The penicillin-binding proteins (PBPs) of Haemophilus influenzae were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Eight major PBPs, ranging in molecular weights from 90,000 to 27,000, were detected. The pattern of molecular weights was different from that determined fro Escherichia coli or Pseudomonas aeruginosa. A study on the binding of several beta-lactam antibodies to the PBPs at their minimal inhibitory concentrations and at lower and higher concentrations revealed that all had highest affinity for PBP 2. Amdinocillin (mecillinam) was an exception; it had highest affinity for PBP 3. The morphological effects of several penicillins, cephalosporins, and amdinocillin on H. influenzae were similar to those reported for E. coli.
L-Sorbose is oxidized to 2-keto-L-gulonic acid (KGA) via the following sequence of reactions which we call the "sorbosone pathway": L-sorbose in equilibrium L-sorbosone leads to KGA. The first step is reversible and is mediated by enzymes found in a soluble fraction obtained from Pseudomonas putida ATCC 21812. Although no cofactor requirements were found for the forward reaction, the reverse reaction clearly required NADH. Enzymes for this NADH-dependent synthesis of L-sorbose could be differentiated on the basis of molecular weights. The second step in the sorbosone pathway is catalyzed by a particulate enzyme found in extracts from P. putida and Gluconobacter melanogenus IFO 3293. The rate limiting reaction in the sorbosone pathway is the synthesis of L-sorbosone. In addition to P. putida, Klebsiella pneumoniae (ATCC 27858) and Serratia marcescens (ATCC 27857) also contain the enzymes which catalyze the reactions of the sorbosone pathway. Two of the bacteria studied, P. putida and G. melanogenus, also contain an enzyme involved in the further metabolism of KGA to L-idonic acid. This enzyme, referred to as KGA-reductase, is found in the soluble fraction of cell-free extracts and is dependent on NADH or NADPH.
Studies on the binding of Ro 13-9904, a new broad-spectrum cephalosporin, showed that it had higher affinities for PBPs 1b, 2, and 3 of Escherichia coil (3> 1b>2) than cefazolin, cephaloridine, cephalothin or cephalexin.With Haemophilus inf uenzae, Ro 13-9904 showed highest affinities for PBPS4 and 5 followed by PBP 2. It inhibited total cell wall synthesis at lower concentrations than the other 8-lactam antibiotics tested.
It has been suggested that replication of DNA in general should start at genetically specific molecular sites.' Unique origins of replication exist in Bacillus subtilis2 and Escherichia coli.3In the present communication, I report preliminary results concerning the origin of replication in the DNA of phage lambda.Lambda DNA extracted from phage particles has regions of dissimilar nucleotide composition.4 When sheared to small fragments and banded in a density gradient of Cs2SO4-HgCl2, the DNA is resolved into two major peaks.' The denser fragments, rich in adenine and thymine (AT), come from a continuous right-hand section of the molecule representing 56 per cent of its length. The less dense fragments, rich in guanine and cytosine (GC), originate from the remaining left-hand section. A given sample of lambda DNA subjected to analysis of the sort indicated may be characterized by the ratio R _ DNA content of denser band DNA content of less-dense band which measures 1.27 for DNA extracted from phage particles. If DNA synthesis starts in the AT-rich section, replicating DNA should give a ratio R that exceeds 1.27 by an amount related to, among other things, the distance between the point of origin and the boundary between sections. However, if replication starts at random sites, R should be the same for replicating and nonreplicating DNA. The experimental approach suggested by these considerations is similar in principle to the genetic method used by Sueoka and Yoshikawa2 to locate the origin of replication in B. subtilis.The following experiments showed that lambda DNA synthesized during the first few minutes after infection (when it is reasonable to assume that most of the DNA is replicating) contains an excess of AT-rich sections. Materials and Methods.-Bacterial and phage strains: A clear-plaque (Cl) mutant of lambda was used to infect E. coli HF4704 T-, Hcr-(provided by Dr. R. L. Sinsheimer). DNA synthesis is arrested in this host by treatment with mitomycin C and restored by infection with phage 6X1746 or lambda.7 Culture medium: Experimental cultures were grown in a medium containing, per liter, 0.05 mole of tris(hydroxymethyl)aminomethane (Trizma-Base, Sigma Chemical Co.); 1.0 ml of 0.66 M KH2PO4; 1.0 ml of 1.0 M MgSO4; 1.0 ml of 1.0% CaCl2; 0.1 ml of 0.5% FeSO 6H20; 0.8 gm NH4C1; 0.2 gm Na2SO4; 1.0 gm KCl; 10 ml of 40% glycerol;and 20 ml of 10% "vitamin-free" casein hydrolysate (Nutritional Biochemicals Corp.).The pH of the medium was adjusted with HCl to 7.4. Thymidine was added as required. Experimental cultures: Bacterial cultures were grown with aeration at 370C in the specified medium supplemented with 10 Ag/ml of thymidine. When they reached 2 X 108 cells/ml, they were treated with 50 lg/ml of mitomycin C for 10 min in the dark without aeration. Then the cells were collected on a membrane filter (B4, Schleicher & Schuell Co.), washed with medium devoid of thymidine, and resuspended in culture medium 1345
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