FINE-SCALE GENETIC AND PHENOTYPIC STRUCTURE IN NATURAL POPULATIONS OF BACILLUS SUBTILIS AND BACILLUS LICHENIFORMIS: IMPLICATIONS FOR BACTERIAL EVOLUTION AND SPECIATION

The genetic and phenotypic structure of sympatric populations of wild bacteria traditionally identified as Bacillus subtilis and B. licheniformis was analyzed. Small soil samples were taken from a single, tiny site in the Sonoran Desert of Arizona, USA, to provide a true population analysis, in contrast to many analyses of genetic structure using bacterial strain collections of widely heterogeneous origin. Genetic analyses of isolates used multilocus enzyme electrophoresis, mismatches in restriction fragment length polymorphism, and variants from Southern hybridization with B. subtilis DNA probes. Phenotypic analyses of isolates used the API test system for detection of growth and acid production on specific carbon sources. The two species were distinct both phenotypically and genetically, despite their known potential for genetic exchange in laboratory experiments. Genic and genotypic diversity were high in both species, and only 16% of observed allozyme variants might possibly be common to both species. Hence, there is probably modest genetic exchange, if any, between the species in nature. Clear hierarchies of population-genetic structure were found for both species. Different types of genetic data yield concordant population structures for B. subtilis. For both species, two-locus and multilocus statistical analyses of linkage demonstrated modest to strong disequilibrium at the species level but truly panmictic subunits within each species. The evidence for extensive genetic recombination within these fine-scale subdivisions is unequivocal, indicating that the sexuality of these bacteria can be well expressed in nature. The relation of these results to processes of bacterial evolution and speciation is discussed.     

RECOMBINATION AND MIGRATION RATES IN NATURAL POPULATIONS OF BACILLUS SUBTILIS AND BACILLUS MOJAVENSIS

We have investigated the rates of recombination and migration in native populations of two closely related, naturally competent Bacillus species. Native soil isolates of Bacillus subtilis and Bacillus mojavensis were obtained from three continents and, within North America, from populations at a range of geographical distances from one another. The rate of recombination within populations of each species was estimated from restriction-site data for three genes. Recombination was shown to occur within each species at about the same rate as neutral mutation, whatever the geographical scale or phylogenetic scale over which strains were sampled. The rate of migration between populations was estimated by a cladistic analysis and was shown to be high (i.e., Nm > 1), even among populations on different continents. The level of migration within each species is sufficient to prevent neutral geographical divergence within species.     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Further studies on recombination in diploid clones from Bacillus subtilis protoplast fusion

Summary Diploid prototrophs were obtained from protoplast fusion of Bacillus subtilis strains. They are unstable but upon further cultivation they stabilize retaining diploidy but are genetically inactive. It has been suggested that recombination between the parental chomosomes is involved in the production of stable prototrophs and recombinants. In this work the occurrence of this recombination was searched for by determining genetic linkages in transformation experiments. In prototrophs two alleles: hisH2 and trpE8 carried originally on each parental chromosome, were shown to be 48% co-transformable in a stable clone whereas they were only cotransformed in 10% of the unstable colonies. For Trp^- recombinants (the most frequent type of a Leu^- Met^- Thr^- x Ade^- Ura^- Trp^- fusion pair) lysed protoplasts were used as donor DNA for the transformations. High values of co-transfer for Ura⁺ Met⁺ were obtained. These results confirm the occurrence of recombination in stable diploid clones, prototrophs or recombinants.     

GENOME RELATIONS OF AGROPYRON SERICEUM,HORDEUM JUBATUM AND THEIR HYBRIDS

Cytogenetic investigation of microsporogenesis in Agropyron sericeum, Hordeum jubatum, their spontaneous hybrid, Agrohordeum pilosilemma, its amphiploid, and the backcross of the amphiploid to A. sericeum, B₁, elucidated the genome relationships of A. sericeum and H. jubatum. The tetraploid parental species share a partially homologous genome which affects the pairing relationships evidenced in their hybrids. The genome formulae assigned to these plants are: A. sericeum, A“A”BB; H. jubatum, AAA'A‘; Agrohordeum pilosilemma, AA'A“B; the amphiploid, AAA'A‘A”A“BB; and B₁, AA'A”A“BB. Observed pairing configurations were compatible with the expected maximum pairing configurations predicted under the assumption of genetic control of pairing with dosage effects. This is interpreted as further support for the hypothesis that pairing in the hybrids of H. jubatum is controlled by the A genome, one dose of A allowing homeologous pairing and two doses of A promoting homeologous association.     

Illegitimate recombination in Bacillus subtilis: nucleotide sequences at recombinant DNA junctions

Summary The illegitimate integration of plasmid pGG20 (the hybrid between Staphylococcus aureus plasmid pE194 and Escherichia coli plasmid pBR322) into the Bacillus subtilis chromosome was studied. It was found that nucleotide sequences of both parental plasmids could be involved in this process. The recombinant DNA junctions between plasmid pGG20 and the chromosome were cloned and their nucleotide sequences were determined. The site of recombination located on the pBR322 moiety carried a short region (8 bp) homologous with the site on the chromosome. The nucleotide sequences of the pE194 recombination sites did not share homology with chromosomal sequences involved in the integration process. Two different pathways of illegitimate recombination in B. subtilis are suggested.     

Hydrolysis of Proteins by Successive Incubation with Proteinase and Aminopeptidases Mixture

1. A trial test was attempted of complete hydrolysis of peptides and proteins into amino acids by enzymes. “Neutral proteinase” of Bacillus subtilis or “Alkalophilic proteinase” of a Streptomyces sp. was used for preliminary digestion of substrate, and a mixture of three aminopeptidases of Bacillus subtilis was employed for subsequent hydrolysis of proteinase digest.

2. The oxidized insulin B chain was hydrolyzed completely by the method. Several proteins including enzymes which contained no or less cystine and cysteine were also hydrolyzed almost completely.

3. On the other hand, certain glycoproteins were hydrolyzed to leave a few glycopeptides in which all glycomoieties of the proteins were retained. The implications of the results are discussed.     

CHROMATOGRAPHIC COMPARISON OF DICENTRA SPECIES AND HYBRIDS

Ten species of Dicentra were examined by two-dimensional descending paper chromatography. The flavonoid components, including anthocyanins, formed patterns which were specific to a species or a group of closely related species, and these confirmed certain natural relationships within the genus. Some of the components were hydrolyzed and close structural relationships among them were revealed. They were further characterized by spray reactions and R_F measurements in a variety of solvent systems. The inheritance of the components, especially of the anthocyanins, was studied in the hybrids. All parental substances appeared in some hybrids, but in others some parental components were missing. “Hybrid substances” which had not been present in either parent were found in certain hybrids. This is thought to represent either reconstruction of ancestral biosynthetic pathways, probably through genic complementation, or extension of existing synthetic routes through some type of interaction between parental genomes.     

MORPHOLOGICAL VARIABILITY IN GENETICALLY DEFINED CATEGORIES OF ANURAN HYBRIDS

Hybridization phenomena in anurans have traditionally been studied through morphological comparisons, under the assumption that various hybrids (e.g., F₁‘s, backcrosses) are predictably intermediate to parental species. We critically evaluate this assumption by examining morphology in genetically categorized hybrids between the treefrogs Hyla cinerea and H. gratiosa. A total of 202 frogs from a hybridizing population in Alabama were assayed for allozyme and mitochondrial DNA genotype and for a large suite of osteological characters. Discriminant analyses demonstrated distinct morphological separation between the genetically “pure” parental species. Morphometric analyses of genetically identified hybrids showed: 1) an extreme range of phenotypic expression within F₁ and backcross classes, and 2) no apparent directional parental bias on the F₁ phenotype. Had morphology alone been used as a guide, over 40 percent of the individuals with known hybrid ancestry would have been misclassified as “pure” parental species, and about 25 percent of the backcross individuals would not have been distinguished from F₁‘s. These results exemplify the utility of joint comparisons of morphology and genotypic constitution in studies of natural hybridization, and they emphasize the limitations inherent in describing hybrid classes solely by morphological criteria.     

CHROMATOGRAPHIC COMPARISON OF TRAGOPOGON SPECIES AND HYBRIDS

Approximately 1,700 plants representing five species of Tragopogon (Compositae) and their F₁ and F₂ hybrids were analyzed by two-dimensional descending paper chromatography. Each species, or population within a species, was chromatographically distinct. Often, however, the differences were more quantitative than qualitative. The chromatographic data generally supported the species relationships which had been determined from previous morphologic, hybridization, and fertility studies. Inheritance of the flavonoid compounds was usually additive in the F₁'s. Segregation and recombination of the genes controlling the synthesis of these compounds sometimes approximated 3:1 or 9:7 ratios in the F₂'s. Occasionally parental compounds were missing from some of the hybrids. “Hybrid” compounds which had not been found in either parent were absent from the F₁ but did occur in several F₂ populations. Two linkage groups were present. The first contains genes controlling the synthesis of three compounds and the second, four compounds.     

Role of Ni-tolerant Bacillus spp. and Althea rosea L. in the phytoremediation of Ni-contaminated soils

In our current study, four nickel-tolerant (Ni-tolerant) bacterial species viz, Bacillus thuringiensis 002, Bacillus fortis 162, Bacillus subtilis 174, and Bacillus farraginis 354, were screened using Ni-contaminated media. The screened microbes exhibited positive results for synthesis of indole acetic acid (IAA), siderophore production, and phosphate solubilization. The effects of these screened microbes on Ni mobility in the soil, root elongation, plant biomass, and Ni uptake in Althea rosea plants grown in Ni-contaminated soil (200 mg Ni kg^?1) were evaluated. Significantly higher value for water-extractable Ni (38 mg kg^?1) was observed in case of Ni-amended soils inoculated with B. subtilis 174. Similarly, B. thuringiensis 002, B. fortis 162, and B. subtilis 174 significantly enhanced growth and Ni uptake in A. rosea. The Ni uptake in the shoots and roots of B. subtilis 174-inoculated plants enhanced up to 1.7 and 1.6-fold, respectively, as compared to that in the un-inoculated control. Bacterial inoculation also significantly improved the root and shoot biomass of treated plants. The current study presents a novel approach for bacteria-assisted phytoremediation of Ni-contaminated areas.     

DEVELOPMENTAL INSTABILITY AND HIGH MERISTIC COUNTS IN INTERSPECIFIC HYBRIDS OF SALMONID FISHES

Interspecific hybrids have been proposed to have reduced developmental stability in comparison to their parental species because the parental genomes have not undergone selection for the maintenance of developmental stability when they occur together. We present data from four interspecific hybrids of salmonid fishes that support this view. Natural hybrids of bull trout (Salvelinus confluentus) with brook trout (Salvelinus fontinalis) and laboratory hybrids of rainbow trout (Salmo gairdneri) with Yellowstone (Salmo clarki bouvieri), westslope (S. c. lewisi), and coastal (S. c. clarki) cutthroat trout all have higher levels of fluctuating asymmetry than either of their parental species raised in the same environment. Thus, the hybrids have reduced developmental stability. The hybrids do not have meristic counts intermediate to the counts of the parental species. The hybrids usually have counts as high as the species with the higher count for those characters that differ between the parental species and often have higher counts for those characters that do not differ between the parental species. We suggest that the tendency for interspecific hybrids to have high meristic counts may be related to differences between the species in the length and timing of the developmental periods during which the counts of the characters are determined.     

Phenotypic diversity and technological properties of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> species isolated from <Emphasis Type="Italic">okpehe</Emphasis>, a traditional fermented condiment

The Bacillus subtilis wild strains isolated from okpehe, a traditional fermented condiment used as seasoning in Nigeria, the reference and typed strains were investigated for their phenotypic diversity and their technological parameters with a view to obtain adequate data that would enable selection of appropriated starter cultures for vegetable protein fermentation in West Africa. All the 7 strains studied demonstrated diverse phenotypic characteristics and they were identified as Bacillus subtilis, based on the API 50 CHB combined with API 20E profile. Specific sugars that indicated a good hydrolytic potential of the wild strains were fermented. The highest proteinase activity of 90 AU/ml determined quantitatively was observed in the strain Bacillus subtilis BFE 5372, the proteinase was identified by the APIZYM gallery as chymotrypsin. Highest amylase activity of 13 AU/ml was noticed in strain Bacillus subtilis DSM 347 while only 4 strains produced polyglutamic acid with the strain Bacillus subtilis BFE 5359 producing the highest polyglutamate activity of 2.5 mm. Although strain Bacillus subtilis BFE 5301 did not release detectable polyglutamate, the strain demonstrated antagonism against different bacteria and the antimicrobial substance produced by strain Bacillus subtilis BFE 5301 was confirmed as a bacteriocin since its activities were lost after treatment with chymotrypsin and pepsin. The data generated showed the technological parameters that can aid selection of wild strains such as Bacillus subtilis BFE 5301, BFE 5359 and BFE 5372 for optimization of condiment production.     

Production of cyclomaltodextrin glucanotransferase of Bacillus circulans var. alkalophilus ATCC21783 in B. subtilis

Summary The cyclomaltodextrin glucanotransferase (CGTase, E.C. 2.4.1.19) gene from an alkalophilic Bacillus circulans var. alkalophilus ATCC21783 was cloned into Escherichia coli and B. subtilis. When cloned from E. coli to B. subtilis, the entire insert containing the CGTase gene was, depending on the plasmid construction, either unstable or the recombinant B. subtilis did not secrete the enzyme in significant amounts. To achieve efficient enzyme production in B. subtilis, the gene was placed under the control of the B. amyloliquefaciens -amylase promoter. In one of the constructions, both the promoter and the signal sequence of the gene were replaced with those of B. amyloliquefaciens, whereas in another construction only the promoter area was exchanged. The recombinant B. subtilis clones transformed with these plasmid constructions secreted CGTase into the culture medium 14 times as much as did the parental strain in shake flask cultures. In fermentor cultures in an industrially feasible medium the enzyme production was substantially higher, yielding 1.2 g/l of CGTase, which is about 33 times the amount of the enzyme produced by the parental strain in corresponding fermentations. Both of the plasmid constructions were stable when grown over 50 generations without antibiotic selection.     

Cytogenetic study of a female Lemur coronatus × Lemur macaco hybrid

Two species of lemur, Lemur macaco and Lemur coronatus, which do not hybridize in the wild, have produced a first, “definite” female hybrid in captivity. Its karyotype contains one haploid set from each parent. The analogy with the parental chromosomes is such that the pairing of the corresponding chromosomal arms leads to the formation of an open chain and a ring. The difficulty in obtaining hybrids between these two species could reflect the existence of a prezygotic barrier. The presence of multivalents, with probably a negative action on the gametogenesis, would introduce a postzygotic barrier.     

The Bacillus subtilis addAB genes are fully functional in Escherichia coli

An Escherichia coli recBCD deletion mutant was transformed with plasmids containing the Bacillus subtilis add genes. The transformants had relatively high ATP-dependent exonuclease- and ATP-dependent helicase activities, and their viability, the ability to repair u.v.-damaged DNA and the recombination in conjugation were nearly completely restored. The B. subtilis Add enzyme did not show Chi-activity in phage lambda recombination. The individual B. subtilis Add proteins were not able to form an enzymatically active complex with the E. coli RecB,C,D proteins, and they could not complement the recB,C,D deficiency. Evidence is presented that only two subunits are involved in the B. subtilis ATP-dependent exonuclease. This is in contrast to E. coli in which the RecBCD enzyme consists of three subunits.     

First Evidence for Homologous Recombination-mediated Large DNA Inversion on the Bacillus subtilis 168 Chromosome

A Bacillus subtilis 168 strain carrying an inversion of about 1600kb-long chromosomal DNA was isolated. Physical and genetic analyses demonstrated that the inversion was generated as a result of homologous recombination between two homologous sequences integrated at the met and leuB loci. This is the first clear evidence of a large stable chromosomal inversion induced by homologous recombination in B. subtilis.     

Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis

To study plasmid instability in Bacillus subtilis the pUB110-derived hybrid plasmid pLB2 (3.6 kb) and the bifunctional replicon pLB5 (5.9 kb), able to replicate in B. subtilis and Escherichia coli, were constructed. In both vectors homologous B. subtilis, or heterologous E. coli DNA fragments of various lengths were inserted. Irrespective of the source of the cloned DNA, the segregational stability of the recombinant plasmids in B. subtilis was severely affected by the DNA inserts. In contrast, no instability was observed in E. coli. In B. subtilis a steep inverse relationship existed between the size of the inserts and the level of stability. Increased size of the pLB plasmids resulted in strongly reduced copy numbers. This seems to be the primary cause of the size-dependent segregational instability.     

The Effect of Bacillus subtilis on in vitro Growth and Pathogenicity of Agrobacterium tumefaciens

Five isolates of Bacillus subtilis isolated from the soil, were found to be antagonistic to 6 isolates of Agrobacterium tumefaciens in vitro. Inoculation of B. subtilis in wounded castor bean plants 30 min before or simultaneous inoculation with A. tumefaciens resulted in excellent control of the crown gall symptoms on the host within 50 days of inoculation. Application of B. subtilis 30 min after inoculation with A. tumefaciens did not result in appreciable disease reduction. Treatment of the tested plants by B. subtilis did not induce any phytotoxic injury or growth retarding side effects. It appears that B. subtilis could potentially be incorporated for crown gall control. However, further tests are needed to test this biological control agent with other plant species especially fruits, nuts and vine nursery stock.