Defined Medium for Moraxella (Branhamella) catarrhalis

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1986, p /86/ $02.00/0 Copyright ) 1986, American Society for Microbiology Vol. 52, No. 3 Defined Medium for Moraxella (Branhamella) catarrhalis ELLIOT JUNI,* GLORIA A. HEYM, AND MARC AVERY Department of Microbiology and Immunology, The University of Michigan Medical School, Ann Arbor, Michigan Received 28 October 1985/Accepted 4 June 1986 A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine. Moraxella (Branhamella) catarrhalis is a gram-negative aerobic coccus commonly found in the nasal mucosa and occasionally associated with a variety of inflammations (4). It grows readily on common laboratory media and has been shown to grow in media containing vitamin-free casein hydrolysate (1). A previous study of the nutrition of a single strain of M. catarrhalis resulted in the conclusion that all the amino acids present in casein hydrolysate might be required for adequate growth of this organism (7). Nutritional studies of various species of Moraxella have shown that some species can grow in simple mineral salts media containing a single organic carbon source, whereas other species failed to grow in media containing casein hydrolysate supplemented with purines, pyrimidines, and vitamins, and still other species required a mixture of heart infusion broth and yeast extract supplemented with serum or oleic acid (1). While investigating the nutrition of M. nonliquefaciens in our laboratory, we devised a general procedure for determination of the growth factor requirements of heterotrophic bacteria (10). Because most strains of M. catarrhalis are competent for genetic transformation (4) and appear to require only amino acid growth factors, we undertook a determination of the nutritional requirements of this organism to facilitate further genetic and physiological studies. Because of a clear genetic relationship between the rodshaped moraxellae and coccal strains formerly designated Branhamella catarrhalis, it has been proposed that the latter strains be designated as Moraxella (Branhamella) catarrhalis, the term Branhamella being considered a subgenus of the genus Moraxella (2). This terminology is used in the latest edition of Bergey's Manual (3). MATERIALS AND METHODS Bacterial strains. The 24 strains of M. catarrhalis examined in this study are listed in Table 1. Strains were obtained * Corresponding author. 546 from the American Type Culture Collection, Rockville, Md., or were isolated from plates streaked with throat and nasal swabs obtained from a local hospital. The friable consistency of most strains of M. catarrhalis grown on semisolid medium made it difficult to prepare uniform suspensions. One strain (strain B4), carried for many years in the stock culture collection of the Department of Microbiology and Immunology, University of Michigan, proved to suspend fairly readily in a buffered salt solution and was used as the chief test strain in the nutritional studies described below. Media and chemicals. S-715 mineral salts solution was prepared by dissolving the following components in distilled water to a final volume of 1 liter: Na2HPO4, 11.2 g; KH2PO4, 4.0 g; NH4Cl, 2 g; MgSO4, 0.2 g; CaCl2, 1 ml of a 1% solution; and FeSO4-7H2O, 0.5 ml of a freshly prepared 0.1% solution. S-715 mineral salts solution was sterilized by membrane filtration. Double-strength medium B4, a defined medium used in the studies described in this report, was prepared by addition of the following components to distilled water to a final volume of 1 liter: S-715 mineral salts solution, 500 ml; 60% DL-sodium lactate, 20 ml; monopotassium aspartate, 5 g; L-proline, 4 g; L-arginine hydrochloride, 1 g; glycine, 2 g; L-methionine, 0.2 g; Tween 80, 2 ml of a 20% solution. Double-strength medium B4 was sterilized by membrane filtration. The ph of this medium was 7.1. Semisolid medium B4 plates were prepared by adding 200 ml of double-strength medium B4 (equilibrated at 60 C) and 2.0 ml of sterile 2% ferric ammonium citrate to 200 ml of melted 3% sterile agar (equilibrated at 60 C), mixing, and pouring approximately 20 plates. Amino acids, Tween 80, and ferric ammonium citrate were obtained from Sigma Chemical Co. DL-Lactic acid (sodium salt, 60%) was obtained from Fisher Scientific Co., Pittsburgh, Pa. Vitamin-free, salt-free casein hydrolysate was obtained from ICN Nutritional Biochemicals, Cleveland, Ohio. Heart infusion broth and GC medium broth were obtained from Difco Laboratories, Detroit, Mich. Determination of essential and stimulatory growth factors. Required and stimulatory growth factors were determined as described previously (10).

2 VOL. 52, 1986 MEDIUM FOR MORAXELLA (BRANHAMELLA) CATARRHALIS 547 TABLE 1. Growth of M. catarrhalis strains on medium B4 plates and on B4 plates lacking a single component of the medium Growth on medium lacking the following componenta: Strain No Arginine component Tween 80 Arginine (contains Glycine Aspartate Proline Methionine missing ornithine) ATCC B (±) ATCC B ()+ + + B B B () B () Bll () B B B ()++ B B B () B () B B B ATCC ATCC ATCC ATCC B a , Best growth of isolated colonies; -, no growth; (±), colonies just visible to the naked eye after 2 days at 34 C. Measurement of growth. Growth in liquid media took place at 34WC in a shaking water bath in a 500-ml Erlenmeyer flask with an attached side arm for determination of culture absorbance in a colorimeter, as described previously (10). Inocula were prepared by removing cells from the surface of a heart infusion plate, with a bacteriological loop, following overnight incubation (12 to 16 h) at 34 C. The cells were suspended in 5 ml of sterile S-715 mineral salts solution contained in a sterile screw-cap colorimeter tube to give a reading of approximately 300 in a Klett-Summerson colorimeter with a 660 (red) filter. Each growth flask was inoculated with 0.5 ml of freshly prepared suspension to give a total culture volume of 10 ml. Measurement of bacterial growth was carried out as described previously (10). RESULTS Essentiality of the components of a defined medium. Using the methods developed in our laboratory for determining growth factor requirements of heterotrophic bacteria (10), we showed that M. catarrhalis B4 could grow well in a defined medium (medium B4) containing a mineral salts mixture, sodium lactate as a partial carbon source, proline as a partial carbon and partial nitrogen source, potassium aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Tween 80 (0.02%) appeared to shorten the lag period in some experiments (data not shown) and was included routinely in most of the studies to be presented. Growth of strain B4 in medium B4 is shown in Fig. 1. The requirement for each component of medium B4 was ascertained by inoculating strain B4 in medium devoid of that particular compound (Fig. 1). No growth was observed during the 10-h period of incubation when arginine was omitted (Fig. 1). In the absence of glycine, slow growth began after a lag period of approximately 4 h (Fig. 1). Omission of methionine resulted in a small increase in the lag period and a somewhat smaller final endpoint of growth (Fig. 1). The absence of either aspartate or proline resulted in an increased lag period and a lower growth rate, but omission of both aspartate and proline resulted in a complete lack of growth (Fig. 1). Omission of sodium lactate resulted in a significantly lower growth rate (Fig. 1). Utilizable nitrogen sources. The absence of growth upon omission of both aspartate and proline from medium B4 (Fig. 1) suggested that these amino acids serve as nitrogen sources for growth of strain B4 in this medium. Growth was not influenced by the presence or absence of ammonium chloride in medium B4 (data not shown). In a separate study, we examined the possible role of proline, aspartate, and glutamate, either singly or in combination, as nitrogen sources for growth of strain B4 in the otherwise defined medium B4. The presence of any of these three amino acids alone resulted in good, but not optimal, growth; the presence of only proline resulted in the best growth rate stimulated by single amino acids of this group (Fig. 2). Although the growth rate observed when both aspartate and glutamate were present together was about the same as that obtained for proline alone, significantly better growth rates were seen when proline was included with either aspartate or glutamate (Fig. 2). Growth with glutamate and proline was slightly better than with aspartate and proline, and the best growth rate and final cell yield were obtained when all three amino acids were added simultaneously (Fig. 2). No growth occurred in the absence of these three amino acids (Fig. 2). Optimal concentrations of nutrients in medium B4. Because the absence of arginine from medium B4 resulted in a complete lack of growth (Fig. 1), an experiment was performed to determine the concentration of arginine required

3 548 JUNI ET AL. z100 ~80 U w ~60 -J40 15L TIME-HOURS FIG. 1. Essentiality of the components of medium B4 for growth of strain B4. Growth of strain B4 in medium B4 lacking nothing (complete medium B4) (0), aspartate (0), proline (OI), lactate (G), methionine (A), glycine (A), aspartate and proline (V), or arginine (V). for optimal growth (Fig. 3). For each of the suboptimal concentrations of arginine used in this experiment, growth leveled off below the maximum yield, presumably when the arginine was depleted. The optimal arginine concentration was found to be 400,ug/ml (Fig. 3). By contrast, in similar studies in which the concentration of glycine was varied below the optimal concentration (1 mg/ml), the maximum growth rate was maintained until the added glycine was depleted, at which point growth continued at a rate approximately one-fourth the maximum (Fig. 4). Serine could not replace the relative requirement for glycine in medium B4. The optimal proline concentration for growth of strain B4 in medium B4, with or without aspartate, was found to be 4 mg/ml (data not shown). Utilizable carbon sources. Using slightly modified medium B4 containing a relatively low concentration of proline (500 ug/ml), we found that sodium lactate (0.6%), monosodium glutamate (0.5%), disodium succinate (0.4%), disodium malate (0.4%), or proline (0.4%) could serve as a carbon source for growth of strain B4 (data not shown). Virtually no growth took place in the modified medium B4 in the absence of a utilizable carbon source. Sodium acetate (0.25%), potassium aspartate (0.25%) (normally present in medium B4), and potassium gluconate (0.4%) could not serve as carbon sources in this medium. Of all the carbon sources tested, sodium lactate gave the best growth rate (data not shown). Other growth-stimulatory factors. The addition of any one of six growth factor pools, each containing six different growth factors (10), failed to stimulate the growth of strain B4 in medium B4. Simultaneous addition of pools containing all 20 naturally occurring amino acids did result in significant stimulation of growth, thus indicating that such stimulation required the presence of several amino acids at the same time. By a process of successive elimination, it was deter- APPL. ENVIRON. MICROBIOL. mined that the growth-stimulating amino acids, each present at a concentration of 200 plg/ml, were asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine. Simultaneous addition of these seven stimulatory amino acids to medium B4 resulted in a small but significant increase in growth rate, an effect that was demonstrated to be reproducible (Fig. 5). Figure 5 also shows the growth of strain B4 in heart infusion broth, in GC medium broth, and in a mineral salts solution containing 1% vitamin-free casein hydrolysate. Nutritional requirements of other strains of M. catarrhalis. The nutritional requirements of 24 strains of M. catarrhalis were examined by inoculating a series of plates of semisolid medium B4, each lacking a single component of the complete medium. All strains were able to grow on plates lacking Tween 80 (Table 1). One strain (B24) failed to grow when Tween 80 was included in the medium. This strain was found to mutate spontaneously to the ability to grow in the presence of Tween 80. With the exception of strain ATCC 23246, all strains failed to grow when arginine was not included in the medium. All strains, except strain B24 and strain ATCC 25238, grew when ornithine was added in place of arginine. More than half the strains could not grow in the absence of glycine, and those that could grow without glycine grew extremely poorly on such a medium, with the exception of strain ATCC 23246, which appeared to be indifferent to the presence of glycine. Except for the strain sensitive to Tween 80 (strain B24), all the other strains were able to grow on the medium lacking TIME- HOURS FIG. 2. Nitrogen sources for growth of strain B4 in a defined medium. Growth in medium B4 devoid of proline and aspartate but supplemented with 0.25% monopotassium aspartate, 0.25% monosodium glutamate, and 0.2% proline (0), 0.25% monopotassium aspartate and 0.2% proline, components that result in complete medium B4 (0), 0.25% monosodium glutamate and 0.2% proline (0), 0.2% proline (N), 0.25% monopotassium aspartate and 0.25% monosodium glutamate (A), 0.25% monosodium glutamate (A), 0.25% monopotassium aspartate (V), or no additions (V).

4 VOL. 52, 1986 MEDIUM FOR MORAXELLA (BRANHAMELLA) CATARRHALIS 549 aspartate or methionine, and only one strain (ATCC 25239) was unable to grow on the medium lacking proline. When strain B4 was streaked heavily on the defined medium lacking arginine, the appearance of small numbers of spontaneous mutant colonies growing well on this medium was observed after several days of incubation. Unlike the wild-type strain, these arginine-independent mutants, purified and maintained on defined medium lacking arginine, grew very poorly on heart infusion plates. Upon incubation of several of these arginine-independent strains on heart infusion agar for 2 to 4 days, large spontaneous revertant colonies could be seen. Isolation and purification of some of these colonies showed that they were identical to the wildtype strain and were unable to grow on the defined medium lacking arginine. Addition of the seven stimulatory amino acids discussed above to medium B4 plates improved the growth of all strains to some extent, except for the Tween 80-sensitive strain (strain B24), which failed to grow on any medium containing Tween 80. Strain ATCC was shown to be able to grow on medium B4 simultaneously lacking glycine, arginine, and methionine. Similarly, this strain could grow on this medium, which normally contains aspartate and proline, if proline, aspartate, or glutamate, either singly or in combination, were included. However, it failed to grow in the absence of all three amino acids. DISCUSSION Although Baumann et al. (1) consistently used a biotin casein hydrolysate medium for growth of several strains of z 0, w -J e ~ ~~~ t a _ r'.ls., TIME-HOURS FIG. 3. Growth of strain B4 in a defined medium as a function of arginine concentration. Strain B4 was grown in medium B4 devoid of the arginine normally present in this medium but supplemented with the following concentrations of arginine (micrograms per milliliter): 400 (0), 200 (0), 100 (O), 50 (-), 20 (A), 10 (A), 5 (V), or 0 (V). 0 z a w H Y: w -J FIG. 4. TIME- HOURS Growth of strain B4 in a defined medium as a function of glycine concentration. Strain B4 was grown in medium B4 devoid of the glycine normally present in this medium but supplemented with the following concentrations of glycine: 1 mg/ml (0), 700,u.g/ml (0), 400 j±g/ml (O), 200,ug/ml (-), 100,ug/ml (A), 50 j.lg/ml (A), 20,ug/ml (V), or 0 (V). M. catarrhalis, it was never shown that any of the strains examined did, in fact, require biotin. None of the strains of M. catarrhalis that we studied was stimulated by addition of biotin to medium B4. Guirard and Snell (9) listed a growth medium for M. catarrhalis that contained 18 amino acids. The results of our studies indicate that good growth of 24 strains of M. catarrhalis occurred in liquid or semisolid medium containing mineral salts, sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. This same medium lacking methionine and Tween 80 also supported good growth of all 24 strains (Table 1). The lack of effect of ammonium chloride on growth of M. catarrhalis in medium B4, coupled with the finding of an absolute requirement for either glutamate, aspartate, or proline for growth in this medium (Fig. 2), suggests that this organism is incapable of carrying out primary amination reactions of the kind required as a first step for the synthesis of a-amino acids (5). Alternatively, it is possible that primary amination reactions do take place but that M. catarrhalis contains an unregulated system for breakdown of glutamate which limits the net de novo synthesis of glutamate. In such a case, other sources of glutamate, e.g., the breakdown of proline (8) or transamination of a-ketoglutarate with aspartate (14), could supply sufficient glutamate to satisfy the biosynthetic requirements for this amino acid. Although all strains of M. catarrhalis studied showed an absolute requirement for arginine (Table 1), the demonstration of spontaneous mutation of strain B4 to arginine independence is evidence that this strain possesses the genetic

5 550 JUNI ET AL. 2' F- wj -lw TIME - HOURS FIG. 5. Growth of strain B4 in defined and in complex media. Strain B4 was grown in medium B4 (0), medium B4 plus the amino acids asparagine, monosodium glutamate, glutamine, histidine, leucine, lysine, and phenylalanine, each present at a concentration of 200.g/ml (0), heart infusion broth (O), GC medium broth (U), or 1% vitamin-free, salt-free casein hydrolysate in one-quarter strength S-715 mineral salts solution (A). determinants required for arginine synthesis and that at least one of the steps in the arginine biosynthetic pathway must normally be inoperative. The fact that growth of arginineindependent revertant strains on complex media was severely inhibited indicates that the revertant strains belong to the general class of nutrient-sensitive mutants (12). Such nutrient sensitivity may account for the lack of appearance and proliferation of mutants of this kind during normal growth in the host or on complex laboratory media. The concentration of glycine required for optimal growth (1 mg/ml) appeared to be unusually high (Fig. 4). The fact that glycine concentrations as high as 400,ug/ml, for example, failed to support optimal growth (Fig. 4) may imply that glycine is broken down rapidly during growth. It is possible that glycine can be synthesized by M. catarrhalis, but that glycine breakdown may result in the requirement of exogenous glycine for optimal growth. Enzyme systems for glycine cleavage have been described for other bacteria (11, 13) Ṫhe finding that all but two of the strains of M. catarrhalis examined could grow in medium B4 with ornithine in place of arginine indicates that for most of these strains the pathway from ornithine to arginine (6) must be intact. Although the presence of 0.02% Tween 80 appeared to shorten the lag period of strain B4 growth in some experiments, elimination of this component from medium B4 plates failed to affect significantly the growth of any but one of the strains studied (Table 1). It is suggested, therefore, that Tween 80 not be included in media used for growth of M. catarrhalis. APPL. ENVIRON. MICROBIOL. Our studies show that growth of strain B4 in medium B4 compares very favorably with growth in three complex media (Fig. 5). When the seven supplementary amino acids were included with the other components of medium B4, growth occurred at the highest rate and with the greatest final cell yield for all the media investigated (Fig. 5). Compared with defined media, complex media shortened the initial lag period (Fig. 5). Of all 24 strains of M. catarrhalis examined, ATCC was unusual in that neither arginine nor glycine was required for its growth in medium B4 (Table 1). In studies of interstrain transformation of the streptomycin resistance marker, it was shown by use of a single recipient strain that the ratio of interstrain to intrastrain transformation varied from 3.6 x 10-3 to 4.6 x 10-3 when DNA from strain ATCC was used, whereas ratios from 3.0 x 10-1 to 9.4 x 10-1 were observed when DNA samples from many other strains of M. catarrhalis were tested (4). It would thus appear that ATCC is not closely related to other strains of M. catarrhalis. B0vre and Hagen (4) have suggested that strain ATCC may represent a separate and as yet undescribed species of Moraxella. ACKNOWLEDGMENT This investigation was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Baumann, P., M. Doudoroff, and R. Y. Stanier Study of the Moraxella group. I. Genus Moraxella and the Neisseria catarrhalis group. J. Bacteriol. 95: B0vre, K Proposal to divide the genus Moraxella Lwoff 1939 emend. Henriksen and B0vre 1968 into two subgenera, subgenus Moraxella (Lwoff 1939) B0vre 1979 and subgenus Branhamella (Catlin 1970) B0vre Int. J. Syst. Bacteriol. 29: B0vre, K Genus II. Moraxella Lwoff 1939, 173 emend. Henriksen and B0vre 1968, 391, p In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 4. B0vre, K., and N. Hagen The family Neisseriaceae: rod-shaped species of the genera Morazella, Acinetobacter, Kingella, and Neisseria, and the Branhamella group of cocci, p In M. P. Starr, H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel (ed.), The prokaryotes, vol. II. Springer- Verlag KG, Berlin. 5. Brown, C. M., D. S. Macdonald-Brown, and J. L. Meers Physiological aspects of microbial inorganic nitrogen metabolism. Adv. Microb. Physiol. 11: Cunin, R Regulation of arginine biosynthesis in prokaryotes, p In K. M. Hermann and R. L. Sommerville (ed.), Amino acids, biosynthesis and genetic regulation. Addison-Wesley Publishing Co., Reading, Mass. 7. Fitting, C., and H. W. Scherp Observations on the metabolism of a strain of Neisseria catarrhalis. J. Bacteriol. 59: Frank, L., and G. Ranhand Proline metabolism in Escherichia coli. III. The proline catabolic pathway. Arch. Biochem. Biophys. 107: Guirard, B. M., and E. E. Snell Biochemical factors in growth, p In P. Gerhardt, and R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.), Manual of methods for general bacteriology. American Society for Microbiology, Washington, D.C. 10. Juni, E., G. A. Heym, and R. A. Bradley General approach to bacterial nutrition: growth factor requirements of Moraxella nonliquefaciens. J. Bacteriol. 160: Meedel, T. H., and L. I. Pizer Regulation of one-carbon

6 VOL. 52, 1986 MEDIUM FOR MORAXELLA (BRANHAMELLA) CATARRHALIS 551 biosynthesis and utilization in Escherichia coli. J. Bacteriol. 13. Sagers, R. D., and I. C. Gunsalus Intermediary metabo- 118: lism of Diplococcus glycinophilus. I. Glycine cleavage and 12. Meuris, P., F. Lacroute, and P. R. Slonimski Etude one-carbon interconversions. J. Bacteriol. 81: systdmatique de mutants inhibes par leurs propres metabolites 14. Tan-Wilson, A. L Alanine biosynthesis and the general chez la levure Saccharomyces cerevisiae. I. Obtention et transaminases, p In K. M. Herrmann and R. L. Somcaracterisation des differentes classes de mutants. Genetics merville (ed.), Amino acids, biosynthesis and genetic regula- 56: tion. Addison-Wesley Publishing Co., Reading Mass.