Eucalyptus breeding is typically conducted by selection in open-pollinated progenies. As mating is controlled only on the female side of the cross, knowledge of outcrossing versus selfing rates is essential for maintaining adequate levels of genetic variability for continuous gains. Outcrossing rate in an open-pollinated breeding population of Eucalyptus urophylla was estimated by two PCR-based dominant marker technologies, RAPD and AFLP, using 11 open-pollinated progeny arrays of 24 individuals. Estimated outcrossing rates indicate predominant outcrossing and suggest maintenance of adequate genetic variability within families. The multilcous outcrossing rate (t) estimated from RAPD markers (0.93$0.027), although in the same range, was higher ('0.01) than the estimate based on AFLP (0.89$0.033). Both estimates were of similar magnitude to those estimated for natural populations using isozymes. The estimated Wright's fixation index was lower than expected based on t possibly resulting from selection against selfed seedlings when sampling plants for the study. An empirical analysis suggests that 18 is the minimum number of dominant marker loci necessary to achieve robust estimates of t. This study demonstrates the usefulness of dominant markers, both RAPD and AFLP, for estimating the outcrossing rate in breeding and natural populations of forest trees. We anticipate an increasing use of such PCR-based technologies in mating-system studies, in view of their high throughput and universality of the reagents, particularly for species where isozyme systems have not yet been optimized.

Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spoOJ and to make wild-type sporulation catabolite resistant suggested that spoOJ had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spoOJ locus (spoOJ::Tn917fQHU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spoOJ::Tn917Q1HU261 to sporulate in medium with glucose. Sequencing the spoOJ locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spoOJ mutations. Analysis of the deduced amino acid sequence of the spoOJ locus indicated that the spoOJ gene product contains an ot-helix-turn-a-helix unit similar to the motif found in Cro-like DNA-binding proteins.Bacillus subtilis sporulation is a model system that is used to study procaryotic gene expression and cellular differentiation as responses to environmental stimuli. Sporulation is subject to catabolite repression; i.e., the presence of glucose or other readily metabolized carbon sources inhibits sporulation by wild-type cells (33). Initiation of sporulation is controlled by at least seven genes, spoOA, spoOB, spoOE, spoOF, spoOH, spoOJ, and spoOK (23). Glucose represses transcription of spoOA and spoOF (3, 43). However, it is not known how availability of nutrients regulates initiation of sporulation. Several spoO genes have been sequenced (5,9,14,15,30,41). It is evident from this work that the spoOH gene codes for a sigma subunit of RNA polymerase and that some spoO genes are responsible for sensing environmental conditions. The spoOA and spoOF gene products are homologous to the effector molecules of the two-component response regulator systems that have been described for a variety of bacteria (15,41 PMB12 and SP10 are also able to suppress the Spo-and oligosporogenic phenotypes of various spoOJ mutations (6,20,34). As a result, both PMB12-infected and SP10-infected spoOJ mutants sporulate at a significantly higher frequency than uninfected spoOJ mutants. The observations that PMB12-and SP10-infected bacteria display catabolite-resistant sporulation and that these bacteriophages suppress spoOJ mutations suggest that spoOJ has a role in catabolite repression of sporulation.The spoOJ locus was originally thought to be represented by two mutations, spoOJ87 and spoOJ93 (17). However, spoIIA and spaIID are expressed in a strain with spoOJ87, whereas spoOJ93 blocks expression of these genes (7, 13). The wild-type alleles of both mutations have been cloned into bacteriophage 0105 vectors (10,12 spoOJ93 (45).The data presented in this report provide additional evidence for the involvement of spoOJ in catabolite repressi...

The previously characterized bacteriophage SP10 enhanced the frequency of wild-type sporulation by Bacillus subtilis W23 and 3-13. Comparison of SP10 with the spore-converting bacteriophage PMB12 indicated that both bacteriophages significantly increased the sporulation frequency of an oligosporogenic mutant that contained spoOJ::Tn917 flHU261. SP10 and PMB12 caused wild-type bacteria to sporulate in a liquid medium that initially contained enough glucose to inhibit the sporulation and expression of cL-amylase by uninfected bacteria. SP10 also induced the expression of a-amylase in the presence of glucose, whereas PMB12 had no detectable effect. These observations were consistent with the conclusion that SP10 is a spore-converting bacteriophage and that SP10 and PMB12 relieve glucose-mediated catabolite repression of sporulation by different mechanisms.Bacteriophage genomes are frequently "trapped" in spores; i.e., lytic bacteriophage development is repressed in a sporulating cell and the bacteriophage DNA segregates into the developing spore (8). After germination of the spore, the bacteriophage genome may direct lytic development in some progeny of the newly emerged cell. Bacteria that are infected with a spore-converting bacteriophage sporulate at higher frequencies than uninfected cells, resulting in more frequent entrapment of the spore-converting bacteriophage DNA. Spore-converting bacteriophages have been isolated for Bacillus pumilus, B. subtilis, B. thuringiensis, and Clostridium perfringens (4,5,10,19,21). All of the known spore-converting bacteriophages are pseudotemperate; i.e., they form turbid plaques and enter an unstable carrier state in the host cell.In addition to enhancing wild-type sporulation, sporeconverting bacteriophages significantly increase the sporulation frequencies of Spoc mutants (4,5,10,11,19 cross-reactive with the B. pumilus spore-converting bacteriophage PMB1, but could not demonstrate SP10-enhanced sporulation for any of more than 50 independently isolated B. subtilis W23 Spo-mutants. The data in the present report establish SP10 as a spore-converting bacteriophage. Comparison of PMB12 and SP10 in a common host strain provided evidence for the conclusion that these bacteriophages relieved glucose-mediated repression of sporulation by different mechanisms. MATERIALS AND METHODSBacterial strains, bacteriophages, and culture conditions. The bacterial strains used in this study are described in Table 1. Penassay broth (Difco Laboratories) was used for routine liquid cultures. Nutrient broth-salts medium (NBS) was used to propagate SP10 and was prepared as previously described (2). Sporulation experiments were performed in phosphatebuffered 2XSG, which was the same as 2XSG (13), except that FeCl2 was omitted and the medium contained 14 g of K2HPO4 and 6 g of KH2PO4 per liter (pH 7.0). All liquid cultures were incubated at 37°C with gyratory shaking at approximately 100 rpm.SP10 plaque assays were performed by standard techniques (1), with tryptone semisoft agar (4) overlaid onto t...

Two of eighteen strains of Bacillus subtilis examined contained covalently closed circular duplex deoxyribonucleic acid (DNA) of homogeneous size and buoyant density. Strain ATCC 15841 contained about 16 copies per chromosome of plasmid pPL1, a circular DNA element having a molecular weight of about 4.7 times 10(6) and a buoyant density of 1.700. Strain ATCC 7003 contained about one to two copies per chromosome of plasmid pPL2. pPL2 had a molecular weight of about 46 times 10(6) and a buoyant density of 1.696. Strain ATCC 7003 appeared to be closely related to B. subtilis 168 by genetic, physiological, and biochemical criteria. Strain ATCC 15841 appeared to be much less closely related. B. pumilus ATCC 12140 contained two size classes of covalently closed circular duplex DNA. The plasmids pMB1 and pMB2 had molecular weights of about 6.8 times 10(6) and 5.3 times 10(6), respectively, and were present in several copies per chromosome.

Bacillus pumilus NRS 576 harbored an estimated two copies per chromosome of a covalently closed, circular (CCC) deoxyribonucleic acid (DNA) molecule, the 576 plasmid. The 576 plasmid has a buoyant density of 1.698 g/cm3 and a molecular weight of about 28 x 106. Plasmid copy number remained about the same in both exponentially growing and stationary-phase cells. Spontaneous variants of NRS 576 that formed spores at an elevated frequency were designated as W mutants. W mutants appeared to have lost the 576 plasmid on the basis of the following: W mutants (38 tested) lacked detectable CCC DNA, and the majority of the plasmid homologous sequences in bulk NRS 576 DNA were absent from bulk W mutant DNA. B. pumilus ATCC 7065 harbored at least 10 copies per chromosome of a CCC DNA element, the 7065 plasmid. The 7065 plasmid has a buoyant density of 1.696 g/cm3 and a molecular weight of about 6 x 106. Although the copy number of the plasmid appeared to remain the same in exponentially growing and stationary-phase cells, an additional CCC form of higher molecular weight was detected in stationary-phase cells.

Industrial wastewater bioreactors are potentially important sources of novel biocatalysts. However, the microbial populations in these bioreactors are not well characterized. The microbial community in an industrial wastewater bioreactor was surveyed by extracting DNA from a sample of activated sludge, followed by PCR amplification and sequencing of cloned 16S rRNA genes. A total of 407 cloned 16S rRNA gene sequences were compared with 88 bacterial isolates cultured from the same sample of sludge using a variety of standard media. Most of the bacteria detected by the PCR-based approach were beta-subdivision Proteobacteria, whereas most of the cultured bacteria were gamma-subdivision Proteobacteria. Only a few types of bacteria were detected by both approaches. These observations indicate that multiple techniques are necessary to characterize the microbial diversity in any complex ecosystem.

Exploration of metabolically diverse rhodococci is generally hampered by the lack of genetic tools. A small cryptic plasmid (pAN12) isolated from Rhodococcus erythropolis strain AN12 was sequenced. Plasmid pAN12 encodes proteins that share homology to replication proteins and putative cell division proteins. Based on in vitro transposon mutagenesis, we determined that the Rep protein of pAN12 is essential for plasmid replication in Rhodococcus spp., and the putative cell division protein Div is important for plasmid stability. The pAN12 replicon is able to replicate in R. erythropolis strains AN12 and CW23 (ATCC 47072) and is compatible with the nocardiophage Q4 replicon present on a Rhodococcus shuttle plasmid pDA71. pAN12 appears to belong to the pIJ101/pJV1 family of rolling circle replication plasmids. Expression of an isoprenoid pathway gene ( dxs) on the pAN12-derived multicopy shuttle vector increased production of carotenoid pigments in R. erythropolis ATCC 47072.

Pulsed-Weld gel electrophoresis (PFGE) revealed three previously uncharacterized megaplasmids in the genome of Rhodococcus erythropolis AN12. These megaplasmids, pREA400, pREA250, and pREA100, are approximately 400, 250, and 100 kb, respectively, based on their migration in pulsed-Weld gels. Genetic screening of an AN12 transposon insertion library showed that two megaplasmids, pREA400, and pREA250, are conjugative. Mobilization frequencies of these AN12 megaplasmids to recipient R. erythropolis SQ1 were determined to be approximately 7 £ 10 ¡4 and 5 £ 10 ¡4 events per recipient cell, respectively. It is known for other bacterial systems that a relaxase encoded by the traA gene is required to initiate DNA transfer during plasmid conjugation. Sequences adjacent to the transposon insertion in megaplasmid pREA400 revealed a putative traA-like open reading frame. A targeted gene disruption method was developed to generate a traA mutation in AN12, which allowed us to address the role of the traA gene product for Rhodococcus megaplasmid conjugation. We found that the AN12 traA mutant is no longer capable of transferring the pREA400 megaplasmid to SQ1. Furthermore, we conWrmed that the conjugation defect was speciWcally due to the disruption of the traA gene, as pREA400 megaplasmid conjugation defect is restored with a complementing copy of the traA gene.