Development of a Synthetic Minimal Medium for Listeria monocytogenes

A defined solid and liquid minimal medium, HTM, which containedmethionine and cysteine as the sole amino acids, was developedfor Listeria monocytogenes. Complex broth-grown L. monocytogeneshad to adapt to HTM by inducing amino acid biosyntheis. HTMis the simplest minimal medium available for growth of L. monocytogenes.

INTRODUCTION

Top

Introduction

Listeria monocytogenes grows well on complex bacteriologicalmedia such as brain heart infusion (BHI) broth (Difco Laboratories,Detroit, Mich.). Several defined chemical media containing differentamino acids and vitamins for L. monocytogenes strains A4413,NCTC7973, Scott A, and 19303 were reported (2, 6, 10, 11, 12,13, 14). None of them were able to support growth of all strains.Liquid culture is a poor criterion for definition of a minimalmedium. A much more rigorous test is the ability to form colonieson a solidified minimal medium. All the defined media abovewere solidified and examined for their ability to support coloniesof L. monocytogenes strain 10403 (serotype 1/2a), but none werecapable.

MOPS is an efficient pH buffer.

Top

Mops is an efficient...

Modified Welshimer's broth (MWB) (Table 1) (10) was reportedto support some growth of 10403 in liquid culture (8) but failedto serve as solid medium. MWB contains a high concentrationof phosphate salts for buffering and as a phosphate source,and this could be toxic to the bacterium. We used 100 mM MOPS(3-N-[morpholino]propanesulfonic acid; pH 7.4) as a bufferingagent and reduced the phosphate concentration to 1/10 the originallevel. Growth in liquid modified MWB was dramatically improved(30°C, agitation at 250 rpm), and the solidified mediumcould support good colony formation by strain 10403 at 37°C.

View this table:
[in this window]
[in a new window]
TABLE 1. L. monocytogenes minimal medium^a

Only cysteine and methionine are essential amino acids for 10403.

Top

Only cysteine and methionine...

Previous reports indicated that L. monocytogenes could not synthesizeall 20 amino acids (2, 10, 11, 12, 13, 14). We systematicallydeleted the amino acids from modified MWB and found that onlymethionine and cysteine were essential (Fig. 1A). Cysteine canbe synthesized by the reduction of sulfate to sulfide, whichis condensed with O-acetylserine to form cysteine. However,a genome survey indicated that genes for reducing sulfate tosulfide equivalent are absent from L. monocytogenes strain EGD-e(3; http://genolist.pasteur.fr/ListiList/). Alternatively, thiosulfatecan be condensed with O-acetylserine to form sulfocysteine,which is reduced to cysteine. We found that thiosulfate couldnot replace cysteine.

View larger version (88K):
[in this window]
[in a new window]
FIG. 1. Essential amino acid requirements for strain 10403 (A) and strain EGD-e (B). Amino acid-free HTM was supplemented with 0.1 mg of each amino acid ml^-1.

L. monocytogenes can use inorganic nitrogen sources.

Top

L. monocytogenes can use...

It was reported that L. monocytogenes could not use inorganicnitrogen sources (10), but we found that we could replace glutaminewith ammonium [(NH₄)₂SO₄ and (NH₄)NO₃] but not nitrate (NaNO₃),implying a lack of nitrate reductase.

Biotin, lipoic acid, riboflavin, and thiamine are essential.

Top

Biotin, lipoic acid, riboflavin,...

The requirement for all vitamins in MWB was examined. We removedeach of the four vitamins in turn from the medium and streakedany resultant colonies on the same medium to ensure completevitamin depletion. Riboflavin and lipoic acid were found tobe essential, and thiamine and biotin were required for healthycolony formation. There may have been trace amounts of biotinand thiamine in the seaweed-derived agar (4). Examination ofthe EGD-e genome revealed a lack of the genes encoding the pathwaysof synthesis of the four cofactors (http://genolist.pasteur.fr/ListiList/).

L. monocytogenes carbohydrate catabolism appears limited.

Top

L. monocytogenes carbohydrate...

The following filter-sterilized (0.22-µm pore size) carbonsources were tested at 1%: glycerol, arabinose, ribose, xylose,glucose, fructose, mannose, mannitol, fucose, galactose, rhamnose,maltose, sucrose, lactose, starch, and Casamino Acids. Onlyglucose, glycerol, fructose, and mannose could support growth.The pH of the media, as shown by incorporating 1% BDH universalpH 4.0 to 11.0 indicator, was reduced from between 7.0 and 7.5to 4.5, indicating that fermentation rather than respirationoccurred. The failure of Casamino Acids to be used, as previouslynoted by Premaratne et al. (10), indicates that amino acid catabolicpathways are not present in this bacterium. The final minimalmedium was named HTM (Hsiang-Ning Tsai medium; Table 1).

HTM can support the growth of L. monocytogenes strains 10403, EGD-e, and L028 and Listeria innocua 33090.

Top

Htm can support the...

Strain EGD-e could grow without methionine (Fig. 1B) and againfailed to use thiosulfate as a cysteine substitute, while strainL028 could grow, albeit poorly, in the absence of methionine.HTM could support the growth of L. innocua 33090 but not strainScott A. Strain EGD-e, as expected, contains all the met genes,but it cannot synthesize cysteine from methionine (Fig. 1B),indicating the lack of the trans-sulfuration pathway (5). Therequirement for methionine by 10403 implies a lesion(s) in amet gene(s).

Adaptation to HTM.

Top

Adaptation to htm.

When a culture of BHI-grown cells was diluted in phosphate-bufferedsaline (PBS) or liquid HTM, the efficiency of plating (eop)on solid HTM was reduced 100-fold compared to that on BHI agar.When the HTM-grown colonies were resuspended in PBS or HTM anddiluted, the eop on solid BHI and HTM was the same. This showedthe cells had adapted to growth on HTM. To test if this adaptationwas physiological or genetic, the cells that had previouslygrown on HTM and then grown on BHI were resuspended, diluted,and spread on HTM agar again. The eop was the same as the firsttime they were presented with HTM, i.e., 100-fold less thanon BHI agar (Fig. 2). This showed that the cells had not retainedthe ability to grow on HTM during subculture on BHI; thus theadaptation was physiological and not genetic.

View larger version (9K):
[in this window]
[in a new window]
FIG. 2. Adaptation on HTM medium. Numbers indicated the eops on HTM agar compared to that on BHI agar.

Amino acid supplements improved growth in liquid HTM.

Top

Amino acid supplements improved...

Strain 10403 grew slowly with a long lag period in liquid HTM(Table 2). Neither washing and resuspending the inocula in HTMnor applying anaerobic conditions nor incubating at 37°Cimproved the growth. Addition of the trace elements ZnCl₂ (80µg liter^-1), CuCl₂ (20 µg liter^-1), MnCl₂ (20 µgliter^-1), Na₂B₄O₇ (20 µg liter^-1), and Mo₇O₂₄ (20 µgliter^-1) (9) did not improve growth, contrary to previous reports(1, 2, 7, 11).

View this table:
[in this window]
[in a new window]
TABLE 2. Effect of iron and amino acid supplements on the growth of L. monocytogenes in liquid HTM^c

We examined the effects on growth of 0.2, 2, and 20 µgof ferric ammonium citrate, ferric citrate, and hemin ml^-1.Ferric citrate and hemin at 0.2 µg ml^-1 accelerated thegrowth rate (doubling time in hours) and decreased the lag period(in hours), with little effect on yield (Table 2). Hemin at20 µg ml^-1 greatly reduced the yield and increased thelag period, with little effect on the growth rate, demonstratingtoxicity. A mixture of amino acids abolished the long lag phaseand decreased the doubling time, with little increase in theyield (Table 2). Iron supplementation in addition to amino acidsupplementation had little effect on growth, with the exceptionof hemin at 20 µg ml^-1, which again inhibited growth.

The abolition of the lag phase with an amino acid mixture impliedthat there was no missing essential component(s) but rathergreatly delayed initiation of amino acid biosynthetic pathwaysin HTM upon transfer from BHI. This also explained the poorplating efficiency and the physiological adaptation on solidHTM.

L. monocytogenes appears to be sensitive to extreme nutrientstarvation and high phosphate levels, which implies a constitutionless robust than those of many other bacteria. So far, HTM isthe simplest minimal medium for L. monocytogenes and is currentlythe medium of choice for amino acid feeding for the proteomeanalysis of strain EGD-e by the European Listeria Genome Consortium(U. Kaerst, personal communication).

ACKNOWLEDGMENTS

We thank Yvonne Paterson for great help in reviewing the paperand Tsan-Kuang Lee for help with the statistical analysis.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom. Phone: 44 (0)24 7652 3559. Fax: 44 (0)24 7652 3701. E-mail: David.Hodgson{at}warwick.ac.uk.

Present address: Department of Microbiology, School of Medicine,University of Pennsylvania, Philadelphia, PA 19104.

REFERENCES

Top