Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots.

The lectin on the surface of 4- and 5-dold pea roots was located by the use of indirect immunofluorescence. Specific antibodies raised in rabbits against pea seed isolectin 2, which crossreact with root lectins, were used as primary immunoglobulins and were visualized with fluorescein- or tetramethylrhodamine-isothiocyanate-labeled goat antirabbit immunoglobulin G. Lectin was observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. On both types of cells, lectin was concentrated in dense small patches rather than uniformly distributed. Lectin-positive young hairs were grouped opposite the (proto)xylematic poles. Older but still-elongating root hairs presented only traces of lectin or none at all. A similar pattern of distribution was found in different pea cultivars, as well as in a supernodulating and a non-nodulating pea mutant. Growth in a nitrate concentration which inhibits nodulation did not affect lectin distribution on the surface of pea roots of this age. We tested whether or not the root zones where lectin was observed were susceptible to infection by Rhizobium leguminosarum. When low inoculum doses (consisting of less than 10(6) bacteria·ml(-1)) were placed next to lectin-positive epidermal cells and on newly formed root hairs, nodules on the primary roots were formed in 73% and 90% of the plants, respectively. Only a few plants showed primary root nodulation when the inoculum was placed on the root zone where lectin was scarce or absent. These results show that lectin is present at those sites on the pea root that are susceptible to infection by the bacterial symbiont.

Mapping of a gene for a major outer membrane protein of Escherichia coli K12 with the aid of a newly isolated bacteriophage.

A method is described for the enrichment of phages which can adsorb to a specific determinant of bacterial cell surfaces. A phage was isolated which absorbs to E. coli cells containing the "major outer membrane= protein c but not to strains that are lacking this protein. With the aid of this phage a gene, meoA which is responsible for the lack of protein c was mapped at 48 min on the linkage map of E. coli K12.

Distribution of lipids in cytoplasmic and outer membranes of Escherichia coli K12.

The lipid composition of cytoplasmic and outer membranes of Escherichia coli K12 was studied. Compared with the cytoplasmic membrane, the outer membrane is enriched in both saturated fatty acids and phosphatidylethanolamine. This is also the case when the fatty acid composition of the phospholipids is changed, either by varying the growth temperature or by using mutants with alterations in their fatty acid metabolism. Phosphatidylethanolamine of the outer membrane contains relatively more saturated fatty acids than phosphatidylethanolamine of the cytoplasmic membrane.

Freeze etch morphology of outer membrane mutants of Escherichia coli K12.

Freeze etching showed that the loss of each of the major outer membrane proteins b, c or d in mutants of Escherichia coki K12 does not influence the morphology of fracture faces of the outer membrane. Mutants that possess a heptose-deficient lipopolysaccharide and which in addition are deficient in one or more major outer membrane proteins exhibit a reduction in the number of intramembranous particles of the outer membrane. Moreover it was shown that lipid phase transitions induce a lateral lipid protein separation in the outer membrane, similar to that found in the cytoplasmic membrane.

Properties of a D-glutamic acid-requiring mutant of Escherichia coli.

Some properties of a d-glutamic acid auxotroph of Escherichia coli B were studied. The mutant cells lysed in the absence of d-glutamic acid. Murein synthesis was impaired, accompanied by accumulation of uridine-5'-diphosphate-N-acetyl-muramyl-l-alanine (UDP-MurNac-l-Ala), as was shown by incubation of the mutant cells in a cell wall medium containing l-[(14)C]alanine. After incubation of the parental strain in a cell wall medium containing l-[(14)C]glutamic acid, the acid-precipitable radioactivity was lysozyme degradable to a large extent. Radioactive UDP-MurNac-pentapeptide was isolated from the l-[(14)C]glutamic acid-labeled parental cells. After hydrolysis, the label was exclusively present in glutamic acid, the majority of which had the stereo-isomeric d-configuration. Compared to the parent the mutant incorporated less l-[(14)C]glutamic acid from the wall medium into acid-precipitable material. Lysozyme degraded a smaller percentage of the acid-precipitable material of the mutant than of that of the parent. No radioactive uridine nucleotide precursors could be isolated from the mutant under these conditions. Attempts to identify the enzymatic defect in this mutant were not successful. The activity of UDP-MurNac-l-Ala:d-glutamic acid ligase (ADP; EC 6.3.2.9) (d-glutamic acid adding enzyme) is not affected by the mutation. Possible pathways for d-glutamic acid biosynthesis in E. coli B are discussed.

Temperature-sensitive mutant of Escherichia coli K-12 with an impaired D-alanine:D-alanine ligase.

The temperature-sensitive Escherichia coli mutant strain ST-640 lyses at the restrictive temperature except when an osmotic stabilizer or a high concentration of d-alanine is present. The presence of dl-alanyl-dl-alanine does not prevent lysis. The rate of murein synthesis, followed in a wall medium, is decreased at both 30 and 42 C. d-Alanyl-d-alanine and uridine diphosphate-N-acetyl-muramyl (UDP-MurNAc)-pentapeptide are synthesized in decreased amounts, accompanied by accumulation of UDP-MurNAc-tripeptide at 42 C but not at 30 C. Uridine nucleotide precursors leak into the medium, especially out of the mutant cells. This leakage is prevented when NaCl is present. The d-alanine: d-alanine ligase (ADP) (EC 6.3.2.4) of the mutant strain, assayed in crude extracts, is temperature sensitive. The impaired ligase is relatively resistant to d-cycloserine and other inhibitors of the enzyme. Combined genetic and enzymatic results show that the low ligase activity is due to a mutation in the ddl gene, the structural gene for d-alanine: d-alanine ligase.

Inhibition of peptidoglycan synthesis by the antibiotic diumycin A.

Diumycin A, a new antibiotic, was found to inhibit cell wall synthesis by Staphylococcus aureus, a phenomenon accompanied by accumulation of uridine-5'-diphosphate-N-acetyl-muramyl-pentapeptide. The antibiotic inhibited in vitro peptidoglycan synthesis by particulate preparations of Bacillus stearothermophilus and Escherichia coli by preventing the utilization of N-acetyl-glucosamine-N-acetyl-muramyl-pentapeptide. In contrast to vancomycin, the antibiotics diumycin, prasinomycin, moenomycin, 11.837 RP, and enduracidin do not inhibit particulate d-alanine carboxypeptidase.

Studies on Escherichia coli enzymes involved in the synthesis of uridine diphosphate-N-acetyl-muramyl-pentapeptide.

The specific activities of l-alanine:d-alanine racemase, d-alanine:d-alanine ligase, and the l-alanine, d-glutamic acid, meso-diaminopimelic acid, and d-alanyl-d-alanine adding enzymes were followed during growth of Escherichia coli. The specific activities were nearly independent of the growth phase. d-Alanine:d-alanine ligase was inhibited by d-alanyl-d-alanine, d-cycloserine, glycine, and glycyl-glycine. l-Alanine:d-alanine racemase was found to be sensitive to d-cycloserine, glycine, and glycyl-glycine. The l-alanine adding enzyme was inhibited by glycine and glycyl-glycine.

Temperature-sensitive mutants of Escherichia coli K-12 with low activities of the L-alanine adding enzyme and the D-alanyl-D-alanine adding enzyme.

A number of properties of temperature-sensitive mutants in murein synthesis are described. The mutants grow at 30 C but lyse at 42 C. One mutant possesses a temperature-sensitive d-alanyl-d-alanine adding enzyme, has an impaired rate of murein synthesis in vivo at both 30 and 42 C, and contains elevated levels of uridine diphosphate-N-acetyl-muramyl-tripeptide (UDP-MurNAc-l-Ala-d-Glu-m-diaminopimelic acid) at 42 C. The other mutant possesses an l-alanine adding enzyme with a very low in vitro activity at both 30 and 42 C. Its in vivo rate of murein synthesis is almost normal at 30 C but is much less at 42 C. When the murein precursors were isolated after incubation of the cells in the presence of (14)C-l-alanine, they contained only a fraction of the radioactivity that could be obtained from a wild-type strain. A genetic nomenclature for genes concerned with murein synthesis is proposed.

Temperature-sensitive mutants of Escherichia coli K-12 with low activity of the diaminopimelic acid adding enzyme.

Five temperature-sensitive lysis mutants were found to possess very low diaminopimelic acid (Dpm) adding enzyme activity in vitro. Murein synthesis at 42 C was impaired, and uridine-5'-diphosphate-N-acetyl-muramyl-l-Ala- d-Glu (UDP-MurNAc-dipeptide) was accumulated. In the presence of NaCl, the mutants could grow at 42 C. NaCl had no influence on the Dpm adding enzyme activity in vitro. The growth rate of most temperature-resistant revertants was decreased, but their Dpm adding enzyme activity remained very low. Two revertants had a rather normal growth rate. Their Dpm adding enzyme activity was significantly increased, but much lower than in the wild type. The influence of growth rate on the viability of the mutants is discussed.

Murein synthesis and identification of cell wall precursors of temperature-sensitive lysis mutants of Escherichia coli.

A group of temperature-sensitive lysis mutants of Escherichia coli K-12 was studied. Mutants impaired in the synthesis of uridine diphosphate-N-acetylmuramyl (UDP-MurNAc)-pentapeptide or in the synthesis of murein amino acids were found. Their rate of murein synthesis at the restrictive temperature was decreased. A large number of mutants did not differ from the parent strain with respect to the rate of murein synthesis and the precursor pattern. The behavior of these mutants is discussed. It was impossible to accumulate UDP-MurNAc-pentapeptide in E. coli by the antibiotics penicillin and vancomycin. The hypothesis is put forward that the amount of this murein precursor is regulated by feedback inhibition.

In vivo and in vitro action of new antibiotics interfering with the utilization of N-acetyl-glucosamine-N-acetyl-muramyl-pentapeptide.

Recent literature on the antibiotics enduracidin, moenomycin, prasinomycin, and 11.837 RP suggested an interaction with murein synthesis. Incubation of sensitive strains from Bacillus cereus and Staphylococcus aureus in a "wall medium" containing labeled l-alanine showed that all four antibiotics inhibited the incorporation of alanine into murein and gave rise to accumulation of radioactive uridine diphosphate-N-acetyl-muramyl (UDP-MurNAc)-pentapeptide. Peptidoglycan was synthesized when the particulate enzyme of B. stearothermophilus was incubated with the murein precursors UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-MurNAc-pentapeptide. The newly formed polymer was less accessible for lysozyme and more strongly bound to the acceptor than the same product from the Escherichia coli particulate enzyme. After incubation in the presence of penicillin, a greater part of the peptidoglycan was lysozyme sensitive and more loosely bound to the acceptor. The antibiotics enduracidin, moenomycin, prasinomycin, and 11.837 RP inhibited peptidoglycan synthesis by the B. stearothermophilus particulate enzyme. The rate of synthesis of GlcNAc-MurNAc(-pentapeptide)-P-P-phospholipid was independent from the addition of these antibiotics, but its utilization was strongly inhibited. With the present results, it is not possible to distinguish the mechanisms of action of enduracidin, moenomycin, prasinomycin, and 11.837 RP from the mechanisms of action of vancomycin and ristocetin.