Construction and use of derivatives of transposon Tn4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis

Abstract

Previous attempts to introduce transposon Tn4001 into Mycoplasma pulmonis and Mycoplasma arthritidis have not been successful, possibly due to functional failure of the transposon's gentamicin resistance determinant. Tn4001C and Tn4001T were constructed, respectively, by insertion of a chloramphenicol acetyltransferase gene and the tetM tetracycline resistance determinant into Tn4001. Both Tn4001C and Tn4001T transposed in M. pulmonis, and Tn4001T transposed in M. arthritidis. The incorporation of a Tn4001T derivative that contained lacZ into either Mycoplasma species resulted in transformants with readily detectable LacZ activity. Tn4001T may be of general utility for use as a mycoplasma cloning vehicle because tetM functions in all species of Mycoplasma examined thus far.

FIG. 1

Schematic diagram of Tn4001mod in plasmid pISM2062, Tn4001C in pIVC-1, Tn4001T in pIVT-1, and Tn4001T-lac in pIVT-lac. Dark regions indicate Tn4001mod sequences. Unshaded regions indicate the cat, tetM, and arcA-lacZ genes. The hatched region of Tn4001C indicates the vsa promoter region. Thin lines in Tn4001T and Tn4001T-lac indicate streptococcal sequences originating from pJI3 that flank tetM. Arrows represent direction of gene transcription. Abbreviations: S, SmaI; B, BamHI; H, HindIII; K, KpnI; Term, putative transcription terminator derived from M. arthritidis sequences upstream of arcA. The bar represents the 1.5-kb PCR product used to probe transformants containing Tn4001T or Tn4001T-lac.

FIG. 2

Southern analysis of mycoplasmal transformants. (A) Autoradiogram of a Southern blot hybridized with a cat-specific probe. Lanes 1 through 5 are HindIII-digested genomic DNAs from independent transformants of M. pulmonis containing Tn4001C. (B) Autoradiogram of a Southern blot hybridized with the probe derived from sequences upstream of tetM (Fig. 1). Lanes 1 through 5 are HindIII-digested genomic DNAs from five independent isolates of M. arthritidis transformed with pIVT-lac.

FIG. 3

Schematic diagrams of the region of the M. arthritidis chromosome containing the arcA gene (top) and the chimeric arcA-lacZ gene (bottom). Coding regions are indicated by thick lines, with arrows indicating direction. Regions shaded in black originate from the M. arthritidis chromosome. The stippled region is derived from the pZErO vector used for gene construction. The unshaded region is the lacZ coding region derived from pISM2062.2lac. The location and direction of primers used for PCR amplification are illustrated as follows: +, degenerate primers used for the initial amplification of an internal portion of arcA, and × and ○, primers used to amplify the 5′ and 3′ ends, respectively, of arcA by inverse PCR. The location of the putative transcription terminator (Term), the 15-nucleotide poly(A) tract, and the arcA SD sequence are also shown. For clarity, the 3-kb lacZ coding region is drawn at one-half scale.

FIG. 4

Blue (LacZ⁺) M. pulmonis (A) and M. arthritidis (C) colonies transformed with pIVT-lac, assayed on mycoplasma agar plates supplemented with X-Gal. Control colonies (LacZ⁻) arising from cells that had not been transformed are shown for M. pulmonis and M. arthritidis in panels B and D, respectively.