Cloning and characterization of a second acid phosphatase from Sinorhizobium meliloti strain 104A14.

Sinorhizobium meliloti has two nonspecific periplasmic acid phosphatases. The NapD enzyme has been previously described, and a second acid phosphatase, NapE, is described in this report. NapE was partially purified from an S. meliloti napD mutant and characterized with respect to molecular mass and substrate range. As predicted from SDS-PAGE analysis, the subunit molecular mass of NapE is approximately 35.8 kDa and gel filtration experiments estimated the native molecular mass to be approximately 70 kDa, indicating that the active enzyme is a homodimer. NapE demonstrated significant activity with p-nitrophenyl phosphate, phenyl phosphate, and alpha-naphthyl-phosphate. The pH optimum was between 4.5 and 5.0. The gene encoding NapE was also sequenced and the inferred amino acid sequence from the predicted ORF was found to be 60% identical and 75% similar to that encoded by napD. An S. meliloti napE mutant was constructed and assessed for symbiotic competence. This mutant did not differ from the wild-type parent strain in nodulation and symbiotic efficiency.

The role of airway stents in the management of pediatric tracheal, carinal, and bronchial disease.

OBJECTIVE: A variety of stents are available to aid in the management of complex tracheal, carinal and bronchial stenoses. We reviewed our multi-institutional experience with airway stents in children. METHODS: Thirty-three children (age, 13 days-18 years) from four institutions have had a total of 40 stents placed to aid in the management of complex airway stenoses. Three stent types were utilized: 29 silastic stents, five expandable metal stents and six customized carinal stents (four patients had two stents and one patient had four stents). Thirty children had tracheal stents, six children had bronchial stents, and two infants had carinal stents (three children had stenting of more than one area and two had stenting of all three locations). Twenty-eight patients (age, 5 months-18 years; mean, 8.06 years; SEM, 1.13 years) had stents placed after a variety of airway reconstructive procedures. Four underwent stenting in a non-operative setting and one as preoperative stabilization. RESULTS: Twenty-seven patients survived. One patient died early due to bleeding. Five patients died late: two due to bleeding, one from mediastinitis, and two patients with functional airways died late from unrelated problems. Complications are related to stent type and location. Carinal stents can migrate; several techniques are available to help manage this problem. Wire stents are essentially non-removable requiring periodic dilation. Silastic stents stimulate granulation tissue formation requiring periodic bronchoscopic removal. CONCLUSION: Tracheal stenting can aid in the management of pediatric airway problems. Complications are common, but can be managed with appropriate intervention.

The ++Sinorhizobium meliloti lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa.

While screening for Sinorhizobium meliloti Pho regulatory mutants, a transposon mutant was isolated that constitutively expressed higher levels of acid and alkaline phosphatase enzymes. This mutant was also found to form pseudonodules on alfalfa that were delayed in appearance relative to those formed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organisms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellular polysaccharide (EPS) than the wild-type strain. The mutant fluoresced brightly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysis of purified total EPS showed that the glucose-to-galactose ratio in the lon mutant total EPS was 5.0 +/- 0.2 (mean +/- standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 +/- 0.5. These data suggested that in addition to EPSI, the lon mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to be produced by S. meliloti, but typically is expressed only under conditions of phosphate limitation. (13)C nuclear magnetic resonance analysis showed no major differences between EPS purified from the mutant and wild-type strains. Normal growth, EPS production, and the symbiotic phenotype were restored in the mutant strain when the wild-type lon gene was present in trans. The results of this study suggest that the S. meliloti Lon protease is important for controlling turnover of a constitutively expressed protein(s) that, when unregulated, disrupts normal nodule formation and normal growth.

Characterization of two inducible phosphate transport systems in Rhizobium tropici.

Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (P(i)). To better understand phosphorus movement between the bacteroid and the host plant, P(i) transport was characterized in R. tropici. We observed two P(i) transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The K(m) and V(max) values for the low-affinity system were estimated to be 34 +/- 3 microM P(i) and 118 +/- 8 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively, and the K(m) and V(max) values for the high-affinity system were 0.45 +/- 0.01 microM P(i) and 86 +/- 5 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively. Both systems were inducible by P(i) starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but P(i) transport through both systems was eliminated by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide; the P(i) transport rate was correlated with the intracellular ATP concentration. Also, P(i) movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P(i) transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.

Allograft replacement of the aortic valve versus the miniroot and valve.

Between February 1986 and April 1992, 27 patients ranging in age from 8 to 65 years (median, 18 years) underwent allograft replacement of the aortic valve with one death (operative mortality, 3.7%). The indications for operation were aortic regurgitation in 14 patients, aortic stenosis in 7, aortic stenosis/regurgitation in 4, and endocarditis in 2. Associated lesions included annuloectasia in 4 patients (1 with Marfan's syndrome), sinus of Valsalva aneurysm in 3, coronary artery disease in 4, and ventricular septal defect in 2. The freehand technique was used in 12 patients and the miniroot replacement in 15 patients. The postoperative transvalvular gradient was 17 mm Hg in freehand valves and 7 mm Hg in the miniroot valves. Initial postoperative study showed no or trivial aortic regurgitation in all 26 survivors except 1 freehand patient who had mild aortic regurgitation. At late study up to 6 years postoperatively all 10 of our available freehand patients showed progressive regurgitation, and it was severe in 4 patients. By contrast only half of the miniroot patients showed progressive regurgitation, and in none was it severe. Allograft replacement of the aortic valve can be done in children and adults with low mortality rate, short hospital stay, and excellent early function. Subsequent follow-up studies suggest that the miniroot replacements are superior to the freehand valves with lower transvalvular gradients, less valvular regurgitation, and delayed progression of valvular regurgitation.

Pancreaticoduodenectomy in the treatment of congenital fibrosarcoma of the duodenum.

A 2-mo-old female with congenital fibrosarcoma causing duodenal obstruction had a pancreaticoduodenectomy. She is doing well with the aid of oral pancreatic enzyme supplements 14 mo after her pancreaticoduodenectomy with no evidence of tumor recurrence or pancreatic endocrine dysfunction.