Construction of chlorobenzene-utilizing recombinants by progenitive manifestation of a rare event

Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-O, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absence of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene or 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant--unlike its parent, R5-3--did not grow on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown on 4-methylbenzoate than in the same cells grown on benzene.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of mispair and nonpair correction in hybrid DNA on base ratios (G + C content) and total amounts of DNA

Base ratios and total DNA amounts can vary substantially between and within higher taxa and genera, and even within species. Gene conversion is one of several mechanisms that could cause such changes. For base substitutions, disparity in conversion direction is accompanied by an equivalent disparity in base ratio at the heterozygous site. Disparity in the direction of gene conversion at meiosis is common and can be extreme. For transitions (which give purine [R]/pyrimidine [Y] mispairs) and for transversions giving unlike R/R and Y/Y mispairs in hybrid DNA, this disparity could give slow but systematic changes in G + C percentage. For transversions giving like R/R and Y/Y mispairs, it could change AT/TA and CG/GC ratios. From the extent of correction direction disparity, one can deduce properties of repair enzymes, such as the ability (1) to excise preferentially the purine from one mispair and the pyrimidine from the other for two different R/Y mispairs from a single heterozygous site and (2) to excise one base preferentially from unlike R/R or Y/Y mispairs. Frame-shifts usually show strong disparity in conversion direction, with preferential cutting of the nonlooped or the looped-out strand of the nonpair in heterozygous h-DNA. The opposite directions of disparity for frame-shifts and their intragenic suppressors as Ascobolus suggest that repair enzymes have a strong, systematic bias as to which strand is cut. The conversion spectra of mutations induced with different mutagens suggest that the nonlooped strand is preferentially cut, so that base additions generally convert to mutant and deletions generally convert to wild-type forms. Especially in nonfunctional or noncoding DNA, this could cause a general increase in DNA amounts. Conversion disparity, selection, mutation, and other processes interact, affecting rates of change in base ratios and total DNA.      

Genetic exchange in soil between introduced chlorobenzoate degraders and indigenous biphenyl degraders.

Pseudomonas aeruginosa JB2, a chlorobenzoate degrader, was inoculated into soil having indigenous biphenyl degraders but no identifiable 2-chlorobenzoate (2CBa) or 2,5-dichlorobenzoate (2,5DCBa) degraders. The absence of any indigenous chlorobenzoate degraders was noted by the failure to obtain enrichment cultures with the addition of 2CBa, 3CBa, or 2,5DCBa and by the failure of soil DNA to hybridize to the tfdC gene, which encodes ortho fission of chlorocatechols. In contrast, DNA extracted from inoculated soils hybridized to this probe. Bacteria able to utilize both biphenyl and 2CBa as growth substrates were absent in uninoculated soil, but their presence increased with time in the inoculated soils. This increase was related kinetically to the growth of biphenyl degraders. Pseudomonas sp. strain AW, a dominant biphenyl degrader, was selected as a possible parental strain. Eight of nine recombinant strains, chosen at random, had high phenotypic similarity (90% or more) to the inoculant; the other, strain JB2-M, had 78% similarity. Two hybrid strains, P. aeruginosa JB2-3 and Pseudomonas sp. JB2-M, were the most effective of all strains, including strain AW, in metabolizing polychlorinated biphenyls (Aroclor 1242). Repetitive extragenic palindromic-PCR analysis of putative parental strains JB2 and AW and the two recombinant strains JB2-3 and JB2-M showed similar fragments among the recombinants and JB2 but not AW. These results indicate that the bph genes were transferred to the chlorobenzoate-degrading inoculant from indigenous biphenyl degraders.     

Enhanced mineralization of polychlorinated biphenyls in soil inoculated with chlorobenzoate-degrading bacteria.

An Altamont soil containing no measurable population of chlorobenzoate utilizers was examined for the potential to enhance polychlorinated biphenyl (PCB) mineralization by inoculation with chlorobenzoate utilizers, a biphenyl utilizer, combinations of the two physiological types, and chlorobiphenyl-mineralizing transconjugants. Biphenyl was added to all soils, and biodegradation of 14C-Aroclor 1242 was assessed by disappearance of that substance and by production of 14CO2. Mineralization of PCBs was consistently greatest (up to 25.5%) in soils inoculated with chlorobenzoate degraders alone. Mineralization was significantly lower in soils receiving all other treatments: PCB cometabolizer (10.7%); chlorobiphenyl mineralizers (8.7 and 14.9%); and mixed inocula of PCB cometabolizers and chlorobenzoate utilizers (11.4 and 18.0%). However, all inoculated soils had higher mineralization than did the uninoculated control (3.1%). PCB disappearance followed trends similar to that observed with the mineralization data, with the greatest degradation occurring in soils inoculated with the chlorobenzoate-degrading strains Pseudomonas aeruginosa JB2 and Pseudomonas putida P111 alone. While the mechanism by which the introduction of chlorobenzoate degraders alone enhanced biodegradation of PCBs could not be elucidated, the possibility that chlorobenzoate inoculants acquired the ability to metabolize biphenyl and possibly PCBs was explored. When strain JB2, which does not utilize biphenyl, was inoculated into soil containing biphenyl and Aroclor 1242, the frequency of isolates able to utilize biphenyl and 2,5-dichlorobenzoate increased progressively with time from 3.3 to 44.4% between 15 and 48 days, respectively. Since this soil contained no measurable level of chlorobenzoate utilizers yet did contain a population of biphenyl utilizers, the possibility of genetic transfer between the latter group and strain JB2 cannot be excluded.     

Deterministic Three-Half-Order Kinetic Model for Microbial Degradation of Added Carbon Substrates in Soil

The kinetics of mineralization of carbonaceous substrates has been explained by a deterministic model which is applicable to either growth or nongrowth conditions in soil. The mixed-order nature of the model does not require a priori decisions about reaction order, discontinuity period of lag or stationary phase, or correction for endogenous mineralization rates. The integrated equation is simpler than the integrated form of the Monod equation because of the following: (i) only two, rather than four, interdependent constants have to be determined by nonlinear regression analysis, (ii) substrate or product formation can be expressed explicitly as a function of time, (iii) biomass concentration does not have to be known, and (iv) the required initial estimate for the nonlinear regression analysis can be easily obtained from a linearized form rather than from an interval estimate of a differential equation. ¹⁴CO₂ evolution data from soil have been fitted to the model equation. All data except those from irradiated soil gave better fits by residual sum of squares (RSS) by assuming growth in soil was linear (RSS = 0.71) as opposed to exponential (RSS = 2.87). The underlying reasons for growth (exponential versus linear), no growth, and relative degradation rates of substrates are consistent with the basic mechanisms from which the model is derived.     

Nitrogen fixation, denitrification, and pleomorphic growth in a highly pigmented Spirillum lipoferum.

A strain of Spirillum lipoferum with intense red pigmentation was isolated from the roots of Cynodon dactylon "Coastal." This isolate vigorously reduced acetylene when grown in N-free, Na-malate, semisolid agar, and it was identical to S. lipoferum strain SP7 by standard taxonomic tests. Both S. lipoferum SP7 and the C. dactylon root isolate displayed the unique features of being denitrifiers as well as N2 fixers. The N2-dependent growth curve was biphasic: cells in younger cultures showed the characteristic spiral shape and motility, but those in older cultures developed larger, nonmotile, cystlike forms. Nitrogenase activity seemed associated only with younger spiral forms. The red pigment may be a b- or c-type cytochrome. The strong red color, which this strain develops, could be used as a marker in evaluating soil inoculation experiments.     

Nitrogen fixation, denitrification, and pleomorphic growth in a highly pigmented Spirillum lipoferum.

A strain of Spirillum lipoferum with intense red pigmentation was isolated from the roots of Cynodon dactylon "Coastal." This isolate vigorously reduced acetylene when grown in N-free, Na-malate, semisolid agar, and it was identical to S. lipoferum strain SP7 by standard taxonomic tests. Both S. lipoferum SP7 and the C. dactylon root isolate displayed the unique features of being denitrifiers as well as N2 fixers. The N2-dependent growth curve was biphasic: cells in younger cultures showed the characteristic spiral shape and motility, but those in older cultures developed larger, nonmotile, cystlike forms. Nitrogenase activity seemed associated only with younger spiral forms. The red pigment may be a b- or c-type cytochrome. The strong red color, which this strain develops, could be used as a marker in evaluating soil inoculation experiments.     

Degradation of mono-, di-, and trihalogenated benzoic acids by Pseudomonas aeruginosa JB2.

Pseudomonas aeruginosa JB2 was isolated from a polychlorinated biphenyl-contaminated soil by enrichment culture containing 2-chlorobenzoate as the sole carbon source. Strain JB2 was subsequently found also to grow on 3-chlorobenzoate, 2,3- and 2,5-dichlorobenzoates, 2,3,5-trichlorobenzoate, and a wide range of other mono- and dihalogenated benzoic acids. Cometabolism of 2,4-dichlorobenzoate was also observed. Chlorocatechols were the central intermediates of all chlorobenzoate catabolic pathways. Degradation of 2-chlorobenzoate was routed through 3-chlorocatechol, whereas 4-chlorocatechol was identified from the metabolism of both 2,3- and 2,5-dichlorobenzoate. The initial attack on chlorobenzoates was oxygen dependent and most likely mediated by dioxygenases. Although plasmids were not detected in strain JB2, spontaneous mutants were detected in 70% of glycerol-grown colonies. The mutants were all of the following phenotype: benzoate+, 3-chlorobenzoate+, 2-chlorobenzoate-, 2,3-dichlorobenzoate-, 2,5-dichlorobenzoate-. While chlorocatechols were oxidized by the mutants at wild-type levels, oxidation of 2-chloro- and 2,3- and 2,5-dichlorobenzoates was substantially diminished. These findings suggested that strain JB2 possessed, in addition to the benzoate dioxygenase, a halobenzoate dioxygenase that was necessary for the degradation of chlorobenzoates substituted in the ortho position.