trmD Operon Research Articles

AamA-mediated epigenetic control of genome-wide gene expression and phenotypic traits in Acinetobacter baumannii ATCC 17978.

Individual deletions of three genes encoding orphan DNA methyltransferases resulted in the occurrence of growth defect only in the aamA (encoding AcinetobacterAdenine Methylase A) mutant of A. baumannii strain ATCC 17978. Our single-molecule real-time sequencing-based methylome analysis revealed multiple AamA-mediated DNA methylation sites and proposed a potent census target motif (TTTRAATTYAAA). Loss of Dam led to modulation of genome-wide gene expression, and several Dam-target sites including the promoter region of the trmD operon (rpsP, rimM, trmD, and rplS) were identified through our methylome and transcriptome analyses. AamA methylation also appeared to control the expression of many genes linked to membrane functions (lolAB, lpxO), replication (dnaA) and protein synthesis (trmD operon) in the strain ATCC 17978. Interestingly, cellular resistance against several antibiotics and ethidium bromide through functions of efflux pumps diminished in the absence of the aamA gene, and the complementation of aamA gene restored the wild-type phenotypes. Other tested phenotypic traits such as outer-membrane vesicle production, biofilm formation and virulence were also affected in the aamA mutant. Collectively, our data indicated that epigenetic regulation through AamA-mediated DNA methylation of novel target sites mostly in the regulatory regions could contribute significantly to changes in multiple phenotypic traits in A. baumannii ATCC 17978.

A novel ribosome-associated protein is important for efficient translation in Escherichia coli

Previously, we showed that strains which have been deleted for the 21K gene (hereafter called yfjA), of the trmD operon, encoding a 21-kDa protein (21K protein) have an approximately fivefold-reduced growth rate in rich medium. Here we show that such mutants show an up to sevenfold reduced growth rate in minimal medium, a twofold-lower cell yield-to-carbon source concentration ratio, and a reduced polypeptide chain growth rate of beta-galactosidase. Suppressor mutations that increased the growth rate and translational efficiency of a delta yfjA mutant were localized to the 3' part of rpsM, encoding ribosomal protein S13. The 21K protein was shown to have affinity for free 30S ribosomal subunits but not for 70S ribosomes. Further, the 21K protein seems to contain a KH domain and a KOW motif, both suggested to be involved in binding of RNA. These findings suggest that the 21K protein is essential for a proper function of the ribosome and is involved in the maturation of the ribosomal 30S subunits or in translation initiation.

Functional analysis of the ffh-trmD region of the Escherichia coli chromosome by using reverse genetics.

We have analyzed the essentiality or contribution to growth of each of four genes in the Escherichia coli trmD operon (rpsP, 21K, trmD, and rplS) and of the flanking genes ffh and 16K by a reverse genetic method. Mutant alleles were constructed in vitro on plasmids and transferred by recombination to the corresponding lambda phage clone (lambda 439) and from the phage clone to the E. coli chromosome. An ability to obtain recombinants only in cells carrying a complementing plasmid indicated that the mutated gene was essential, while an ability to obtain recombinants in plasmid-free cells indicated nonessentiality. In this way, Ffh, the E. coli homolog to the 54-kDa protein of the signal recognition particle of mammalian cells, and ribosomal proteins S16 and L19 were shown to be essential for viability. A deletion of the second gene, 21K, of the trmD operon reduced the growth rate of the cells fivefold, indicating that the wild-type 21-kDa protein is important for viability. A deletion-insertion in the same gene resulted in the accumulation of an assembly intermediate of the 50S ribosomal subunit, as a result of polar effects on the expression of a downstream gene, rplS, which encodes ribosomal protein L19. This finding suggests that L19, previously not considered to be an assembly protein, contributes to the assembly of the 50S ribosomal subunits. Strains deleted for the trmD gene, the third gene of the operon, encoding the tRNA (m1G37)methyltransferase (or TrmD) showed a severalfold reduced growth rate. Since such a strain grew much slower than a strain lacking the tRNA(m(1)G37) methyltransferase activity because of a point mutation, the TrmD protein might have a second function in the cell. Finally, a 16-kDa protein encoded by the gene located downstream of, and convergently transcribed to, the trmD operon was found to be nonessential and not to contribute to growth.

Sequences of the Serratia marcescens rplS and trmD genes.

The trmD and rplS genes from the Serratia marcescens trmD operon are presented. These genes are about 85% similar to those of Escherichia coli and encode very nearly identical proteins. However, there are an additional 22 nt around the beginning of trmD in S. marcescens which in addition uses a GUG start codon.

Importance of mRNA folding and start codon accessibility in the expression of genes in a ribosomal protein operon of Escherichia coli

The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kilodalton protein (21K) of unknown function, the tRNA(m 1G37)methyltransferase (TrmD), and r-protein L19, in that order. The synthesis of the 21K and TrmD proteins is 12 and 40-fold lower, respectively, than that of the two r-proteins, although the corresponding parts of the mRNA are equally abundant. This translational control of expression of at least the 21K protein gene ( 21K), is mediated by a negative control element located between codons 18 and 50 of 21K. Here, we present evidence for a model in which mRNA sequences up to around 100 nucleotides downstream from the start codon of 21K fold back and basepair to the 21K translation initiation region, thereby decreasing the translation initiation frequency. Mutations in the internal negative control element of 21K that would prevent the formation of the proposed mRNA secondary structure over both the Shine-Dalgarno (SD) sequence and the start codon increased expression up to about 20-fold, whereas mutations that would disrupt the base-pairing with the SD-sequence had only relatively small effects on expression. In addition, the expression increased 12-fold when the stop codon of the preceding gene, rpsP, was moved next to the SD-sequence of 21K allowing the ribosomes to unfold the postulated mRNA secondary structure. The expression increased up to 150-fold when that stop codon change was combined with the internal negative control element base-substitutions that derepressed translation about 20-fold. The negative control element of 21K does not seem to be responsible for the low expression of the trmD gene located downstream. However, a similar negative control element native to trmD can explain at least partly the low expression of trmD. Possibly, the two mRNA secondary structures function to decouple translation of 21K and trmD from that of the respective upstream cistron in order to achieve their independent regulation.

A regulatory element within a gene of a ribosomal protein operon of Escherichia coli negatively controls expression by decreasing the translational efficiency

The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kDa protein (21K) of unknown function, the tRNA(m1G37)methyltransferase (TrmD), and r-protein L19, in this order. Previously we have shown that the steady-state amount of the two r-proteins exceeds that of the 21K and TrmD proteins 12- and 40-fold, respectively, and that this differential expression is solely explained by translational regulation. Here we have constructed translational gene fusions of the trmD operon and lacZ. The expression of a lacZ fusion containing the first 18 codons of the 21K protein gene is 15-fold higher than the expression of fusions containing 49 or 72 codons of the gene. This suggests that sequences between the 18th and the 49th codon may act as a negative element controlling the expression of the 21K protein gene. Evidence is presented which demonstrates that this regulation is achieved by reducing the efficiency of translation.

Differentially expressed trmD ribosomal protein operon of Escherichia coli is transcribed as a single polycistronic mRNA species

The trmD operon is a four-cistron operon in which the first and fourth genes encode ribosomal proteins S16 ( rpsP) and L19 ( rplS), respectively. The second gene encodes a 21,000 M r polypeptide of unknown function and the third gene ( trmD) encodes the enzyme tRNA(m 1G37)methyltransferase, which catalyzes the formation of 1-methylguanosine (m 1G) next to the 3′ end of the anticodon (position 37) of some tRNAs in Escherichia coli. Here we show under all regulatory conditions studied, transcription initiates at one unique site, and the entire operon is transcribed into one polycistronic mRNA. Between the promoter and the first gene, rpsP, an attenuator-like structure is found ( ΔG = −18 kcal; 1 cal = 4.184 J), followed by four uridine residues. This structure is functional in vitro, and terminates more than two-thirds of the transcripts. The different parts of the trmD operon mRNA decay at a uniform rate. The stability of the trmD mRNA is not reduced with decreasing growth rate, which is in contrast to what has been found for other ribosomal protein mRNAs. Furthermore, earlier experiments have shown the existence of differential expression as well as non-co-ordinate regulation within the operon. Our results are consistent with the regulation of the trmD operon being due to some mechanism(s) operating at the post-transcriptional level, and do not involve differential degradation of different mRNA segments, internal promoters or internal terminators.

Non-autogenous control of ribosomal protein synthesis from the trmD operon in Escherichia coli

The trmD operon of Escherichia coli encodes the ribosomal proteins S16 and L19, the tRNA(m 1G37)methyltransferase and a 21,000 M r protein of unknown function. Here we demonstrate that, in contrast to the expression of other ribosomal protein operons, the amount of trmD operon mRNA and the rate of synthesis of the proteins encoded by the operon respond to increased gene dosage. The steady-state level of the mRNA was about 18 times higher, and the relative rate of synthesis of the ribosomal proteins S16 and L19, the tRNA(m 1G37)methyltransferase and the 21,000 M r protein was 15, 9, 25 and 23 times higher, respectively, in plasmid-containing cells than in plasmid-free cells. Overproduced tRNA(m 1G37)methyltransferase and 21,000 M r protein were as stable as E. coli total protein, whereas the two ribosomal proteins were degraded to a large extent. The steady-state amount of S16 and L19 in the plasmid-containing cells exceeded that in plasmid-free cells by threefold and twofold, respectively. No significant effect on the synthesis of the trmD operon proteins from the chromosomally located genes was observed when parts of the operon were expressed on different plasmids. Taken together, these results suggest that the expression of the trmD operon is not subject to transcriptional or translational feedback regulation, and demonstrate that not all ribosomal protein operons are regulated in the same manner. We propose that ribosomal protein operons that do not encode proteins that bind directly to rRNA are not under autogenous control. Metabolic regulation at the transcriptional level and protein degradation are plausible mechanisms for the control of expression of such operons.

Noncoordinate translation-level regulation of ribosomal and nonribosomal protein genes in the Escherichia coli trmD operon.

The trmD operon of Escherichia coli contains the genes for the ribosomal protein S16, a 21-kilodalton polypeptide of unknown function, the tRNA(1-methylguanosine)methyltransferase, and the ribosomal protein L19, in that order. As reported elsewhere, the operon is transcribed as a single polycistronic mRNA species, and there is no significant difference in the steady-state amounts of different parts of the mRNA (A.S. Byström, A. von Gabain, and G.R. Björk, submitted for publication). Furthermore, accumulation of all parts of the transcript is altered in a stringently controlled manner upon starvation for valyl-tRNA. Here we show that the rate of synthesis of the trmD operon proteins increased with increasing growth rate and that the amount in steady state, at a specific growth rate (k = 1.0), of the tRNA(1-methylguanosine)methyltransferase was 260 molecules per gene copy, which is about 40 times lower than the amount of the two ribosomal proteins, whereas the 21-kilodalton protein was synthesized to the amount of about 850 molecules per gene copy. The lower steady-state amount of the two nonribosomal proteins was not due to a higher turnover rate. Synthesis of the 21-kilodalton and TrmD proteins responded differently from that of the two ribosomal proteins during conditions which provoked amino acid starvation, although accumulation of the entire mRNA molecule responds similarly to the rate of synthesis of the two ribosomal proteins. We conclude that the observed differential and noncoordinate expression is achieved by regulation at the level of mRNA translation.

The structural gene (trmD) for the tRNA(m1G)methyltransferase is part of a four polypeptide operon in Escherichia coli K-12.

The trmD gene, which is the structural gene for the tRNA(m1G)-methyltransferase, is shown to be part of a polycistronic operon. A 4.6 kb SalI-EcoRI chromosomal DNA fragment contains the trmD gene (Byström and Björk 1982). Subclonings, deletion mapping and Tn5 insertions into plasmid pBY03 have established the gene organization of the trmD area on the Escherichia coli chromosome. The different plasmid derivatives were analysed for expression of protein products using the minicell system. Such analyses established the organisation of genes encoding six polypeptides to be SalI1-48 K-13 K-25 K-31 K-15 K-16 K-EcoRI1. The 31 K polypeptide was shown to be the tRNA(m1G)methyltransferase. The trmD operon encodes for four polypeptides; 13 K-25 K-31 K(trmD)-15 K and the direction of transcription is from 13 K (promoter proximal) to 15 K (promoter distal). However, there might be a weak internal promoter between the trmD gene and the gene encoding the 15 K product. The level of expression from this operon in the minicell system does not seem to follow normal polarity since we observed high expression of 13 K, 25 K, and 15 K products but low expression of the internal trmD gene.