Contact-Dependent Growth Inhibition Causes Reversible Metabolic Downregulation in Escherichia coli

Intercellular communication mechanisms enable bacteria to coordinate biological phenomena such as DNA uptake, differentiation for fruiting body development, light production, and swarming (7, 8). These cell-to-cell interactions enable individual bacteria to form a multicellular community, such as in a biofilm on a solid surface, under specific environmental conditions (20, 52). Similarly to multicellular organisms, bacteria have signal transduction mechanisms to facilitate cellular cross talk, including two-component regulatory systems and other cell surface ligand-receptor interactions that control cellular processes. We previously described a cross talk phenomenon designated as contact-dependent growth inhibition (CDI) in which one bacterial isolate (CDI+) blocks the growth of another bacterium when mixed together (4). CDI requires two contiguous genes, cdiB and cdiA, which encode proteins that are in the two-partner secretion (TPS) family. Overlapping the stop codon of cdiA is a downstream open reading frame designated cdiI, which encodes a 79-amino-acid protein that provides immunity to growth inhibition from cells expressing cdiBA (4). Evidence strongly indicates that cell-to-cell contact is required for growth inhibition. First, separation of CDI+ inhibitor cells and target cells by a 0.4-μm nitrocellulose membrane blocked CDI, distinguishing CDI from the class of soluble bacterial growth inhibitors known as bacteriocins. Second, to address the possibility that a very short-lived bacteriocin-like molecule might be released from CDI+ inhibitor cells, we separated inhibitor-target cell aggregates by fluorescence-activated cell sorting. Target cells within aggregates with CDI+ inhibitor cells lost viability, as measured by growth on LB medium, more rapidly than did unbound target cells, supporting the conclusion that cell-to-cell contact is required for CDI (4). In our previous work, analysis of CDI was carried out with a bipartite system using Escherichia coli inhibitor cells containing cdiB, -A, and -I on a multicopy plasmid that constitutively expressed CDI activity, cocultured with E. coli K-12 target cells. Mixing inhibitor cells with E. coli K-12 target cells resulted in a 5- to 6-log decrease in target cell number after only 1 to 2 h (4). Because this bipartite CDI assay contains both inhibitor and target cells, monitoring of target cell growth in real time is not possible. Thus, we have not been able to determine if CDI is a reversible process or a nonreversible toxin-like system. This is important in assessing the role of CDI in the biology of E. coli as well as its potential role in many gram-negative bacteria, including uropathogenic E. coli, Burkholderia pseudomallei, and Yersinia pestis that contain genes with significant sequence identity to cdiB and cdiA (4). Recently, in collaboration with J. Malinverni and T. Silhavy (Princeton University), we showed that the target cell receptor for CDI is BamA, an essential outer membrane protein (OMP). Homologues of bamA are conserved in genomes from bacteria to mitochondria (3). BamA is a key component of the β-barrel assembly machine required for biogenesis of many other OMPs (34, 43, 53). Our results indicated that the BamA receptor facilitates CdiB/CdiA-dependent cell-to-cell binding and growth inhibition since antibodies to BamA blocked formation of inhibitor-target cell aggregates and CDI (3). The ligand for BamA is not known, but it seems probable that it is CdiA, which is at the surface of inhibitor cells (4), and may form a short 40- to 50-nm fiber, such as filamentous hemagglutinin in Bordetella pertussis, based on sequence similarities (42). AcrB is also required for sensitivity of target cells to CDI, which acts downstream of the BamA receptor (3). AcrB is a protein that exports small molecules, including drugs/antibiotics, through the inner membrane, with further transport through the outer membrane in conjunction with AcrA and TolC (47, 55, 56). This export machine requires proton motive force (Δp) as an energy source (46). Markedly, only BamA and AcrB, independent from their respective export machines, are required for CDI (3). Based on these results, we developed a model in which CdiA of inhibitor cells bind to BamA on target cells, transmitting a signal into target cells such as a CdiA peptide (Fig. ​(Fig.1A).1A). This signal could enter cells through an AcrB portal to interact with an unidentified cytosolic target. It is also possible that AcrB could play an indirect role in CDI-mediated growth inhibition. FIG. 1. Construction of a contact-dependent autoinhibition system in Escherichia coli. (A) CDI model. CdiB and CdiA (CdiBA) expressed by one cell (inhibitor cell) binds to BamA in the outer membrane (OM) of an adjacent cell (target cell) (3). A signal, such as ... Here, we describe the development of a CDI autoinhibition system in which growth inhibition is regulated by controlled expression of the CdiI immunity protein, enabling examination of a single population of cells undergoing CDI. Using this system, we show that cells undergoing CDI have significantly reduced cellular respiration, Δp, and steady-state ATP levels. Notably, this metabolic downregulation, as well as cellular growth inhibition, is reversible. To the best of our knowledge, this is the first report of a natural, reversible system controlling bacterial metabolism and growth.