Changes with growth rate in the membrane lipid composition of and amino acid utilization by continuous cultures of Campylobacter jejuni

Journal of Applied Microbiology 1997, 82, 631-640

Changes with growth rate in the membrane lipid composition of and amino acid utilization by continuous cultures of Campylobacter jejuni

S. Leach, P. Harvey and R. Wait Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, UK

5901/09/96: received 2 September 1996, revised and accepted 4 October 1996

s. LEACH, P. HARVEY AND R . WAIT. 1997. Methods and media (defined and complex) are described which permit studies designed to determine the influence of single environmental factors on the survival and virulence of Camp,ylobactev jejuni. T h e effect of growth rate on selected physiological traits (amino acid utilization, membrane lipid composition, motility, cell morphology) was studied in continuous culture. In both media, growth was at the expense of amino acid (serine, aspartate, glutamate and proline) catabolism. Slow growth in the complex medium shifted amino acid utilization from more (serine and aspartate) to less preferred substrates (glutamate, proline and possibly amino acids from the proteolysis of peptones). Low growth rates promoted the conversion of unsaturated 1 l-octadecenoic acid substituted phosphatidyl ethanolamines to corresponding 1 l-methylene substituted species, a feature correlated with stationary phase and exposure to environmental stress in other organisms. During continuous growth, cells lost motility although they still possessed flagella. Slow growth resulted in longer cells. Future studies will investigate the independent effects of nutrient stress and growth rate on the virulence and persistence of cells.

INTRODUCTION

Infection with Campylobacter jq'uni and closely related species accounts for the greatest proportion (ca 34%) of reported cases of gastrointestinal illness in the UK, with z 1% of the population estimated to become infected each year (Skirrow and Blaser 1992; Anon. 1995). In common with much of the rest of the developed world, the number of reported cases has risen every year throughout the last decade. The clinical and economic importance of this pathogen is therefore sub- stantial. The main sources of infection appear to be raw meat (particularly poultry), raw milk, untreated water and contact with ill pets (Adak et al. 1995).

The organism is normally considered to be microaerophilic, with atmospheric oxygen tensions usually proving to be toxic (Hoffman et al. 1979). However, the development of aerotolerance has been described for some strains on laboratory subculture (Jones et al. 1993). The organism is often referred to as thermophilic since growth is absent below about 30"C, and optimal at 42°C. Campylobartev jejuni appears

Cowespondenre to: Dr S. Leach, Centre jiw .4&ed .MirrobioloLy and Research, Porton Down, Salisbwy, .Uiltshrre SP4 OJG, UK.

0 1997 The Society for Applied Bacteriology

therefore best adapted to growth in the intestines of warm- blooded animals, behaving as a commensal in most animal species but relatively uniquely as a pathogen in man (Ketley 1995). Nevertheless, the organism clearly persists sufficiently well in the environment, for example, on food surfaces and in water, to maintain the colonization of domestic animals and the transmission of disease to man. Some studies have reported transition to a so-called viable but non-culturable (VNC) state in response to suboptimal conditions (Rollins and Colwell 1986). The status of this condition, however, remains uncertain since the resuscitation of putative VNC forms in animals (Jones et ul. 1991; Stern et nl. 1994) is not reproducible (Beumer et al. 1992; Medema et al. 1992).

Some of the presumptive virulence determinants of the organism have been reviewed, but it is not clear which are the most important and whether others remain to be identified (Ketley 1995). Identifying the mechanisms by which the organism can adapt its metabolism to the range of different environments it is likely to encounter both within and outside its host will prove fundamental to our understanding of the disease-causing potential of the organism. Such studies should augment those focused purely on the elucidation of

632 S. LEACH E T A L

virulence determinants. Despite this, knowledge of the organism’s metabolism is largely fragmentary and based on studies of batch cultures where it is impossible to distinguish between biochemical differences due to growth rate and those due to environmental change. Many studies have identified biochemical characteristics relevant to typing methods. Some have concentrated on the more fundamental aspects of the organism’s physiology (Hoffman and Goodman 1982; Westfall et al. 1986) and its responses to different growth conditions (Field et ul. 1986; McCardell et (11. 1986; Jones et ul. 1993; Schwartz et al. 1994). Such studies would be greatly facilitated by the application of continuous culture tech- niques, particularly in conjunction with a chemically defined growth medium, enabling much closer control of the organism’s external environment and the independent manipu- lation of growth rate.

The aim of this study was to establish microaerobic continuous cultures of Camp. j+mi and investigate their amino acid utilization, motility, cell morphology and membrane lipid composition. Growth at the expense of amino acid catabolism has been described in batch culture (Smibert 1978; Elharrif and Megraud 1986). Alterations of cellular morphology have similarly been described; for example, from the short spiral form associated with exponential growth to a coccal form in stressed and stationary phase cultures (Buck et al. 1983; Moran and Upton 1987b), and longer, more filamentous cells under iron restricted growth (Field et al. 1986) and late exponential phaselstationary phase cultures (Griffiths 1993). Several studies have identified flagella as virulence determinants required for motility and adhesion (Nuijten et al. 1992; Grant et al. 1993). In several organisms, membrane lipid composition has been shown to alter in response to culture conditions, particularly temperature and growth phase (Cronan 1968; Thomas and Batt 1969; Guckert et ul. 1986).

In addition, two liquid culture media were compared in these studies: one a complex peptone-based broth more akin to the organism’s nutritionally complex natural environment and the other a chemically defined medium more suitable for subsequently establishing specific nutrient limitations.

METHODS

Cell growth

Cum~y1r)barterjejuni 81 116 (NCTC 11828) was obtained from the National Collection of Type Cultures (Colindale, UK). Other Camp. jejuni were fresh clinical and environmental isolates kindly donated by Dr T. Humphrey (Exeter PHL). Laboratory stocks of cells were stored frozen onto beads (Prolab) at -70°C with a back up store kept over liquid nitrogen. Cells were revived by plating a single bead onto Columbia Blood Agar (prepared as bilayer plates) with 10%

defibrinated horse blood (CBA, Oxoid). Inocula were prepared by harvesting cells from one or two 24-h-old CBA plates with a sterile loop and resuspending them in z 5ml continuous culture growth medium (see below). Agar plates were incubated in a controlled, gaseous environment (Don Whitley CO, incubator; 37”C, 5941 C02, 4% 0,).

The continuous culture equipment used was based on Anglicon controllers (Brighton Systems, Newhaven, UK) supplemented by L H series 500 fermentation units (LH Engineering, Maidenhead, UK). Probes (Eh and pH: sup- plied by Ingold or Russell; galvanic 0, by Uniprobe) and their appropriate controllers were calibrated as described in the manufacturer’s instructions. Culture volume was maintained at a level of 500 ml by a gravity-fed effluent weir, the temperature was maintained at 37°C by feedback control to a heating element and the dissolved oxygen tension (DOT) at 2-196 of air saturation by control to a solenoid which switched oxygen into a continuous stream of anaerobic gas mix (809i) N,/lO?/o COJlOob H,). Cells were grown at dilution rates (D) equal to 0.30, 0.15 and 0.08 h-’, which are equivalent to mean generation times of 2.3, 4.6 and 8.6 h, respectively. Approximately 5 ml samples were removed from the chemostat vessel by means of the sample port and sub- jected to microbiological and chemical analysis.

Media for continuous culture

Two different growth media were compared. The first was a complex medium (designated Cp mod4) and contained (g 1.’): Bactotryptone (Difco), 10; Proteose peptone (Oxoid), 10; glucose, 1; yeast extract (Difco), 2; NaCl, 5; sodium pyruvate, 1.1 . This medium was sterilized by autoclaving (15 psi for 30 min) in 20 1 volumes. The second was a chemically defined medium originally devised for the culture of legionellae (ACES buffered chemically defined, ABCD; Pine et al. 1986) and contained (mg 1-I): ACES, 10 000; KH2P04, 220; Na2S04, 150; CaCl2.2H20, 0.56; MgS04.7H,0, 214; NH4V03, 1.17; ZnS04.7H20, 28.75; CoC12.6H20, 0.48; CuS04.5H20, 0.03; MnC1,.4H20, 0.02; Na2M00+2H20, 1.21; NiS04.6H,0, 0.53; 100 mg each of alanine, arginine, asparagine, aspartic acid, glutamine, glycine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine; serine, 2000; sodium pyruvate, 1000; potassium a-ketoglutarate, 1000; inositol, 2; thiamine HCl, 2; calcium pantothenate, 2; nicotinamide, 1; biotin, 0.1; thioctic acid, 0.1; coenzyme A, 0.1; cysteine HC1, 500; glutathione, 500; tyrosine, 50; FeSO4.7H,0, 40; haemin, 2. All amino acids were the L isomers. This medium was filter sterilized in 20 1 volumes (045 and 0.22 pm pore size cartridge filters, Sartorius).

Measurement of cell density and purity

The optical density (650 nm, 1 cm light path) of the cultures was monitored with an SP 6-550 u.v.-vis spectrophotometer

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 631 -640

GROWTH OF CAMP. JEJUNl IN CONTINUOUS CULTURE 633 ~~~~

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology82, 631-640

(Pye-Unicam). Samples were routinely examined by microscopy (Gram stain of acetone fixed cells; phase contrast optics on wet films) and by subculture onto CBA in order to ensure purity.

Cellular protein determinations

Cells from 1 ml aliquots of culture were harvested separately into screw-capped Eppendorf tubes and carefully washed once in PBS using a microfuge (1 1 OOOg, Microcentaur, MSE). The pelleted cells in each tube were disrupted by heating to 100°C for 5 min in 100 pl of 1 mol 1-' NaOH to release cellular protein. The resulting solution was neu- tralized by the addition of Sop1 of 2 mol 1-' HCl and the protein content determined using a commercial Lowry kit with BSA as the standard (Sigma).

Polar lipid extraction

Polar lipids were extracted by a modification of the procedure of Bligh and Dyer (1959). Between 0.5 ml and 1.5 ml of culture was centrifuged at 11 000 rev m i d for 5 min and the pellets washed with PBS and freeze dried. The lyophilized pellets were transferred to 5 ml screw-capped glass tubes and suspended in 2ml of a monophasic mixture of chloroform/methanol/distilled water (1:2:0.8) and vortex mixed for 2min. A further 0.6ml of chloroform was added and the mixture was vortexed for 0.5 min, after which 0.6ml of distilled water was added, and the tubes were again vortesed. Separation of the phases was encouraged by low speed centrifugation, after which the lower (mainly chloroform) layer was removed with a Pasteur pipette to a fresh tube, and washed with ultra-high quality water (UHQ, Elgastat). The water washes were discarded, and the chloroform phase was evaporated in a vacuum centrifuge and redissolved in 30pl of chloroform, prior to analysis by fast-atom bombardment mass spectrometry.

The fatty acid compositions of the polar lipid extracts were determined by G U M S after mild acid methanolysis with 1 ml of methano1:toluene:sulphuric acid (1.0:1.0:0.025; 16 h at 60°C) (Wait and Hudson 1985).

Fast-atom bombardment mass spectrometry (FAB- MS) FAB mass spectra were recorded in both positive and negative ion modes with an MS80RFA (Kratos, Manchester, UK) spectrometer equipped with an Ion Tech FAB gun using xenon atoms as the bombarding particles. The liquid matrices used were either 3-nitrobenzyl alcohol or a 1:l mixture of glycerol and dithiothreitol/dithioerythritol(5: 1) and 1 p1 was mixed with an equal volume of sample solution on the stain- less steel target. The instrument was operated at 1 kV acce-

lerating voltage and scanned at 10 s per decade over the range 1800-100. Between five and 10 scans were acquired and averaged by the DS90 data system. The resolution was 1000 (10% valley) in all cases. Positive ion collision-induced dis- sociation was performed using helium as the collision gas, at a pressure sufficient to attenuate the precursor ion beam to 30% of its initial intensity. The products of collision in the field-free region between the source and the electrostatic analyser were recorded by means of computer-controlled constant neutral loss scans.

Gas chromatography/mass spectrometry (GC/MS)

G U M S was performed with the same instrument interfaced to a Carlo-Erba 5160 chromatograph fitted with a 25m x 0.2mm BP5 fused silica column (SGE Ltd, Milton Keynes, UK). Helium was used as carrier gas and samples were introduced by splitless injection. Spectra were recorded in electron ionization mode at an ionization energy of 70 eV.

Amino acid determinations

Spent culture medium was cleared of bacterial cells and debris by centrifugation (1 1 OOOg for 5 min; Microcentaur, MSE) followed by membrane filtration (0.22 pm, Millipore). The concentration of amino acids in the clarified spent and fresh culture media was determined using an amino acid analyser system with post-column ninhydrin detection (LKB 4100), and quantified by reference to external standards.

Electron microscopy

Electron micrographs (Philips E M 100T, 100 kV recorded on Ilford Technical film) of the cells were prepared by fixing them first in 290 formalin overnight, repeatedly washing them in phosphate-buffered saline (PBS) and then applying them to formvar/carbon filmed, 400 mesh copper specimen grids. The cells were then negatively stained with 1% sodium silico- tungstate, pH 7.

Deaminase assays

Semi-quantitative deaminase assays were performed using cell suspensions washed in PBS. Twenty-one separate cell suspensions of ca 5 x 10' cells ml-' in PBS, pH 7.3 (adjusted with NaOH), were prepared in duplicate, each suspension containing a different amino acid (aspartate, glutamate, glutamine, asparagine, isoleucine, serine, arginine, proline, alanine, lysine, methionine, histidine, leucine, tryptophan, hydroxyproline, cystine, valine, glycine and threonine, 10 mg ml-'; phenylalanine and tyrosine, saturated solutions) and incubated at 37°C. The concentration of ammonium in these suspensions was determined at time zero and at 30 min and

634 S. LEACH E T A L

60 min using a modification of the indophenol blue method (Cook 1976). One ml of each of the colour development reagents was added to 10 pl aliquots of the cell suspensions. With sample volumes larger than 10 pl, the amino acids themselves interferred significantly and to varying degrees with the development of the chromophore. Calibration curves for ammonia (0-6 pg) in the presence of each of the amino acids were prepared and were all approximately linear giving O.D.,,, values ranging from 0.8 to 1.6 with 6 pg of ammonia. Suspensions liberating ammonia equivalent to at least 1 p g per lop1 (0.1 mg ml-') after 60 min incubation were scored as positive for deaminase. Controls in the absence of bacteria or amino acid demonstrated no significant ammonia release.

Determination of nutrient limitations

Aqueous solutions of medium components were added as pulses directly to vessels containing established steady-state cultures of Camp. jejuni via a membrane filter (0.22 pm Milli- pore) and the optical density measured at regular intervals thereafter. Alternatively the concentration of individual components was increased in the bulk medium feeding the culture and the effects on optical density determined. In either case a rise in optical density indicated the addition of growth- limiting nutrient.

RESULTS

Cell growth

Campyloharter jejuni (NCTC 11828) grew well in continuous culture in both of the media employed; however, initial batch phase growth was considerably longer in the defined medium, on account of a more prolonged lag phase. After the optical density of the culture had reached about 0.4, initiation of a continuous supply of fresh medium routinely maintained the growing cultures for several hundreds of hours. Steady-state conditions were reached at each dilution rate after z 15 mean cell doubling times (i.e. h" 130 h at D = 0.08 h-', 69 h at D z 0 . 1 5 h-' and 35h at D = 0 . 3 h-'). This was reflected in the steady optical density and cellular protein content of the cultures in each case (Table 1). Maximum specific growth rates (pmax) in the two media were calculated during the initial exponential batch phase growth of the cultures and found to be 0.37 h-' in the complex medium and 0 4 1 h-' in the defined medium. These represent mean doubling times of 1.87 h and 1.69 h, respectively. Continuous cultures of the fresh isolates (9752, 799, CH1, 9519) were also well supported by the chemically defined medium, attaining equivalent steady-state optical densities of z 1.3 at D = 0.08 h-'.

In the complex medium, the steady-state optical density and cellular protein concentration decreased markedly as the dilution rate was increased. In the defined medium, yield was

Table 1 Changes in the steady-state biomass concentration of continuous cultures of Cumpylobacter jejuni in response to growth rate and growth medium

Dilution Cellular protein Medium rate (h-') 0.D.6sot (pg ml I)"?

Cp mod4 0.48 0.25 ND 0.3 0.44 156 (3) 0.15 0.56 216 (6) 0.08 0.73 309 (6)

Cp mod4+cysll 0.08 0.90 348 (2)

ABCD 0.15 1.28 ND 0.08 1.20 464 (4)

ABCD+aa.f 0.08 1.82 ND ABCD+ser.$ 0.08 1.83 ND

"Figures in parentheses are the number of replicate determinations. tcoefficients of variation (cv) < 159'0; ND, not determined. fAmino acid concentration of medium increased by 5096. $Serine concentration of medium increased by 509'0. IjCystine.HC1 added to medium (0.35 mmol I-').

not influenced by the change in dilution rate between 0.08 h-' and 0.15 h-', and was considerably higher than in the complex medium at equivalent dilution rate (Table 1). There was a strong positive correlation between the steady-state optical density of cultures and their cellular protein content (regression analysis: 0.D.6,0 = 0.04 + 2 4 x cellular protein [mg ml-'I; R2 = 98%; P = 0.0001) regardless of culture medium or growth rate in these experiments.

Morphology and motility

During preliminary batch phase growth and initial growth into steady state, cultures were composed predominantly of rapidly motile cells as determined by phase contrast microscopy of wet films. However, once in steady state, motility was rapidly lost in both media. Despite loss of motility the bacteria continued to produce full length flagella as determined by electron microscopy (Fig. 1). Non-motile cultures produced colonies of non-motile cells on the CBA purity plates. These non-motile colonies produced further non-motile colonies when subcultured.

In the complex medium at the lowest growth rate, cellular morphology was heterogeneous particularly with respect to cell length (data not shown). The cells became progressively shorter (Fig. 1) and less heterogeneous at the higher growth rates. In the defined medium, cell morphology was less heterogeneous and cell length was not noticeably influenced by the change in dilution rate between 0-08 h-' and 0.15 h-'. All cultures had a small proportion of coccal cells (< lo%),

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 631-640

GROWTH OF CAMP. JEJUNl IN CONTINUOUS CULTURE 635

Fig. 1 Transmission electron micrographs of Campylobucter jejuni NCTC 11828 from continuous cultures in complex medium; D = (a) 0.08 h-I, (b) 0.15 h-' and (c) 0.3 h-'. Bars, 1 pm

which was not influenced by the changes in growth rate or medium described here.

Cells harvested from either medium at the lowest dilution rate deaminated only aspartate, asparagine, serine, glutamate and glutamine, defined as the production of 1 pg ammonia per 10 p1 of incubation mixture. Amino acid utilization and deaminase activity

In the complex medium, the concentrations of aspartate and serine were the most heavily depleted regardless of growth rate (Table 2 ) . As the growth (dilution) rate was decreased, glutamate and proline also became increasingly depleted. In the chemically defined medium, serine, aspartate, glutamate and proline were the most heavily depleted amino acids at the lowest dilution rate (0.08 h-'). In both media, the other amino acids were considerably less depleted. Indeed, a number were present at a higher concentration in the spent medium than in the fresh medium. This was the case for glycine, isoleucine, leucine, lysine, histidine, alanine and d i n e in the complex medium at the lower growth rates. In the chemically defined medium this was true only for glycine.

Nutrient limitation

Supplementation of the complex medium with pulses of acet- ate, aspartate or phosphate had no effect on biomass yield (D=0.08 h-') as determined by optical density (data not shown). Addition of a pulse of cystine (10 ml of a 0.35 mmol 1-' solution), by contrast, resulted in a prompt, transient rise in optical density (0.16, 0.D.,50). Reformulation of the medium by the addition of 0.35 mmol 1F' cystine.HC1 resulted in a sustained increase in biomass yield (Table 1).

With the defined medium, increasing the concentration of the amino acids in the bulk medium reservoir by 50% resulted in a sustained increase in the optical density of the culture


636 S. LEACH E T A / .

Table 2 Changes in the amino acid Cp mod4 broth ABCD broth utilization of continuous cultures of

Cump.yloharter jejuni in response to

medium Dilution rate (h-') 0.3 0.15 0.08 0.08 growth rate and growth

Amino acid (mmol 1 ') Fresh Spent Spent Spent Fresh Spent ~

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Cysteine Cystine Methioninc Isoleucine Leucinc Tyrosine Phenplalanine Lpsine Histidine Arginine

0.74 0.99 1.54 1.71 0.36 1.22 2.53 1-88 0.15 N D 0.94 0.83 2.38 0.53 1.94 2.06 0.30 2.33

N D 1.12 ND 0.88 0.32 1.10 2.28 1.65 0.05 N D 0.83

2.74 0.37 1.67 149 0.22 2.11

0.80

ND 0.83 ND 0.41 0.35 1.21 2.34 1.72 0.10 ND 0.77 0.78 2.39 0.44 1.53 1.58 0.22 1.98

ND 0.39 0.94 0.65 ND 13.04 ND 0.30 ND 0.56 1.58 1.3 2.85 0.91 2.22 0.82 0.10 0.60 N D 0.35 0.74 0.61 1.05 0.73 2.98 0.78 0.49 0.29 1.57 0.57 3.21 043 1.75 0.35 1.21 0.67

N D 0.37 0.28 ND N D 1.84 0.93 0.37 0.55 0.33 0.26 0.29 0.23 0.11 0.25 0.07 0.25 0.25

The figures in thc body of the table represent the means of duplicate or triplicate determinations of free amino acids in the media on separate samples harvested at the same dilution rate on separate days (the cv for these repeat determinations averaged 150.h). ND, Not detected.

(Table l) , indicating that an amino acid was growth limiting. Pulsed addition of serine to the defined medium (10 ml of a 120 mmol 1 ' solution) resulted in transient rises (0.05-0.07, 0.D.,,5,,) in optical density which were unaffected by the addition of other amino acids such as tryptophan, tyrosine or lysine (data not shown). Increasing the concentration of serine by 5000 in the bulk-defined medium resulted in an increased steady-state optical density equivalent to that achieved previously with all the amino acids used in combination (Table 1).

Phospholipid composition

Positive ion FAB-MS of the extracted phospholipids gave signals between m / c (madcharge) 664 and 758 which were assigned as the protonated molecules of a family of 1,2-diacyl- sn-glycero-3-0-phosphoethanolamines. This identification was confirmed by collisional activation experiments which indicated a facile loss of 141 (the mass of the ethanolamine phosphate head-group), a process characteristic of phospho-

ethanolamines. The acyl substitution pattern (Table 3) of the intact lipids was presumptively identified by GC-MS of methanolysates of the polar lipid extracts and by reference to previously published data on the fatty acid composition of Camp. jejuni (Wait and Hudson 1985). In the negative ion mode of the mass spectrometer, additional signals were observed at m / z 773 and 747, which were assigned as 1,2- diacyl-sn-3-0-phosphoglycerols substituted, respectively, with 18:1/18:1 and 18:1/16:0 fatty acids.

The profile of the phosphoethanolamine (PE) species changed strikingly in response to alterations of growth rate (Table 3). In the complex medium at higher growth rates (Dx0.15 h-' and 0.3 h ') the major species was 1-palmitoyl-2-vaccenoyl-sn-glycero-3-0-phosphoethanolamine (MH' = m / c 7 18) and the proportion of 11,12-methyleneoctadecanoic acid-containing PEs (i.e. those with MH' at m / z 730, 732 and 758) was low. When the dilution rate was reduced to 0.08 h-', however, the proportion of vaccenic acid-substituted species declined and there was a shift towards PEs substituted with 11,12-methyleneoctadecanoic acid (MH' = m / z 730, 732 and 758). This tendency was even more pronounced in

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GROWTH OF CAMP. JEJUNl IN CONTINUOUS CULTURE 637 ~ ~ ~ ~ ~ ~

Table 3 Changes in phosphatidyl ethanolamine profile of Cumpylnbucterjejunz in response to growth rate and growth medium

Acyl substituents* 14:0/16:1 14:0/16:0 16:0/16: 1 16:1/18: 1 16:0/18:1 16:1/19cyc 16:0/19cyc 18: 1/18: 1 18: 1/19cyc

m/z 662 664 690 716 718 730 732 744 758

Medium Dilution rate

Complex 0.3 36 62 39 42 100 ND 26 42 N D Complex 0.15 49 82 45 44 100 N D 31 42 N D Complex 0.08 48 100 41 22 51 41 67 25 23 ABCD defined 0.08 33 72 43 32 84 39 100 45 43

Figures in the body of the table represent relative intensity normalized to the most abundant ion in the FAB-MS spectrum. ND, Not detected. *14:0, Tetradecanoic acid; 16:0, Hexadecanoic acid; 16: 1, Hexadecenoic acid; 18:1, Octadec-1 1-enoic acid; 19cyc, 11,lZ- methyleneoctadecanoic acid

the defined medium at low dilution rate, in which the most abundant PE was 1-palmitoyl-2-methyleneoctadecanoyl-sn- glycero-3-0-phosphoethanolamine (MH' = m / z 732) .

DISCUSSION

This is, to our knowledge, the first published report of the growth of Camp. jejuni in continuous culture. The growth- limiting nutrient in the chemically defined medium was serine which was presumably the primary carbon and energy source. Determination of the limiting nutrient in a complex medium is often difficult, but in this case appeared to be cystine. However, carbon substrate utilization was mixed and changed with growth rate making the determination of limiting nutrient less certain, although pulses of the other substrates had no effect on yield. Optimization of the defined medium by elimination of unnecessary components was not undertaken since this would preclude the comparative culture of other, auxotrophic strains (Tenover and Patton 1987). The defined medium supported cultures of all of the fresh isolates so far examined.

In both media, the most heavily depleted amino acids were serine, aspartate, glutamate and proline, indicating their use as carbon and energy sources, although in the defined medium serine was the main carbon and energy source on account of its high concentration in the fresh medium. Camp.vloburter ,jejuni deaminated only serine, aspartate, glutamate, asparagine and glutamine. These results are in accordance with those of previous batch culture studies (Karmali et al. 1986; Westfall et a/. 1986; Tenover and Patton 1987) which have demonstrated these same amino acids to be among the carbon substrates utilized by Camp. jejuni.

In the complex medium, the different carbon substrates were not utilized with equal preference. Aspartate and serine were metabolized at the highest dilution rate, with glutamate

and proline (in that order) used only as the dilution rate was decreased. This sequential metabolism of amino acids presumably reflects the inhibition of uptake or utilization of the less preferred (glutamate and proline) by the more fav- oured substrates (aspartate and serine) which would be at a higher residual concentration at the higher dilution rates (Harder and Dijkhuizen 1976). As the residual concentrations of aspartate and serine declined at the lower dilution rates this inhibition would have been lifted. An equivalent response in batch culture often results in diauxic growth. This pro- gressive utilization and incorporation of carbon substrates was presumably responsible for the increased biomass at the lower dilution rates. Alternatively, higher yields at lower growth rates might have been expected if the cultures had been grown close to their critical dilution rate. However, this was not the case as determined by the high p,,,,, of the organism relative to the dilution rates. Since the relationship between the O.D. and the cellular protein content of the cultures remained linear throughout, it seems improbable that a simple accumulation of storage polymers accounts for the increased yield at low growth rate. Indeed, Cump. jejuni has so far not been reported to accumulate storage polymers. A more balanced accumulation of biomass seems more likely to have taken place.

Interestingly, cell length also increased at the lower dilution rates. This was unexpected since slow growth and entry into stationary phase in batch cultures more commonly lead to smaller cells (Kolter et ul. 1993). This phenomenon in Cump. jejuni appears to be a response to slow growth rate in conjunction with conditions of carbon excess, since it only occurred with the cystine limited, carbon excess cultures in the complex medium. Increases in the cell length of Camp. jejuni have previously been reported to occur in response to iron restriction in batch culture (Field et al. 1986). Although iron restriction may be a specific trigger for this event, it is


638 S. LEACH E T A L .

clear that a similar effect can be elicited purely by changes in growth rate and carbon substrate utilization. It seems more likely then that the effects of iron were indirect, probably as a result of slower growth rate in conjunction with the excess of carbon substrate attendant on iron limitation. Since coccal cell formation is a common response of Camp. jejuni to environmental stress (Moran and Upton 1987a, b), particularly oxidative stress, the low and unchanging proportion of coccal cells in the continuous cultures indicate such environmental stresses were constant and probably slight.

Intriguingly, several of the amino acids in the complex medium were at a higher concentration in the spent medium than in the incoming fresh medium, particularly at low growth rate. This may be the result of increased cell death and autolysis at the lower dilution rate releasing free amino acids, but there was no other evidence to indicate this was occurring. An alternative source could be the increased proteolysis of peptides in the complex medium. It has been suggested that Camp. jq.uni expresses protease activity in stationary phase cultures concomitantly with enterotoxin production (Saha and Sanyal 1990). Such activity would explain the restriction of the phenomenon to the peptone- and yeast extract-containing complex medium. Furthermore, the biomass of the cultures per unit concentration of free amino acid consumed (Table 4) is much higher for cultures grown in the complex medium than would be predicted from the equivalent growth yield in the defined medium. This was particularly the case at the low dilution rate where the excess of amino acids in the spent culture medium was most marked. This would be consistent with the action of proteases and the presence in the complex medium of peptides that were increasingly utilized at the low dilution rate as the free amino acids were depleted.

The loss of motility in the steady-state cultures was not altogether unexpected (Nuijten et ul. 1989; Diker et ul. 1992),

Table 4 Apparent growth yields of continuous cultures of Cumpjllnbuctrr jejuni, assuming that only free amino acids are utilized

~ _____ ~~~

Growth yields" relative to consumption of

Medium rate (h-') Carbon Amino acids Dilution

Cp mod4 0.30 1.08 0.40 0.15 1.25 0.48 0.08 1 4 2 0.56

ABCD 0.08 0.83 0.3 1

*Apparent growth yields calculated as mg of cellular protein produced per mg of carbon or amino acid (serine, aspartate, glutamate and proline) consumed.

since in well mixed homogeneous continuous cultures there would be no gradient to confer selective advantage on motile cells. It was surprising, however, that the cultures retained full length flagella. Lesions in associated motility determinants, such as flagellar motor components or chemotactic elements, must have been involved. Strain variation or more prolonged culture might, however, lead to a loss of flagella themselves.

The predominant phospholipids identified by positive ion FAB-MS were phosphatidyl ethanolamines with masses consistent with the acyl substitution patterns, 14:0/16: 1, 14:0/ 16:0, 16:0/ 16: 1, 16: 1 / 18: 1, 16:0/ 18: 1, 16: 1/19cyc, 16:0/19cyc, 18:1/18:1 and 18:1/19cyc. The profile of fatty acid constituents in these intact phosphatidyl ethanolamines was consistent with that described for the hydrolysed free fatty acids of Camp. jejuni from agar plate cultures (Lambert et ul. 1987), where 16:O and 18:l predominated with lesser amounts of 19cyc, 14:O and 16:l. In the continuous cultures, however, there were clear shifts in the substitution patterns of the intact phospholipids as a function of growth rate. Most strikingly, the proportion of cyclopropane (19cyc) substituted species increased at the expense of the corresponding 18:l precursors at the lowest dilution rate, suggesting an enhanced addition of methylene groups across the double bonds of the vaccenoyl groups to form 11,12-methyleneoctadecanoic acid. The overall pattern of change mirrored that reported for batch cultures of Esrherirhza [oli where the proportion of cyclopropane fatty acids increases in late logarithmic and stationary phase or when cells are exposed to stresses such as low pH or high temperature (Cronan 1968). Similar responses have also been reported for Streptococcus lurtis (Thomas and Batt 1969) and Iibibrio rholerae (Guckert et al. 1986) in response to starvation.

The formation of cyclopropane fatty acids in E. roli occurs by the action of a soluble, cytoplasmic cyclopropane fatty acid (CFA) synthase. The growth phase-dependent synthesis of CFA in E. coli results from the action of an alternative RpoS (KatF)-dependent promoter of the structural gene coding for CFA synthase, in conjunction with a marked in zizio instability of the CFA synthase itself (Wang and Cronan 1994). In the chemostat studies reported here, Camp. jejuni also presumably produced a CFA synthase in response to low growth rate that was specific for the 18: 1 fatty acid substrate. Increased CFA synthesis in response to low growth rate and stress factors has been suggested to have a role in increasing membrane stability during periods of low metabolic rate (Wang and Cronan 1994).

Further chemostat studies are now planned to determine the influence of individual environmental variables on the biochemistry of Camp. jq'uni in relation to its virulence and survival mechanisms. The data derived from this current study will provide useful baseline information to facilitate this. Variables such as temperature, pH and oxygen tension


GROWTH OF CAMP. JEJUNl IN CONTINUOUS CULTURE 639

would clearly affect the growth rate of batch cultures. With the chemostat cultures, particularly when using the defined medium, it will be possible to determine the influence of these variables independently of growth rate effects.

ACKNOWLEDGEMENTS

The authors wish to thank Dr B. Dowsett for the electron microscopy and preparation of micrographs and Professor P. D. Marsh (CAMR) for his help and encouragement with the work and the manuscript.

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Changes with growth rate in the membrane lipid composition of and amino acid utilization by continuous cultures of Campylobacter jejuni