- Volume 138, Issue 1, 1992

Summary: Five bacterial species were transformed using particle gun-technology. No pretreatment of cells was necessary. Physical conditions (helium pressure, target cell distance and gap distance) and biological conditions (cell growth phase, osmoticum concentration, and cell density) were optimized for biolistic transformation of Escherichia coli and these conditions were then used to successfully transform Agrobacterium tumefaciens, Erwinia amylovora, Erwinia stewartii and Pseudomonas syringae pv. syringae. Transformation rates for E. coli were 104 per plate per 0·8 μg DNA. Although transformation rates for the other species were low (<102 per plate per 0·8 μg DNA), successful transformation without optimization for each species tested suggests wide utility of biolistic transformation of prokaryotes. E. coli has proven to be a useful model system to determine the effects of relative humidity, particle size and particle coating on efficiency of biolistic transformation.

Summary: In Escherichia coli, constituents of the main recombination pathway are provided by the genes recA (RecA protein) and recBCD (RecBCD enzyme). Recombination in conjugation experiments and repair of UV damage of E. coli mutants deleted for recA, for recBCD or for recA plus recBCD were restored, although to different degrees, by the cloned recA and recBCD genes from Serratia marcescens or Proteus mirabilis. When both recombination enzymes were from the same species, repair and recombination efficiencies had the order E. coli > S. marcescens > P. mirabilis. However, the P. mirabilis recA plus recBCD genes resulted in higher levels of repair and recombination than those obtained with one component from P. mirabilis (recA or recBCD) and the other from E. coli or S. marcescens. The data provide evidence for the similarity of RecABCD pathways of recombination among enteric bacteria and suggest an in vivo advantage of an intraspecies combination of P. mirabilis RecA protein and RecBCD enzyme over interspecies combinations. This could point to a cooperation between these basic recombination enzymes. The molecular processes which could be involved are discussed.

Summary: The pattern and extent of DNA sequence variability at the rplX locus (encoding ribosomal protein L24) has been investigated in nine strains of Bacillus subtilis. Overall, there is a very low level of nucleotide diversity, even at silent sites, which is probably due to selection among synonymous codons. By analogy with Escherichia coli, there may also be some effect of the relative proximity of rplX to the chromosomal origin of replication. The small number of nucleotide substitutions are non-randomly distributed: all of the synonymous changes are in valine codons. From the sequence differences the strains can be divided into two groups, which are not coincident with their previous classification; this observation is consistent with recombination among strains.

Summary: Metabolites such as diaminopimelate and some aromatic derivatives, not synthesized in mammalian cells, are essential for growth of bacteria. As a first step towards the design of a new human live vaccine that uses attenuated strains of Leptospira interrogans, the asd, aroD and dapD genes, encoding aspartate β-semialdehyde dehydrogenase, 3-dehydroquinase and tetrahydrodipicolinate N-succinyltransferase, respectively, were cloned by complementation of Escherichia coli mutants. The complete nucleotide sequence of the asd gene was determined and found to contain an open reading frame capable of encoding a protein of 349 amino acids with a calculated M r of 38007. Comparison of this deduced L. interrogans aspartate β-semialdehyde dehydrogenase amino acid sequence with those of the same enzyme from Saccharomyces cerevisiae and Corynebacterium glutamicum revealed 46% and 36% identity, respectively. By contrast, the identity between the L. interrogans enzyme and the Streptococcus mutans or E. coli enzymes was less than 31%. Highly conserved sequences within aspartate semialdehyde dehydrogenase from the five organisms were observed at the amino and carboxyl termini, and around the cysteine of the active site.

Summary: The nucleotide (nt) sequence of a DNA segment containing the majority of a gene cloned from Bacillus thuringiensis DSIR517 encoding a 130 kDa insecticidal crystal protein has been determined. Sequence analysis reveals an open reading frame (ORF) of 3453 nt. The ATG initiation codon, which is preceded by a potential ribosome-binding site sequence, was confirmed by N-terminal amino acid sequencing. The ORF extends beyond the 3” terminus of the cloned fragment; however, the high degree of homology between the deduced amino acid sequence of this ORF and other Cry proteins suggests the clone lacks only five C-terminal amino acids. Making this assumption, the ORF of 3468 nt encodes a protein of 1156 amino acids with an estimated molecular mass of 129700 Da. Analysis of the deduced amino acid sequence reveals a number of features characteristic of Cry proteins. Alignment of the Cry 517 protein sequence with other Cry proteins suggests it is most closely related to the crylA-E genes but sufficiently different to form a new cryI gene subclass.

Summary: An insertion sequence element of Mycobacterium avium subsp. silvaticum was isolated and its complete nucleotide squence determined. IS902 is 1470 bp in size and is repeated 10-12 times per genome. An open reading frame of 1200 bp was identified, encoding a protein product of M r 43932. This protein is highly similar to the predicted proteins of IS900 of Mycobacterium paratuberculosis, IS116 of Streptomyces clavuligerus and IS110 of Streptomyces coelicolor. IS902 lacks terminal inverted repeats and flanking direct repeats but displays insertion site specificity.

Summary: Methicillin-resistant (Mcr) staphylococci contain chromosomal DNA that is absent from Mcs cells. This extra DNA harbours the methicillin resistance determinant mec and often other resistance determinants. The mec region can differ substantially in structure among different isolates. We present studies on the mec region of a group of Staphylococcus aureus isolates prevalent in Australia and London. Southern hybridization analyses of a prototype Australian isolate, ANS46, and an isogenic Mcs deletion mutant, ANS62, allowed the physical map of the region to be extended to 55 kb. The DNA corresponding to the deletion, which includes mec and resistance determinants for mercury, cadmium (Cd) and tetracycline, amounted to 41 kb. It was bounded precisely at one end by the macrolides-lincosamides-streptogramin B (MLS)-resistance transposon, Tn554. Near the other end was an element with homology to Tn554, ΨTn554, which carried the Cdr determinant. The mec region of an American Mcr isolate, R35, was found to be virtually the same as that of ANS46, except that it lacked Tn554. Another class of American Mcr isolates, prevalent since 1987, differs markedly from ANS46 in mec region organization. However, this other American class also contains an insertion of Tn554 in the mec region, and the attachment site for this insertion was found to have significant homology to attachment sites for the Tn554 and ΨTn554 insertions in the mec region of the Australian strain. These results suggest possible roles of Tn554 and Tn554-like elements in the evolutionary variation of the mec region.

Summary: Many species of pseudomonads produce fluorescent siderophores involved in iron uptake. We have investigated the DNA homology between the siderophore synthesis genes of an opportunist animal pathogen, Pseudomonas aeruginosa, and three plant-associated species Pseudomonas syringae, Pseudomonas putida and Pseudomonas sp. B10. There is extensive homology between the DNA from the different species, consistent with the suggestion that the different siderophore synthesis genes have evolved from the same ancestral set of genes. The existence of DNA homology allowed us to clone some of the siderophore synthesis genes from P. aeruginosa, and genetic mapping indicates that the cloned DNA lies in a locus previously identified as being involved in siderophore production.

Summary: The recent discovery of the phenomenon that some prokaryotes fragment their 23S rRNA during post-transcriptional processing of precursor rRNA has been shown to be particularly prevalent among strains and species of Salmonella. Some strains of Salmonella cleaved 23S rRNA at multiple sites producing several fragments. The cleavage patterns of 23S rRNA differed among Salmonella strains and sometimes among the rRNA operons in the same strain. Fragmentation of 23S rRNA was not observed in strains of the closely related species Escherichia coli. Fragmentation of 23S rRNA occurred in Brucella and Agrobacterium but the cleavage pattern was not as diverse as that demonstrated in Salmonella. Introduction of cleavage sites into precusor 23S rRNA of Salmonella is probably a recent evolutionary event.

Summary: pSLH8, an insertional-inactivation cloning vector for Escherichia coli has been constructed by inserting a pigment gene (probably encoding an indole dioxygenase) from Rhodococcus sp. ATCC 21145 into pUC18. Wild-type E. coli colonies containing pSLH8 produce insoluble indigo and turn dark blue on unsupplemented LB agar. Insertion of DNA fragments into the unique BamHI, EcoRI, EcoRV, HindIII, PstI, SphI and SstI polylinker cloning sites disrupts the reading frame of the fully sequenced pigment gene and results in unpigmented colonies. pSLH8 may be an attractive alternative to pUC18 and similar plasmids because it does not require specifically mutated host strains or an expensive substrate for colour development.

Summary: Saccharomycopsis fibuligera can utilize starch but not tributyrin as the sole carbon source, whereas Yarrowia lipolytica can utilize tributyrin but not starch. S. fibuligera mutant strain 193 met was crossed to Y. lipolytica A his1 to produce hybrids. The intergeneric hybrids were able to utilize both starch and tributyrin as sole carbon source. They were mitotically unstable and gave segregants during prolonged vegetative culture. The majority of these segregants had the phenotype of the S. fibuligera parent, with a very low number of genetic recombinants. A more stable hybrid was produced by protoplast fusion between a mutant, ade met, obtained from UV treatment of a Met− mitotic segregant of the hybrid 14i and Y. lipolytica B lys5 leu2 ade1 xpr2. Sixteen of the 40 mitotic segregants of this hybrid were recombinants. Furthermore when a haploid mitotic segregant of S. fibuligera 193 met, met glu, was crossed to Y. lipolytica B leu2 ade1 xpr2 more stable hybrids were also obtained. Eleven of the 35 mitotic segregants of the hybrid SA11 were recombinants. Such high frequencies of recombinant sectors from these hybrids should provide a system to establish genetic mapping analysis for the novel Saccharomycopsis-Yarrowia recombinant strains.

The P2 outer membrane protein of Haemophilus influenzae belongs to a class of apparently ubiquitous proteins in Gram-negative bacteria that function as porins. Murine hybridomas raised to the P2 protein and synthetic peptides were used to investigate the structural and antigenic relationships among P2 proteins of encapsulated and non-encapsulated H. influenzae. Three monoclonal antibodies (mAbs), P2-17, P2-18 and P2-19, recognizing epitopes on the P2 protein, as shown by Western immunoblotting of outer membrane preparations, and purified and recombinant P2 proteins are described. The epitopes reactive with the mAbs were widely distributed among H. influenzae strains since 70-100% of strains of encapsulated and non-encapsulated isolates collected worldwide were recognized by individual mAbs. None of the mAbs reacted with H. parainfluenzae or other bacterial species. The peptide composition of P2 epitopes was determined by analysis of mAb reactivity with a series of overlapping synthetic peptides that covered the amino acid sequences of H. influenzae type b. The domains recognized by these mAbs were completely distinct. mAb P2-18, reactive with an epitope conserved among all H. influenzae P2 porin molecules which were screened, recognized a peptide corresponding to the N-terminal segment (residues 1-14). The P2-17- and P2-19-specific epitopes were located between residues 28 and 55, and 101 and 129, respectively. None of the epitopes were exposed on the cell surface since no mAbs bound to intact live bacteria. The identification of epitopes that are shared among P2 proteins from different strains of encapsulated and non-encapsulated H. influenzae strains suggests that the P2 porin protein from all H. influenzae strains has been highly conserved and that these epitopes may represent amino acid residues critical to the porin's structure.