scholarly journals Detoxification of endogenous serine prevents cell lysis upon glucose depletion in bacteria

2018 ◽  
Author(s):  
Michelle A. Kriner ◽  
Arvind R. Subramaniam
Keyword(s):  
Amino Acid ◽  
Growth Medium ◽  
Nutrient Availability ◽  
Dominant Role ◽  
Protective Role ◽  
Growth Media ◽  
E Coli ◽  
Glucose Depletion ◽  
Glycine Cleavage ◽  
Serine Deaminase

AbstractThe amino acid serine, despite its diverse metabolic roles, can become toxic when present in excess. Indeed, many bacteria rapidly deaminate exogenously supplied serine into pyruvate and ammonia, even at the expense of biomass production. Here we report a surprising case in which endogenously produced serine must be detoxified in order for the bacteriumEscherichia colito survive. Specifically, we show thatE. colicells lacking thesdaCBoperon, which encodes a serine transporter and a serine deaminase, lyse upon glucose depletion when serine is absent from the growth medium. Lysis can be prevented by omission of glycine or by inhibition of the glycine cleavage system, suggesting that activation of glycine catabolism upon glucose depletion causes a transient increase in intracellular serine levels. Heterologous expression of the serine transporter SdaC is sufficient to prevent lysis, indicating a dominant role for serine export, rather than deamination, in mitigating serine toxicity. Since lysis can be modulated by altering alanine availability, we further propose that mis-incorporation of serine instead of alanine into peptidoglycan crosslinks is the cause of lysis. Together, our results reveal that SdaC-mediated detoxification of intracellularly produced serine plays a protective role during sudden shifts in nutrient availability in bacteria.Author summaryThe amino acid serine is a building block used to make many types of macromolecules, yet bacteria actively degrade serine that is provided in growth media. Serine degradation is thought to prevent toxic serine accumulation, but the biological role of this process is not fully understood. We observed that cells lacking thesdaCBoperon, which encodes a serine transporter and an enzyme that converts serine to pyruvate, suddenly lyse upon depletion of glucose from the growth medium. This surprising phenotype occurs only in media lacking serine, suggesting thatsdaCBis required to detoxify intracellularly produced serine. Expression of the serine transporter SdaC is sufficient to prevent lysis, providing the first evidence that serine export can be an essential function of this protein. Our results reveal that sudden shifts in nutrient availability can increase the intracellular concentration of useful metabolites to toxic levels and suggest that increasing intracellular serine levels by manipulating SdaC activity may be a possible antimicrobial strategy.

scholarly journals Different Cellular Origins and Functions of Extracellular Proteins from Escherichia coli O157:H7 and O104:H4 as Determined by Comparative Proteomic Analysis

2016 ◽  
Vol 82 (14) ◽  
pp. 4371-4378 ◽  
Author(s):  
Nazrul Islam ◽  
Attila Nagy ◽  
Wesley M. Garrett ◽  
Dan Shelton ◽  
Bret Cooper ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Medium ◽  
Foodborne Pathogen ◽  
The United States ◽  
Extracellular Proteins ◽  
Growth Media ◽  
Environmental Matrices ◽  
Content Type ◽  
E Coli ◽  
Relative Abundances

ABSTRACTExtracellular proteins play important roles in bacterial interactions with the environmental matrices. In this study, we examined the extracellular proteins fromEscherichia coliO157:H7 and O104:H4 by tandem mass spectrometry. We identified 500 and 859 proteins from the growth media ofE. coliO157:H7 and O104:H4, respectively, including 371 proteins common to both strains. Among proteins that were considered specific toE. coliO157:H7 or present at higher relative abundances in O157:H7 medium, most (57 of 65) had secretion signal sequences in their encoding genes. Noticeably, the proteins included locus of enterocyte effacement (LEE) virulence factors, proteins required for peptidyl-lipoprotein accumulation, and proteins involved in iron scavenging. In contrast, a much smaller proportion of proteins (37 of 150) that were considered specific to O104:H4 or presented at higher relative abundances in O104:H4 medium had signals targeting them for secretion. These proteins included Shiga toxin 2 subunit B and O104:H4 signature proteins, including AAF/1 major fimbrial subunit and serine protease autotransporters. Most of the abundant proteins from the growth medium ofE. coliO104:H4 were annotated as having functions in the cytoplasm. We provide evidence that the extensive presence of cytoplasmic proteins inE. coliO104:H4 growth medium was due to biological processes independent of cell lysis, indicating alternative mechanisms for this potent pathogen releasing cytoplasmic contents into the growth milieu, which could play a role in interaction with the environmental matrices, such as pathogenesis and biofilm formation.IMPORTANCEIn this study, we compared the extracellular proteins from two of the most prominent foodborne pathogenicE. coliorganisms that have caused severe outbreaks in the United States and in Europe.E. coliO157:H7 is a well-studied Shiga toxigenic foodborne pathogen of the enterohemorrhagic pathotype that has caused numerous outbreaks associated with various contaminated foods worldwide.E. coliO104:H4 is a newly emerged Shiga toxigenic foodborne pathogen of the enteroaggregative pathotype that gained notoriety for causing one of the most deadly foodborne outbreaks in Europe in 2011. Comparison of proteins in the growth medium revealed significant differences in the compositions of the extracellular proteins for these two pathogens. These differences may provide valuable information regarding the cellular responses of these pathogens to their environment, including cell survival and pathogenesis.

scholarly journals Characterization of L-serine deaminases, SdaA (PA2448) and SdaB (PA5379), and their potential role inPseudomonas aeruginosapathogenesis

2018 ◽  
Author(s):  
Sixto M. Leal ◽  
Elaine Newman ◽  
Kalai Mathee
Keyword(s):  
Immune System ◽  
Amino Acid ◽  
Carbon Source ◽  
Bacterial Infections ◽  
Sole Carbon Source ◽  
Opportunistic Pathogen ◽  
Host Immune System ◽  
E Coli ◽  
Serine Deaminase

ABSTRACTRegardless of the site of infectivity, all pathogens require high energetic influxes. This energy is required to counterattack the host immune system and in the absence the bacterial infections are easily cleared by the immune system. This study is an investigation into one highly bioenergetic pathway inPseudomonas aeruginosainvolving the amino acid L-serine and the enzyme L-serine deaminase (L-SD).P. aeruginosais an opportunistic pathogen causing infections in patients with compromised immune systems as well as patients with cystic fibrosis. L-SD has been linked directly to the pathogenicity of several organisms including but not limited toCampylobacter jejuni, Mycobacterium bovis,Streptococcus pyogenes, andYersinia pestis. We hypothesized thatP. aeruginosaL-SD is likely to be critical for its virulence. The genome sequence analysis revealed the presence of two L-SD homologs encoded bysdaAandsdaB.We analyzed the ability ofP. aeruginosato utilize serine and the role of SdaA and SdaB in serine deamination by comparing mutant strains ofsdaA(PAOsdaA) andsdaB(PAOsdaB) with their isogenic parentP. aeruginosaPAO1. We demonstrate thatP. aeruginosais unable to use serine as a sole carbon source. However, serine utilization is enhanced in the presence of glycine. Both SdaA and SdaB contribute to L-serine deamination, 34 % and 66 %, respectively. Glycine was also shown to increase the L-SD activity especially from SdaB. Glycine-dependent induction requires the inducer serine. The L-SD activity from both SdaA and SdaB is inhibited by the amino acid L-leucine. These results suggest thatP. aeruginosaL-SD is quite different from the characterizedE. coliL-SD that is glycine-independent but leucine-dependent for activation. Growth mutants able to use serine as sole carbon source were isolated. In addition, suicide vectors were constructed which allow for selective mutation of thesdaAandsdaBgenes on anyP. aeruginosastrain of interest. Future studies with a double mutant will reveal the importance of these genes for pathogenicity.

scholarly journals Comparation Between Mac conkey and Coconut Water Medium as a Growth Medium for Escherichia coli

2021 ◽  
Vol 3 (1) ◽  
pp. 19-25
Author(s):  
Endah Prayekti ◽  
Suliati Suliati ◽  
Dwi Agustin Wulandari
Keyword(s):  
Escherichia Coli ◽  
Growth Medium ◽  
Coconut Water ◽  
Growth Media ◽  
Water Medium ◽  
E Coli ◽  
Commercial Media ◽  
Randomized Design ◽  
Significant Difference ◽  
Water Media

Escherichia coli is the bacteria that can cause diarrhea in humans and often used as a parameter of stool environmental pollution. Culture of E. coli from the sample often requires Mac Conkey as commercial media which is able to distinguish it from other bacteria in the Enterobacteriaceae group. Commercial media such as Mac Conkey certainly has a price that is quite expensive because of its ability as a growth medium for Enterobacteriaceae. Therefore, in the study tested natural ingredients that can be used for growth media, such as coconut water. The purpose of this study was to compare the ability of Mac Conkey media and coconut water to support the growth of E. coli. This research is an experimental study with a completely randomized design. The concentration of coconut water tested was 0%, 20%, 40%, 60%, 80%, and 100%. The results showed that at the concentration of coconut water 20% to 60% the number of E. coli colonies on coconut water media was slightly below the Mac Conkey Agar media, while in coconut water a concentration of 80% showed a greater number of colonies than Mac Conkey. The Mann Whitney test showed a significant difference between the number of colonies on 80% coconut water media and Mac Conkey Agar, which was equal to 0.004 (p < 0.05). Based on these results, coconut water has the potential to be used as a growth medium for E. coli.

relA Gene control of bacterial glycogen synthesis

1978 ◽  
Vol 56 (6) ◽  
pp. 403-406 ◽  
Author(s):  
William A. Bridger ◽  
William Paranchych
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Stationary Phase ◽  
Nutrient Availability ◽  
Glycogen Content ◽  
Glycogen Synthesis ◽  
Gene Control ◽  
E Coli ◽  
Stimulation Of ◽  
In Cells

Starvation of Escherichia coli K12 for an amino acid results in the stimulation of bacterial glycogen synthesis in cells containing the relA+ gene, but not in cells carrying the relA− allele. Similarly, a large difference in glycogen content is demonstrable between relA+ and relA− cells in stationary phase. It is concluded that guanosine 5′,3′-bis(diphosphate) (ppGpp) or some related relA -dependent metabolite is involved in the regulation of bacterial glycogen synthesis. Detection of significant basal levels of glycogen in a relA− strain of E. coli and in unstarved relA+E. coli indicates that relA control is not absolutely required for glycogen synthesis but serves as a signal for modulation in response to nutrient availability.

scholarly journals Effects of FUdR on gene expression in the C. elegans bacterial diet OP50

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Grace McIntyre ◽  
Justin Wright ◽  
Hoi Tong Wong ◽  
Regina Lamendella ◽  
Jason Chan
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Expression Patterns ◽  
Growth Media ◽  
Bacterial Gene ◽  
Intestinal Microbes ◽  
E Coli ◽  
C Elegans ◽  
Bacterial Gene Expression ◽  
General Transport

Abstract Objective Many C. elegans aging studies use the compound 5-fluro-2ʹ-deoxyuridine (FUdR) to produce a synchronous population of worms. However, the effects of FUdR on the bacterial gene expression of OP50 E. coli, the primary laboratory C. elegans food source, is not fully understood. This is particularly relevant as studies suggest that intestinal microbes can affect C. elegans physiology. Therefore, it is imperative that we understand how exposure to FUdR can affect gene expression changes in OP50 E. coli. Results An RNAseq dataset comprised of expression patterns of 2900 E. coli genes in the strain OP50, which were seeded on either nematode growth media (NGM) plates or on FUdR (50 µM) supplemented NGM plates, was analyzed. Analysis showed differential gene expression in genes involved in general transport, amino acid biosynthesis, transcription, iron transport, and antibiotic resistance. We specifically highlight metabolic enzymes in the l-histidine biosynthesis pathway as differentially expressed between NGM and FUdR exposed OP50. We conclude that OP50 exposed to FUdR results in differential expression of many genes, including those in amino acid biosynthetic pathways.

scholarly journals A Novel Tetrahydrofolate-Dependent O-Demethylase Gene Is Essential for Growth of Sphingomonas paucimobilis SYK-6 with Syringate

2004 ◽  
Vol 186 (9) ◽  
pp. 2757-2765 ◽  
Author(s):  
Eiji Masai ◽  
Miyuki Sasaki ◽  
Yasunori Minakawa ◽  
Tomokuni Abe ◽  
Tomonori Sonoki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Extract ◽  
Growth Defect ◽  
Sphingomonas Paucimobilis ◽  
Reading Frame ◽  
E Coli ◽  
Growth Deficiency ◽  
Glycine Cleavage ◽  
Derivative Strain

ABSTRACT Sphingomonas paucimobilis SYK-6 degrades syringate to 3-O-methylgallate (3MGA), which is finally converted to pyruvate and oxaloacetate via multiple pathways in which protocatechuate 4,5-dioxygenase, 3MGA dioxygenase, and gallate dioxygenase are involved. Here we isolated the syringate O-demethylase gene (desA), which complemented the growth deficiency on syringate of a Tn5 mutant of the SYK-6 derivative strain. The desA gene is located 929 bp downstream of ferA, encoding feruloyl-coenzyme A synthetase, and consists of a 1,386-bp open reading frame encoding a polypeptide with a molecular mass of 50,721 Da. The deduced amino acid sequence of desA showed 26% identity in a 325-amino-acid overlap with that of gcvT of Escherichia coli, which encodes the tetrahydrofolate (H4folate)-dependent aminomethyltransferase involved in glycine cleavage. The cell extract of E. coli carrying desA converted syringate to 3MGA only when H4folate was added to the reaction mixture. DesA catalyzes the transfer of the methyl moiety of syringate to H4folate, forming 5-methyl-H4folate. Vanillate and 3MGA were also used as substrates for DesA; however, the relative activities toward them were 3 and 0.4% of that toward syringate, respectively. Disruption of desA in SYK-6 resulted in a growth defect on syringate but did not affect growth on vanillate, indicating that desA is essential to syringate degradation. In a previous study the ligH gene, which complements the growth deficiency on vanillate and syringate of a chemical-induced mutant of SYK-6, DC-49, was isolated (S. Nishikawa, T. Sonoki, T. Kasahara, T. Obi, S. Kubota, S. Kawai, N. Morohoshi, and Y. Katayama, Appl. Environ. Microbiol. 64:836-842, 1998). Disruption of ligH resulted in the same phenotype as DC-49; its cell extract, however, was found to be able to convert vanillate and syringate in the presence of H4folate. The possible role of ligH is discussed.

scholarly journals Effects of FUdR on gene expression in the C. elegans bacterial diet OP50

2021 ◽  
Author(s):  
Grace McIntyre ◽  
Justin Wright ◽  
Hoi Tong Wong ◽  
Regina Lamendella ◽  
Jason P Chan
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Expression Patterns ◽  
Coli Strain ◽  
Growth Media ◽  
Bacterial Gene ◽  
Intestinal Microbes ◽  
E Coli ◽  
C Elegans ◽  
Aging Studies

Abstract Objective: Many C. elegans aging studies use the compound, 5-fluro-2’-deoxyuridine (FUdR), to produce a synchronous population of worms in aging studies. However, it is not fully clear what effects FUdR have on the bacterial gene expression in the E. coli strain OP50, the primary laboratory C. elegans food source. This is particularly relevant as studies indicate that intestinal microbes can affect C. elegans physiology. Here, we examine how exposure to FUdR can affect gene expression changes in OP50 E. coli. Results: RNAseq datasets were used to compare the expression patterns of E. coli genes in the strain OP50 seeded on either nematode growth media (NGM) plates or on FUdR (50µM) supplemented NGM plates. Analysis showed differential expression of genes involved in general transport, amino acid biosynthesis, transcription, iron transport, and antibiotic resistance. We specifically highlight metabolic enzymes in the L-histidine biosynthesis pathway may be regulated by FUdR exposed OP50. We conclude that OP50 exposed to FUdR results in many differentially expressed genes, including those in amino acid biosynthetic pathways.

scholarly journals Deficiency in l-Serine Deaminase Interferes with One-Carbon Metabolism and Cell Wall Synthesis in Escherichia coli K-12

2010 ◽  
Vol 192 (20) ◽  
pp. 5515-5525 ◽  
Author(s):  
Xiao Zhang ◽  
Ziad W. El-Hajj ◽  
Elaine Newman
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Division ◽  
Amino Acid ◽  
Cell Wall Synthesis ◽  
E Coli ◽  
Amino Acid Mixtures ◽  
Glycine Cleavage ◽  
High Level ◽  
K 12

ABSTRACT Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes l-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S l-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of l-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C1 units and interferes with cell wall synthesis. We suggest that at high concentrations, l-serine decreases synthesis of UDP-N-acetylmuramate-l-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high l-serine is overcome in several ways: by a large concentration of l-alanine, by overproducing MurC together with a low concentration of l-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.

scholarly journals Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rajdeep Banerjee ◽  
Erin Weisenhorn ◽  
Kevin J. Schwartz ◽  
Kevin S. Myers ◽  
Jeremy D. Glasner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Urinary Tract ◽  
Amino Acid ◽  
Regulatory Network ◽  
Iron Homeostasis ◽  
Iron Limitation ◽  
Content Type ◽  
E Coli ◽  
General Stress ◽  
K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

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

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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