Cultivation methods have contributed to our present knowledge about the presence and diversity of microbes in naturally occurring communities. However, it is well established that only a small fraction of prokaryotes have been cultivated by standard methods and, therefore, the prokaryotes that are cultivated may not ref lect the composition and diversity within those communities. Of the two prokaryotic phylogenetic domains, Bacteria and Archaea, members of the former have been shown to be ubiquitous in nature, with ample evidence of vast assemblages of uncultured organisms. There is also now increasingly compelling evidence that the Archaea, which were once thought to occupy a limited number of environments, are also globally widespread. Here we report the use of molecular phylogenetic techniques, which are independent of microbial cultivation, to conduct an assessment of Archaea in a soil microbial community. Small subunit ribosomal RNA genes of Archaea were amplified from soil and cloned. Phylogenetic and nucleotide signature analyses of these cloned small subunit ribosomal RNA gene sequences revealed a cluster of Archaea from a soil microbial community that diverge deeply from the crenarchaeotal line of descent and has the closest affiliation to the lineage of planktonic Archaea. The identification and phylogenetic classification of this archaeal lineage from soil contributes to our understanding of the ecological significance of Archaea as a component of microbial communities in non-extreme environments.

We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, ␣-Proteobacteria, and CFB groups; greater representation among the ␤-and ␥-Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea. In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives. The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation. This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division.

The bacterium Photorhabdus luminescens is a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora. The nematode requires the bacterium for infection of insect larvae and as a substrate for growth and reproduction. The nematodes do not grow and reproduce in insect hosts or on artificial media in the absence of viable P. luminescens cells. In an effort to identify bacterial factors that are required for nematode growth and reproduction, transposon-induced mutants of P. luminescens were screened for the loss of the ability to support growth and reproduction of H. bacteriophora nematodes. One mutant, NGR209, consistently failed to support nematode growth and reproduction. This mutant was also defective in the production of siderophore and antibiotic activities. The transposon was inserted into an open reading frame homologous to Escherichia coli EntD, a 4-phosphopantetheinyl (Ppant) transferase, which is required for the biosynthesis of the catechol siderophore enterobactin. Ppant transferases catalyze the transfer of the Ppant moiety from coenzyme A to a holo-acyl, -aryl, or -peptidyl carrier protein(s) required for the biosynthesis of fatty acids, polyketides, or nonribosomal peptides. Possible roles of a Ppant transferase in the ability of P. luminescens to support nematode growth and reproduction are discussed.Photorhabdus luminescens (Enterobacteriaceae) bacteria are symbiotic with entomopathogenic rhabditid nematodes of the family Heterorhabditidae, with which they cooperate in infecting a wide variety of insect larvae (38, 45; for reviews, see references 25 and 26). The nematode requires P. luminescens for insect pathogenicity (34), while the bacteria depend on the nematodes for transmission between insect prey. The infective juvenile (IJ)-stage nematodes specifically retain symbiotic P. luminescens cells in their gut mucosa, and transmission of the bacteria is a requisite for insect pathogenicity (31,32,34). The nematodes require P. luminescens cells as a substrate for growth and reproduction (2,21,22,30). It was suggested previously that symbiotic P. luminescens cells provide favorable nutritional conditions for Heterorhabditis bacteriophora nematodes to grow and reproduce (45).During prolonged laboratory culture, P. luminescens strains show a tendency to undergo an apparent phase variation phenomenon (8, 9, 36). The native form of the bacteria, termed primary phase, is isolated from the IJ stage of the nematode. The secondary-phase variants appear at high frequency during prolonged culturing, while more rare is the generation of primary-phase cells from secondary phase (6). The secondaryphase cells differ from the primary-phase cells in colony morphology, cell size, and dye uptake characteristics (6, 7, 9, 52). Also, typical primary-phase characteristics such as bioluminescence, pigment synthesis, phospholipase and siderophore activities, and production of intracellular crystalline inclusion proteins are depressed or absent in secondary-phase cells. The mechanism and role of phase variati...

Summary. In order to study the extent and nature of differences among various S-allele-associated proteins in N. alata, we carried out comparative studies of seven such proteins. We first isolated and sequenced cDNA clones for the Sz-, SF11-, S~-, and Sa-alleles, and then we compared the deduced amino acid sequences both of these four S-proteins and of three previously published $2-, $3-, and S6-proteins. This comparison revealed (1) an average homology of 53.8% among the seven proteins and (2) two homology classes, with Sz and SF~ in one class and $1, $2, $3, and $6 in the other class. There are 60 conserved residues, including 9 cysteines. Of the 144 variable residues, 50 were identified as hypervariable based on a calculation of their Similarity Indices. Although conserved, variable, and hypervariable residues are dispersed throughout the protein, some are clustered to form five conserved, five hypervariable, and a number of variable regions. Those variable sites which contain residues conserved within one class of S-proteins but different between classes might provide a clue to the evolutionary relationship of these two classes of S-proteins. The hypervariable residues, which account for sequence variability, may contribute to allelic specificity.

The entomopathogenic bacterium Photorhabdus luminescensexhibits phase variation when cultured in vitro. The variant forms ofP. luminescens are pleiotropic and are designated phase I and phase II variants. One of the characteristic phenotypes of phase I cells is the production of two types of intracellular protein inclusions. The genes encoding the protein monomers that form these inclusions, designated cipA and cipB, were cloned and characterized. cipA and cipB encode hydrophobic proteins of 11,648 and 11,308 Da, respectively. The deduced amino acid sequences of CipA and CipB have no significant amino acid sequence similarity to any other known protein but have 25% identity and 49% similarity to each other. Insertional inactivation ofcipA or cipB in phase I cells of P. luminescens produced mutants that differ from phase I cells in bioluminescence, the pattern and activities of extracellular products, biochemical traits, adsorption of dyes, and ability to support nematode growth and reproduction. In general, the cip mutants were phenotypically more similar to each other than to either phase I or phase II variants.