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Biosynthesis of riboflavin (RF), the precursor of the redox cofactors FMN and FAD, was thought to be well understood in bacteria, with all the pathway enzymes presumed to be known and essential. Our previous research has challenged this view by showing that, in the bacterium Sinorhizobium meliloti, deletion of the ribBA gene encoding the enzyme that catalyzes the initial steps on the RF biosynthesis pathway only causes a reduction in flavin secretion rather than RF auxotrophy. This finding led us to hypothesize that RibBA participates in the biosynthesis of flavins destined for secretion, whereas S. meliloti has another enzyme that performs this function for internal cellular metabolism. Here, we identify and biochemically characterize a novel formamidase (SMc02977) involved in the production of RF for intracellular functions in S. meliloti. This catalyst, which we named Sm-BrbF, releases formate from the early RF precursor 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate to yield 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate. We show that homologs of this enzyme are present in many bacteria, are highly abundant in the Rhizobiales order, and that sequence homologs from Brucella abortus and Liberobacter solanacearum complement the RF auxotrophy of the Sm1021ΔSMc02977 mutant. Furthermore, we show that the B. abortus enzyme (Bab2_0247, Ba-BrbF) is also an 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate formamidase, and that the bab2_0247 mutant is a RF auxotroph exhibiting a lower level of intracellular infection than the wildtype strain. Finally, we show that Sm-BrbF and Ba-BrbF directly interact with other RF biosynthesis pathway enzymes. Together, our results provide novel insight into the intricacies of RF biosynthesis in bacteria.

Bacteria in the genus Brucella are important human and veterinary pathogens. The abortion and infertility they cause in food animals produce economic hardships in areas where the disease has not been controlled, and human brucellosis is one of the world's most common zoonoses. Brucella strains have also been isolated from wildlife, but we know much less about the pathobiology and epidemiology of these infections than we do about brucellosis in domestic animals. The brucellae maintain predominantly an intracellular lifestyle in their mammalian hosts, and their ability to subvert the host immune response and survive and replicate in macrophages and placental trophoblasts underlies their success as pathogens. We are just beginning to understand how these bacteria evolved from a progenitor alphaproteobacterium with an environmental niche and diverged to become highly host-adapted and host-specific pathogens. Two important virulence determinants played critical roles in this evolution: (i) a type IV secretion system that secretes effector molecules into the host cell cytoplasm that direct the intracellular trafficking of the brucellae and modulate host immune responses and (ii) a lipopolysaccharide moiety which poorly stimulates host inflammatory responses. This review highlights what we presently know about how these and other virulence determinants contribute to Brucella pathogenesis. Gaining a better understanding of how the brucellae produce disease will provide us with information that can be used to design better strategies for preventing brucellosis in animals and for preventing and treating this disease in humans.

Bacteria in the genus Brucella are important human and veterinary pathogens. The abortion and infertility they cause in food animals produce economic hardships in areas where the disease has not been controlled, and human brucellosis is one of the world’s most common zoonoses. Brucella strains have also been isolated from wildlife, but we know much less about the pathobiology and epidemiology of these infections than we do about brucellosis in domestic animals. The brucellae maintain predominantly an intracellular lifestyle in their mammalian hosts, and their ability to subvert the host immune response and survive and replicate in macrophages and placental trophoblasts underlies their success as pathogens. We are just beginning to understand how these bacteria evolved from a progenitor alphaproteobacterium with an environmental niche and diverged to become highly host-adapted and host-specific pathogens. Two important virulence determinants played critical roles in this evolution: (i) a type IV secretion system that secretes effector molecules into the host cell cytoplasm that direct the intracellular trafficking of the brucellae and modulate host immune responses and (ii) a lipopolysaccharide moiety which poorly stimulates host inflammatory responses. This review highlights what we presently know about how these and other virulence determinants contribute to Brucella pathogenesis. Gaining a better understanding of how the brucellae produce disease will provide us with information that can be used to design better strategies for preventing brucellosis in animals and for preventing and treating this disease in humans.

The neurotransmitter gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the human brain; however, it is becoming more evident that this non-proteinogenic amino acid plays multiple physiological roles in biology. In the present study, the transport and function of GABA is studied in the highly infectious intracellular bacterium Brucella abortus. The data show that 3H-GABA is imported by B. abortus under nutrient limiting conditions and that the small RNAs AbcR1 and AbcR2 negatively regulate this transport. A specific transport system, gts, is responsible for the transport of GABA as determined by measuring 3H-GABA transport in isogenic deletion strains of known AbcR1/2 regulatory targets; however, this locus is unnecessary for Brucella infection in BALB/c mice. Similar assays revealed that 3H-GABA transport is uninhibited by the 20 standard proteinogenic amino acids, representing preference for the transport of 3H-GABA. Metabolic studies did not show any potential metabolic utilization of GABA by B. abortus as a carbon or nitrogen source, and RNA sequencing analysis revealed limited transcriptional differences between B. abortus 2308 with or without exposure to GABA. While this study provides evidence for GABA transport by B. abortus, questions remain as to why and when this transport is utilized during Brucella pathogenesis.

Brucella is a zoonotic pathogen requiring iron for its survival and acquires this metal through the expression of several high-affinity uptake systems. Of these, the newly discovered ferrous iron transporter, FtrABCD, is proposed to take part in ferrous iron uptake. Sequence homology show, FtrA, the proposed periplasmic ferrous-binding component, is a P19-type protein (a periplasmic protein from C. jejuni which shows Cu(2+) dependent iron affinity). Previous structural and biochemical studies on other P19 systems have established a Cu(2+) dependent Mn (2+) affinity as well as formation of homodimers for these systems. The Cu(2+) coordinating amino acids from these proteins are conserved in Brucella FtrA, hinting towards similar properties. However, there has been no experimental evidence, till date, establishing metal affinities and the possibility of dimer formation by Brucella FtrA. Using wild-type FtrA and Cu(2+)-binding mutants (H65A, E67A, H118A, and H151A) we investigated the metal affinities, folding stabilities, dimer forming abilities, and the molecular basis of the Cu(2+) dependence for this P19-type protein employing homology modeling, analytical gel filtration, calorimetric, and spectroscopic methods. The data reported here confirm a Cu(2+)-dependent, low-μM Mn (2+) (Fe (2+) mimic) affinity for the wild-type FtrA. In addition, our data clearly show the loss of Mn(2+) affinity, and the formation of less stable protein conformations as a result of mutating these conserved Cu(2+)-binding residues, indicating the important roles these residues play in producing a native and functional fold of Brucella FtrA.

Brucella abortus is a Gram-negative bacterium that causes abortion and infertility in food animals and a chronic debilitating febrile disease in humans known as brucellosis. As with all pathogenic bacteria , the Brucella spp. require sufficient metal nutrition during the course of an infection. Host-mediated 'metal withdrawal' defenses actively restrict the bioavailability of metals which requires invading bacteria to employ high affinity metal acquisition systems to overcome these metal-limiting conditions. While obtaining sufficient metals during host infection is critical to the survival of these bacteria , avoiding metal toxicity is equally important. Excess accumulation of one metal relative to others can lead to protein mis-metallation when surplus metal ions outcompete other metal species for their native binding sites. To prevent metal toxicity , bacteria respond to high intracellular metal concentrations by means of metal-responsive transcriptional regulators that downregulate metal import systems , and exporters that remove excess intracellular metal. Manganese (Mn) is an essential micronutrient for Brucella strains , and the purpose of this study was to better define the cellular components that maintain Mn homeostasis and prevent Mn toxicity in these bacteria. The Mn-responsive repressor Mur downregulates the expression of mntH , the gene encoding the sole high affinity Mn importer in Brucella in response to increased intracellular levels of Mn. But phenotypic analysis of a B. abortus mur mutant suggests that Mur plays a minimal role in preventing Mn toxicity. Instead , an ortholog of the cation diffusion facilitator (CDF) type metal exporter EmfA , which prevents Mn toxicity in the close phylogenetic relative Rhizobium etli , is critical for preventing Mn toxicity in Brucella. The experimental findings of this study indicate that EmfA-mediated resistance to Mn toxicity plays a critical role in the virulence of Brucella strains , and suggests that the primary function of EmfA may be to maintain the proper intracellular balance of Mn in these bacteria during the course of infection.

The Gram-negative cell envelope is a remarkable structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. Multiple molecular systems function to maintain integrity of this essential barrier between the interior of the cell and its surrounding environment. We show that a conserved DUF1849 family protein, EipB, is secreted to the periplasmic space of Brucella species, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual 14-stranded β-spiral structure that resembles the LolA and LolB lipoprotein delivery system, though the overall fold of EipB is distinct from LolA/LolB. Deletion of eipB results in defects in Brucella cell envelope integrity in vitro and in maintenance of spleen colonization in a mouse model of Brucella abortus infection. Transposon disruption of ttpA, which encodes a periplasmic protein containing tetratricopeptide repeats, is synthetically lethal with eipB deletion. ttpA is a reported virulence determinant in Brucella, and our studies of ttpA deletion and overexpression strains provide evidence that this gene also contributes to cell envelope function. We conclude that eipB and ttpA function in the Brucella periplasmic space to maintain cell envelope integrity, which facilitates survival in a mammalian host. IMPORTANCE Brucella species cause brucellosis, a global zoonosis. A gene encoding a conserved DUF1849-family protein, which we have named EipB, is present in all sequenced Brucella and several other genera in the class Alphaproteobacteria. The manuscript provides the first functional and structural characterization of a DUF1849 protein. We show that EipB is secreted to the periplasm where it forms a spiral-shaped antiparallel β protein that is a determinant of cell envelope integrity in vitro and virulence in an animal model of disease. eipB genetically interacts with ttpA, which also encodes a periplasmic protein. We propose that EipB and TtpA function as part of a system required for cell envelope homeostasis in select Alphaproteobacteria.

Brucella is a zoonotic pathogen requiring iron for its survival and acquires this metal through the expression of several high-affinity uptake systems. Of these, the newly discovered ferrous iron transporter, FtrABCD, is proposed to take part in ferrous iron uptake. Sequence homology shows that, FtrA, the proposed periplasmic ferrous-binding component, is a P19-type protein (a periplasmic protein from C. jejuni which shows Cu2+ dependent iron affinity). Previous structural and biochemical studies on other P19 systems have established a Cu2+ dependent Mn2+ affinity as well as formation of homodimers for these systems. The Cu2+ coordinating amino acids from these proteins are conserved in Brucella FtrA, hinting towards similar properties. However, there has been no experimental evidence, till date, establishing metal affinities and the possibility of dimer formation by Brucella FtrA. Using wild-type FtrA and Cu2+-binding mutants (H65A, E67A, H118A, and H151A) we investigated the metal affinities, folding stabilities, dimer forming abilities, and the molecular basis of the Cu2+ dependence for this P19-type protein employing homology modeling, analytical gel filtration, calorimetric, and spectroscopic methods. The data reported here confirm a Cu2+-dependent, low-μM Mn2+ (Fe2+ mimic) affinity for the wild-type FtrA. In addition, our data clearly show the loss of Mn2+ affinity, and the formation of less stable protein conformations as a result of mutating these conserved Cu2+-binding residues, indicating the important roles these residues play in producing a native and functional fold of Brucella FtrA.

Pyruvate kinase plays a central role in glucose catabolism in bacteria, and efficient utilization of this hexose has been linked to the virulence of Brucella strains in mice. The brucellae produce a single pyruvate kinase which is an ortholog of the Bradyrhizobium manganese (Mn)-dependent pyruvate kinase PykM. A biochemical analysis of the Brucella pyruvate kinase and phenotypic analysis of a Brucella abortus mutant defective in high-affinity Mn import indicate that this enzyme is an authentic PykM ortholog which functions as a Mn-dependent enzyme in vivo. The loss of PykM has a negative impact on the capacity of the parental 2308 strain to utilize glucose, fructose, and galactose but not on its ability to utilize ribose, xylose, arabinose, or erythritol, and a pykM mutant displays significant attenuation in C57BL/6 mice. Although the enzyme pyruvate phosphate dikinase (PpdK) can substitute for the loss of pyruvate kinase in some bacteria and is also an important virulence determinant in Brucella, a phenotypic analysis of B. abortus 2308 and isogenic pykM, ppdK, and pykM ppdK mutants indicates that PykM and PpdK make distinctly different contributions to carbon metabolism and virulence in these bacteria. IMPORTANCE Mn plays a critical role in the physiology and virulence of Brucella strains, and the results presented here suggest that one of the important roles that the high-affinity Mn importer MntH plays in the pathogenesis of these strains is supporting the function of the Mn-dependent kinase PykM. A better understanding of how the brucellae adapt their physiology and metabolism to sustain their intracellular persistence in host macrophages will provide knowledge that can be used to design improved strategies for preventing and treating brucellosis, a disease that has a significant impact on both the veterinary and public health communities worldwide.

The YbeY endoribonuclease is one of the best-conserved proteins across the kingdoms of life. In the present study, we demonstrated that YbeY in Brucella abortus is linked to a variety of important activities, including proper cellular morphology, mRNA transcript levels, and virulence. Deletion of ybeY in B. abortus led to a small-colony phenotype when the bacteria were grown on agar medium, as well as to significant aberrations in the morphology of the bacterial cell as evidenced by electron microscopy. Additionally, compared to the parental strain, the Δ ybeY strain was significantly attenuated in both macrophage and mouse models of infection. The Δ ybeY strain also showed increased sensitivities to several in vitro -applied stressors, including bile acid, hydrogen peroxide, SDS, and paraquat. Transcriptomic analysis revealed that a multitude of mRNA transcripts are dysregulated in the Δ ybeY strain, and many of the identified mRNAs encode proteins involved in metabolism, nutrient transport, transcriptional regulation, and flagellum synthesis. We subsequently constructed gene deletion strains of the most highly dysregulated systems, and several of the YbeY-linked gene deletion strains exhibited defects in the ability of the bacteria to survive and replicate in primary murine macrophages. Taken together, these data establish a clear role for YbeY in the biology and virulence of Brucella ; moreover, this work further illuminates the highly varied roles of this widely conserved endoribonuclease in bacteria. IMPORTANCE Brucella spp. are highly efficient bacterial pathogens of animals and humans, causing significant morbidity and economic loss worldwide, and relapse of disease often occurs following antibiotic treatment of human brucellosis. As such, novel therapeutic strategies to combat Brucella infections are needed. Ribonucleases in the brucellae are understudied, and these enzymes represent elements that may be potential targets for future treatment approaches. The present work demonstrates the importance of the YbeY endoribonuclease for cellular morphology, efficient control of mRNA levels, and virulence in B. abortus . Overall, the results of this study advance our understanding of the critical roles of YbeY in the pathogenesis of the intracellular brucellae and expand our understanding of this highly conserved RNase.

It is well established that high-affinity zinc importers play essential roles in bacterial virulence, but the studies described by Moreau et al. in this issue (G. B. Moreau, A. Qin, and B. J. Mann, J Bacteriol 200:e00587-17, 2018, https://doi.org/10.1128/JB.00587-17 ) demonstrate that we probably still have much to learn about how these transporters function and how the genes that encode them are regulated in different bacterial pathogens.

Metal homeostasis in bacterial cells is a highly regulated process requiring intricately coordinated import and export, as well as precise sensing of intracellular metal concentrations. The uptake of zinc (Zn) has been linked to the virulence ofBrucella abortus; however, the capacity ofBrucellastrains to sense Zn levels and subsequently coordinate Zn homeostasis has not been described. Here, we show that expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by the Zn-sensing Fur family transcriptional regulator, Zur, by direct interactions between Zur and the promoter region ofznuABC. Moreover, the MerR-type regulator, ZntR, controls the expression of the gene encoding the Zn exporter ZntA by binding directly to its promoter. Deletion ofzurorzntRalone did not result in increased zinc toxicity in the corresponding mutants; however, deletion ofzntAled to increased sensitivity to Zn but not to other metals, such as Cu and Ni, suggesting that ZntA is a Zn-specific exporter. Strikingly, deletion ofzntRresulted in significant attenuation ofB. abortusin a mouse model of chronic infection, and subsequent experiments revealed that overexpression ofzntAin thezntRmutant is the molecular basis for its decreased virulence.IMPORTANCEThe importance of zinc uptake forBrucellapathogenesis has been demonstrated previously, but to date, there has been no description of how overall zinc homeostasis is maintained and genetically controlled in the brucellae. The present work defines the predominant zinc export system, as well as the key genetic regulators of both zinc uptake and export inBrucella abortus. Moreover, the data show the importance of precise coordination of the zinc homeostasis systems as disregulation of some elements of these systems leads to the attenuation ofBrucellavirulence in a mouse model. Overall, this study advances our understanding of the essential role of zinc in the pathogenesis of intracellular bacteria.

Summary Iron transport has been linked to the virulence of Brucella strains in both natural and experimental hosts. The genes designated BAB2_0837–0840 in the Brucella abortus 2308 genome sequence are predicted to encode a CupII-type ferrous iron transporter homologous to the FtrABCD transporter recently described in Bordetella. To study the role of the Brucella FtrABCD in iron transport, an isogenic ftrA mutant was constructed from B. abortus 2308. Compared with the parental strain, the B. abortus ftrA mutant displays a decreased capacity to use non-haem iron sources in vitro, a growth defect in a low iron medium that is enhanced at pH 6, and studies employing radiolabelled FeCl3 confirmed that FtrABCD transports ferrous iron. Transcription of the ftrA gene is induced in B. abortus 2308 in response to iron deprivation and exposure to acid pH, and similar to other Brucella iron acquisition genes that have been examined the iron-responsiveness of ftrA is dependent upon the iron response regulator Irr. The B. abortus ftrA mutant exhibits significant attenuation in both cultured murine macrophages and experimentally infected mice, supporting the proposition that ferrous iron is a critical iron source for these bacteria in the mammalian host.

Virulence of pathogenic bacteria is often determined by their ability to adapt to stress.The Brucella abortus general stress response (GSR) system is required for chronic mammalian infection and is regulated by phosphorylation and proteolysis.The B. abortus GSR signaling pathway has multiple layers of post-translational control and is a determinant of chronic infection.This study provides new, molecular level insight into chronic Brucella infection. Brucella spp. are adept at establishing a chronic infection in mammals. We demonstrate that core components of the α-proteobacterial general stress response (GSR) system, PhyR and σ(E1), are required for Brucella abortus stress survival in vitro and maintenance of chronic murine infection in vivo. ΔphyR and ΔrpoE1 null mutants exhibit decreased survival under acute oxidative and acid stress but are not defective in infection of primary murine macrophages or in initial colonization of BALB/c mouse spleens. However, ΔphyR and ΔrpoE1 mutants are attenuated in spleens beginning 1 month postinfection. Thus, the B. abortus GSR system is dispensable for colonization but is required to maintain chronic infection. A genome-scale analysis of the B. abortus GSR regulon identified stress response genes previously linked to virulence and genes that affect immunomodulatory components of the cell envelope. These data support a model in which the GSR system affects both stress survival and the interface between B. abortus and the host immune system. We further demonstrate that PhyR proteolysis is a unique feature of GSR control in B. abortus. Proteolysis of PhyR provides a mechanism to avoid spurious PhyR protein interactions that inappropriately activate GSR-dependent transcription. We conclude that the B. abortus GSR system regulates acute stress adaptation and long term survival within a mammalian host and that PhyR proteolysis is a novel regulatory feature in B. abortus that ensures proper control of GSR transcription.

Magnesium, copper and cobalt are essential micronutrients for Brucella strains, but relatively little is known about how the brucellae acquire the levels of these metals they need and avoid their toxicity. This chapter will review the information that is available in the literature and can be derived from surveys of currently available genome sequences regarding magnesium, copper and cobalt homeostasis in Brucella.

The Brucella BhuQ protein is a homolog of the Bradyrhizobium japonicum heme oxygenases HmuD and HmuQ. To determine if this protein plays a role in the ability of Brucella abortus 2308 to use heme as an iron source, an isogenic bhuQ mutant was constructed and its phenotype evaluated. Although the Brucella abortus bhuQ mutant DCO1 did not exhibit a defect in its capacity to use heme as an iron source or evidence of increased heme toxicity in vitro , this mutant produced increased levels of siderophore in response to iron deprivation compared to 2308. Introduction of a bhuQ mutation into the B. abortus dhbC mutant BHB2 (which cannot produce siderophores) resulted in a severe growth defect in the dhbC bhuQ double mutant JFO1 during cultivation under iron-restricted conditions, which could be rescued by the addition of FeCl 3 , but not heme, to the growth medium. The bhuQ gene is cotranscribed with the gene encoding the iron-responsive regulator RirA, and both of these genes are repressed by the other major iron-responsive regulator in the alphaproteobacteria, Irr. The results of these studies suggest that B. abortus 2308 has at least one other heme oxygenase that works in concert with BhuQ to allow this strain to efficiently use heme as an iron source. The genetic organization of the rirA-bhuQ operon also provides the basis for the proposition that BhuQ may perform a previously unrecognized function by allowing the transcriptional regulator RirA to recognize heme as an iron source.

Hfq is an RNA-binding protein that functions in post-transcriptional gene regulation by mediating interactions between mRNAs and small regulatory RNAs (sRNAs). Two proteins encoded by BAB1_1794 and BAB2_0612 are highly over-produced in a Brucella abortus hfq mutant compared with the parental strain, and recently, expression of orthologues of these proteins in Agrobacterium tumefaciens was shown to be regulated by two sRNAs, called AbcR1 and AbcR2. Orthologous sRNAs (likewise designated AbcR1 and AbcR2) have been identified in B. abortus 2308. In Brucella, abcR1 and abcR2 single mutants are not defective in their ability to survive in cultured murine macrophages, but an abcR1 abcR2 double mutant exhibits significant attenuation in macrophages. Additionally, the abcR1 abcR2 double mutant displays significant attenuation in a mouse model of chronic Brucella infection. Quantitative proteomics and microarray analyses revealed that the AbcR sRNAs predominantly regulate genes predicted to be involved in amino acid and polyamine transport and metabolism, and Northern blot analyses indicate that the AbcR sRNAs accelerate the degradation of the target mRNAs. In an Escherichia coli two-plasmid reporter system, overexpression of either AbcR1 or AbcR2 was sufficient for regulation of target mRNAs, indicating that the AbcR sRNAs from B. abortus 2308 perform redundant regulatory functions.

Similar to other bacteria,Brucellastrains require several biologically essential metals for their survivalin vitroandin vivo. Acquiring sufficient levels of some of these metals, particularly iron, manganese and zinc, is especially challenging in the mammalian host, where sequestration of these micronutrients is a well-documented component of both the innate and acquired immune responses. This review describes theBrucellametal transporters that have been shown to play critical roles in the virulence of these bacteria in experimental and natural hosts.

MntH is the only high-affinity manganese transporter identified in Brucella . A previous study showed that MntH is required for the wild-type virulence of Brucella abortus 2308 in mice (Anderson ES, et al., Infect. Immun. 77: 3466–3474, 2009) and indicated that the mntH gene is regulated in a manganese-responsive manner in this strain by a Mur homolog. In the study presented here, the transcriptional start site for mntH in B. abortus 2308 was determined by primer extension analysis. Specific interactions between Mur and the mntH promoter region were demonstrated in an electrophoretic mobility shift assay (EMSA), and a Mur binding site was identified in the −55 to −24 region of the mntH promoter by DNase I footprint analysis. The specificity of the interaction of Mur with the putative Mur box was further evaluated by EMSA employing oligonucleotides in which the consensus nucleotides in this region were substituted. These studies not only confirm a direct role for Mur in the Mn-responsive regulation of mntH expression in Brucella abortus 2308 but also identify the cis -acting elements upstream of mntH that are responsible for this regulation.

The type IV secretion system encoded by the virB operon is required for full virulence of Brucella sp., and the present study links the RNA chaperone Hfq to wild-type expression of virB in Brucella abortus 2308. Studies employing virB-lacZ fusions, quantitative reverse transcription-PCR, and immunoblot analysis showed that both transcription and translation of virB are decreased in an isogenic hfq mutant compared to those in the parental strain. These results led to the hypothesis that Hfq regulation of virB is mediated through an intermediate transcriptional regulator. Subsequent experiments determined that expression of the gene encoding the putative Brucella quorum-sensing regulator BabR (also known as BlxR), a known virB regulator, is also controlled by Hfq at the posttranscriptional level, and a cis-acting element in the 5′ untranslated region of the babR transcript responsible for this regulation was identified. Consistent with its role as a virB regulator, recombinant Brucella BabR binds to the virB promoter region in electrophoretic mobility shift assays. However, experiments employing a babR mutant strain determined that BabR is a repressor, not an activator, of virB transcription. These findings suggest that Hfq regulates virB expression through both BabR-dependent and BabR-independent pathways.

Gram-negative bacteria belonging to the Brucella species cause chronic infections that can result in undulant fever, arthritis, and osteomyelitis in humans. Remarkably, Brucella sp. genomes encode a protein, named TcpB, that bears significant homology with mammalian Toll/IL-1 receptor domains and whose expression causes degradation of the phosphorylated, signal competent form of the adapter MyD88-adapter–like (MAL). This effect of TcpB is mediated through its box 1 region and has no effect on other TLR adapter proteins such as MyD88 or TIR-domain containing adapter protein-inducing IFNβ. TcpB also does not affect a mutant, signal-incompetent form of MAL that cannot be phosphorylated. Interestingly, the presence of TcpB leads to enhanced polyubiqitination of MAL, which is likely responsible for its accelerated degradation. A Brucella abortus mutant lacking TcpB fails to reduce levels of MAL in infected macrophages. Therefore, TcpB represents a unique pathogen-derived molecule that suppresses host innate-immune responses by specifically targeting an individual adapter molecule in the TLR signaling pathway for degradation.

Brucella strains produce abortion and infertility in their natural hosts and a zoonotic disease in humans known as undulant fever. These bacteria do not produce classical virulence factors, and their capacity to successfully survive and replicate within a variety of host cells underlies their pathogenicity. Extensive replication of the brucellae in placental trophoblasts is associated with reproductive tract pathology in natural hosts, and prolonged persistence in macrophages leads to the chronic infections that are a hallmark of brucellosis in both natural hosts and humans. This review describes how Brucella strains have efficiently adapted to their intracellular lifestyle in the host.

The gene designated BAB1_1460 in the Brucella abortus 2308 genome sequence is predicted to encode the manganese transporter MntH. Phenotypic analysis of an isogenic mntH mutant indicates that MntH is the sole high-affinity manganese transporter in this bacterium but that MntH does not play a detectable role in the transport of Fe 2+ , Zn 2+ , Co 2+ , or Ni 2+ . Consistent with the apparent selectivity of the corresponding gene product, the expression of the mntH gene in B. abortus 2308 is repressed by Mn 2+ , but not Fe 2+ , and this Mn-responsive expression is mediated by a Mur-like repressor. The B. abortus mntH mutant MWV15 exhibits increased susceptibility to oxidative killing in vitro compared to strain 2308, and a comparative analysis of the superoxide dismutase activities present in these two strains indicates that the parental strain requires MntH in order to make wild-type levels of its manganese superoxide dismutase SodA. The B. abortus mntH mutant also exhibits extreme attenuation in both cultured murine macrophages and experimentally infected C57BL/6 mice. These experimental findings indicate that Mn 2+ transport mediated by MntH plays an important role in the physiology of B. abortus 2308, particularly during its intracellular survival and replication in the host.

Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacI q - lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alpha- and gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZ α. In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10- to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens , a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation.

Phenotypic evaluation of isogenic mutants derived from Brucella abortus 2308 indicates that the AlcR homolog DhbR (2,3- d i h ydroxy b enzoic acid [2,3-DHBA] biosynthesis r egulator) modulates the expression of the genes involved in 2,3-DHBA production, employing 2,3-DHBA or brucebactin as a coinducer.

Recent breakthroughs in next-generation sequencing technologies have led to the identification of small noncoding RNAs (sRNAs) as a new important class of regulatory molecules. In prokaryotes, sRNAs are often bound to the chaperone protein Hfq, which allows them to interact with their partner mRNA(s). We screened the genome of the zoonotic and human pathogen Brucella suis 1330 for the presence of this class of RNAs. We designed a coimmunoprecipitation strategy that relies on the use of Hfq as a bait to enrich the sample with sRNAs and eventually their target mRNAs. By deep sequencing analysis of the Hfq-bound transcripts, we identified a number of mRNAs and 33 sRNA candidates associated with Hfq. The expression of 10 sRNAs in the early stationary growth phase was experimentally confirmed by Northern blotting and/or reverse transcriptase PCR. IMPORTANCE Brucella organisms are facultative intracellular pathogens that use stealth strategies to avoid host defenses. Adaptation to the host environment requires tight control of gene expression. Recently, small noncoding RNAs (sRNAs) and the sRNA chaperone Hfq have been shown to play a role in the fine-tuning of gene expression. Here we have used RNA sequencing to identify RNAs associated with the B. suis Hfq protein. We have identified a novel list of 33 sRNAs and 62 Hfq-associated mRNAs for future studies aiming to understand the intracellular lifestyle of this pathogen.

Brucella abortus 2308 derivatives with mini-Tn 5 insertions in purE , purL , and purD display significant attenuation in the BALB/c mouse model, while isogenic mutants with mini-Tn 5 insertions in pheA , trpB , and dagA display little or no attenuation in cultured murine macrophages or mice. These experimental findings confirm the importance of the purine biosynthesis pathways for the survival and replication of the brucellae in host macrophages. In contrast to previous reports, however, these results indicate that exogenous tryptophan and phenylalanine are available for use by the brucellae in the phagosomal compartment.

Members of the bacterial genus Brucella are facultative intracellular pathogens that reside predominantly within membrane-bound compartments within two host cell types, macrophages and placental trophoblasts. Within macrophages, the brucellae route themselves to an intracellular compartment that is favourable for survival and replication, and they also appear to be well-adapted from a physiological standpoint to withstand the environmental conditions encountered during prolonged residence in this intracellular niche. Much less is known about the interactions of the Brucella with placental trophoblasts, but experimental evidence suggests that these bacteria use an iron acquisition system to support extensive intracellular replication within these host cells that is not required for survival and replication in host macrophages. Thus, it appears that the brucellae rely upon the products of distinct subsets of genes to adapt successfully to the environmental conditions encountered within the two cell types within which they reside in their mammalian hosts.

Sinorhizobium meliloti , a legume symbiont, and Brucella abortus , a phylogenetically related mammalian pathogen, both require the bacterial-encoded BacA protein to establish chronic intracellular infections in their respective hosts. We found that the bacterial BacA proteins share sequence similarity with a family of eukaryotic peroxisomal-membrane proteins, including the human adrenoleukodystrophy protein, required for the efficient transport of very-long-chain fatty acids out of the cytoplasm. This insight, along with the increased sensitivity of BacA-deficient mutants to detergents and cell envelope-disrupting agents, led us to discover that BacA affects the very-long-chain fatty acid (27-OHC28:0 and 29-OHC30:0) content of both Sinorhizobium and Brucella lipid A. We discuss models for how BacA function affects the lipid-A fatty-acid content and why this activity could be important for the establishment of chronic intracellular infections.

Brucella strains encounter oxygen deprivation during their intracellular replication in host cells, and the capacity of these bacteria to utilize NO(3) as an alternative electron acceptor for respiration plays an important role in their successful adaption to their intracellular niche. In this issue of Molecular Microbiology, Carrica et al (2012). report that NtrY and NtrX comprise a redox-responsive two-component regulator in Brucella abortus 2308 that responds to decreasing levels of O(2) and induces the expression of this strain's denitrification genes. Thus, NtrYX joins the increasing number of genetic regulators that contribute to the metabolic versatility required for the virulence of Brucella strains in their mammalian hosts.

The gene encoding a Lon protease homologue has been cloned from Brucella abortus. The putative Brucella abortus Lon shares > 60% amino acid identity with its Escherichia coli counterpart and the recombinant form of this protein restores the capacity of an Escherichia coli lon mutant to resist killing by ultraviolet irradiation and regulate the expression of a cpsB::lacZ fusion to wild-type levels. A σ32 type promoter was identified upstream of the predicted lon coding region and Northern analysis revealed that transcription of the native Brucella abortus lon increases in response to heat shock and other environmental stresses. ATP-dependent proteolytic activity was also demonstrated for purified recombinant Lon. To evaluate the capacity of the Brucella abortus Lon homologue to function as a stress response protease, the majority of the lon coding region was removed from virulent strain Brucella abortus 2308 via allelic exchange. In contrast to the parent strain, the Brucella abortus lon mutant, designated GR106, was impaired in its capacity to form isolated colonies on solid medium at 41°C and displayed an increased sensitivity to killing by puromycin and H2O2. GR106 also displayed reduced survival in cultured murine macrophages and significant attenuation in BALB/c mice at 1 week post infection compared with the virulent parental strain. Beginning at 2 weeks and continuing for 6 weeks post infection, however, GR106 and 2308 displayed equivalent spleen and liver colonization levels in mice. These findings suggest that the Brucella abortus Lon homologue functions as a stress response protease that is required for wild-type virulence during the initial stages of infection in the mouse model, but is not essential for the establishment and maintenance of chronic infection in this host.

PHE1 is a htrA cycL double gene deletion mutant of virulent Brucella abortus strain 2308 (S2308) which has previously been evaluated in the murine and caprine models of bovine brucellosis. This report describes the results of studies conducted with this mutant in the natural bovine host. Six sexually mature, non-gravid heifers were inoculated via the conjunctival sac with 1 x 10(10) colony forming units (CFU) of either the parental S2308 or the htrA cycL gene deletion mutant, PHE1. At 4, 7 and 11 days post-inoculation, PHE1 was found to colonize the bovine host at lower levels than S2308. In a second experiment, eight heifers in mid-gestation were infected with 1 x 10(7) CFU of either strain via the conjunctival sac. The virulent S2308 caused abortions or weak calves in 4/4 cows, while all four cows infected with PHE1 had healthy calves. Furthermore, PHE1 exhibited decreased resistance to killing by cultured bovine neutrophils and macrophages compared to the parental strain. These studies demonstrate that the B. abortus htrA cycL gene deletion mutant PHE1 is highly attenuated in the bovine host when compared to the virulent parental S2308.

A DNA fragment encoding an approximately 18 kDa protein from Brucella abortus strain 2308 was cloned and expressed in Escherichia coli. This recombinant protein, designated BA18K, reacted in Western blot analysis with sera obtained from experimentally and naturally infected animals including mice, goats, dogs and humans. Restriction enzyme analysis of the plasmid (pBA28) encoding BA18K revealed the presence of an approximately 8.7 kbp Sau3A genomic DNA fragment within the vector and subsequent subcloning and Western blot analysis limited the region encoding BA18K to an approximately 3.0 kbp Pst 1 DNA fragment. DNA sequence analysis of this region identified an open reading frame capable of encoding a protein of 177 amino acids with a predicted relative molecular mass of 17529. Comparison of the deduced amino acid sequence of BA18K with those in the protein sequence databases yielded no homology with previously described proteins from other bacterial genera. These searches did, however, indicate that BA18K is identical to the previously described outer membrane protein (OMP) from B. abortus strain 544 designated Omp 19.

The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella . Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 ( nolR ), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [ nolR ]), and in Brucella , as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella , and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants.

An htrA deletion mutant was created in the bovine vaccine strain, B. abortus S19, by replacing the majority of the htrA gene with a kanamycin resistance gene. Antibiotic selection for a double crossover event yielded kanamycin-resistant, ampicillin-sensitive colonies confirmed by Southern and western blot analysis to be HtrA deficient. The B. abortus S19 htrA mutant was significantly more susceptible than the parental strain to killing by H2O2 (P < 0.001) and O(2)- generated by the redox cycling agent paraquat (P < 0.05) in disk sensitivity assays. Deletion of the htrA gene from S19 produced a bimodal effect on the spleen colonization profile of this strain in BALB/c mice. At one week post-infection, the B. abortus S19 htrA mutant colonized the spleens of experimentally infected BALB/c mice at significantly lower levels (P < 0.01) than the parental strain. Enhanced clearance (P < 0.05) was also observed at later timepoints, i.e. 4 and 7 weeks post infection, however at 2 and 3 weeks post infection, the mutant and parental strains colonized the mice at equivalent levels. The temporal development of specific delayed type hypersensitivity and antibody responses in BALB/c mice infected with the mutant or parental strain were equivalent. These results suggest that the htrA gene product contributes to successful host colonization by S19. However, deletion of this gene does not radically alter the overall, characteristic spleen colonization profile of this vaccine strain in the BALB/c mouse model, nor compromise the capacity of this strain to elicit Brucella cellular or humoral immune responses in this experimental host.

Bacterial stress response proteins of the high temperature requirement A (HtrA) family are serine proteases which appear to play an important role in scavenging oxidatively damaged proteins from the cell before they reach toxic levels. An isogenic htrA deletion mutant, designated RWP5, was constructed from virulent Brucella melitensis 16M via gene replacement to determine whether the B. melitensis HtrA protein functions as a stress response protein, and to evaluate the contribution of this protein to virulence. Unlike the parental strain, RWP5 would not form isolated colonies on solid media at 40°C or grow on Schaedler agar without blood supplementation. RWP5 also grew poorly in broth culture in contrast to 16M. The B. melitensis htrA mutant was significantly more sensitive ( P 2 O 2 , and puromycin than the parental strain, and a significant reduction ( P In vitro and in vivo evaluation of RWP5 reisolates obtained from the spleens of mice at 4 and 16 weeks post-infection demonstrated that mouse passage did not significantly alter these characteristic in vitro and in vivo properties of RWP5. These results support a stress response function for the B. melitensis HtrA protein and suggest that this protein contributes to the pathogenesis of B. melitensis early in infection. The basis for the recovery of RWP5 at later timepoints in infected mice is presently unknown; however, the results presented here suggest that it is not caused by a stable genetic change resulting from mouse passage.

The organic hydroperoxide resistance protein Ohr has been identified in numerous bacteria where it functions in the detoxification of organic hydroperoxides, and expression ofohris often regulated by a MarR-type regulator called OhrR. The genes annotated as BAB2_0350 and BAB2_0351 in theBrucella abortus2308 genome sequence are predicted to encode OhrR and Ohr orthologs, respectively. Using isogenicohrandohrRmutants andlacZpromoter fusions, it was determined that Ohr contributes to resistance to organic hydroperoxide, but not hydrogen peroxide, inB. abortus2308 and that OhrR represses the transcription of bothohrandohrRin this strain. Moreover, electrophoretic mobility shift assays and DNase I footprinting revealed that OhrR binds directly to a specific region in the intergenic region betweenohrandohrRthat shares extensive nucleotide sequence similarity with so-called “OhrR boxes” described in other bacteria. While Ohr plays a prominent role in protectingB. abortus2308 from organic hydroperoxide stress inin vitroassays, this protein is not required for the wild-type virulence of this strain in cultured murine macrophages or experimentally infected mice.

Irr and RirA, rather than Fur, serve as the major iron-responsive regulators in the alphaproteobacteria. With only a few exceptions, however, the relative contributions of these transcriptional regulators to the differential expression of specific iron metabolism genes in Brucella strains are unclear. The gene encoding the outer membrane heme transporter BhuA exhibits maximum expression in Brucella abortus 2308 during growth under iron-deprived conditions, and mutational studies indicate that this pattern of bhuA expression is mediated by the iron-responsive regulator Irr. Specifically, a bhuA-lacZ transcriptional fusion does not produce elevated levels of β-galactosidase in response to iron deprivation in the isogenic irr mutant BEA5, and, unlike the parental strain, B. abortus BEA5 cannot utilize heme as an iron source in vitro and is attenuated in mice. A derivative of the bhuA-lacZ transcriptional fusion lacking the predicted Irr binding site upstream of the bhuA promoter does not produce elevated levels of β-galactosidase in response to iron deprivation in the parental B. abortus 2308 strain, and a direct and specific interaction between a recombinant version of the Brucella Irr and the bhuA promoter region was observed in an electrophoretic mobility shift assay. Despite the fact that it lacks the heme regulatory element linked to the iron-responsive degradation of its counterpart in Bradyrhizobium japonicum , readily detectable levels of Irr were found only in B. abortus 2308 cells by Western blot analysis following growth under iron-deprived conditions.

Brucella strains are exposed to potentially toxic levels of H 2 O 2 both as a consequence of their aerobic metabolism and through the respiratory burst of host phagocytes. To evaluate the relative contributions of the sole catalase KatE and the peroxiredoxin AhpC produced by these strains in defense against H 2 O 2 -mediated toxicity, isogenic katE , ahpC , and katE ahpC mutants were constructed and the phenotypic properties of these mutants compared with those of the virulent parental strain B. abortus 2308. The results of these studies indicate that AhpC is the primary detoxifier of endogenous H 2 O 2 generated by aerobic metabolism. KatE, on the other hand, plays a major role in scavenging exogenous and supraphysiologic levels of H 2 O 2 , although this enzyme can play a supporting role in the detoxification of H 2 O 2 of endogenous origin if AhpC is absent. B. abortus ahpC and katE mutants exhibit wild-type virulence in C57BL/6 and BALB/c mice, but the B. abortus ahpC katE double mutant is extremely attenuated, and this attenuation is not relieved in derivatives of C57BL/6 mice that lack NADPH oxidase (cybb) or inducible nitric oxide synthase (Nos2) activity. These experimental findings indicate that the generation of endogenous H 2 O 2 represents a relevant environmental stress that B. abortus 2308 must deal with during its residence in the host and that AhpC and KatE perform compensatory roles in detoxifying this metabolic H 2 O 2 .

The gene annotated BAB2_1150 in the Brucella abortus 2308 genome sequence is predicted to encode a homolog of the well-characterized heme transporter ShuA of Shigella dysenteriae and accordingly has been given the designation bhuA ( B rucella h eme u tilization). Phenotypic analysis of an isogenic bhuA mutant derived from B. abortus 2308 verified that there is a link between BhuA and the ability of the parent strain to use heme as an iron source in in vitro assays. Maximum expression of bhuA in B. abortus 2308 is observed during stationary phase when this strain in cultivated in low-iron minimal medium, and a comparison of the growth characteristics of the B. abortus bhuA mutant and 2308 in this medium suggested that heme serves as an important iron source for the parent strain during stationary phase. The B. abortus bhuA mutant HR1703 exhibits significant attenuation in cultured murine macrophages compared to strain 2308, and unlike its parent strain, the B. abortus bhuA mutant is unable to maintain a chronic spleen infection in experimentally infected BALB/c mice. These experimental findings suggest that heme and/or heme-containing proteins represent important iron sources for B. abortus 2308 during its residence in the mammalian host and that BhuA is required for efficient utilization of these iron sources.