Introduction

Brucella is a genus of gram-negative bacteria of the α-2 subdivision of the class Proteobacteria [1] that includes the causal agents of brucellosis, a zoonosis producing important economical loses and human suffering in many developing countries [2]. Currently, the genus contains twelve nominal species often showing host preference. Those spp. that were identified early (frequently referred to as the classical Brucella spp.) are Brucella abortus, preferentially infecting cattle, B. melitensis, usually infecting sheep and goats, B. suis, infecting pigs, hares, reindeer ad several wild rodents, B. canis, found in dogs, B. neotomae, isolated from desert wood rats, and Brucella ovis, a non-zoonotic species that is restricted to sheep and causes a condition known as B. ovis ovine epididymitis [3]. More recently, brucellae have been isolated from marine mammals, voles and other wild life vertebrates, and new species proposed [4,5,6,7,8].

Conventional identification of the classical Brucella spp. and biovars has traditionally relied on dye and phage sensitivity, H2S production, urease activity, requirement of 5–10% CO2 atmospheres (0.04% in normal air) for growth (CO2-dependence) and surface antigens [9]. Even though these methods are being rapidly replaced by molecular tests, antigenic structure and CO2-dependence remain of immediate practical interest as these properties affect the conditions for primary isolation and propagation in vitro and the implementation of diagnostic tests. Antigenically, the classical Brucella spp. are divided in two groups: the rough (R) species (B. canis and B. ovis), which carry R-type lipopolysaccharides (LPS), and the smooth (S) brucellae, which carry S-LPS [3, 10]. Concerning CO2-dependence, this is a trait of B. ovis and most strains of B. abortus biovars 1–4 [3, 10]. In both cases, CO2-independent variants may appear with low frequency [11] and, for obvious practical reasons, some of these variants have been used for B. ovis and B. abortus antigen production [3, 10]. Similarly, B. abortus vaccines have been developed on CO2-independent backgrounds [10].

The classical Brucella spp. are facultative intracellular parasites able to circumvent early proinflammatory responses and endowed with a type IV secretion system involved in the control of intracellular trafficking [12,13,14,15]. Moreover, it is postulated that these bacteria have progressively adapted their metabolism to the nutrients encountered within cells as an essential part of their intracellular strategy [16,17,18,19,20,21]. In this regard, despite being a notorious phenotype of practical importance, Brucella CO2-dependence has deserved no attention since the demonstration over 60 years ago that CO2-rich atmospheres are not required to reduce oxygen tension and that CO2 is used as a nutrient per se (reviewed in [11]). Indeed, CO2 assimilation requires carbonic anhydrases (CAs), a group of critically important ubiquitous enzymes distributed into six evolutionary distinct classes named α to η, with the β class present in bacteria [22]. In heterotrophs, CAs are involved in C acquisition via assimilatory and anaplerotic reactions linked to several biosynthetic processes [23], and CO2-dependence has been related to defects in CA function in several microorganisms [24,25,26,27,28,29,30]. However, to the best of our knowledge, the role of CAs in CO2-dependence has not been investigated in Brucella spp. where the information is limited to recent investigations in search for targets for new drugs [31,32,33,34]. These investigations have described that B. suis (thus CO2-independent) strain 1330 has two ORFs (BRA0788 and BR1829) that code for β CAs (henceforth Bs1330CAI and Bs1330CAII). Both CAs are predicted to contain all the amino acid residues involved in the catalytic site and, more important, their activity was verified upon purification and found to be better for Bs1330CAII [31,32,33]. The demonstration that these B. suis ORFs actually code for enzymes with the predicted activity, together with the availability of the genome sequences of both CO2-independent and -dependent Brucella spp. and biovars, open the way to investigate the mechanisms underlying CO2-dependence in Brucella. The aim of the work described here was twofold: to investigate the genetic background behind the Brucella CO2-independent and -dependent phenotypes and, for B. ovis, a species that shows constant CO2-dependence, to construct a CO2-independent strain suitable for vaccine and antigen production.

Materials and methods

Bacterial strains and growth conditions

The bacterial strains and plasmids used in this study are listed in Table 1 and Additional files 1 and 2. B. abortus and B. suis strains were grown in standard Peptone-Glucose (Tryptic soy broth, TSB) or in this medium supplemented with agar (TSA). B. ovis strains were grown in TSB supplemented with yeast extract (0.5%) and fetal bovine serum 5% (TYSB) or this medium supplemented with agar (TYSA). For the studies in mice, strains were growth in Blood Agar Base supplemented with fetal bovine serum 5% (BABS). In addition, two minimal media were used. The components for 1 L of the defined medium of Gerhardt (Glutamate–Lactate–Glycerol) [35] are: glycerol (30 g), lactic acid (5 g), glutamic acid (1.5 g), thiamine (0.2 mg), nicotinic acid (0.2 mg), pantothenic acid (0.04 mg), biotin (0.0001 mg), K2HPO4 (10 g), Na2S2O3∙5H2O (0.1 g), MgSO4 (10 mg), MnSO4 (0.1 mg), FeSO4 (0.1 mg) and NaCl (7.5 g). The pH was adjusted to 6.8–7. The second minimal medium was a modification of Plommet’s [17, 36] and 1 L of this medium is composed of thiamine (0.2 g), nicotinic acid (0.2 g), pantothenic acid (0.07 g), biotin (0.1 mg), K2HPO4 (2.3 g), KH2PO4 (3 g), Na2S2O3 (0.1 g), MgSO4 (0.01 g), MnSO4 (0.1 mg), FeSO4 (0.1 mg); NaCl (5 g), (NH4)2SO4 (0.5 g) and 1 g/L of glucose. Incubation was at 37 °C, with (5%) or without CO2. When needed, media were supplemented with 5% sucrose (Sigma), kanamycin (Km) at 50 μg/mL, nalidixic acid (Nal) at 25 μg/mL, polymyxin (Pmx) at 1.5 μg/mL, chloramphenicol (Cm) at 20 μg/mL, spectinomycin (Spc) at 100 μg/mL or ampicillin (Amp) at 100 μg/mL (all from Sigma). All strains were stored in skimmed milk or TYSB-DMSO at −80 °C.

Table 1 Characteristics of the Brucella strains used in CO 2 -dependence studies
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Sequence analyses

Genomic sequences of B. suis 1330, B. suis 513 (not annotated), B. abortus 544 (not annotated) and B. abortus 292 were obtained from the databases at National Center for Biotechnology Information (NCBI), Kyoto Encyclopedia of Genes and Genomes (KEGG) or The Broad Institute. The genomic sequence of B. abortus 2308W was obtained from the European Nucleotide Archive (ENA) and compared with its sibling 2308 sequence in KEGG. When genomic sequences were not available (B. ovis PA, B. ovis REO198, B. abortus AB0339, AB0339-CO mut2 , B. abortus AB0127, AB0127-CO mut2 , B. abortus AB0130, AB0130-CO mut2 and BoPA-CO mut2 ) ORFs were PCR amplified and then sequenced. DNA sequencing was carried out by “Servicio de Secuenciación de CIMA (Centro de Investigación Médica Aplicada, Pamplona, Spain)”. Sequence alignments were performed with Clustal Omega.

DNA manipulations

Plasmid and chromosomal DNA were extracted with QIAprep Spin Miniprep (Qiagen) and Ultraclean Microbial DNA Isolation kit (Mo Bio Laboratories), respectively. When needed, DNA was purified from agarose gels using QIAquick Gel Extraction Kit (Qiagen). Primers (Additional file 3) were synthesized by Sigma (Haverhill, United Kingdom). Restriction modification enzymes were used under the conditions recommended by the manufacturer.

Construction of B. abortus 2308W and B. suis mutants by gene disruption

For the construction of the CAI mutants, an internal region of 323 bp was amplified with oligonucleotides CAI-F1-ins (5′-GAATTTCTATGGATCGGCTGTT-3′) and CAI-R2-ins (5′-CGGTCCTGCGTGTTTTCTAT-3′). The resulting fragment containing an internal region of the ORF was cloned into pCR2.1-TOPO® vector (Invitrogen) to generate plasmid pCR2.1Ba2308WCAI (Additional file 2) and then, sequenced to verify the insertion. After sequencing, this fragment was cloned into the BamHI and XbaI sites of the suicide vector pJQKm [37]. The resulting plasmid pJQKmBa2308WCAI (Additional file 2) was transformed into competent E. coli S17 λpir [38, 39] and transferred into B. abortus 2308W, B. suis 1330 and B. suis 513 by conjugation, where a single crossover led to disruption of the wild type locus. Integrative mutants were selected on a medium containing kanamycin and nalidixic acid or polymyxin and called B. abortus 2308W::pJQKm-CAI, B. suis 513::pJQKm-CAI and B. suis 1330::pJQKm-CAI (Additional file 1). Since the orientation of the insert in the pJQKm vector was known after sequencing, gene disruption was confirmed by detecting PCR products with primers CAI-Fw and M13Fw and primers CAI-Rv and M13Rv.

The CAII mutants were constructed in a similar way. A 302 bp internal fragment of CAII was amplified with oligonucleotides CAII-F1-Ins (5′-CAATGTGGCCAATCTCATTC-3′) and CAII-R2-ins (5′-GCGAATAGCGGATCGAAATA-3′). The resulting fragment was cloned into pCR2.1-TOPO® vector (Invitrogen) to generate plasmid pCR2.1Ba2308W CAII (Additional file 2), sequenced to verify the insertion and subsequently cloned into the BamHI and XbaI sites of the suicide vector pJQKm [37]. The mutants were named B. suis 513::pJQKm-CAII and B. suis 1330::pJQKm-CAII (Additional file 1). Since the orientation of the insert in the pJQKm vector was known after sequencing, the site of the insertion was confirmed by independent PCR rounds with primers CAII-Fw and M13Fw, and primers CAII-Rv and M13Rv. After several attempts, no mutant in B. abortus 2308W CAII was obtained either under normal or CO2-enriched conditions.

Selection of CO2-independent spontaneous mutants

To obtain CO2-independent spontaneous mutants from CO2-dependent bacteria, B. ovis PA and three B. abortus isolates (one biovar 1 and two biovar 3) were plated on TYSA or TSA and incubated at 37 °C without CO2. After 5 days, one colony was picked and the genes encoding CAI and CAII were PCR amplified using primers CAI-Fw and CAI-Rv, and CAII-Fw and CAII-Rv (see Additional file 3). DNA sequencing with these primers and CAI-F1-Sec and CAII-F1-Sec primers (Additional file 3) allowed identification of mutations by comparison with the nucleotide sequence of the parental CO2-dependent strains.

Construction of the plasmid carrying CAIIba2308W and introduction into B. abortus 292 and 544

For the construction of the expression plasmid encoding Ba2308WCAII oligonucleotides CAII-Fw-Gw (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCGCTGCCGTGTTTGAAATCA-3′) and CAII-Rv-Gw (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAAAGTTCAGGGCGTTTGAA-3′) that contain sequences attB (underlined) were used to amplify CAII and the promoter from B. abortus 2308W. The resulting PCR product was cloned into pDONR223 to generate plasmid pDONOR223Ba2308WCAII (Additional file 2). After sequence verification, the ORF encoding CAII was transferred from pDONOR223Ba2308WCAII to pRH001 [40]. The resulting plasmid, pRH001Ba2308WCAII (Additional file 2) was transformed into competent E. coli S17 λpir and introduced into Brucella strains by conjugation. The clones that had acquired the plasmid were selected by kanamycin resistance and confirmed by PCR using primers CAII-Fw-Gw and CAII-Rv-Gw, and M13F-M13R. The strains were called B. abortus 292 pRH001Ba2308WCAII and B. abortus 544 pRH001Ba2308WCAII (Additional file 1).

Construction of miniTn7T-KmR plasmids carrying CAI or CAII and introduction into Brucella strains

Using DNA from B. abortus 2308W, oligonucleotides CAII-IF-F1 (5′-CCGGGCTGCAGGAATTCGCTGCCGTGTTTGAAATCA-3′) and CAII-IF-R2 (5′-AGCTTCTCGAGGAATTTCAAAGTTCAGGGCGTTTGAA-3′) amplified a 966 bp region containing CAII and the promoter region. This fragment was cloned into the linearized vector (EcoRI) pUC18R6KT-miniTn7T-KmR [41] using the In-Fusion HD Enzyme Premix (Clontech). The resulting plasmid was called pUC18R6KT-miniTn7T-KmRBa2308WCAII (Additional file 2) and transformed into E. coli PIR1 and subsequently to E. coli S17 λpir. Then, it was transferred into Brucella by a tetraparental conjugation [42]. The resulting constructs (B. abortus 292 Tn7Ba2308WCAII, B. abortus 544 Tn7Ba2308WCAII and B. ovis PA Tn7Ba2308WCAII; Additional file 1) were confirmed by PCR for the correct insertion and orientation of the mini-Tn7 between genes glmS and recG. Primers GlmS_B (5′-GTCCTTATGGGAACGGACGT-3′) and Ptn7-R (5′-CACAGCATAACTGGACTGATT-3′) were used to confirm insertion downstream glmS; Ptn7-L (5′-ATTAGCTTACGACGCTACACCC-3′) and RecG (5′-TATATTCTGGCGAGCGATCC-3′) insertion upstream recG and GlmS_B and RecG presence of transposon.

Oligonucleotides CAI-IF-F1 (5′-CCGGGCTGCAGGAATTTGTGGAATTGCACCGACAC-3′) and CAI-IF-R2 (5′-AGCTTCTCGAGGAATTCAATTATTCTGCCGGTTGG-3′) amplified a 987 bp fragment from B. suis 513 DNA containing CAI and the promoter. This fragment was subsequently cloned into the linearized vector (EcoRI) pUC18R6KT-miniTn7T-KmR using the In-Fusion HD Enzyme Premix (Clontech). The resulting plasmid was called pUC18R6KT-miniTn7T-KmRBs513CAI (Additional file 2) and was transformed into E. coli PIR1 and then to E. coli S17 λpir. After, the plasmid was introduced into the different Brucella strains by a tetraparental conjugation [42]. The strains were called B. abortus 2308W Tn7Bs513CAI, B. abortus 292 Tn7Bs513CAI and B. abortus 544 Tn7 Bs513CAI. The insertion of the transposon was confirmed by PCR (see above and Additional file 3).

When necessary, constructs without kanamycin resistance cassette were obtained following the protocol set up by Martínez-Gómez et al. [43].

Growth measurements

The strains were inoculated into 10 mL of TSB or TYSB in a 50 mL flask and incubated at 37 °C for 18 h with or without orbital shaking, in an atmosphere with 5% CO2 in the case of CO2-dependent strains. Then, these bacteria were harvested by centrifugation, resuspended in 10 mL of the test medium at an optical density at 600 nm (OD600nm) of 0.1, and incubated under the same conditions for 18 h. These exponentially growing bacteria were harvested by centrifugation, resuspended at an OD600nm of 0.1 (equivalent to 0.05 readings in the Bioscreen apparatus) in the test medium in appropriate multiwell plates (200 μL/well) and cultivated in a Bioscreen C (Lab Systems) apparatus with continuous shaking at 37 °C. Absorbance values at 420–580 nm were automatically recorded at 30 min-intervals. All experiments were performed in triplicate. Controls with medium and no bacteria were included in all experiments.

Studies in mice

Seven-week-old female BALB/c mice (Harlan Laboratories; Bicester, United Kingdom) were accommodated in the facilities of “Centro de Investigación y Tecnología Agroalimentaria de Aragón” (CITA; Registration code ES502970012025) for 2 weeks before and during the experiments, with water and food ad libitum under P3 biosafety containment conditions. The animal handling and other procedures were in accordance with the current European (directive 86/609/EEC) and Spanish (RD 53/2013) legislations, supervised by the Animal Welfare Committee of the CITA (2014-20).

To prepare inocula, BABS-grown bacteria were harvested, adjusted spectrophotometrically (OD600nm = 0.170) in sterile buffered saline (BSS; 0.85% NaCl, 0.1% KH2PO4, 0.2% K2HPO4; pH 6.85) and diluted in the same diluent up to approximately 5 × 107 CFU/mL. For each bacterial strain, five mice were intraperitoneally inoculated with 0.1 mL/mouse, the exact doses assessed retrospectively by plating dilutions of the inocula. The number of CFU in spleen was determined at 3 and 8 weeks post-inoculation. For this, the spleens were aseptically removed and individually weighed and homogenized in 9 volumes of BSS. Serial tenfold dilutions of each homogenate were performed, and each dilution was plated by triplicate. Plates were incubated at 37 °C, without CO2, for 5 days. The identity of the spleen isolates was confirmed by PCR. The individual number of CFU/spleen was normalized by logarithmic transformation, and the mean log CFU/spleen values and the standard deviations (n = 5) were calculated. Statistical comparisons were performed by Student’s t-test.

Results

ORF sequences suggest a critical role of CAII in CO2-independence

We first analyzed whether the sequences of Bs1330CAI and Bs1330CAII, respectively encoded by BRA0788 and BR1829 of B. suis 1330 and with proved CA activity, had orthologues in reference and collection strains representative of the CO2-independent and -dependent Brucella phenotypes (Table 1). This analysis showed that all these brucellae carry Bs1330CAI and Bs1330CAII orthologues, with the peculiarities summarized below (for further details, see Additional files 4 and 5).

B. suis 513 Bs1330CAI orthologue differed from Bs1330CAI only at position 40 (serine instead of leucine) and carried a CAII identical to Bs1330CAII. The B. abortus 2308W Bs1330CAI orthologue differed from Bs1330CAI in that the serine at position 40 and valine in position 76 were both substituted by glycine. Similarly, the Bs1330CAII orthologue had an extra amino acid (alanine) at position 114. B. abortus 292 and 544, both CO2-dependent, contained a Bs1330CAI orthologue with the same serine and valine substitutions as strain 2308W, and a cytosine insertion at position 338 of the Bs1330CAII orthologue leading to a frameshift affecting almost 50% of the protein. In the B. ovis PA Bs1330CAI orthologue, a deletion of 24 nucleotides at positions 217–240 results in a protein lacking amino acids 74–81, and the insertion of a guanine at the Bs1330CAII orthologue originates a frameshift and a protein defective in the last 40 amino acids. B. ovis REO198, which is CO2-independent, is identical to B. ovis PA with respect to the Bs1330CAI orthologue. However, the lack of a guanine in the Bs1330CAII orthologue three positions after the 521 position guanine of its B. ovis PA counterpart restores the reading frame and should allow synthesis of a protein identical to that of B. suis 1330 (Additional files 4 and 5). Altogether, the observations strongly suggest that the CO2-dependence of B. ovis PA, B. abortus 292 and 544 is caused by the lack of an active CAII (activity defined empirically as that allowing growth in a normal atmosphere) and, conversely, that mutations in B. ovis CAII could account for the spontaneous emergence of CO2-independent strains in at least B. ovis. On the other hand, these analyses did not allow inferring the relevance of CAI, which was apparently complete in B. abortus and B. suis.

CAII is mutated in spontaneous CO2-independent mutants

We examined first the validity of the hypothesis on the relevance of CAII for growth in normal air by comparing the putative CA genes of several spontaneous CO2-independent mutants that appeared during routine laboratory manipulations with their parental counterparts (Table 1). For the B. ovis PA CO2-independent mutant (BoPA-CO mut2 ), we observed that while the guanine in the CAII gene causing the above-described frameshift was absent, the CAI gene had not undergone any changes. The CO2-independent mutants of three recent B. abortus isolates (one biovar 1 and two biovar 3) [44] lacked a guanine at position 340 of the CAII gene that was however present in the CO2-dependent parental isolates. Altogether, these results support the starting hypothesis that CAII mutations are involved in the emergence of CO2-independent mutants and indirectly suggest that CAI is less relevant in the uptake of CO2.

An active CAI is enough by itself to support CO2-independent growth of B. suis but not of B. abortus

To compare the physiological importance of CAI and CAII we first carried out mutagenesis in different backgrounds and tested the mutants for CO2-independence. Using B. abortus 2308W, we found that its CAI mutant (B. abortus 2308W::pJQKm-CAI) kept the CO2-independent phenotype, proving that CAII by itself can sustain growth under normal atmospheric conditions (Figure 1). In contrast, and despite repeated attempts, we failed to obtain a similar mutant in CAII, suggesting that the B. abortus 2308W CAI cannot supply bicarbonate at a rate high enough for growth under normal atmospheric conditions. This is in keeping with the identity of CAI sequence between B. abortus 2308W on one hand and B. abortus 292 and 544 on the other, and supports the idea that CAI is not active in these three B. abortus strains.

Figure 1
figure 1

CAI is dispensable for CO2-independent growth of B. abortus 2308W. Growth of B. abortus 2308W and the corresponding insertion mutant in CAI were tested under normal atmospheric conditions in Peptone-Glucose. Each point represents the mean ± standard error (error bars are within the size of the symbols) of technical triplicates. The experiment was repeated three times with similar results.

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Köhler et al. [34] reported recently the failure to obtain B. suis 1330 (biovar 1) double CAI–CAII mutants, which together with the analysis of the purified CAI [31,32,33] strongly suggests that CAI is active in this strain. Thus, we hypothesized that CAI could be inactive in some Brucella strains but active in others. Indeed, this possibility was consistent with the observation that the valine in position 76 in B. suis 1330 Bs1330CAI was substituted by glycine in B. abortus 292 and 544 (both CO2-dependent) as well as in strain 2308W (see above and Additional file 4) where CAII seemed essential for CO2-independence. To validate our hypothesis, we first constructed pJQKm insertion mutants in the Bs1330CAI and Bs1330CAII genes of B. suis 1330 (biovar 1) and found that both were CO2-independent (Figure 2). Then, we obtained similar pJQKm insertion mutants in B. suis 513 (biovar 5). Again, both mutants kept the CO2-independent phenotype of the parental strain, proving that CAI was also active in B. suis 513 despite the difference in position 40 (serine instead of leucine) with respect to Bs1330CAI (see above and Additional files 4 and 5).

Figure 2
figure 2

CAI and CAII are functional in B. suis 1330 and 513. Growth of B. suis 1330 and B. suis 513 and the corresponding insertion mutants in CAI (B. suis 1330::pJQKm-CAI; B. suis 513::pJQKm-CAI) and CAII (B. suis 1330::pJQKm-CAII; B. suis 513::pJQKm-CAII) were tested under normal atmospheric conditions in Peptone-Glucose. Each point represents the mean ± standard error (error bars are within the size of the symbols) of technical triplicates. The experiment was repeated three times with similar results.

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Once we knew that CAII was essential for CO2-independent growth of B. abortus 2308 W and that CAI was active in B. suis 1330 and 513, we tested whether a functional CAII or CAI could accomplish the same role in CO2-dependent B. abortus strains. For this, we first constructed a low-copy plasmid (pRH001Ba2308WCAII) carrying the gene encoding B. abortus 2308W CAII (which we had proven to be active) under the control of its own promoter. When we introduced this plasmid into B. abortus 292 and 544 (both CO2-dependent), the pRH001Ba2308WCAII constructs were able to grow in a normal atmosphere (Figure 3). Then, to circumvent any gene dosage artifacts associated with plasmid constructs, we introduced a miniTn7 carrying Ba2308WCAII (Tn7Ba2308WCAII) [42] into a neutral site of the genomes of B. abortus 292 and 544. We found that, like the strain origin of the CAII gene, the two constructs grew in a normal atmosphere (Figure 3). Then, we did similar experiments with a miniTn7 carrying Bs513CAI (which we had proven to be active) and its promoter (Tn7Bs513CAI). In this case, however, we found that the B. abortus 292 and 544 Tn7Bs513CAI constructs failed to grow without CO2 enrichment (data not shown) leading to the conclusion that an active CAI was not enough by itself to support CO2-independent growth of B. abortus.

Figure 3
figure 3

B. abortus 292 and 544 carrying a functional CAII become CO2-independent. Growth of B. abortus strains 292 and 544 and the derivative strains carrying plasmid pRH001Ba2308WCAII or a stable Tn7Ba2308WCAII insertion in the genome were tested under normal atmospheric conditions in Peptone-Glucose. Each point represents the mean ± standard error (error bars are within the size of the symbols) of technical triplicates. The experiment was repeated three times with similar results.

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The CO2-independence mediated by CAI is conditioned by nutrient availability

While the above-described experiments show that an active CAII but not an active CAI was enough to bypass CO2-dependence in B. abortus, it was not immediately obvious why CAI by itself was enough to support growth of B. suis 1330 and B. suis 513. However, the B. suis CAII (and CAI) mutants were tested for CO2-independence in a medium rich in peptones and glucose, conditions that are likely to downplay the role of the anabolic pathways where CA activity is important. Therefore, we reasoned that, depending upon the metabolic abilities of Brucella spp. and biovars, experiments in complex media could be not stringent enough to reveal differences between CAI and CAII activities. To analyze this, we took advantage of the almost prototrophic characteristics of B. suis 513, a strain that only requires a few vitamins and grows efficiently with limited C supplies [45]. When we tested B. suis 513::pJQKm-CAII insertion mutant on Glutamate–Lactate–Glycerol (a gluconeogenic medium [21, 45]) or Glucose as the only C sources, we found that the mutant failed to grow under a normal atmosphere (Figure 4). This result, which shows that B. suis 513 CAI cannot meet the biosynthetic demands of this strain in simple media, strongly suggest that CAI is not active enough in less prototrophic species such as B. abortus even in complex media and, therefore, that it adds little to the role of CAII. In keeping with this, we found that a B. abortus 2308 W construct carrying Bs513CAI and its parental strain did not differ in growth rates (Figure 5).

Figure 4
figure 4

The CO2-independence mediated by CAI is conditioned by nutrient availability. Growth of B. suis strain 513 and the CA insertion mutants (B. suis 513::pJQKm-CAI; B. suis 513::pJQKm-CAII) were tested under normal atmospheric conditions in Glutamate–Lactate–Glycerol and Glucose. Each point represents the mean ± standard error (error bars are within the size of the symbols) of technical triplicates. The experiment was repeated three times with similar results.

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Figure 5
figure 5

Insertion of the gene of a functional CAI in B. abortus 2308W does not increase growth rates. Growth of B. abortus 2308W and its derivative strain carrying B. suis 513 CAI in the genome (B. abortus 2308W Tn7Bs513CAI) were tested under normal atmospheric conditions in Peptone-Glucose. Each point represents the mean ± standard error (error bars are within the size of the symbols) of technical triplicates. The experiment was repeated three times with similar results.

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An active CAII reverts the CO2-dependence of B. ovis PA and does not alter its multiplication in the mouse model

Among the brucellae, B. ovis is notorious for its CO2-dependence and fastidious nutritional requirements caused in all likelihood by its comparatively genome degradation [46]. Since the above-described experiments not only proved the chief role of CAII in the B. abortus and B. suis biovars tested but also provided molecular tools for relieving CO2-dependence in B. abortus, we introduced Tn7Ba2308WCAII into the B. ovis PA chromosome and examined the construct for CO2-dependence. As can be seen in Figure 6A, B. ovis PA Tn7Ba2308WCAII grew under normal atmospheric conditions. Then, we used the Tn7Ba2308WCAII construct to test whether the CO2-dependence and/or this genetic manipulation would alter the virulence of B. ovis in the standard mouse model. We found that the introduction of a functional CAII into B. ovis PA did not affect the multiplication (acute phase) and permanence (chronicity) of the bacteria in the spleens of BALB/c mouse (Figure 6B).

Figure 6
figure 6

An active CAII reverts the CO2-dependence of B. ovis PA and does not alter its multiplication in the mouse model. A Growth of B. ovis PA and its derivative strain carrying B. abortus 2308W CAII in the genome (B. ovis PA Tn7Ba2308WCAII) under normal atmospheric conditions in Peptone-Glucose-Yeast Extract-Serum. Each point represents the mean ± standard error (error bars are within the size of the symbols) of technical triplicates. The experiment was repeated three times with similar results. B Bacterial loads of B. ovis PA and B. ovis PA Tn7Ba2308WCAII in the spleens of BALB/c mice at 3 and 8 weeks post-infection. No statistical differences were found (Student’s t-test).

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Discussion

The metalloenzymes generically designated as CAs catalyze the reversible hydration of CO2 into bicarbonate, the substrate of key anaplerotic and biosynthetic enzymes [23]. Many aerobic microorganisms can obtain enough bicarbonate from ambient air (about 0.04% CO2) and not surprisingly CA mutants of at least Ralstonia eutropha, Streptococcus pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Corynebacterium glutamicum are unable to grow under these conditions [25,26,27,28,29,30]. All these observations made very likely the existence of a relationship between CA deficiencies and the CO2-dependence that is characteristic of some Brucella biovars and species, a hypothesis proved in this work. If we define CA inactivity as that not high enough to make the bacteria able to grow in a normal atmosphere, we have demonstrated that while B. suis 1330 and 513 genomes encode two active CAs (CAI and CAII) only CAII is active in B. abortus 2308W and none in representative strains of the CO2-dependent B. abortus biovars or in B. ovis PA. Indeed, the fact that the gene encoding CAII is conserved in B. abortus 2308W is in agreement with previous in vitro enzymatic analysis that show that B. suis 1330 CAII is a better catalyst for the conversion of CO2 to bicarbonate (with an enzymatic activity 1.85 times higher) than Bs1330CAI [32].

It has to be stressed that the functional definition of activity used here pertains to the particular physiology of each strain. Although the number of strains tested was necessarily limited, we found evidence supporting the hypothesis that CAI is insufficient to support growth of Brucella when the medium is limited to simple C substrates, or even in rich media for those brucellae that display comparatively reduced biosynthetic abilities. Indeed, whereas growth on the minimal media used here requires bicarbonate being incorporated by the reactions catalyzed by enzymes such as phosphoenolpyruvate carboxylase, pyruvate carboxylase, carbamoyl phosphate synthetase, 5-aminoimidazole ribotide carboxylase and enoyl-CoA carboxylases/reductases, growth on rich media does not entail an intense biosynthesis of amino acids and nucleic acid precursors and, therefore, most if not all of the linked pathways should pose no stringent demands for bicarbonate. In the context of this hypothesis, B. suis, the fast-growing B. suis biovar 5 (strain 513) in particular, on one hand, and B. ovis, on the other, would respectively represent two opposite situations. The existence of Brucella strains carrying inactivated CAs strongly suggests that this enzymatic activity is not necessary for the persistency in nature of at least B. abortus and B. ovis. Indeed, the presence of mutations inactivating the metabolic genes may result from the absence of a positive selective pressure, reflecting an adaptation during which the cognate functions become dispensable because of the nutritional environment. Such a Brucella adaptation would not be a novelty in intracellular parasites because, while a majority of the genome-sequenced Proteobacteria retain a CA gene, intracellular genera such as Buchnera and Rickettsia contain CA-defective representatives [47]. It remains to be investigated whether such a CA dispensability represents a high CO2 tension in their niche, as described for Symbiobacterium thermophilum [48], the exploitation of host CAs or the presence of nutrients bypassing metabolic steps connected to CA activity.

It is important to highlight the practical implications of this work. Unraveling the genetic background of Brucella CO2-dependence allowed us to construct a B. ovis CO2-independent mutant with practical implications on the diagnosis and control of B. ovis infection. Because of the non-zoonotic nature of B. ovis, this disease may not always deserve the attention of official programs and it is often overlooked. Control and eventual eradication of B. melitensis brucellosis of small ruminants is based on the use of diagnostic tests detecting antibodies to the S-LPS [49] and vaccination with B. melitensis Rev 1, a vaccine that also protects against B. ovis. However, Rev 1 may interfere in serological diagnosis [49] and it is virulent for humans [50] and resistant to streptomycin (an antibiotic of choice to treat human brucellosis). Owing to these drawbacks, Rev 1 vaccination is discontinued and finally banned in those regions or countries where B. melitensis prevalence is considered low enough to implement an exclusively test and slaughter strategy. Withdrawal of Rev 1 vaccination leaves animals unprotected against B. ovis, thus favoring the emergence of the disease in areas where B. melitensis is almost or totally eradicated. Moreover, B. ovis has remained endemic in many areas where B. melitensis is not present and Rev 1 vaccination was never implemented [51, 52]. Accordingly, research on B. ovis-specific vaccines is an area of increasing interest as these vaccines would neither pose risk of zoonotic infection nor interfere in those B. melitensis serological tests detecting S-LPS O-polysaccharide antibodies (i.e., rose bengal and complement fixation tests) [53,54,55,56]. The CO2 requirement represents a significant obstacle in the development of a B. ovis live attenuated vaccine for large-scale production. We have demonstrated that the B. ovis PA Tn7Ba2308WCAII described here not only grows under normal atmospheric conditions but also retains the virulence in at least the accepted laboratory model, thus representing an appropriate tool for the development of such CO2-independent attenuated vaccines. For instance, B. ovis PA Tn7Ba2308WCAII can be used as the background to apply the strategy proposed by Conde-Álvarez et al. [57] based on the deletion of LPS core glycosyltransferases that results in a truncated structure that by uncovering innate immunity targets triggers a potent protective Th1 response. In fact, Soler-Lloréns et al. [56] recently demonstrated that deletion of two of such glycosyltransferases in B. ovis PA results in attenuation and suitable vaccine properties in the mouse model. Similarly, the B. ovis PA Tn7Ba2308WCAII construct could be used to produce the R-specific antigen currently used in B. ovis serological tests. This antigen is made of vesicles rich in outer membrane proteins and R-LPS, and both types of components have been shown to be important for optimal sensitivity [58, 59]. Currently, this R antigen is obtained from B. ovis REO198 taking advantage of the unusual CO2-independence of this strain (Table 1). Yet, B. ovis REO198 LPS carries a core oligosaccharide defect that damages the diagnostic epitopes of the R LPS and it is thus likely to yield suboptimal results in serodiagnosis [60]. If this is confirmed, it could be advantageously replaced by B. ovis PA Tn7Ba2308WCAII. Research is in progress to evaluate the attenuation and protection against B. ovis of B. ovis PA Tn7Ba2308WCAII core glycosyltransferase mutants as well as the diagnostic properties of R antigens obtained from this strain. In summary, the evidence presented in this work not only clarifies the biochemical basis of an important Brucella phenotype but also provides a tool for large-scale production of B. ovis diagnostic antigens and vaccines.