Characterization of mutations in the metY-nusA-infB operon that suppress the slow growth of a DeltarimM mutant

Abstract

The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes. A DeltarimM mutant shows a sevenfold-reduced growth rate and a reduced translational efficiency, probably as a result of aberrant assembly of the ribosomal 30S subunits. The slow growth and translational deficiency can be partially suppressed by increased synthesis of the ribosome binding factor RbfA. Here, we have identified 14 chromosomal suppressor mutations that increase the growth rate of a DeltarimM mutant by increasing the expression of rbfA. Nine of these mutations were in the nusA gene, which is located upstream from rbfA in the metY-nusA-infB operon; three mutations deleted the transcriptional terminator between infB and rbfA; one was an insertion of IS2 in infB, creating a new promoter for rbfA; and one was a duplication, placing a second copy of rbfA downstream from a promoter for the yhbM gene. Two of the nusA mutations were identical, while another mutation (nusA98) was identical to a previously isolated mutation, nusA11, shown to decrease termination of transcription. The different nusA mutations were found to increase the expression of rbfA by increasing the read-through of two internal transcriptional terminators located just downstream from the metY gene and that of the internal terminator preceding rbfA. Induced expression of the nusA(+) gene from a plasmid in a nusA(+) strain decreased the read-through of the two terminators just downstream from metY, demonstrating that one target for a previously proposed NusA-mediated feedback regulation of the metY-nusA-infB operon expression is these terminators. All of the nusA mutations produced temperature-sensitive phenotypes of rimM(+) strains. The nusA gene has previously been shown to be essential at 42 degrees C and below 32 degrees C. Here, we show that nusA is also essential at 37 degrees C.

FIG. 1

Genetic organization of the metY-nusA-infB operon region on the chromosomes of wild-type E. coli (A) and strains that contain a duplication covering the 3′ part of infB to the 5′ half of yhbM (B; shaded region). P₋₁, P₁, P₂, and P indicate the locations of promoters; T, T₁, T₂, and T₃ indicate different transcriptional terminators; while R E and R III show sites for the RNA-processing enzymes RNase E and RNase III, respectively. The horizontal arrows represent transcriptional products. For references and explanations of gene symbols, see the introduction.

FIG. 2

Locations and natures of different alterations in NusA that suppress the slow growth of a ΔrimM102 mutant. The linear functional map of NusA shown has been modified from reference , integrating information from reference . S1 and KH represent different motifs found to be important for binding to RNA (2, 11, 49).

FIG. 3

Transcriptional analyses of the metY-nusA-infB operon in different mutants. (A) Quantitation of metY-nusA-infB operon mRNAs in wild-type (wt) and nusA mutant strains by Northern blot analysis. Five micrograms of total RNA was subjected to electrophoresis in an agarose gel containing formaldehyde, transferred to a Hybond N filter, and probed with a radiolabeled PCR fragment corresponding to the p15a gene. The strains used (with the relevant genetic markers in parentheses) are indicated above the respective lanes. The sizes of the ³²P-end-labeled fragments of the 1-kb DNA ladder (GIBCO BRL Life Technologies Inc., Gaithersburg, Md.) are indicated. The 6.7-kb transcript results from read-through of the metYt₁ and metYt₂ terminators between metY and p15a and the infBt₃ terminator just upstream from rbfA, while the 4.8-kb transcript terminates at the infBt₃ terminator (Fig. 1). The amounts of these transcripts (determined by quantitation of the radioactivity using a PhosphorImager from Molecular Dynamics, Inc.) in the different nusA mutants were normalized to those for the nusA⁺ strain MW100. The read-through (RT) of the infBt₃ terminator was calculated as the amount of radioactivity in the 6.7-kb band divided by the sum of the radioactivity in the 4.8- and 6.7-kb bands. (B) Northern blot analysis showing the effect of deletions in the infBt₃ transcriptional terminator on the amount of the 6.7-kb transcript relative to that of the 4.8-kb transcript. (C) Identification of the 5′ end of the mRNA resulting from transcription initiation at a new promoter created by the insertion of IS2 in infB. Primer extension analyses of mRNA and DNA sequencing of a PCR fragment covering the 3′ part of infB and the 5′ part of rbfA from a wild-type strain were performed using a ³²P-end-labeled primer binding to positions −54 to −73 relative to the start codon of rbfA. The primer extension product obtained for strain JML125 (infB::IS2) corresponds to an mRNA 5′ end at the A 6 nt downstream from the −10 region of the proposed promoter.

FIG. 4

Synthesis of individual proteins at 37°C in the ΔrimM102 mutant and three different suppressor strains. Total cell extracts of the indicated strains labeled with [³⁵S]methionine were separated on 2-D gels. Only the relevant part of each gel is shown. The indicated proteins are as follows: 1, RbfA; 2, H-NS; 3, 4, and 5, ribosomal protein S6. The position of RbfA on the gels was determined previously (4), and the identities of the other proteins were obtained by comparing the gels with those of VanBogelen et al. (50).

FIG. 5

Transcriptional fusions integrated into the lacI-lacZ region of the chromosome. The fusion point in strain GOB434 is 245 bp downstream from that in strain GOB435. P₋₁, P₁, and P₂ indicate promoters of the metY operon, and P_tet is the promoter for the tetracycline resistance gene of plasmid pBR322; T₁ and T₂ indicate the terminators between metY and p15a, and T_rplS is the terminator of the trmD operon. Two different RNase III-processing sites are indicated, one (left-hand arrow) native to the region between metY and p15a and the other (right-hand arrow) present in the lacZ fusion vector used (22). For a description of the construction of the different fusions, see Materials and Methods.

FIG. 6

Transcriptional read-through of terminators in different nusA mutants. The read-through of the terminators T₁ and T₂ between metY and p15a (A) and that of the attenuator upstream from rpsP (B) were calculated as the ratio of the β-galactosidase activity from a lacZ fusion containing the respective terminator(s) to the activity from a fusion lacking the terminator(s) in the genetic backgrounds indicated. The standard deviations are shown as error bars.

FIG. 7

Deletion of the chromosomal nusA gene. (A) Plasmid pJML001 carries a metY-nusA-infB operon fragment containing an in-frame deletion of nusA cloned into the temperature-sensitive allelic replacement vector pMAK705. (B) Genetic organization of the chromosomal metY-nusA-infB operon containing the nusA deletion and that of the complementing nusA⁺ plasmid after resolution of cointegrates formed between plasmid pJML001 and the chromosome of the wild-type strain, MW100. P represents the P₋₁ and P₁ promoters, and T represents the infBt₃ terminator.