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SUMMARYThe rate of photosynthesis in the third pair of leaves of Perilla friitesceiis declined gradually from the time of completion of expansion of the leaves until approximately i week from abscission when there was a more rapid decrease. The decrease in rate of photosynthesis first became apparent in the parts of the lamina proximal to the petiole and gradually extended to the more distal regions. The rate of respiration of the leaf remained approximately constant until i week from abscission w hen there was a transient rise prior to tbe final decrease in rate, Tbe gradual decline in pbotosyntbesis was accompanied hy a similar decline in tbe RNA and protein content of tbe leaf altbough the cbloropbyll content did not cbange during tbis period, ^Measurements of pbotosyntbesis by means of' ''^CO, incorporation sbowed a similar pattern of cbange and further revealed that tbe rate of incorporation of label into protein declined more rapidly than did tbe total protein content, Tbe data are interpreted as suggesting tbe cessation of synthesis of proteins essential for pbotosyntbesis ratber tban an increased rate of proteolysis as tbe cause of tbe senescence cbanges.

Pseudomonas cepacia has an inducible P-lactamase which is responsible for its novel ability to catabolize I-lactam compounds. The gene encoding this enzyme, penA, was cloned from a genomic library of P. cepacia 249 on the broad-host-range cosmid pLAFR. This separated the penA gene from the gene encoding a second ,-lactamase in P. cepacia 249. Expression of penA was inducible in an Escherichia coli host strain by low levels of penicillin. The 33,500-molecular-weight enzyme had penicillinase activity not inhibited by clavulanic acid or sulbactam and was highly active against piperacillin and azlocillin. In comparison with other inducible I-lactamases produced by gram-negative organisms, the penA enzyme had many properties which were similar to those of the penicillinase produced by Alcaligenes faecalis. It was unlike the ampC-type cephalosporinase produced by Pseudomonas aeruginosa.Pseudomonas cepacia has become an increasingly important pathogen in certain patient populations, particularly in immunocompromised hosts and children with cystic fibrosis (12). These infections are difficult to treat owing to the intrinsic resistance of this species to most antibiotics and the lack of clinical response to the few antimicrobial agents with in vitro activity against P. cepacia (11). Inducible P-lactamase activity, a general property of P. cepacia, has been associated with increased resistance to ,-lactam antibiotics in clinical isolates of P. cepacia (7). However, the specific enzyme responsible for this resistance has not been identified. Beckman and Lessie (1) found that inducible penicillinase activity in P. cepacia was associated with the ability of this species to hydrolyze penicillin and to utilize 3-lactam compounds as a source of carbon. This novel property has not been associated with the 1-lactamases of other gramnegative bacteria.In P. cepacia 249, two separate ,-lactamases have been identified: a penicillinase responsible for approximately 80% of the total 3-lactamase activity of the strain and a second enzyme with primarily cephalosporinase activities (15). It is not known if the cephalosporinase, like the penicillinase, is preserved throughout the species. The precise role of these enzymes in the in vivo development of resistance to Plactam antibiotics is unclear. However, it has become quite apparent to clinicians that P. cepacia infections are much less responsive to 3-lactam antibiotic therapy than Pseudomonas aeruginosa infections in the same patient population.Although P. aeruginosa produces a number of 3-lactamases, differences in the properties of the ,-lactamases produced by these two species may be in part responsible for their differing response to antibiotics.In this study, we sought to characterize the inducible penicillinase found in P. cepacia. In addition to comparing its physical properties with those of other 1-lactamases, we were interested in studying the regulation of penicillinase expression in this species. Using cloning techniques we isolated the ,-lactamase from P. cepacia 249 which ...

We have identified three transposable gene-activating elements from Pseudomonas cepacia on the basis of their abilities to increase expression of the lac genes of the broad-host-range plasmid pGC91.14 (pRPl::Tn951). When introduced into auxotrophic derivatives of P. cepacia 249 (ATCC 17616), this plasmid failed to confer the ability to utilize lactose. The lac genes of Tn951 were poorly expressed in P. cepacia and were not induced by isopropyl-I3-D-thiogalactopyranoside. Lac' variants of the pGC91.14-containing strains which formed I8-galactosidase at high constitutive levels as a consequence of transposition of insertion sequences from the P. cepacia genome to sites upstream of the lacZ gene of Tn951 were isolated. Certain of the elements also increased gene expression in other bacteria. For example, IS407 strongly activated the lacZ gene of Tn951 in Pseudomonas aeruginosa and Escherichia coli, and IS406 (but not IS407) did so in Zymomonas mobilis. The results indicate that IS elements from P. cepacia have potential for turning on the expression of foreign genes in a variety of gram-negative bacteria.Although Pseudomonas cepacia is notable for its extraordinary nutritional versatility (4,20,26,27,29), most soil isolates of this bacterium are unable to utilize lactose as a sole source of carbon and energy (4,20,21). Cornelis and co-workers identified a 17-kilobase (kb) transposon, Tn951 (Tnlac), in Yersinia enterolitica which carried lacI, lacZ, and lacY genes seemingly identical to those of the Escherichia coli lac operon and isolated several variants of the broadhost-range plasmid pRP1 (30) containing this element (12)(13)(14). We transferred one of these, pGC91.14 ( Fig. 1) into auxotrophic derivatives of P. cepacia 249 (ATCC 17616) by conjugation from E. coli JC3272. Although unable to utilize lactose themselves, the pGC91.14-containing transconjugants gave rise to Lac' variants (M. S. Wood, C. Lory, and T. G. Lessie, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, H23, p. 143). In these strains, transposable gene-activating elements from the P. cepacia genome had inserted into Tn951 and increased expression of its lac genes. The results were similar to those we reported earlier for activation of the bla gene of Tnl in P. cepacia (20,28), which restored ability of 1-lactamase-deficient strains of this bacterium to utilize penicillin as a sole source of carbon and energy.We report here data describing the transposition of three P. cepacia insertion sequences, IS406, IS407, and IS415, to different sites within Tn951 and their effect on formation of lac-specific mRNA and of 3-galactosidase. We also describe the influence of P. cepacia insertion sequences on lac gene expression in other gram-negative bacteria. MATERIALS AND METHODSBacterial strains and plasmids. Table 1 lists strains and plasmids used in this study. Bacteria were grown in inorganic salts medium (33) supplemented with 1% (wt/vol) yeast extract or Casamino Acids or 0.5% of the specified carbon sources. Plasmid pGC91.14 was transferred from E. coli J...