Pesticinogenic and Ca2+-dependent strains of Yersiniapestis harbored plasmids of about 6 and 45 megadaltons, respectively. In addition, most isolates examined possessed a cryptic 65-megadalton plasmid.
Cultures of wild-type Caulobacter crescentus and strains with fla mutations representing 24 genes were pulse-labeled with 14C-amino acids and analyzed by immunoprecipitation to study the synthesis of flagellar components. Most fla mutants synthesize flagellin proteins at a reduced rate, suggesting the existance of some mechanism to prevent the accumulation of unpolymerized flagellin subunits. Two strains contain deletions that appear to remove a region necessary for this regulation. The hook protein does not seem to be subject to this type of regulation and, in addition, appears to be synthesized as a faster-sedimenting precursor. Mutations in a number of genes result in the appearance of degradation products of either the flagellin or the hook proteins. Mutations inflaA,-X,-Y, or-Z result in the production of filaments (stubs) that contain altered ratios of the flagellin proteins. In some flaA mutants, other flagellin-related proteins were assembled into the stub structures in addition to the flagellins normally present. Taken together, these analyses have begun to provide insight into the roles of individual fla genes in flagellum biogenesis in C. crescentus. Caulobacter crescentus undergoes a unique developmental cycle in which the cell spends approximately one-third of its life as a motile swarmer cell and the remainder as a nonmotile stalked cell (18, 19). The swarmer cell is essen
To obtain amidases with novel substrate specificity, the cloned gene for penicillin amidase of Escherichia coli ATCC 11105 was mutagenized and mutants were selected for the ability to hydrolyze glutaryl-(L)-leucine and provide leucine to Leu-host cells. Cells with the wild-type enzyme did not grow in minimal medium containing glutaryl-(L)-leucine as a sole source of leucine. The growth rates of Leu-cells that expressed these mutant amidases increased as the glutaryl-(L)-leucine concentration increased or as the medium pH decreased. Growth of the mutant strains was restricted by modulation of medium pH and glutaryl-(L)-leucine concentration, and successive generations of mutants that more efficiently hydrolyzed glutaryl-(L)-leucine were isolated. The kinetics of glutaryl-(L)-leucine hydrolysis by purified amidases from two mutants and the respective parental strains were determined. Glutaryl-(L)-leucine hydrolysis by the purified mutant amidases occurred most rapidly between pH 5 and 6, whereas hydrolysis by wild-type penicillin amidase at this pH was negligible. The second-order rate constants for glutaryl-(L)-leucine hydrolysis by two "second-generation" mutant amidases, 48 and 77 M-' s-l, were higher than the rates of hydrolysis by the respective parental amidases. The increased rates of glutaryl-(L)-leucine hydrolysis resulted from both increases in the molecular rate constants and decreases in apparent Km values. The results show that it is possible to deliberately modify the substrate specificity of penicillin amidase and successively select mutants with amidases that are progressively more efficient at hydrolyzing glutaryl-(L)-leucine.
Chromosomal insertions of TnS in Caulobacter crescentus displayed complete stability upon transduction and proved useful in strain building on complex media. RP4-primes constructed in vitro containing C. crescentus genomic sequences in the HindIlI site of the kanamycin resistance gene failed to show enhanced or directed chromosome mobilization abilities. One of these kanamycin-sensitive RP4 derivatives, pVS1, was used as a mobilization vector in conjugation experiments on complex media where chromosomal TnS transfer to the recipient was selected. pVS1-mediated transfer of TnS-induced auxotrophic mutations occurred at frequencies of 10-6 to 10-8 per donor cell. During conjugations with TnS-encoded kanamycin resistance as the selected marker, TnS remained in its donor-associated locus in 85 to 100% of the transconjugants. A collection of eight temperature-sensitive donor strains bearing TnS insertion mutations from various regions of the C. crescentus genetic map were used to provide a rapid means for the determination of the map location of a new mutation. Use of the techniques described in this paper allowed an expansion of the C. crescentus genetic map to include the relative locations of 32 genes.
Homogeneous preparations of pesticin, a bacteriocin produced by Yersinia pestis, neither significantly inhibited net synthesis of deoxyribonucleic acid, ribonucleic acid, or protein in Escherichia coli 4) nor caused detectable degradation of deoxyribonucleic acid in vivo. Accordingly, its mode of action does not resemble that of colicin E2 as suggested by others. However, incorporation of cell wall-specific label (['4C]diaminopimelic acid) into trichloroacetic acid-insoluble material of growing cells was inhibited by pesticin which also promoted release of such radioactivity from both resting cells and purified mureinlipoprotein. Sodium dodeycl sulfate-polyacrylamide gel electrophoresis ofreaction mixtures containing appropriately labeled mureinlipoprotein showed that [3H]N-acetylglucosamine comigrated either with ['4C]diaminopimelic acid in the murein peptide or with ["C]isoleucine of the Braun lipoprotein. As judged by these findings and pesticindependent release of reducing equivalents but not 4-hydroxy-2-acetamido sugars, the bacteriocin possesses N-acetylglucosaminidase activity. Hydrolysis of mureinlipoprotein occurred over a broad pH, with an optimum of 4.7. Mureinlipoproteins from a variety of pesticin-sensitive and -resistant organisms were hydrolyzed by the bacteriocin, indicating that its antibacterial specificity resides at the level of absorption. 495 on August 1, 2020 by guest
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