JD258, a Tn5 insertion mutant of Myxococcus xanthus, was shown to have major defects in three development-associated properties: expression of the developmentally regulated tps gene, spore formation, and production of multicellular fruiting bodies. The defects in tps gene expression and sporulation could be substantially corrected, at the phenotypic level, by mixing JD258 with wild-type cells (extracellular complementation). By this criterion, JD258 appeared to be a new member of a group of conditional developmental mutants that were previously characterized and placed in four extracellular complementation groups (A to D) based on the ability of mutants in one group to stimulate development in mutants belonging to a different group (D. C. Hagen, A. P. Bretscher, and D. Kaiser, Dev. Biol. 64:284-296, 1978). Mutants from groups A, B, C, and D all displayed extracellular complementation activity when mixed with JD258. These results, and other aspects of the phenotype of JD258, indicate that this mutant defines a fifth extracellular complementation group, group E. The M. xanthus esg locus identified by the Tn5 insertion in JD258 was cloned in Escherichia coli and used for further genetic analysis of the locus. These studies indicated that the esg locus resides within a 2.5-kb region of the M. xanthus chromosome and that the locus contains at least two genetic complementation groups. Our results are consistent with a model in which the esg locus controls the production of a previously unrecognized extracellular signal that must be transmitted between cells for the completion of M. xanthus development.

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.

The group A streptococcal sequela acute rheumatic fever (ARF) has been associated with immunological cross-reactivity between streptococcal and heart proteins. To identify Streptococcus pyogenes genes that encode a myosin cross-reactive antigen(s) recognized by ARF sera, a genomic library from an emm deletion strain (T28/51/4) was screened with a single ARF serum. A positively identified lambda EMBL3 clone (T.2.18) produced a protein which reacted with myosin-specific antibodies affinity purified from individual ARF sera. The recombinant protein was initially estimated to be 60 kDa in size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, upon sequence analysis it had a molecular mass equivalent to 67 kDa. Sera from patients with streptococcal infections, acute glomerulonephritis, and ARF were reactive with the recombinant 67-kDa protein. However, individual sera from healthy persons were negative or demonstrated low levels of reactivity with the 67-kDa antigen. The gene encoding the 67-kDa myosin-cross-reactive antigen was subcloned, and its nucleotide sequence was determined by using a combined strategy of DNA sequencing of the cloned gene and N-terminal amino acid sequencing of the protein expressed in Escherichia coli. The amino-terminal sequence deduced from the nucleotide sequence of an open reading frame was identical to that determined from the 67-kDa protein expressed in E. coli. The gene encoded 590 amino acids with a calculated molecular weight of 67,381. No cleavable signal peptide was detected with the 67-kDa protein expressed in E. coli. The deduced amino acid sequence of the 67-kDa protein did not exhibit significant similarity to any known streptococcal proteins. However, it was found to be 19% identical and 62% similar over 151 amino acid residues to the beta chain of mouse major histocompatibility complex class II antigen (I-Au). Similar degrees of homology to the beta chains of other murine and human class II haplotypes were found. Mouse anti-IA sera reacted with the recombinant 67-kDa protein about five times more strongly than normal mouse sera in the enzyme-linked immunosorbent assay. Southern hybridization experiments using a probe for the gene encoding the 67-kDa protein showed that the gene was present and conserved among pathogenic groups A, C, and G of streptococci. These data suggest that the streptococcal protein, which is distinct from the M protein, may have structural features in common with the beta chain of the class II antigens, as well as myosin, and may play an important role in the pathogenesis of streptococcal infections.

To differentiate between relapse of infection and reinfection of the urinary tract due to Klebsiella pneumoniae, 33 K. pneumoniae isolates collected from 20 patients with spinal cord injury (SCI) over 2 years were typed by genomic fingerprinting by repetitive-element PCR. Clinical isolates obtained from the same patients with recurrent episodes of urinary tract infection (UTI) revealed identical genomic fingerprints indicating relapse of UTI due to K. pneumoniae, despite appropriate antibiotic therapy. Seventeen isolates obtained from 8 of the 20 SCI patients shared a common genotype, termed RD6. Among non-SCI patients residing in other nursing units, the RD6 genotype was found in 5 of 10 patients with K. pneumoniae UTI but in only 1 of 20 patients with K. pneumoniae infection that did not involve the urinary tract, suggesting a strong association of this genotype with UTI. All RD6 isolates exhibited strong adherence (>50 adherent bacteria per cell) to HEp-2 cells, whereas other K. pneumoniae isolates generally did not adhere to or adhered very weakly to HEp-2 cells (<5 adherent bacteria per cell). Adherence was inhibited either by 4% D-mannose or by anti-type 1 fimbrial rabbit serum. These results suggest that the capacity of K. pneumoniae RD6 isolates to cause UTI may be mediated by its striking adherence to mammalian cells.

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.