Isolation of Bacillus megaterium mutants that produce high levels of heterologous protein, and their use to construct a highly mosquitocidal strain.

A xylose-regulated plasmid expression system for producing high levels of recombinant proteins in Bacillus megaterium has recently been described [Appl Microbiol Biotechnol 35:594, 1991]. Using an antibiotic resistance protein as the expressed protein, we have been able to select mutant plasmids that produce increased levels of heterologous protein. The mutant plasmids show increased segregational stability and have lost the ability to be transformed into Escherichia coli. The same selection protocol has been used to isolate a mutant strain producing high levels of the Bacillus sphaericus mosquitocidal binary toxin. This strain shows toxicity to Culex quinquefasciatus larvae that is comparable toB. sphaericus 2362 and higher than a B. megaterium strain with the original expression plasmid. This approach may be generally useful for high-level regulated protein expression in B. megaterium.

Cloning and expression of the Bacillus sphaericus 2362 mosquitocidal genes in a non-toxic unicellular cyanobacterium, Synechococcus PCC6301.

Genes encoding the mosquitocidal binary toxin of Bacillus sphaericus 2362 were introduced into Synechococcus PCC6301, a cyanobacterium that can tolerate a number of potential variations in the mosquito breeding environment, and can serve as a food source for mosquito larvae. The toxin genes, preceded by a Synechococcus rbcL promoter, were located on a mobilizable Escherichia coli Synechococcus shuttle vector, which was introduced into Synechococcus PCC6301 at frequencies of 10(-5)-10(-7) exconjugants/recipient, depending on the selective conditions used. Recombinant Synechococcus exhibited significant toxicity against 2-day-old and 6-day-old Culex quinquefasciatus larvae, the concentration required to kill 50% of larvae (LC50) being 2.1 x 10(5) and 1.3 x 10(5) cells/ml respectively. Mosquitocidal activity decreased tenfold after 20 generations of non-selective growth.

Changes in biologic phenotype of human immunodeficiency virus during treatment of patients with didanosine.

Progression to AIDS in patients harboring human immunodeficiency virus type-1 (HIV-1) isolates expressing a syncytium-inducing (SI) phenotype is faster than in those in whom the virus expresses a non-SI (NSI) phenotype. Zidovudine monotherapy does not appear to alter this outcome. To examine the role of didanosine (ddI) monotherapy in phenotype expression, HIV-1 isolates from 73 patients receiving ddI for up to 72 weeks were analyzed. After 12 weeks, the number of isolates expressing an NSI phenotype was 29% higher than at the start of treatment. Patients receiving high-dose ddI (375 mg twice daily) were significantly more likely to express the NSI phenotype at 12 weeks than patients who received low-dose ddI (100 mg twice daily), even after adjustment for phenotype and CD4 cell count at baseline, suggesting that ddI may be selective against the faster-replicating virus. ddI at 375 mg twice daily significantly increases the probability of an NSI phenotype over the short term in patients with advanced HIV disease.

Primary structure of the V3 region of gp120 from sequential human immunodeficiency virus type 1 isolates obtained from patients from the time of seroconversion.

V3 loop sequences were compared from 5 human immunodeficiency virus type 1 (HIV-1)-infected patients over time. Three patients remained asymptomatic and 2 became symptomatic with large decrease in CD4 cell counts. The patient isolates were previously evaluated for phenotypic and antigenic properties and had different sensitivities to serum neutralization and changes in phenotype. This study showed a number of amino acid changes for the 2 symptomatic patients, each of whom progressed to AIDS during the study. The only amino acid substitution consistently associated with reduced CD4 cell counts, cytopathic effect, and progression to AIDS was Arg at position 11. Specific amino acid changes could not be correlated with increasing serum neutralization resistance or cytotropism changes. Increased loop charge was associated with a switch from macrophage to T cell tropism and a decrease in the number of CD4 cells. The study shows the importance of naturally occurring mutations in the V3 loop in controlling the biologic properties of HIV-1.

Organization of the fimbrial gene region of Bacteroides nodosus: class I and class II strains.

The fimbrial subunit genes of Bacteroides nodosus may be divided into two distinct classes, based on the sequence of the major subunit gene fimA (accompanying paper--Mattick et al., 1991). The genetic organization of the fibrial gene region in these two classes is also distinct. Upstream of fimA in both classes in opposite transcriptional orientation is the gene aroA which encodes amino acid biosynthetic enzyme 5-enolpyruvylshikimate-3-phosphate synthase. However, downstream of fimA the two classes are quite different until homology is restored at a bidirectional transcription termination signal separating the fimbrial operon from a gene clpB, which appears to encode the regulatory subunit of an ATP-dependent protease. Between aroA and clpB class I strains contain, apart from fimA, only one other gene (fimB). Sequence and polymerase chain reaction analyses indicate that fimB does not have a separate promoter but rather is co-transcribed with fimA at a level attenuated by the strength of the transcription termination signal in the intergenic region. In class II strains fimA is followed by a more extended region containing three genes, which appear to have the same transcriptional arrangement as fimB. The second of these genes (fimD) may represent a functional analogue of fimB although there is no close sequence homology. The first gene (fimC) has no obvious similarity to either fimB or fimD. Beyond fimD, at the 3' end of the class II-specific region, is a variant fimbrial subunit gene (fimZ) which is virtually identical in serogroups D and H and which appears to represent a duplicate, possibly redundant, gene closely related to the progenitor of the more divergent structural subunit fimA gene found in these strains. Comparisons of the predicted fimZ product with those of fimA in class I and class II strains, as well as of the boundaries of the class-specific regions, suggest that the class II sequences evolved in another type 4 fimbriate species and were subsequently substituted in the B. nodosus genome by lateral transfer. Analysis of the sequences flanking fimA in different strains indicates that recombinational exchange of both fimA and the entire operon has also occurred between strains, and is possibly a mechanism for disseminating structural diversity in the population.

Transcription of the fimbrial subunit gene and an associated transfer RNA gene of Pseudomonas aeruginosa.

Gene fimA encoding the structural subunit of the fimbriae of Pseudomonas aeruginosa PAK is located in the centre of a 1.2-kb HindIII genomic DNA fragment [see also Sastry et al., J. Bacteriol. 164 (1985) 571-577], which in turn is located within a 6.2-kb EcoRI fragment. Immediately downstream from fimA is a putative threonine tRNA gene [Dalrymple and Mattick, Biochem. Int. 13 (1986) 547-553]. Northern blotting experiments showed that fimA is transcribed to an mRNA of approx. 650 nucleotides, which also includes the threonine tRNA sequence but no other protein-coding region. There was no indication that this mRNA is processed to release the tRNA sequence. However, the tRNA did appear to be expressed independently from its own promoter in the region 3' to fimA. When these sequences were introduced into Pseudomonas putida, we found that the level of expression of fimA from the cloned 6.2-kb EcoRI fragment was approx. 30-fold greater than that from the smaller HindIII fragment, whereas that of the specific tRNA species was unaltered. The size of the fimA transcript was also unaltered. These results provide evidence that the fimA gene is subject to specific transcriptional activation in vivo and that this activation involves sequences flanking the 1.2-kb HindIII fragment.

The nucleotide sequence for the large subunit of ribulose 1,5-bisphosphate carboxylase from a unicellular cyanobacterium, Synechococcus PCC6301.

The gene for the large subunit (LSU) of ribulose 1,5-bisphosphate carboxylase from a unicellular cyanobacterium, Synechococcus PCC6301, was cloned using the spinach LSU gene as a hybridization probe. The coding region of the Synechococcus LSU gene consists of 1419 nucleotides and shows 70% homology to the spinach nucleotide sequence. The derived amino acid sequence (472 amino acids) shows 81% homology to the spinach LSU and 78% to the maize LSU. Regions containing active-site residues are highly conserved among spinach, maize, and Synechococcus. In contrast, the first 13 amino acids are poorly conserved (30% homology), supporting the hypothesis that this region is proteolytically removed. The 5'-flanking region of the Synechococcus LSU gene contains sequences which correspond to bacterial consensus sequences for the -35 region and Pribnow box. Two 11-bp sequences in the 5' region show high homology to sequences in spinach and maize. One of these encompasses a possible ribosome-binding site. The 3'-flanking region contains a 35-bp sequence capable of giving rise to a terminator structure.

Temporal genetic mapping in the blue-green alga Anacystis nidulans using ethyl methanesulphonate.

Cultures of the blue-green alga Anacytis nidulans were synchronized with respect to DNA synthesis as well as cell division. Application of ethyl methanesulphonate at different stages of replication resulted in a peak of mutation frequency for different genetic markers; this peak can be accounted for in terms of the involvement of repair processes. A temporal map of 19 markers has been constructed by this method. Comparison of gene position obtained by temporal mapping indicates that either bidirectional replication or unidirectional replication from more than one origin occurs.