J Mol Microbiol Biotechnol 2008;14:22–30 DOI: 10.1159/000107966
Low Carbamoyl Phosphate Pools May Drive Lactobacillus plantarum CO2-Dependent Growth Phenotype Françoise Bringel Stéphane Vuilleumier Florence Arsène-Ploetze UMR 7156 Université Louis Pasteur, CNRS Génétique Moléculaire, Génomique, Microbiologie, Département Microorganismes, Génomes, Environnement, Strasbourg, France
Key Words Lactic acid bacteria ! Lactobacillus plantarum ! Pyrimidine nucleotide metabolism ! Arginine biosynthesis ! Inorganic carbon ! Carbamoyl phosphate ! Capnophile ! Uracil-sensitive
Abstract Lactobacillus plantarum is often found in nutrient-rich habitats with elevated levels of inorganic carbon (IC), and IC-dependent growth is commonly encountered in natural isolates of this species. High CO2-requiring (HCR) prototrophs are unable to grow under conditions of low IC unless arginine and pyrimidines are provided. Prototrophy is restored under high IC conditions, that is in 4% CO2-enriched air or bicarbonate-supplemented medium. Bicarbonate is required for the synthesis of carbamoyl phosphate (CP), a precursor of both arginine and pyrimidine biosynthesis. We hypothesize that at low IC levels, intracellular CP pools limit growth through the limitation of arginine and nucleotide supplies. HCR mutants obtained in the laboratory can be classified into 3 functional groups: mutants with impaired CP synthesis, increased CP consumption or increased CP requirements relative to wild type. This classification provides a framework for investigating the origin of the HCR phenotype in natural environmental isolates of Lactobacillus species, and to investigate the hypothesis that a low level of carbamoyl phosphate is a major determinant of the CO2-dependent growth phenotype often observed in L. plantarum isolates. Copyright © 2008 S. Karger AG, Basel
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Introduction
Lactic acid bacteria (LAB) are low-GC Gram-positive heterotrophic anaerobes that tolerate oxygen but lack a complete respiratory metabolism. Lactic acid is the major metabolic end product of their carbohydrate fermentation [Kandler and Weiss, 1986]. LAB are generally regarded as safe due to their ubiquitous presence in food and their contribution to the healthy microflora of human mucosal surfaces. They are associated with nutrientrich niches and manufactured products in which elevated levels of inorganic carbon (IC) are often found. In the gastrointestinal tract of mammals, CO2 is produced by metabolic activity in the intestinal mucosa. Lactobacilli present on plant surfaces are subject to changes in CO2 exposure during the day. In ecological niches with mixed or sequential microbial populations, CO2-producing organisms such as heterofermentative LAB, Saccharomyces cerevisiae or Propionibacterium are usually associated with homofermentative lactobacilli (for instance in sourdough, beer, kefir and certain types of cheeses). In the food industry, packaging under a CO2-controlled atmosphere increases the shelf life of fresh meat, fish and vegetables, since high atmospheric CO2 concentrations inhibit microbial growth, although the extent of the inhibition depends on the microorganism, medium composition and IC concentration that are present [Dixon and Kell, 1989].
Françoise Bringel, UMR 7156 Université Louis Pasteur CNRS Génétique Moléculaire, Génomique, Microbiologie Département Microorganismes, Génomes, Environnement, 28 rue Goethe FR–67083 Strasbourg Cedex (France), Tel. +33 3 90 24 18 15, Fax +33 3 90 24 20 28 E-Mail
[email protected]
Uracil
Bicarbonate CO2
pyrP
Out Uracil Bicarbonate pyrAa1Ab1
Asp pyrB
Carbamoyl pyrCDEF UMP aspartate
CP
carAB
ATP + Gln
PRPP
In
upp
Pyrimidine nucleotides
argF
Orn
Citrulline
argGH
Arginine
Proteins
Fig. 1. Intracellular CP pools fuel arginine and uridine mono-
nificantly decreases pyrAa1Ab1 transcription mediated by PyrR 2 [Arsène-Ploetze et al., 2006a]. Utilization of preformed uracil requires the high-affinity uracil transmembrane transporter protein PyrP, and activity of the uracil phosphoribosyltransferase enzyme encoded by the upp gene, which converts uracil to UMP [Arsène-Ploetze et al., 2006b]. In turn, the UMP pool controls expression of the pyr genes involved in de novo pyrimidine biosynthesis. The RNA-binding protein PyrR1 senses the concentration of UMP in the cell and regulates pyr gene expression through an attenuator mechanism. This regulation mechanism was demonstrated in Bacillus subtilis [Switzer et al., 1999], and has been proposed for L. plantarum to only partially control the pyr regulon in reponse to pyrimidine availability [Arsène-Ploetze et al., 2006a; Nicoloff et al., 2005].
Unlike the oxygen concentration, the concentration of CO2 is much lower today (less than 0.05% of total atmospheric gases) than it was when the first microorganisms appeared more than 3.5 billion years ago [Kasting, 1993]. The 2 major forms of IC are CO2 and bicarbonate in physiological neutral liquids. IC is used as a carbon source by autotrophs [Shively et al., 1998], as a terminal electron acceptor by methanogens [Deppenmeier, 2002], as a substrate by most organisms in carboxylation reactions such as anaplerotic reactions and amino acid and pyrimidine biosynthesis [Krebs, 1941], and, as more recently discovered, in the metabolism of toxic compounds [Ensign et al., 1998]. Some microorganisms, called capnophiles, require an increase in the carbon dioxide levels for optimum growth. Lactobacillus plantarum is a capnophilic homofermentative LAB [Arsène-Ploetze and Bringel, 2004]. Air enriched with 5–10% CO2 stimulates growth of L. plantarum on solid media [Kandler and Weiss, 1986]. Intracellular CO2 levels may be naturally low in L. plantarum grown under laboratory conditions, since the
two CO2-producing steps of the Krebs cycle (isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) are inoperative in this bacterium [Morishita and Yajima, 1995]. In fact, IC has been proposed to be the growth-limiting factor in L. plantarum in normal air [Nicoloff et al., 2005] by limiting the carboxylation reaction leading to carbamoyl phosphate (CP) [Anderson and Meister, 1965], which is shared by arginine and pyrimidine biosynthetic pathways (fig. 1). In addition, high CO2-requiring (HCR) arginine and pyrimidine prototrophs have been commonly found in lactobacilli isolated from natural habitats: approximately 30% of L. plantarum and Lactobacillus pentosus isolates and 80% of Lactobacillus paraplantarum strains [Bringel and Hubert, 2003]. Intracellular CP homeostasis sustains arginine and uridine monophosphate (UMP) biosynthesis (fig. 1), which provide building blocks in protein and nucleic acid synthesis and whose presence drives growth of L. plantarum. In conditions of elevated CP pools, UMP and arginine synthesis are not growth limiting. On the other
High CO2-Dependent Growth under Altered CP Metabolism
J Mol Microbiol Biotechnol 2008;14:22–30
phosphate (UMP) synthesis. CP metabolism and regulation in response to pyrimidine availability, inorganic carbon levels and arginine concentration. Pyrimidine and arginine biosynthesis are represented with black and gray lines and characters, respectively. CP is the common intermediate for both biosyntheses. Two functional CP synthases (CPS) are present in L. plantarum [Nicoloff et al., 2000]. CPS-A, the arginine-repressed CPS, is encoded by the carAB operon [Nicoloff et al., 2004] under the control of 2 repressors, ArgR1 and ArgR 2, which are active in presence of the corepressor arginine. CPS-A activity is dependent on high CO2 levels [Nicoloff et al., 2000]. CPS-P, the pyrimidine-regulated CPS, is encoded by the pyrAa1Ab1 genes in the pyrR 1-B-C-Aa1-Ab1-D-F-E operon [Elagöz et al., 1996], which is repressed when pools of UMP are high [Arsène-Ploetze et al., 2006a]. IC enrichment sig-
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carAB
pyrAa1Ab1
CPS-A
CPS-P Shortage of the main CPS: CPS-P
Inefficient CPS-A
1
pyrAa 1Ab1 mutants pyrAa 1Ab1 downshift transcription Regulator PyrR 2 mutant Regulator PyrR 1 mutant and altered pyr operon cisregulatory elements Unknown mechanism PyrR1/PyrR2 independent
Fig. 2. Types of mutations that lower intracellular CP pools in L. plantarum. Black boxes represent pyrimidine metabolism, gray boxes arginine metabolism. Numbers in the 3 large arrows refer to the 3 groups of mutants that have been defined: group 1 = mutations that cause low CP synthesis by targeting CPS-P or CPS-A activity; group 2 = mutations that cause CP flux deviation into arginine synthesis and group 3 = mutations that cause impaired re-cycling of pyrimidines. CPS = CP synthase.
High-level consumption
Impaired recycling
Altered uracil salvage pathway in upp mutants Little UMP derived from RNA degradation High levels of pyrimidine compounds other than UMP (uracil?)
J Mol Microbiol Biotechnol 2008;14:22–30
High CO2level-dependent wild-type enzyme activity Low wild-type transcription of the carAB operon
Low CP pools
3
hand, low CP pools result in UMP and arginine shortage and growth inhibition. Although direct measurements of intracellular concentrations of CP, a labile compound, have not yet been successful in Gram-positive bacteria, we have investigated in detail a collection of characterized mutants derived from a prototroph strain, L. plantarum CCM 1904, that are unable to grow under low partial pressure of CO2 in the absence of both arginine and pyrimidines and thus display the so-called HCR phenotype. The genetic elements carrying mutations leading to an HCR phenotype have been obtained in different genetic screens and have been identified and described previously [Arsène-Ploetze et al., 2006a, b; Nicoloff and Bringel 2003; Nicoloff et al., 2000, 2004, 2005]. Genetic and physiological data obtained with the HCR mutant collection, taken together with additional data shown in this paper, provide evidence that high CO2 nutritional needs occur in strains with genetic defects in CP metabolism, causing altera24
Low-level synthesis
carAB mutations
2 Flux deviation
Pumping into arginine synthesis Loss of arginine repression argR1/argR2 mutants Increased amounts of ArgF
tions either in CP synthesis or in the activity of pathways affecting the intracellular concentration of CP. On the basis of our investigations of L. plantarum mutants with CO2-dependent growth phenotypes, we therefore propose in the present review that low intracellular CP pools impede growth of L. plantarum under conditions of low inorganic carbon. Monitoring CP Metabolism in L. plantarum Correlates with CO2-Dependent Nutritional Needs
Extensive genetic characterization of genes involved in CP metabolism [Arsène-Ploetze et al., 2006a, b; Bringel et al., 1997; Elagöz et al., 1996; Nicoloff et al., 2000, 2004, 2005] has been performed in the genetic background of the prototroph strain CCM 1904 and its mutants displaying the HCR phenotype. In some LAB, CP may be produced from arginine catabolism via the argiBringel/Vuilleumier/Arsène-Ploetze
nine deiminase (ADI) pathway. The ADI pathway is not functional in most L. plantarum strains [Bringel and Hubert 2003; Liu et al., 1995], although it has been found in some isolates [Spano et al., 2004]. Indeed, genes with similarity to known genes involved in arginine catabolism were not detected in the genome of the sequenced L. plantarum strain WCFS1 [Kleerebezem et al., 2003] or in strain CCM 1904 [Nicoloff et al., 2000]. The ADI pathway is therefore not further considered here. In this review, we classify the genetic elements that confer CO2 level-dependent growth in L. plantarum into 3 functional groups (fig. 2). Group 1: Mutations That Reduce CP Synthesis CP synthases (CPS) synthesize CP from glutamine, ATP and bicarbonate (EC 6.3.5.5). The CPS enzyme is formed by 2 different polypeptides. The smaller subunit CarA catalyzes the hydrolysis of glutamine to glutamate and ammonia, whereas the larger subunit CarB contains 2 homologous active sites that are responsible for the overall synthesis of CP from bicarbonate, ammonia and 2 molecules of MgATP. Prokaryotes such as Escherichia coli harbor a single CPS regulated by both the pyrimidine and the arginine levels in the cell. Alternatively, in Bacillaceae and L. plantarum, 2 CPS that are specifically pyrimidine and arginine regulated are present. The arginine-repressed CPS-A is encoded by the carAB operon [Bringel et al., 1997]. The pyrimidine-repressed CPS-P is encoded by the pyrAa1Ab1 genes that are cotranscribed within the pyr operon for de novo pyrimidine biosynthesis [Elagöz et al., 1996]. The respective part of each of these CPS in CP biosynthesis and how their expression modulates the ability of L. plantarum to grow at low IC conditions is highlighted in the following. In the prototroph strain CCM 1904, no CP-producing system other than CPS-A and CPS-P is significant from a physiological point of view, since deletion of both CPS enzymes results in arginine and pyrimidine auxotrophy [Nicoloff et al., 2000]. To analyze the requirement for CPS-P in CP synthesis necessary to sustain growth for arginine and pyrimidine synthesis, the growth requirement for the !carAB mutant FB335 with only a functional CPS-P was tested. Mutant FB335 has a prototroph phenotype, demonstrating that the CPS-P produces enough CP for both pathways. In contrast, the !pyrAa1Ab1 mutant FB331 that only harbors a functional CPS-A is unable to grow when both arginine and uracil are omitted from the growth medium. In presence of added pyrimidines alone, FB331 grows, demonstrating that CPS-A provides only enough CP for arginine biosynthesis [Nicoloff et al., High CO2-Dependent Growth under Altered CP Metabolism
2000]. Thus, CPS-P is a major supplier of CP synthesis in the cell. The minor CP production from CPS-A could be explained by limiting amounts of CPS-A in case of low carAB expression. This hypothesis was confirmed in FB331 derivatives that acquired the ability to grow on minimal medium upon increased transcription of the carAB operon due to loss of arginine-mediated repression (mutants either in 1 of the 2 arginine repressor-encoding genes, argR1 and argR 2, or in the repressor-binding site ‘ARG box’ located in the operator region of the operon carAB [Nicoloff et al., 2004]). We conclude that wild-type carAB transcription is growth limiting in minimal medium in L. plantarum strains that lack CPS-P. In contrast to CPS-P, CPS-A is only significantly active in cells exposed to CO2-enriched air or bicarbonatesupplemented medium [Nicoloff et al., 2000]. This was first evidenced under conditions in presence of uracil with no synthesis of CPS-P. Growth under such conditions depends on CPS-A, and therefore on CO2 concentration, for CP synthesis fuelling arginine biosynthesis. In other words, uracil inhibits L. plantarum growth in air but not in CO2-enriched air, a phenotype designated as uracil sensitivity. The CO2 level-dependent uracil sensitivity observed in wild-type prototrophs may be due to the low affinity of CPS-A for its substrate and/or to regulation of the CP pool by cellular IC levels. CPS-P is the major provider of CP in the cell so that low expression of genes pyrAa1Ab1 would be expected to limit growth. Indeed, the HCR phenotype is correlated with mutations that decrease pyrAa1Ab1 transcription. Mutations were introduced in the regulatory region of the pyr operon encoding CPS-P. These mutations led to constitutive repression of pyr gene expression. The resulting strain (AE1023) displayed the HCR phenotype with measured UTP and CTP pool sizes lower than those in the wild-type prototroph [Nicoloff et al., 2005]. It was proposed, therefore, that constitutively low expression of pyr genes prevents growth at the low CO2 levels found in ordinary air [Nicoloff et al., 2005]. Other mutations were found in genetic elements involved either in the pyrimidine-specific PyrR1-mediated transcription regulation or in the PyrR 2-mediated response at low IC levels. PyrR1 and PyrR 2 display 62% amino acid identity. The IC- and pyrimidine-mediated regulations were studied in pyrR1 and pyrR 2 mutants [Arsène-Ploetze et al., 2006a; Nicoloff et al., 2005]. PyrR1 is an RNA-binding protein that regulates the pyr genes in response to pyrimidine availability by a mechanism of transcriptional attenuation [Nicoloff et al., 2005]. Pyrimidine-dependent transcription of pyrAa1Ab1 requires PyrR1, as a 100-fold repression was J Mol Microbiol Biotechnol 2008;14:22–30
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Table 1. Evaluation of arginine and pyrimidine excretion ability in pyrR and argR mutants
Strain
Genotype
name
reference
CCM 1904 AE1026 FB335 U17 FB331 FB331-12
Bringel and Hubert, 2003 Nicoloff et al., 2005 Nicoloff et al., 2000 Nicoloff et al., 2005 Nicoloff et al., 2000 Nicoloff et al., 2004
1 2
arginine1
pyrimidine2
0
