- Volume 136, Issue 5, 1990

Excision of the transposon Tn5 from sites of insertion in plasmid DNA was shown to occur at high frequency in Pseudomonas aeruginosa. Plasmids with Tn5 insertions conjugated poorly into P. aeruginosa and adversely affected growth compared to the respective parental plasmids. The kanamycin-resistance phenotype of Tn5 was expressed poorly in P. aeruginosa and kanamycin-sensitive strains were common during the manipulation of the P. aeruginosa transconjugants. Examination of plasmid DNA isolated from kanamycin-sensitive P. aeruginosa transconjugants revealed excision of Tn5 sequences. A plasmid containing a selectable marker (mercury resistance) inactivated by a Tn5 insertion was constructed, and Tn5 excised precisely, permitting the expression of the mercury-resistance marker at high frequency (10−3) in P. aeruginosa and at the expected low frequency (10−7) in Escherichia coli. The recombinational mechanism that promotes frequent Tn5 excision in P. aeruginosa operated in the absence of the P. aeruginosa recA gene product. Fragments of Tn5 were also examined for excision and instability in P. aeruginosa. A plasmid containing the terminal 485 bp of inverted repeat sequences from Tn5, but lacking the transposase or kanamycin-resistance genes, also showed precise excision of Tn5 DNA at high frequency (10−2) in P. aeruginosa. Unlike plasmids containing a complete Tn5 insertion, this plasmid transferred to P. aeruginosa at normal frequencies and growth of the host was not severely impaired. In contrast, plasmids containing either IS50 element transferred to P. aeruginosa at greatly reduced frequencies, and transconjugants containing the IS50R element (which contains the active transposase gene) were small and especially difficult to maintain. P. aeruginosa transconjugants harbouring a plasmid containing only the DNA between the IS50 elements (which included the kanamycin-resistance gene) were of normal size and stably maintained. These observations suggest that frequent and precise excision of Tn5 in P. aeruginosa required the long inverted repeat sequences at the termini of Tn5. The adverse effects conferred by IS50 on the transconjugant formation and growth of P. aeruginosa were apparently not required to promote Tn5 excision.

A method comprising enzyme separation by SDS-PAGE and subsequent use of peptidyl aminomethylcoumarins as substrates has been used to study proteinases of the protozoan parasite Trypanosoma brucei. The application of this method has allowed investigation of the substrate specificities of individual proteinases in cell lysates without the need for enzyme purification. The results show that T. brucei contains a group of cysteine proteinases, probably four in number, with substrate and inhibitor specificities similar to those of cathepsin L. A second group of proteinases, larger enzymes with significantly different substrate specificities and sensitivity to inhibitors, was also detected. Peptidyl diazomethanes inhibited the cysteine proteinases and also parasite growth, offering promise that peculiarities in the substrate specificity of trypanosomal cysteine proteinases could be exploited by compounds of this type.

Pyrimidine biosynthesis in Pseudomonas fluorescens strain A126 was investigated. In this study, de novo pyrimidine biosynthetic pathway mutant strains were isolated using both conventional mutagenesis and transposon mutagenesis. The resulting mutant strains were deficient for either aspartate transcarbamoylase, dihydroorotase or orotate phosphoribosyltransferase activity. Uracil, uridine or cytosine could support the growth of every mutant strain selected. In addition, the aspartate transcarbamoylase mutant strains could utilize orotic acid to sustain their growth while the orotidine-5′-monophosphate decarboxylase mutant strains grew slowly upon uridine 5′-monophosphate. The wild-type strain and the mutant strains were used to study possible regulation of de novo pyrimidine biosynthesis in P. fluorescens. Dihydroorotase specific activity more than doubled after the wild-type cells were grown in orotic acid relative to unsupplemented minimal-medium-grown cells. Starving the mutant strains of pyrimidines also influenced the levels of several de novo pyrimidine biosynthetic pathway enzyme activities.

The electrophoretic mobility (EPM) of a variety of Gram-negative and Gram-positive bacteria was measured with a Penkem S3000 analyser. Under standard growth conditions and neutral pH all cells displayed a negative EPM. The polysaccharide capsules of Escherichia coli strains Kl, K5, K29 and K30 generated the highest EPM; to a lesser and varying degree O-antigens with charged groups and core lipopolysaccharides also contribute to the net EPM. Very little negative EPM was measured in suspension cultures of the gliding bacterium Cytophaga U67. No difference in the EPM was observed between rapidly growing and stationary-phase E. coli B. De-energization of the cell membranes by carbonyl cyanide m-chlorophenylhydrazone (CCCP) did not affect the EPM of wild-type and deep rough mutants of E. coli; and the EPM of Cytophaga U67 and Acholeplasma laidlawii remained unaltered by CCCP when measured in their respective growth media. Extrusion of filamentous bacteriophage f1 from cells of its host, E. coli A95, caused a shift to a higher negative EPM. We also measured a variety of Grampositive strains, all of which displayed different EPMs. When membrane fractions of E. coli were adsorbed to latex spheres, characteristic differences between the EPM of beads coated with either inner or outer membrane were observed. The results suggest that the rapid EPM analysis is a useful tool to study the net electric charge of microorganisms and to examine changes of surface properties during interaction of cells with viruses, proteins (antibody) and charged antibiotics.

Nucleotide sequencing of DNA in the region of the Streptococcus mutans chromosome adjacent to the previously characterized gtfA gene revealed the presence of two long open reading frames. One of these corresponds to the dexB gene and has been shown to encode an intracellular exodextranase (dextran glucosidase) which has short isomaltosaccharides as preferred substrates. Comparison with other published sequences showed that the dexB gene product shares regions of similarity with enzymes, from a variety of sources, which attack other glucose polymers.

Nineteen strains of Bacteroides fragilis were examined by negative staining for surface structures. One strain (ATCC 23745) possessed peritrichous fibrils, 16 strains carried peritrichous fimbriae and two strains carried no surface structures. The fimbriae had a diameter of 2·1±0·25 nm and appeared to be ‘curly’. Only a small proportion (4 to 41%, depending on the strain) of cells in a population carried fimbriae or fibrils. Strain A312 showed phase variation of fimbriae as expression of fimbriae was repressed at 20 °C and in early exponential phase at 37 °C. The fibrils on strain ATCC 23745 did not exhibit phase variation in response to changes in incubation temperature, growth phase or growth in two different media. Capsules were demonstrated by the Indian ink method on 18 of the 19 strains, varying in size from strain to strain and within the same population. Cultures often contained both capsulate and noncapsulate cells. All strains possessed an electron dense ruthenium red staining layer between 7·9 and 23·9 nm in width attached to the outer membrane. Cell surface hydrophobicity quantified by the hexadecane partition assay gave low values ranging from 6·6 to 52·1%. Only a few strains were able to haemagglutinate and these were only weakly active. There was no correlation between cell surface hydrophobicity, haemagglutinating activity and surface structures.