Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum

Abstract

We have used a combination of nucleotide and N-terminal-amino-acid-sequence analyses to determine the primary structure of the 37-kilodalton (kDa) endoflageliar outer layer, or sheath, protein. Initially, a Agtll clone (designated AA34) expressing a portion of the 37-kDa protein was selected from a Treponema pallidum genomic library with a murine monoclonal antibody (H9-2) directed against an epitope of the 37-kDa protein.The insert from AA34 provided a probe with which a chimeric plasmid (pRI4) encoding all but the nine N-terminal amino acids of the entire protein was selected from a T. palldium(pBR322) genomic library. The nine N-terminal amino acids determined by amino acid sequencing were combined with the DNA sequence encoded by pRI4 to determine the primary structure of the entire 37-kDa protein; the combined sequence made up a polypeptide with a calculated molecular mass of 36,948 Da. Approximately one-third of the deduced sequence was confirmed by N-terminal amino acid analysis of tryptic peptides from the purified 37-kDa protein. Repeated attempts to clone upstream portions of the gene (flaA) by using a variety of strategies were unsuccessful, suggesting that unregulated expression of the intact sheath protein or of its most amino-terminal portions is toxic in Escherichia coli. These studies should provide the basis for further molecular investigations of the endoflagellar apparatus and of treponemal motility.

Cloning, sequencing, and expression of two class B endoflagellar genes of Treponema pallidum subsp. pallidum encoding the 34.5- and 31.0-kilodalton proteins

“…In these two groups, referred to as class A and class B, the single sheath protein having a molecular mass of 37 kDa was designated class A, while the three proteins having molecular masses of 34.5, 33, and 31 kDa, which were related to the core structure of the filament, were designated class B. The T. pallidum endoflagellar genes encoding the class A 37-kDa protein, designated flaA, and a class B protein, designatedflaB2, have recently been cloned and sequenced by Isaacs et al (15) and Pallesen and Hindersson (25), respectively. The designation of the cloned class B gene as flaB2 was based on a comparison of the deduced amino acid sequence with the N-terminal amino acid sequence determined by Norris for the generation of all of the multiple polypeptides.…”

“…Alternatively, one could speculate on the existence of a heterogeneous population of endoflagella in which individual filaments are composed of the 37-kDa subunits (sheath) and only one of the class B (core) subunits. However, it is interesting that the endoflagellar gene which encodes the T. pallidum class A 37-kDa protein has recently been reported to possess a consensus E. coli -35 and -10 promoter sequence (15). Therefore, it is possible that this INFECT.…”

A novel flagellar sheath protein, FcpA, determines filament coiling, translational motility and virulence for the Leptospira spirochete

“…In contrast to the flagellar filaments of enterobacteria, which are composed of a single flagellin protein (Macnab, 1996;Berg, 2003), spirochete PF are a multi-protein complex that comprise of a core, composed of the FlaB family of flagellin-like proteins, and sheath proteins (Li et al, 2000b;Charon and Goldstein, 2002;Wolgemuth et al, 2006). Although FlaA, a protein family conserved across spirochetes (Li et al, 2000b;Wolgemuth et al, 2006;Li et al, 2008;Liu et al, 2010), was found to form a flagellar sheath for Brachyspira hyodysenteriae (Li et al, 2000a), Treponema pallidum (Cockayne et al, 1987;Isaacs et al, 1989) and Spirochaeta aurantia (Brahamsha and Greenberg, 1989) this observation was not confirmed for Borrelia burgdorferi (Motaleb et al, 2004) and L. interrogans (Lambert et al, 2012). Spirochetes appear thus to differ in the composition and organization of their flagella.…”

Membrane Topology and Cellular Location of theTreponema pallidumGlycerophosphodiester Phosphodiesterase (GlpQ) Ortholog

“…A murine monoclonal antibody specific for E. coli alkaline phosphatase (PhoA) was obtained from Caltag Laboratories (Burlingame, Calif.). Rabbit antisera directed against T. pallidum endoflagella (TpEf) (34) and native 47-kDa lipoprotein (Tp47) (22) were described previously.…”