Abstract
BackgroundSince the RpoN-RpoS regulatory network was revealed in the Lyme disease spirochete Borrelia burgdorferi a decade ago, both upstream and downstream of the pathway have been intensively investigated. While significant progress has been made into understanding of how the network is regulated, most notably, discovering a relationship of the network with Rrp2 and BosR, only three crucial virulence factors, including outer surface protein C (OspC) and decorin-binding proteins (Dbps) A and B, are associated with the pathway. Moreover, for more than 10 years no single RpoS-controlled gene has been found to be critical for infection, raising a question about whether additional RpoS-dependent virulence factors remain to be identified.Methodology/Principal FindingsThe rpoS gene was deleted in B. burgdorferi; resulting mutants were modified to constitutively express all the known virulence factors, OspC, DbpA and DbpB. This genetic modification was unable to restore the rpoS mutant with infectivity.Conclusions/SignificanceThe inability to restore the rpoS mutant with infectivity by simultaneously over-expressing all the three virulence factors allows us to conclude RpoS also regulates essential genes that remain to be identified in B. burgdorferi.
Highlights
The Lyme disease spirochete Borrelia burgdorferi has three s factors, including the major factor, RpoD (s70), and two alternative factors, RpoN (s54) and RpoS (s38)
A pioneering study by Norgard and colleagues published in 2001 successfully associated the two alternative factors to form a regulatory network, in which RpoS expression depends on RpoN and controls expression of at least three important surface lipoproteins including outer surface protein C (OspC), and decorin-binding proteins (Dbps) A and B [1], This started the era of intensively investigating both up- and down-stream of the RpoN-RpoS pathway [2]
Because the parental clone 13A and DrpoS lost lp25, the plasmid that carries the gene bbe22 coding for a nicotinamidase essential for survival of B. burgdorferi in the mammalian environment, the recombinant plasmids pBBE22 and pME22, which harbor a copy of bbe22, were used as shuttle vectors [19,27]. pME22 was modified from pBBE22 by replacing the restriction enzyme site XbaI with two sites, Acc65I and PstI
Summary
The Lyme disease spirochete Borrelia burgdorferi has three s factors, including the major factor, RpoD (s70), and two alternative factors, RpoN (s54) and RpoS (s38). A 1418-bp promoterless dbpBA operon, including the entire dbpBA coding region, the space between the two gene, 25 bps of upstream sequence (from the dbpB start codon) and 218 bps of downstream sequence (from the dbpA stop codon), was amplified from borrelial DNA with use of the primer P8F and P8R, digested with BamHI and PstI, purified, and cloned into pME22-ospC9, to complete construction of pME22-C9B9A9 (Fig. 2B).
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