Impact on Bacterial Community in Midguts of the Asian Corn Borer Larvae by Transgenic Trichoderma Strain Overexpressing a Heterologous chit42 Gene with Chitin-Binding Domain

This paper is the first report of the impact on the bacterial community in the midgut of the Asian corn borer (Ostrinia furnacalis) by the chitinase from the transgenic Trichoderma strain. In this study, we detected a change of the bacterial community in the midgut of the fourth instar larvae by using a culture-independent method. Results suggested that Proteobacteria and Firmicutes were the most highly represented phyla, being present in all the midgut bacterial communities. The observed species richness was simple, ranging from four to five of all the 16S rRNA clone libraries. When using Trichoderma fermentation liquids as additives, the percentages of the dominant flora in the total bacterial community in larval midgut changed significantly. The community of the genus Ochrobactrum in the midgut decreased significantly when the larvae were fed with the fermentation liquids of the transgenic Trichoderma strain Mc4. However, the Enterococcus community increased and then occupied the vacated niche of the Ochrobactrum members. Furthermore, the Shannon–Wiener (H) and the Simpson (1-D) indexes of the larval midgut bacterial library treated by feeding fermentation liquids of the transgenic Trichoderma strain Mc4 was the lowest compared with the culture medium, fermentation liquids of the wild type strain T30, and the sterile artificial diet. The Enterococcus sp. strain was isolated and characterized from the healthy larvae midgut of the Asian corn borer. An infection study of the Asian corn borer larvae using Enterococcus sp. ACB-1 revealed that a correlation existed between the increased Enterococcus community in the larval midgut and larval mortality. These results demonstrated that the transgenic Trichoderma strain could affect the composition of the midgut bacterial community. The change of the midgut bacterial community might be viewed as one of the factors resulting in the increased mortality of the Asian corn borer larvae.     

Properties of NAD (P) H Azoreductase from Alkaliphilic Red Bacteria Aquiflexum sp. DL6

Azoreductase plays a key role in bioremediation and biotransformation of azo dyes. It initializes the reduction of azo bond in azo dye metabolism under aerobic or anaerobic conditions. In the present study, we isolated an alkaliphilic red-colored Aquiflexum sp. DL6 bacterial strain and identified by 16S rRNA method. We report nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-dependent azoreductase purified from Aquiflexum sp. DL6 by a combination of ammonium sulfate precipitation and chromatography methods. The azoreductase was purified up to 30-fold with 37 % recovery. The molecular weight was found to be 80 kDa. The optimum activity was observed at pH 7.4 and temperature 60 °C with amaranth azo dye as a substrate. The thermal stability of azoreductase was up to 80 °C. The azoreductase has shown a wide range of substrate specificity, including azo dyes and nitro aromatic compounds. Metal ions have no significant inhibitory action on azoreductase activity. The apparent K _m and V _max values for amaranth azo dye were 1.11 mM and 30.77 U/mg protein respectively. This NAD (P) H azoreductase represents the first azoreductase to be characterized from alkaliphilic bacteria.     

The Function of Cytoplasmic Flavin Reductases in the Reduction of Azo Dyes by Bacteria

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an “azo reductase.” The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic “azo reductases,” which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.     

Biochemical degradation pathway of reactive blue 13 by Candida rugopelliculosa HXL-2

A strain HXL-2 from a lab-scale sequence batch reactor (SBR) was identified as Candida rugopelliculosa based on its physiological, biochemical characteristics, and 26S rDNA D1/D2 gene phylogenetic analysis. About 90% of the 50 mg/L Reactive blue 13(RB13) was degraded in 48 h after inoculation with strain HXL-2. The optimum efficiency of pH on decolorization was obtained at pH 5.The optimum efficiency temperature of C. rugopelliculosa HXL-2 decolorization RB13 was obtained at 28 °C. The color removal efficiency was obtained at 80.3% when the feed concentration reached 2000 mg/L. We first detected naphthalene-like compound is produced as degradation intermediate after the cleavage of RB13 azo bond, detected 1-chloro-3-aniline- 2,4,6-triazine. We proposed degradation pathway of Reactive blue 13 by Candida sp and proved RB13 degradation pathway by Candida sp. has some difference from RB13 degradation pathway by Pseudomonas sp.     

Bacterial decolorization and degradation of the reactive dye Reactive Red 180 by Citrobacter sp. CK3

A bacterial strain, CK3, with remarkable ability to decolorize the reactive textile dye Reactive Red 180, was isolated from the activated sludge collected from a textile mill. Phenotypic characterization and phylogenetic analysis of the 16S rDNA sequence indicated that the bacterial strain belonged to the genus Citrobacter. Bacterial isolate CK3 showed a strong ability to decolorize various reactive textile dyes, including both azo and anthraquinone dyes. Anaerobic conditions with 4 g l^?1 glucose, pH = 7.0 and 32 °C were considered to be the optimum decolorizing conditions. Citrobacter sp. CK3 grew well in a high concentration of dye (200 mg l^?1), resulting in approximately 95% decolorization extent in 36 h, and could tolerate up to 1000 mg l^?1 of dye. UV–vis analyses and colorless bacterial cells suggested that Citrobacter sp. CK3 exhibited decolorizing activity through biodegradation, rather than inactive surface adsorption. It is the first time that a bacterial strain of Citrobacter sp. has been reported with decolorizing ability against both azo and anthraquinone dyes. High decolorization extent and facile conditions show the potential for this bacterial strain to be used in the biological treatment of dyeing mill effluents.     

Identification ofEnterococcus sp. from midgut of silkworm based on biochemical and 16S rDNA sequencing analysis

Enterococcus sp. was isolated from the midgut of silkworm against the germination ofNosema bombycis spores. Identification was based on the biochemical characteristics, 16S rDNA sequences analysis and species-specific probes ofEnterococcus spp. The isolated strains fermented sorbitol and arabinose but did not ferment raffinose.Enterococcus sp. was clustered together withEnterococcus mundtii ATCC 43188 and 100% sequence homology was found by 16S rDNA sequences BLAST analysis and constructing the phylogenetic tree. Comparison of the sequences of the 16S rDNA species-specific probes ofEnterococcus spp. with the 16S rDNA sequence of isolate revealed similar segment to the species-specific probe ofE. mundtii. So, we can make conclusion the 16S rDNA segment ofEnterococcus sp. can hybridise with species-specific probe ofE. mundtii. Enterococcus mundtii was detected for the first time in the intestine of silkworm.     

Molecular Cloning and Characterization of the Gene Coding for the Aerobic Azoreductase from Xenophilus azovorans KF46F

The gene coding for an aerobic azoreductase was cloned from Xenophilus azovorans KF46F (formerly Pseudomonas sp. strain KF46F), which was previously shown to grow with the carboxylated azo compound 1-(4′-carboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole source of carbon and energy. The deduced amino acid sequence encoded a protein with a molecular weight of 30,278 and showed no significant homology to amino acid sequences currently deposited at the relevant data bases. A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase. The enzyme was heterologously expressed in Escherichia coli and the azoreductase activities of resting cells and cell extracts were compared. The results suggested that whole cells of the recombinant E. coli strains were unable to take up sulfonated azo dyes and therefore did not show in vivo azoreductase activity. The turnover of several industrially relevant azo dyes by cell extracts from the recombinant E. coli strain was demonstrated.     

Antibacterial activity of soil bacteria isolated from Kochi,India and their molecular identification

The present study, deal about the antibiosis activity of soil bacteria, isolated from 10 different locations of rhizosphere and diverse cultivation at Kochi, Kerala, India. The bacteria were isolated by standard serial dilution plate techniques. Morphological characterization of the isolate was done by Gram’s staining and found that all of them gram positive. Isolated bacteria were tested against 6 human pathogens viz., Escherichia coli, Enterococcus sp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Acinetobacter sp. Primary screening was carried out by perpendicular streaking and seed overlay method. Based on the result of primary screening most potential isolates of S1A1 and S7A3 were selected for secondary screening. Both the isolates showed positive results against Enterococcus sp. and S.aureus. The maximum antagonistic activity of 20.98 and 27.08?mm zone of inhibition was recorded at S1A1 against Enterococcus sp. and S. aureus respectively, at 180?µl concentration. Molecular identification was carried out by 16S rRNA sequence. The 16S rRNA was amplified from the DNA samples by using PCR. The amplified 16S rRNA PCR products were purified and sequenced. The sequences were subjected to NCBI BLAST. The isolates S1A1 and S7A3 BLAST results showed 99% and 95% respectively, similarity with the available database sequence of Bacillus amyloliquefaciens. The sequences were deposited in GenBank and the accession numbers KY864390 (S1A1) and KY880975 (S7A3) were obtained.     

Enterocin T, a novel class IIa bacteriocin produced by Enterococcus sp. 812

Enterococcus sp. 812, isolated from fresh broccoli, was previously found to produce a bacteriocin active against a number of Gram-positive bacteria, including Listeria monocytogenes. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was inactivated by protease K. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 4,521.34 Da. N-terminal amino acid sequencing yielded a partial sequence, NH₂-ATYYGNGVYXDKKKXWVEWGQA, by Edman degradation, which contained the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The obtained partial sequence showed high homology with some enterococcal bacteriocins; however, no identical peptide or protein was found. This peptide was therefore considered to be a novel bacteriocin produced by Enterococcus sp. 812 and was termed enterocin T.     

A New Alkali-Thermostable Azoreductase from Bacillus sp. Strain SF

A screening for dye-decolorizing alkali-thermophilic microorganisms resulted in a Bacillus sp. strain isolated out of the wastewater drain of a textile finishing company. An NADH-dependent azoreductase of this strain, Bacillus sp. strain SF, was found to be responsible for the decolorization of azo dyes. This enzyme was purified by a combination of ammonium sulfate precipitation and anion-exchange and affinity chromatography and had a molecular mass of 61.6 kDa and an isoelectric point at pH 5.3. The pH optimum of the azoreductase depended on the substrate and was within the range of pHs 8 to 9, while the temperature maximum was reached at 80°C. Decolorization only took place in the absence of oxygen and was enhanced by FAD, which was not consumed during the reaction. A 26% similarity of this azoreductase to chaperonin Cpn60 from a Bacillus sp. was found by peptide mass mapping experiments. Substrate specificities of the azoreductase were studied by using synthesized model substrates based on di-sodium-(R)-benzyl-azo-2,7-dihydroxy-3,6-disulfonyl-naphthaline. Those dyes with NO₂ substituents, especially in the ortho position, were degraded fastest, while analogues with a methyl substitution showed the lowest degradation rates.     

Microaerophilic–aerobic sequential decolourization/biodegradation of textile azo dyes by a facultative Klebsiella sp. strain VN-31

Four different azo dyes were decolourized and biodegraded in a sequential microaerophilic–aerobic treatment by a facultative Klebsiella sp. strain VN-31, a bacterium isolated from activated sludge process of the textile industry. Dye decolourization was performed under microaerophilic conditions until no colour was observed (decolourization percentage >94%). The medium was then aerated to promote the biodegradation of the amines produced. The presence of aromatic amine in the microaerophilic stage and its absence in the aerobic stage demonstrate azo bond reduction and an oxidative biodegradation process, respectively. Total Organic Carbon (TOC) reduction for the growth medium plus dyes was ～50% in the microaerophilic stage and ～80% in the aerobic stage. The degradation products were also characterized by FT-IR and UV–vis techniques and their toxicity measured using Daphnia magna. The results provide evidence that the successive microaerophilic/aerobic stages, using a single Klebsiella sp. strain VN-31 in the same bioreactor, were able to form aromatic amines by the reductive break down of the azo bond and to oxidize them into non-toxic metabolites.     

Stable Coexistence of Five Bacterial Strains as a Cellulose-Degrading Community

A cellulose-degrading defined mixed culture (designated SF356) consisting of five bacterial strains (Clostridium straminisolvens CSK1, Clostridium sp. strain FG4, Pseudoxanthomonas sp. strain M1-3, Brevibacillus sp. strain M1-5, and Bordetella sp. strain M1-6) exhibited both functional and structural stability; namely, no change in cellulose-degrading efficiency was observed, and all members stably coexisted through 20 subcultures. In order to investigate the mechanisms responsible for the observed stability, “knockout communities” in which one of the members was eliminated from SF356 were constructed. The dynamics of the community structure and the cellulose degradation profiles of these mixed cultures were determined in order to evaluate the roles played by each eliminated member in situ and its impact on the other members of the community. Integration of each result gave the following estimates of the bacterial relationships. Synergistic relationships between an anaerobic cellulolytic bacterium (C. straminisolvens CSK1) and two strains of aerobic bacteria (Pseudoxanthomonas sp. strain M1-3 and Brevibacillus sp. strain M1-5) were observed; the aerobes introduced anaerobic conditions, and C. straminisolvens CSK1 supplied metabolites (acetate and glucose). In addition, there were negative relationships, such as the inhibition of cellulose degradation by producing excess amounts of acetic acid by Clostridium sp. strain FG4, and growth suppression of Bordetella sp. strain M1-6 by Brevibacillus sp. strain M1-5. The balance of the various types of relationships (both positive and negative) is thus considered to be essential for the stable coexistence of the members of this mixed culture.     

Freeze-Thaw Tolerance and Clues to the Winter Survival of a Soil Community

Although efforts have been made to sample microorganisms from polar regions and to investigate a few of the properties that facilitate survival at freezing or subzero temperatures, soil communities that overwinter in areas exposed to alternate freezing and thawing caused by Foehn or Chinook winds have been largely overlooked. We designed and constructed a cryocycler to automatically subject soil cultures to alternating freeze-thaw cycles. After 48 freeze-thaw cycles, control Escherichia coli and Pseudomonas chlororaphis isolates were no longer viable. Mixed cultures derived from soil samples collected from a Chinook zone showed that the population complexity and viability were reduced after 48 cycles. However, when bacteria that were still viable after the freeze-thaw treatments were used to obtain selected cultures, these cultures proved to be >1,000-fold more freeze-thaw tolerant than the original consortium. Single-colony isolates obtained from survivors after an additional 48 freeze-thaw cycles were putatively identified by 16S RNA gene fragment sequencing. Five different genera were recognized, and one of the cultures, Chryseobacterium sp. strain C14, inhibited ice recrystallization, a property characteristic of antifreeze proteins that prevents the growth of large, potentially damaging ice crystals at temperatures close to the melting temperature. This strain was also notable since cell-free medium derived from cultures of it appeared to enhance the multiple freeze-thaw survival of another isolate, Enterococcus sp. strain C8. The results of this study and the development of a cryocycler should allow further investigations into the biochemical and soil community adaptations to the rigors of a Chinook environment.     

Characterization of a salt resistant bacterial strain <Emphasis Type="Italic">Proteus</Emphasis> sp. NA6 capable of decolorizing reactive dyes in presence of multi-metal stress

Microbial biotechnologies for the decolorization of textile wastewaters have attracted worldwide attention because of their economic suitability and easiness in handling. However, the presence of high amounts of salts and metal ions in textile wastewaters adversely affects the decolorization efficiency of the microbial bioresources. In this regard, the present study was conducted to isolate salt tolerant bacterial strains which might have the potential to decolorize azo dyes even in the presence of multi-metal ion mixtures. Out of the tested 48 bacteria that were isolated from an effluent drain, the strain NA6 was found relatively more efficient in decolorizing the reactive yellow-2 (RY2) dye in the presence of 50 g L^?1 NaCl. Based on the similarity of its 16S rRNA gene sequence and its position in a phylogenetic tree, this strain was designated as Proteus sp. NA6. The strain NA6 showed efficient decolorization (>90 %) of RY2 at pH 7.5 in the presence of 50 g L^?1 NaCl under static incubation at 30 °C. This strain also had the potential to efficiently decolorize other structurally related azo dyes in the presence of 50 g L^?1 NaCl. Moreover, Proteus sp. NA6 was found to resist the presence of different metal ions (Co⁺², Cr⁺⁶, Zn⁺², Pb⁺², Cu⁺², Cd⁺²) and was capable of decolorizing reactive dyes in the presence of different levels of the mixtures of these metal ions along with 50 g L^?1 NaCl. Based on the findings of this study, it can be suggested that Proteus sp. NA6 might serve as a potential bioresource for the biotechnologies involving bioremediation of textile wastewaters containing the metal ions and salts.     

Efficient decolorization and detoxification of sulphonated azo dye Ponceau 4R by using single and mixed bacterial consortia

Abstract

The decolorization of toxic azo dye Ponceau 4R by three strains of bacteria Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1 individually and in consortia was studied. At optimal conditions, up to 95%, 93% and 87% of the dye was decolorized by the strains AK1, AK2 and VKY1, respectively, in 24?h at 200?mg/L of the dye. Decolorization of the dye was optimized for different parameters such as the concentration of dye, pH, temperature and NaCl concentration. These strains were able to decolorize Ponceau 4R up to an initial concentration of 800?mg/L in the pH range of 5–10, temperature 25–55?°C and NaCl concentration up to 30?g/L. The dye decolorization efficiency of these strains was further enhanced by using different consortia of AK1, AK2 and VKY1 in various combinations. The complete decolorization of the dye by a consortium was achieved within 18?h at 200?mg/L. The cell-free extract of these strains grown on this dye exhibited a remarkable activity of azoreductase which is involved in the breakage of the azo bond. The steady-state kinetics of azoreductase, validated the ping pong Bi-Bi mechanism of enzyme action. UV–Vis spectra, HPLC, FTIR and LC-MS analysis of the dye decolorized samples showed the formation of 4-aminonaphthalene-1-sulphonic acid and 5-amino-6-hydroxynaphthalene-2, 4-disulphonic acid as the products of azo bond breakage. The phytotoxicity test of decolorized sample revealed a considerable reduction in the toxicity in comparison with the parent dye.     

Enhanced decolorization of sulfonated azo dye methyl orange by single and mixed bacterial strains AK1, AK2 and VKY1

Abstract

Methyl orange, a sulfonated azo dye having various industrial applications was decolorized by three bacteria Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1. The effect of various factors such as dye concentration, pH, temperature and NaCl concentration on decolorization was investigated. At 200?mg/L methyl orange concentration, the strains AK1, AK2 and VKY1 exhibited maximum decolorizing potential of 93, 95 and 96%, respectively, at temperature 35?°C and pH 7.0 within 18?h of incubation. These strains decolorized the dye over a wide range of pH (5–10), temperature (15–55?°C), and NaCl concentration (5–20?g/L). Further, these strains decolorize up to 800?mg/L concentrations of methyl orange within 24?h. The dye decolorization efficiency was further increased by using different consortia of these three strains which could decolorize the dye completely within 12?h of incubation. The cell-free extracts of the strains AK1, AK2 and VKY1 grown on methyl orange exhibited the azoreductase activity of 0.4794, 1.56 and 1.01?µM/min/mg protein, respectively. HPLC and FTIR analysis of the dye decolorized sample indicated the formation of 4-aminobenzenesulfonic acid and N,N-dimethyl-p-phenylenediamine as breakdown products of azo bond. The high decolorization potential of these bacterial strains individually and in consortia has potential application in remediation of dye effluent.     

Degradation of azo dyes containing aminonaphthol by Sphingomonas sp strain 1CX

Sphingomonas sp strain 1CX was isolated from a wastewater treatment plant and is capable of aerobically degrading a suite of azo dyes, using them as a sole source of carbon and nitrogen. All azo dyes known to be decolorized by strain 1CX (Orange II, Acid Orange 8, Acid Orange 10, Acid Red 4, and Acid Red 88) have in their structure either 1-amino-2-naphthol or 2-amino-1-naphthol. In addition, an analysis of the structures of the dyes degraded suggests that there are certain positions and types of substituents on the azo dye which determine if degradation will occur. Growth and dye decolorization occurs only aerobically and does not occur under fermentative or denitrification conditions. The mechanism by which 1CX decolorizes azo dyes appears to be through reductive cleavage of the azo bond. In the case of Orange II, the initial degradation products were sulfanilic acid and 1-amino-2-naphthol. Sulfanilic acid, however, was not used by 1CX as a growth substrate. The addition of glucose or inorganic nitrogen inhibited growth and decoloration of azo dyes by 1CX. Attempts to grow the organism on chemically defined media containing several different amino acids and sugars as sources of nitrogen and carbon were not successful. Phylogenetic analysis of Sphingomonas sp strain 1CX shows it to be related to, but distinct from, other azo dye-decolorizing Sphingomonas spp strains isolated previously from the same wastewater treatment facility. Received 19 May 1999/ Accepted in revised form 11 August 1999     

Synergistic Degradation of Linuron by a Bacterial Consortium and Isolation of a Single Linuron-Degrading Variovorax Strain

The bacterial community composition of a linuron-degrading enrichment culture and the role of the individual strains in linuron degradation have been determined by a combination of methods, such as denaturing gradient gel electrophoresis of the total 16S rRNA gene pool, isolation and identification of strains, and biodegradation assays. Three strains, Variovorax sp. strain WDL1, Delftia acidovorans WDL34, and Pseudomonas sp. strain WDL5, were isolated directly from the linuron-degrading culture. In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7. Of these five strains, only Variovorax sp. strain WDL1 was able to use linuron as the sole source of C, N, and energy. WDL1 first converted linuron to 3,4-dichloroaniline (3,4-DCA), which transiently accumulated in the medium but was subsequently degraded. To the best of our knowledge, this is the first report of a strain that degrades linuron further than the aromatic intermediates. Interestingly, the rate of linuron degradation by strain WDL1 was lower than that for the consortium, but was clearly increased when WDL1 was coinoculated with each of the other four strains. D. acidovorans WDL34 and C. testosteroni WDL7 were found to be responsible for degradation of the intermediate 3,4-DCA, and H. sulfonivorans WDL6 was the only strain able to degrade N,O-dimethylhydroxylamine. The role of Pseudomonas sp. strain WDL5 needs to be further elucidated. The degradation of linuron can thus be performed by a single isolate, Variovorax sp. strain WDL1, but is stimulated by a synergistic interaction with the other strains isolated from the same linuron-degrading culture.     

Enrichment and Isolation of Naphthalenesulfonic Acid-Utilizing Pseudomonads

Naphthalenesulfonate-degrading bacteria were obtained by continuous enrichment from a naphthalene-degrading population from sewage. In addition to naphthalene, Pseudomonas sp. A3 can utilize 2-naphthalenesulfonate (2NS) and Pseudomonas sp. C22 can utilize both 1-naphthalenesulfonate (1NS) and 2NS as sole carbon sources. In a mixture of 1NS and 2NS, the former substrate is utilized by strain C22 only after complete consumption of 2NS. During exponential growth, approximately 10% of the organic carbon of naphthalenesulfonates is temporarily excreted. These unidentified metabolites can readily be used by other bacteria, which, by supplying strain C22 with vitamins, allow optimal growth in stable mixed cultures. The degradative capability of Pseudomonas sp. A3 for 2NS was irreversibly lost under nonselective growth conditions and could be transferred from the wild type to a distinguishable cured strain of the wild type.