Alkaline phosphatase localization and spheroplast formation of <i>Pseudomonas aeruginosa</i>

Cheng, K.-J.; Ingram, J. M.; Costerton, J. W.

doi:10.1139/m70-218

Cited by 68 publications

(52 citation statements)

References 25 publications

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“…Myxospore breakage was monitored by phase-contrast microscopy. Samples were then centrifuged at 5,000 x g for 5 min to remove debris from the cell extract, and supernatants (0.3-ml samples) were assayed for alkaline phosphatase (4). For determination of the alkaline phosphatase activity during starvation-induced cell development, exponential-phase cells were concentrated by centrifugation, suspended in CF broth to 3,000 or 4,500 Klett units, and incubated at 30°C or heat shocked, respectively.…”

Section: Methodsmentioning

confidence: 99%

Acceleration of starvation- and glycerol-induced myxospore formation by prior heat shock in Myxococcus xanthus

Killeen

Nelson

1988

J Bacteriol

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The effect of heat shock on Myxococcus xanthus was investigated during both glycerol-and starvationinduced development. Cells heat shocked at 40°C for 1 h prior to a development-inducing signal displayed an accelerated rate of myxospore formation at 30°C. Additionally, M. xanthus cells heat shocked prior to glycerol induction formed a greater total number of myxospores when sporulation was complete than did control cells maintained at 30°C. However, in starvation-induced fruiting cells the total number of myxospores in control and heat-shocked populations was about equal when fruiting body and myxospore formation was complete. When extended heat shock (3 h) was applied to cells prior to development, no acceleration of myxospore formation was observed. Heat shock elicited the premature expression of many developmentally regulated proteins. Cell fractionation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography revealed the subcellular location and molecular weights of the 18 glycerol-induced and 9 starvation-induced developmental proteins. Comparison with previously identified M. xanthus heat shock proteins showed that nine of the developmental proteins found in glycerol-induced cells and three of the developmental proteins found in starvation-induced cells were heat shock proteins. Furthermore, heat shock increased the activity of alkaline phosphatase, a developmentally regulated enzyme, in vegetative cells, glycerol-induced cells, and starvation-induced cells.All organisms respond to elevated temperatures by altering their pattern of growth and protein synthesis (16). Upon exposure to elevated temperatures, cells rapidly cease growth, repress the synthesis of most vegetative polypeptides, and coordinately synthesize a small number of different proteins (16). These heat shock proteins (HSPs) may also be induced by other environmental stresses, such as exposure to various alcohols, oxidants (16), and abnormal proteins (1).In many diverse organisms, such as Saccharomyces cerevisiae (13), Drosophila melanogaster (22), and mice (12), some heat shock genes are expressed during particular stages of cellular development. The association of HSPs with certain periods of cellular differentiation suggests that these proteins may be involved in both normal developmental processes and stress responses.Myxococcus xanthus is a gram-negative, rod-shaped, gliding bacterium (9). Although it is a procaryote, it exhibits a complex program of multicellular development. When subjected to starvation on a solid surface, cells migrate inwards towards localized aggregation centers to form raised mounds of cells. Within these mounds, individual cells differentiate to form round, environmentally resistant myxospores. These mounds of cells and spores are termed fruiting bodies (15). This process of fruiting body formation is cell density dependent, includes the temporal expression of proteins such as myxobacterial hemagglutinin (5) and proteins U (11) and S (8), and extends over a 48-to 72-h period (6...

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Section: Methodsmentioning

confidence: 99%

Acceleration of starvation- and glycerol-induced myxospore formation by prior heat shock in Myxococcus xanthus

Killeen

Nelson

1988

J Bacteriol

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“…The procedures described by Nelson et al (22) were used to obtain the periplasmic, cytoplasmic, and membrane fractions of M. xanthus cells. The fractions were checked for cross contamination by assaying each for specific enzyme markers: alkaline phosphatase (periplasmic [5]), succinic dehydrogenase (membrane [17]), and glutamic dehydrogenase (cytoplasmic [7]). All fractions showed less than 10% cross contamination.…”

Section: Methodsmentioning

confidence: 99%

Heat shock proteins of vegetative and fruiting Myxococcus xanthus cells

Nelson¹,

Killeen²

1986

J Bacteriol

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The heat shock response of Myxococcus xanthus was investigated and characterized. When shifted from 28 to 40°C, log-phase cells rapidly ceased growth, exhibited a 50% reduction in CFU, and initiated the synthesis of heat shock proteins (HTPs). Heat-shocked log-phase M. xanthus cells labeled with [35S]methionine were found to produce 18 major HTPs. The HTPs, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, were characterized with regard to molecular mass, subcellular location (periplasm, membrane, or cytoplasm), and temperature required for expression. Most HTPs were expressed at 36°C, the optimum growth temperature of M. xanthus. Cells preincubated at 36°C for 1 h before being shifted to 40°C demonstrated increased thermotolerance compared with cells shifted directly from 28 to 40°C. The HTPs produced by heat-shocked starvation-induced fruiting cells and glycerol-induced sporulating cells were also analyzed and characterized. Thirteen HTPs were detected in fruiting cells shifted from 28 to 40°C. Six of these HTPs were not seen in vegetative M. xanthus cells. Log-phase cells induced to sporulate by the addition of glycerol produced 17 HTPs after being shifted to 40°C. These HTPs were found to be a mixture of HTPs detected in heat-shocked log-phase cells and heat-shocked fruiting cells.Myxococcus xanthus is a gram-negative, gliding, rodshaped bacterium that is commonly found in soil where it grows by preying upon other microorganisms and by degrading complex organic matter (14,29). When subjected to starvation on a solid surface, vegetative swarms of M. xanthus reverse their outward growth and migrate inwards to localized centers of aggregation, forming raised mounds of cells. Within these mounds, individual cells convert to ovoid or round, environmentally resistant myxospores. These mounds of myxospores are termed fruiting bodies (19). Rapid, synchronous myxospore formation may also be induced in vegetative M. xanthus cells by the addition of glycerol (to 0.5 M), dimethyl sulfoxide, or a variety of alcohols (6,15,30). In contrast to fruiting body formation which occurs over 48 to 72 h and requires a solid surface, glycerol-induced myxospores form within 3 h, do not aggregate, and do not require a solid surface (6). Glycerol-induced myxospores also lack some of the proteins characteristic of starvation-induced fruiting cells (15).The intent of this investigation was to examine the response of M. xanthus to heat shock. It has been shown that organisms from all three urkingdoms respond to elevated temperatures by altering their patterns of growth and protein synthesis. Upon experiencing elevated temperatures, cells rapidly cease growth, repress the synthesis of most proteins, and begin to preferentially synthesize a small number of new proteins (21). This has been termed the heat shock response. Studies have shown that some of the genes coding for heat shock proteins (HTPs) are highly conserved across all three urkingdoms (1, 21). In this paper, we demonstrate that M. xanthus e...

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“…In previous papers (3)(4)(5) we reported that all of the cell-bound alkaline phosphatase is removed from P. aeruginosa by 0.2 M Mg2+. In the present study we have shown that the LPS (23).…”

Section: Discussionmentioning

confidence: 90%

“…The LPS was hydrolyzed in 0.02 N H2SO4 in a boiling water bath as described by Osborn et al (22). The KDO was determined by the thiobarbituric acid procedure of Weissbach (37) (4,5). Magnesiumlysozyme spheroplasts were prepared similarly, except that the 20% sucrose was replaced by 0.2 M MgCl2 and 0.01 M Tris (pH 8.4).…”

mentioning

confidence: 99%

Susceptibility of Whole Cells and Spheroplasts of Pseudomonas aeruginosa to Actinomycin D

Cheng

Costerton

Singh

et al. 1973

Antimicrob Agents Chemother

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Alkaline phosphatase localization and spheroplast formation of Pseudomonas aeruginosa

Cited by 68 publications

References 25 publications

Acceleration of starvation- and glycerol-induced myxospore formation by prior heat shock in Myxococcus xanthus

Acceleration of starvation- and glycerol-induced myxospore formation by prior heat shock in Myxococcus xanthus

Heat shock proteins of vegetative and fruiting Myxococcus xanthus cells

Susceptibility of Whole Cells and Spheroplasts of Pseudomonas aeruginosa to Actinomycin D

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