SUMMARY Bacterial cell wall synthesis is the target for some of our most powerful antibiotics and has thus been the subject of intense research focus for more than 50 years. Surprisingly, we still lack a fundamental understanding of how bacteria build, maintain and expand their cell wall. Due to technical limitations, directly testing hypotheses about the coordination and biochemistry of cell wall synthesis enzymes or architecture has been challenging, and interpretation of data has therefore often relied on circumstantial evidence and implicit assumptions. A number of recent papers have exploited new technologies, like single molecule tracking and real-time, high resolution temporal mapping of cell wall synthesis processes, to address fundamental questions of bacterial cell wall biogenesis. The results have challenged established dogmas and it is therefore timely to integrate new data and old observations into a new model of cell wall biogenesis in rod-shaped bacteria.

The Bacillus subtilis GlmR (formerly YvcK) protein is essential for growth on gluconeogenic carbon sources. Mutants lacking GlmR display a variety of phenotypes suggestive of impaired cell wall synthesis including antibiotic sensitivity, aberrant cell morphology and lysis. To define the role of GlmR, we selected suppressor mutations that ameliorate the sensitivity of a glmR null mutant to the beta-lactam antibiotic cefuroxime or restore growth on gluconeogenic carbon sources. Several of the resulting suppressors increase the expression of the GlmS and GlmM proteins that catalyze the first two committed steps in the diversion of carbon from central carbon metabolism into peptidoglycan biosynthesis. Chemical complementation studies indicate that the absence of GlmR can be overcome by provision of cells with N-acetylglucosamine (GlcNAc), even under conditions where GlcNAc cannot re-enter central metabolism and serve as a carbon source for growth. Our results indicate that GlmR facilitates the diversion of carbon from the central metabolite fructose-6-phosphate, which is limiting in cells growing on gluconeogenic carbon sources, into peptidoglycan biosynthesis. Our data suggest that GlmR stimulates GlmS activity, and we propose that this activation is antagonized by the known GlmR ligand and peptidoglycan intermediate UDP-GlcNAc. Thus, GlmR presides over a new mechanism for the regulation of carbon partitioning between central metabolism and peptidoglycan biosynthesis.

Orientia tsutsugamushiis an obligate intracellular bacterium that infects mononuclear and endothelial cells to cause the emerging global health threat scrub typhus. The ability ofO. tsutsugamushito survive in monocytes facilitates bacterial dissemination to endothelial cells, which can subsequently lead to several potentially fatal sequelae. As a strict intracellular pathogen that lives in the cytoplasm of host cells,O. tsutsugamushihas evolved to counter adaptive immunity. How the pathogen does so and the outcome of this strategy in monocytes versus endothelial cells are poorly understood. This report demonstrates thatO. tsutsugamushireduces cellular levels of NOD-, LRR-, and CARD-containing 5 (NLRC5), a recently identified specific transactivator of major histocompatibility complex class I (MHC-I) component gene expression, to inhibit MHC-I biosynthesis. Importantly, the efficacy of this approach varies with the host cell type infected. In nonprofessional antigen-presenting HeLa and primary human aortic endothelial cells, theO. tsutsugamushi-mediated reduction of NLRC5 results in lowered MHC-I component transcription and, consequently, lower total and/or surface MHC-I levels throughout 72 h of infection. However, in infected THP-1 monocytes, which are professional antigen-presenting cells, the reductions in NLRC5 and MHC-I observed during the first 24 h reverse thereafter.O. tsutsugamushiis the first example of a microbe that targets NLRC5 to modulate the MHC-I pathway. The differential ability ofO. tsutsugamushito modulate this pathway in nonprofessional versus professional antigen-presenting cells could influence morbidity and mortality from scrub typhus.

Phosphohexomutase superfamily enzymes catalyze the reversible intramolecular transfer of a phosphoryl moiety on hexose sugars. Bacillus subtilis phosphoglucomutase PgcA catalyzes the reversible interconversion of glucose 6-phosphate (Glc-6-P) and glucose 1-phosphate (Glc-1-P), a precursor of UDP-glucose (UDP-Glc). B. subtilis phosphoglucosamine mutase (GlmM) is a member of the same enzyme superfamily that converts glucosamine 6-phosphate (GlcN-6-P) to glucosamine 1-phosphate (GlcN-1-P), a precursor of the amino sugar moiety of peptidoglycan. Here, we present evidence that B. subtilis PgcA possesses activity as a phosphoglucosamine mutase that contributes to peptidoglycan biosynthesis. This activity was made genetically apparent by the synthetic lethality of pgcA with glmR, a positive regulator of amino sugar biosynthesis, which can be specifically suppressed by overproduction of GlmM. A gain-of-function mutation in a substrate binding loop (PgcA G47S) increases this secondary activity and suppresses a glmR mutant. Our results demonstrate that bacterial phosphoglucomutases may possess secondary phosphoglucosamine mutase activity, and that this dual activity may provide some level of functional redundancy for the essential peptidoglycan biosynthesis pathway.