TANDEM GENES OF CHLAMYDIA-PSITTACI THAT ENCODE PROTEINS LOCALIZED TO THE INCLUSION MEMBRANE

Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole, termed an inclusion. To identify chlamydial pr oteins that are unique to the intracellular phase of the life cycle, a lambda expression library of Chlamydia psittaci DNA was differentiall y screened with convalescent antisera from infected guinea pigs and an tisera directed at formalin-fixed purified chlamydial elementary bodie s (EBs). One library clone was identified that harboured two open read ing frames (ORFs) with coding potential for similar-sized proteins of approximate to 20 kDa. These proteins were subsequently termed IncB an d IncC. Sequencing of the cloned insert revealed a strong Escherichia coli-like promoter sequence immediately upstream of incS and a 36nt in tergenic region between the ORFs. Sequence analysis of the region upst ream of incB and incC revealed two ORFs that had strong homologies to an amino acid transporter and a sodium-dependent transporter. Immunobl otting with antisera directed at IncB or IncC demonstrated that these proteins are present in C. psiffaci-infected HeLa cells but are absent or below the level of detection in purified EBs. Reverse transcriptas e-polymerase chain reactions provided evidence that incB and incC are transcribed in an operon, Immunofluorescence microscopy demonstrated t hat IncB and IncC are each localized to the inclusion membrane of infe cted cells. No primary sequence similarity is evident between IncA, In cB or IncC, but each contains a large hydrophobic domain of similar si ze and character as in IncA. Analysis of the recently completed C. tra chomatis serovar D genome database has revealed C. trachomatis ORFs en coding homologues to incS and incC, indicating that these genes are co nserved among the chlamydiae.