A Mycobacterial Enzyme Essential for Cell Division Synergizes with Resuscitation-Promoting Factor

The final stage of bacterial cell division requires the activity of one or more enzymes capable of degrading the layers of peptidoglycan connecting two recently developed daughter cells. Although this is a key step in cell division and is required by all peptidoglycan-containing bacteria, little is known about how these potentially lethal enzymes are regulated. It is likely that regulation is mediated, at least partly, through protein–protein interactions. Two lytic transglycosylases of mycobacteria, known as resuscitation-promoting factor B and E (RpfB and RpfE), have previously been shown to interact with the peptidoglycan-hydrolyzing endopeptidase, Rpf-interacting protein A (RipA). These proteins may form a complex at the septum of dividing bacteria. To investigate the function of this potential complex, we generated depletion strains in M. smegmatis. Here we show that, while depletion of rpfB has no effect on viability or morphology, ripA depletion results in a marked decrease in growth and formation of long, branched chains. These growth and morphological defects could be functionally complemented by the M. tuberculosis ripA orthologue (rv1477), but not by another ripA-like orthologue (rv1478). Depletion of ripA also resulted in increased susceptibility to the cell wall–targeting β-lactams. Furthermore, we demonstrate that RipA has hydrolytic activity towards several cell wall substrates and synergizes with RpfB. These data reveal the unusual essentiality of a peptidoglycan hydrolase and suggest a novel protein–protein interaction as one way of regulating its activity

Interaction and Modulation of Two Antagonistic Cell Wall Enzymes of Mycobacteria

Bacterial cell growth and division require coordinated cell wall hydrolysis and synthesis, allowing for the removal and expansion of cell wall material. Without proper coordination, unchecked hydrolysis can result in cell lysis. How these opposing activities are simultaneously regulated is poorly understood. In Mycobacterium tuberculosis, the resuscitation-promoting factor B (RpfB), a lytic transglycosylase, interacts and synergizes with Rpf-interacting protein A (RipA), an endopeptidase, to hydrolyze peptidoglycan. However, it remains unclear what governs this synergy and how it is coordinated with cell wall synthesis. Here we identify the bifunctional peptidoglycan-synthesizing enzyme, penicillin binding protein 1 (PBP1), as a RipA-interacting protein. PBP1, like RipA, localizes both at the poles and septa of dividing cells. Depletion of the ponA1 gene, encoding PBP1 in M. smegmatis, results in a severe growth defect and abnormally shaped cells, indicating that PBP1 is necessary for viability and cell wall stability. Finally, PBP1 inhibits the synergistic hydrolysis of peptidoglycan by the RipA-RpfB complex in vitro. These data reveal a post-translational mechanism for regulating cell wall hydrolysis and synthesis through protein–protein interactions between enzymes with antagonistic functions

Bacterial Growth and Cell Division: a Mycobacterial Perspective

Summary: The genus Mycobacterium is best known for its two major pathogenic species, M. tuberculosis and M. leprae, the causative agents of two of the world's oldest diseases, tuberculosis and leprosy, respectively. M. tuberculosis kills approximately two million people each year and is thought to latently infect one-third of the world's population. One of the most remarkable features of the nonsporulating M. tuberculosis is its ability to remain dormant within an individual for decades before reactivating into active tuberculosis. Thus, control of cell division is a critical part of the disease. The mycobacterial cell wall has unique characteristics and is impermeable to a number of compounds, a feature in part responsible for inherent resistance to numerous drugs. The complexity of the cell wall represents a challenge to the organism, requiring specialized mechanisms to allow cell division to occur. Besides these mycobacterial specializations, all bacteria face some common challenges when they divide. First, they must maintain their normal architecture during and after cell division. In the case of mycobacteria, that means synthesizing the many layers of complex cell wall and maintaining their rod shape. Second, they need to coordinate synthesis and breakdown of cell wall components to maintain integrity throughout division. Finally, they need to regulate cell division in response to environmental stimuli. Here we discuss these challenges and the mechanisms that mycobacteria employ to meet them. Because these organisms are difficult to study, in many cases we extrapolate from information known for gram-negative bacteria or more closely related GC-rich gram-positive organisms

An <i>M. smegmatis</i> strain depleted of RipA is sensitive to a cell wall–targeting antibiotic.

<p>The <i>ripA</i> depletion strain of <i>M. smegmatis</i> was spread on LB agar plates containing different amounts of anhydrotetracycline inducer (ng/ml concentrations) to regulate the amount of <i>ripA</i> expressed. A filter disc with 10 µl antibiotic was placed in the center of plate and the diameter of inhibition of growth was measured after 4 days of growth. Antibiotic concentration on disc: carbenicillin (100 mg/ml) and cycloserine (100 mg/ml).</p

Depletion of RipA results in branching and chaining of <i>M. smegmatis</i>.

<p>(A) Micrographs of <i>M. smegmatis</i> strains with membranes imaged by staining with TMA-DPH. i. <i>ripA</i> depletion strain chains and branches when depleted of <i>ripA</i> for 24 hours (no inducer), ii. Wild-type. Arrowheads indicate regions where the cell wall appears pinched or lysed. Bacteria were visualized with 100× objective. (B) Micrograph of a branch of <i>ripA</i> depleted <i>M. smegmatis</i> or wild-type strains. Membranes (green) and DNA (red) were stained with TMA-DPH or SYTO 9, respectively, revealing apparent functional septation and DNA segregation. Bacteria were visualized with 100× objective.</p

Depletion of RipA results in a reversible arrest in growth of <i>M. smegmatis.</i>

<p>(A) Diagram showing the strategy used to replace the native promoter of the <i>ripA-B</i> operon (Prip) with a tetracycline-inducible promoter (Ptet) through homologous recombination (strategy and diagram adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000001#ppat.1000001-Ehrt1" target="_blank">[28]</a>). OriE: <i>E. coli</i> origin of replication. (B) <i>ripA</i> depletion strain of <i>M. smegmatis</i> was grown with inducer (Tet), then inoculated into media with decreasing amounts of inducer and followed by OD₆₀₀ over time. Cultures with none or 2 ng/ml inducer grew in tight clumps that resulted in underestimation by OD₆₀₀. Data are represented as mean+/−SEM. (C) Series of DIC micrographs of the <i>ripA</i> depletion strain of <i>M. smegmatis</i>. Depicts bacteria first grown with 50 ng/ml tetracycline inducer (i), then transferred to media lacking inducer for 24 hours (ii), and finally transferred to media with inducer for 24 hours (iii). Bacteria were visualized with a 100× objective. (D) Depletion strains of <i>M. smegmatis</i> grown on plates containing a gradient of inducer created by placing 10 µl of 10 ng/ml Tet on a paper disc in the center of the plate, resulting in a concentration of inducer highest at the middle of the plate and lowest at the edges. Colonies formed in a Tet-dependent manner for the RipA-depletion strain, while colonies from the RpfB-depletion strain grew independent of inducer.</p

Recombinant RpfB and RipA combine to synergistically hydrolyze peptidoglycan.

<p>(A) Diagram of the structure of peptidoglycan, indicating where RpfB and RipA are predicted to have hydrolytic activity. One unit of the peptidoglycan is magnified to show the structure of NAG and NAM as well as the amino acids that are part of the DAP-DAP crosslinkage. Lines connecting NAG to NAM represent β-1,4-glycosidic bonds, while residues connecting NAM to NAM depict peptide cross-linkages. NAG: N-acetylglucosamine, NAM: N-acetylmuramic acid. (B–D) N-terminal GST fusion proteins were expressed and purified from <i>E. coli</i>. Equal molar amounts of individual or combinations of proteins were incubated with insoluble FITC-labeled substrate: <i>M. smegmatis</i> cell wall (B), Streptomyces peptidoglycan (C), or <i>M. luteus</i> cell wall (D). The extent of hydrolysis was determined by measuring the amount of soluble FITC remaining after centrifugation, and thus released during hydrolysis of the insoluble substrate. GST alone, as well as buffer alone, were used to determine background release of FITC and were subtracted from final values. Data are from representative experiments, each done in triplicate. Data are represented as mean +/−SEM.</p

<i>M. tuberculosis ripA</i> allele complements depletion.

<p>(A) Predicted domains of <i>M. tuberculosis</i> RipA-like proteins and <i>L. monocytogenes</i> p60. The NLPC_P60 putative domain in p60 was previously shown to have endopeptidase activity against cell wall material and defines the family. (B) Gene neighbors and predicted operons of <i>rip</i> genes in <i>M. tuberculosis</i>. Operons are indicated with black arrows, <i>rip</i> genes are in black text, gene numbers are given above arrows and further annotation for some genes is provided below arrows. One gene on either side of the <i>rip</i>-operon is shown with gray arrows. (C) A <i>ripA-smeg</i> depletion strain containing the <i>M. tuberculosis ripA</i> allele, <i>ripA-mtb,</i> on an episomal construct with its native promoter grew like wildtype when depleted of <i>ripA-smeg</i> (no tetracycline), while a strain carrying an empty plasmid formed chains when <i>ripA-smeg</i> was depleted. The <i>M. tuberculosis</i> allele of the <i>ripA</i> paralogue, <i>rv1478</i>, was not able to complement <i>M. smegmatis</i> depleted of <i>ripA</i>. All strains grew like wildtype in the presence of inducer, though the RipA and RipA-like strains grew slightly shorter than empty vector in the presence of Tet. Membranes were visualized with TMA-DPH. Bacteria were visualized with 100× objective.</p