Study of pH Changes in Media during Bacterial Growth of Several Environmental Strains

The effect of pH on bacterial cell-growth and the evolution of extracellular pH triggered by bacterial growth has been monitored for three bacterial strains, Escherichia coli ATCC 25922 and Pseudomonas putida KT2440 as reference strains, and Pseudomonas pseudoalcaligenes CECT 5344 because of its capacity to assimilate cyanide as the sole nitrogen source under alkaline conditions. In a first instance, the influence of the initial pH in the growth curve has been texted in LB-medium adjusted to pH 6, 7 and 8, for E. coli and P. putida, and 7.5, 8.25 and 9 for P. pseudoalcaligenes. Although the initial pH were different, the pH of the extracellular medium at the end of the stationary phase converged to a certain pH that is specific for each bacterium. Similar experiments were carried out in minimal medium with glucose as the carbon source. In this case, the pHs of the culture of both Pseudomonadaceae strains were almost constant, whereas it suddenly dropped during the exponential growth phase of E. coli. When the initial pH was 6 the extracellular pH fell sharply to 4.5, which irreversibly prevented further cellular growth. Nevertheless, at higher initial pH values subsequent cellular growth of E. coli restored the medium to the initial pHs values. Finally, in all cases the evolution of the pH has been shown to depend on the carbon source used. Among the sources used, cellular growth with glucose or glycerol did not affect the extracellular pH, whereas citrate caused the alkalinization of the media. This phenotype is in concordance with computational predictions, at least in the case of the genome-scale metabolic model of Pseudomonas putida KT2440.

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Carbon Source Influence on Extracellular pH Changes along Bacterial Cell-Growth

Genes ◽

10.3390/genes11111292 ◽

2020 ◽

Vol 11 (11) ◽

pp. 1292

Author(s):

Rubén Sánchez-Clemente ◽

M. Isabel Guijo ◽

Juan Nogales ◽

Rafael Blasco

Keyword(s):

Cell Growth ◽

Carbon Source ◽

Bacterial Cell ◽

Carbon Sources ◽

Extracellular Ph ◽

Interspecies Interactions ◽

Ph Change ◽

Pseudomonas Putida Kt2440 ◽

Ph Changes ◽

Initial Ph

The effect of initial pH on bacterial cell-growth and its change over time was studied under aerobic heterotrophic conditions by using three bacterial strains: Escherichia coli ATCC 25922, Pseudomonas putida KT2440, and Pseudomonas pseudoalcaligenes CECT 5344. In Luria-Bertani (LB) media, pH evolved by converging to a certain value that is specific for each bacterium. By contrast, in the buffered Minimal Medium (MM), pH was generally more stable along the growth curve. In MM with glucose as carbon source, a slight acidification of the medium was observed for all strains. In the case of E. coli, a sudden drop in pH was observed during exponential cell growth that was later recovered at initial pH 7 or 8, but was irreversible below pH 6, thus arresting further cell-growth. When using other carbon sources in MM at a fixed initial pH, pH changes depended mainly on the carbon source itself. While glucose, glycerol, or octanoate slightly decreased extracellular pH, more oxidized carbon sources, such as citrate, 2-furoate, 2-oxoglutarate, and fumarate, ended up with the alkalinization of the medium. These observations are in accordance with pH change predictions using genome-scale metabolic models for the three strains, thus revealing the metabolic reasons behind pH change. Therefore, we conclude that the composition of the medium, specifically the carbon source, determines pH change during bacterial growth to a great extent and unravel the main molecular mechanism behind this phenotype. These findings pave the way for predicting pH changes in a given bacterial culture and may anticipate the interspecies interactions and fitness of bacteria in their environment.

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CONSERVATION OF NUCLEIC ACIDS DURING BACTERIAL GROWTH

The Journal of General Physiology ◽

10.1085/jgp.38.2.145 ◽

1954 ◽

Vol 38 (2) ◽

pp. 145-148 ◽

Cited By ~ 60

Author(s):

A. D. Hershey

Keyword(s):

Characteristic Feature ◽

Nucleic Acids ◽

Bacterial Growth ◽

Cellular Growth ◽

E Coli

In experiments of 6 hours duration, no replacement of phosphorus or purine and pyrimidine carbon in DNA, nor flow of these atoms from RNA to DNA, could be detected in rapidly growing cultures of E. coli. The slow replacement that has been demonstrated for many substances in non-proliferating tissues of other organisms, though it may occur also in bacteria, is not greatly accelerated under conditions of rapid cellular growth, and therefore cannot be a characteristic feature of synthetic processes.

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The Glycerol-Dependent Metabolic Persistence of Pseudomonas putida KT2440 Reflects the Regulatory Logic of the GlpR Repressor

mBio ◽

10.1128/mbio.00340-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 43

Author(s):

Pablo I. Nikel ◽

Francisco J. Romero-Campero ◽

Joshua A. Zeidman ◽

Ángel Goñi-Moreno ◽

Víctor de Lorenzo

Keyword(s):

Carbon Source ◽

Pseudomonas Putida ◽

Molecular Mechanisms ◽

Transcriptional Response ◽

Time Dependent ◽

Lag Phase ◽

Gradual Change ◽

Pseudomonas Putida Kt2440 ◽

Genetic Changes ◽

Content Type

ABSTRACTThe growth of the soil bacteriumPseudomonas putidaKT2440 on glycerol as the sole carbon source is characterized by a prolonged lag phase, not observed with other carbon substrates. We examined the bacterial growth in glycerol cultures while monitoring the metabolic activity of individual cells. Fluorescence microscopy and flow cytometry, as well as the analysis of the temporal start of growth in single-cell cultures, revealed that adoption of a glycerol-metabolizing regime was not the result of a gradual change in the whole population but rather reflected a time-dependent bimodal switch between metabolically inactive (i.e., nongrowing) and fully active (i.e., growing) bacteria. A transcriptional Φ(glpD-gfp) fusion (a proxy of the glycerol-3-phosphate [G3P] dehydrogenase activity) linked the macroscopic phenotype to the expression of theglpgenes. Either deletingglpR(encoding the G3P-responsive transcriptional repressor that controls the expression of theglpFKRDgene cluster) or altering G3P formation (by overexpressingglpK, encoding glycerol kinase) abolished the bimodalglpDexpression. These manipulations eliminated the stochastic growth start by shortening the otherwise long lag phase. Provision ofglpRintransrestored the phenotypes lost in theΔglpRmutant. The prolonged nongrowth regime ofP. putidaon glycerol could thus be traced to the regulatory device controlling the transcription of theglpgenes. Since the physiological agonist of GlpR is G3P, the arrangement of metabolic and regulatory components at this checkpoint merges a positive feedback loop with a nonlinear transcriptional response, a layout fostering the observed time-dependent shift between two alternative physiological states.IMPORTANCEPhenotypic variation is a widespread attribute of prokaryotes that leads,inter alia, to the emergence of persistent bacteria, i.e., live but nongrowing members within a genetically clonal population. Persistence allows a fraction of cells to avoid the killing caused by conditions or agents that destroy most growing bacteria (e.g., some antibiotics). Known molecular mechanisms underlying the phenomenon include genetic changes, epigenetic variations, and feedback-based multistability. We show that a prolonged nongrowing state of the bacterial population can be brought about by a distinct regulatory architecture of metabolic genes when cells face specific nutrients (e.g., glycerol).Pseudomonas putidamay have adopted the resulting carbon source-dependent metabolic bet hedging as an advantageous trait for exploring new chemical and nutritional landscapes. Defeating such naturally occurring adaptive features of environmental bacteria is instrumental in improving the performance of these microorganisms as whole-cell catalysts in a bioreactor setup.

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Cloning and expression of a trehalose synthase from Pseudomonas putida KT2440 for the scale-up production of trehalose from maltose

Canadian Journal of Microbiology ◽

10.1139/cjm-2014-0330 ◽

2014 ◽

Vol 60 (9) ◽

pp. 599-604 ◽

Cited By ~ 8

Author(s):

Tengfei Wang ◽

Shiru Jia ◽

Kun Dai ◽

Hongjuan Liu ◽

Ruiming Wang

Keyword(s):

Escherichia Coli ◽

High Pressure ◽

Pseudomonas Putida ◽

Scale Up ◽

Activity Analysis ◽

Cloning And Expression ◽

Pseudomonas Putida Kt2440 ◽

Trehalose Synthase ◽

E Coli ◽

Optimal Activity

Trehalose synthase (TreS) is considered to be a potential biocatalyst for trehalose production. We aimed to scale-up produce the TreS protein in Escherichia coli and further investigate the bioconversion capacity of TreS. The treS gene from Pseudomonas putida KT2440 was amplified and expressed in E. coli BL21 (DE3). The recombinant TreS showed a molecular mass of 67 kDa. Activity analysis suggested that TreS had optimal activity at a temperature of 55 °C, a pH of 7.4, with a substrate concentration of 30%. High-pressure liquid chromatography results indicated that this enzyme had the ability to catalyze 59% maltose into trehalose, with about 5.1% glucose as by-product. Purification analysis showed that trehalose crystals with a purity of 98% were obtained by cooling trehalose solution. The TreS from P. putida KT2440 might be a candidate for trehalose production. Further study is needed to improve the trehalose conversion rate.

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Technical-Economical Approach for PHB Production by Ralstonia Eutropha Strain Using Concentrated Vinasse as Carbon Source and Other Biotechnological Applications

10.21203/rs.3.rs-942291/v1 ◽

2021 ◽

Author(s):

Manuella Silverio ◽

Rosane Piccoli ◽

João Reis ◽

José Gregorio Gomez ◽

Antonio Baptista

Keyword(s):

Carbon Source ◽

Ralstonia Eutropha ◽

Culture Media ◽

Dry Weight ◽

Global Market ◽

Cellular Growth ◽

Production Costs ◽

E Coli ◽

Phb Production ◽

Threonine Production

Abstract The Brazilian ethanol industry is one of the most important in the global market, however these important industrial activities have been generating significant amounts of vinasse and its management has become costly for distilleries. In this study, the aim was to evaluate concentrated and in natura vinasse as basal culture media for biotechnological processes. Different bacteria and processes were assessed: L-threonine production by E. coli THR14, with glucose as carbon source; PHB production by halophilic strain Halomonas sp. HG03, with sucrose as carbon source; and PHB biosynthesis by R. eutropha L359PCJ, which used glycerol from vinasse as carbon source. Strains were evaluated firstly in shake flasks cultivations using vinasse-based media. E. coli THR14 had no statistical difference for biomass and L-threonine concentrations among control and vinasse-based treatments (up to 50% v v-1 of in natura vinasse). Halomonas sp. HG03 and R. eutropha L359PCJ were cultivated in mineral media diluted by in natura (50% and 75% v v-1) and concentrated (50% and 75% v v-1) vinasses. Higher vinasse concentrations resulted in higher cellular growth rather than PHB accumulation for both bacteria. In vinasse-based treatments, Halomonas sp. HG03 had PHB content between 19.6 – 75.2% and R. eutropha L359PCJ, 48.4 – 68.5%. 50% (v v-1) of concentrated vinasse was the most attractive condition for PHB production by both bacteria. Further experiments in CSTR bioreactors used this nutritional condition and R. eutropha L359PCJ had PHB content of 66.3%, concentrations of residual cell dry weight (rCDW) = 9.4 g L-1 and PHB = 18.6 g L-1, with YX/S = 0.16 g gGLYCEROL-1, YP/S = 0.32 g gGLYCEROL-1 and 0.25 gPHB Lh-1. Halomonas sp. HG03 had PHB content of 45.7%, rCDW = 9.8 g L-1, PHB = 8.3 g L-1 and YX/S = 0.18 g gSUCROSE-1, YP/S = 0.16 g gSUCROSE-1 and 0.12 gPHB Lh-1. Finally, cost reductions of PHB production by R. eutropha L359PCJ with concentrated vinasse-based medium were evaluated in silico by using SuperPro Designer. As a partial source of glycerol and other nutrients for PHB production by R. eutropha L359PCJ, vinasse reduced overall production costs by 13%. Simulated processes that used concentrated vinasse-based media combined with improvements of PHB productivity and higher cellular densities had production costs between US$ 3.9 – 7.5/kgPHB and 2.6 – 7.3 years of payback time.

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Cloning, Sequencing, and Phenotypic Characterization of the rpoS Gene from Pseudomonas putida KT2440

Journal of Bacteriology ◽

10.1128/jb.180.13.3421-3431.1998 ◽

1998 ◽

Vol 180 (13) ◽

pp. 3421-3431 ◽

Cited By ~ 78

Author(s):

María Isabel Ramos-González ◽

Søren Molin

Keyword(s):

Pseudomonas Putida ◽

Carbon Starvation ◽

Phenotypic Characterization ◽

Gram Negative Bacteria ◽

Pseudomonas Putida Kt2440 ◽

Gram Negative ◽

Acid Sensitivity ◽

E Coli ◽

Short Period ◽

In Cells

ABSTRACT A gene homologous to the rpoS gene of Escherichia coli was cloned from a Pseudomonas putida KT2440 gene bank by complementation of the rpoS-deficient strainE. coli ZK918. The rpoS gene of P. putida complemented the acid sensitivity and catalase deficiency of the rpoS mutant of E. coli and stimulated expression of the RpoS-controlled promoter,bolAp 1. The gene was sequenced and found to be highly similar to the rpoS genes of other gram-negative bacteria. Like in other gram-negative bacteria, a homolog of thenlpD gene was found upstream to the rpoS gene. A transcriptional fusion of the promoter of the P. putida rpoS gene to the luxAB genes from Vibrio harveyi was constructed and used as an inactivated allele ofrpoS for gene replacement of the wild-type copy in the chromosome of P. putida. The resultantrpoS mutant of P. putida, C1R1, showed reduced survival of carbon starvation and reduced cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol. Survival in soil amended with m-methylbenzoate was also reduced in the mutant strain P. putida C1R1. The RpoS protein ofP. putida controls the expression of more than 50 peptides, which are normally expressed in cells after a short period of carbon starvation.

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Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00781-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5238-5246 ◽

Cited By ~ 9

Author(s):

Dongfei Han ◽

Ji-Young Ryu ◽

Robert A. Kanaly ◽

Hor-Gil Hur

Keyword(s):

Escherichia Coli ◽

Pseudomonas Putida ◽

Hypothetical Protein ◽

Aldehyde Group ◽

Agrobacterium Vitis ◽

T7 Promoter ◽

Content Type ◽

E Coli ◽

Cell Extracts ◽

Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

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Influence of nutrient availability on in vitro growth of major bovine mastitis pathogens

Journal of Dairy Research ◽

10.1017/s0022029921000133 ◽

2021 ◽

Vol 88 (1) ◽

pp. 80-88

Author(s):

Remo Stürmlin ◽

Josef J. Gross ◽

Olga Wellnitz ◽

Lea A. Wagner ◽

Camille Monney ◽

...

Keyword(s):

Bacterial Growth ◽

Nutrient Availability ◽

Bovine Mastitis ◽

Milk Composition ◽

B Vitamins ◽

E Coli ◽

Streptococcus Uberis ◽

Whole Milk ◽

Mastitis Pathogens

AbstractThe aim of the present study was to investigate the effects of milk composition changes on the in vitro growth of bovine mastitis pathogens. Nutritional requirements of three major bovine mastitis pathogens Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Streptococcus uberis (S. uberis) were investigated in vitro. We used ultra-high temperature (UHT) treated milk with different contents of fat, protein, and carbohydrates to test the influence of the availability of various milk constituents on pathogen growth characteristics. Additionally, the bacterial growth was investigated under experimentally modified nutrient availability by dilution and subsequent supplementation with individual nutrients (carbohydrates, different nitrogen sources, minerals, and different types of B vitamins) either to milk or to a conventional medium (thioglycolate broth, TB). Varying contents of fat, protein or lactose did not affect bacterial growth with the exception of growth of S. uberis being promoted in protein-enriched milk. The addition of nutrients to diluted whole milk and TB partly revealed different effects, indicating that there are media-specific growth limiting factors after dilution. Supplementation of minerals to diluted milk did not affect growth rates of all studied bacteria. Bacterial growth in diluted whole milk was decreased by the addition of high concentrations of amino acids in S. aureus, and by urea and additional B vitamins in E. coli and S. aureus. The growth rate of S. uberis was increased by the addition of B vitamins to diluted whole milk. The present results demonstrate that growth-limiting nutrients differ among pathogen types. Because reduced bacterial growth was only shown in diluted milk or TB, it is unlikely that alterations in nutrient availability occurring as a consequence of physiological changes of milk composition in the cow's udder would directly affect the susceptibility or course of bovine mastitis.

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