A segment of Myxococcus xanthus ops DNA functions as an upstream activation site for tps gene transcription.

A segment of DNA located between 131 and 311 base pairs (bp) upstream from the transcriptional start of the Myxococcus xanthus ops gene (-131 to -311) was shown to function as an upstream activation site (UAS) for developmentally regulated transcription from the tps gene promoter region. The activation of early developmental transcription by the ops UAS was independent of orientation and could be increased by the addition of a second copy of the UAS. The ops UAS segment continued to function when placed 1.5 kbp upstream from the transcription initiation site. DNA from the tps promoter region was required for transcriptional activation by the ops UAS, and a specific requirement for the sequence of tps DNA between -34 and -66 was demonstrated. Several specific ops UAS DNA-protein complexes were observed after incubation of this DNA segment with an extract of early developmental M. xanthus cells. Extracts of vegetative cells contained much less ops UAS-specific DNA-binding activity. When the distance between the tps and ops genes was increased from 2 to 15 kbp by insertion of a transduced segment of DNA, the amount of developmentally induced tps RNA was found to be about one-third that found in wild-type M. xanthus. Our observations suggest that the regulatory region of the ops gene functions not only to control ops gene expression but also to increase early developmental expression of the tps gene located about 2 kbp downstream on the M. xanthus chromosome.

Tn5-mediated transposition of plasmid DNA after transduction to Myxococcus xanthus.

After coliphage P1-mediated transfer of Tn5-containing plasmid DNA from Escherichia coli to Myxococcus xanthus, transductants were identified which contained plasmid sequences integrated at many sites on the bacterial chromosome. The unaltered plasmid DNA sequences in these transductants were apparently flanked by intact Tn5 or IS50 sequences. These results suggest that Tn5-mediated transposition has occurred and provide a method for integrating plasmid DNA into the M. xanthus chromosome without the requirement for homologous recombination.

Localization of the cis-acting regulatory DNA sequences of the Myxococcus xanthus tps and ops genes.

The cis-acting regulatory regions of the tps and ops genes of Myxococcus xanthus were localized by analyzing the expression of fusions of these genes with the lacZ gene. A 201-base-pair (bp) fragment of tps DNa extending 95 bp upstream (-95) from the transcriptional start was sufficient to direct developmentally regulated expression of fusion gene activity. The segment of tps DNA between -95 and -81 contained information necessary for developmental regulation. A segment of ops DNa extending upstream to -131 directed a very low level of ops-lacZ fusion expression, but the inclusion of DNA to -208 greatly increased the amount of developmentally regulated expression. M. xanthus DNA upstream from -108 in the tps gene and -311 in the ops gene was required for maximal expression of gene fusion activity. The upstream regulatory regions of both the tps and ops genes seem to be involved in positive transcriptional regulation. Two mutations, a deletion of 1 bp at -8 in the tps gene and a 3-bp substitution at -27 to -29 in the ops gene, greatly increased the level of vegetative expression of gene fusion activity, suggesting that both genes may also be subject to negative regulation in M. xanthus.

Identification of the RNA products of the ops gene of Myxococcus xanthus and mapping of ops and tps RNAs.

The expression of the ops gene, like that of the highly homologous and closely linked tps gene, is induced during development of the fruiting bacterium Myxococcus xanthus. The RNA products of the ops gene have been identified and compared with tps RNA. The ops RNA was observed in developmental cells only after spore formation had commenced, and it was necessary to use a sporulation-defective mutant strain or to disrupt spores to isolate this RNA. RNA from the ops gene was not observed in vegetative cells but was readily detected in cells subjected to glycerol-induced sporulation. In contrast, a large amount of developmental tps RNA was observed in cells well before sporulation had occurred; low levels of tps RNA were observed in vegetative cells; and only a slight increase in tps RNA was found during glycerol-induced sporulation. Several ops and tps RNAs were observed in this study, and the positions of these RNAs were mapped on the M. xanthus genome. The 5' ends of both the ops and tps RNAs mapped predominantly to positions about 50 bases upstream from the respective translational initiation sites. The 3' ends of RNAs from both genes were heterogeneous. The four ops RNAs were 620, 775, 845, and 1,230 bases in length, while the tps RNAs were 612, 695, 730, and 935 bases.

Differential expression of protein S genes during Myxococcus xanthus development.

Protein S, the most abundant protein synthesized during development of the fruiting bacterium Myxococcus xanthus, is coded by two highly homologous genes called protein S gene 1 (ops) and protein S gene 2 (tps). The expression of these genes was studied with fusions of the protein S genes to the lacZ gene of Escherichia coli. The gene fusions were constructed so that expression of beta-galactosidase activity was dependent on protein S gene regulatory sequences. Both the gene 1-lacZ fusion and the gene 2-lacZ fusion were expressed exclusively during fruiting body formation (development) in M. xanthus. However, distinct patterns of induction of fusion protein activity were observed for the two genes. Gene 2 fusion activity was detected early during development on an agar surface and could also be observed during nutritional downshift in dispersed liquid culture. Gene 1 fusion activity was not detected until much later in development and was not observed after downshift in liquid culture. The time of induction of gene 1 fusion activity was correlated with the onset of sporulation, and most of the activity was spore associated. This gene fusion was expressed during glycerol-induced sporulation when gene 2 fusion activity could not be detected. The protein S genes appear to be members of distinct regulatory classes of developmental genes in M. xanthus.

Gene expression during development of Myxococcus xanthus. Analysis of the genes for protein S.

Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10(3) base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of beta-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.

Early RNAs in SP82- and SP01-infected Bacillus subtilis may be processed.

Transcription of SP82 and SP01 DNAs in vitro by Bacillus subtilis RNA polymerase yielded mostly large RNA species, with many in excess of 1,500 bases in length, whereas most of the RNAs synthesized in vivo early in infection were much smaller. Addition of an extract from uninfected B. subtilis to reaction mixtures containing RNAs synthesized in vitro generated additional discrete RNAs whose mobilities on polyacrylamide gels matched the mobilities of some of the smaller RNAs synthesized in vivo.

Bacillus subtilis bacteriophages SP82, SPO1, and phie: a comparison of DNAs and of peptides synthesized during infection.

The genomes of Bacillus subtilis phages phie, SPO1, and SP82 were compared by DNA-DNA hybridization, analysis of DNA fragments produced by digestion with restriction endonucleases, comparison of the arrays of peptides synthesized during infection, and phage neutralization. DNA-DNA hybridization experiments indicated that about 78% of the SP82 DNA was homologous with SPO1 DNA, whereas 40% of the phie DNA was homologous to either SPO1 or SP82 DNA. Agarose gel electrophoresis was used to compare the molecular weights of DNA fragments produced by cleavage of SP82, SPO1, and phie DNAs with the restriction endonucleases Hae III, Sal I, Hpa II, and Hha I. Digestion of the DNAs with Hae III and Sal I produced only a few fragments, whereas digestion with Hpa II and Hha I yielded 29 to 40 fragments, depending on the DNA and the enzyme. Comparing the Hpa II fragments, 51% of the SP82 fragments had mobilities which matched those of SPO1 fragments, 32% of the SP82 fragments matched the phie fragments, and 34% of the SPO1 fragments matched the phie fragments. Comparing the Hha I digestion products, 62% of the SP82 fragments had mobilities matching the SPO1 fragments, 24% of the SP82 fragments matched the phie fragments, and 22% of the SPO1 fragments matched the phie fragments. Analysis of peptides by electrophoresis on one-dimensional sodium dodecyl sulfate-polyacrylamide slab gels showed that approximately 70 phage-specific peptides were synthesized in the first 24 min of each infection. With mobility and the intervals of synthesis as criteria, 66% of the different SP82 peptides matched the SPO1 peptides, 34% of the SP82 peptides matched the phie peptides, and 37% of the SPO1 peptides matched the phie peptides. Phage neutralization assays using antiserum to SP82 yielded K values of 510 for SP82, 240 for SPO1, and 120 for phie.

Bacterial xanthine oxidase from Arthrobacter S-2.

Arthrobacter S-2, originally isolated by enrichment on xanthine, produced high levels of xanthine oxidase activity, requiring as little as a 20-fold purification to approach homogeneity with some preparations. Molecular oxygen, ferricyanide, and 2,6-dichlorophenol-indophenol served as electron acceptors, but nicotinamide adenine dinucleotide did not. The enzyme was relatively specific when compared with previously studied xanthine-oxidizing enzymes, but at least one purine was observed to be oxidized at each of the three positions of the purine ring that have been subject to oxidation by this type of enzyme. The enzyme had a relatively high Km for xanthine (1.3 X 10(-4) M), and substrate inhibition was not observed with this compound, in contrast to the enzyme from cow's milk. In fact, an opposite effect was observed, and double-reciprocal plots with xanthine as the variable substrate showed a concave downward deviation at high concentrations. At 2.5 mM xanthine the enzyme had a specific activity approximately 50 times that of the most active preparations of the milk enzyme. The spectrum of the Arthrobacter enzyme resembled that of milk xanthine oxidase, suggesting a similarity of the prosthetic centers of the two enzymes. The bacterial enzyme was relatively small and may be dimeric, with approximate native and subunit molecular weights of 146,000 and 79,000, respectively.

Distribution of xanthine oxidase and xanthine dehydrogenase specificity types among bacteria.

A diverse collection of xanthine-metabolizing bacteria was examined for xanthine-, 1-methylxanthine-, and 3-methylxanthine-oxidizing activity. Both particulate and soluble fractions of extracts from aerobically grown gram-negative bacteria exhibited oxidation of all three substrates; however, when facultative gram-negative bacteria were grown anaerobically, low particulate and 3-methylxanthine activities were detected. Gram-positive and obligately anaerobic bacteria showed no particulate activity or 3-methylxanthine oxidation. Substrate specificity studies indicate two types of enzyme distributed among the bacteria along taxonomic lines, although other features indicate diversity of the enzyme within these two major groups. The soluble and particulate enzymes from Pseudomonas putida and the enzyme from Arthrobacter S-2 were examined as type examples with a series of purine and analogues differing in the number and position of oxygen groups. Each preparation was active with a variety of compounds, but the compounds and position attacked by each enzyme was different, both from the other enzymes examined and from previously investigated enzymes. The soluble enzyme from Pseudomonas was inhibited in a competitive manner by uric acid, whereas the Arthrobacter enzyme was not. This was correlated with the ability of Pseudomonas, but not Arthrobacter, to incorporate radioactivity from [2-14C]uric acid into cellular material.