SummaryType IV pilus genes have been shown to be required for social gliding motility in Myxococcus xanthus. We report the discovery of four additional pil genes: pilD, a homologue of type IV prepilin leader peptidases; and pilG, pilH and pilI, which have no known homologues in other type IV pilus systems. pilH encodes an ATP-binding cassette (ABC) transporter homologue, the first such homologue to be required for the biogenesis of any bacterial pilus type. pilG and pilI are co-transcribed with pilH and appear to be functionally related to pilH. Null mutants of pilG, pilH and pilI all lack social motility, are deficient in pilus production, have elevated sporulation efficiencies and display similar developmental abnormalities. In addition, all three mutations reduced the amount of PilA found in the supernatant after cells were sedimented from liquid culture. We suggest that the products of these three genes form a single ABC exporter complex, in which pilI is an integral membrane protein with membrane-spanning domains, and pilG is an accessory factor. The complex may participate in pilus assembly and/or the export of PilA pilin.

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Random segments of Myxococcus xanthus DNA were cloned in yeast artifical chromosomes (YACs) to construct a physical map of the genome. EcoRI restriction maps of 409 YAC clones with inserts averaging 111 kilobase pairs (kb) were determined. Comparison to the map of a 300-kb region of M. xanthus obtained from clones in Escherichia coli indicates that segments of DNA cloned in YACs are stably maintained in yeast and that their sequences accurately reflect the structure of the Myxococcus genome. The 409 YAC inserts were ordered within 60 map segments (contigs) by aligning their EcoRI restriction maps and by hybridization with 18 gene-specific DNA probes. These 60 map segments may represent the entire Myxococcus genome and could be used to organize its genetic information. This study illustrates the utility of YACs for cloning large segments of DNA and for reliable long-range genomic mapping.The study of many organisms is hindered by the absence of a long-range genetic map. A physical map can substitute for a genetic map, providing a long-range structural framework on which genetic loci can be positioned. A physical map consists of landmarks such as restriction sites, transposon insertion sites, or of partial DNA sequences that have been physically ordered.Several strategies for constructing physical genomic maps have been described that utilize a large number of randomly cloned segments of DNA to create a continuous array of overlapping segments (1-3). Random segments are ordered by finding regions that overlap, a process that ultimately requires comparing every segment with every other segment. This ordering step is difficult because an entire genome must be covered by mapping a large number of cloned fragments, each of which constitutes a small fraction of the genome. One way around this difficulty is to start with a smaller number of larger clones, thereby decreasing the number of comparisons required. Since DNA fragments as large as 800 kilobase pairs (kb) can be cloned in yeast artificial chromosomes (YACs; refs. 4 and 5), they should permit mapping a genome with fewer clones than would be required with Escherichia coli vectors. Although such an approach has been proposed (4), it remains to be demonstrated that accurate genomic maps can be constructed from information obtained with YAC clones. Before YACs can be confidently used for mapping in general and for the random cloning approach in particular, it must be shown that genomic DNA can be maintained in YACs without deletion or rearrangement and is selected with little bias favoring one genomic region over another.Myxococcus xanthus is a microbe whose genetic character is poorly understood, yet is of interest because it is the most primitive organism known that exhibits multicellular development with cellular differentiation (6). Many mutations that alter its development have been isolated and studied genetically. Both specialized and generalized transduction are available to transfer genomic segments of <50 kb, but no reliable method for long-range g...

The amino acid sequence of the Dsg protein is 50%o identical to that of translation initiation factor IF3 of Escherichia coli, the product of its infC gene. dsg mutants are able to initiate the developmental process, but their development goes awry when the cells have formed loose asymmetric aggregates, at about 6 to 10 h. These mutants are unable to transcribe a particular set of developmentally regulated genes (3). The dsg mutation also affects the cells during vegetative growth. Mutants form compact, heaped, smooth-edged colonies, unlike the flat, spreading wild-type colonies. These mutants are also defective in the phase variation of colony color. In the accompanying paper (5), we describe the sequence of the dsg gene, which contains a missense mutation in dsg mutants. The amino acid sequence of Dsg is 50% and 51% identical to that of translation initiation factor IF3 of both Escherichia coli and Bacillus stearothermophilus, respectively. We also show that like IF3, Dsg begins with a unique initiation codon and contains conserved residues which are critical for the function of IF3. The Dsg protein extends 66 more amino acids to its C terminus than the IF3 proteins of E. coli, other enteric bacteria, and B. stearothermophilus.An important role of IF3 in translation initiation is to help the ribosome select the initiation codon on the mRNA. Thus, IF3 ensures the fidelity of translation by preventing initiation at an incorrect location in the message or in the wrong reading frame (12,13,26 codon. The IF3 protein, which is associated with the 30S ribosomal subunit during initiation, inspects the codons near the Shine-Dalgarno site for one which will pair correctly with the anticodon on the initiator tRNA, fMet-tRNA et, which is also associated with the 30S subunit. When the proper initiation codon is located, the 50S ribosomal subunit joins the 30S initiation complex to form the 70S ribosome, and the initiation factors are released (8).The infC gene, which encodes IF3 in E. coli, begins with the atypical AUU initiation codon (21). Recent work shows that IF3 selects against initiation at AUU codons and for initiation at AUG, GUG, and UUG codons, the typical initiators. In fact, this atypical AUU initiation codon is involved in the translational autoregulation of infC expression (2,9,26). When IF3 levels are high, the mRNA encoding infC is not translated because IF3 does not recognize AUU for the initiation of translation. However, when IF3 levels are low, the infC message is translated because, in the absence of IF3, the ribosome shows low selectivity for the typical initiation codons. Translation of infC from AUU continues until the levels of IF3 are high enough to restrict translation from an AUU initiation codon, thus regulating the cellular level of the IF3 protein according to its function (1, 2, 9).In this work, we show that Dsg reacts with anti-IF3 serum. Using anti-Dsg antibodies, we show that Dsg is present at constant levels at all times during vegetative growth and development, as would be expected of ...

The dsg mutants of Myxococcus xanthus are defective in fruiting body development and sporulation, yet they grow normally. The deduced amino acid sequence of the dsg gene product is 50 and 51% identical to the amino acid sequence of translation initiation factor IF3 of both Escherichia coli and Bacillus stearothermophilus, respectively. However, the Dsg protein has a carboxy-terminal extension of 66 amino acids, which are absent from its E. coli and B. stearothermophilus homologs. The Shine-Dalgarno sequence GGAGG and 5 bases further upstream are identical in M. xanthus and several enteric bacteria, despite the wide phylogenetic gap between these species. The infC gene, which encodes IF3 in enteric bacteria, starts with the atypical translation initiation codon AUU, which is known to be important for regulating the cellular level of IF3 in E. coli. Translation of the Dsg protein overexpressed from the M. xanthus dsg gene in E. coli cells initiates at an AUC codon, an atypical initiation codon in the AUU class. The dsg mutants DK429 and DK439 carry the same missense mutation that changes Gly-134 to Glu in a region of amino acid identity.