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Decreasing the Hyphal Branching Rate of Saccharopolyspora erythraea NRRL 2338 Leads to Increased Resistance to Breakage and Increased

Antibiotic Production
J. N. Wardell,1 S. M. Stocks,2 C. R. Thomas,2 M. E. Bushell1 Microbial Products Laboratory, School of Biological Sciences, University of Surrey, Guildford, GU2 7XH, United Kingdom; telephone: 44-1483-300-800; fax: 44-1483-300-394; e-mail: 2 School of Centre for Bioprocess Engineering, The University of Birmingham, Birmingham, B15 2TT, United Kingdom
Received 5 February 2001; accepted 7 October 2001

Abstract: Mutation and selection for increased resistance to cell-wall synthesis inhibitors led to alterations in the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338. Mutants with decreased branching frequency exhibited increased hyphal strength (estimated by in vitro micromanipulation). As the hyphal strength was increased, this led to a greater proportion of hyphal particles in liquid culture with a hyphal fragment diameter of greater than 88 lm. This, in turn, coincided with proportionately increased antibiotic production. 2002
Wiley Periodicals, Inc. Biotechnol Bioeng 78: 141146, 2002; DOI 10.1002/bit.10210

Keywords: antibiotic production; hyphal morphology; erythromycin production

INTRODUCTION We have reported previously that antibiotic production in liquid culture is correlated with mycelial fragment diameter (minimum diameter of a circle that can bound a hyphal fragment) in actinomycete cultures (Bushell et al., 1997a; Martin and Bushell, 1996). Our ndings implied that there is a critical size (8090 lm), below which mycelial fragments are not productive, despite having the same growth rate as larger particles (Bushell et al., 1997a). This phenomenon appears to account for loss of biosynthesis in liquid cultures in species able to produce antibiotic on agar (Pickup et al., 1993), dierences in productivity in dierent small-scale culture vessels (Bushell et al., 1997a), and morphology-related eects on productivity in bioreactor culture (Martin and Bushell, 1996). Manipulating the size distribution of mycelial fragments by using an ultrasonic lter improved the spe-

Correspondence to: M. E. Bushell Contract grant sponsors: Biotechnology and Biological Sciences Research Council (UK)

cic antibiotic productivity by 33% (Wardell and Bushell, 1999). Chemostat culture experiments have demonstrated that more highly branched mycelial fragments (obtained at high dilution rates, simulating conditions of nutrient excess; Kretschmer et al., 1981) are more resistant to breakage than unbranched elements obtained at low dilution rates (Wardell and Bushell, 1999). We have obtained evidence that resistance to hyphal breakage depends on the activity of a peptidoglycan synthesising enzyme, phospho-N-acetyl muramyl pentapeptide translocase (Pickup and Bushell, 1995). Relationships between culture performance in bioreactors and mycelial morphology have been studied mainly in the fungi. Metz et al. (1981) demonstrated the utility of hyphal fragment length and hyphal growth unit (Caldwell and Trinci, 1973) for assessing the impact of bioreactor conditions on mycelial morphology, and Ujcova et al. (1980) established the use of nucleotide release as an indicator of hyphal breakage. Values for these parameters were shown to depend on both growth rate and stirrer speed in Penicillium chrysogenum. It is possible that clumps formed by actinomycetes fragment rapidly towards the end of rapid growth in batch fermentations (BelmarBeiny and Thomas, 1991; Sebastine et al., 1999; Stocks and Thomas, 2001) and that in most antibiotic-producing cultures, freely dispersed forms may predominate for most of the culture. Fragmentation in Streptomyces clavuligerus is aected by stirrer speed although growth and productivity are not signicantly inuenced (BelmarBeiny and Thomas, 1991). Most previous studies on the mechanical properties of individual cells have used measuring forces that resist compression (Thomas et al., 2000). Recently, however, we have described a procedure for measuring the

2002 Wiley Periodicals, Inc.

breaking strain involved in pulling apart individual hyphae (Stocks and Thomas, 2001).

MATERIALS AND METHODS Strains and Culture Media Saccharopolyspora erythraea NRRL 2338 was used throughout. Antibiotic concentrations were measured routinely by bioassay using a strain of Arthrobacter citreus (GL1) obtained from the Shell Laboratories culture collection, Sittingbourne, Kent (see Antibiotic Assays). The chemically dened (carbon limited) antibiotic production medium for S. erythraea contained the following major nutrients: (g L)1 in reverse osmosispuried water) glucose 15, NaNO3 11.12, KH2PO4 3.0, K2HPO4 7.0; and the following trace components: (in g L)1) MgSO4 7H2O 0.25, FeSO4 7H2O 0.025, CuCl2 0.00053, CoCl2 0.00055, CaCl2 2H2O 0.0138, ZnCl2 0.0104, MnCl2 0.0062, and Na2MoO4 0.0003. The pH was adjusted to 7.0 prior to lter sterilization using a 0.2-lm lter (Sartorius). S. erythraea maintained on tomato puree oatmeal agar (g L)1: tomato puree, 10; oatmeal, 10; agar, 20), and A. citreus was maintained on nutrient agar. Both were grown in 250-mL baed Erlenmeyer asks containing 25 mL of nutrient broth at 30C on a rotary shaker at 250 rpm. After 48 h agitation (S. erythraea), 1 mL was removed and used to inoculate preculture asks containing 23 mL of dened medium. After 48 h of further incubation, the precultures were used at approximately 1% (v/v) as an inoculum for ask or bioreactors. Bioreactor Culture An LH 500 series bioreactor with a working volume of 1.5 L, was used. Agitation was provided by two impellers (an upper marine impellor and a disc turbine impeller) rotating at 1000 rpm. Sterile air was supplied through a sparger at a ow rate of 1.2 L h)1. The temperature was controlled at 30C. Dissolved oxygen concentration in the bioreactor was monitored using a galvanic dissolved oxygen electrode (Uniprobe). Results presented here are taken only from cultures in which agitation conditions resulted in dissolved oxygen tensions of at least 60% (v/v) saturation. The pH was monitored throughout but was not controlled. The cultures, which contained a phosphate-buering system (see Strains and Culture Media), increased in pH over the course of an experiment by no more than 0.5 pH unit. Foaming was eliminated by including 0.01% Breox FMT30 antifoam (Water Management and Gamlen) in the culture medium. Antibiotic Assays A bioassay using Arthrobacter citreus was performed using procedures described previously (Huck et al.,

1991). Challenge strain seed cultures and assay plates were incubated at 30C, and the diameters of the zones of inhibition were recorded after 48 h. Examination of a number of samples using high-performance liquid chromatography (Tsuji and Goetz, 1978) conrmed that the bioassay values corresponded to erythromycin concentrations. Morphology Measurements The manual procedure described previously for estimating maximum particle diameter (Bushell et al., 1997a) was automated using PC Image image analysis software (Foster Findlay Associates, Newcastle, UK). This software incorporated a procedure for estimating the diameter of a minimum circle into which a mycelial fragment could be tted. A modication of the technique of Packer and Thomas (1993) was used to prepare slides for analysis. Fresh culture samples (40 lL) were spread over the surface of the slide and air dried. When dried, the slides were heat xed and stained with methylene blue solution (0.3 g methylene blue, 30 mL 95% (v/v) methanol in 100 mL of water) for 1 min. The slides were thoroughly rinsed in reverse osmosis (RO) water for two min and allowed to air dry. Six slides were prepared for each sample. The slides were examined and photographed using a Leitz orthoplan microscope at a magnication of 200. Sucient elds were photographed to allow 150 200 separate fragments to be measured and counted. All fragments capable of being measured by the software were included in the analysis. Those rejected consisted of aggregates of hyphal fragments. The possible consequences of this rejection procedure on the interpretation of the image analyses has been discussed by us previously (Wardell and Bushell, 1999). The software also estimated the total length and number of tips within each mycelial fragment (BLT procedure, S. Nandy, Foster Findlay).

Isolation of Mutants Spore suspension (0.5 mL of approximately 1.2 108 cfu ml)1) was diluted 1:20 in sterile saline (0.85% w/v + 0.01% v/v Tween 80) to give 10 mL spore suspension with a count of ~5 106 cfu mL)1. This was transferred to a sterile glass Petri dish and exposed to ultraviolet irradiation (8 W at 10 cm) at 254 nm for 50 without the lid (resulting in a 98% reduction in viable count or 98% kill). After exposure, the spore suspension was decimally diluted to 10)3 in sterile saline and the dilutions vortex mixed for 10 s to disperse the spores. Each dilution (0.5 mL) was inoculated into 10-mL volumes of nutrient broth containing either 2 or 10 the minimum inhibitory concentration (MIC) (against the unmutated



Table I. Erythromycin production in shake ask culture of mutants selected for resistance to inhibitors of peptidoglycan biosynthesis. Mean maximum erythromycin production relative to NRRL 2338a 15% (0.75%) 175% (8.75%) 125% (6.25%)

Mutant class bac-r tun-r pen-r


Inhibitor Bacitracin Tunicamycin Penicillin

Target of inhibitor in peptidoglycan biosynthesis Addition of the lipid carrier to the UDP-M-penta-peptide Migration of the lipid-UDP-M-penta-peptide complex across the cell membrane Cross-linking linear molecules to form the peptidoglycan net

Standard error of mean.

strain, NRRL 2338) of the following antibiotics: bacitracin, cycloserine, penicillin, vancomycin, and tunicamycin together with a control. The broths were incubated at 30C in the dark for 2 weeks and then at ambient temperature in the light. After 4 weeks incubation, the resultant broth cultures were subcultured onto nutrient agar containing the appropriate selective antibiotic.

ducer. Free hyphal ends were trapped against a at surface using a probe. The force transducer was then moved at a xed rate, the individual hypha strained as a result, and the force resisting motion recorded. Maximum force resisting motion was taken as the force at which breakage occurred.

Reproducibility and Statistical Analysis Hyphal Breaking Force Measurements The procedure described by Stocks and Thomas (2001) was used throughout. Hyphae were xed to a tungsten lament using adhesive and mounted on a force transAll experimental data presented in this work were obtained from single cultures. Experiments were performed in triplicate to ensure that the trends and relationships observed in the culture parameters meas-

Figure 1. Time courses of production of erythromycin (circles) and biomass (squares) in batch bioreactor cultures of S. erythraea NRRL 2338 and mutants resistant to cell-wall synthesis inhibitors in C-limited medium.



ured were reproducible. Individual assays were replicated four-fold. Experiments were rejected where a Chisquared test indicated signicant dierences between replicates. Biomass concentrations varied by no more than 5% between replicates. Where error bars (which represent standard error determinations) are not shown, they were too small to be visible on the gures. RESULTS AND DISCUSSION Isolation of Mutants Populations of mutants resistant to bacitracin, tunicamycin, and penicillin were obtained (2030 examples of each depending on antibiotic selection). We were unable to isolate mutants resistant to concentrations of cycloserine or vancomycin greater than the MICs for NRRL 2338. Mutants were screened to determine their eects on antibiotic production (Table I), and single examples of mutants showing the greatest resistance to each inhibitor were selected for further study. Erythromycin Production in Batch Bioreactor Culture Maximum biomass concentrations obtained in cultures of the mutants did not dier signicantly from those of NRRL 2338 (Fig. 1). However, signicantly greater antibiotic titers were observed in cultures of the tun-r and pen-r mutants whereas those of the bac-r mutant produced signicantly less antibiotic than NRRL 2338 (Figs. 1 and 2). The kinetics of production were similar to those observed in our previous studies (Bushell et al., 1997b; Wilson and Bushell, 1995). Hyphal Fragment-Bounding Circle Diameter When the fragment size distribution was examined, a relationship between the proportion of larger particles (minimum bounding circle greater than 88 lm) and

maximum antibiotic tilter emerged (Fig. 3), supporting our assertion that hyphal particles with a hyphal fragment-bounding circle diameter of greater than 88 lm make the signicant contribution to antibiotic production (Martin and Bushell, 1996). Cultures of tun-r (which supported the greatest antibiotic production: Fig. 2) had a signicantly higher proportion of >88 lm hyphal fragments compared to the wild type (Fig. 3a). Pen-r, which supported the next highest titer, had proportionately less >88 lm fragments than tun-r but more that the wild type (Fig 3b). Bac-r, which produced signicantly less antibiotic than the wild type, had few detectable >88 lm fragments (Fig. 3c). Force Required to Break the Hyphae by Micromanipulation Because of the diculties of measuring actinomycete mechanical properties (Stocks and Thomas, 2001) few data are available. The wild type was well characterised throughout the fermentation (Fig. 4a). There appeared to be a reasonably linear increase in breaking force throughout the rapid growth phase. With less data, there appeared to be a similar rate of change in breaking force for the pen-r and bac-r mutants (Fig. 4b). Using this rate

Figure 2. Maximum titer of erythromycin obtained in batch bioreactor cultures of S. erythraea NRRL 2338 and mutants resistant to cell-wall synthesis inhibitors in C-limited medium.

Figure 3. Distribution of hyphal fragment sizes obtained in batch bioreactor cultures of S. erythraea NRRL 2338 (open bars) and mutants (closed bars) resistant to cell-wall synthesis inhibitors in C-limited medium after the phase of rapid growth.



Figure 4. (a) Variation of hyphal breaking force with time during batch bioreactor cultures of S. erythraea NRRL 2338 in C-limited medium; (b) hyphal breaking force measurements taken during the phase of rapid growth during batch bioreactor cultures of S. erythraea NRRL 2338 (squares), pen-r (inverted triangles), bac-r (circles), and tun-r (triangle) in C-limited medium.

of change and assuming a linear increase with time, the breaking force of the tun-r mutant when maximum biomass concentration was attained was estimated at 1130 nN. Figure 5a shows the breaking forces at the time when antibiotic was rst detected. It appears that the bac-r mutant was weakest, the wild type and pen-r mutant were similar, and that the extrapolated value for tun-r was the largest. These observations are consistent with the fragment size distribution data. Bac-r, the mutant with the hyphae most susceptible to breakage, had the lowest proportion of the >88 lm (breakage susceptible) hyphal fragments. Tun-r, the highest titer mutant, was the most resistant to breakage and had the highest proportion of >88 lm hyphal fragments. Thus, the weaker the hyphae (breaking force), the greater the rate of breakage into smaller, non-producing hyphae in the bioreactor.

Figure 5. (a) Force required to break individual hyphae of S. erythraea (in micromanipulation experiments) obtained from batch bioreactor cultures of NRRL 2338 and mutants resistant to cell-wall synthesis inhibitors in C-limited medium. Samples were taken when maximum biomass concentration was attained except for tun-r for which the value was estimated by extrapolation from 46 h; (b) mean hyphal growth unit (total hyphal length/no, of tips of individual hyphal fragments) of mycelium obtained from batch bioreactor cultures of S. erythraea NRRL 2338 and mutants resistant to cell-wall synthesis inhibitors at times corresponding to the measurements/estimates in (a).

Branching Rates The mean hyphal branching rate of pen-r and tun-r mycelium was lower (higher hgu) than that of NRRL

2338 (Fig. 5b), whereas that of bac-r was higher. We speculate that the correlation of these data with the breaking force data (Fig. 5a) are consistent with the introduction of branch points, resulting in areas of weakness in the cell wall. This is consistent with viability (dierential staining) measurements of S. erythraea cultures (S. M. Stocks, unpublished data), during which we found that hyphal breakage took place at non-viable regions of the hyphae, near branch points. However, the physiology of branch formation in actinomycetes is not well known and requires further investigation. Nevertheless, it is encouraging that a widely accepted mechanism in fungi requires wall turnover as a prerequisite to tip elongation and branch formation (Bartnicki-Garcia, 1973; Rast et al., 1991), and we speculate that this may also apply in actinomycetes.



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