bvg Repression of Alcaligin Synthesis in Bordetella bronchiseptica Is Associated with Phylogenetic Lineage

JOURNAL OF BACTERIOLOGY, Nov. 1995, p. 6058 6063 Vol. 177, No. 21 0021-9193/95/$04.00 0 Copyright 1995, American Society for Microbiology bvg Repression of Alcaligin Synthesis in Bordetella bronchiseptica Is Associated with Phylogenetic Lineage PETER C. GIARDINA, 1,2 * LISA-ANNE FOSTER, 2 JAMES M. MUSSER, 3 BRIAN J. AKERLEY, 4 JEFF F. MILLER, 4 AND DAVID W. DYER 1 Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 1 ; Department of Microbiology and Immunology, State University of New York at Buffalo, Buffalo, New York 2 ; Section of Molecular Pathobiology, Department of Pathology, Baylor College of Medicine, Houston, Texas 3 ; and Department of Microbiology and Immunology, University of California School of Medicine, Los Angeles, California 4 Received 23 May 1995/Accepted 15 August 1995 Recent studies have shown that Bordetella bronchiseptica utilizes a siderophore-mediated transport system for acquisition of iron from the host iron-binding proteins lactoferrin and transferrin. We recently identified the B. bronchiseptica siderophore as alcaligin, which is also produced by B. pertussis. Alcaligin production by B. bronchiseptica is repressed by exogenous iron, a phenotype of other microbes that produce siderophores. In this study, we report that alcaligin production by B. bronchiseptica RB50 and GP1SN was repressed by the Bordetella global virulence regulator, bvg, in addition to being Fe repressed. Modulation of bvg locus expression with 50 mm MgSO 4 or inactivation of bvg by deletion allowed strain RB50 to produce alcaligin. In modulated organisms, siderophore production remained Fe repressed. These observations contrasted with our previous data indicating that alcaligin production by B. bronchiseptica MBORD846 and B. pertussis was repressed by Fe but bvg independent. Despite bvg repression of alcaligin production, strain RB50 was still able to acquire Fe from purified alcaligin, suggesting that expression of the bacterial alcaligin receptor was not repressed by bvg. We tested 114 B. bronchiseptica strains and found that bvg repression of alcaligin production was strongly associated with Bordetella phylogenetic lineage and with host species from which the organisms were isolated. Bordetella bronchiseptica is a gram-negative coccobacillus that colonizes the upper respiratory tract of several mammals (19). Colonization may lead to upper respiratory tract disease such as atrophic rhinitis in swine, an illness characterized by nasal turbinate atrophy, snout disfiguration, and weight loss (33, 40). In some cases, death results from pneumonia caused by secondary bacterial infections. B. bronchiseptica also predisposes swine to subsequent infection by Pasteurella multocida, which can severely exacerbate atrophic rhinitis (9 11). B. bronchiseptica causes infectious canine tracheobronchitis (kennel cough) in dogs (41, 50) and respiratory infections in other domesticated mammals (8, 19). Humans can sustain respiratory and other infections caused by B. bronchiseptica; these infections most commonly occur in immunocompromised individuals (15, 34, 48, 49). Our laboratory has been interested in examining how Fe transport by B. bronchiseptica contributes to pathogenesis. In order to successfully colonize a mammalian host, a pathogen must be able to obtain Fe, an essential cofactor in many biological oxidation-reduction reactions (43, 44). Although Fe is an essential nutrient for growth of virtually all microorganisms, the concentration of free Fe in the mammalian body is normally too low to support microbial growth (44). In mammals, most Fe is sequestered intracellularly. Extracellular Fe is bound to lactoferrin (LF; in exocrine secretions) or transferrin * Corresponding author. Mailing address: Oklahoma University Health Sciences Center, Department of Microbiology and Immunology, P.O. Box 73190, Oklahoma City, OK 73190. Phone: (405) 271-1201. Fax: (405) 271-3117. Electronic mail address: pgiardin@zena. uokhsc.edu. Present address: Department of Molecular Microbiology and Immunology, Washington University, St. Louis, MO 63110. (in plasma and tissue fluids) (44). This iron sequestration effectively limits Fe availability to most invading microorganisms and suppresses growth. This phenomenon has been termed nutritional immunity (43). To obtain essential Fe, many pathogenic bacteria have evolved the ability to synthesize and secrete small Fe-binding compounds, termed siderophores, that sequester Fe for use by the bacterial cell (32). Many studies have demonstrated that siderophore-mediated iron transport is critical in supporting pathogenesis. For example, the ability of enteroinvasive Escherichia coli to synthesize and secrete aerobactin correlates directly with the ability of these organisms to cause disease (47). B. bronchiseptica secretes an Fe-chelating compound, alcaligin (28), that is able to remove Fe from LF and transferrin (1, 6, 17, 20). The metabolic pathway for alcaligin biosynthesis and secretion, and the uptake mechanism for the Fe chelate, are not understood. However, it is clear that, similar to the control of siderophore synthesis by other bacteria, alcaligin production is regulated by Fe availability (17). That is, when B. bronchiseptica is Fe starved, the organism produces alcaligin. Release of this siderophore is not detected when the organism is grown in Fe-replete conditions. Studies from our laboratory and others previously suggested that Bordetella alcaligin production was independent of bvg control (20). The bvg operon (36), encoding a complex twocomponent regulator (42), controls the expression of many Bordetella virulence determinants. This operon includes BvgS, a transmembrane sensory protein, and BvgA, a cytoplasmic, transcriptional activator of several Bordetella virulence determinants, including filamentous hemagglutinin (35), fimbriae (46), adenylate cyclase toxin (18), and dermonecrotic toxin (25). BvgA also regulates its own expression (36, 37) and the synthesis of certain outer membrane proteins of unknown 6058

VOL. 177, 1995 REGULATION OF ALCALIGIN PRODUCTION 6059 TABLE 1. Bacterial strains and plasmids used Strain or plasmid Description Reference Bordetella strains MBORD846 bvg pig isolate 1 MBA4 MBORD846 bvgs 54 This study RB50 bvg rabbit isolate 12 RBA2 RB50 frlab 2 RB53 RB50 bvgs-c3; bvg(con) 12 RB54 bvgas derivative of RB50 12 RBA1 RB54 frlab 2 GP1SN bvg guinea pig isolate 4 DM107 GP1SN bvgas 4 Plasmids pba291 prk415 containing frlab 2 prk415 Cloning vector function (16, 45). The present report demonstrates that in certain strains of B. bronchiseptica, alcaligin production is repressed by bvg. We further discovered that bvg repression of alcaligin synthesis strongly correlated with phylogenetic lineage as defined by multilocus enzyme electrophoresis (MLEE) analysis (30, 31) and with the host species from which a particular B. bronchiseptica strain was isolated. MATERIALS AND METHODS Bacteria, plasmids, and media. B. bronchiseptica strains and plasmids used in the initial phase of this study are described in Table 1. All strains were routinely cultured on Bordet-Gengou (BG) agar base (Difco Laboratories, Detroit, Mich.) containing 15% defibrinated sheep blood (Crane Laboratories, Syracuse, N.Y.). Chelex-treated Stainer-Scholte medium (CSSM) and Chelex-treated defined medium (CDM) in which N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES) buffer was substituted with Tris buffer (ph 7.4) were prepared as previously described (1, 17, 39). All experiments involving LF were carried out in CDM supplemented with 10 mm sodium bicarbonate. Bovine LF (Sigma Chemical Co., St. Louis, Mo.) was prepared as previously described (1, 17). Ferrous sulfate was prepared daily and used as a supplemental Fe source at a final concentration of 10 M, where indicated. Ferric nitrate (ph 4.0) was used at a final concentration of 10 M. Purified desferri-alcaligin was kindly provided by Chris Moore and Bradley Gibson (University of California, San Francisco) (28). When indicated, media were supplemented with 50 mm MgSO 4 to modulate the expression of bvg-dependent virulence determinants. All glassware was washed in 3 N nitric acid and rinsed with 10 changes of deionized distilled water as previously described (1, 17); alternatively, tissue culture-grade, sterile plasticware was used to eliminate Fe contamination. Growth conditions. For all growth assays, B. bronchiseptica strains were cultured on BG blood agar for 48 h at 37 C. The organisms were transferred to 10 ml of fresh growth medium (CSSM or CDM) with a Dacron swab (Baxter Scientific Products, McGaw Park, Ill.) and incubated overnight at 37 C with rotary shaking at 250 rpm. The organisms were sedimented by centrifugation, washed with a 10-ml volume of fresh medium, and suspended in fresh medium. The optical density was adjusted to 10 to 20 Klett units, using a Klett-Summerson colorimeter (green filter), or to 0.05 to 0.07 U at 660 nm in a Spectronic 21D spectrophotometer (Milton Roy Company). An Fe source was added, incubation was continued at 37 C with rotary shaking, and growth was periodically assessed by optical density measurement. A sample from each culture was transferred to BG blood agar to determine the presence of bvg phase variants. To determine if a culture produced alcaligin, the cells were sedimented by centrifugation, and the culture supernatants were filter sterilized. All culture supernatants were then assayed for the presence of alcaligin with the Csaky assay as previously described (1, 14). To determine the effect of phenotypic modulation of bvg on alcaligin synthesis, cells were grown for 48 h on BG blood agar supplemented with 50 mm MgSO 4. Organisms from nonhemolytic colonies were cultured as described above except that the growth medium was supplemented with 50 mm MgSO 4. Following the experiment, bacteria were again cultured on BG blood agar without MgSO 4,to confirm that nonhemolytic organisms were phenotypically modulated and not phase variants that had sustained a genetic mutation in the bvg operon. RB53, a bvg(con) mutant of RB50 in which the modulation of bvg is prevented by a point mutation in bvgs, was used as a negative control (12, 27). Survey of strains for alcaligin production. Raised, hemolytic colonies (bvg ) were transferred to CSSM and incubated at 37 C in a rotary shaker for 20 h. Cells were sedimented by centrifugation, and the supernatants were analyzed in duplicate for the presence of alcaligin with the Csaky assay (14). Experiments were performed twice each in the presence and absence of 50 mm MgSO 4. In certain instances, strains that did not produce detectable alcaligin after 20 h of culture were incubated for an additional 24 to 28 h prior to collection of culture supernatants for the Csaky assay. Strains that did not produce detectable hydroxamate under these conditions were also examined by using the more sensitive chrome azurol S assay (38). RESULTS Strain-dependent repression of alcaligin production by bvg. Our previous work showed that alcaligin production by B. bronchiseptica MBORD846 was repressed when the organism was grown in the presence of as little as 10 M Fe. Moreover, alcaligin production was not controlled by the bvg operon (17). Similar results have been noted by other authors (20). The availability of defined mutants in the B. bronchiseptica bvg operon (3, 12) led us to reexamine this issue, in order to confirm that alcaligin production was independent of bvg regulation. Surprisingly, B. bronchiseptica RB50 did not secrete detectable alcaligin in the nonmodulated (Bvg ) state, even when Fe starved (Table 2). However, when RB50 was phenotypically modulated by growth with 50 mm MgSO 4 in CSSM, the organism produced copious amounts ( 50 M) of alcaligin in response to Fe stress (Table 2). Fe-starved RB50 grew to comparable extents (as measured by optical density), whether or not the organism was modulated (data not shown). Thus, excretion of alcaligin by modulated RB50 was not due to a nonspecific effect on the growth of the organism. Strain RB54, lacking a functional bvg locus (bvg), produced alcaligin in response to Fe stress (Table 2), irrespective of the presence of MgSO 4. Strain RB53, which is constitutively bvg and does not respond to modulating signals (12, 28), did not secrete alcaligin under any tested conditions, including Fe starvation in the presence of 50 mm MgSO 4. We also tested a second isogenic pair of B. bronchiseptica strains, GP1SN (bvg ) and DM107, a bvgas deletion mutant of GP1SN (bvg) (4). GP1SN and DM107 behaved similarly to RB50 and RB54. That is, the wild-type strain (GP1SN) did not produce alcaligin, while the bvg derivative (DM107) produced alcaligin in an Fe-repressible manner (data not shown). These data were in contrast to earlier reports indicating that alcaligin production was independent of bvg, including our observations on B. bronchiseptica MBORD846 (20). Modulation of strain MBORD846 with 50 mm MgSO 4 did not alter the amount of alcaligin produced compared with cells grown under nonmodulating conditions. To confirm that alcaligin production by strain MBORD846 was independent of bvg, we constructed a bvg deletion derivative of this strain, designated MBA4, as previously described (3, 12). Strain MBA4 produced alcaligin at levels comparable to those produced by MBORD 846, indicating that a deletion of the bvg locus did not enhance alcaligin production in this strain (data not shown). Alcaligin synthesis remained responsive to Fe stress. These observations suggested that in some strains of B. bronchiseptica (such as RB50), alcaligin production was under dual control by bvg and Strain (genotype) TABLE 2. Alcaligin production is repressed by bvg Alcaligin production MgSO 4 MgSO 4 Fe Fe Fe Fe RB50 (bvg ) RB53 [bvg(con)] RB54 (bvg)

6060 GIARDINA ET AL. J. BACTERIOL. FIG. 1. RB50 used LF as an iron source when purified desferri-alcaligin is added:, LF added; å, LF and purified desferri-alcaligin added. (A) Strain RB50; (B) strain RB54. Fe repression, while in other strains (such as MBORD846), alcaligin synthesis was susceptible only to Fe repression. Strain RB50 can use purified alcaligin for growth with LF. In general, siderophore receptor expression in bacteria is regulated by the same mechanisms that regulate siderophore production (22). If this is also true for B. bronchiseptica, then expression of the alcaligin receptor by RB50 should be repressed by bvg. Therefore, we cultured B. bronchiseptica RB50 with bovine LF and determined whether purified alcaligin could mediate transfer of Fe from LF to the organism. The results are shown in Fig. 1. B. bronchiseptica RB50 (Bvg, not modulated) did not grow in the presence of bovine LF (Fig. 1). In contrast, strain RB54 grew easily in CDM containing LF (Fig. 1). When we added purified desferri-alcaligin to these cultures, strain RB50 grew with bovine LF, while growth of RB54 with LF was slightly enhanced (Fig. 1). Therefore, RB50 was capable of growth with LF if exogenous alcaligin was provided to the organism. These data suggested that although alcaligin production in strain RB50 was repressed by the bvg locus, the ability to metabolize ferri-alcaligin complexes was not repressed by bvg. Alcaligin synthesis is not controlled by frl. Akerley et al. have shown that motility in strain RB50 is inhibited by bvg through repression of frl, a positive regulator of the fla operon (2, 3). Because control of alcaligin production in strain RB50 is phenotypically similar to control of motility (both are repressed by bvg), bvg-mediated repression of motility may act through the action of bvg on frl. We therefore examined if alcaligin production was affected by frl mutations that block activation of motility. These experiments were done under nonmodulating conditions. The results are shown in Table 3. While RBA2 (bvg frl) did not make alcaligin when Fe stressed, strain RBA1 (bvg frl) produced alcaligin in normal amounts, and siderophore synthesis was derepressed by Fe limitation. Introduction of a plasmid-borne copy of frl (pba291) into RBA1 had no effect on alcaligin production (Table 3). These results indicated that alcaligin production was not dependent on the frl transactivator and that bvg repression of alcaligin production was not mediated through an effect on frl. Phylogeny of bvg repression of alcaligin production. Is the phenotypic repression of alcaligin production in strains RB50 and GP1SN unusual, or does strain MBORD846 represent a laboratory artifact that has spontaneously lost bvg repression of alcaligin synthesis? Musser et al. (30, 31) identified 21 distinct electrophoretic types (ETs) of B. bronchiseptica, marking phylogenetic lineages that were grouped into five MLEE clusters, A to E. We examined 114 of these strains for bvg repression of alcaligin synthesis and compared the observed phenotype with the lineage of each strain. A complete list of these strains, including the ET, host, country of origin, and relevant phenotype is available from D.W.D. ET1, ET1a, ET3, and ET4 represent clones from MLEE cluster A, clones ET6, ET6a, and ET8 are members of MLEE cluster B, and ET14 and ET16 are clones assigned to MLEE cluster D. Since MLEE clusters C and E were each represented by one phylogenetic lineage and together contained only three strains (31), they were not examined in this study. We identified two groups of strains based on the conditions under which alcaligin was produced. Group 1 strains (total of 46) resembled strain MBORD846 in that they produced alcaligin in response to Fe stress but independently of bvg. Strains in group 2 (total of 64), like strain RB50, were bvg repressed for alcaligin production. Supernatants from a minority of strains (total of four) were devoid of iron-binding activity in the chrome azurol S assay and did not appear to secrete siderophore under any tested conditions (data not shown). The association of bvg regulation of alcaligin production with MLEE cluster is shown in Table 4. The majority (75%) of strains in MLEE cluster A were in group 1 (bvg independent), while the majority of MLEE cluster B (88%) and D (100%) were in group 2 (bvg repressed). This relationship remained consistent when bvg regulation was compared with phylogenetic lineage (Table 5). Individual ETs from cluster A were TABLE 3. Alcaligin production is independent of frl Strain (genotype) Fe Alcaligin production Fe RB50 (bvg frl ) RB54 (bvg frl ) RBA2 (bvg frl) RBA1 (bvg frl) RBA1(pRK415) RBA1(pBA291)

VOL. 177, 1995 REGULATION OF ALCALIGIN PRODUCTION 6061 TABLE 4. bvg repression of alcaligin production is associated with phylogenetic cluster No. (%) of strains tested MLEE cluster within the cluster Group 1 Group 2 A 43 (75) 14 (25) B 2 (12) 15 (88) D 0 (0) 29 (100) predominantly in group 1, while ETs from clusters B and D were predominantly group 2. Equally striking was the association between the independence of alcaligin production from bvg repression with the ability of B. bronchiseptica to infect pigs (Table 6). Among the 30 tested pig strains, 94% (28 strains) were found to produce alcaligin in a bvg-independent manner (group 1). The majority of strains isolated from other hosts were in group 2 (bvg repressed). Taken together, these data indicated that the mode of regulation of alcaligin was strongly associated with phylogenetic lineage and with the host species from which each strain was obtained. DISCUSSION MLEE cluster TABLE 5. bvg repression of alcaligin is associated with B. bronchiseptica phylogenetic lineage ET No. (%) of total strains tested within the phylogenetic lineage Group 1 (bvg-independent) Group 2 (bvg-repressed) Total tested A 1 38 (74.5) 13 (25.5) 51 1a 1 1 2 3 4 (100) 4 4 1 1 B 6 1 8 (89) 9 6a 1 1 8 1 6 (86) 7 D 14 1 1 16 28 (100) 28 ND a ND 1 5 6 Total 46 64 110 a ND, not determined. The bvg operon has been shown to directly or indirectly modify the expression of a variety of virulence determinants in B. bronchiseptica and B. pertussis in response to environmental signals. In this study, we have demonstrated that in certain strains of B. bronchiseptica, alcaligin biosynthesis is repressed by bvg as well as by Fe and that derepression of alcaligin synthesis can be achieved by genetic mutation of the bvg operon or by exposing the organisms to modulating conditions in culture. The rabbit isolate, RB50, failed to secrete alcaligin in iron-limited media unless phenotypically modulated by MgSO 4 (Table 2). Further, strain RB53, a bvg(con) mutant of RB50 that is unresponsive to modulating conditions, failed to produce alcaligin under any tested conditions. In the isogenic bvg deletion mutant, RB54, alcaligin production was iron repressible but independent of bvg. This eliminated the possibility that the Mg 2 ion may have spuriously interfered with repression of alcaligin synthesis by Fe rather than by bvg. Similar results were obtained for GP1SN, a guinea pig isolate (data not shown). These data conflicted with what we (17) and others (20) previously reported with regard to alcaligin production in Bordetella species. Little is known about the mode of bvg repression at the molecular level. Akerley et al. (4) demonstrated that motility in B. bronchiseptica RB50 is bvg repressed, which is phenotypically identical to regulation of alcaligin production in this strain. Although motility and alcaligin production were phenotypically regulated similarly in strain RB50, we found that frl was not involved in regulation of alcaligin biosynthesis (Table 3). Nonetheless, we cannot rule out the possibility that regulation of alcaligin is mediated through an activator similar to FrlAB. Interestingly, the ferri-alcaligin uptake system was expressed at some level when Fe stressed, even in the absence of modulating factors. When Fe stressed, RB50 was able to use purified alcaligin as an iron source in the absence of detectable alcaligin production (Fig. 1). This is unlike the regulation of most other siderophore uptake systems that are coordinately regulated with siderophore production. We surveyed 114 strains of B. bronchiseptica, previously defined by MLEE analysis (30, 31), for alcaligin production under various conditions. Among these strains, we found two distinct groups that differed in whether alcaligin synthesis was bvg repressed. These data suggested a relationship between phylogenetic lineage, mammalian host species, and mode of regulation of alcaligin production. Musser and coworkers earlier demonstrated a relationship between B. bronchiseptica phylogenetic lineage and the mammalian hosts from which these organisms were isolated (30, 31). For example, ET1 strains, which we showed to be predominantly bvg independent for alcaligin production, preferentially infect pigs as opposed to other hosts. Therefore, the events that occurred during the differentiation of the ET1 phylogenetic lineage may have included a mutation that rendered alcaligin synthesis independent of bvg. Akerley et al. have recently suggested that bvg repression is important for suppressing the expression of determinants that would otherwise interfere with colonization by B. bronchiseptica (2). This suggests that alcaligin production may be essential for B. bronchiseptica colonization of pigs but perhaps not critical in other hosts. Further, the combined population genetic data suggest that the Bordetella species capable of infecting mammals (B. bronchiseptica, B. pertussis, and B. parapertussis) are actually biovars of a single genetic species (23, 29 31). Thus, differences in expression of alcaligin production in B. bronchiseptica isolates from different phylogenetic lineages are similar to variation in the expression of other virulence determinants between these three species. For example, B. pertussis and B. parapertussis Host TABLE 6. bvg repression of alcaligin is associated with mammalian host species Predominant phenotype a % of total (no. of strains within the predominant phenotype/total no. tested) Pig Group 1 94 (28/30) Cat Group 2 90 (9/10) Dog Group 2 80 (20/25) Guinea pig Group 2 82 (9/11) Horse Group 2 86 (6/7) Koala Group 2 100 (3/3) Rabbit Group 2 59 (10/17) a Group 1, alcaligin production is bvg independent; group 2, alcaligin production is bvg repressed.

6062 GIARDINA ET AL. J. BACTERIOL. harbor the fla operon found in B. bronchiseptica strains, although the former organisms are not motile under any conditions (24). Beattie et al. have shown that the vrg-6 locus is essential for B. pertussis virulence in a murine model (7). Although vrg-6 is not expressed in B. bronchiseptica, the latter organism harbors a silent copy of this locus (7). Most strikingly, B. parapertussis and B. bronchiseptica have intact genes for the pertussis toxin (ptx) operon (5, 21, 26), although only B. pertussis secretes the toxin. In B. parapertussis and B. bronchiseptica, the ptx operon is functionally silent due to promoter mutations (26). Also, single base substitutions found in the silent B. parapertussis locus were found in the silent B. bronchiseptica locus (26), suggesting that these changes took place in a common progenitor. B. bronchiseptica retains the complex secretion machinery necessary to transport the multicomponent toxin into the extracellular space (13, 26). Collectively, these data are consistent with the idea that the phylogenetic lineages of mammalian Bordetella species have only recently diverged from one another. As B. pertussis, B. parapertussis, and ET1 strains of B. bronchiseptica are highly host specific, the population genetic data further suggest that subtle allelic variation among the phylogenetic lineages of mammalian Bordetella species probably contribute significantly to host specificity. Our data suggest that release of bvg repression of alcaligin production by ET1 B. bronchiseptica strains may have been a significant event in the development of the ability of these strains to specifically colonize swine. Conversely, bvg repression of alcaligin synthesis may be important for maintaining colonization of other hosts. Since alcaligin synthesis by human Bordetella strains (i.e., B. pertussis strains) is similarly unresponsive to bvg repression (17), alcaligin production may also be critical for the colonization of the human respiratory tract. ACKNOWLEDGMENTS This work was supported by grant 91372046731 from the U.S. Department of Agriculture (D.W.D.) and by grant A131548 from the National Institute of Allergy and Infectious Disease (J.E.M.). REFERENCES 1. Agiato, L. A., and D. W. Dyer. 1991. Siderophore production and membrane protein alterations by Bordetella pertussis in response to iron starvation. Infect. Immun. 60:117 123. 2. Akerley, B. J., P. A. Cotter, and J. F. Miller. 1995. Ectopic expression of the flagellar regulon alters development of the Bordetella-host interaction. Cell 80:611 620. 3. Akerley, B. J., and J. F. Miller. 1993. Flagellin gene transcription in Bordetella bronchiseptica is regulated by the BvgAS virulence control system. J. Bacteriol. 175:3468 3479. 4. Akerley, B. J., D. 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