Bacterial methionine biosynthesis

Ferla, Matteo P.; Patrick, Wayne M.

doi:10.1099/mic.0.077826-0

Cited by 181 publications

(190 citation statements)

References 85 publications

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“…In E. coli, the conversion of aspartate to methionine involves the activities of MetA and MetB, which are absent in C. burnetii. However, C. burnetii might use one of several alternative pathways for methionine biosynthesis, as it contains several met genes (40). A predicted methionine-specific ABC transporter (CBU0107 to CBU0109) likely compensates for an inability to synthesize sufficient levels of methionine (41,42).…”

Section: Discussionmentioning

confidence: 99%

Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium

Sandoz

Beare

Cockrell

et al. 2016

Appl Environ Microbiol

101

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Host cell-free (axenic) culture of Coxiella burnetii in acidified citrate cysteine medium-2 (ACCM-2) has provided important opportunities for investigating the biology of this naturally obligate intracellular pathogen and enabled the development of tools for genetic manipulation. However, ACCM-2 has complex nutrient sources that preclude a detailed study of nutritional factors required for C. burnetii growth. Metabolic reconstruction of C. burnetii predicts that the bacterium cannot synthesize all amino acids and therefore must sequester some from the host. To examine C. burnetii amino acid auxotrophies, we developed a nutritionally defined medium with known amino acid concentrations, termed ACCM-D. Compared to ACCM-2, ACCM-D supported longer logarithmic growth, a more gradual transition to stationary phase, and approximately 5-to 10-fold greater overall replication. Small-cell-variant morphological forms generated in ACCM-D also showed increased viability relative to that generated in ACCM-2. Lack of growth in amino acid-deficient formulations of ACCM-D revealed C. burnetii auxotrophy for 11 amino acids, including arginine. Heterologous expression of Legionella pneumophila argGH in C. burnetii permitted growth in ACCM-D missing arginine and supplemented with citrulline, thereby providing a nonantibiotic means of selection of C. burnetii genetic transformants. Consistent with bioinformatic predictions, the elimination of glucose did not impair C. burnetii replication. Together, these results highlight the advantages of a nutritionally defined medium in investigations of C. burnetii metabolism and the development of genetic tools. IMPORTANCEHost cell-free growth and genetic manipulation of Coxiella burnetii have revolutionized research of this intracellular bacterial pathogen. Nonetheless, undefined components of growth medium have made studies of C. burnetii physiology difficult and have precluded the development of selectable markers for genetic transformation based on nutritional deficiencies. Here, we describe a medium, containing only amino acids as the sole source of carbon and energy, which supports robust growth and improved viability of C. burnetii. Growth studies confirmed that C. burnetii cannot replicate in medium lacking arginine. However, genetic transformation of the bacterium with constructs containing the last two genes in the L. pneumophila arginine biosynthesis pathway (argGH) allowed growth on defined medium missing arginine but supplemented with the arginine precursor citrulline. Our results advance the field by facilitating studies of C. burnetii metabolism and allowing non-antibiotic-based selection of C. burnetii genetic transformants, an important achievement considering that selectable makers based on antibiotic resistance are limited.C oxiella burnetii is a wide-ranging bacterial pathogen that causes the zoonosis Q fever (1, 2). Humans are generally infected by inhalation of contaminated aerosols generated by domestic livestock, with sheep, goats, and dairy cattle being the prim...

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Section: Discussionmentioning

confidence: 99%

Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium

Sandoz

Beare

Cockrell

et al. 2016

Appl Environ Microbiol

101

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“…1B), and we therefore also considered its importance for the results obtained with S. Typhimurium lacking PurN or PurT. Methionine is synthesized by two homocysteine-methylating enzymes, MetE and MetH (27). The apparent redundancy of the reaction should enable the strain to synthesize methionine even when either the metE or metH gene is mutated, and previous predictions of redundancy in S. Typhimurium predicted that the two enzymes form a cut set (6).…”

Section: Purn and Purt Are Redundant In Vitro But Not In Vivo Purn Andmentioning

confidence: 99%

The In Vitro Redundant Enzymes PurN and PurT Are Both Essential for Systemic Infection of Mice in Salmonella enterica Serovar Typhimurium

et al. 2016

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Metabolic enzymes show a high degree of redundancy, and for that reason they are generally ignored in searches for novel targets for anti-infective substances. The enzymes PurN and PurT are redundant in vitro in Salmonella enterica serovar Typhimurium, in which they perform the third step of purine synthesis. Surprisingly, the results of the current study demonstrated that singlegene deletions of each of the genes encoding these enzymes caused attenuation (competitive infection indexes [CI] of <0.03) in mouse infections. While the ⌬purT mutant multiplied as fast as the wild-type strain in cultured J774A.1 macrophages, net multiplication of the ⌬purN mutant was reduced approximately 50% in 20 h. The attenuation of the ⌬purT mutant was abolished by simultaneous removal of the enzyme PurU, responsible for the formation of formate, indicating that the attenuation was related to formate accumulation or wasteful consumption of formyl tetrahydrofolate by PurU. In the process of further characterization, we disclosed that the glycine cleavage system (GCV) was the most important for formation of C 1 units in vivo (CI ‫؍‬ 0.03 ؎ 0.03). In contrast, GlyA was the only important enzyme for the formation of C 1 units in vitro. The results with the ⌬gcvT mutant further revealed that formation of serine by SerA and further conversion of serine into C 1 units and glycine by GlyA were not sufficient to ensure C 1 formation in S. Typhimurium in vivo. The results of the present study call for reinvestigations of the concept of metabolic redundancy in S. Typhimurium in vivo. Salmonella enterica is a common cause of foodborne disease worldwide. Annually, more than 93 million people have been estimated to suffer from nontyphoid salmonellosis, and more than 155,000 people succumb to the disease (1). Infection of mice with S. enterica serovar Typhimurium does not cause diarrhea but results in a systemic, life-threatening condition in which bacteria predominantly localize to cells of the immune system in the liver and spleen. For this reason, S. Typhimurium infection of mice is used as a model for systemic salmonellosis, including infection with the host-specific serovar S. Typhi (2).The diversity of intracellular and extracellular host niches occupied by S. Typhimurium is reflected in a high degree of metabolic flexibility (3). This flexibility is achieved through component and system-level redundancy in the metabolic network (4), and recent years have seen several studies of S. Typhimurium in order to understand the importance of metabolic redundancy for its pathogenic lifestyle (5-8). These studies have used genome-scale metabolic modeling to predict essential and combined lethal metabolic reactions; the latter group consists of two or more nonessential reactions which, when considered as one unit, are found to be essential. Such combinations are referred to as minimal cut sets in metabolic modeling (9).A list of 102 cut sets of metabolic reactions in S. Typhimurium was recently produced using a novel genome-scale metabolic model. Eac...

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“…Methionine is an essential sulfur-containing amino acid crucial for maintaining levels of cysteine, glutathione and sulfate. Most bacteria possess biosynthetic pathways for methionine [98], and it is possible that glyphosate disrupts their ability to supply this critical nutrient to the host.…”

Section: Glyoxal Methylglyoxal and Glyoxylatementioning

confidence: 99%