Factors Reducing and Promoting the Effectiveness of Proline as an Osmoprotectant in Escherichia coli K12

Milner, Jocelyn L.; McClellan, Denise J.; Wood, Janet M.

doi:10.1099/00221287-133-7-1851

Cited by 39 publications

(43 citation statements)

References 36 publications

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“…The importance of using iso-osmolar solutions to stop the reaction is evident from the observation that the "activated" uptake of glycine betaine uptake was no longer observed when the cells were exposed to a hypo-osmotic LiCl (100 instead of 500 mM) solution during the washing step. The fact that washing with a hypo-osmotic solution can obscure osmoregulatory solute transport was also established in other studies (1,10). This is also the reason why in the earlier study by Molenaar et al (11) no osmotic activation of glycine betaine transport was observed.…”

supporting

confidence: 50%

“…These studies prompted us to reinvestigate the regulation of glycine betaine transport in Lactococcus lactis, which is thought not to respond to any form of osmotic stress whereas the pool sizes during growth in different media do (11). Osmotic regulation of transport through alterations in activity has not only been shown in lactic acid bacteria but is also well documented for other bacteria (1,10,13,15). In this study, we examined the regulation of glycine betaine uptake in two well-defined L. lactis strains that in many respects are paradigmatic of our knowledge of the physiology, energetics, and genetics of lactic acid bacteria.…”

mentioning

confidence: 99%

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Glycine Betaine Transport in Lactococcus lactis Is Osmotically Regulated at the Level of Expression and Translocation Activity

Heide

Poolman

2000

J Bacteriol

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Microorganisms react upon hyperosmotic stress by accumulating compatible solutes. Here we report that Lactococcus lactis uses a transport system for glycine betaine that, contrary to earlier observations (D. Molenaar et al., J. Bacteriol. 175:5438-5444, 1993), is osmotically regulated at the levels of both expression and transport activity.In their natural habitats, microorganisms are often exposed to changes in the concentrations of the solutes in their environment whereas the internal concentrations of nutrients need to be relatively constant (2,8,13). A sudden increase in the osmolarity of the environment results in the movement of water from the cell to the outside medium, which causes turgor pressure loss, intracellular solute concentration changes, and cell volume changes. Such hyperosmotic conditions are detrimental to any living cell. Bacteria counteract hyperosmotic stress by accumulating compatible solutes by uptake and/or synthesis. These solutes can be accumulated to high intracellular concentrations without affecting vital cellular processes, and they restore the osmotic balance of the cell. Upon hypoosmotic stress, these compatible solutes are released from the cell, which prevents too high a turgor pressure that may ultimately lead to bursting of the cell.Lactic acid bacteria have a limited capacity to synthesize compatible solutes, and a range of studies indicate that glycine betaine, carnitine, and proline are the most important compatible solutes in this group of organisms (3,4,11,12,19). The role of these compounds in osmoregulation in other (micro)-organisms is also well established (2,5,9,13,15,16), but in those cases, additional molecules play a major role as well. There is a considerable amount of data that indicate that in Lactobacillus plantarum and Listeria monocytogenes, glycine betaine, carnitine, and proline are taken up via semiconstitutive transport systems that are activated upon hyperosmotic stress, whereas these compounds are rapidly released by channel-like activities upon osmotic downshock (3, 19). These studies prompted us to reinvestigate the regulation of glycine betaine transport in Lactococcus lactis, which is thought not to respond to any form of osmotic stress whereas the pool sizes during growth in different media do (11). Osmotic regulation of transport through alterations in activity has not only been shown in lactic acid bacteria but is also well documented for other bacteria (1,10,13,15). In this study, we examined the regulation of glycine betaine uptake in two well-defined L. lactis strains that in many respects are paradigmatic of our knowledge of the physiology, energetics, and genetics of lactic acid bacteria. L. lactis MG1363 is a plasmid-free derivative of ML3 that was studied previously (11), whereas IL1403 is a plasmid-free strain for which the genome sequence will soon become available.Regulation of glycine betaine uptake by osmotic upshock. Cells were grown in CDM (14) (with or without proline) plus 25 mM glucose and 500 mM KCl, harvested by centrifugati...

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confidence: 50%

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confidence: 99%

Glycine Betaine Transport in Lactococcus lactis Is Osmotically Regulated at the Level of Expression and Translocation Activity

Heide

Poolman

2000

J Bacteriol

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“…Using a typical value for the volume of an E. coli cell (1 ϫ 10 Ϫ15 liters), the intracellular concentration of hydroxyproline at 450 mM NaCl is ϳ150 mM. This concentration represents an ϳ4-fold increase over the extracellular concentration of 40 mM and is comparable with the increases typically found for proline under osmotic shock conditions (29). Significantly, this concentration approaches the estimated lower limit for the K m of hydroxyproline activation by ProRS.…”

Section: Trans-4-hydroxyproline Incorporation In Bacteriamentioning

confidence: 48%

Co-translational Incorporation ofTrans-4-Hydroxyproline into Recombinant Proteins in Bacteria

Buechter¹,

Paolella²,

Leslie³

et al. 2003

Journal of Biological Chemistry

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Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. Amino acids not specified by the genetic code are common in native proteins and can be essential to both structure and function. Proteins that contain novel non-natural amino acid analogues, furthermore, are of value in structure and function studies and may possess beneficial therapeutic properties. Noncoded amino acids in proteins arise either by enzymatic modification of a transfer RNA (tRNA) aminoacylated with one of the 20 coded amino acids (e.g. selenocysteine from serine) or by post-translational modifications. Reproduction of these reactions when recombinant proteins are being expressed in heterologous hosts is often either inefficient or not possible. Because of these difficulties, many proteins that contain noncoded amino acids are not available in quantities sufficient for detailed biophysical and biochemical studies.Current methodology to make proteins that contain noncoded amino acids in in vitro systems is limited to the production of relatively small amounts of protein. Use of in vitro acylated suppressor tRNAs to insert amino acid analogues at termination codons in genes translated in vitro results in sitespecific incorporation (1) but suffers from inefficiency and low yield. These problems occur in part because of limitations in producing acylated tRNA, constraints in achieving appropriate concentrations of other translational components, and variability in suppression efficiency. In vivo approaches that use an aminoacyl-tRNA synthetase with both altered amino acid and tRNA recognition and suppressor tRNAs are promising (2-4), but experimental hurdles inherent to this approach have not yet been fully overcome.In a general strategy to produce proteins containing novel amino acids, the use of DNA coding triplets to insert the amino acid analogue should be more efficient than use of termination codons. If an aminoacyl-tRNA synthetase is sufficiently promiscuous, it will aminoacylate cognate wild-type tRNA with an amino acid analogue, and the misacylated-tRNA can be used as usual by the translational machinery. This phenomenon has been exploited to produce proteins in prokaryotic systems that contain, for example, analogues of phenylalanine (5) and tryptophan (6). Two requirements must be met for success in these experiments: (1) the analogue must be acylated onto a tRNA at a demonstrable rate, and (2) the analogue must accumulate in the cell at concentrations high enough to give adequate acylation. The first requirement can, in theory, be met either by exploiting the promiscuity of a wild-type synthetase or through mutagenesis to alter the substrate specificity of a wild-type syntheta...

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“…The put operon includes divergently transcribed genes putP and putA. PutP is a Na+-proline symporter which does not accept glycine betaine as substrate (Milner et al, 1987). PutA is the bifunctional enzyme which catalyses the oxidation of proline to glutamic acid (Maloy, 1987).…”

Section: Construction Of Putpa-and Prop-def Icient Mutantsmentioning

confidence: 99%

Osmoregulatory transporter ProP influences colonization of the urinary tract by Escherichia coli

et al. 1998

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Osmoregulatory transporters Prop and ProU mediate the use of betaines as osmoprotectants by Escherichia coli. Glycine betaine and proline betaine are present in mammalian urines. Betaine uptake may therefore facilitate the growth of Em coli in the urinary tract, an environment of fluctuating osmolality. Prop transporter activity was approximately threefold higher in a pyelonephritis isolate, E. coli HU734, than in E. coli K-12. The growth rate of E. coli HU734 in aerated minimal salts medium was reduced twofold by 0 2 M NaCl in the absence and by 055 M NaCl in the presence of glycine betaine.Maximal growth rate stimulation was achieved when glycine betaine was added a t a concentration as low as 25 pM. Deletion of the prop locus impaired the growth rate of E. coli HU734 in human urine but not in minimal medium supplemented with NaCl (04 M), with or without glycine betaine (0-1 mM). The expression of pyelonephritis-associated (P) pili was reduced when E. coli HU734 was cultured in a rich culture medium (LB) of elevated salinity. The prop lesion had no influence on P pilus expression in witm or on the recovery of bacteria from the kidneys of inoculated mice. However, it did reduce their recovery from the bladders of inoculated mice 100-fold. These data provide the first direct evidence that osmoprotective betaine accumulation and transporter Prop are pertinent to both growth in human urine and colonization of the murine urinary tract by uropathogenic E. coli.

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Factors Reducing and Promoting the Effectiveness of Proline as an Osmoprotectant in Escherichia coli K12

Cited by 39 publications

References 36 publications

Glycine Betaine Transport in Lactococcus lactis Is Osmotically Regulated at the Level of Expression and Translocation Activity

Glycine Betaine Transport in Lactococcus lactis Is Osmotically Regulated at the Level of Expression and Translocation Activity

Co-translational Incorporation ofTrans-4-Hydroxyproline into Recombinant Proteins in Bacteria

Osmoregulatory transporter ProP influences colonization of the urinary tract by Escherichia coli

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