Parental Mutant Strains Research Articles

Growth and sporulation defects in Bacillus subtilis mutants with a single rrn operon can be suppressed by amplification of the rrn operon.

The genome of Bacillus subtilis strain 168 encodes ten rRNA (rrn) operons. We previously reported that strains with only a single rrn operon had a decreased growth and sporulation frequency. We report here the isolation and characterization of suppressor mutants from seven strains that each have a single rrn operon (rrnO, A, J, I, E, D or B). The suppressor mutants for strain RIK656 with a single rrnO operon had a higher frequency of larger colonies. These suppressor mutants had not only increased growth rates, but also increased sporulation frequencies and ribosome levels compared to the parental mutant strain RIK656. Quantitative PCR analyses showed that all these suppressor mutants had an increased number of copies of the rrnO operon. Suppressor mutants were also isolated from the six other strains with single rrn operons (rrnA, J, I, E, D or B). Next generation and capillary sequencing showed that all of the suppressor mutants had tandem repeats of the chromosomal locus containing the remaining rrn operon (amplicon). These amplicons varied in size from approximately 9 to 179 kb. The amplifications were likely to be initiated by illegitimate recombination between non- or micro-homologous sequences, followed by unequal crossing-over during DNA replication. These results are consistent with our previous report that rrn operon copy number has a major role in cellular processes such as cell growth and sporulation.

Role of the GlgX protein in glycogen metabolism of the cyanobacterium, Synechococcus elongatus PCC 7942

The putative glgX gene encoding isoamylase-type debranching enzyme was isolated from the cyanobacterium, Synechococcus elongatus PCC 7942. The deduced amino acid sequence indicated that the residues essential to the catalytic activity and substrate binding in bacterial and plant isoamylases and GlgX proteins were all conserved in the GlgX protein of S. elongatus PCC 7942. The role of GlgX in the cyanobacterium was examined by insertional inactivation of the gene. Disruption of the glgX gene resulted in the enhanced fluctuation of glycogen content in the cells during light–dark cycles of the culture, although the effect was marginal. The glycogen of the glgX mutant was enriched with very short chains with degree of polymerization 2 to 4. When the mutant was transformed with putative glgX genes of Synechocystis sp. PCC 6803, the short chains were decreased as compared to the parental mutant strain. The result indicated that GlgX protein contributes to form the branching pattern of polysaccharide in S. elongatus PCC 7942.

Genetic Nature, Stability, and Improved Virulence of Hybrids from Protoplast Fusion in Beauveria

Genetic improvement of two different strains of the entomopathogenic fungus Beauveria bassiana for more effective control of Ostrinia nubilalis and Leptinotarsa decemlineata was obtained by crosses with the insecticidal toxin-producing strain Beauveria sulfurescens. Protoplast fusion between diauxotrophic mutants resulted in the recovery of some stable prototrophic fusion products. The low levels of virulence of the wild type strain B. bassiana 28 isolated originally from L. decemlineata were enhanced both on L. decemlineata and O. nubilalis for one of the hybrids obtained (FP 8) from the cross B. bassiana 28xB. sulfurescens 2. Fusion product 25 obtained from the cross between B. sulfurescens and the highly pathogenic strain B. bassiana 147 showed a three-day reduction in the LT50 towards O. nubilalis. Southern blot hybridization with nine probe-enzyme combinations were conducted on genomic DNAs from the original wild strains, parental mutant strains, and fusion products. Additive banding patterns or unique banding pattern of either parental strain was observed in five hybrids, indicating their status as recombinant and/or partially diploid. Combination of RFLP markers indicative of both parental genomes was never observed with fusion product FP 25. The stability of the virulence following passage through insect-host and stability of molecular structure for the fusion products FP 8 and FP 25 suggest that asexual genetic recombination by protoplast fusion may provide an attractive method for the genetic improvement of biocontrol efficiency in entomopathogenic fungi.

PROTOPLAST FUSION AND GENETIC COMPLEMENTATION OF PIGMENT MUTATIONS IN THE RED MICROALGA PORPHYRZDZUM SP.1

ABSTRACT Protoplasts from two green pigment mutants of Porphyridium sp. (UTEX 637) containing a low phycoerythrin level were fused by exposure to polyethylene glycol (MW 6000) combined with a short heat shock (45° C, 5 min). Following regeneration on agar plates, red colonies arose in which complementation of the phycoerythrin deficiency had occurred. The complementation frequency was estimated to be 0.2%. Eight progeny showing red pigmentation were isolated and purified by consecutive transfers on agar plates. Characterization of the fusion progeny revealed that their phycobiliprotein and chlorophyll contents per cell were higher than those of their parental mutant strains and, in most strains, similar to that of the wild type. The fusion products proved to be stable over many growth cycles. The DNA content of the wild type and of the parental mutant strains was about 0.05 pg‐cell−1. Fusion progeny strains showed a variable DNA content: a few fusants contained the same amount of DNA as the wild type and the parental strains, while others had about 50% more DNA per cell. The DNA content of one of the progeny strains (CF1c) was double that of the wild type (0.1 pg. cell−1). Cells of this fusion progeny contained one nucleus per cell, which suggests that nuclear fusion and the formation of a stable diploid followed cell fusion. Analysis of phycobilisome components of CF1c revealed complementation of linker polypeptides associated with phycoerythrin (γ subunits). CF1c contained, like the wild‐type strain, four linker polypeptides; all of these were absent in one parental strain and one was absent in the second. To the best of our knowledge, this is the first report of protoplast fusion, formation of somatic hybrids, and the apparent completion of a parasexual cycle in a red microalga.

Susceptibility of Cryptococcus neoformans to amphotericin B.

Four mutants strains of Aspergillus fennelliae resistant to various polyene antibiotics have been analyzed for their sterol content. The mutant strains contained Delta(7,22,24(28))-ergostatrien-3beta-o1 and Delta(7,22)-ergostadien-3beta-o1 as the major sterols, whereas the wild types contained ergosterol, indicating that the mutants contain metabolic blocks for C(5)-C(6) dehydrogenation and C(24)-C(28) reduction in the biosynthesis of ergosterol. Revertant sectors arising from the mutant colonies contained either more Delta(7,22)-ergostadien-3beta-o1 than the parent mutant strain or showed the reappearance of ergosterol. The revertant sectors also showed lower resistance to amphotericin B and a higher rate of sporulation than the parent mutant strain. These results confirm our previous observations that the presence of ergosterol is directly related to the drug susceptibility and the normal rate of sporulation in this species.