Potential of Agrobacterium tumefaciens and Octopine-Utilizing Fluorescent Pseudomonas Strains To Attach to Susceptible Potato Tissues.

The binding characteristics of two octopine-catabolizing pseudomonads, Pseudomonas fluorescens B99A and E175D, which were isolated from crown galls, have been examined. The binding of strain B99A to potato disks was very weak, followed a Freundlich isotherm, and was temperature and pH independent. Strain E175D displayed strong attachment and followed a Langmuir isotherm. Despite these fundamental differences in binding characteristics, when each strain was placed in competitive binding assays with either Agrobacterium tumefaciens B6 or A. tumefaciens ATCC 15955, the number of bound pseudomonad cells decreased compared with those obtained in independent trials. Furthermore, the binding of A. tumefaciens cells was increased. In prebinding experiments, in which the potato disks were bound with the pseudomonads before exposure to the agrobacteria, the number of bound pseudomonad cells again decreased. This implies that increased desorption was occurring. In these prebinding studies, the numbers of bound A. tumefaciens ATCC 15955 increased, but the number of bound A. tumefaciens B6 remained the same. The mechanism for this observed synergism on the binding of agrobacterial cells and the depression in bound pseudomonad cells is believed to be alterations in the electrostatic or ionic charges on the plant and bacterial cell surfaces. The synergistic effect on A. tumefaciens undermines the use of these pseudomonads as potential biocontrol agents for crown gall.

Modulation of in vivo and in vitro transcription of bacteriophage phi 29 early genes.

The majority of early transcripts of the phi 29 bacteriophage are produced throughout the lytic cycle but the levels of a class of transcripts from the right end of the phi 29 genome are significantly reduced late in the infection. We have isolated a phage early protein which selectively interferes with the initiation in vitro of transcription from promoters at the right end of the phi 29 genome. The amino acid sequence of the purified inhibitory protein correlates to the sequence predicted from the phi 29 gene 6 reading frame. In addition the inhibitory protein was not detectable in cells infected with phage mutated in gene 6 and the decrease in transcription did not occur in vivo when nonpermissive cells were infected with phi 29(sus6). The results indicate that the gene 6 protein modulates transcription from the right side of the phi 29 genome.

Inhibition of in vitro peptidoglycan biosynthesis in Escherichia coli by guanosine 5'-diphosphate 3'-diphosphate.

Physiological concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the synthesis of lipid intermediates and peptidoglycan catalyzed by a particulate enzyme preparation from Escherichia coli. The inhibition of these reactions was dependent on the concentrations of ppGpp and MgCl2 in the assay. The degree of inhibition of lipid intermediate synthesis decreased as the molar ratio of MgCl2 to ppGpp was increased, and no inhibition was observed above a MgCl2 to ppGpp ratio of 2.5. The synthesis of peptidoglycan was more sensitive to inhibition by ppGpp, and significant inhibition occurred under conditions where lipid intermediate synthesis was unaffected (i.e., at MgCl2 to ppGpp ratios of 2.5 or more). A variety of other nucleotides did not inhibit the synthesis of lipid intermediates and peptidoglycan.

Involvement of the relA gene product and feedback inhibition in the regulation of DUP-N-acetylmuramyl-peptide synthesis in Escherichia coli.

The regulation of uridine diphosphate-N-acetylmuramyl-peptide (UDP-MurNAc-peptide) synthesis was studied by labeling Escherichia coli strains auxotrophic for lysine and diaminopimelate with [3H]diaminopimelate for 15 min under various conditions. The amounts of [3H]diaminopimelate incorporated into UDP-MurNAc-tripeptide and -pentapeptide by a stringent (rel+) strain were the same in the presence or absence of lysine. Chloramphenicol-treated rel+ cells showed a 2.8-fold increase in labeled UDP-MurNAc-pentapeptide. An isogenic relaxed (relA) strain deprived of lysine showed a 2.7-fold increase in UDP-MurNAc-pentapeptide. Thus, UDP-MurNAc-pentapeptide synthesis is regulated by the relA gene. D-Cycloserine treatment of rel+ and relA strains caused a depletion of intracellular UDP-MurNAc-pentapeptide. Labeled UDP-MurNAc-tripeptide accumulated in D-cycloserine-treated cells of the rel+ and relA strains, suggesting that UDP-MurNAc-pentapeptide is a feedback inhibitor of UDP-MurNAc-peptide synthesis. In lysine-deprived cells, D-cycloserine treatment caused 41- and 71-fold accumulations of UDP-MurNAc-tripeptide in rel+ and relA strains, respectively. A 124-fold increase in UDP-MurNAc-tripeptide occurred in lysine-deprived rel+ cells treated with both chloramphenicol and D-cycloserine. These results indicate that both the relA gene product and feedback inhibition are involved in regulating UDP-MurNAc-peptide synthesis during amino acid deprivation.

Site of inhibition of peptidoglycan biosynthesis during the stringent response in Escherichia coli.

The site of inhibition of peptidoglycan synthesis during the stringent response in Escherichia coli was determined in strains which were auxotrophic for both lysine and diaminopimelic acid (DAP). Cells were labeled with [(3)H]DAP for 30 to 60 min in the presence and absence of required amino acids, and the cellular distribution of [(3)H]DAP was determined. In both stringent (rel(+)) and relaxed (relA) strains, amino acid deprivation did not inhibit the incorporation of [(3)H]DAP into the nucleotide precursor and lipid intermediate fractions. The amount of [(3)H]DAP incorporated into the peptidoglycan fraction by the amino acid-deprived relA strain was over 70% of the amount incorporated in the presence of required amino acids. In contrast, the amounts of labeled peptidoglycan in amino acid-deprived rel(+) strains were only 20 to 44% of the amounts synthesized in the presence of amino acids. These results indicate that a late step in peptidoglycan synthesis is inhibited during the stringent response. The components of the lipid intermediate fraction synthesized by rel(+) strains in the presence and absence of required amino acids were quantitated. Amino acid deprivation did not inhibit the synthesis of either the monosaccharide-pentapeptide or the disaccharide-pentapeptide derivatives of the lipid intermediate. Thus, the reaction which is most likely inhibited during the stringent response is the terminal one involving the incorporation of the disaccharide-pentapeptide into peptidoglycan.

Effect of amino acid deprivation and chloramphenicol treatment on cell sizes of rel+ and relA- strains of Escherichia coli.

The effects of inhibition of protein synthesis on the cell size distributions of rel+ and relA- derivatives of Escherichia coli K-12 were determined. Amino acid deprivation resulted in a reduction in the cell sizes of rel+ strains but not of relA- strains. Treatment with chloramphenicol (CAM) did not alter the size distributions of either rel+ or relA- strains except when they were rel+ dap-. CAM treatment of rel+ dap- strains resulted in an increase in cell size. It is proposed that these results reflect differences in the structures of the cell envelopes of rel+ and relA- bacteria.

Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12.

[3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli.