Identification of functional promoters in the msp2 expression loci of Anaplasma marginale and Anaplasma phagocytophilum.
Organisms in the family Anaplasmataceae are important tick-borne pathogens of livestock worldwide and cause recently emergent infections in humans. Despite their medical importance, very little is known about how these organisms regulate gene expression in the mammalian host, the tick vector, or during transition between the host and vector. However, it is clear that gene regulation, in addition to recombinatorial mechanisms, is essential for these small genome pathogens to adapt to distinctly different environments. In this study, we identify and establish the function of three promoter elements in the locus encoding major outer membrane protein expression sites in both Anaplasma marginale and Anaplasma phagocytophilum. Gene expression from this locus involves both classical and atypical polycistronic transcripts. The identified promoter elements have a structure similar to that defined in Escherichia coli and are functional in driving protein expression in a prokaryotic cell-free transcription and translation system and in recombinant E. coli. The two strongest promoters identified in vitro and with recombinant E. coli were also shown to be functional in A. marginale infected cells, as determined by quantification of downstream transcripts. The promoters in both A. marginale and A. phagocytophilum have similar structure and activity, supporting the conclusion that the two loci are syntenic with conservation of function. In addition, they share structural elements within the promoters that appear to be likely sites for regulation. These data enhance our understanding of how expression of these variable outer membrane proteins may be controlled in the key stages of tick-borne transmission and infection.
Barbet
Anthony F
AF
Department of Pathobiology, College of Veterinary Medicine, University of Florida, Box 110880, Gainesville, FL 32611-0880, USA. barbeta@mail.vetmed.ufl.edu
Agnes
Joseph T
JT
Moreland
Annie L
AL
Lundgren
Anna M
AM
Alleman
A Rick
AR
Noh
Susan M
SM
Brayton
Kelly A
KA
Munderloh
Ulrike G
UG
Palmer
Guy H
GH
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000235:genetics
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C103348:msp2 protein, Anaplasma marginale; C488283:msp2 protein, Anaplasma phagocytophilum; D049452:Green Fluorescent Proteins