the function of micf rna - journal of biological chemistry · the journal of biological chemistry 0...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No. 30, Issue of October 25, pp. 17961-17970, 1989 Printed in U. S. A.

The Function of micF RNA micF RNA IS A MAJOR FACTOR IN THE THERMAL REGULATION OF OmpF PROTEIN IN ESCHERICHIA COLI *

(Received for publication, June 16, 1989)

Janet Andersent 11, Steven A. ForstTQ, Kun Zhao$, Masayori InouyelI, and Nicholas Delihas$** From the $Department of Microbiology, School of Medicine, State University of New York, Stony Brook, New York 1 I794 and the TDepartment of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854-5635

The role of chromosomally derived micF RNA as a repressor of outer membrane protein OmpF of Esche- richia coli was examined for various growth conditions. Levels of micF RNA as determined by Northern analyses are found to increase in response to cell growth at high temperature, in high osmolarity or in the presence of ethanol. After a switch to higher growth temperature, the levels of ompF mRNA and of newly synthesized OmpF decrease with time in E. coli strain, MC4100 but these decreases are not observed in isogenic micF deletion strain, SM3001. In addition, while levels of ompF mRNA are substantially reduced in both strains in response to high osmolarity or ethanol at 24 “C, the reduced levels in the parental strain are still 4-5-fold lower compared with the micF deletion strain. These findings indicate that chromosomally derived micF RNA plays a major role in the thermal regulation of OmpF and represses OmpF synthesis in response to several environmental signals by decreasing the levels of ompF mRNA. Analyses of the effect of a multicopy micF plasmid on the levels of OmpF and ompF mRNA after an increase in temperature indicated that multicopies of micF RNA markedly inhibited OmpF synthesis but did not accentuate ompF mRNA decrease. These data suggest that multicopy micF inhibits OmpF synthesis primarily through translational inactivation of ompF mRNA and that a limiting factor in addition to micF RNA is necessary to destabilize ompF mRNA.

The chromosomal micF RNA gene of Escherichia coli pro- duces an antisense RNA against the mRNA for the major outer membrane protein OmpF (Mizuno et al., 1984; Andersen et al., 1987). The gene for the repressor RNA was discovered from the observation that cloning a restriction fragment which contained the micF gene on a multicopy plasmid inhibited OmpF synthesis dramatically in the transformed cells (Mizuno et al., 1983b; Mizuno et al., 1984). The predominant

tion Grants DBM 85-02213 and DBM 88-03122 (to N. D.) and * This work was supported, in part, by National Science Founda-

National Institutes of Health Grant GM 19043 and American Cancer Society Grant N1387N (to M. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported on National Research Service Award GM 1553. )I Recipient of Public Health Service Training Grant 5T32 CA

09176 awarded by the National Cancer Institute, Department of Health and Human Services and a recipient of a Sigma-Xi Grants- in-Aid for Research Award for 1988-1989.

** To whom correspondence should be addressed.

transcript from the chromosomal micF RNA gene was char- acterized and shown to be a 4.5 S RNA (93 nucleotides) which is triphosphorylated at its 5’ end (Andersen et al., 1987).

Small antisense RNAs have been found to function as repressors in several systems (for review see Inouye and Delihas, 1988; Inouye, 1988). micF RNA is unique as a repressor RNA in that its gene (47’) is distal from the gene coding for OmpF (21’) on the E. coli chromosome and yet its primary sequence shows extensive complementarity with the 5‘ end of ompF mRNA (Mizuno et al., 1984; Andersen et al., 1987). micF RNA is believed to negatively regulate expression of OmpF, in part, by hybridizing to ompF mRNA at its ribosome-binding domain (Mizuno et al., 1984; Andersen et al., 1987).

The micF gene is upstream from the gene for another outer membrane porin protein, OmpC (Inokuchi et al., 1982; Mizuno et al., 1983a; Mizuno et al., 1984). OmpF and OmpC are coordinately regulated at the level of transcription in response to the osmolarity of the growth medium (Hall and Silhavy, 1981~). I t was then intriguing to find a gene near ompC whose amplification could suppress the synthesis of OmpF.

Earlier, Matsuyama and Mizushima (1985) reported that the micF gene does not play any role in the osmoregulation of ompF from gel analyses of the steady state amount of OmpF in a micF deletion strain and its parental strain. However, later, in a kinetic analysis of newly synthesized OmpF after a shift from low to high osmolarity growth conditions (Aiba et al., 1987), it was found that the micF gene facilitated the rapid and complete repression of OmpF production indicating that chromosomally derived micF RNA indeed plays a role in the osmoregulation of OmpF porin.

In another study, Misra and Reeves (1987) demonstrated that activation of the micF RNA gene was necessary for the suppression of OmpF which occurs as a result of a mutation in the tolC locus. They also determined by means of 0mpF:lacZ operon and protein fusions that suppression of OmpF via micF/tolC occurs at a post-transcriptional level. Also, Cohen et al. (1988) have recently found that the marA (multiple- antibiotic-resistant) locus represses OmpF primarily by a post-transcriptional mechanism, and this repression correlates with increased micF expression. These studies provide genetic evidence to support the hypothesis that micF RNA is a repressor of OmpF and acts post-transcriptionally.

OmpF protein levels have been shown to vary in response not only to the osmolarity of the growth medium, but also to the growth temperature (Lugtenberg et al., 1976; Lundrigan and Earhart, 1984). OmpF creates the larger passive diffusion pore and is most abundant during growth conditions of low temperature and low osmolarity which would be found outside the human body (Lutenberg et al., 1976; van Alphen and

17961

17962 The Function of micF RNA TABLE I

Strains and plasmids Strain Genotype Ref.

E. coli K-12 MC4100 F-, A lacU169 araD139 Casadaban (1976)

SM3001 MC4100 A micFl JA221/F'recA

rpsL relA thi flbB Matsuyama and Mizushima (1985)

hsdR leuB6 lacy thi recA A Nakamura and Inouye (1982) trpE5/F'lacIq lac+ proAB lacZ YA

pho(Am) supC(Ts) str sc122 trp(Am) lac(Am) mal(Am) Beckman and Cooper (1973)

K165 SC122 htpR- Neidhardt and VanBogelen (1981) Plasmid Relevant properties Ref.

pKI0041 Ap'; vector pBR322; cloned This study genes, micF and ompC (see text)

gene, ompF

genes, micF and ompC up to EcoRI site

gene, three tandem micF genes

pGR201

pJANO12

Ap'; vector pBR322; cloned Ramakrishnan et al. (1985)

Ap'; vector pBR322 cloned This study

pAM336 Ap'; vector pUC1S; cloned Andersen et al. (1987)

Lugtenberg, 1977; Kawaji et al., 1979; Hall and Silhavy, 1981a; Lundrigan and Earhart, 1984). While much is known concerning osmoregulation of porin production, relatively little is known concerning the modulation of porins in response to changes in temperature (Lugtenberg et al., 1976; Hall and Silhavy, 1981c; Lundrigan and Earhart, 1984; Forst and In- ouye, 1988).

In this report, we have investigated micF RNA as a repressor of OmpF synthesis by directly observing the effect of micF RNA on steady state levels of ompF mRNA and on the translational activity of the mRNA pool in uiuo. Comparisons of the levels of ompF mRNA and newly synthesized OmpF in the micF deletion strain (SM3001), and its isogenic parental (MC4100) grown under various conditions, have led to the discovery that chromosomally derived micF RNA is a major factor in the thermal regulation of OmpF production and that micF RNA participates in the decrease of ompF mRNA levels in response to various stress conditions. Given that micF RNA affects ompF expression a t a post-transcriptional level, these findings have ramifications with respect to the regulation of messenger RNA half-life.

MATERIALS AND METHODS

Cell Strains and Plasmids-The E. coli K12 strains and plasmids bearing the micF RNA gene used in this study are listed in Table I. Reference strains were transformed with pBR322 to maintain ampicillin resistance so that these cells could be grown in the same medium as cells transformed with plasmids hearing the micF gene.

Cell Growth and RNA Extraction-Overnight cultures were grown under temperature conditions and in growth medium identical to initial experimental conditions in order to establish the cells a t steady state. In experiments where proteins were labeled, the cells were grown in M9 X medium (Miller, 1972) supplemented with 0.4% glucose, 2 rg/ml thiamine, 20 pg/ml ampicillin and, if strain JA221 were grown, 40 pg/ml each of leucine and tryptophan. Otherwise nutrient broth (Difco Laboratories) containing 20 pg/ml ampicillin was used for cell growth. Low osmolarity signifies medium without additional salt or sucrose; high osmolarity signifies medium with sucrose added to 20%. Cells were grown at several temperatures: 22- 24 and 30 "C (low temperature); 37 and 42 "C (high temperature).

Temperature shift experiments were accomplished by the addition of an equal volume of hot medium (50 or 60 "C) to cultures actively growing at room temperature (24 "C) so that a temperature of 37 or 42 "C (respectively) was quickly reached, or the addition of an equal

volume of cold medium (4 "C) to cultures actively growing at 37 'C so that a temperature of 20-24 "C was quickly reached. The cultures after temperature shift were grown at the new temperature until harvested. Shifts to high osmolarity were carried out by the addition of concentrated (60%) sucrose in nutrient broth (or M9 X ] V i ) to growing cultures resulting in a final sucrose concentration of 20%. Additionally, cells actively growing at 24 "C were exposed to 10% ethanol, 100 p~ copper sulfate, chloramphenicol at a final concentration of 10 pg/ml, sufficient 10 N NaOH to raise the pH to 8.8, or 0.05-1.0 mM H,On. After exposure, the cells were incubated at 24 "C for 1-2 h.

Before harvesting cells, sodium azide was added to the growth medium to a final concentration of 0.02 M, and cells were quick chilled in liquid nitrogen to 0 "C. Cells were then pelleted by centrifugation. RNA was purified from pelleted cells as described in Andersen et al. (1987) except the RNA was not column purified to segregate low molecular weight species. RNA concentrations were determined by ultraviolet (UV) absorption at A,,,. The stability and abundance of ribosomal and tRNAs allowed the samples to be normalized based on UV absorption readings at AZM. RNA samples (2.5 fig) were electrophoresed on an agarose gel containing 50 mM Tris-borate, pH 8.3, 1 mM EDTA (TBE), ethidium bromide, and visualized under UV light to assay the quality of the extracted RNA and the accuracy of determined ribosomal and tRNA concentrations (data not shown).

Northern Blot Analyses-To assay relative levels of the 4.5 S micF RNA, an equal quantity (25-50 pg) of total RNA for each sample was denatured and electrophoresed as follows: 1) heating in 7 M urea and TBE at 50 "C for 5 min and electrophoresis in a 6% polyacrylamide slab gel containing 7 M urea and TBE or 2) heating in 12% formaldehyde, 20 mM MOPS,' pH 7 , 5 mM NaOAc, 0.5 mM EDTA at 50 "C for 5 min followed by the addition of formamide to 50% and running dyes xylene cyanole ff and bromphenol blue and electrophoresis in a 1.5% agarose gel containing 6% formaldehyde, 20 mM MOPS, pH 7, 5 mM NaOAc, and 0.5 mM EDTA. To assay the relative levels of ompF mRNA from cells grown under various growth conditions, RNA samples used to assay micF RNA levels were normalized to 20-30 r g and electrophoresed in agarose gels. In order to visualize micF RNA transcribed from the chromosomal gene, more total RNA (100 rg) needed to be loaded onto the gels for each sample. Resulting RNA bands were viewed by UV shadowing in order to assess the uniformity of ribosomal and transfer RNA application to the gel. Although, the use of polyacrylamide gels in Northern blot analyses resulted in better resolution of micF RNA from other small RNAs, the efficiency of retrieved counts/minute (cpm) from the membrane was poor. Agarose gels, which provided a better yield of cpm, were also used in some

The abbreviation used is: MOPS, 4-morpholinepropanesulf~nic acid.

The Function of micF RNA 17963

analyses of chromosomal micF RNA levels. Also, in order to compen- sate for problems in Northern blotting, such as uneven sample application, samples were harvested and/or loaded in duplicate or triplicate, where possible, and the results averaged. Column purified low molecular weight RNAs (6 pg) isolated from cells containing pAM336 (Andersen et al., 1987), and 3"labeled E. coli 5 S RNA and fMet-tRNA (England and Uhlenbeck, 1978) were electrophoresed on the polyacrylamide gels as markers and as controls. The low molecular weight sample allowed comparisons to be made between the various blots.

The procedures used for electroelution, Northern hybridization, and filter washing are described by the manufacturers' instructions for Nytran Filters (Schleicher and Schuell), except that to reduce background levels in later experiments, the first set of washes was done at 50 "C and the two subsequent washes at 42 "C. The banded RNAs were transfered from polyacrylamide gels to a Nytran filter (0.1 pm) by electroblotting in 10 mM Tris acetate, pH 7.8, 5 mM NaOAc and 0.5 mM EDTA for 1 h using a current of 0.5 amps. If the RNAs were electrophoresed on agarose gels, transfer to Nytran was accomplished by diffusion in 1.5 M NaCl, 0.15 M sodium citrate, pH 7 (10 X SSC, where SSC is standard sodium citrate) over a 5-h period (Maniatis et al., 1982).

Several types of labeled probes were used in Northern blot analyses: nick-translated (Maniatis et al., 1982) or randomly primed (Boehrin- ger-Mannheim kit) double-stranded DNA, or a complementary DNA oligomer kinased at its 5' end with 32P (Maniatis et al., 1982). The probe was then ethanol precipitated with 10 pg of salmon sperm DNA. 1-5 X lo7 counts, as determined by counting trichloroacetic acid precipitates of a fraction of freshly labeled probe, were incubated with prepared membranes.

To visualize the relative levels of micF RNA, the filters were probed with either a 0.9-kilobase nick-translated EcoRI/HindIII restriction fragment from pAM336 which contains the tripartite micF RNA genes, or with a 5' 32P-labeled 54-mer complementary to the central portion of 4.5 S micF RNA (positions 11-64). To visualize the relative levels of ompF mRNA, a 0.6-kilobase restriction fragment containing the 5' end of the ompF structural gene was cut from pGR201 with PuuII and XbaI and labeled with [w3*P]dCTP (Amersham Corp.) by random priming. This probe appears to be specific for ompF mRNA under the conditions used in our Northern analyses (see above) since 1) no signal was observed for a strain deficient in OmpF, 2) an enhanced signal was observed for cells harboring a multicopy ompF plasmid, and 3) OmpF protein data correlates with the Northern blot results. Also, Northern blot results for the highly homologous ompC mRNA showed a different pattern for the same RNA samples (data not shown) and correlated with that observed for OmpC protein data.

The blots were autoradiographed on Kodak SB-5 or X-Omat AR diagnostic x-ray film with Du Pont Cronex intensifying screens (if needed). Using autoradiographs as templates, uniform bands were sliced from the Nytran membranes, placed in scintillation mixture (Ecolume from ICN Radiochemicals) and counted for greater than 5 X lo3 total counts. To obtain an average background count, pieces of the membrane were cut the same size as the sample bands in several areas of low radioactivity and counted.

Protein Radiolabeling, Purification, and Gel Ekctrophoresk- Pulse-labeling of E. coli proteins with [35S]methionine, the prepara- tion of outer membrane proteins and electrophoretic procedures (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are as previously described (Forst et al., 1989). In addition, unlabeled outer membrane protein gels were stained with Coomassie Brilliant Blue for analysis.

Gel Scans-Autoradiographs of labeled protein gels, dried Coo- massie Blue-stained gels, and gel photographs were scanned on the LKB Ultrascan XL Laser Densitometer. The scans were analyzed with the LKB 2400 Gelscan Software package. Lanes were scanned two to three times each and the results averaged.

RESULTS

The Levels of micF RNA under Various Growth Condi- tiom-Northern blot analysis shows that chromosomal micF RNA levels are near background in cells (strain MC4100, recA') grown to early log phase in low osmolarity at 24 "C (Fig. 1, A and B, lane To) but dramatically increase within 2 h if cells are subjected to 37 "C, 10% ethanol at 24 "C or 20% sucrose at 24 "C (Fig. 1B). A minor increase is seen in the levels of micF RNA if the cells were grown at 24 "C for an

A. To 24; 37OC E s ""-

-4.5s micF RNA -

8. 1,200

1,000

000

CPM 600

400

200

FIG. 1. Steady state levels of chromosomally derived micF RNA in cells grown under various stress conditions. Panel A, autoradiograph of a Northern blot analysis of RNA harvested from MC4100 transformed with pBR322 grown to early log phase at 24 "C in M9 X *h medium, divided into aliquots which were either harvested (To lune) or grown for another 2 h a t 24 "C (lune 24 "C), 37 "C (lune 37 "C), in 10% ethanol a t 24 "C (lane E), or in 20% sucrose at 24 "C (lune S). RNAs were separated by electrophoresis on an agarose gel before transfer to Nytran filters. The probe was a 32P-5'-labeled single-stranded DNA oligonucleotide (54-mer) complementary to the central portion of micF RNA (positions 11-64). For each tested stress condition, cells were harvested in triplicate and the three separate samples were normalized for 100 pg of total RNA before loading onto the agarose gel. Panel B, bar graph showing the cpm retrieved from uniform bands cut from the membrane using the autoradiograph shown in panel A as a template. The cpm of the triplet sets were averaged.

additional 2 h (Fig. lA, lane 24 "C) and may reflect a response to cell concentration. However, the levels are about 10-fold higher at 37 "C, 17-fold higher in ethanol, and 5-fold higher at high osmolarity when compared with the levels in the 24 "C sample. The increased micF RNA levels seen in ethanol or at high osmolarity could not be due to increased cell concentration since the cells grew slower under these conditions than in the 24 "C control sample. Also it is unlikely that increased micF RNA levels at high temperature are due to increased cell concentration (see below), although increasing cell concentration may account for a small portion of increased micF RNA at high temperature.

The increase of micF RNA levels in response to increased temperature or stress appears to be due, at least in part, to transcriptional regulation of the gene. An analysis of P-galactosidase activity of the lacZ gene fused to the micF gene on a multicopy plasmid (plasmid B, Mizuno et al., 1983b) in cells grown at 24 "C and then subjected to 37 "C, ethanol or sucrose for 1 h showed elevated activity (data not shown).

17964 The Function of micF RNA

4.5s W F RNA-

80

60

CPM 40

20

C

B.

- 5s RNA

- fmet tRNA

n

FIG. 2. Steady state levels of plasmid derived micF RNA in cells grown under various stress conditions. Panel A, autoradiograph of a Northern blot analysis of RNA harvested from JA221 cells harboring the multicopy plasmid pKI0041 which contains the micF gene (see Table I and text). The cells were grown as described in Fig. 1 except that the cells grew under the stress conditions for 75 min. RNA samples are from cells grown to early log phase at 24 "C and harvested before changing growth conditions (lane To); cells which continued to grow at 24 "C for an additional 75 min (lane 24 "C); cells grown in 100 p~ CuS04 at 24 "C (lane C3'); cells grown in 10% ethanol a t 24 "C (lane EtOH); cells grown in 10 pg/ml chloramphenicol a t 24 "C (lane Chl); cells grown at pH 8.8 at 24 "C (lane pH 8.8); cells grown in 20% sucrose at 24 "C (lane S); cells grown at 37 "C (lane 37 "C). The lane marked LMW contains 6 pg of low molecular weight RNAs, obtained from cells harboring pAM336, and purified over a Sephadex G-100 column in order to concentrate the quantity of micF RNA (Andersen et al., 1987). The lane marked M contains 3"labeled (England and Ohlenbeck, 1978) marker RNAs, E. coli 5 S RNA and met-tRNA. RNAs were separated by electrophoresis on a polyacrylamide gel before transfer to Nytran filter. The probe was a nick-translated 0.9-kilobase restriction fragment containing the tripartite micF RNA genes cut from pAM336 with Hind111 and EcoRI. Panel B, bar graph showing the cpm retrieved from uniform micF RNA bands cut from the membrane using the autoradiograph shown in panel A as a template.

Comparisons were made of the change in levels of multicopy plasmid-derived micF RNA in response to the stress conditions described above for those of the chromosomal micF RNA (Fig. 2). The plasmids were transformed in a recA- strain of E. coli, JA221. Plasmid pKI0041, a pBR322 deriva- tive, contains the region of the E. coli chromosome at 47' which includes the micF RNA and ompC genes. The plasmid- encoded ompC gene has a point mutation which causes its transcript not to be translated.' This plasmid was used in our study to prevent an inadvertent exclusion of upstream sequences necessary for transcriptional regulation of the micF RNA gene (such as the OmpR-binding sequence (Norioka et al., 1987)). Levels of plasmid-derived micF RNA also are elevated after the cells are grown for 2 h at 37 "C @-fold) or at 24 "C in the presence of 10% ethanol (12-fold) or 20% sucrose (3-fold) (Fig. 5). In addition, multicopy micF RNA levels were elevated in 100 PM Cu2+ (%fold), but not in cells grown in the presence of chloramphenicol (Fig. 2) another heat shock inducer (Nover, 1984). H202 (data not shown) and

* M. Inouye and K. Ikenaka, personal communication.

A. HEAT INDUCTION: 2 2 "C-37OC HEAT

LMW TO ' f0" 45" 90" 3' 6' 15' M

- 5s RNA

- 4.55 &F RNA

- fmet tRNA

B. COLD REPRESSION: 37 OC- 2OoC COLD

LMW To ' 1' 5' 10' 30' 60' 120'WN M

1, - 5s RNA

- 4.5s C F RNA a - fmet tRNA

C. AT 2 4 "C SUCROSE

LMW To ' I' 5' IO' 30' 60' O/N M

- 5s RNA

- 4.5s &F RNA

- fmet tRNA

FIG. 3. Autoradiographs of Northern blot analyses of total RNA harvested from cells bearing pKI0041 and harvested at time points after sudden introduction of heat, cold, or high osmolarity. The amount of total RNA electrophoresed for each sample was 50 pg. The lanes marked LMW and M and the probe are as described in Fig. 2. In panel A, the temperature was raised from 22 to 37 "C by the addition of equal volume of medium equilibrated to 50 "C. The lane marked To contains RNA from cells harvested just before the increase in temperature and 10" - 15' denote time in seconds or minutes after the temperature shift up. In panel B, the temperature was lowered from 37 to 20 "C by the addition of equal volume medium equilibrated to 4 "C. Lunes marked 1'-120' denote time in minutes after temperature shift down. The lane marked To is as described for panel A. The lane marked O/N contains RNA from cells grown at 20 "C overnight in low osmolarity. Panel C, autoradiographs of Northern blot analyses of RNA derived from cells harboring pKI0041 grown at 24 "C in nutrient broth and harvested at time points (1 '-60' and overnight, O/N) after a sudden shift in osmolarity with sucrose (final concentration of 20%). The lane marked TO is as described for panel A .

basic pH (Fig. 2) which induce genes controlled by the oxi- dative stress (oxyR gene) and SOS (Zed gene) regulons, respectively (Christman et al., 1985; Schuldiner et al., 1986; Taglicht et al., 1987; VanBogelen et al., 1987), also failed to cause an increase in the levels of micF RNA.

The stress-induced increase in the levels of micF RNA from the gene on pKI0041 is similar, although somewhat less pronounced, to that observed for micF RNA from the chromosomal gene (compare with Fig. l ) , i.e. the data show that higher temperatures, increased osmolarity, or 10% ethanol results in proportionately similar increased levels of micF RNA. While the response of the micF gene on plasmid pKI0041 mimics the response of the chromosomal gene, such was not the case when similar analyses were done with plasmids containing the micF RNA gene within the CX28 restriction fragment, e.g. osmoregulation was limiting (data not

The Function of micF RNA

A. MC 4100 A. mic-/mic+ 0.83 0.83 4.0

To 24OC 37OC E S -""

8. SM 3001 To 24OC 37OC E S -""

FIG. 4. Northern blot analyses of ompF mRNA levels in MC4100 (micF+) and SM3001 (micF-) grown under several stress conditions. MC4100 (A ) and SM3001 ( B ) were grown a t 24 'C in minimal medium to early log phase (lane To) and aliquots of the cells were harvested for RNA extraction and Northern analysis (or pulse-labeled for protein analysis, see Fig. 5B) . The rest of the two cultures were divided into several batches each of which continued to grow for 2 h either a t 24 "C (lanes 24 "C), 37 "C (lanes 37 "C), in 10% ethanol a t 24 'C (lane E ) , or in 20% sucrose at 24 "C (lane S); (lane marked To shows initial ompF mRNA levels in the early log phase sample). At the end of the 2 h, aliquots of each batch of cells were harvested for RNA analyses (or pulse-labeled for protein, see Fig. 58). Each growth condition shows triplicate samples. (RNA samples analyzed for ompF mRNA in panel A were also analyzed for micF RNA as shown in Fig. 1 and for ompC mRNA, data not shown.)

shown). The CX28 fragment begins about 140 base pairs upstream of the micF gene and thus excludes sequences such as the OmpR-binding domain (Norioka et al., 1986), a cis- acting element important to ompC transcription. Preliminary data indicate that micF RNA levels are dependent on OmpR.3

Time Course of Change i n the Level of micF RNA-Because micF RNA levels increase in response to heat and ethanol (a known heat shock inducer), we were interested to see if micF RNA levels transiently increased after a temperature increase and, also whether the 4.5 S species was the principle transcript from the gene under these conditions. Polyacrylamide gels were used to obtain the best resolution of micF RNA, and total RNA from cells harboring plasmid pKI0041 was analyzed to maximize cpm retrieved from a membrane. The levels of micF RNA increase rapidly (within 3 min) after a sudden shift up in the temperature from 22 to 37 "C (Fig. 3A) and the increase in the levels of the RNA is not transient, i.e. the levels do not rise sharply and then decrease again. In addition, the increase occurs too rapidly for it to result from higher cell concentration. Results with extended time points reveal that 37 "C steady state levels are reached within the cell doubling time (data not shown). After a sudden decrease in temperature from 37 to 20 "C, the levels of micF RNA decrease to less than half of the zero time (To) level a t about 30 min (Fig. 3B). Again, steady state levels are not reached until the doubling time. After a sudden increase in osmolarity (to 20% sucrose) a t 24 "C (Fig. 3C), micF RNA levels rise slowly so that after 60 min, the level is still less than half that found in the cells after being grown overnight at 24 "C in 20% sucrose. In all

' J. Andersen and N. Delihas, unpublished observations.

U z U E

0 a n 0

600 , 400

200

0

0

B.

1

17965

3.9 4.7

n

24°C 37°C E S

mic-/mic+ 0.84 0.80 2.9 ND ND

ND n 0 24°C 37°C E S

FIG. 5. Quantitation of levels of ompF mRNA and of newly synthesized OmpF in MC4100 (micF+) and SM3001 (micF-) grown under several stress conditions. Panel A shows a bar graph of the average cpm of the probe which hybridized to ompF mRNA in the triplicate samples of the Northern analyses shown in Fig. 4. The dark bars show the cpm for the MC4100 samples and the light bars show the cpm for SM3001 samples. 24 "C, 37 "C, E, and S are as described in Fig. 4, however, To has been marked as 0 in this figure. The ratios of the cpm for strain SM3001 (mic-) and for strain MC4100 (mic+) are shown above each set of dark and light bar graphs. Panel B shows the ratio of OmpF to OmpA based on the calculated relative % areas determined by scans of the autoradiographs of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pulse-labeled outer membrane proteins from the batches of cells described in Fig. 4. The dark bars show the ratio for the MC4100 samples and the light bars show the ratio for SM3001.24 "C, 37 "C, E, and S are as described in Fig. 4 and To has been marked as 0 in this figure. The ratios of OmpF relative % area for strain SM3001 (mic-) and of MC4100 strain (mic') are shown above each set of dark and light bar graphs. The ratio could not be determined for the E and S samples as discussed in the text.

analyses, only the 4.5 S micF RNA was detected. The levels of micF RNA were examined at 24 and 37 "C in

mutant E. coli strain K165 (hptR-), which is deficient in heat shock regulatory factor sigma-32 (Neidhardt and VanBogelen, 1981; Grossman et al., 1984) and in its isogenic parental SC122 (hptR +). Both strains had 8-%fold higher levels of micF RNA at 37 "C than at 24 "C (data not shown). Therefore, the increase in micF RNA levels due to temperature increase do


A. 24OC To 2' 5' 10' 20' 30' 40' 60' 90' 120'

"""-' *"" - " JOmPC "-.""""- - Omp F

C. I-MC4100 I [-SM3004.-d

o b c d e f g h i j k o b c d e f g h i j k

-0mpFmRNA

FIG. 6. Kinetic analysis of changes in newly synthesized OmpF and corresponding steady state levels of ompF mRNA in E. coli strains MC4100 (mi#+) and SM3001 (mi#-) after a shift in temperature from 24 to 42 "C. Panels A and B show the change in polyacrylamide gel pattern of 3sSS-pulse-labeled outer membrane proteins from MC4100 and SM3001, respectively, with time after the temperature shift. The lane marked To, shows the pattern of the newly synthesized proteins in cells just before the temperature shift after growth to early log phase at 24 "C in minimal medium. The lane marked 24 "C shows the pattern in an aliquot of cells grown at 24 "C for 2 additional h (instead of a t 42 "C). Lanes marked 2'-120' denote time in minutes when cell samples were pulse labeled and harvested after being shifted to 42 "C growth. OmpC, OmpF, and OmpA bands are as marked. Panel C shows a Northern analysis of ompF mRNA from cell samples corresponding to those shown in panels A and B. The left side shows the change with time in steady state levels of ompF mRNA in MC4100 (mi#+) and the right side in SM3001 (rnicF-) after the temperature shift. The lanes marked a correspond to the 24 "C protein samples, the lanes marked b correspond to the To samples, and the lanes marked c-k correspond to the lanes marked 2'-120', respectively, as explained above for A and B.

not appear to be either directly or indirectly dependent on the heat shock regulon hptR.

ompF mRNA and OmpF Levels during Various Stress Con- ditions-The effect of micF RNA on the steady state levels of ompF mRNA and on the levels of newly synthesized OmpF was examined for various environmental growth conditions (Figs. 4 and 5). E. coli strains MC4100 (micF') and SM3001 (micF-) were first grown a t 24 "C in minimal medium to early log phase (marked To or 0) and then, aliquots of cells were grown for an additional 2 h a t 24 "C, a t 37 "C, in 10% ethanol a t 24 "C (E), or in 20% sucrose a t 24 "C (S). Cells were harvested for RNA extraction. RNA samples were normalized based on UV absorption readings and analyzed by Northern blot for ompF mRNA (Fig. 4), and for micF RNA levels (see Fig. 1). Radioactive bands from each blot were cut uniformly from the membranes using the autoradiographs as templates and counted to quantitate the RNA results (Fig. 5A). In addition, aliquots of cells from the same batches harvested for RNA analysis were pulse-labeled with [3sS]methionine for protein analysis. Outer membrane proteins were isolated, normalized for cpm, and resolved on sodium dodecyl sulfate- polyacrylamide gel electrophoresis. Autoradiographs of gels

from outer membrane protein analyses were scanned to quantitate the change in OmpF levels (Fig. 5B).

In the parental strain, MC4100, ompF mRNA levels decrease under conditions where micF RNA levels increase, i.e. a t 37 "C, in 10% ethanol a t 24 "C (E) , and in 20% sucrose a t 24 "C (S). In contrast, ompF mRNA levels are elevated 4-5- fold in SM3001 (micF-) compared with parental MC4100 (Fig. 5A), when these cells are grown under conditions where micF RNA levels are high. In fact, the levels of ompF mRNA are slightly higher in the deletion strain after growth at 37 "C for 2 h when compared with the levels after growth a t 24 "C for 2 h (compare light bars in Fig. 5A, lanes 24 and 37 "C). This result suggests that the thermal regulation of ompF mRNA is primarily dependent on the micF gene.

In addition, ompF mRNA levels are reduced in both strains in response to osmolarity and ethanol due to factors other than micF RNA. In SM3001, (micF-), the levels of ompF mRNA are down by about %fold after growth in ethanol and down by about 10-fold after growth in high osmolarity for 2 h a t 24 "C compared with the initial level of the mRNA (compare light bars in Fig. 5A, lanes E and S with lane 0). Interestingly, the levels of ompF mRNA also decrease in both

The Function of micF RNA 17967

A 24OC To 2' 5' 10' 15' 30' 60' 120' 37OC

A-A MC4100 0-0 SM 3001

0 0.1 ' E I I I "E 6

0.1 I 1 I I I I I

0 20 40 60 80 100 120

Time ( minutes 1 FIG. 7. Quantitation of kinetic analysis. Panel A shows a

graph of the change with time of the OmpF to OmpA ratio based on measured relative percent areas from laser scans of autoradiograph from Fig. 6, A and B. The OmpF/OmpA ratios for MC4100 (micF+) are shown by solid triangles, while those of SM3001 (micF-) are shown by solid circles. Panel B shows the relative cpm of the 32P- labeled probe hybridized to ompF mRNA shown in the Northern blot analysis of Fig. 6C. The graphs for SM3001 and MC4100 have been normalized a t zero time.

To 24OC 42OC "-

-ompF mRNA

FIG. 8. Complementation of micF deletion by plasmid carrying the micF gene. Autoradiograph of Northern analysis showing the levels of ompF mRNA in SM3001 (mi#-) transformed with pKI0041 after growth a t 24 and 42 "C. Lanes marked 7'0 show ompF mRNA in cells just before the temperature shift; lanes marked 24 "C show the mRNA in cells grown for 2 additional h a t 24 "C; lanes marked 42 "C show the mRNA in cells grown a t 42 "C for 2 additional h. The samples were loaded in triplicate. ompF mRNA levels are almost 10-fold lower a t 42 than at 24 "C (compare with Figs. 6C and 7B).

40 t 0 200 E L

.c Reference Strain

0 - 0

I ' I I I I I

0 20 40 60 80 I00 120

Time (minutes 1 FIG. 9. Kinetic analyses of the effect of micF multicopy

plasmid on the levels of newly synthesized OmpF. Panel A is an autoradiograph showing the change with time of the pattern of newly synthesized outer membrane proteins in strain JA221 transformed with pBR322 after a sudden shift in temperature from 24 to 37 "C. Panel B shows the change for JA221 transformed with plasmid pKI0041 which carries the micF RNA gene. Lanes marked 24 "C and To are as described for Fig. 6. Lanes marked 1'-120' denote times in minutes after the temperature shift a t which the cells were pulse labeled with [35S]methionine and harvested. The lanes marked 37 "C shows the protein pattern from cells that were diluted from an overnight grown a t 37 "C and then grown to mid-log stage a t 37 "C. OmpC, OmpF, and OmpA bands are as marked. Panel C is a graph showing the change with time of the relative % area of OmpF determined by scans of the autoradiograms from panels A and B. The OmpF areas for the reference strain, (JA221/pBR322) are graphed with solid circles. The OmpF areas for the strain harboring a multicopy plasmid with the micF gene (JA221/pKI0041) is graphed with solid triangles.

MC4100 and SM3001 after cell growth for 2 h a t 24 "C. This effect seems to be independent of the presence of the micF gene since the ratio of ompF mRNA in the micF- and micF' strains are similar (e.g. both are 0.83) in the initial and 24 "C samples (Fig. 5A, lunes 0 and 24 "C). Growth under various stress conditions appears to affect the overall levels of o m p F mRNA to a different degree.

Fig. 5B shows that the corresponding levels of newly synthesized OmpF are about 3-fold higher in SM3001 (micF-) than in MC4100 grown a t 37 "C. While a scan of the autoradiograph did not detect OmpF in MC4100 grown in 20% sucrose a t 24 "C, a small amount (ratio of % area OmpF/


200

180

- -

160 -

140

120

-

-

' O 0 I 80

"0 20 40 60 80 io0 120 Time (minutes 1

FIG. 10. The change of plasmid derived micF RNA levels with time after a temperature shift from 24 to 37 "C. The graph shows the cpm of the 32P-labeled probe hybridized to micF RNA during Northern analysis of RNA from JA221 harboring the micF multicopy plasmid, pKI0041. The cell samples were coordinately harvested with the cells that were pulse labeled for protein analysis in Fig. 9.

TABLE I1 Relative percent areas of porins in untransformed and transformed

JA221 at several growth temDeratures Ornu + F C A F/A CIA FIC

JA221 24 "C 36.8 14.7 28.2 1.3 0.5 2.5 30 "C 23.0 20.4 24.7 0.9 0.8 1.1 37 "C 13.2 29.0 26.2 0.5 1.1 0.5 42 "C 5.3 28.0 34.3 0.2 0.8 0.2

JA221/pKI0041 24 "C 23.0 10.3 36.5 0.6 0.3 2.2 30 "C 18.3 17.1 31.5 0.6 0.5 1.1 37 "C 3.0 31.5 36.1 0.1 0.9 0.1 42 "C - a 25.8 36.6 NDb 0.7 N D

Levels were too low to be determined. ND, not determined.

OmpA = about 0.05) was detected in SM3001. Addition of ethanol to the growth medium resulted in the lack of uptake of [35S]methionine by the cells so that the pulse-labeled outer membrane pattern could not be observed for ethanol.

Kinetics of OmpF and of ompF mRNA Change after Tem- perature Increase-The change in levels of newly synthesized OmpF and in steady state levels of ompF mRNA with time after a shift in temperature from 24 to 42 "C in MC4100 (parental) and SM3001 (micF deletion strain) was also examined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pulse-labeled outer membrane proteins display the change with time in pulse-labeled OmpF in MC4100 and SM3001 (Fig. 6 , A and& respectively), and an autoradiograph for the Northern hybridization (Fig. 6C) shows the corresponding steady state levels of ompF mRNA. Quantitation of these results shows that the levels of newly synthesized OmpF drop 2-%fold within 10 min in MC4100 but only slightly in

SM3001 after the temperature increase (Fig. 7A). In addition, the steady state levels of ompF mRNA drop almost 10-fold within 30 min in MC4100 but remained constant with time in SM3001 (Fig. 7 B ) . (Differences in the magnitude of decrease between OmpF protein and the mRNA may be due to changes in efficiency of translation at the two temperatures.) Interestingly, newly synthesized OmpF levels decrease faster than the steady state pool of ompF mRNA (compare Fig. 7, A and B ) , suggesting that the first part of the inhibition of OmpF synthesis found in the micF' strain occurs through inhibition of translation of the mRNA, that is, translational inactivation. The second part of the inhibition occurs through a destabilization of the mRNA which leads to its chemical decay.

Complementation of micF Gene in Strain SM3001"Ther- mal regulation of ompF mRNA levels can be rescued in SM3001 by transforming the cells with a multicopy plasmid carrying the micF gene (Fig. 8). SM3001 was transformed with pKI0041 which has the micF gene and a mutated ompC gene or with pJAN012 which was created by removing an EcoRI fragment from pKI0041, thus deleting the 3' portion of the ompC gene. Both plasmids were able to rescue the phenotype of diminished ompF mRNA levels at 42 "C. North- ern analysis shows that the levels of ompF mRNA in SM3001 harboring pKI0041 are 9.3 times higher when the cells are grown at 24 "C than when grown at 42 "C which approximates what was seen for parental MC4100 after a temperature shift from 24 to 42 "C (compare Fig. 8 with Figs. 6C and 7B) . A similar result was observed using pJAN012 (data not shown).

Multicopy Effect of micF-A kinetic analysis of changes in levels of newly synthesized OmpF in E. coli strain JA221 (recA-) harboring control plasmid pBR322 (Fig. 9A) or harboring multicopy plasmid pK10041 (Fig. 9B), after a sudden shift from growth at 24 "C to 37 "C, shows that multicopy inhibition of OmpF synthesis correlates with heightened expression of the micF RNA gene. Within 15 min, the levels of OmpF decrease 3-4-fold in the cells harboring pK10041 (Fig. 9C). The levels of micF RNA rise dramatically within the same period of time (Fig. 10). In contrast, the levels of newly synthesized OmpF in the reference strain JA221 (transformed with pBR322) only drop about 20% after a temperature shift from 24 to 37 "C, (a similar value to that seen for MC4100 (25%) under the same conditions (data not shown)).

From the protein data, one would expect that multicopies of micF RNA would also decrease ompF mRNA levels beyond that resulting from chromosomal micF RNA after a temperature increase. Surprisingly, however, the rate and magnitude of decrease with time in the steady state levels of ompF mRNA in the cells harboring the multicopy micF plasmid (JA221/ pKI0041) and the cells harboring the control plasmid (JA221/ pBR322) were the same (data not shown). This implies that a rate-limiting factor besides micF RNA is involved also in the decrease of ompF mRNA levels seen after temperature increase.

JA221/pKI0041 did have about 2-fold less ompF mRNA at each time point than JA221/pBR322. The lower abundance of ompF mRNA in JA221/pKI0041 resulted in less OmpF even when micF RNA levels were low, i.e. there was about 2- fold less OmpF in the cells harboring pKI0041 than in the reference strain (JA221/pBR322) when grown at 24 "c, (see Fig. 9C, 0 time).

Similar findings were obtained in a comparison between the steady state levels of OmpF and ompF mRNA in untransformed JA221 and JA221 harboring pKI0041 grown under different temperature conditions at low osmolarity. Table I1 shows the results from densitometer tracings of the Coomassie

The Function of micF RNA

A. 8. C.

17969

&F RNA

J /

42 r pnpx mRNA

0- Multicopy

2 -

&F

OmpF/OmpC Ratio

0

0- 0- . O ( I 20 25 30 35 40 45 20 25 30 35 40 45 20 25 30 35 40 45

Temperature "C

FIG. 11. The effect of multicopy micF on the steady state levels of ompF mRNA and the OmpF/OmpC ratio. Panel A shows the change in steady state levels of micF RNA as a function of temperature at low osmolarity (L) . The cpm of the four samples were normalized to those of the 24 "C sample. Panel B shows the change of ompF mRNA in untransformed JA221 (solid triangles) and in JA221 cells harboring plasmid pKI0041 which carries the micF RNA gene (solid circles). The cpm of the JA221/pKI0041 samples were on the average 2.1-fold lower than the cpm of samples from untransformed JA221 (for the same Northern analysis). The samples were normalized to the untransformed JA221 sample at 42 "C to illustrate the similarity of the temperature-dependent change of ompF mRNA levels in these cells. Panel C shows the graph of the ratios of OmpF to OmpC a t low osmolarity condition calculated from % area of peaks listed in Table 11. Since the gel scan showed no distinct OmpF peak at 42 "C for cells harboring pKI0041, a line has been extended beyond the 37 "C point to 42 "C (open circle) which follows the change in the slope produced by multicopy inhibition and this shows approximately a 10-fold reduction in the OmpF/OmpC ratio at the 42 "C point when compared with the ratio of OmpF/OmpC in untransformed JA221 at 42 "C. This may reuresent a conservative estimate of the change in OmpF/OmpC ratio caused by multicopies of micF RNA i t 4 i "C.

Blue-stained outer membrane protein pattern from untransformed JA221 and JA221/pKI0041 grown at different temperatures in nutrient broth. OmpF and OmpC are about 1.5- 2-fold less abundant compared with OmpA at 24 and 30 "C, when micF RNA levels are low (Fig. l lA), in cells harboring plasmid pKI0041 than in untransformed cells grown under the same low temperature. Also, ompF mRNA in cells harboring multicopy pKI0041 are about 2-fold less than in the untransformed cells a t each temperature point investigated (data not shown). However, when we normalize for the differ- ence in the mRNA levels, the temperature-dependent changes in relative ompF mRNA levels observed in untransformed JA221 and JA221/pKI0041 do not appear to be affected by multicopies of micF RNA (Fig. 11B).

Contrary to our expectations, then, multicopy micF RNA did not amplify the reduction of ompF mRNA levels at the higher temperatures. In contrast, multicopy inhibition of OmpF synthesis by micF does occur at 37 and 42 "C and is reflected in OmpF/OmpC ratios (Fig. 1lC) calculated from the results in Table 11. There is a &fold decrease in the OmpF/OmpC ratio at 37 "C in JA221/pKI0041 and a deple- tion of OmpF a t 42 "C. This effect cannot be fully explained by the decreased levels of ompF mRNA (about a factor of 2) which appears to be due to the multicopy plasmid. Also, the plasmid copy number was not affected by growth temperature (data not shown). The variance in the OmpF/OmpC ratio occurs only at the higher temperatures when micF RNA levels are elevated (compare Figs. 11, A and C). These data suggest that multicopies of micF RNA repress OmpF production primarily at the level of protein synthesis by rendering ompF mRNA translationally inactive but do not further decrease ompF mRNA levels beyond that resulting from chromosomal micF RNA.

DISCUSSION

The goals of the current study were to identify the function of chromosomally derived micF RNA, to define environmental growth conditions in which micF RNA primarily acts, and to broach the mechanism by which the small RNA inhibits OmpF synthesis. The findings of this paper show that chromosomally encoded micF RNA plays a major role in the thermal regulation of OmpF synthesis and in decreasing the levels of ompF mRNA in response to high temperature. micF RNA also plays a partial role in repressing OmpF synthesis in response to other stress conditions, such as high osmolarity and exposure to ethanol.

The work of Misra and Reeves (1987) and Cohen et al. (1988) both show that micF RNA influences OmpF synthesis at a post-transcriptional level. In addition, our analyses of p- galactosidase activity in cells having a lac2 operon fusion to the chromosomal ompF promoter (strain MH513, (Hall and Silhavy, 1981a)) revealed that the repressed levels of ompF mRNA seen at high temperature are not due to decreased transcription of the ompF gene; in fact, the analyses suggest that transcription may increase slightly a t higher temperatures (data not shown). Since micF RNA causes the levels of ompF mRNA to decrease, we believe that the interaction of micF RNA with ompF mRNA leads to a destabilization of the mRNA. In addition, kinetic analyses of the change in OmpF synthesis and in the steady state levels of ompF mRNA levels after a temperature shift, show that the levels of newly synthesized OmpF decrease faster than the steady state pool of ompF mRNA. This suggests that micF RNA can first inhibit the translation of ompF mRNA presumably by hybridizing to its ribosome-binding site before the messenger is destabilized and degraded. Differences in functional (ie. translational)


and chemical half-lives have been observed for other mRNA (Schwartz et al., 1970; Gupta and Schlessinger, 1976).

micF RNA appears to repress OmpF in response to several stress conditions by destabilizing ompF mRNA. When the cells are subjected to high osmolarity and ethanol, however, OmpF is also repressed by other factors besides micF RNA (see Fig. 5). For example, it is known that the ompB locus ( e m 2 and ompR) negatively regulates transcription of ompF in response to high osmolarity (Hall and Silhavy, 1981a; Hall and Silhavy, 1981b). OmpF regulation in response to ethanol is not fully understood. The factors responsible for regulation of OmpF in response to osmolarity or ethanol do not appear to be directly involved in the regulation of OmpF in response to temperature. However, the micF gene may be transcriptionally regulated by some of these factors.

Analysis of the effect of multicopy micF RNA on changes in ompF mRNA levels in response to a sudden shift in temperature revealed that multicopy micF RNA primarily inhibits the translation of the mRNA, but does not cause additional changes in the rate or magnitude in decrease of the mRNA compared with that seen for chromosomally encoded micF RNA. The multicopy plasmid data suggests then that blocking translation of ompF mRNA is not sufficient to cause the messenger to be destabilized.

There is a decrease in both OmpF and OmpC porins seen in the cells harboring a micF multicopy plasmid at 24 and 30 "C compared with OmpA (Fig. 9C and Table 11). Also, there is an overall reduction in the amount of ompF mRNA in the transformed cells harboring a multicopy micF plasmid by about a factor of 2. The reduction of ompF mRNA seen in cells harboring these plasmids may be due to the sequestering of a porin gene-specific transcription factor by sequences on the multicopy plasmid.

Taken together, the multicopy micF and chromosomal micF effects on the levels of ompF mRNA suggest that micF RNA is necessary but not sufficient to destabilize ompF mRNA, and acts with another factor, a limiting one, to destabilize the messenger. micF RNA may need a cognate protein for the destabilization of ompF mRNA and may function in a ribo- nucleoprotein particle possibly similar to processing enzyme ribonuclease P (Reich et al., 1988). Alternatively, the putative hybrid formed between micF RNA and ompF mRNA may be recognized specifically by a limiting cellular ribonuclease.

The present work shows that the levels of the 4.5 S micF RNA are suppressed during low temperature (24 "C) and low osmolarity growth conditions but increase 10-fold or greater in response to high temperature (37 and 42 "C) . The levels of micF RNA increase in response to increased osmolarity of the growth medium as well as to the addition of ethanol and copper, agents known to induce heat shock response. micF expression, however, is not under the htpR heat shock regulon. The response of micF RNA levels to various environmental stimuli places the micF gene, which codes for a small RNA and not a protein, with other regulatory genes that respond to stress factors and/or environmental signals (Nikaido and Vaara, 1987; Cohen et al., 1988; Miller et al., 1989).

micF RNA is one of several small RNAs known to have nonribosomal functions in prokaryotes (for review see Inouye and Delihas, 1988). Several of these small RNAs are antisense RNAs that act as repressors of gene expression. Their genes overlap and/or are divergent from target mRNA genes, auto-

matically making them complementary to portions of the respective mRNAs. Thus far, micF RNA appears to be unique as a repressor RNA in that its gene is distal from the target ompF gene. Yet micF RNA exhibits a high degree of complementarity to the 5"untranslated portions of ompF mRNA and thus is capable of functioning as an antisense RNA in repressing OmpF synthesis. The existence of micF RNA suggests that genes for other repressor RNAs may exist which are distal from the gene of their target RNA. micF RNA may be part of a class of regulatory cellular RNAs involved in mRNA destabilization.

Acknowledgments-We thank Drs. Shoji Mizushima and Shin-ichi Matsuyama for the micF deletion strain and Drs. Kazuhiro Ikenaka and Girija Ramakrishnan for plasmid constructions. We thank Drs. Frederick Neidhardt and Ruth A. VanBogelen for the gift of E. coli strains SC122 and K165. We also thank Matthew Schmidt for help with Northern blots, Craig De Loughery for help with the lucZ fusion analyses, and Dr. Michael G. Schmidt for help with the gel scans. We thank Dr. James Coyer for his critical reading of the manuscript. We appreciate the information that Jessica Jordan from Schleicher & Schuell gave us on the uses of Nytran in Northern blotting and we thank Karen Henrickson for the beautiful art work.

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