Transcriptional analysis of the conserved ftsZ gene cluster in Mycoplasma genitalium and Mycoplasma pneumoniae

Abstract

Several experimental approaches were used to construct a detailed transcriptional profile of the phylogenetically conserved ftsZ cell division gene cluster in both Mycoplasma genitalium and its closest relative, Mycoplasma pneumoniae. We determined initiation and termination points for the cluster, as well as an absolute steady-state RNA level for each gene. Transcription of this cluster in both these organisms was shown to be highly strand specific. While the four genes in this cluster are cotranscribed, their transcription unit also includes two genes of close proximity yet disparate function. A transcription initiation point immediately upstream of these two genes was detected in M. genitalium but not M. pneumoniae. In M. pneumoniae, transcription of the six genes terminates at a poly(U)-tailed hairpin. In M. genitalium, this transcription terminates at two closely spaced points by an unknown mechanism. Real-time reverse transcription-PCR analysis of this cluster in M. pneumoniae shows that mRNA levels for all six genes vary at most fivefold and form a gradient of decreasing quantity with increasing distance from the promoter at the beginning of the cluster. mRNA from coding regions was approximately 20- to 100-fold more abundant than that from intergenic regions. We estimated the most abundant mRNA we detected at 0.6 copy per cell. We conclude that groups of functionally related genes in M. genitalium and M. pneumoniae are often preceded by promoters but rarely followed by terminators. This causes functionally unrelated genes to be commonly cotranscribed in these organisms.

FIG. 1.

(A) To-scale schematic of the ftsZ gene cluster and surrounding genes in M. pneumoniae and M. genitalium. Each gene is identified by its name in each of these species, as well as by the COG to which it belongs (27). Common names are indicated for some genes. Numbered bars show where we attempted to detect transcription across gene junctions with RT-PCR. A solid line represents a positive result, and a dashed line represents a relatively weak result. (B) Agarose gel visualization of RT-PCR products as numbered in panel A. Targets 1 to 6 are strand-specific reactions. Target 7 represents long-range RT-PCR across two gene junctions. Lanes + are positive PCR controls with genomic DNA as template. Lanes F represent use of the forward primer in the reverse transcription reaction, which will detect negative-strand RNA; lanes R represent use of the reverse primer in the reverse transcription reaction, which will detect positive-strand RNA. Lanes N are negative controls for DNA contamination done without the reverse transcriptase enzyme.

FIG. 2.

Primer extension determination of the first transcribed nucleotides for MPN314. Autoradiographic results shown were obtained with primer 1; the use of primer 2 produced the same results but only with significantly more template RNA, probably due to primer-specific factors. The same oligonucleotide was used in both the sequencing (lanes labeled G, A, T, and C) and the primer extension (lane PE) reactions to generate the sequence complementary to the mRNA. For primer extension, the primer was 5′ phosphorylated to include a radiolabel, causing its mobility to shift about 1 nt downward relative to the sequencing products. The two results are shown mapped in large capital letters onto the relevant sequence. The predicted −35 and −10 promoter regions and +1 nt (marked with gray boxes) are as determined by the mycoplasma promoter matrix (34). The coding region for MPN314 is italicized. The positions of the two primers are underlined.

FIG. 3.

(A) To-scale schematic showing regions of RNase protection analysis of and surrounding the ftsZ gene clusters of M. genitalium and M. pneumoniae. Probe positions are shown by numbered arrows, where direction indicates polarity. Mapped transcription initiation points are marked by short arrows; mapped transcription termination points are marked by diamonds. RT-PCR targets are marked for reference as in Fig. 1. (B) Autoradiographic results are shown only for those probes that mapped transcript termini; full protection of the probe was observed for many junctions (see the supplemental material). Genomic coordinates are listed for each probe as obtained from GenBank sequences NC_000912 (M. pneumoniae) and NC_000908 (M. genitalium). Three reactions are shown with each probe. As numbered, they are as follows: 1, probe hybridized with mycoplasma RNA and digested with an RNase A/T1 mix (except for MG probe 8, which was digested with RNase I); 2, probe hybridized with a corresponding amount of yeast RNA and digested with an RNase A/T1 mix (RNase I for MG probe 8); and 3, probe hybridized with the same amount of yeast RNA but incubated without RNases. The ladder is composed of 100-, 200-, 300-, 400-, 500-, 750-, and 1,000-nt fragments (RNA Century Marker Plus template set; Ambion). The size of the probe, including 12 to 13 nt of T7 promoter sequence, is labeled. The size of each digested species is also labeled. This was determined by using ImageJ software (National Institutes of Health) to mark a coordinate for each of the ladder fragments and the resulting fragments and then using DNASIZE (25) to calculate the size of the unknown fragment. Accuracy of this assay was determined to be within 30 nt (from −20 to +10 nt) of the calculated result.

FIG. 4.

Quantitative Taqman real-time RT-PCR results for nine regions of analysis within and flanking the M. pneumoniae ftsZ gene cluster. These regions are represented as numbered black arrows on a to-scale diagram. Results have been normalized to the average value of the 313/314 amplicon (1); each point on the graph represents the result for one run. The average value for each amplicon is represented by a labeled bar whose length is to scale. The average for each amplicon comes from at least three repeated experiments and is relative to a standard curve of a recombinant RNA. The trendline shows an exponential fit to the average results for amplicons 2 to 8.

FIG. 5.

Summary of results of transcriptional analyses of and surrounding the phylogenetically conserved ftsZ gene clusters of M. pneumoniae and M. genitalium. Each diagram is to scale and shows the cluster in the context of surrounding genes of disparate function. This demonstrates how gene clusters may be identified merely as groups of closely spaced genes, regardless of functional information. Transcripts that can be deduced from each type of experimental approach are shown as long arrows below the diagrams. A transcription initiation point is represented by a short arrow, termination at a poly(U)-tailed hairpin by a stem-loop symbol, and termination at a sequence of unknown significance by a vertical line. If an experiment does not define the termini of a transcript, then the region that can be inferred is extended by dotted lines.