Cti in Solvent Resistance of Pseudomonas putida DOT-T1E

Pseudomonas putida DOT-T1E is a solvent-resistant strain that is able to grow in the presence of high concentrations of toluene. We have cloned and sequenced the cti gene of this strain, which encodes the cis/trans isomerase, termed Cti, that catalyzes the cis-trans isomerization of esterified fatty acids in phospholipids, mainly cis-oleic acid (C(16:1,9)) and cis-vaccenic acid (C(18:1,11)), in response to solvents. To determine the importance of this cis/trans isomerase for solvent resistance a Cti-null mutant was generated and characterized. This mutant showed a longer lag phase when grown with toluene in the vapor phase; however, after the lag phase the growth rate of the mutant strain was similar to that of the wild type. The mutant also showed a significantly lower survival rate when shocked with 0.08% (vol/vol) toluene. In contrast to the wild-type strain, which grew in liquid culture medium at temperatures up to 38.5 degrees C, the Cti-null mutant strain grew significantly slower at temperatures above 37 degrees C. An in-frame fusion of the Cti protein with the periplasmic alkaline phosphatase suggests that this constitutively expressed enzyme is located in the periplasm. Primer extension studies confirmed the constitutive expression of Cti. Southern blot analysis of total DNA from various pseudomonads showed that the cti gene is present in all the tested P. putida strains, including non-solvent-resistant ones, and in some other Pseudomonas species.     

BopB is a type III secreted protein in Bordetella bronchiseptica and is required for cytotoxicity against cultured mammalian cells

The cytotoxicity of Bordetella bronchiseptica to infected cells is known to be dependent on a B. bronchiseptica type III secretion system. Although the precise mechanism of the type III secretion system is unknown, BopN, BopD and Bsp22 have been identified as type III secreted proteins. In order to identify other proteins secreted via the type III secretion machinery in Bordetella, a type III mutant was generated, and its secretion profile was compared with that of the wild-type strain. The results showed that the wild-type strain, but not the type III mutant, secreted a 40-kDa protein into the culture supernatant. This protein was identified as BopB by the analysis of its N-terminal amino acid sequence. Severe cytotoxicity such as necrosis was induced in L2 cells by infection with the wild-type B. bronchiseptica. In contrast, this effect was not observed by the BopB mutant infection. The haemolytic activity of the BopB mutant was greatly impaired compared with that of the wild-type strain. The results of a digitonin assay strongly suggested that BopB was translocated into HeLa cells infected with the wild-type strain. Taken together, our results demonstrate that Bordetella secretes BopB via a type III secretion system during infection. BopB may play a role in the formation of pores in the host plasma membrane which serve as a conduit for the translocation of effector proteins into host cells.     

Isolation and characterization of solvent-tolerant Pseudomonas putida strain T-57, and its application to biotransformation of toluene to cresol in a two-phase (organic-aqueous) system

Pseudomonas putida T-57 was isolated from an activated sludge sample after enrichment on mineral salts basal medium with toluene as a sole source of carbon. P. putida T-57 utilizes n-butanol, toluene, styrene, m-xylene, ethylbenzene, n-hexane, and propylbenzene as growth substrates. The strain was able to grow on toluene when liquid toluene was added to mineral salts basal medium at 10–90% (v/v), and was tolerant to organic solvents whose log  P _ow (1-octanol/water partition coefficient) was higher than 2.5. Enzymatic and genetic analysis revealed that P. putida T-57 used the toluene dioxygenase pathway to catabolize toluene. A cis-toluene dihydrodiol dehydrogenase gene (todD) mutant of T-57 was constructed using a gene replacement technique. The todD mutant accumulated o-cresol (maximum 1.7 g/L in the aqueous phase) when cultivated in minimal salts basal medium supplemented with 3% (v/v) toluene and 7% (v/v) 1-octanol. Thus, T-57 is thought to be a good candidate host strain for bioconversion of hydrophobic substrates in two-phase (organic-aqueous) systems.

     

Stimulation ofEscherichia coli adenylate cyclase by lactose in strains carrying mutations in lactose permease

When a wild-type strain ofEscherichia coli contains lactose permease, the accumulation of cyclic AMP (cAMP) by intact cells isinhibited by lactose. This inhibitory effect of lactose is observed in a strain with a mutant cAMP phosphodiesterase and therefore involves a regulation of adenylate cyctase activity. Some E. coli strains carrying mutations in lactose permease show an effect opposite to that of the wild-type strain; the accumulation of cAMP by intact cells isstimulated by lactose, but only when the mutant permease is present. Insertion of lactose permease into the membrane of ceils can produce a change in the specific activity of adenylate cycIase; induction of the wild-type transporter is correlated with a decrease in the specific activity, while implantation of a mutant form of lactose permease can lead to an increase in the specific activity. From these data, it is suggested that the state of the lactose transporter in the cell membrane influences the activity of adenytate cyclase.     

Membrane proteins associated with amino acid transport by yeast (Saccharomyces cerevisiae).

Cells of the wild-type yeast (Saccharomyces cerevisiae) strain Y185, grown under conditions that de-repress the formation of a general amino acid permease ('Gap') system, bind delta-N-chloroacetyl[1-(14)C]ornithine; L- and D-amino acid substrates of the general amino acid permease system protect against this binding. The protein responsible is released from the cells by homogenization or by preparation of protoplasts; it is not released by osmotic shock. This protein is virtually absent from the wild-type strain when it is grown under conditions that repress the general amino acid permease system, and is also absent from a Gap- mutant Y185-His3, selected by its resistance to D-amino acids. This mutant and repressed wild-type cells also fail to form a number of membrane proteins elaborated by de-repressed wild-type cells. It is possible that all these proteins are components of the general amino acid permease system.     

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.     

Demonstration of the physiological role of autolysis by a comparative study with a wild-type and its non-autolytic mutant of Micrococcus lysodeikticus (luteus) cultivated with externally added proteolytic enzymes

The log phase cells of autolytic Microccus lysodeikticus (luteus) IFO 3333 did not autolyze when grown in the presence of trypsin although the growth curve and morphology of the cells were not influenced. A non-autolytic mutant was obtained by subculture of the wild-type strain IFO 3333 on an agar slant containing 1% glucose. The mutant (strain MT) was wild-type IFO 3333 which occurred singly or in irregular masses. The mutant MT grown in a culture medium containing trypsin caused remarkable alteration in cell morphology: large cell packets consisting of a number of "unit tetrads" arranged regularly in three dimensions were formed by the addition of trypsin to the medium. The findings suggest that inhibition of the separation of divided cells is brought about by inactivation or suppression of a cell wall autolytic enzyme which plays an important role in the separation step and is accessible to externally added trypsin in the mutant cells but not in the wild-type cells. The possibility that there are two kinds or phases of autolytic enzymes "a physiological autolytic enzyme" and "a useless autolytic enzyme", is discussed.     

DNA degradation in wild-type and repair-deficient strains of salmonella typhimurium exposed to ultraviolet light of photodynamic treatment.

Five mutants of Salmonella typhimurium strain LT2 trp DI (ColEI)+, initiallly detected because they released little or no colicin when tested on solid medium, proved to be sensitive to ultraviotet light (u.v.). Further testing indicated that one of the mutants was deficient in genetic recombination and was probably a recA-type mutant, while three of the others were deficient in DNA polymerase activity and appeared to be typical polA mutants. The fifth mutant was less sensitive than the others to methyl methanesulphonate, showed reduced proficiency in genetic recombination, and was of approximately normal u.v. mutability. This mutant may be a counterpart of the class known as uvrD in Escherichia coli. All five mutants degraded significantly more of their DNA following exposure to u.v. than did the wild-type strain. The recA-type mutant and the possible uvrD mutant also degraded significantly more of their DNA spontaneously than did the wild-type. Treatment with visible light and acridine orange (photodynamic treatment) cause no significant degradation of DNA in the wild-type strain, a highly significant increase in the extent of DNA degradation in a polA mutant, and a decrease in the extent of degradation in the recA-type mutant.     

The TOL Plasmid pWW0 xylN Gene Product from Pseudomonas putida Is Involved in m-Xylene Uptake

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. The last gene of the upper operon, xylN, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to FadL, an outer membrane protein involved in fatty acid transport in Escherichia coli. To analyze the role of the xylN gene product, xylN on TOL plasmid pWW0 was disrupted by inserting a kanamycin resistance gene, and the phenotypes of P. putida harboring the wild-type and xylN mutant TOL plasmids were characterized. The growth of P. putida harboring the wild-type TOL plasmid was inhibited by a high concentration of m-xylene, while that of P. putida harboring the xylN mutant TOL plasmid was not. The apparent K(s) value for the oxidation of m-xylene in intact cells of the xylN mutant was fourfold higher than that of the wild-type strain, although the TOL catabolic enzyme activities in cell extracts from the two strains were almost identical. We therefore presume that the xylN gene product is a porin involved in the transport of m-xylene and its analogues across the outer membrane. Western blot analysis confirmed the localization of XylN in the outer membrane.     

Survival of nonspecific porin-deficient mutants of Escherichia coli in black sea water

AIMS: The aim of this study was to investigate the links between survival of Escherichia coli in sea water microcosms in the laboratory and the presence of porins in the outer membrane. The E. coli strains studied were a wild-type strain and a series of outer membrane protein (omp) mutants. METHODS AND RESULTS: Bacteria were suspended in natural or filtered-autoclaved sea water microcosms and numbers determined over an incubation period by plate count and by count of cells capable of respiration. CONCLUSIONS: The type of omp mutation has a significant impact in bacterial survival. The double OmpC-OmpF mutant and the OmpR mutant (which was incapable of synthesizing OmpC and OmpF) survived poorly compared with single omp mutants and the wild-type strain. This suggests that these proteins are important in determining the entry of E. coli into the survival mode. The EnvZ mutant, which lacks the protein by which the cell senses some changes in the environment, survived as well as the wild-type strain when compared by plate counts and by respiring cell count. The loss of the EnvZ protein has no effect on survival but it could prevent the organism sensing the changes in the environment through which entry into the survival state is triggered. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is another piece in the puzzle as to how bacteria survive stress conditions.