Agents that cause Lyme disease, relapsing fever, leptospirosis, and syphilis belong to the phylum Spirochaetae-a unique lineage of bacteria most known for their long, spiral morphology. Despite the relevance to human health, little is known about the most fundamental aspects of spirochete growth. Here, using quantitative microscopy to track peptidoglycan cell-wall synthesis, we found that the Lyme disease spirochete Borrelia burgdorferi displays a complex pattern of growth. B. burgdorferi elongates from discrete zones that are both spatially and temporally regulated. In addition, some peptidoglycan incorporation occurs along the cell body, with the notable exception of a large region at the poles. Newborn cells inherit a highly active zone of peptidoglycan synthesis at midcell that contributes to elongation for most of the cell cycle. Concomitant with the initiation of nucleoid separation and cell constriction, second and third zones of elongation are established at the 1/4 and 3/4 cellular positions, marking future sites of division for the subsequent generation. Positioning of elongation zones along the cell is robust to cell length variations and is relatively precise over long distances (>30 μm), suggesting that cells "sense" relative, as opposed to absolute, cell length to establish zones of peptidoglycan synthesis. The transition from one to three zones of peptidoglycan growth during the cell cycle is also observed in relapsing fever Borrelia. However, this mode of growth does not extend to representative species from other spirochetal genera, suggesting that this distinctive growth mode represents an evolutionary divide in the spirochete phylum.L yme disease is a multisystem disorder that results in flu-like symptoms and, if left untreated, can develop into arthritis, carditis, and severe neurological complications. In recent years, the incidence and geographical range of Lyme disease have rapidly risen (1, 2), making it the most reported vector-borne disease in the United States. In North America, the primary causative agent of Lyme disease is the spirochetal bacterium Borrelia burgdorferi sensu stricto. Whereas most research efforts have focused on host invasion, immune response, and the gene regulatory mechanisms involved in pathogen transmission, comparatively little attention has been paid to the basic biology of this important pathogen (3). In particular, how this bacterium grows and divides remains unknown, despite the fact that these processes are essential for its proliferation. Our knowledge gap in the principles fundamental to cell growth and division extends to the entire spirochete phylum, which, besides B. burgdorferi, includes many important disease-causing agents, such as those responsible for syphilis, relapsing fever, and leptospirosis (4).Spirochetes are unusual bacteria in many respects. For example, most spirochetes are very thin (∼0.2 μm) and long (up to 150 μm) and have a spiral or undulated morphology. Despite similar morphological features, the phylum displays extensive niche dive...

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall and disrupt the bacterial life cycle by cleaving the linkage between the NAG and NAM carbohydrates. Lab exercises focused on the effects of lysozyme on the bacterial cell wall are frequently incorporated in biochemistry classes designed for undergraduate students in diverse fields as biology, microbiology, chemistry, agronomy, medicine, and veterinary medicine. Such exercises typically do not include structural data. We describe here a sequence of computer tasks designed to illustrate and reinforce both physiological and structural concepts involved in lysozyme effects on the bacterial cell-wall structure. This lab class usually lasts 3.5 hours. First, the instructor presents introductory concepts of the bacterial cell wall and the effect of lysozyme on its structure. Then, students are taught to use computer modeling to visualize the three-dimensional structure of a lysozyme in complex with bacterial cell-wall fragments. Finally, the lysozyme inhibitory effect on a bacterial culture is optionally proposed as a simple microbiological assay. The computer lab exercises described here give students a realistic understanding of the disruptive effect of lysozymes on the bacterial cell wall, a crucial component in bacterial survival. V C 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):83-90, 2018.

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