Small, heat-stable, DNA-binding proteins from Caulobacter crescentus.

From a heterogenous cell population of Caulobacter crescentus a deoxyribonucleoprotein fraction (DNP) was obtained by the modified technique of Sjåstad et al. (1982). Under the electron microscope DNP had a smooth fibrillar structure. The chemical properties of the proteins associated with the DNA were similar to those of other bacteria. An abundant, heat-stable, basic and DNA-binding protein, termed HCc, which has a molecular weight of 13.4 kD may be an analogue of the HU-histone-like protein from Escherichia coli.

Mutants of Caulobacter crescentus resistant to beta-lactam antibiotics.

A number of mutants of Caulobacter crescentus CB15 resistant ot ampicillin were isolated. The mutants differred in their resistance to several beta-lactam antibiotics. No differences in composition of the penicillin-binding proteins of the mutants compared to the parental strain, or in the affinity of these proteins to penicillin or ampicillin were found. The mutants were found to differ from the parent and also in many cases from each other in outer membrane protein composition.

Characterization of a Helical Morphological Mutant of Caulobacter crescentus.

Screening of poorly growing colonies following EMS treatment of Caulobacter crescentus culture revealed several mutants with altered cell morphology. One of these (CB15-UW.5) grew as elongated, helically twisted cells which were more susceptible to autolysis than the wild type. The mutant cells were found to have higher transglycosylase activity, this being reflected by shorter chain length of their murein. A second difference between the murein of the mutant and the parent was lower trimer content in the former, although in both cases the degree of crosslinkage was similar.

Autolysis of Caulobacter crescentus grown in the presence of glycine.

Caulobacter crescentus was grown in complex medium supplemented with low (0.05%) concentration of glycine, a component of the murein peptide side chains of this bacterium. Murein synthesized in the presence of glycine was poorly crosslinked and the rate of its synthesis was slowed down compared to the control cells. The glycine-grown cells were considerably more sensitive to the chelating agent EDTA and Tris buffer than the control cells and also lysed faster when incubated with beta-lactam antibiotics. No changes in phospholipid composition in the presence of glycine were observed and the outer membrane protein composition of the glycine-grown cells was altered only in the amount of 130 000 protein which forms the surface array of C. crescentus. The effects of glycine can thus be tentatively put down to the reduced crosslinkage of murein synthesized in its presence.

Mutants of Escherichia coli K12 sensitive to acidic pH.

Four mutants of Escherichia coli K12 were isolated on the basis of their sensitivity to pH 5.4. Under non-permissive conditions their growth was reversibly inhibited. At pH 7.0 these mutants showed a highly pleiotropic phenotype, which included altered phage and detergent sensitivities and leakage of periplasmic proteins. The findings suggest a defect in the outer membrane, perhaps in lipopolysaccharide. Two mutants mapped in or near the rfa locus, while the other two were removed from this region.

Purification and some properties of ATP-dependent deoxyribonuclease of Caulobacter crescentus.

An ATP-dependent deoxyribonuclease has been partially purified from extracts of Caulobacter crescentus cells in a procedure involving ion-exchange and affinity chromatography. The enzyme was purified approximately 350-fold and was free of contaminating nucleolytic and ATPase activity. The nuclease hydrolyzes linear, double-stranded DNA with subsequent release of short oligonucleotides, mostly from one to four bases in length. The release of nucleotides is accompanied by hydrolysis of ATP, 7.6 nmol ATP being consumed for each nmol of acid-soluble products of DNA degradation. The enzyme shows an absolute requirement for divalent cations and in most active at pH 7.6 to 8.8. The molecular weight of the nuclease, estimated by gel filtration and sucrose density gradient centrifugation, is 280 000.

Isolation of mutants of Caulobacter crescentus deficient in ATP-dependent deoxyribonuclease activity.

Caulobacter crescentus mutants sensitive to UV radiation, mitomycin C and methyl methane-sulfonate were isolated and tested for ATP-dependent deoxyribonuclease activity. Two mutants were identified of which one had less than 5 per cent of the wild-type level of the nuclease activity. This mutant in cross with another auxotrophic partner gave highly reproducible two-fold reduction in number of recombinants compared to a control cross. The suggested causes for reduced recombination frequency when one of the partners has residual ATP-dependent deoxyribonuclease activity are discussed.

Aggregation deficient Escherichia coli K-12 female mutants with altered lipopolysaccharide.

Two F- mutants deficient in conjugation with F-donors have been characterized. They map at about 83 minut position, show resistance to T3 and T7 bacteriophages, and form mating aggregates in the liquid medium with lowered efficiency. Mutants have no detectable alterations in their outer membrane protein composition.