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Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes L-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S L-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of L-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C 1 units and interferes with cell wall synthesis. We suggest that at high concentrations, L-serine decreases synthesis of UDP-Nacetylmuramate-L-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high L-serine is overcome in several ways: by a large concentration of L-alanine, by overproducing MurC together with a low concentration of L-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.The Escherichia coli genome contains three genes, sdaA, sdaB, and tdcG, specifying three very similar 4Fe4S L-serine deaminases. These enzymes are very specific for L-serine for which they have unusually high K m values (3, 32). Expression of the three genes is regulated so that at least one of the gene products is synthesized under all common growth conditions (25). This suggests an important physiological role for the enzymes. However, why E. coli needs to deaminate L-serine has been a long-standing problem of E. coli physiology, the more so since it cannot use L-serine as the sole carbon source.We showed recently that an E. coli strain devoid of all three L-serine deaminases (L-SDs) loses control over its size, shape, and cell division when faced with complex amino acid mixtures containing L-serine (32). We attributed this to starvation for single-carbon (C 1 ) units and/or S-adenosylmethionine (SAM). C 1 units are usually made from serine via serine hydroxymethyl transferase (GlyA) or via glycine cleavage (GCV). The L-SDdeficient triple mutant strain is starved for C 1 in the presence of amino acids, because externally provided glycine inhibits GlyA and a very high internal L-serine concentration along with several other amino acids inhibits glycine cleavage. While the parent cell can defend itself by reducing the L-serine level by deamination, this crucial reaction is missing in the ⌬sdaA ⌬sdaB ⌬tdcG triple mutant. We therefore consider these to be "defensive" serine deaminases.The fact that an inability to deaminate L-serine leads to a high concentration of L-serine and inhibition of GlyA is not surprising. However, it is not obvious why a high level of L-serine inhibits cell division and causes swelling, lys...

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DNA replication is carried out by a multi-protein machine called the replisome. In Saccharomyces cerevisiae, the replisome is composed of over 30 different proteins arranged into multiple subassemblies, each performing distinct activities. Synchrony of these activities is required for efficient replication and preservation of genomic integrity. How this is achieved is particularly puzzling at the lagging strand, where current models of the replisome architecture propose turnover of the canonical lagging strand polymerase, Pol δ, at every cycle of Okazaki fragment synthesis.Here we established single-molecule fluorescence microscopy protocols to study the binding kinetics of individual replisome subunits in live S. cerevisiae. Our results show long residence times for most subunits at the active replisome, supporting a model where all subassemblies bind tightly and work in a coordinated manner for extended periods, including Pol δ, hence redefining the architecture of the active eukaryotic replisome..

Bacteria have been traditionally classified in terms of size and shape and are best known for their very small size. Escherichia coli cells in particular are small rods, each 1–2 μ. However, the size varies with the medium, and faster growing cells are larger because they must have more ribosomes to make more protoplasm per unit time, and ribosomes take up space. Indeed, Maaløe’s experiments on how E. coli establishes its size began with shifts between rich and poor media. Recently much larger bacteria have been described, including Epulopiscium fishelsoni at 700 μm and Thiomargarita namibiensis at 750 μm. These are not only much longer than E. coli cells but also much wider, necessitating considerable intracellular organization. Epulopiscium cells for instance, at 80 μm wide, enclose a large enough volume of cytoplasm to present it with major transport problems. This review surveys E. coli cells much longer than those which grow in nature and in usual lab cultures. These include cells mutated in a single gene (metK) which are 2–4 × longer than their non-mutated parent. This metK mutant stops dividing when slowly starved of S-adenosylmethionine but continues to elongate to 50 μm and more. FtsZ mutants have been routinely isolated as long cells which form during growth at 42°C. The SOS response is a well-characterized regulatory network that is activated in response to DNA damage and also results in cell elongation. Our champion elongated E. coli is a metK strain with a further, as yet unidentified mutation, which reaches 750 μm with no internal divisions and no increase in width.

Although Escherichia coli is a very small (1-to 2-m) rod-shaped cell, here we describe an E. coli mutant that forms enormously long cells in rich media such as Luria broth, as long indeed as 750 m. These extremely elongated (eel) cells are as long as the longest bacteria known and have no internal subdivisions. They are metabolically competent, elongate rapidly, synthesize DNA, and distribute cell contents along this length. They lack only the ability to divide. The concentration of the essential cell division protein FtsZ is reduced in these eel cells, and increasing this concentration restores division. IMPORTANCEEscherichia coli is usually a very small bacterium, 1 to 2 m long. We have isolated a mutant that forms enormously long cells, 700 times longer than the usual E. coli cell. E. coli filaments that form under other conditions usually die within a few hours, whereas our mutant is fully viable even when it reaches such lengths. This mutant provides a useful tool for the study of aspects of E. coli physiology that are difficult to investigate with small cells. We think of bacterial cells as small organisms. Cells in a growing Escherichia coli culture look like rigid rods 0.5 m wide by 2 m long. Each rod elongates to twice its original length by making new peptidoglycan in many disperse areas by using an enzyme complex including penicillin-binding protein 2 (PBP2). The organism then localizes peptidoglycan synthesis to midcell by using a different enzyme complex, based on PBP3. This changes the direction of cell wall synthesis; the cell wall invaginates from both sides, thus forming two identical daughter cells (1, 2, 3).E. coli cells are small because their division controls are set accordingly (4). However, they can be much longer if a shift to PBP3 cannot be made but conditions still permit PBP2 function. This occurs under many circumstances. Mutants with conditional temperature-sensitive mutations in the gene coding for the essential cell division protein FtsZ have been isolated. These mutations result in the inhibition of division, accompanied by elongation into short-lived filaments, when cells are shifted from 37°C to 42°C (5).Filaments also form when the SOS response is triggered under conditions of DNA damage (6) or when the cells are treated with the antibiotic aztreonam, which blocks division irreversibly by inhibiting the FtsI protein, involved in the formation of the septal ring (7). The filaments formed under these conditions, and others, are not viable and lyse within a few hours. As a result, it was largely believed that E. coli could not sustain a large cell size.However, in this paper, we describe a mutant that forms very long viable (i.e., colony-forming) cells. This strain was derived from our earlier mutant carrying a deletion in metK, which codes for S-adenosylmethionine (SAM) synthetase; SAM is the major donor of methyl groups (8, 9). Our metK deletion mutant, MEW649, had to be exogenously provided with both SAM and methionine in order to grow, as well as with a SAM transporte...

Introduction Racial disparities in Alzheimer's disease (AD) and all‐cause dementia (DEMENTIA) incidence may exist differentially among men and women, with unknown mechanisms. Methods A retrospective cohort study examining all‐cause and AD dementia incidence was conducted linking Third National Health and Nutrition Examination Survey (NHANES III) to Centers for Medicare & Medicaid Services Medicare data over ≤26 years of follow‐up (1988 to 2014). Cox regression and generalized structural equation models (GSEMs) were constructed among men and women ≥60 years of age at baseline (N = 4592). Outcomes included onset ages of all‐cause and AD dementia, whereas the main exposures were race/ethnicity contrasts (RACE_ETHN). Potential mediators) included socio‐economic status (SES), lifestyle factors (dietary quality [DIET] nutritional biomarkers [NUTR], physical activity [PA], social support [SS], alcohol [ALCOHOL], poor health [or HEALTH], poor cognitive performance [or COGN]. In addition to RACE_ETHN, the following were exogenous covariates in the GSEM and potential confounders in Cox models: age, sex, urban‐rural, household size, and marital status. Results Non‐Hispanic Black (NHB) women had a higher risk of DEMENTIA versus non‐Hispanic White (NHW) women in GSEM, consistent with Cox models (age‐adjusted model: hazard ratio [HR] = 1.34, 95% confidence interval [CI]: 1.10 to 1.61). The total effect of this RACE_ETHN contrast in women was explained by four main pathways: (1) RACE_ETHN→ poor cognitive performance (COGN, +) → DEMENTIA (+); (2) RACE_ETHN → SES (−) → COGN (−) → DEMENTIA (+); (3) RACE_ETHN → SES (−) → physical activity (PA, +) → COGN (−) → DEMENTIA (+); and (4) RACE_ETHN → SES (−) → DIET (+) → COGN (−) → DEMENTIA (+). A reduced AD risk in Mexican American (MA) women versus NHW women upon adjustment for SES and downstream factors (HR = 0.53, 95% CI: 0.35 to 0.80). For the non‐White versus NHW contrast in incident DEMENTIA, pathways involved lower SES, directly increasing cognitive deficits (or indirectly through lifestyle factors), which then directly increases DEMENTIA . Discussion Socioeconomic and lifestyle factors explaining disparities between NHB and NHW in dementia onset among women are important to consider for future observational and intervention studies.

An E. coli K-12 mutant deficient in S-adenosylmethionine (SAM) synthesis, i.e DmetK, but expressing a rickettsial SAM transporter, can grow in glucose minimal medium if provided with both SAM and methionine. It uses the externally provided (R)-enantiomer of SAM as methyl donor to produce most but not all of its methionine, by methylation of homocysteine catalysed by homocysteine methyltransferase (MmuM). The DmetK cells are also altered in growth and are twice as long as those of the parent strain. When starved of SAM, the mutant makes a small proportion of very long cells suggesting a role of SAM and of methylation in the onset of crosswall formation.