IS256 is a highly active insertion sequence (IS) element of multiresistant staphylococci and enterococci. Here we show that, in a Staphylococcus epidermidis clinical isolate, as well as in recombinant Staphylococcus aureus and Escherichia coli carrying a single IS256 insertion on a plasmid, IS256 excises as an extrachromosomal circular DNA molecule. First, circles were identified that contained a complete copy of IS256. In this case, the sequence connecting the left and right ends of IS256 was derived from flanking DNA sequences of the parental genetic locus. Second, circle junctions were detected in which one end of IS256 was truncated. Nucleotide sequencing of circle junctions revealed that (i) either end of IS256 can attack the opposite terminus and (ii) the circle junctions vary significantly in size. Upon deletion of the IS256 open reading frame at the 3 end and site-directed mutageneses of the putative DDE motif, circular IS256 molecules were no longer detectable, which implicates the IS256-encoded transposase protein with the circularization of the element.IS256 is an insertion sequence (IS) element of staphylococci and enterococci that was originally identified as a bordering component of the composite aminoglycoside resistance-mediating transposon Tn4001 (2,8,9). IS256, together with 32 other elements, forms a family of ISs that also includes eukaryotic relatives (3). Information about IS256 is very limited (10), despite its wide distribution. Recently, it was demonstrated that the flanking IS256 copies of Tn4001 form tandem dimers and IS circles in an Escherichia coli genetic background (17). In previous studies we have been able to show that IS256 is associated with genetic rearrangements, causing phenotypic changes in its natural host Staphylococcus epidermidis. Multiresistant S. epidermidis as well as Staphylococcus aureus are the most common causes of nosocomial infections associated with implanted biomaterials. In these clinical staphylococcal isolates IS256 proved to be highly active and caused a variety of genetic aberrations, such as reversible gene inactivations, DNA rearrangements, and large chromosomal deletions that affected the expression of virulence-and resistance-associated genes (12,(27)(28)(29)(30). Specifically, biofilm formation, which is one of the major factors in S. epidermidis pathogenesis, is influenced by IS256. The element causes phase variation of biofilm expression in S. epidermidis by inactivation of the icaADBC operon, which encodes enzymes responsible for biofilm synthesis (28). Apparently, the icaC gene represents a hot spot for IS256 insertions. It was shown that the element creates 8-and 7-bp target site duplications during transposition (28; S. H. Cho and W. Ziebuhr, unpublished data). The spontaneous ica:: IS256 insertions are reversible and, accordingly, nucleotide sequencing of biofilm-forming revertants confirmed the precise excision of the element, including the initially duplicated target sequences. In these initial studies, however, the molecular mechanisms th...

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SummaryThe adenylyl cyclase Rv1625c from Mycobacterium tuberculosis codes for a protein with six transmembrane spans and a catalytic domain, i.e. it corresponds to one half of the pseudoheterodimeric mammalian adenylyl cyclases (ACs). Rv1625c is active as a homodimer. We investigated the role of the Rv1625c membrane domain and demonstrate that it efficiently dimerizes the protein resulting in a 7.5-fold drop in K m for ATP. Next, we generated a duplicated Rv1625c AC dimer by a head-to-tail concatenation. This produced an AC with a domain order exactly as the mammalian pseudoheterodimers. It displayed positive cooperativity and a 60% increase of v max compared with the Rv1625c monomer. Further, we probed the compatibility of mycobacterial and mammalian membrane domains. The second membrane anchor in the Rv1625c concatamer was replaced with membrane domain I or II of rabbit type V AC. The mycobacterial and either mammalian membrane domains are compatible with each other and both recombinant proteins are active. A M. tuberculosis Rv1625c knockout strain was assayed in a mouse infection model. In vitro growth characteristics and in vivo organ infection and mortality were unaltered in the knockout strain indicating that AC Rv1625c alone is not a virulence factor.

cAMP generation in bacteria is often stimulated by sudden, but lasting, changes in extracellular conditions, whereas intracellular cAMP concentrations quickly settle at new levels. As bacteria lack G‐proteins, it is unknown how bacterial adenylate cyclase (AC) activities are modulated. Mycobacterium tuberculosis has 15 class III AC genes; therefore, we examined whether mycobacteria contain a factor that may regulate AC activities. We identified mycobacterial polyphosphates with a mean chain length of 72 residues as highly potent inhibitors of dimeric class IIIa, class IIIb and class IIIc ACs from M. tuberculosis and other bacteria. The identity of the inhibitor was established by phosphatase degradation, 31P‐NMR, acid or base hydrolysis, PAGE and comparisons with commercial standards, and functional substitution by several polyphosphates. The data indicate that each AC dimer occupies 8–15 phosphate residues on a polyphosphate strand. Other polyionic polymers such as polyglutamate, polylysine and hyaluronic acid do not affect cyclase activity. Notably, the structurally unrelated class I AC Cya from Escherichia coli is unaffected. Bacterial polyphosphate metabolism is generally viewed in the context of stress‐related regulatory networks. Thus, regulation of bacterial class III ACs by polyphosphates could be a component of the bacterial stress response.