We isolated a replication-thermosensitive mutant of the broad-host-range replicon pVWVOl. The mutant pVE6002 is fuly thermosensitive above 35°C in both gram-negative and gram-positive bacteria. Four clustered mutations were identified in the gene encoding the replication protein of pVE6002. The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position. Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed. The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range. Using pVE6002 as the delivery vector for a transposon TnlO derivative in BaciUus subtilis, we observed transposition frequencies of about 1%.
Oxygen is a major determinant of both survival and mortality of aerobic organisms. For the facultative anaerobe Lactococcus lactis, oxygen has negative effects on both growth and survival. We show here that oxygen can be beneficial to L. lactis if heme is present during aerated growth. The growth period is extended and long-term survival is markedly improved compared to results obtained under the usual fermentation conditions. We considered that improved growth and survival could be due to the capacity of L. lactis to undergo respiration. To test this idea, we confirmed that the metabolic behavior of lactococci in the presence of oxygen and hemin is consistent with respiration and is most pronounced late in growth. We then used a genetic approach to show the following. (i) The cydA gene, encoding cytochrome d oxidase, is required for respiration and plays a direct role in oxygen utilization. cydA expression is induced late in growth under respiration conditions. (ii) The hemZ gene, encoding ferrochelatase, which converts protoporphyrin IX to heme, is needed for respiration if the precursor, rather than the final heme product, is present in the medium. Surprisingly, survival improved by respiration is observed in a superoxide dismutase-deficient strain, a result which emphasizes the physiological differences between fermenting and respiring lactococci. These studies confirm respiratory metabolism in L. lactis and suggest that this organism may be better adapted to respiration than to traditional fermentative metabolism.The toxic cellular effects of oxygen are a major factor in aging and mortality (3, 28). Oxygen toxicity is attributed to the activity of reactive oxygen species that attack proteins, lipids, and nucleic acids (15). Effects of oxygen have been extensively studied by use of bacterial models, principally with the facultatively respiring bacterium Escherichia coli (see references 7 and 13 for reviews). In this model, respiration itself is implicated as a source of oxidative damage in E. coli (8, 9, 18, 20, 27, and 36). It has been suggested that the shutdown of respiration in nutrient-limited conditions may reduce reactive oxygen species levels and thereby improve E. coli survival. Recent evidence further suggests that survival is favored by shifting cells to anaerobic conditions during entry into stationary phase (9).Current information on the effects of oxygen is mainly based on respiring organisms. As such, the question of what anaerobes do in the presence of oxidative stress has been explored little. It is presumed that these organisms cope with stress in much the same way as aerobes, except that their defense systems, which may include superoxide dismutases (SODs) and catalases, may be more limited. However, there has been no demonstration to date that responses of anaerobes to an oxidative environment are predictable from the behavior of respiring bacteria.The effects of oxygen have been examined with Lactococcus lactis, a gram-positive facultative anaerobe with a fermentative metabolism that ca...
The role of recA in Lactococcus lactis, a microaerophilic fermenting organism, was examined by constructing a recA-disrupted strain. This single alteration had a surprisingly pleiotropic effect. In addition to its roles in homologous recombination and DNA repair, recA is also involved in responses to oxygen and heat stresses. We found that oxygen stress induced by aeration causes reductions in growth and stationary-phase survival of the recA strain. Toxicity is a consequence of hydroxyl radical production via the Fenton Reaction and is alleviated by catalase or Ferrozine addition. These results suggest that oxygen radicals are not efficiently eliminated and accumulate in lactococcal cultures, and that RecA is needed to deal with the damage they incur. Unexpectedly, thermal stress arrested growth of the recA strain. Immunological data indicate that the recA mutant is deficient in heat-shock proteins DnaK, GroEL, and GrpE. Poor growth at elevated temperature is therefore due to a diminished heat-shock response in the recA strain. In contrast, levels of a novel heat-shock protein, HfIB, are elevated. In Escherichia coli, HfIB downregulates the heat-shock response by promoting degradation of the transcription factor sigma 32. We propose that recA regulates the heat-shock response via HfIB. This work provides the first evidence showing that two major pathways of stress response, induced by heat shock and DNA damage, are interactive.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.