A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c 551 (PA c 551 ) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c 551 with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c 552 (HT c 552 ), which has similar main chain folding to PA c 551 , but is more stable to heat. Thermodynamic properties of PA c 551 with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c 551 with HT c 552 . In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c 552 . We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c 551 can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c 552 . The higher stability of HT c 552 may in part be attributed to some of these substitutions.

The genes for nitrous oxide (N 2 O) reduction, nosRZDFYL, are clustered on the chromosome of Pseudomonas aeruginosa. Promoter assays using transcriptional fusions to lacZ revealed that the structural gene for nitrous oxide reductase, nosZ, is transcribed with the upstream nosR gene. The nosR gene product is not required for the activity of the nosR promoter. A sequence similar to the consensus FNR-binding motif was found 41?5 bp upstream from the major transcriptional start point of nosR. Mutation of the motif significantly reduced the promoter activity. DNR, an FNR-related transcriptional regulator required for the expression of denitrification genes in P. aeruginosa, is necessary for the transcription of nosR, indicating that the motif is recognized by DNR. Nitrite (NO 2 2 ), nitric oxide (NO) and NO-generating reagents induced nosR promoter activity, but N 2 O did not. The NO 2 2 -induced nosR promoter activity was reduced by mutation of the NO 2 2 reductase gene. However, a low concentration of NO 2 2 induced the promoter activity in a NO reductase mutant. These results indicate that NO is the inducer molecule for transcription of the nos genes.

Mesophilic cytochrome c 551 of Pseudomonas aeruginosa (PA c 551 ) became as stable as its thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c 552 (HT c 552 ), through only five amino acid substitutions. The five residues, distributed in three spatially separated regions, were selected and mutated with reference to the corresponding residues in HT c 552 through careful structure comparison. Thermodynamic analysis indicated that the stability of the quintuple mutant of PA c 551 could be partly attained through an enthalpic factor. The solution structure of the mutant showed that, as in HT c 552 , there were tighter side chain packings in the mutated regions. Furthermore, the mutant had an increased total accessible surface area, resulting in great negative hydration free energy. Our results provide a novel example of protein stabilization in that limited amino acid substitutions can confer the overall stability of a natural highly thermophilic protein upon a mesophilic molecule.Heat-stable proteins from thermophilic bacteria usually exhibit main chain foldings similar to those of mesophilic counterparts. Mutational studies on mesophilic proteins modeled with respect to thermophilic counterparts have proved that specific side chain interactions in the thermophiles are partially responsible for the higher stability (1-3). Thermodynamic analysis has also indicated that a thermophilic protein can be stabilized through global interaction throughout the molecule (4). It remains enigmatic as to how many amino acid substitutions contribute to the stability of a natural thermophilic protein (5). In some cases, a mesophilic protein only acquires the stability of the thermophilic counterpart after substantial exchanges of a linear sequence; groups of individual mutations are not sufficient (6). Multiple mutations in mesophilic proteins that completely increase the stability to the levels of thermophilic counterparts would provide important information about relationships between local side chain interactions and overall protein stability and demonstrate that the thermophilic character can depend on a limited number of strong noncovalent interactions.Cytochrome c is a powerful tool for characterizing protein stability because structural information on a variety of cytochromes c is available, and heterologous expression systems for holopoteins have been established (7). Cytochrome c 551 (PA c 551 )1 from a mesophile, Pseudomonas aeruginosa, and cytochrome c 552 (HT c 552 ) from a thermophile, Hydrogenobacter thermophilus, are 82-and 80-amino acid proteins, respectively, each with a covalently attached heme. These proteins exhibit 56% sequence identity (8) and almost the same main chain foldings (9), but HT c 552 exhibits much higher stability compared with PA c 551 (10). On a structural comparison between HT c 552 (9) and PA c 551 (11), we identified three distal regions responsible for the higher stability of the former (9). The single mutation Val-78 to Ile (V78I), and two double mutations Phe-7 to Ala/Val-13...

Thermal stability was measured for variants of cytochrome c-551 (PA c-551) from a mesophile, Pseudomonas aeruginosa, and a thermophilic counterpart, Hydrogenobacter thermophilus cytochrome c-552 (HT c-552), by differential scanning calorimetry (DSC) at pH 3.6. The mutated residues in PA c-551, selected with reference to the corresponding residues in HT c-552, were located in three spatially separated regions: region I, Phe7 to Ala/Val13 to Met; region II, Glu34 to Tyr/Phe43 to Tyr; and region III, Val78 to Ile. The thermodynamic parameters determined indicated that the mutations in regions I and III caused enhanced stability through not only enthalpic but also entropic contributions, which reflected improved packing of the side chains. Meanwhile, the mutated region II made enthalpic contributions to the stability through electrostatic interactions. The obtained differences in the Gibbs free energy changes of unfolding [Delta(DeltaG)] showed that the three regions contributed to the overall stability in an additive manner. HT c-552 had the smallest heat capacity change (DeltaC(P)), resulting in higher DeltaG values over a wide temperature range (0-100 degrees C), compared to the PA c-551 variants; this contributed to the highest stability of HT c-552. Our DSC measurement results, in conjunction with mutagenesis and structural studies on the homologous mesophilic and thermophilic cytochromes c, provided an extended thermodynamic view of protein stabilization.

A 44-year-old man with no significant past medical history presented to the emergency department with a left-sided abdominal bulge that developed after he hit his abdomen against the blunt end of a bicycle motocross handlebar. Abdominal computed tomography showed a left abdominal wall hernia containing a loop of small intestine. The herniated intestine was easily reducible. We made the diagnosis of traumatic abdominal wall hernia ("handlebar" hernia) caused by direct blunt trauma, and performed laparoscopic-assisted herniorrhaphy. Laparoscopy revealed tears in the abdominal wall muscles (transverse abdominal, and internal and external oblique muscles) and bowel injury (Type Ⅰ a). The hernia defect was closed in 2 layers of muscle, along with mesh repair. The patient was discharged on postoperative day 5.Traumatic abdominal wall hernia (TAWH) caused by a handlebar is very rare, and we report our case herein, along with a review of the literature.