A cultural divide on the use of chemostats.
Explore the source record for details and available documents.
Bacteria↗
Biomedical subjects
T Ferenci
Publications and source records attributed to T Ferenci.
Hungry bacteria--definition and properties of a nutritional state.
Bacteria are sometimes neither starving nor under nutrient-excess conditions. When growing with suboptimal levels of nutrients, hungry bacteria express appropriate cellular responses. This review discusses approaches to defining the hunger response in both molecular and growth kinetic terms. The gene expression changes unique to hunger conditions are described, using Escherichia coli as the primary example. Metabolite changes with hunger and starvation and the differing role of the stationary phase regulator RpoS also lead to the hypothesis proposed in this review that bacteria undertake distinct approaches to hunger and starvation. Indeed, an understanding of the difference between hunger and starvation and the incompatibility between hunger and starvation responses explains some of the paradoxical mutational adaptations, such as rpoS inactivation, found in natural populations.
Bacteria↗
An analysis of multifactorial influences on the transcriptional control of ompF and ompC porin expression under nutrient limitation.
Expression of the major outer-membrane porins in Escherichia coli is transcriptionally controlled during nutrient limitation. Expression of ompF was more than 40-fold higher under glucose limitation than under nitrogen (ammonia) limitation in chemostat cultures at the same growth rate. In contrast, ompC expression was higher under N limitation. The basis of regulation by nutrient limitation was investigated using mutations affecting expression of porin genes. The influence of cyaA, rpoS, ackA and pta, as well as the two-component envZ-ompR system, was studied under glucose and N limitation in chemostat cultures. A major contributor to low ompF expression under N limitation was negative control by the RpoS sigma factor. RpoS levels were high under N limitation and loss of RpoS resulted in a 19-fold increase in ompF transcription, but little change was observed with ompC. Lack of RpoS under glucose limitation had a lesser stimulatory effect on ompF expression. Porin production was minimally dependent on EnvZ under N limitation due to OmpR phosphorylation by acetyl phosphate. Evidence obtained with pta and ackA mutants suggested that the acetyl phosphate level also regulates porins independently and indirectly via RpoS and other pathways. pta-envZ double mutants had a residual level of porin transcription, implicating alternative means of OmpR phosphorylation under nutrient limitation. Another critical factor in regulation was the level of cAMP, as a cyaA mutant hardly expressed ompF under glucose limitation but boosted ompC. In addition, the role of DNA-binding proteins encoded by hns and himA was tested under glucose limitation: the hns mutation reduced the glucose-limitation peak, but the himA mutation suppressed the hns effect, suggesting a complex web of interrelationships between the DNA-binding proteins. Indeed, multiple inputs and no single regulator were responsible for the high peak of ompF expression under glucose limitation.
Adenylyl Cyclases↗
Experimental analysis of molecular events during mutational periodic selections in bacterial evolution.
A fundamental feature of bacterial evolution is a succession of adaptive mutational sweeps when fitter mutants take over a population. To understand the processes involved in mutational successions, Escherichia coli continuous cultures were analyzed for changes at two loci where mutations provide strong transport advantages to fitness under steady-state glucose limitation. Three separate sweeps, observed as classic periodic selection events causing a change in the frequency of neutral mutations (in fhuA causing phage T5 resistance), were identified with changes at particular loci. Two of the sweeps were associated with a reduction in the frequency of neutral mutations and the concurrent appearance of at least 13 alleles at the mgl or mlc loci, respectively. These mgl and mlc polymorphisms were of many mutational types, so were not the result of a mutator or directed mutation event. The third sweep observed was altogether distinct and involved hitchhiking between T5 resistance and advantageous mgl mutations. Moreover, the hitchhiking event coincided with an increase in mutation rates, due to the transient appearance of a strong mutator in the population. The spectrum of mgl mutations among mutator isolates was distinct and due to mutS. The mutator-associated periodic selection also resulted in mgl and fhuA polymorphism in the sweeping population. These examples of periodic selections maintained significant genotypic diversity even in a rapidly evolving culture, with no individual "winner clone" or genotype purging the population.
Adenosine Triphosphatases↗
Global adaptations resulting from high population densities in Escherichia coli cultures.
The scope of population density effects was investigated in steady-state continuous cultures of Escherichia coli in the absence of complications caused by transient environmental conditions and growth rates. Four distinct bacterial properties reflecting major regulatory and physiological circuits were analyzed. The metabolome profile of bacteria growing at high density contained major differences from low-density cultures. The 10-fold-elevated level of trehalose at higher densities pointed to the increased role of the RpoS sigma factor, which controls trehalose synthesis genes as well as the general stress response. There was an eightfold difference in RpoS levels between bacteria grown at 10(8) and at 10(9) cells/ml. In contrast, the cellular content of the DNA binding protein H-NS, controlling many genes in concert with RpoS, was decreased by high density. Since H-NS and RpoS also influence porin gene expression, the influence of population density on the intricate regulation of outer membrane composition was also investigated. High culture densities were found to strongly repress ompF porin transcription, with a sharp threshold at a density of 4.4 x 10(8) cells/ml, while increasing the proportion of OmpC in the outer membrane. The density-dependent regulation of ompF was maintained in rpoS or hns mutants and so was independent of these regulators. The consistently dramatic changes indicate that actively growing, high-density cultures are at least as differentiated from low-density cultures as are exponential- from stationary-phase bacteria.
Adaptation, Physiological↗
Substrate specificity and signal transduction pathways in the glucose-specific enzyme II (EII(Glc)) component of the Escherichia coli phosphotransferase system.
Escherichia coli adapted to glucose-limited chemostats contained mutations in ptsG resulting in V12G, V12F, and G13C substitutions in glucose-specific enzyme II (EII(Glc)) and resulting in increased transport of glucose and methyl-alpha-glucoside. The mutations also resulted in faster growth on mannose and glucosamine in a PtsG-dependent manner. By use of enhanced growth on glucosamine for selection, four further sites were identified where substitutions caused broadened substrate specificity (G176D, A288V, G320S, and P384R). The altered amino acids include residues previously identified as changing the uptake of ribose, fructose, and mannitol. The mutations belonged to two classes. First, at two sites, changes affected transmembrane residues (A288V and G320S), probably altering sugar selectivity directly. More remarkably, the five other specificity mutations affected residues unlikely to be in transmembrane segments and were additionally associated with increased ptsG transcription in the absence of glucose. Increased expression of wild-type EII(Glc) was not by itself sufficient for growth with other sugars. A model is proposed in which the protein conformation determining sugar accessibility is linked to transcriptional signal transduction in EII(Glc). The conformation of EII(Glc) elicited by either glucose transport in the wild-type protein or permanently altered conformation in the second category of mutants results in altered signal transduction and interaction with a regulator, probably Mlc, controlling the transcription of pts genes.
Amino Acid Substitution↗
OmpF changes and the complexity of Escherichia coli adaptation to prolonged lactose limitation.
The disaccharide lactose has no specific diffusion pathway across the outer membrane of Escherichia coli. At least three classes of spontaneous mutation affecting outer membrane permeability arose with each of three independent E. coli populations adapting to prolonged lactose limitation in chemostats. Both structural and regulatory mutations affecting OmpF porin predominated in isolates after 210-280 generations of culture. Six types of ompF mutation were found, including in-frame deletions and substitutions at Arg82 and Asp113, all affecting the channel constriction residues of OmpF. Isolates had increased susceptibility to antibiotics and were affected in the quantity of OmpF, LamB and OmpA proteins. A minimum of three or four mutations was evident in all isolates after 280 generations in a lactose-limited environment, in addition to lac mutations defined in previous studies.
Adaptation, Physiological↗
'Growth of bacterial cultures' 50 years on: towards an uncertainty principle instead of constants in bacterial growth kinetics.
Ever since Monod's efforts to study bacterial cultures in quantitative terms, the growth of Escherichia coli on sugars like glucose has appeared an attractive subject for the mathematical description of nutrient conversion into biomass. But instead of simplicity, it is becoming evident that bacterial adaptations affect 'constants' such as K(s) (growth affinity constant) and are, in turn, a complex function of nutrient concentration. Instead of a single affinity, bacteria exhibit a continuum of nutrient scavenging abilities peaking at micromolar sugar levels; there is lower affinity with millimolar or submicromolar glucose in the medium. Similar problems arise in defining parameters such as Y (growth yield constant), because nutrient-limited growth at low exponential growth rates induces a continuum of hunger and starvation responses. Autocatalytic changes to the environment caused by growth (as well as external factors) ensure that bacteria present an ever-adapting interface to the outside world. The regulatory interaction between the organism and environment means that no universal kinetic constants describe bacterial growth.
Bacteria↗
Regulation by nutrient limitation.
Growth with suboptimal nutrient levels elicits adaptations not observed with either starving (resting) or unstressed bacteria. The hunger response results in patterns of gene expression optimising scavenging capabilities through novel control mechanisms. In Escherichia coli, recent results indicate that outermembrane permeability (porin and glycoporin regulation) as well as transport involving the phosphotransferase system and ABC-type high affinity transporters change under glucose limitation. Many other adaptations in expression and metabolic capabilities at subsaturating growth rates are still poorly understood, even in E. coli.
ATP-Binding Cassette Transporters↗
Adaptive mgl-regulatory mutations and genetic diversity evolving in glucose-limited Escherichia coli populations.
The mutational adaptation of E. coli to low glucose concentrations was studied in chemostats over 280 generations of growth. All members of six independent populations acquired increased fitness through the acquisition of mutations at the mgl locus, increasing the binding protein-dependent transport of glucose. These mutations provided a strong fitness advantage (up to 10-fold increase in glucose affinity) and were present in most isolates after 140 generations. mgl constitutivity in some isolates was caused by base substitution, short duplication, small deletion and IS1 insertion in the 1041 bp gene encoding the repressor of the mgl system, mglD (galS). But an unexpectedly large proportion of mutations were located in the short mgl operator sequence (mglO), and the majority of mutations were in mglO after 280 generations of selection. The adaptive mglO substitutions in several independent populations were at exactly the positions conserved in the two 8 bp half-sites of the mgl operator, with the nature of the base changes also completely symmetrical. Either mutations were directed to the operator or the particular operator mutations had a selective advantage under glucose limitation. Indeed, isolates carrying mglO mutations showed greater rates of transport for glucose and galactose at low concentrations than those carrying mglD null mutations. mglO mutations avoid cross-talk by members of the GalR-Lacl repressor family, reducing transporter expression and providing a competitive advantage in a glucose-limited environment. Another interesting aspect of these results was that each adapted population acquired multiple mgl alleles, with several populations containing at least six different mgl-regulatory mutations co-existing after 200 generations. The diversity of mutations in the mglO/mglD region, generally in combination with mutations at other loci regulating glucose uptake (malT, mlc, ptsG), provided evidence for multiple clones in each population. Increased fitness was accompanied by the generation of genetic diversity and not the evolution of a single winner clone, as predicted by the periodic selection model of bacterial populations.
Adaptation, Physiological↗
The generation of multiple co-existing mal-regulatory mutations through polygenic evolution in glucose-limited populations of Escherichia coli.
The multicomponent glucose transport system of Escherichia coli was used to study the polygenic basis of increased fitness in prolonged nutrient-limited, continuous cultures. After 280 generations of glucose-limited growth, nearly all bacteria in four independent chemostat populations exhibited increased glucose transport and contained multiple, stable mutations. Fitter bacteria increased outer membrane permeability for glucose through overexpression of the LamB glycoporin. Three classes of mutation influenced LamB levels as well as regulation of other mal genes. Low-level mal/lamB constitutivity resulted from mlc mutations acquired in all populations as well as changes at another uncharacterized locus. Larger increases in transporter content resulted from widespread acquisition of a regulatory malT-con mutation in fit isolates. The malT mutations sequenced from 67 adapted isolates were all single base substitutions resulting in amino acid replacements in the N-terminal third of the MalT activator protein. Analysis of malT-con sequences revealed a mutational spectrum distinct from that found in plate-selected malT mutants, suggesting that mutational pathways were affected by environmental factors. A second major finding was the remarkable allele diversity in malT within a population derived from a single clone, with at least 11 different alleles co-existing in a population. The multiplicity of alleles (as well as those found in adaptive mgl changes in the accompanying study) suggest that the periodic selection events observed previously in such populations are not a major factor in reducing genetic diversity. A simple model is presented for the generation of genetic heterogeneity in bacterial populations undergoing polygenic selection.
Adaptation, Physiological↗
Mutational adaptation of Escherichia coli to glucose limitation involves distinct evolutionary pathways in aerobic and oxygen-limited environments.
Mutational adaptations leading to improved glucose transport were followed with Escherichia coli K-12 growing in glucose-limited continuous cultures. When populations were oxygen limited as well as glucose limited, all bacteria within 280 generations contained mutations in a single codon of the ptsG gene. V12F and V12G replacements in the enzyme IIBC(Glc) component of the glucose phosphotransferase system were responsible for improved transport. In stark contrast, ptsG mutations were uncommon in fully aerobic glucose-limited cultures, in which polygenic mutations in mgl, mlc, and malT (regulating an alternate high-affinity Mgl/LamB uptake pathway) spread through the adapted population. Hence the same organism adapted to the same selection (glucose limitation) by different evolutionary pathways depending on a secondary environmental factor. The clonal diversity in the adapted populations was also significantly different. The PtsG V12F substitution under O(2) limitation contributed to a universal "winner clone" whereas polygenic, multiallelic changes led to considerable polymorphism in aerobic cultures. Why the difference in adaptive outcomes? E. coli physiology prevented scavenging by the LamB/Mgl system under O(2) limitation; hence, ptsG mutations provided the only adaptive pathway. But ptsG mutations in aerobic cultures are overtaken by mgl, mlc, and malT adaptations with better glucose-scavenging ability. Indeed, when an mglA::Tn10 mutant with an inactivated Mgl/LamB pathway was introduced into two independent aerobic chemostats, adaptation of the Mgl(-) strain involved the identical ptsG mutation found under O(2)-limited conditions with wild-type or Mgl(-) bacteria.
Adaptation, Physiological↗
Assessing the effect of reactive oxygen species on Escherichia coli using a metabolome approach.
A two-dimensional thin-layer chromatographic analysis of [14C]-labelled metabolites in Escherichia coli was employed to follow metabolic shifts in response to superoxide stress. Steady-state challenge with paraquat at concentrations inducing SoxRS-controlled genes resulted in several alterations in metabolite pools, including a striking increase in valine concentration. Elevated valine levels, together with increased glutathione and alkylperoxidase, are proposed to constitute an intracellular protection mechanism against reactive oxygen species. As shown by this example of metabolome analysis, novel cellular responses to environmental challenge can be revealed by following the total complement of metabolites in a cell.
Escherichia coli↗
Regulation of porin-mediated outer membrane permeability by nutrient limitation in Escherichia coli.
OmpF and OmpC porins were differentially regulated by nutrient limitation and growth rate in glucose- or nitrogen-limited chemostat cultures of Escherichia coli. Transcriptional and translational ompF fusions showed a sharp peak of expression under glucose limitation at D = 0.3 h-1, with lower amounts at lower and higher growth rates. The peak of OmpR-dependent transcriptional stimulation of ompF under glucose limitation in minimal salts media was about 20-fold above nutrient excess levels and 3-fold higher than that achieved with low osmolarity. Analysis of outer membrane protein levels and results of growth competition experiments with porin mutants were consistent with the enhanced role of OmpF under glucose limitation, but not N limitation. In contrast, OmpC was the major porin under N limitation but was increasingly expressed under glucose limitation at very low growth rates approaching starvation, when OmpF was downregulated. In summary, outer membrane permeability under N-limited, sugar-rich conditions is largely based on OmpC, whereas porin activity is a complex, highly sensitive function of OmpF, OmpC, and LamB glycoporin expression under different levels of glucose limitation. Indeed, the OmpF level was more responsive to nutrient limitation than to medium osmolarity and suggested a significant additional layer of control over the porin-regulatory network.
Bacterial Proteins↗
Effect of slow growth on metabolism of Escherichia coli, as revealed by global metabolite pool ("metabolome") analysis.
Escherichia coli growing on glucose in minimal medium controls its metabolite pools in response to environmental conditions. The extent of pool changes was followed through two-dimensional thin-layer chromatography of all 14C-glucose labelled compounds extracted from bacteria. The patterns of metabolites and spot intensities detected by phosphorimaging were found to reproducibly differ depending on culture conditions. Clear trends were apparent in the pool sizes of several of the 70 most abundant metabolites extracted from bacteria growing in glucose-limited chemostats at different growth rates. The pools of glutamate, aspartate, trehalose, and adenosine as well as UDP-sugars and putrescine changed markedly. The data on pools observed by two-dimensional thin-layer chromatography were confirmed for amino acids by independent analysis. Other unidentified metabolites also displayed different spot intensities under various conditions, with four trend patterns depending on growth rate. As RpoS controls a number of metabolic genes in response to nutrient limitation, an rpoS mutant was also analyzed for metabolite pools. The mutant had altered metabolite profiles, but only some of the changes at slow growth rates were ascribable to the known control of metabolic genes by RpoS. These results indicate that total metabolite pool ("metabolome") analysis offers a means of revealing novel aspects of cellular metabolism and global regulation.
Adenosine↗
A novel levansucrase-levanase gene cluster in Bacillus stearothermophilus ATCC12980.
A 3.1 kb fragment of Bacillus stearothermophilus ATCC12980 DNA permitted growth of Escherichia coli on sucrose. The fragment encoded genes for a levansucrase (surB) and also a levanase (surC). SurB and SurC shared 97% and 43% amino acid identity with the corresponding SacB and SacC proteins of Bacillus subtilis, whose sacB and sacC genes are organised very differently.
Amino Acid Sequence↗
Gene organisation and regulatory sequences in the sucrose utilisation cluster of Bacillus stearothermophilus NUB36.
The nucleotide sequence of the surP and surT genes in a sucrose-utilisation cluster cloned from Bacillus stearothermophilus NUB36 was determined. The surP gene encoded a protein of 466 amino acid residues and shared 60-62% amino acid identity with the sucrose-specific enzyme II components of the phosphotransferase system of Bacillus subtilis, Salmonella typhimurium and Klebsiella pneumoniae. SurP, like other sucrose EIIs, lacked the hydrophilic domain containing the first (IIA) phosphorylation site. The surT gene encoded a 278 amino acid polypeptide which showed 63.1% and 54% amino acid identity to the B. subtilis antiterminators SacT and SacY, respectively. A region containing a palindromic structure preceding surP was highly homologous to the regulatory transcription termination regions of the sacPA and sacB operons of B. subtilis and the bgl operon of Escherichia coli. Hence the sucrose gene cluster of B. stearothermophilus NUB36 is very similar to the B. subtilis sacPA operon in terms of gene order and regulatory organisation.
Amino Acid Sequence↗
Rate constants of sugar transport through two LamB mutants of Escherichia coli: comparison with wild-type maltoporin and LamB of Salmonella typhimurium.
Two LamB (maltoporin) point mutants of Escherichia coli (R8H and Y118F) and wild-type LamB of Salmonella typhimurium were reconstituted into artificial lipid bilayer membranes. Ion transport through wild-type LamB of S. typhimurium and the LamB mutants was inhibited by the addition of carbohydrates of maltose and maltooligosaccharide type in a dose-dependent fashion. The sugar-induced block of the channel function could be used for the study of current noise through the different wild-type and mutant LamB-channels. The analysis of the power density spectra allowed the evaluation of the on and off-reactions (k1 and k-1) of sugar-binding to the binding site inside the channels. Wild-type LamB of S. typhimurium had approximately the same sugar-binding kinetics as has been observed for LamB of E. coli. The results suggest that the binding site inside the channel interacts with a maximum of three glucose residues within the maltooligosaccharides. The LamB mutants R8H and Y118F showed kinetics for sugar binding substantially different from that of wild-type LamB. In particular, the on-rate, k1, for the binding of different sugars of the maltooligosaccharide series to the mutant R8H was approximately 500-times smaller than for wild-type LamB, which resulted in substantially smaller stability constant of sugar binding to the channel. Similarly, the off-rate constant, k-1, for sugar binding to the mutant Y118F decreased about 20-fold, which led to a strong increase of the affinity of carbohydrates to the site. The role of the amino residues acid R8 and Y118 in the transport of maltose and maltooligosaccharides through LamB-channels is discussed on the basis of the net flux of sugars through the channels.
Alkanes↗