Gene transfer in enteric bacteria through the formation of R-prime plasmids by an RP4: :mini-Mu element.

Gene transfer in seven pathogenic enteric bacteria was studied using an RP4: :mini-Mu element, the plasmid pULB113. From the E. coli K-12 host strain the plasmid could be efficiently transferred to these enteric bacteria, but its transfer back to E. coli K-12 was not as efficient, being detected only in Shigella dysenteriae 1, S. flexneri and the 'smooth' variant of S. sonnei. In these three species, transposition of chromosomal fragments into the plasmid to produce R-prime plasmid was also detected at a frequency of approximately 10(-5). Transposition was random as suggested by the recovery at approximately the same frequency (10(-5) to 10(-6)) of R-primes involving 20 different auxotrophic markers from widely separated chromosomal locations. Formation of R-prime plasmids expressing toxicity in the E. coli K-12 recipient strain was also efficient in S. dysenteriae 1 but the toxin-activity was rapidly lost from these R-primes. In our experiments, the plasmid pULB113 incorporated relatively small amounts of chromosomal DNA as determined by restriction endonuclease digestion. For a Thy+ R-prime that we analyzed, the amount of cloned DNA was approximately 15 kb.     

A DSS for job scheduling under process interruptions

The primary objective of this research is to solve the job-shop scheduling problems (JSSPs), by minimizing the makespan, with and without process interruptions. In this paper, we first developed a genetic algorithm for solving JSSPs, and then improved the algorithm by integrating it with two simple priority rules and a hybrid rule. The performance of the developed algorithm was tested by solving 40 benchmark problems and comparing their results with that of a number of well-known algorithms. In addition, we have studied the job-shop scheduling under process interruptions such as machine unavailability and breakdown. For convenience of implementation, we have developed a decision support system (DSS). In the DSS, we built a graphical user interface for user friendly data inputs, model choices, and output generation. An overview of the DSS and an analysis of the experimental results are provided. The incorporation of priority rules, and a hybrid rule, not only improves the solutions but also reduces the overall computational time. The experimental results show that when the machine unavailability information is known in advance, the effect on the schedule is very little compared to the sudden machine breakdown scenario.     

Affinities of phosphorylated substrates for the E. coli tryptophan synthase α-subunit: Roles of Ser-235 and helix-8′ dipole

The roles of Ser-235 and helix-8′ (residues 235–242) in the functional binding and turnover of phosphorylated substrates by the α-subunit of the E. coli tryptophan synthase (TSase) α₂β₂-holoenzyme complex are examined. Previous crystallographic analyses indicated that this region was one of several near the phosphate moiety of the physiological substrate, indole-3-glycerol phosphate (IGP). The peptidyl amido group of Ser-235 was suggested to H-bond to the phosphate group; a helix macrodipole binding role was suggested for helix-8′. The activities and substrate K_ms of mutant α-subunits altered in this region by site-specific mutagenesis are reported here. Substitutions at Ser-235 by an acidic (glutamic acid, mutant SE235), basic (lysine, mutant SK235), or a nonpeptidyl amido-containing residue (proline, mutant SP235) exhibit 40- to 180-fold K_m increases for IGP and D -glyceraldehyde-3-phosphate; no K_m defects for indole were observed. k_cat values for SP235, SE235, and SK235 are 100, 70, and 40%, respectively, of the wild-type value. Steric considerations may explain the results with the SE235 and SK235 mutant α-subunits; however, the SP235 results are consistent with the suggested phosphate binding role for the Ser-235 peptidyl amide group during catalysis. A helix-8′ dipole role was explored following proline substitutions separately at the first six (of eight) residues. Proline substitutions at positions-1 through -4 in helix-8′ have normal indole K_ms and catalytic activities in all four TSase reactions, suggesting no major global structural changes in these proteins. By these criteria, substitutions at positions-5 and -6 lead to significant structural alterations. K_m increases for phosphorylated substrates are substantial (up to 40-fold) and are dependent upon the presence of L -serine at the β-subunit active site. In the absence of L -serine, substitution only at the first position results in binding defects; in the presence of L -serine, substitutions at the first, second and third positions show binding defects of decreasing magnitude, sequentially. Substitutions at the fourth and fifth position have no effect on substrate binding. It is suggested that during catalysis a helix dipole effect on binding may be exerted but only via inter-subunit-induced conformational changes due to ligand (L -serine) binding to the β-subunit. © 1995 Wiley-Liss, Inc.