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)     

Effect of Protease Inhibitors on Early Events of Apoptosis

Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization ofin situDNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence ofin situDNA strand breaks. DNA stability was estimated by the measure of its sensitivityin situto denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4′,6-diamidino-2-phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of ≥50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNAin situ,and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to ≥50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.     

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.     

Sulfation of hypertensive and hypotensive drugs by monkey brain phenol sulfotransferase

The substrate specificity and affinity of two forms of phenol sulfotransferase (PST) from Rhesus macaque brain cortex were studied. Catecholamines, their methylated metabolites (normetanephrine, metanephrine) and methylated precursor, -methylDOPA, were examined as substrates for both the cationic (PST I) and the anionic (PST II) forms of the enzyme. Sulfation of hypertensive drugs (phenylephrine, octopamine, metaraminol), hypotensive drugs (-methylDOPA, minoxidil), and related agents without a free hydroxy group on the benzene ring were also studied. Results indicated that both PST forms sulfated -methylDOPA and minoxidil, but only PST II transferred the sulfate group to catecholamines and most of the adrenergic agents examined.     

THE RELATIONSHIPS BETWEEN THE PRIMARY VASCULAR SYSTEM AND PHYLLOTACTIC PATTERNS OF ANAGALLIS ARVENSIS (PRIMULACEAE)

The various primary vascular systems of shoots of Anagallis arvensis L. (Primulaceae) can be distinguished in relation to the number of leaves (two, three, or four) at each node. In this study, shoot segments (single intemodes and the nodes above them) were examined. The arrangement of segments within shoots was also recorded. The vasculature forms a closed system with the number of sets of bundles usually equal to twice the number of leaves. Irregularities are found in the following features of the system: the number of bundles composing leaf half-traces; occurrence of anastomosing bundles; the number of intemodes through which bundles extend; levels of leaf attachment to the stem at the node; and distribution of parenchyma within the vascular cylinder, which determines the number of bundles in sets and the number of bundle sets. The irregularities occur with different frequencies for segments exhibiting different phyllotactic patterns. Comparison of these frequencies leads to the following conclusions: anastomosing bundles occur only in decussate or trimerous shoot segments, whereas sets of bundles united within intemodes and displaced leaves occur only in tetramerous or trimerous ones; decrease of the number of bundles per leaf and displacement of leaves at the nodal level are correlated; variation between segments exhibiting the same phyllotactic pattern is greatest for trimerous, less for tetramerous, and least for decussate segments; the vascular system of decussate shoot segments is more stable than that of the other systems; and trimerous segments seem to be intermediate between the other two segment types.     

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.