Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Catalytic (C) subunit of cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) requires phosphorylation at Thr-197 for expression of full activity, and this residue is found phosphorylated in both the enzyme isolated from animal tissues and in recombinant C subunit expressed in Escherichia coli (26,33,38). In addition to lowering the K m values for both ATP and peptide substrates, the Thr-197 phosphate causes a distinctive reduction in the mobility of the protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (33). Although C subunit is also phosphorylated at Ser-338 in both bacteria and mammalian cells and can be phosphorylated on additional Ser residues, these phosphorylations do not appear to affect C-subunit activity and have only minor effects on the SDS-PAGE mobility of the protein (6,26,33,38).Thr-197 falls in the activation loop region contained within subdomain VIII that also is associated with activating phosphorylation sites in many other protein kinases, including CDC2 kinase, the mitogen-activated protein (MAP) kinases, the MAP kinase kinases, and most protein tyrosine kinases (12,13,38). The sequence in this region is fully conserved in mammalian C subunits, including C␣, C␤, and C␥ isoforms (3, 27, 37). Activation of protein tyrosine kinases by phosphorylation in this region appears to be by autophosphorylation (13), while that of CDC2, MAP kinases, and MAP kinase kinases is by heterologous enzymes (8, 12). C-subunit phosphorylation in E. coli is apparently an intermolecular autophosphorylation reaction, and the purified recombinant protein is capable of autophosphorylation with concomitant activation (33,38). In the present report, we present evidence that the phosphorylation of C subunit in intact mammalian cells is catalyzed by a heterologous PKA kinase. Furthermore, we describe an activity from extracts of a PKA-deficient mutant of S49 mouse lymphoma cells that appears to phosphorylate C subunit specifically at Thr-197. MATERIALS AND METHODSExpression and radiolabeling of recombinant C subunits. Wild-type and mutant forms of recombinant murine C␣ subunit were expressed from the pET-8c expression vector in E. coli BL21(DE3) as described previously (33). Construction of the wild-type and Thr-1973Ala plasmids has been described elsewhere (33). The Lys-723Met mutation was introduced by replacement of an NcoIBstEII restriction fragment from sequences amplified from pMT-C␣K72M-EV (17), using an upstream PCR primer modified to introduce an NcoI restriction site overlapping the C-subunit initiati...

Stenotrophomonas maltophilia has recently emerged as an important nosocomial pathogen in immunocompromised patients, in transplant recipients, and in persons with cystic fibrosis (CF). While this organism is nonpathogenic in healthy individuals, it is increasingly associated with morbidity and mortality in susceptible populations. Recent studies have indicated that for approximately 10% of CF patients with moderate lung disease, S. maltophiliacan be cultured from respiratory tract secretions. Identification ofS. maltophilia can be problematic, and analysis of isolates from the Burkholderia cepacia Research Laboratory and Repository showed that several isolates presumptively identified asB. cepacia by clinical microbiology laboratories were in fact S. maltophilia. To overcome the problems associated with definitive identification, we developed species-specific PCR (SS-PCR) primers, designated SM1 and SM4, directed to the 23S rRNA gene, and tested their utility to accurately identify S. maltophilia directly from sputum. The SS-PCR was developed and tested against a panel of 112 S. maltophilia isolates collected from diverse geographic locations. To test for specificity, 43 isolates from 17 different species were analyzed. PCR with the SM1-SM4 primer pair and isolated genomic DNA as a template resulted in amplification of a band from all S. maltophilia isolates and was uniformly negative for all other species tested, yielding a sensitivity and a specificity of 100% for the SS-PCR. The utility of the SS-PCR to directly identify S. maltophilia in sputum was examined. Thirteen expectorated sputum samples from CF patients were analyzed by SS-PCR. Three samples were PCR positive, in complete concordance with the conventional laboratory culture. Thus, we have developed an SS-PCR protocol that can rapidly and accurately identifyS. maltophilia isolates and which can be used for the direct detection of this organism in CF patient sputum.

Definitive identification of the species in the Burkholderia cepacia complex by routine clinical microbiology methods is difficult. Phenotypic tests to identify B. multivorans andB. vietnamiensis have been established; more recent work indicates B. stabilis may also be identified by growth characteristics and biochemical tests. However, attempts to identify genomovars I and III have, thus far, proved unsuccessful. Previously, we demonstrated the utility of two primer pairs, directed to the rRNA operon, to specifically identify the B. cepacia complex in a PCR. One of these primer pairs, G1-G2, only amplified a DNA fragment from genomovars I and III and B. stabilis in a PCR with genomic DNA isolated from prototypical strains representing the five genomovars. Sequence analysis of the rRNA operon for all the genomovars indicated that this primer pair targeted a region shared by these isolates. Further analysis revealed a region of heterogeneity between genomovar III and B. stabilis internal to the amplified product of G1-G2. Primers designed to target this region were tested with prototypical strains following an initial amplification with the G1-G2 primer pair. New primers specific for the prototypical genomovar III and B. stabilis were designated SPR3 and SPR4, respectively. Analysis of 93 isolates representing 18 genomovar I, 13B. multivorans, 36 genomovar III, 11 B. stabilis, and 15 B. vietnamiensis isolates was performed. DNA from all isolates of genomovars I and III and B. stabilis was amplified by G1-G2. Genomovar III isolates yielded a product with SPR3/G1 while B. stabilis amplified with SPR4-G1. Genomovar I isolates were amplified by either SPR3-G1 or SPR4-G1, but not both. B. multivorans yielded a product with SPR3-G1 but not G1-G2, and B. vietnamiensis isolates were negative in all PCRs. Thus using an algorithm with G1-G2, SPR3-G1, and SPR4-G1 primers in a PCR analysis, genomovar III isolates can be separated from B. stabilis and the identity of B. multivorans and B. vietnamiensis can be confirmed.

Burkholderia gladioli colonizes the respiratory tracts of patients with cystic fibrosis and chronic granulomatous disease. However, due to the high degree of phenotypic similarity between this species and closely related species in the Burkholderia cepacia complex, accurate identification is difficult. Incorrect identification of these species may have serious repercussions for the management of patients with cystic fibrosis. To develop an accurate procedure for the identification of B. gladioli , a molecular method to discriminate between this species and other species commonly isolated from the sputa of patients with cystic fibrosis was investigated. The 23S ribosomal DNA was cloned from several clinical isolates of B. gladioli , and the nucleotide sequence was determined. Computer-assisted sequence comparisons indicated four regions of the 23S rRNA specific for this species; these regions were used to design three primer pairs for species-specific PCR. Two of the primer pairs showed 100% sensitivity and specificity for B. gladioli when tested against a panel of 47 isolates comprising 19 B. gladioli isolates and 28 isolates of 16 other bacterial species. One of the primer pairs was further assessed for species specificity by using a panel of 102 isolates obtained from the Burkholderia cepacia Research Laboratory and Repository. The species-specific PCR was positive for 70 of 74 isolates of B. gladioli and was negative for all other bacterial species examined. Overall, this primer pair displayed a sensitivity and specificity of 96% (89 of 93) and 100%, respectively. These data demonstrate the potential of species-specific PCR for the identification of B. gladioli .