Comparative genome analysis of rice-pathogenic Burkholderia provides insight into capacity to adapt to different environments and hosts

Background

In addition to human and animal diseases, bacteria of the genus Burkholderia can cause plant diseases. The representative species of rice-pathogenic Burkholderia are Burkholderia glumae, B. gladioli, and B. plantarii, which primarily cause grain rot, sheath rot, and seedling blight, respectively, resulting in severe reductions in rice production. Though Burkholderia rice pathogens cause problems in rice-growing countries, comprehensive studies of these rice-pathogenic species aiming to control Burkholderia-mediated diseases are only in the early stages.

Results

We first sequenced the complete genome of B. plantarii ATCC 43733^T. Second, we conducted comparative analysis of the newly sequenced B. plantarii ATCC 43733^T genome with eleven complete or draft genomes of B. glumae and B. gladioli strains. Furthermore, we compared the genome of three rice Burkholderia pathogens with those of other Burkholderia species such as those found in environmental habitats and those known as animal/human pathogens. These B. glumae, B. gladioli, and B. plantarii strains have unique genes involved in toxoflavin or tropolone toxin production and the clustered regularly interspaced short palindromic repeats (CRISPR)-mediated bacterial immune system. Although the genome of B. plantarii ATCC 43733^T has many common features with those of B. glumae and B. gladioli, this B. plantarii strain has several unique features, including quorum sensing and CRISPR/CRISPR-associated protein (Cas) systems.

Conclusions

The complete genome sequence of B. plantarii ATCC 43733^T and publicly available genomes of B. glumae BGR1 and B. gladioli BSR3 enabled comprehensive comparative genome analyses among three rice-pathogenic Burkholderia species responsible for tissue rotting and seedling blight. Our results suggest that B. glumae has evolved rapidly, or has undergone rapid genome rearrangements or deletions, in response to the hosts. It also, clarifies the unique features of rice pathogenic Burkholderia species relative to other animal and human Burkholderia species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1558-5) contains supplementary material, which is available to authorized users.     

Use of Bifidobacterium dentium as an Indicator of the Origin of Fecal Water Pollution

A new, simple, and specific protocol to discriminate between human and animal fecal pollution is described. The procedure is based on the detection of certain Bifidobacterium species in the samples. Two 16S rRNA gene-targeted probes are described. One of these probes (BDE) has as its target a region of the 16S rRNA gene of Bifidobacterium dentium, a Bifidobacterium species of exclusively human origin. The other probe (BAN) is based on the sequence of a region of 16S rRNA gene for several Bifidobacterium species related with animal origins. The specificity of both probes was evaluated by using 24 Bifidobacterium species, and their threshold detection limit was established by DNA-DNA hybridization. DNA-DNA hybridization with the BDE probe showed it to be specific for B. dentium, whereas that with the BAN probe showed it to be specific for B. animalis, B. asteroides, B. coryneforme, B. cuniculi, B. globosum, B. magnum, B. minimum, and B. subtile. A simple and specific protocol was also developed for the detection of their target species in environmental samples (sewage and feces). DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats. Thus, this probe is not suitable for the identification of any animal fecal pollution. Whereas all samples with human fecal pollution showed a positive DNA-DNA hybridization result with the BDE probe, none of those with animal fecal pollution did. Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin.     

Polyphasic characterisation of Burkholderia cepacia complex species isolated from children with cystic fibrosis

Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recAsequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recAsequencing to identify Bcc species. The recAsequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%),Burkholderia vietnamiensis (30.6%), B. cenocepaciaIIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes.     

Burkholderia denitrificans sp. nov., isolated from the soil of Dokdo Island, Korea

A novel, Gram-negative, bacterial strain KIS30-44^T was identified from wet forest soil collected on the Korean island of Dokdo. Growth of the strain was observed at 15?C30°C, pH 5?C9, 0?C3% NaCl, and 950 mM KNO₃. KIS30-44^T reduced nitrate to nitrogen gas. Analysis of the 16S rRNA gene sequence showed that KIS30-44^T was phylogenetically related to Burkholderia sacchari, Burkholderia mimosarum, and Burkholderia oxyphila (98.1%, 98.0%, and 98.0% sequence similarity, respectively). The genomic G+C content was 63.5 mol%. KIS30-44^T exhibited less than 52% DNA-DNA relatedness with the type strains of 9 closely related Burkholderia species. The major isoprenoid quinone was Q-8. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unknown aminolipids. The major fatty acids in KIS30-44^T were C_16:0, C_18:1 ??7c and summed feature 3 (iso-C_15:0 2-OH and C_16:1 ??7c), and the strain contained half the amount of C_17:0 cyclo found in the 9 closely related Burkholderia species. The results of these phenotypic, 16S rRNA gene sequence, DNA-DNA hybridization, and chemotaxonomic data indicate that KIS30-44^T represents a novel species within the genus Burkholderia, for which the name Burkholderia denitrificans (Type strain KIS30-44^T =KACC 12733^T =DSM 24336^T) is proposed.     

Comparative Metabolic Systems Analysis of Pathogenic Burkholderia

Burkholderia cenocepacia and Burkholderia multivorans are opportunistic drug-resistant pathogens that account for the majority of Burkholderia cepacia complex infections in cystic fibrosis patients and also infect other immunocompromised individuals. While they share similar genetic compositions, B. cenocepacia and B. multivorans exhibit important differences in pathogenesis. We have developed reconciled genome-scale metabolic network reconstructions of B. cenocepacia J2315 and B. multivorans ATCC 17616 in parallel (designated iPY1537 and iJB1411, respectively) to compare metabolic abilities and contextualize genetic differences between species. The reconstructions capture the metabolic functions of the two species and give insight into similarities and differences in their virulence and growth capabilities. The two reconstructions have 1,437 reactions in common, and iPY1537 and iJB1411 have 67 and 36 metabolic reactions unique to each, respectively. After curating the extensive reservoir of metabolic genes in Burkholderia, we identified 6 genes essential to growth that are unique to iPY1513 and 13 genes uniquely essential to iJB1411. The reconstructions were refined and validated by comparing in silico growth predictions to in vitro growth capabilities of B. cenocepacia J2315, B. cenocepacia K56-2, and B. multivorans ATCC 17616 on 104 carbon sources. Overall, we identified functional pathways that indicate B. cenocepacia can produce a wider array of virulence factors compared to B. multivorans, which supports the clinical observation that B. cenocepacia is more virulent than B. multivorans. The reconciled reconstructions provide a framework for generating and testing hypotheses on the metabolic and virulence capabilities of these two related emerging pathogens.     

Diversity of Cultivated Endophytic Bacteria from Sugarcane: Genetic and Biochemical Characterization of Burkholderia cepacia Complex Isolates

Bacteria were isolated from the rhizosphere and from inside the roots and stems of sugarcane plants grown in the field in Brazil. Endophytic bacteria were found in both the roots and the stems of sugarcane plants, with a significantly higher density in the roots. Many of the cultivated endophytic bacteria were shown to produce the plant growth hormone indoleacetic acid, and this trait was more frequently found among bacteria from the stem. 16S rRNA gene sequence analysis revealed that the selected isolates of the endophytic bacterial community of sugarcane belong to the genera of Burkholderia, Pantoea, Pseudomonas, and Microbacterium. Bacterial isolates belonging to the genus Burkholderia were the most predominant among the endophytic bacteria. Many of the Burkholderia isolates produced the antifungal metabolite pyrrolnitrin, and all were able to grow at 37°C. Phylogenetic analyses of the 16S rRNA gene and recA gene sequences indicated that the endophytic Burkholderia isolates from sugarcane are closely related to clinical isolates of the Burkholderia cepacia complex and clustered with B. cenocepacia (gv. III) isolates from cystic fibrosis patients. These results suggest that isolates of the B. cepacia complex are an integral part of the endophytic bacterial community of sugarcane in Brazil and reinforce the hypothesis that plant-associated environments may act as a niche for putative opportunistic human pathogenic bacteria.     

Diversities in Virulence,Antifungal Activity,Pigmentation and DNA Fingerprint among Strains of Burkholderia glumae

Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG) agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR) for BOX-A1R-based repetitive extragenic palindromic (BOX) or enterobacterial repetitive intergenic consensus (ERIC) sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR.     

Identification of aquatic Burkholderia (Pseudomonas) cepacia by hybridization with species-specific rRNA gene probes.

Burkholderia (Pseudomonas) cepacia is a common environmental bacterium which can be pathogenic for plants and humans. In this study, four strategies were used to identify aquatic isolates: API test strips, hybridization with species-specific DNA probes for the 16S and 23S rRNA genes, fatty acid methyl ester (FAME) profiles, and growth on selective medium (TB-T agar [C. Hagedorn, W. D. Gould, T. R. Bardinelli, and D. R. Gustarson, Appl. Environ. Microbiol. 53:2265-2268, 1987]). Only 59% of the isolates identified as B. cepacia with the API test strips were confirmed as B. cepacia by using fatty acid profiles. The 23S rRNA probe generated a few false-positive results but dramatically underestimated the number of B. cepacia isolates (i.e., 40% of the colonies that did not hybridize to the probe were B. cepacia, as determined by FAME). The 16S rRNA probe generated more false-positive results than the 23S rRNA probe but was effective in identifying the majority of the B. cepacia isolates. The selective medium was only partially successful in recovering B. cepacia. Use of the B. cepacia-specific 16S rRNA probe was the most efficient and accurate way of identifying B. cepacia.     

Comparative genomic analysis of two Burkholderia glumae strains from different geographic origins reveals a high degree of plasticity in genome structure associated with genomic islands

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, a growing disease problem in global rice production. To better understand its genome-scale characteristics, the genome of the highly virulent B. glumae strain 336gr-1 isolated from Louisiana, USA was sequenced using the Illumina Genome Analyser II system. De novo assembled 336gr-1 contigs were aligned and compared with the previously sequenced genome of B. glumae strain BGR1, which was isolated from an infected rice plant in South Korea. Comparative analysis of the whole genomes of B. glumae 336gr-1 and B. glumae BGR1 revealed numerous unique genomic regions present only in one of the two strains. These unique regions contained accessory genes including mobile elements and phage-related genes, and some of the unique regions in B. glumae BGR1 corresponded to predicted genomic islands. In contrast, little variation was observed in known and potential virulence genes between the two genomes. The considerable amount of plasticity largely based on accessory genes and genome islands observed from the comparison of the genomes of these two strains of B. glumae may explain the versatility of this bacterial species in various environmental conditions and geographic locations.     

Burkholderia cepacia Genomovar III Is a Common Plant-Associated Bacterium

A polyphasic taxonomic study involving DNA-DNA hybridization, whole-cell protein electrophoresis, and 16S ribosomal DNA sequence analysis revealed that a group of Burkholderia cepacia-like organisms isolated from the rhizosphere or tissues of maize, wheat, and lupine belong to B. cepacia genomovar III, a genomic species associated with “cepacia syndrome” in cystic fibrosis patients. The present study also revealed considerable protein electrophoretic heterogeneity within this species and demonstrated that the B. cepacia complex consists of two independent phylogenetic lineages.     

Burkholderia semiarida sp. nov. and Burkholderia sola sp. nov., two novel B. cepacia complex species causing onion sour skin

Two putative novel Burkholderia cenocepacia lineages found in the semi-arid region of north-east Brazil causing onion sour skin were studied using genomic approaches to determine their taxonomic position. Four strains belonging to one novel lineage (CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171) and one strain (CCRMBC51) belonging to another novel lineage had their whole genome sequenced to carry out taxogenomic analyses. The phylogenomic tree built using the type (strain) genome server (TYGS) clustered the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171 into the same clade, while grouped the strain CCRMBC51 separately. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analysis showed values above 99.21 % and 93.2 %, respectively, among the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171, while ANI and dDDH values between these strains and the strain CCRMBC51 were below 94.49 % and 56.6 %, respectively. All these strains showed ANI and dDDH values below 94.78 % and 58.8 % concerning type strains of the B. cepacia complex (Bcc) species. The phylogenetic maximum likelihood tree constructed based on the multilocus sequence analysis of core genes (cMLSA) clustered the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171 and the strain CCRMBC51 in two exclusive clades, which did not cluster with any known species of the Bcc. Therefore, combined data from TYGS, ANI, dDDH, and cMLSA demonstrated that the strains represent two novel species of the Bcc, which we classified as Burkholderia semiarida sp. nov. and Burkholderia sola sp. nov., and proposed the strains CCRMBC74^T (=IBSBF 3371 ^T = CBAS 905 ^T) and CCRMBC51^T (=IBSBF3370^T = CBAS 904 ^T) as type strains, respectively.     

Activation of Human Toll-like Receptor 4 (TLR4)·Myeloid Differentiation Factor 2 (MD-2) by Hypoacylated Lipopolysaccharide from a Clinical Isolate of Burkholderia cenocepacia

Lung infection by Burkholderia species, in particular Burkholderia cenocepacia, accelerates tissue damage and increases post-lung transplant mortality in cystic fibrosis patients. Host-microbe interplay largely depends on interactions between pathogen-specific molecules and innate immune receptors such as Toll-like receptor 4 (TLR4), which recognizes the lipid A moiety of the bacterial lipopolysaccharide (LPS). The human TLR4·myeloid differentiation factor 2 (MD-2) LPS receptor complex is strongly activated by hexa-acylated lipid A and poorly activated by underacylated lipid A. Here, we report that B. cenocepacia LPS strongly activates human TLR4·MD-2 despite its lipid A having only five acyl chains. Furthermore, we show that aminoarabinose residues in lipid A contribute to TLR4-lipid A interactions, and experiments in a mouse model of LPS-induced endotoxic shock confirmed the proinflammatory potential of B. cenocepacia penta-acylated lipid A. Molecular modeling combined with mutagenesis of TLR4-MD-2 interactive surfaces suggests that longer acyl chains and the aminoarabinose residues in the B. cenocepacia lipid A allow exposure of the fifth acyl chain on the surface of MD-2 enabling interactions with TLR4 and its dimerization. Our results provide a molecular model for activation of the human TLR4·MD-2 complex by penta-acylated lipid A explaining the ability of hypoacylated B. cenocepacia LPS to promote proinflammatory responses associated with the severe pathogenicity of this opportunistic bacterium.     

Involvement of a Quorum-Sensing-Regulated Lipase Secreted by a Clinical Isolate of Burkholderia glumae in Severe Disease Symptoms in Rice

Burkholderia glumae is an emerging rice pathogen in several areas around the world. Closely related Burkholderia species are important opportunistic human pathogens for specific groups of patients, such as patients with cystic fibrosis and patients with chronic granulomatous disease. Here we report that the first clinical isolate of B. glumae, strain AU6208, has retained its capability to be very pathogenic to rice. As previously reported for rice isolate B. glumae BGR1 (and also for the clinical isolate AU6208), TofI or TofR acyl homoserine lactone (AHL) quorum sensing played a pivotal role in rice virulence. We report that AHL quorum sensing in B. glumae AU6208 regulates secreted LipA lipase and toxoflavin, the phytotoxin produced by B. glumae. B. glumae AU6208 lipA mutants were no longer pathogenic to rice, indicating that the lipase is an important virulence factor. It was also established that type strain B. glumae ATCC 33617 did not produce toxoflavin and lipase and was nonpathogenic to rice. It was determined that in strain ATCC 33617 the LuxR family quorum-sensing sensor/regulator TofR was inactive. Introducing the tofR gene of B. glumae AU6208 in strain ATCC 33617 restored its ability to produce toxoflavin and the LipA lipase. This study extends the role of AHL quorum sensing in rice pathogenicity through the regulation of a lipase which was demonstrated to be a virulence factor. It is the first report of a clinical B. glumae isolate retaining strong rice pathogenicity and finally determined that B. glumae can undergo phenotypic conversion through a spontaneous mutation in the tofR regulator.     

The genome of Burkholderia cenocepacia J2315, an epidemic pathogen of cystic fibrosis patients

Bacterial infections of the lungs of cystic fibrosis (CF) patients cause major complications in the treatment of this common genetic disease. Burkholderia cenocepacia infection is particularly problematic since this organism has high levels of antibiotic resistance, making it difficult to eradicate; the resulting chronic infections are associated with severe declines in lung function and increased mortality rates. B. cenocepacia strain J2315 was isolated from a CF patient and is a member of the epidemic ET12 lineage that originated in Canada or the United Kingdom and spread to Europe. The 8.06-Mb genome of this highly transmissible pathogen comprises three circular chromosomes and a plasmid and encodes a broad array of functions typical of this metabolically versatile genus, as well as numerous virulence and drug resistance functions. Although B. cenocepacia strains can be isolated from soil and can be pathogenic to both plants and man, J2315 is representative of a lineage of B. cenocepacia rarely isolated from the environment and which spreads between CF patients. Comparative analysis revealed that ca. 21% of the genome is unique in comparison to other strains of B. cenocepacia, highlighting the genomic plasticity of this species. Pseudogenes in virulence determinants suggest that the pathogenic response of J2315 may have been recently selected to promote persistence in the CF lung. The J2315 genome contains evidence that its unique and highly adapted genetic content has played a significant role in its success as an epidemic CF pathogen.     

Structural Stability of Burkholderia cenocepacia Biofilms Is Reliant on eDNA Structure and Presence of a Bacterial Nucleic Acid Binding Protein

Cystic fibrosis (CF) is the most common lethal inherited genetic disorder affection Caucasians. Even with medical advances, CF is life-shortening with patients typically surviving only to age 38. Infection of the CF lung by Burkholderia cenocepacia presents exceptional challenges to medical management of these patients as clinically this microbe is resistant to virtually all antibiotics, is highly transmissible and infection of CF patients with this microbe renders them ineligible for lung transplant, often the last lifesaving option. Here we have targeted two abundant components of the B. cenocepacia biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins. Treatment of B. cenocepacia biofilms with antiserum directed at one of these DNABII proteins (integration host factor or IHF) resulted in significant disruption of the biofilm. Moreover, when anti-IHF mediated destabilization of a B. cenocepacia biofilm was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing. Pre-incubation of B. cenocepacia with anti-IHF serum prior to exposure to murine CF macrophages, which are normally unable to effectively degrade ingested B. cenocepacia, resulted in a statistically significant increase in killing of phagocytized B. cenocepacia. Collectively, these findings support further development of strategies that target DNABII proteins as a novel approach for treatment of CF patients, particularly those whose lungs are infected with B. cenocepacia.     

Development and application of an efficient recombineering system for Burkholderia glumae and Burkholderia plantarii

The lambda phage Red proteins Redα/Redβ/Redγ and Rac prophage RecE/RecT proteins are powerful tools for precise and efficient genetic manipulation but have been limited to only a few prokaryotes. Here, we report the development and application of a new recombineering system for Burkholderia glumae and Burkholderia plantarii based on three Rac bacteriophage RecET-like operons, RecEThe_BDU8, RecETh_TJI49 and RecETh1h2e_YI23, which were obtained from three different Burkholderia species. Recombineering experiments indicated that RecETh_TJI49 and RecETh1h2e_YI23 showed higher recombination efficiency compared to RecEThe_BDU8 in Burkholderia glumae PG1. Furthermore, all of the proteins currently categorized as hypothetical proteins in RecETh1h2e_YI23, RecETh_TJI49 and RecEThe_BDU8 may have a positive effect on recombination in B. glumae PG1 except for the h2 protein in RecETh1h2e_YI23. Additionally, RecET_YI23 combined with exonuclease inhibitors Pluγ or Redγ exhibited equivalent recombination efficiency compared to Redγβα in Escherichia coli, providing potential opportunity of recombineering in other Gram-negative bacteria for its loose host specificity. Using recombinase-assisted in situ insertion of promoters, we successfully activated three cryptic non-ribosomal peptide synthetase biosynthetic gene clusters in Burkholderia strains, resulting in the generation of a series of lipopeptides that were further purified and characterized. Compound 7 exhibited significant potential anti-inflammatory activity by inhibiting lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. This recombineering system may greatly enhance functional genome research and the mining of novel natural products in the other species of the genus Burkholderia after optimization of a protocol.     

Phenotypic characterization of trimeric autotransporter adhesin-defective bcaC mutant of Burkholderia cenocepacia: cross-talk towards the histidine kinase BCAM0218

Burkholderia cenocepacia is a virulent species belonging to the Burkholderia cepacia complex (Bcc) and one of the most problematic agents of chronic lung infection in cystic fibrosis patients. B. cenocepacia possesses a large panel of virulence traits that include trimeric autotransporter adhesins (TAAs). Such proteins are obligate homotrimeric anchored in the outer membrane. They are players in the adhesion events that occur between bacteria and biotic/abiotic surfaces. In this study, we constructed two insertional-mutants for TAA bcaC and Histidine kinase (HK) BCAM0218 genes, which are clustered together within the B. cenocepacia K56-2 TAA cluster. The bcaC-mutant affects B. cenocepacia adhesion to extracellular matrix proteins and red blood cells hemagglutination. BcaC contributes to enhancing B. cenocepacia K56-2 adhesion to bronchial epithelial cells. The expression of bcaC seems to affect biofilm formation negatively. Due to a BCAM0218 disruption, the bcaC expression increases significantly, indicating that they are functionally linked. The overexpression of bcaC in the BCAM0218-mutant background rescues at least part of the BcaC functions. Altogether, these findings reveal the multifunctionality of BcaC as a novel B. cenocepacia K56-2 virulence factor and postulate the involvement of a sensor HK (BCAM0218) in the control of this TAA gene.