Promoter-creating mutations in Pseudomonas putida: a model system for the study of mutation in starving bacteria.

A novel experimental system to study mutation in starving bacteria was designed, relying on the activation of a promoterless phenol degradation operon of Pseudomonas putida. The Phe+ (phenol-utilizing) mutants accumulated in the starving culture of P. putida in the presence of phenol but not in the absence of it. We ruled out the possibility that the absence of phenol eliminates Phe+ mutants from the starving population. Sequence analysis of the Phe+ mutants revealed that base substitutions, deletions, and insertion of Tn4652 can result in creation of a sequence similar to the sigma70-specific promoter consensus. One particular C --> A transversion was predominant in the Phe+ mutants that arose in the starving population under selection for phenol use. In contrast, various deletions were the most frequent Phe+ mutants occurring in a culture growing without selection. The accumulation rate of the Phe+ mutants on selective plates was found to be higher for bacteria plated from stationary-phase culture than that from exponentially growing cells. This suggests that some specific processes, occurring predominantly in stationary-phase cells, facilitate generation and/or fixation of such mutations.

Regulation of the catechol 1,2-dioxygenase- and phenol monooxygenase-encoding pheBA operon in Pseudomonas putida PaW85.

In Pseudomonas putida PaW85, the ortho-cleavage pathway is used for catechol degradation. The 11.4-kb XhoI fragment cloned from phenol degradation plasmid pEST1226 into pKT240 (recombinant plasmid pAT1140) contains the inducible pheBA operon that encodes catechol 1,2-dioxygenase (gene pheB) and phenol monooxygenase (gene pheA), the first two enzymes for the phenol degradation pathway. The promoter of the pheBA operon is mapped 1.5 kb upstream of the pheB gene. The plasmid pAT1140, when introduced into P. putida PaW85, enables the bacteria to use the hybrid plasmid-chromosome-encoded pathway for phenol degradation. The synthesis of the plasmid-encoded phenol monooxygenase and catechol 1,2-dioxygenase is induced by cis,cis-muconate. The expression studies of the deletion subclones derived from pAT1140 revealed that the transcription of the pheBA operon is positively controlled by a regulatory protein that is chromosomally encoded in P. putida. cis,cis-Muconate in cooperation with positive transcription factor CatR activates the transcription of the chromosomal ortho-pathway genes catA and catBC in P. putida (R. K. Rothmel, T. L. Aldrich, J. E. Houghton, W. M. Coco, L. N. Ornston, and A. M. Chakrabarty, J. Bacteriol. 172:922-931, 1990). The inability to express the pheBA operon in a P. putida CatR- background and activation of transcription of the pheBA operon in Escherichia coli in the presence of the catR-expressing plasmid demonstrated that the transcription of the pheBA operon in P. putida PaW85 carrying pEST1226 is controlled by the chromosomally encoded CatR.

Sequence of the gene (pheA) encoding phenol monooxygenase from Pseudomonas sp. EST1001: expression in Escherichia coli and Pseudomonas putida.

The plasmid pEST1412 contains the genes, pheA and pheB, encoding phenol monooxygenase (PMO) and catechol 1,2-dioxygenase (C12]), respectively. Thse were originally cloned from the plasmid DNA of Pseudomonas sp. EST1001 [Kivisaar et al., Plasmid 24 (1990) 25-36]. Although pheA and pheB are cotranscribed using the promoter sequences derived from Tn4652 and the level of expression of C120 activities from pEST1412 was equal both in Escherichia coli and in Pseudomonas putida, the level of PMO activity measured in the cell-free extracts of E. coli was lower than that in P. putida. The nucleotide sequence of the 2.0-kb PstI-HindIII fragment of pEST1412 carrying pheA was determined. A 1821-bp ORF was found in this DNA. The structural gene (tfdB) encoding 2,4-dichlorophenol hydroxylase from pJP4 has been sequenced [Perkins et al., J. Bacteriol. 172 (1990) 2351-2359]. Comparison of the deduced amino acid sequences of tfdB and pheA revealed highly conserved regions in the protein products of these genes.

Sequence of the plasmid-encoded catechol 1,2-dioxygenase-expressing gene, pheB, of phenol-degrading Pseudomonas sp. strain EST1001.

Phenol monooxygenase (PMO) and catechol 1,2-dioxygenase (C12O), the two first enzymes of the phenol-degradation pathways, are encoded by a 3.4-kb DNA fragment cloned from Pseudomonas sp. EST1001 plasmid DNA. We have previously shown that activation of the cloned genes in Pseudomonas putida PaW85 is controlled by insertion of the 17-kb transposon, Tn4652, from the host chromosome into the plasmid carrying these genes [Kivisaar et al. Plasmid 24 (1990) 25-36]. Transcription of the DNA encoding PMO (pheA) and C12O (pheB) is activated by a promoter located on a 0.2-kb SacI-ClaI fragment from Tn4652. We have determined the nucleotide sequence of pheB. The 906-bp gene encodes a protein product with a deduced Mr of 33,362. The relationship between the pheB gene and other C12O-encoding genes has been shown: comparison of the pheB sequence with sequences of catA of Alcaligenes calcoaceticus, tfdC of A. eutrophus and clcA of P. putida demonstrated that there are conserved residues in all the four protein products of these genes.

Selection of independent plasmids determining phenol degradation in Pseudomonas putida and the cloning and expression of genes encoding phenol monooxygenase and catechol 1,2-dioxygenase.

Long-term cultivation of the Pseudomonas putida multiplasmid strain EST1020 on phenol resulted in the formation of individual PHE plasmids determining phenol degradation. Four types of PHE plasmids, pEST1024, pEST1026, pEST1028, and pEST1029, are characterized. They all contain a transferrable replicon similar to pWWO-8 with a partly duplicated DNA sequence of the 17-kb transposable element of this plasmid and include various amounts of DNA that carry genes encoding phenol degradation (phe genes). We cloned the genes determining phenol monooxygenase and catechol 1,2-dioxygenase from the Pseudomonas sp. parent strain plasmid DNA into the broad host range vector pAYC32 and studied the expression of the cloned DNA. The formation of a new hybrid metabolic plasmid, pEST1354, was demonstrated in P. putida PaW85 as the result of transposition of the 17-kb genetic element from the chromosome of PaW85 into the plasmid carrying cloned phe genes. The target site for the 17-kb transposon was localized in the vector DNA, just near the cloning site. In subcloning experiments we found two regions in the 17-kb DNA stretch that are involved in the expression of the cloned phe genes.

Hydrolase activities in the rat aorta. I. Effects of diabetes mellitus and insulin treatment.

Vascular disease in diabetics could arise in part from altered vessel wall catebolism. Specific activities of hydrolases in aortic smooth muscle cells from rats with streptozotocin-induced diabetes were measured. Enyzmes included: neutral alpha-glucosidase, alpha-mannosidase, and lysosomal N-acetyl beta-glucosaminidase, beta-galactosidase, cathepsin C, acid alpha-glucosidase, and acid cholesteryl esterase. After 4,8, and 11 weeks of diabetes, activities of all enzymes studied were decreased significantly in diabetic vessels, decreases ranging from 15% for cathepsin C to 62% for alpha-mannosidase. After 3 weeks of diabetes, insulin treatment for 1 week restored enzyme levels to normal. After 7 weeks of diabetes, 1 week of insulin treatment did not restore enzyme levels fully to normal (acid cholesteryl esterase was unchanged); 4 weeks of insulin did. Acid phosphatase and N-acetyl beta-glucosaminidase activities were reduced markedly in histochemical studies of diabetic aortas at all time periods and were restored by insulin treatment. Alloxan-induced diabetes gave results similar to those with streptozotocin. Significant decreases of aortic hydrolase activities, including those of lysosomes, occur in experimental diabetes mellitus and could contribute to accumulation of substrates in vascular smooth muscle cells.

Hydrolase activities in the rat aorta. II. Effects of hypertension alone and in combination with diabetes mellitus.

Hypertension is an important risk factor for atherosclerosis and often occurs in association with diabetes mellitus. Specific activities of hydrolases in homogenates of aortas from rats with renal-clip hypertension, normotension following a period of hypertension, and hypertension combined with streptozotocin-induced diabetes mellitus were measured. Enzymes included: neutral alpha-glucosidase, and lysosomal N-acetyl-beta-glucosaminidase, beta-galactosidase, cathepsin C, acid alpha-glucosidase, and acid cholesteryl esterase. After 6 or 12 weeks of hypertension, specific activities of all enzymes measured were significantly increased, levels ranging from 24% above normal for cathepsin C to 351% above normal for N-acetyl-beta-glucosaminidase. Six weeks of normotension following 6 weeks of hypertension resulted in restoration to normal of four of the six enzyme activities; the remaining two enzymes were significantly below normal levels. Combined hypertension and diabetes mellitus showed smooth muscle cell levels of four of the five hydrolases measured to be significantly lower than those present with hypertension alone. In every instance, histochemical studies of aortas showed acid phosphatase and N-acetyl-beta-glucosaminidase activities which corresponded to the biochemical findings. These findings indicate profound and discrete effects of two clinical risk factors on vascular smooth muscle cell lysosomes.

Production of complete and incomplete antibodies to human red blood cells in dogs and rabbits.

The characteristics of the immune response of dogs and rabbits to single and multiple injections of human red blood cells were analysed. The results show that the primary and secondary response of both species are essentially the same. Following repeated stimulation with this antigen quantitative differences of the immune response between these animals were observed. The rabbit produced substantially greater amounts of IgG complete and incomplete antibody, and proportionally greater amounts of IgM antibody, whereas the dog response was seen to be inhibited.

Arterial lysosomes and connective tissue in primate atherosclerosis and hypertension.

The cellular events that occur in the vessel wall consequent to changes in endothelial permeability result in the progression of vascular disease, particularly atherosclerosis. Female rhesus monkeys were fed an atherogenic diet or were made hypertensive for 6-8 months; and their vessels were then compared with vessels from control monkeys. Length-defined segments of coronary vessels, the thoracic aorta, and the abdominal aorta showed significant increases in total connective tissue in the atherosclerotic and hypertensive groups; pulmonary vessels did not. The diseased aortic segments had increased levels of two lysosomal enzymes, acid phosphatase and beta-N-acetylglucosaminidase; pulmonary vessels were not diseased and did not show these changes. Coronary vessels from the atherosclerotic and hypertensive groups did not show an increase in enzyme levels on biochemical measurements, but focal accumulations of lysosomes were identified by cytochemical techniques. In atherosclerotic lesions, a doubling of cholesterol and more than a tenfold increase in cholesterol ester were found. These connective tissue and lysosomal changes are early features of primate vascular disease and may result from the accumulation of excessive substrate (cholesterol ester) in the lysosomes of vascular smooth muscle cells.

Lysosomes in aortic smooth muscle cells. Effects of hypertension.

Hypertension induces hypertrophy and increased turnover of aortic smooth muscle cells along with an accumulation of connective tissue in the aortic wall. We identified the lysosomes in normal and hypertensive aortic muscle cells by light and electron microscopy, utilizing cytochemical staining for acid phosphatase activity. Lysosomes were found to be more numerous in hypertensive vessels. Biochemical assays of two specific lysosomal enzymes revealed a doubling of acid phosphatase and a more than threefold increase in beta-N-acetyl-glucosaminidase activities in hypertensive aortas.