Molecular characterization of a large Borrelia burgdorferi motility operon which is initiated by a consensus sigma70 promoter

Abstract

A large motility operon, referred to as the flgB operon, was identified, characterized, and mapped at 310 to 320 kb on the linear chromosome of the spirochete Borrelia burgdorferi. This is the first report that a The bacterial motility apparatus is a highly organized but complicated structure composed of the flagellar filament, the hook-basal body complex, membrane-associated energy-transducing components, and proteins involved in export (1, 49, 69). Most of the flagellar genes of Escherichia coli and Salmonella typhimurium (1, 49), Bacillus subtilis (3,9,22,51,52,56,73), and Caulobacter crescentus (4, 19, 69) have been identified and sequenced. More than 50 genes are involved in flagellar structure and assembly, motility, and chemotaxis. These genes are organized into clusters and constitute functional operons. For example, in E. coli there are four motility and chemotaxis clusters (regions I, II, IIIa, and IIIb) consisting of 15 operons (49). These operons together comprise a large flagellar regulon. In contrast, the organization of the B. subtilis motilitychemotaxis genes is quite different (3,9,17,22,51,56,73). Although these genes are not clustered into four regions as is found in E. coli, the orders of certain flagellar genes in B. subtilis and E. coli are similar. The genes in B. subtilis are arranged such that several small flagellar clusters are involved in the last stage of flagellar assembly (17, 51, 52). In addition, there is a large (26-kb) fla/che locus containing more than 30 structural, motility, and chemotaxis genes (9). The genes encoding the proteins for early flagellar assembly are concentrated within this operon. In C. crescentus (4,19), the flagellar regulon is composed of more than 10 small operons with a gene order different from those in S. typhimurium, E. coli, and B. subtilis.The morphological pathway of flagellar assembly has been studied in detail for E. coli and S. typhimurium (1, 49) and C. crescentus (4, 69). Flagellar synthesis is highly coordinated, involving a specific order of gene expression which is achieved by a tightly controlled transcriptional cascade (1,4,19,60). In E. coli and S. typhimurium, the class 1 master proteins FlhD and FlhC are regulated under catabolite repression by glucose (49). Expression of both proteins results in the activation of all seven class 2 operons encoding components of the hook-basal body complex, the export apparatus, and the flagellum-specific sigma factor 28 (FliA) (36,44,49). The majority of the products encoded by class 2 genes are essential for full expression of class 3 genes.28 is utilized to initiate transcription of the seven class 3 operons. The class 3 gene products include the hook-associated proteins, flagellin, the motor proteins (MotA and MotB), and chemotaxis proteins. All class 3 genes and many of the class 2 genes, including fliA itself, are transcribed from 28 -like promoter elements (36,44,49). In B. subtilis, the genes corresponding to the class 2 and class 3 genes, including those from the large fla/che operon, are tra...

Borrelia burgdorferi Uniquely Regulates Its Motility Genes and Has an Intricate Flagellar Hook-Basal Body Structure

“…3a). The flagellum assembly protein FliI (22,45) and the motor protein FliM (63) were present at similar levels in the wild type and SC-E1, while the level of MotB, which is part of the flagellar stator (33), was slightly reduced in SC-E1 compared to the wild type. Taken together, the Western blot analyses indicated that insertion of kan into flgE did not appreciably alter the amounts of proteins encoded by other select motility genes that were located either upstream or downstream of the mutation.…”

“…Monoclonal antibodies to FlaB (H9724), FlaA, and DnaK were provided by A. Barbour (University of California, Irvine), B. Johnson (Centers for Disease Control and Prevention, Atlanta, GA), and J. Benach (State University of New York, Stony Brook), respectively. Polyclonal anti-FliI, anti-CheA2, anti-CheX, and anti-CheY3 were described previously (22,37,48). Polyclonal anti-recombinant CheW3 was provided by C. Li (unpublished data).…”

“…flgE maps near the center of the large flgB motility operon in the B. burgdorferi chromosome (19,22). This operon is approximately 21 kb long and consists of 26 putative open reading frames.…”

“…Homologs of the class 2 transcriptional regulatory genes fliA and flgM are not apparent, and no 28 promoter consensus sequences are evident in the B. burgdorferi genome (19,22,38). Furthermore, when promoter sequences in B. burgdorferi are analyzed, only 70 promoters are evident for initiation of motility and chemotaxis gene expression (19,22,38).…”

Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles ofBorrelia burgdorferi

“…We overexpressed CsrA Bb (ES25) under the control of a constitutive promoter, P flgB (35), with a C-terminal sixhistidine tag in an lp25-deficient, clonal isolate of B. burgdorferi on May 10, 2018 by guest http://iai.asm.org/ (ML23) by using the borrelial shuttle vector pBSV2 (78). Surprisingly, we found that the colonies on BSK-II agar overlays that grew in the presence of kanamycin (conferred by the shuttle vector) were compact compared to those of the parental strain (data not shown).…”