Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis

Brucellosis is a worldwide disease of humans and livestock that is caused by a number of very closely related classical Brucella species in the alpha-2 subdivision of the Proteobacteria. We report the complete genome sequence of Brucella abortus field isolate 9-941 and compare it to those of Brucella suis 1330 and Brucella melitensis 16 M. The genomes of these Brucella species are strikingly similar, with nearly identical genetic content and gene organization. However, a number of insertion-deletion events and several polymorphic regions encoding putative outer membrane proteins were identified among the genomes. Several fragments previously identified as unique to either B. suis or B. melitensis were present in the B. abortus genome. Even though several fragments were shared between only B. abortus and B. suis, B. abortus shared more fragments and had fewer nucleotide polymorphisms with B. melitensis than B. suis. The complete genomic sequence of B. abortus provides an important resource for further investigations into determinants of the pathogenicity and virulence phenotypes of these bacteria.     

Complete genome sequence of Brucella suis VBI22, isolated from bovine milk

Brucella suis is the causative agent of swine brucellosis and is known to be able to infect several different hosts, including cattle, dogs, and horses, without causing disease symptoms. Here we report the complete genome sequence of Brucella suis VBI22, which was isolated from raw milk from an infected cow.     

Differences in chromosome number and genome rearrangements in the genus Brucella

We have studied the genomic structure and constructed the Spe I, Pac I and I- Ceu I restriction maps of the four biovars of the pathogenic bacterium Brucella suis . B . suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B . melitensis , B . abortus , B . ovis and B . neotomae . Two chromosomes were also observed in the genome of B . suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B . suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome-containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B . suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.     

上海地区散发布氏杆菌感染的细菌学及分子鉴定

目的 本研究对我院的1例散发布氏杆菌病患者进行细菌学及分子生物学的分析,并在国内首次尝试了用数目可变串联重复单元(VNTR)分子指纹分析法对其进行了基因分型并和国际流行株进行了分子流行病学比较分析。方法 对临床疑似布氏杆菌病病例作血液细菌培养与生化鉴定,进一步作布氏杆菌特异性基因片段的序列分析鉴定以及利用布氏杆菌基因组中的8个位点构建VNTR指纹图谱,参照国际布氏杆菌VNTR数据库,构建布氏杆菌基因系统树。结果 用细菌学方法确定散发疑似布氏杆菌病病例体内分离到的为布氏杆菌,通过基因序列分析进一步得到证实,但不能鉴定到生物种和生物型。对分离株作VNTR指纹分析提示该散发布氏杆菌病为猪2型布氏杆菌感染所致。结论 通过传统细菌培养方法与布氏杆菌VNTR指纹分析可用于我国布氏杆菌病分子流行病学的系统调查。     

Taxonomic Position in the Genus Brucella of the Causative Agent of Canine Abortion

The gram-negative organism causing abortion in dogs was examined in parallel with cultures representative of the Brucella species and with Bordetella bronchiseptica. The organism fits into the genus Brucella and most closely resembles B. suis on the basis of its growth characteristics. It is of rough colonial morphology and is agglutinated by antisera prepared against rough Brucella. In mouse toxicity tests, no endotoxic activity could be demonstrated. In contrast to most Brucella cultures, it does not utilize erythritol. Electron microscopy showed a cell wall structure similar to that of other gram-negative organisms. The question of whether the organism should be designated Brucella canis, as proposed by Carmichael and Bruner, or Brucella suis biotype 5 is discussed. The authors favor the designation Brucella canis because the organism lacks the lipopolysaccharide antigen associated with the smooth agglutinogen and endotoxin, and it does not utilize erythritol.     

Advanced multiplex PCR assay for differentiation of Brucella species

Two new primer sets of a 766- and a 344-bp fragment were introduced into the conventional Bruce-ladder PCR assay. This novel multiplex PCR assay rapidly and concisely discriminates Brucella canis and Brucella microti from Brucella suis strains and also may differentiate all of the 10 Brucella species.     

Type III secretion homologs are present in Brucella melitensis,B. ovis,and B. suis biovars 1, 2, and 3

Protein sequences from characterized type III secretion (TTS) systems were used as probes in silico to identify several TTS gene homologs in the genome sequence of Brucella suis biovar 1 strain 1330. Four of the genes, named flhB, fliP, fliR, and fliF on the basis of greatest homologies to known flagellar apparatus proteins, were targeted in PCR and hybridization assays to determine their distribution among other Brucella nomen species and biovars. The results indicated that flhB, fliP, fliR and fliF are present in Brucella melitensis, Brucella ovis, and Brucella suis biovars 1, 2 and 3. Similar homologos have been reported previously in Brucella abortus. Using RT-PCR assays, we were unable to detect any expression of these genes. It is not yet known whether the genes are the cryptic remnants of a flagellar system or are actively involved in a process contributing to pathogenicity or previously undetected motility, but they are distributed widely in Brucella and merit further study to determine their role.     

A study of the formation of antibodies in new-born animals following transfer of nucleoprotein fractions

1. (1)
   Мы выделяли РНП и ДНП из селезенки через 24 и 48 час. после введения 1 дозы антигена Brucella suis взрослому кролику. Нуклеопротеины, перенесенные 3–5-дневным крольчатам, не вызывали у реципиентов появления антител к Brucella suis.     
   
   

A large domain swap in the VirB11 ATPase of Brucella suis leaves the hexameric assembly intact

VirB11 ATPases are hexameric assemblies that power type IV secretion systems in bacteria. The hexamer of Brucella suis VirB11 (BsB11), like that of the Helicobacter pylori VirB11 (Hp0525), consists of a double ring structure formed by the N-terminal and C-terminal domains of each monomer. However, the monomer differs dramatically from that of Hp0525 by a large domain swap that leaves the hexameric assembly intact but profoundly alters the nucleotide-binding site and the interface between subunits.     

用MLVA技术和多重PCR对犬种布氏菌基因分型

目的：对犬种布氏菌的遗传关系进行不同分子分型方法的对比研究,为犬布病分子流行病溯源提供有效方法。方法：采用多重PCR和多位点可变数量串联重复序列分析（MLVA）方法对24株犬种布氏菌的遗传关系进行比较研究。结果：多重PCR只鉴定出1株犬种布氏菌,其余23株均鉴定为猪种鲁氏菌,但不能鉴定型别;MLVA方法对已鉴定为猪种的布氏菌仍可再细分为型,87%（20/23）为猪3型,13%（3/20）为猪1型。结论：MLVA可以对布氏菌种（生物型）进行基因分型鉴定,可以作为传统表型鉴定方法的补充。     

Whole genome sequences of four Brucella strains

Brucella melitensis and Brucella suis are intracellular pathogens of livestock and humans. Here we report four genome sequences, those of the virulent strain B. melitensis M28-12 and vaccine strains B. melitensis M5 and M111 and B. suis S2, which show different virulences and pathogenicities, which will help to design a more effective brucellosis vaccine.     

Experimental Brucella suis biovar 4 infection in a moose

A moose (Alces alces gigas) was inoculated with Brucella suis biovar 4 to better understand the effects of brucellosis in this species. Serum antibody titers increased rapidly and peaked within 21 to 56 days. Fever, leukocytosis, recumbency, anorexia and depression were observed starting 42 days post inoculation. Brucella suis biovar 4 was isolated from blood, lymph nodes, liver and spleen.     

BmaC, a novel autotransporter of Brucella suis, is involved in bacterial adhesion to host cells

Brucella is an intracellular pathogen responsible of a zoonotic disease called brucellosis. Brucella survives and proliferates within several types of phagocytic and non-phagocytic cells. Like in other pathogens, adhesion of brucellae to host surfaces was proposed to be an important step in the infection process. Indeed, Brucella has the capacity to bind to culture human cells and key components of the extracellular matrix, such as fibronectin. However, little is known about the molecular bases of Brucella adherence. In an attempt to identify bacterial genes encoding adhesins, a phage display library of Brucella suis was panned against fibronectin. Three fibronectin-binding proteins of B. suis were identified using this approach. One of the candidates, designated BmaC was a very large protein of 340 kDa that is predicted to belong to the type I (monomeric) autotransporter family. Microscopy studies showed that BmaC is located at one pole on the bacterial surface. The phage displaying the fibronectin-binding peptide of BmaC inhibited the attachment of brucellae to both, HeLa cells and immobilized fibronectin in vitro. In addition, a bmaC deletion mutant was impaired in the ability of B. suis to attach to immobilized fibronectin and to the surface of HeLa and A549 cells and was out-competed by the wild-type strain in co-infection experiments. Finally, anti-fibronectin or anti-BmaC antibodies significantly inhibited the binding of wild-type bacteria to HeLa cells. Our results highlight the role of a novel monomeric autotransporter protein in the adhesion of B. suis to the extracellular matrix and non-phagocytic cells via fibronectin binding.     

Characterization of Brucella suis clpB and clpAB mutants and participation of the genes in stress responses

Pathogens often encounter stressful conditions inside their hosts. In the attempt to characterize the stress response in Brucella suis, a gene highly homologous to Escherichia coli clpB was isolated from Brucella suis, and the deduced amino acid sequence showed features typical of the ClpB ATPase family of stress response proteins. Under high-temperature stress conditions, ClpB of B. suis was induced, and an isogenic B. suis clpB mutant showed increased sensitivity to high temperature, but also to ethanol stress and acid pH. The effects were reversible by complementation. Simultaneous inactivation of clpA and clpB resulted in a mutant that was sensitive to oxidative stress. In B. suis expressing gfp, ClpA but not ClpB participated in degradation of the green fluorescent protein at 42 degrees C. We concluded that ClpB was responsible for tolerance to several stresses and that the lethality caused by harsh environmental conditions may have similar molecular origins.     

Homologies of Deoxyribonucleic Acids from Brucella ovis, Canine Abortion Organisms, and other Brucella Species

The bacterium that causes canine abortion has polynucleotide sequences similar, in deoxyribonucleic acid (DNA)-DNA homology studies, to those of Brucella suis and, by inference from previous data, those of B. abortus and B. melitensis as well as B. neotomae. Therefore, the organism causing canine abortion appears to be a member of the genus Brucella. DNA preparations from Serratia marcescens, Alcaligenes faecalis, and Bordetella bronchiseptica, 58, 62, and 66 mole% guanine plus cytosine, respectively, do not have detectable polynucleotide sequence homologies with B. suis DNA which is 56 mole% guanine plus cytosine. B. ovis DNA lacks some of the polynucleotide sequences present in B. suis DNA and appears to be a deletion mutant. However, a large proportion of B. ovis polynucleotides are similar to those of other Brucella species, which supports the inclusion of B. ovis in the genus.     

Brucella suis histidinol dehydrogenase: synthesis and inhibition studies of a series of substituted benzylic ketones derived from histidine

Brucella spp. is the causative agent of brucellosis (Malta fever), which is the most widespread zoonosis worldwide. The pathogen is capable of establishing persistent infections in humans which are extremely difficult to eradicate even with antibiotic therapy. Moreover, Brucella is considered as a potential bioterrorism agent. Histidinol dehydrogenase (HDH, EC 1.1.1.23) has been shown to be essential for the intramacrophagic replication of this pathogen. It therefore constitutes an original and novel target for the development of anti-Brucella agents. In this work, we cloned and overexpressed the HDH-encoding gene from Brucella suis, purified the protein and evidenced its biological activity. We then investigated the inhibitory effects of a series of substituted benzylic ketones derived from histidine. Most of the compounds reported here inhibited B. suis HDH in the lower nanomolar range and constitute attractive candidates for the development of novel anti-Brucella agents.     

Cloning, expression, and purification of Brucella suis outer membrane proteins

Brucella, an aerobic, nonsporeforming, nonmotile Gram-negative coccobacillus, is a NIH/CDC category B bioterror threat agent that causes incapacitating human illness. Medical defense against the bioterror threat posed by Brucella would be strengthened by development of a human vaccine and improved diagnostic tests. Central to advancement of these goals is discovery of bacterial constituents that are immunogenic or antigenic for humans. Outer membrane proteins (OMPs) are particularly attractive for this purpose. In this study, we cloned, expressed, and purified seven predicted OMPs of Brucella suis. The recombinant proteins were fused with 6-His and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based on their ORF sequences and directly cloned into an entry vector. The recombinant entry constructs were propagated in TOP 10 cells, recombined into a destination vector, pET-DEST42, then transformed into Escherichia coli BL21 cells for IPTG-induced protein expression. The expressed recombinant proteins were confirmed with Western blot analysis using anti-6-His antibody conjugated with alkaline phosphatase. These B. suis OMPs were captured and purified using a HisGrab plate. The purified recombinant proteins were examined for their binding activity with antiserum. Serum derived from a rabbit immunized intramuscularly with dialyzed cell lysate of Brucella rough mutant WRR51. The OMPs were screened using the rabbit antiserum and purified IgG. The results suggested that recombinant B. suis OMPs were successfully cloned, expressed and purified. Some of the expressed OMPs showed high binding activity with immunized rabbit antiserum.     

Brucella suis biotype 4: a case of granulomatous nephritis in a barren ground caribou (Rangifer tarandus groenlandicus L.) with a review of the distribution of rangiferine brucellosis in Canada

Severe granulomatous nephritis caused by Brucella suis biotype 4 was found in a barren ground caribou (Rangifer tarandus groenlandicus) from Northwest Territories, Canada. A review of the distribution of human and animal cases of brucellosis in northern Canada indicated that B. suis biotype 4 is distributed widely and is probably enzootic in most Canadian caribou herds.     

Phospholipids of bacteria of Brucella genus

The phospholipid composition of 6 Brucella species (B. melitensis, B. abortus, B. suis, B. ovis. B. canis, B. neotomae) and Australian mouse-derived strains of Brucella N 4, 11, 12 were studied. Comparison of phospholipid composition of Brucella cells with that of serologically related microorganisms revealed that all Brucella biotypes contain phosphatidyl-(N-methyl)ethanolamine and phosphatidylcholine while Y. enterocolitica, Sh. disenteriae, E. coli cells do not contain these two substances. It is concluded that the specific phospholipid pattern of Brucella biotypes may be useful in typing of new Brucella strains.