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Deoxyribonucleic acid hybridization and plasmid studies in the genus Lactobacillus
Using thermal denaturation methodology, the nucleic acid
base composition of deoxyribonucleic acid (DNA) isolated from type,
neotype and stock strains of Lactobacillus species was determined.
The genus was found to be heterogeneous in base composition,
ranging from 33.1 to 50.0 moles % guanine plus cytosine (GC).
Genus members fell into three groups based on DNA GC content.
Group I included species with a GC content between 33.1 and
39. 7 moles %: L. salivarius, L. jensenii, L. acidophillus, L.
piscium sp. nov. (fish isolate), L. helveticus, and L. sanfrancisco
(sourdough bread isolate). Group II included species with a GC
content between 44. 0 and 48. 0 moles %: L. plantarum, L. rhamnosus
(human isolate) and L. casei var. casei. Group III included organisms
with a GC content between 48.4 and 50.0 moles %: L. leichmanii
and L. fermenti. Several well known Lactobacillus species were given varietal status.
These three groups were further divided into homofermentative
and heterofermentative subgroups leading to the formation of six
groups. Speciation of group members was determined from DNA,
DNA hybridization results as follows:
Homofermentative group I - L. salivarius, L. jensenii, L.
acidophillus, L. helveticus, and L. piscium sp. nov.
Heterofermentative group I - L. sanfrancisco
Homofermentative group II - L. plantarum, L. casei and
L. rhamnosus (formerly L. casei var. rhamnosus)
Heterofermentative group II - L. brevis and L. viridescens.
Homofermentative group III - L. leichrnanii, including the
varieties lactis, bulgaricus and delbrueckii
Heteromentative group III - L. fermenti, including the cellobiosus
variety
This classification, while differing significantly from the traditional
taxonomic system for these organisms based on biochemical and
serological properties, nevertheless constitutes a firm basis for
species identification. This is substantiated from these homology
values:
1. With L. salivarius 11 742 as reference organism, the following
homology values for the indicated organisms were obtained:
L. helveticus 15009, 10%; L. jensenii 25258, 0%, L. acidophilus 4356, 9%.
2. With L. piscium as the reference organism, the following
homology values for the indicated organisms were obtained:
L. piscium LRPKI-70, 92 %, L. salivarius 11742, 2, 45%; L.
jensenii 25258, 22%; L. helveticus 8018, 16%; L. jugurt 521,
32%; Erysipelotrix insidiosa 19414, 14%.
3. With L. helveticus 15009 as reference organism, the following
homology values for the indicated organisms were obtained:
L. jugurt 521, 79%; L. jensenii 25258, 0%; L. acidophilus
4356, 0%; L. salivarius 11742, 9%; L. sanfrancisco WRRL-L,
0%; L. piscium 0%;
4. With L. sanfrancisco WRRL B as reference organism, the following
homology values for the indicated organisms were obtained:
L. sanfrancisco WRRL C, 89%; L. sanfracisco WRRL
L, 95%; L. . sanfrancisco WRRL T95 %; L. acidophilus 4536, 23%;
L. salivarius 11742, 12 %; L. jugurt 521, 6%; L. helveticus
8014, 32%; L. brevis NCDO 473, 39%.
5. With L. plantarurn 1491 7 as the reference organism, the following
homology values for the indicated organisms were obtained:
L. casei 393, 1 7%; L. casei 7469, 0%; L. brevis NCDO 473,
0%; L. lactis 1 231 5, 0%; L. fermenti NCDO 215, 2%; S.
inulinus, 0%; L. helveticus 1 5009, 6%; Lactobacillus MSH,
0%; Lactobacillus Sardo 6, 84%; Lactobacillus DS46F, 88%. 6. With. L. casei 393 as the reference organism, the following
homology values for the indicated organisms were obtained:
L. casei 7469, 14%; L. plantarum 14917, 43%; L. brevis NCDO
473, 16%; L. fermenti NCDO 215, 4%; L. helveticus 15009, 17%;
L. salivarius 11 742, 10%; S. inulinus, 21%; L. lactis 1 231 5,
4%; Lactobacillus Sardo 6,36%; Lactobacillus MSH, 41%; Lactobacillus
DS46F, 24%.
7. With L. lactis 1 231 5 as the reference organism, the following
homology values for the indicated organisms were obtained:
L. bulgaricus 11842, 78%; L. delbrueckii 9649, 85%; L. leich.-
manii 4797, 78%; L, fermenti, NCDO 215, 11%; L. cellobiosus
NCDO 9 27, 8%; L. helveticus 1 5009, 30%.
8. With L. fermenti NCDO 215 as the reference organism the following
homology values for the indicated organisms were obtained:
L. cellobiosus NCDO 927, 83%; L. delbrueckii 9649,
0%; L. leichmanii 4797, 0%; L. lactis 1 2315, 6%; L. bulgaricus
11842, 11%; L. helveticus 15009, 21%.
These homology values were obtained from experiments done at the
stringent hybridization conditions of Tm 15 C.
Genome size determinations also were carried out and two
findings emerged: one, lactobacilli have approximately half the
genetic capability of Escherichia coli and two, Sporolactobacillus
inulinus has a genome which is as large as E. coli and similar to members of the Bacillus genus.
Cesium chloride density gradient centrifugation and agarose
gel electrophoresis revealed the presence of supercoiled (plasmid)
DNA in L. rhamnosus MSH. The plasmid was approximately one
million daltons in molecular weight.
DNA hybridization studies of a Lactobacillus associated with
fish kidney disease revealed that it is a new member of the genus.
It was assigned the name L. piscium
Salmonella enterica: Latency
Infection caused by more than 1500 serotypes of Salmonella enterica subsp. enterica is one of the most common food-borne diseases, prevalent worldwide. Concerning public health, Salmonella latent carrier animals represent an important source of transmission of the disease. They are responsible for silent introduction of the bacteria into the food chain and the environment. Most pathogenesis studies of salmonellosis are focused on events that lead to clinical disease. Researchers have been unable to clearly discern the interaction between intracellular microorganisms and their resistant hosts in latency. However, understanding this interaction is essential for the proper employment of the control and eradication strategies. Thus, the objective of this article is to present an overview of some important events that occur during the infection cycle of S. enterica in latent carriers
In search of a combined brucellosis and tuberculosis vaccine for cattle
Bovine brucellosis is caused by Brucella abortus. The bacterial pathogen causes economic losses because it induces abortion in cattle. Vaccination of calves with live B. abortus strain 19 induces a certain level of protection but induces persistent antibodies against cell envelope lipopolysaccharide that make it difficult to Distinguish Infected from Vaccinated Animals (DIVA). Live vaccine B. abortus strain RB51 was developed to eliminate such interfering antibodies and therefore, facilitate the differentiation of infected from vaccinated animals and help in the eradication of the disease. Vaccination with strain RB51 induces levels of protection similar to strain 19 but neither of the two vaccines give complete protection. We have been working to enhance protection induced by strain RB51 vaccine. Protective Brucella antigens can be over-expressed in strain RB51 by introducing a plasmid containing the leuB gene and the genes encoding such antigens. To avoid the expression of antibiotic resistance genes, we produced a leuB deficient strain RB51 and introduced a plasmid containing the leuB gene and the genes to be over-expressed. This new strain maintains the plasmid and has induced significantly high protection levels in mice. In addition, it allowed the construction of an RB51 vaccine strain able to express Mycobacterium bovis protective antigens so that the vaccine could protect against brucellosis and tuberculosis simultaneously
Identification of a Single-Nucleotide Insertion in the Promoter Region Affecting the sodC Promoter Activity in Brucella neotomae
Brucella neotomae is not known to be associated with clinical disease in any host species. Previous research suggested that B. neotomae might not express detectable levels of Cu/Zn superoxide dismutase (SOD), a periplasmic enzyme known to be involved in protecting Brucella from oxidative bactericidal effects of host phagocytes. This study was undertaken to investigate the genetic basis for the disparity in SOD expression in B. neotomae. Our Western blot and SOD enzyme assay analyses indicated that B. neotomae does express SOD, but at a substantially reduced level. Nucleotide sequence analysis of region upstream to the sodC gene identified a single-nucleotide insertion in the potential promoter region. The same single-nucleotide insertion was also detected in the sodC promoter of B. suis strain Thomsen, belonging to biovar 2 in which SOD expression was undetectable previously. Examination of the sodC promoter activities using translational fusion constructs with E. coli β-galactosidase demonstrated that the B. neotomae and B. suis biovar 2 promoters were very weak in driving gene expression. Site-directed mutation studies indicated that the insertion of A in the B. neotomae sodC promoter reduced the promoter activity. Increasing the level of SOD expression in B. neotomae through complementation with B. abortus sodC gene did not alter the bacterial survival in J774A.1 macrophage-like cells and in tissues of BALB/c and C57BL/6 mice. These results for the first time demonstrate the occurrence of a single-nucleotide polymorphism affecting promoter function and gene expression in Brucella
Enzymatic, immunological and phylogenetic characterization of Brucella suis urease
<p>Abstract</p> <p>Background</p> <p>The sequenced genomes of the <it>Brucella </it>spp. have two urease operons, <it>ure</it>-1 and <it>ure</it>-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from <it>Brucella suis </it>strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined.</p> <p>Results</p> <p>Urease encoded by the <it>ure</it>-1 operon of <it>Brucella suis </it>strain 1330 was purified to homogeneity using ion exchange and hydrophobic interaction chromatographies. The urease was purified 51-fold with a recovery of 12% of the enzyme activity and 0.24% of the total protein. The enzyme had an isoelectric point of 5, and showed optimal activity at pH 7.0 and 28–35°C. The purified enzyme exhibited a Michaelis-Menten saturation kinetics with a <it>K</it><sub><it>m </it></sub>of 5.60 ± 0.69 mM. Hydroxyurea and thiourea are competitive inhibitors of the enzyme with K<sub>i </sub>of 1.04 ± 0.31 mM and 26.12 ± 2.30 mM, respectively. Acetohydroxamic acid also inhibits the enzyme in a competitive way. The molecular weight estimated for the native enzyme was between 130–135 kDa by gel filtration chromatography and 157 ± 7 kDa using 5–10% polyacrylamide gradient non-denaturing gel. Only three subunits in SDS-PAGE were identified: two small subunits of 14,000 Da and 15,500 Da, and a major subunit of 66,000 Da. The amino terminal sequence of the purified large subunit corresponded to the predicted amino acid sequence encoded by <it>ureC1</it>. The UreC1 subunit was recognized by sera from patients with acute and chronic brucellosis. By phylogenetic and cluster structure analyses, <it>ureC1 </it>was related to the <it>ureC </it>typically present in the <it>Rhizobiales</it>; in contrast, the <it>ureC2 </it>encoded in the <it>ure</it>-2 operon is more related to distant species.</p> <p>Conclusion</p> <p>We have for the first time purified and characterized an active urease from <it>B. suis</it>. The enzyme was characterized at the kinetic, immunological and phylogenetic levels. Our results confirm that the active urease of <it>B. suis </it>is a product of <it>ure</it>-1 operon.</p
Molecular targets for rapid identification of Brucella spp
BACKGROUND: Brucella is an intracellular pathogen capable of infecting animals and humans. There are six recognized species of Brucella that differ in their host preference. The genomes of the three Brucella species have been recently sequenced. Comparison of the three revealed over 98% sequence similarity at the protein level and enabled computational identification of common and differentiating genes. We validated these computational predictions and examined the expression patterns of the putative unique and differentiating genes, using genomic and reverse transcription PCR. We then screened a set of differentiating genes against classical Brucella biovars and showed the applicability of these regions in the design of diagnostic tests. RESULTS: We have identified and tested set of molecular targets that are associated in unique patterns with each of the sequenced Brucella spp. A comprehensive comparison was made among the published genome sequences of B. abortus, B. melitensis and B. suis. The comparison confirmed published differences between the three Brucella genomes, and identified subsets of features that were predicted to be of interest in a functional comparison of B. melitensis and B. suis to B. abortus. Differentiating sequence regions from B. abortus, B. melitensis and B. suis were used to develop PCR primers to test for the existence and in vitro transcription of these genes in these species. Only B. suis is found to have a significant number of unique genes, but combinations of genes and regions that exist in only two out of three genomes and are therefore useful for diagnostics were identified and confirmed. CONCLUSION: Although not all of the differentiating genes identified were transcribed under steady state conditions, a group of genes sufficient to discriminate unambiguously between B. suis, B. melitensis, and B. abortus was identified. We present an overview of these genomic differences and the use of these features to discriminate among a number of Brucella biovars
Complete Genomic Sequence of Bacteriophage Felix O1â€
Bacteriophage O1 is a Myoviridae A1 group member used historically for identifying Salmonella. Sequencing revealed a single, linear, 86,155-base-pair genome with 39% average G+C content, 131 open reading frames, and 22 tRNAs. Closest protein homologs occur in Erwinia amylovora phage φEa21-4 and Escherichia coli phage wV8. Proteomic analysis indentified structural proteins: Gp23, Gp36 (major tail protein), Gp49, Gp53, Gp54, Gp55, Gp57, Gp58 (major capsid protein), Gp59, Gp63, Gp64, Gp67, Gp68, Gp69, Gp73, Gp74 and Gp77 (tail fiber). Based on phage-host codon differences, 7 tRNAs could affect translation rate during infection. Introns, holin-lysin cassettes, bacterial toxin homologs and host RNA polymerase-modifying genes were absent
Brucella suis urease encoded by ure1 but not ure2 is necessary for intestinal infection of BALB/c mice
BACKGROUND: In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. RESULTS: The deduced amino acid sequence of urease-α subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Δure1K (generated by deleting ureD and ureA in ure1 operon), strain 1330Δure2K (ureB and ureC in ure2 operon), strain 1330Δure2C (ureA, ureB, and ureC in ure2 operon), and strain 1330Δure1KΔure2C (ureD and ureA in ure1 operon and ureA, ureB, and ureC in ure2 operon). When grown in urease test broth, strains 1330, 1330Δure2K and 1330Δure2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Δure1K and 1330Δure1KΔure2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Δure1K and 1330Δure1KΔure2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Δure1KΔure2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Δure1K was completely killed, strain 1330Δure2C was partially killed, but strains 1330 and 1330Δure2K were not killed. CONCLUSION: These findings suggest that the ure1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure2 operon apparently enhances the resistance to low-pH killing in-vitro
Intracellular invasion and survival of Brucella neotomae, another possible zoonotic Brucella species.
In 1967, Brucella neotomae was first isolated from Neotoma lepida, the dessert wood rat, in Utah. With little infection data since its discovery, the zoonotic potential of this Brucella species is largely unknown. Recent reports of isolation from human cerebrospinal fluid, along with current literature suggest that B. neotomae has the ability to infect various hosts and cell types. In this report we extend the knowledge of B. neotomae ATCC 23459's intracellular invasion and survival abilities to a variety of cell lines through gentamicin protection assays. Some of the phagocytic and epithelial cell lines from various mammalian species represent characteristics of some cell types that could be encountered by Brucella in potential hosts. It was found that B. neotomae ATCC 23459 exhibits generally lower intracellular bacterial CFUs compared to the mouse-passaged strain of B. neotomae ATCC 23459, B. suis 1330, and B. abortus 2308. Ultimately, these observations provide a small piece of the puzzle in the investigation of the breadth of B. neotomae's pathogenic potential
Flagellin Is an Effective Adjuvant for Immunization against Lethal Respiratory Challenge with Yersinia pestis
Gram-negative flagellin, a Toll-like receptor 5 (TLR5) agonist, is a potent inducer of innate immune effectors such as cytokines and nitric oxide. In the lung, flagellin induces a localized and transient innate immune response characterized by neutrophil infiltration and the production of cytokines and chemokines. In view of the extraordinary potency of flagellin as an inducer of innate immunity and the contribution of innate responses to the development of adaptive immunity, we evaluated the efficacy of recombinant Salmonella flagellin as an adjuvant in an acellular plague vaccine. Mice immunized intranasally or intratracheally with the F1 antigen of Yersinia pestis and flagellin exhibited dramatic increases in anti-F1 plasma immunoglobulin G (IgG) titers that remained stable over time. In contrast, control mice had low or undetectable antibody responses. The IgG1/IgG2a ratio of antibody titers against F1 in immunized mice is consistent with a Th2 bias. However, no significant antigen-specific IgE production was detected. Interferons, tumor necrosis factor alpha, and interleukin-6 were not essential for the adjuvant effects of flagellin. Preexisting antiflagellin antibodies had no significant effect on the adjuvant activity of flagellin. Importantly, intranasal immunization with flagellin and the F1 antigen was protective against intranasal challenge with virulent Y. pestis CO92, with 93 to 100% survival of immunized mice. Lastly, vaccination of cynomolgus monkeys with flagellin and a fusion of the F1 and V antigens of Y. pestis induced a robust antigen-specific IgG antibody response
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