Small non-coding RNAs and the bacterial outer membrane

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Recent systematic genome searches revealed that bacteria encode a tremendous number of small non-coding RNAs (sRNAs). Whereas most of these molecules remain of unknown function, it has become increasingly clear that many of them will act to modulate gene expression at the post-transcriptional level. Where studied in more detail, sRNAs have often been found to control the expression of outer membrane proteins (OMPs). Enterobacteria such as Escherichia coli and Salmonella are now known to encode at least eight OMP-regulating sRNAs (InvR, MicA, MicC, MicF, OmrAB, RseX and RybB). These sRNAs exert their functions under a variety of growth and stress conditions, including the σE-mediated envelope stress response. An sRNA–OMP network is emerging in which some sRNAs act specifically on a single omp mRNA, whereas others control multiple omp mRNA targets.

Introduction

Small non-coding RNAs (sRNAs) occur in all kingdoms of life, and have become increasingly recognized as a novel class of gene expression regulators. The eubacterial sRNAs constitute a structurally diverse class of molecules that are typically of 50–250 nucleotides in length and do not commonly contain expressed open reading frames (ORFs). Small RNAs have been known in bacteria since the early 1970s, but only the success of recent systematic genome-wide searches for these molecules and their genes has led to their full appreciation [1, 2, 3, 4, 5, 6, 7, 8, 9]. Beginning in 2001, several screens using various methodologies [10•] have increased the number of known sRNAs expressed from the Escherichia coli chromosome to greater than 70. Many of the E. coli sRNA genes are conserved in closely related pathogens, such as Salmonella and Yersinia species [11]. It has been estimated that enterobacterial genomes with an average size ranging from 4–5 Mb might contain 200–300 sRNA genes [12]: a figure approximately equal to 5% of the total number of protein-encoding genes.

Whereas the search for new bacterial sRNAs is ongoing, ∼20 E. coli sRNAs have been assigned cellular functions, and often their mode of action has been described (reviewed in [13•, 14•, 15•]). From these functional studies, it emerges that many sRNAs act as antisense RNAs on trans-encoded mRNAs. Unlike the well-studied cis-encoded antisense RNAs of plasmids and phages [16], these trans-encoded antisense RNAs typically have only short and imperfect complementarity with their target. Base-pairing most often occurs in the 5′ untranslated region (UTR) of the target mRNA, and is aided by the bacterial Sm-like protein, Hfq (host factor for phage Qβ) [17].

The founding member of this class of trans-encoded antisense RNAs, MicF RNA of E. coli, was discovered more than 20 years ago [18]. The micF gene was isolated in a genetic study using its phenotype to repress production of OmpF when present in multiple copies. The 93 nt (nucleotide) MicF RNA forms a ∼20 bp imperfect RNA duplex with the translation-initiation region of ompF mRNA (Figure 1) [19], in order to negatively regulate expression of this outer membrane protein (OMP) at the post-transcriptional level. Much of this regulation, including the growth conditions that lead to MicF expression, is now well understood and has been reviewed [20, 21].

The outer membrane (OM) is a hallmark of Gram-negative bacteria. Together with the peptidoglycan layer and the inner membrane, it forms the bacterial cell envelope. Because of its physical properties, the OM functions as a selective barrier that prevents the entry of many toxic molecules into the cell, and plays a vital role in bacterial survival in diverse environments. However, as membranes are fairly impermeable to hydrophilic solutes, the channels formed by porins such as OmpF facilitate the uptake of nutrients and the excretion of toxic waste-products. Other OMPs that do not function as channels are able to serve as enzymes as well as adhesins. In pathogenic and symbiotic bacteria, the OM represents the bacterial surface that interacts with the eukaryotic host, whilst it also accommodates many proteins that have direct roles in bacterial virulence.

Given the importance of the OM, it does not come as a surprise that the environment-dependent expression of OMPs is extensively coordinated at the level of transcription. However, research in the last two years has shown that aside from MicF, enterobacteria use many additional small RNAs in order to fine-tune the OM composition at the post-transcriptional level. These sRNAs are the subject of this review.

Section snippets

MicC regulates the major porin, OmpC

The OmpC and OmpF porins are amongst the most abundant proteins that are translocated to the OM. These proteins span the OM with amphiphatic antiparallel β-strands that adopt a barrel-like conformation, thereby forming a channel. Of the two, OmpC forms the smaller pore, and plays the predominant role under conditions where nutrients, as well as toxins are abundant, whereas the wider OmpF pore is thought to be important under conditions of limiting nutrients and of low toxin levels. The

Growth rate-dependent control of OmpA expression by MicA RNA

OmpA belongs to a class of proteins that is highly conserved among enterobacteria, it occurs at ∼100 000 copies per cell, and it is thought to anchor the OM to the murein layer of the periplasmic space. The ompA mRNA is abundant and long-lived, and has long served as a model to study RNA processing and decay. It was early noted that ompA mRNA stability varied greatly depending on the growth rate: specifically, this mRNA becomes destabilized at the onset of stationary phase [23]. Over the years,

Two homologous RNAs, OmrA and OmrB, control multiple OMPs

Recent genome searches revealed the existence of two sRNA genes, omrA and omrB sRNAs, in the ∼600 bp intergenic region between aas and galR (these sRNA genes were first denoted rygA/sraE and rygB [1, 2]). Intriguingly, the two sRNAs are of similar length, and are almost identical in their 5′ and 3′ regions, respectively, with these terminal sequences being highly conserved in other bacteria. The 88 nt OmrA RNA accumulates in late stationary phase [1], whereas the 82 nt OmrB RNA is transiently

Suppressor function of RseX RNA under extracytoplasmic stress

Extracytoplasmic or membrane stress as caused by, for example, the accumulation of misfolded proteins in the periplasm, triggers a global response that is mediated by the alternative sigma factor, σE. A key player in the σE induction cascade is the protease, RseP, which participates in the release of active σE from its membrane-bound precursor complex. The rseP gene is essential in E. coli. However, a recent screen for multicopy suppressors that could bypass rseP resulted in a sRNA surprise: a

An emerging sRNA–OMP network to fine-tune the OM composition

With the recent discovery of new sRNAs that are involved in OMP regulation, a network is emerging in which some sRNAs act specifically on a single omp mRNA (MicA and MicF), whereas others have multiple targets (MicC, OmrA, OmrB and RseX). Likewise, the very same omp mRNA could be subject to regulation by more than one sRNA (e.g. ompC is regulated by both MicC and RseX; Figure 3). Work in progress in our, and several other laboratories, however, indicates that this is only the tip of the

σE and a two-component system feed the sRNA–OMP network

Networks make the most sense if each player can be pulled by individual strings, so to speak, thus enabling the integration of multiple input signals. In terms of the network described above, some of the sRNAs that regulate the same OMPs are part of different regulons (Figure 3). The EnvZ–OmpR two-component system, known to regulate major porin gene expression in response to high osmolarity, has been implicated in the differential expression of MicC and MicF [21, 33]. OmpR-dependent

Conclusions and perspective

Small RNA-mediated control of OMP expression, which started with the serendipitous finding of MicF RNA two decades ago, has become an exciting field of research. In many ways, MicF laid the grounding for our current understanding of how bacterial sRNAs modulate the expression of trans-encoded target mRNAs. That OMPs and their mRNAs are usually abundant has been an added advantage of identifying these as prominent sRNA targets.

Currently, about a third of the E. coli sRNAs with known cellular

Update

A first paper describing the σE-dependent transcription of the E. coli micA and rybB genes, as well as the OMP-regulatory function of E. coli RybB sRNA, has been published [36••].

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We extend our gratitude to Susan Gottesman and Gerhart Wagner for sharing unpublished results, and to members of our laboratory (Franziska Mika, Verena Pfeiffer, Cynthia Sharma) for comments on the manuscript. This brief review comes with severe space constraints, and we apologize to those whose work is covered inadequately. Work in the Vogel laboratory is supported by the Max Planck Society, and by DFG grant VO 875/1-1 (Deutsche Forschungsgemeinschaft).

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