- Volume 139, Issue 10, 1993

SUMMARY: Previous studies have shown that gas vesicles isolated from the cyanobacterium Anabaena flos-aquae contain two types of protein, GvpA, a small hydrophobic protein that forms the main ribbed structure, and GvpC, a protein comprising five repeats of a 33-amino-acid-residue motif, which is located on the outer surface of the GvpA shell. GvpC was shown to increase the critical collapse pressure of the gas vesicles; it was thought to do this by forming a series of molecular ties that bind the ribs together. We now show that antibodies raised against GvpC label both the central cylinders and the conical end caps of native gas vesicles but fail to bind to gas vesicles that have been stripped of GvpC. The molar ratio of GvpA to GvpC has been calculated from amino acid analyses of gas vesicle hydrolysates by reference to the abundance of amino acids that occur predominantly or exclusively in one protein or the other; the molar ratio was found to be 25:1 in freshly isolated gas vesicles and 23:1 in gas vesicles saturated with GvpC. We have considered three ways in which the 33-residue repeats of GvpC might interact with the crystallographic unit cell of GvpA molecules in the ribs. The Anabaena GvpC will bind to and restore the strength of gas vesicles isolated from Aphanizomenon and Microcystis that lack their native GvpC.

SUMMARY: Transposon mutagenesis and antibiotic enrichment were employed to isolate a mutant of Rhodobacter sphaeroides Si4 designated strain M22, that had lost the ability to grow on D-mannitol and to produce the enzyme mannitol dehydrogenase (MDH). DNA flanking the transposon in the mutant strain was used as a probe for the identification and cloning of the MDH gene (mtlK). A 5.5 kb EcoRI/Bg/II fragment from R. sphaeroides Si4 was isolated and shown to complement the mutation in R. sphaeroides M22. Successful complementation required that a promoter of the vector-plasmid pRK415 be present, suggesting that the mtlK gene is part of a larger operon. Using oligonucleotides derived from the N-terminal sequence of MDH as probes mtlK was located on the complementing fragment and the gene was sequenced. The mtlK open reading frame encodes a protein of 51 404 Da with an N-terminal sequence identical to that obtained from amino acid analysis of the purified MDH. The MDH of R. sphaeroides Si4 exhibits distant similarity to the mannitol-1-phosphate dehydrogenases from Escherichia coli and Enterococcus faecalis, with 28.1% and 26.3% identity, respectively. Mutant strains deficient in MtlK displayed substantial levels of sorbitol dehydrogenase activity, originally thought to be only a minor activity associated with the MDH enzyme. It is likely that we have uncovered an additional polyol dehydrogenase with activity for sorbitol. The mtlK gene can be used for overexpression of MDH in E. coli in order to obtain sufficient amounts of enzyme for further investigations and applications.

SUMMARY: Two new segregants, PPW1-1 and PPW161-1, of Pseudomonas putida were isolated from the stock cultures PaW85(pWW0) and PaW85(pWW0-161). Strain PPW1-1 had lost its ability to grow on m-xylene but was able to grow on m-toluate. A deletion of the left-hand of transposon Tn4651, including the upper-operon genes, had taken place in plasmid pWWOmut11, isolated from strain PPW1-1. Additional deletions were observed in pWWOmut1 after ‘benzoate-curing’ (plasmids pWW0mut15, pWWOmut19, pWW0mut27). The genes of the upper-operon and beginning of the meta-operon were deleted from pWW0-161mut1, isolated from strain PPW161-1. Despite this deletion, cells of PPW161-1 grew on all normal TOL plasmid substrates. The Tol+ phenotype was stable in cells of PPW161-1 growing on benzoate. We propose that this is because in cells of strain PPW161-1 the catabolic genes deleted from pWW0-161mut1 were integrated into the chromosome at the site where the (chromosomally encoded) ortho-pathway genes are located, resulting in the inability of the cells to use this pathway.

SUMMARY: We report here the formation of plasmid linear multimers promoted by the Red-system of phage lambda using a multicopy plasmid comprised of lambda reda, and red genes, under the control of the lambda cl 857 repressor. Our observations have revealed that the multimerization of plasmid DNA is dependent on the red and recA genes, suggesting a concerted role for these functions in the formation of plasmid multimers. The formation of multimers occurred in a recBCD + sbcB + xthA + lon genetic background at a higher frequency than in the isogenic lon + host cells. The multimers comprised tandem repeats of monomer plasmid DNA. Treatment of purified plasmid DNA with exonuclease III revealed the presence of free double-chain ends in the molecules. Determination of the size of multimeric DNA, by pulse field gel electrophoresis, revealed that the bulk of the DNA was in the range 50240 kb, representing approximately 524 unit lengths of monomeric plasmid DNA. We provide a conceptual framework for Red-system-promoted formation and enhanced accumulation of plasmid linear multimers in lon mutants of E. coli.

SUMMARY: Two starch-degrading enzymes produced by Bacillus acidocaldavius (renamed as Alicyclobacillus acidocaldarius) were identified. According to SDS-PAGE, the apparent molecular masses of the enzymes were 90 and 160 kDa. Eight peptide fragments and the N-terminal end of the 90 kDa polypeptide were sequenced. An oligonucleotide, based on the amino acid sequence of a peptide fragment of the 90 kDa protein, was used to screen a lDgt10 bank of B. acidocaldarius, and the region encoding the 90 kDa protein was cloned. Unexpectedly, the ORF continued upstream of the N terminus of the 90 kDa protein. The entire ORF was 1301 amino acids (aa) long (calculated molecular mass 140 kDa) and it was preceded by a putative ribosomal binding site and a promoter. Computer analysis showed that the 1301 aa protein was closely related to an a-amylase-pullulanase of Clostridium thermohydrosulfuricum. We suggest that the starch-degrading 160 kDa protein of B. acidocaldarius is an a-amylase-pullulanase, and the 90 kDa protein is a cleavage product of the 160 kDa protein. Another ORF, apparently in the same transcription unit, was found downstream from the amylase gene. It encoded a protein that was closely related to the maltose-binding protein of Escherichia coli.

SUMMARY: Salmonella paratyphi B and Salmonella java are biovars of common serotype 1,4,[5],12:b:1,2 which respectively cause human paratyphoid fever and gastroenteritis. In order to define genotypes and phylogenetic relationships in this group, we examined representative strains for restriction fragment length polymorphisms (RFLPs) in and around the 16S ribosomal RNA (rrn) genes, and the five to eleven insertion sites of the Salmonella-specific DNA insertion sequence IS200. One of four 16S rrn profiles was predominant, and was shared by the majority of strains, irrespective of their designation as S. paratyphi B or S. Java. On the other hand, thirteen unique IS200 profiles were found and this technique was able to distinguish, for the first time, distinct genotypes for S. paratyphi B and S.java. One of the S. paratyphi B profiles, Spj-IP1.0, represented a globally-distributed clone. Greater diversity was detected within IS200 profiles of S. java than within those of S. paratyphi B. IS200 profiles described a phylogenetic complex in which strains of both biovars could be placed. They constituted reproducible molecular fingerprints, which could be compared in a band-matching database suitable for molecular epidemiological typing.

SUMMARY: The possible functions of two recently described flagellar genes, fliU and fliV, have been examined. Introduction of gene fliC, encoding the bacterial flagellin protein, into a number of flagellin-deficient Salmonella and Escherichia coli strains failed to complement the mutations in these strains, and the FliC flagellin was accumulated in the bacterial cytoplasm. Complementation with fliU and fliV, which map downstream of fliC, restored motility to some of the mutants which became flagellated. After inactivation of either fliU or fliV, such complementation no longer occurred and the flagellin protein accumulated in the cytoplasm, which suggested that both genes are required for the secretion of flagellin and expression of motility. Expression of these genes from high copy number plasmids resulted in the synthesis of exceptionally long flagella and in detection of the FliV protein on polyacrylamide gels.

SUMMARY: Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique for nucleic acids (NA), was investigated for the species-specific identification of mycobacteria. A set of primers was selected from a highly conserved region of the 16S rRNA sequence of mycobacteria sandwiching a variable sequence to perform amplification of mycobacterial RNA. Species-specific probes for the M. tuberculosis complex, M. avium-paratuberculosis, M. intracellulare and M. leprae were hybridized in-solution with the amplified nucleic acids of 10 pathogenic mycobacteria and 11 closely related bacteria, as well as with human-derived NA in an enzyme-linked gel assay (ELGA). Each probe was shown to hybridize specifically to the amplified single-stranded RNA of the corresponding species. Thirty-two clinical isolates of M. tuberculosis strains from different parts of the world were correctly identified by NASBA using the M. tuberculosi-complex-specificprobe. In combination with the ELGA, NASBA could identify mycobacteria rapidly, i.e. in less than 6 h. The relative simplicity and rapidity of this technique makes it an attractive tool for species-specific identification of mycobacteria.

SUMMARY: PCR primers corresponding to the adhesin genes of Mycoplasma pneumoniae and Mycoplasma genitalium were shown to detect the corresponding organisms specifically. Absence of cross-reaction with seven other mollicute species and six unrelated bacterial species commonly found in humans was demonstrated. Positive control primers directed against human mitochondrial DNA could be mixed with the Mycoplasma primers without loss of specificity or sensitivity. A detection level of 10 c.f.u. of either Mycoplasma species could be readily obtained, even in the presence of 104 human cells. The triplex PCR method developed is very simple and does not require hybridization or the use of radioisotopes and allows detection and differentiation of these mycoplasmas against the background of human DNA found in clinical specimens.

SUMMARY: The two protein exotoxins secreted by Bacillus anthvacis are composed of three distinct components: protective antigen (PA), lethal factor (LF), and (o)edema factor (EF). We have developed a genetic strategy that permits us selectively to inactivate each of the genes coding for PA, EF or LF. This strategy involved the deletion of a portion of the structural gene and the insertion of an antibiotic resistance cassette. With this technique, double mutant strains of B. anthracis producing only one toxin component have been constructed. Characterization of the mutant strains indicated that they produced the expected single toxin protein. Using a simple, two-step protocol, we have purified PA, LF and EF to homogeneity from culture supernatants. These three mutant strains are potentially powerful tools for studying the individual effect of each toxin component in vitro and in vivo.

SUMMARY: Two virulent, archaeal phages, øF1 and øF3, were isolated that were capable of infecting different thermophilic members of the genus Methanobacterium. Both phages exhibited a similar morphology consisting of a polyhedral head and a tail but differed considerably in their host specificities and the size and topology of their genomes. Phage øF1 contained a linear, double-stranded DNA genome of 85 + 5 kb in size and showed a broad host range including M. thermoformicicum strains Z-245, FTF, FF1, FF3 and CSM3, and M. thermoautotrophicum strain ΔH. In contrast, øF3 phage particles contained a circular or terminally redundant linear genome, comprising approximately 36 + 2 kb double-stranded DNA, and could only be propagated on M. thermoformicicum strain FF3. Hybridization experiments did not reveal similarity between the genomes of øF1 and øF3 nor between both phages and genomic DNA from different thermophilic members of the genus Methanobacterium or DNA from phage øM1 of M. thermoautotrophicum Marburg. A physical map of both phage genomes was constructed. The DNA of phage øF1 was found to contain multiple GGCC sites which form the target for the restriction-modification (R/M) system MthTI of M. thermoformicicum THF. In contrast, the DNA of øF1 contained only a single CTAG site recognized by the R/M systems MthZI and MthFI of M. thermoformicicum Z-245 and FTF, respectively. The distribution of these sites correlates well with the capacity of øF1 to infect M. thermoformicicum strains Z-245 and FTF but not strain THF.

SUMMARY: The oxytetracycline-producing Streptomyces rimosus strains R6-65 and R7 (ATCC 10970) are lysogenic for the two narrow-host-range phages RP2 and RP3. Both phages are released at low frequency from the lysogenic strains and form plaques on ‘cured’ S. rimosus strains. RP2 and RP3 are of similar shape with flexible tails and contain double-stranded DNA of about 70% G + C with cohesive ends (group B1 of bacteriophage classification). The two phages also have identical, very slow, growth kinetics in S. rimosus, with a latent phase of about 6 h and a rise period of about 4 h. RP2 and RP3 are heteroimmune and they differ slightly in their size of phage particles and length of DNA (64.7 and 62.4 kb for RP2 and RP3, respectively). The restriction maps of the two phages are completely different, and hybridization experiments showed only one short region of sequence similarity (less than 430 bp); the two phages are thus essentially unrelated. Both phages lysogenize their hosts by recombination via defined attachment (att) sites. The positions of the attP sites have been localized on the restriction maps of RP2 and RP3 to restriction fragments of 800 and 300 bp, respectively. The prophages did not affect the level of oxytetracycline production or the genetic instability of this trait.

SUMMARY: Conditions for the preparation of protoplasts from Streptomyces aureofaciens and transformation with several replicative and integrative plasmids were optimized. Of all the replicative plasmids used, carrying various Streptomyces replication origins, only plasmid pGM9 produced transformants, with a transformation efficiency of 2 × 103 (μg DNA)-1. When plasmid DNA isolated from S. aureofaciens was used, the transformation efficiency increased to 105 transformants (μg DNA)-1. Transformation of protoplasts with integrative plasmids containing part of the hrdA gene gave rise to transformants with an efficiency of approximately 30 transformants (μg DNA)-1. Integration frequency was strongly reduced when plasmid DNA was modified by dam or dcm methylase. By integrative transformation, via a double crossover, a stable null mutant of the hrdA gene was prepared. This mutation appeared to have no obvious effect on growth, morphology, differentiation and production of chlortetracycline.

SUMMARY: Most strains of Helicobacter pylori are naturally competent for uptake of chromosomal DNA. Transformation frequencies for streptomycin resistance or rifampicin resistance markers ranged from 1 10-4 to 1 10-3 per viable cell using a plate transformation procedure. Transformation of a metronidazole resistance marker (MtrR) was demonstrated when either a laboratory-derived mutant or a MtrR clinical isolate were used as the source of donor DNA. MtrR was transformed at a frequency of 3 10-5 per viable cell. All H. pylori strains tested produce large amounts of DNAase, which may reduce DNA available for transformation. Four H. pylori plasmids were isolated. DNA fragments from H. pylori plasmids were deleted or rearranged when cloned in pUC19 and propagated in Escherichia coli DH5a. An H. pylori plasmid, pUOA26 which contained a chloramphenicol resistance determinant from Campylobacter coli, was constructed in H. pylori. This plasmid could be successfully introduced by natural transformation only into H. pylori recipients which contained a homologous resident plasmid. Transformation of pUOA26 into plasmid-free cells of H. pylori was achieved by electroporation. Transformation frequencies were 1 10-4 transformants per viable cell when plasmid DNA was isolated from the same strain; however, introduction of pUOA26 DNA derived from H. pylori 8091 into a different H. pylori strain, NCTC 11639, resulted in transformation at much lower frequencies (? 1 10-7 per viable cell). Changes in plasmid restriction patterns were also noted when pUOA26 was isolated from H. pylori NCTC 11639, suggesting the presence of at least two different DNA restriction and modification systems in H. pylori which may interfere with uptake of plasmid DNA.