Bacillus cereus Fur regulates iron metabolism and is required for full virulence

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 569-577 ◽  
Author(s):  
Duncan R. Harvie ◽  
Susana Vílchez ◽  
James R. Steggles ◽  
David J. Ellar
Keyword(s):  
Bacillus Cereus ◽  
Kanamycin Resistance ◽  
Infection Model ◽  
Predicted Amino Acid Sequence ◽  
Dependent Manner ◽  
Genome Data ◽  
Kanamycin Resistance Cassette ◽  
Allelic Replacement ◽  
Cloned Gene ◽  
Null Strain

A homologue of the Bacillus subtilis fur gene was identified in Bacillus cereus and characterized. The predicted amino acid sequence of the cloned gene was found to be highly similar to other members of the Fur family of transcriptional regulators. The B. cereus fur gene was shown to partially complement an Escherichia coli fur mutant. Purified B. cereus Fur bound specifically to a 19 bp DNA sequence homologous to the B. subtilis Fur box in a metal-dependent manner. Analysis of the available B. cereus genome data identified a number of genes which contain predicted Fur box sequences in the promoter region. Many of these genes are predicted to play a role in bacterial iron uptake and metabolism, but several have also been implicated as having a role in virulence. Fur and iron regulation of a siderophore biosynthesis operon was confirmed in a β-galactosidase assay. A B. cereus fur null strain was constructed by allelic replacement of the chromosomal gene with a copy disrupted with a kanamycin resistance cassette. The Δfur mutant was found to constitutively express siderophores, to accumulate iron intracellularly to a level approximately threefold greater than the wild-type, and to be hypersensitive to hydrogen peroxide. In an insect infection model, the virulence of the fur null strain was found to be significantly attenuated, highlighting the essential role played by Fur in the virulence of this pathogen.

scholarly journals Kanamycin Resistance Cassette for Genetic Manipulation of Treponema denticola

2015 ◽  
Vol 81 (13) ◽  
pp. 4329-4338 ◽  
Author(s):  
Yuebin Li ◽  
John Ruby ◽  
Hui Wu
Keyword(s):  
Genetic Manipulation ◽  
Periodontal Diseases ◽  
Genetic System ◽  
Kanamycin Resistance ◽  
Treponema Denticola ◽  
Oral Pathogen ◽  
Content Type ◽  
Kanamycin Resistance Cassette ◽  
Allelic Replacement ◽  
Kanamycin Cassette

ABSTRACTTreponema denticolahas been recognized as an important oral pathogen of the “red complex” bacterial consortium that is associated with the pathogenesis of endodontal and periodontal diseases. However, little is known about the virulence ofT. denticoladue to its recalcitrant genetic system. The difficulty in genetically manipulating oral spirochetes is partially due to the lack of antibiotic resistance cassettes that are useful for gene complementation following allelic replacement mutagenesis. In this study, a kanamycin resistance cassette was identified and developed for the genetic manipulation ofT. denticolaATCC 35405. Compared to the widely usedermF-ermAMcassette, the kanamycin cassette used in the transformation experiments gave rise to additional antibiotic-resistantT. denticolacolonies. The kanamycin cassette is effective for allelic replacement mutagenesis as demonstrated by inactivation of two open reading frames ofT. denticola, TDE1430 and TDE0911. In addition, the cassette is also functional intrans-chromosomal complementation. This was determined by functional rescue of a periplasmic flagellum (PF)-deficient mutant that had theflgEgene coding for PF hook protein inactivated. The integration of the full-lengthflgEgene into the genome of theflgEmutant rescued all of the defects associated with theflgEmutant that included the lack of PF filament and spirochetal motility. Taken together, we demonstrate that the kanamycin resistance gene is a suitable cassette for the genetic manipulation ofT. denticolathat will facilitate the characterization of virulence factors attributed to this important oral pathogen.

scholarly journals Oligoribonuclease Is Encoded by a Highly Conserved Gene in the 3′-5′ Exonuclease Superfamily

1998 ◽  
Vol 180 (10) ◽  
pp. 2779-2781 ◽  
Author(s):  
Xiaolan Zhang ◽  
Liuqin Zhu ◽  
Murray P. Deutscher
Keyword(s):  
Kanamycin Resistance ◽  
Open Reading Frame ◽  
Sequence Information ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Kanamycin Resistance Cassette ◽  
Wide Range ◽  
Conserved Gene ◽  
Cloned Gene

ABSTRACT Oligoribonuclease, a 3′-to-5′ exoribonuclease specific for small oligoribonucleotides, was purified to homogeneity from extracts ofEscherichia coli. The purified protein is an α2 dimer of 40 kDa. NH2-terminal sequence analysis of the protein identified the gene encoding oligoribonuclease as yjeR(o204a), a previously reported open reading frame located at 94 min on the E. coli chromosome. However, as a consequence of the sequence information, the translation start site of this open reading frame has been revised. Cloning of yjeRled to overexpression of oligoribonuclease activity, and interruption of the cloned gene with a kanamycin resistance cassette eliminated the overexpression. On the basis of these data, we propose thatyjeR be renamed orn. Orthologs of oligoribonuclease are present in a wide range of organisms, extending up to humans.

scholarly journals Potentiating Effect of Mandelate and Lactate on Chemically Induced Germination in Members of Bacillus cereus Sensu Lato

2017 ◽  
Vol 83 (24) ◽  
Author(s):  
Alistair H. Bishop
Keyword(s):  
Bacillus Cereus ◽  
Large Scale ◽  
Germination Rate ◽  
Dependent Manner ◽  
Clostridium Sporogenes ◽  
Specific Chemical ◽  
Content Type ◽  
Ph Range ◽  
Significant Discovery ◽  
The Impact

ABSTRACT Endospores of the genus Bacillus can be triggered to germinate by a limited number of chemicals. Mandelate had powerful additive effects on the levels and rates of germination produced in non-heat-shocked spores of Bacillus anthracis strain Sterne, Bacillus cereus, and Bacillus thuringiensis when combined with l-alanine and inosine. Mandelate had no germinant effect on its own but was active with these germinants in a dose-dependent manner at concentrations higher than 0.5 mM. The maximum rate and extent of germination were produced in B. anthracis by 100 mM l-alanine with 10 mM inosine; this was equaled by just 25% of these germinants when supplemented with 10 mM mandelate. Half the maximal germination rate was produced by 40% of the optimum germinant concentrations or 15% of them when supplemented with 0.8 mM mandelate. Germination rates in B. thuringiensis were highest around neutrality, but the potentiating effect of mandelate was maintained over a wider pH range than was germination with l-alanine and inosine alone. For all species, lactate also promoted germination in the presence of l-alanine and inosine; this was further increased by mandelate. Ammonium ions also enhanced l-alanine- and inosine-induced germination but only when mandelate was present. In spite of the structural similarities, mandelate did not compete with phenylalanine as a germinant. Mandelate appeared to bind to spores while enhancing germination. There was no effect when mandelate was used in conjunction with nonnutrient germinants. No effect was produced with spores of Bacillus subtilis, Clostridium sporogenes, or C. difficile. IMPORTANCE The number of chemicals that can induce germination in the species related to Bacillus cereus has been defined for many years, and they conform to specific chemical types. Although not a germinant itself, mandelate has a structure that is different from these germination-active compounds, and its addition to this list represents a significant discovery in the fundamental biology of spore germination. This novel activity may also have important applied relevance given the impact of spores of B. cereus in foodborne disease and B. anthracis as a threat agent. The destruction of spores of B. anthracis, for example, particularly over large outdoor areas, poses significant scientific and logistical problems. The addition of mandelate and lactate to the established mixtures of l-alanine and inosine would decrease the amount of the established germinants required and increase the speed and level of germination achieved. The large-scale application of “germinate to decontaminate” strategy may thus become more practicable.

AgmR controls transcription of a regulon with several operons essential for ethanol oxidation in Pseudomonas aeruginosa ATCC 17933

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1851-1857 ◽  
Author(s):  
Nicole Gliese ◽  
Viola Khodaverdi ◽  
Max Schobert ◽  
Helmut Görisch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ethanol Oxidation ◽  
Response Regulator ◽  
Regulatory System ◽  
Pyrroloquinoline Quinone ◽  
Kanamycin Resistance ◽  
Kanamycin Resistance Cassette ◽  
Two Component ◽  
Ethanol Dehydrogenase ◽  
Oxidation System

The response regulator AgmR was identified to be involved in the regulation of the quinoprotein ethanol oxidation system of Pseudomonas aeruginosa ATCC 17933. Interruption of the agmR gene by insertion of a kanamycin-resistance cassette resulted in mutant NG3, unable to grow on ethanol. After complementation with the intact agmR gene, growth on ethanol was restored. Transcriptional lacZ fusions were used to identify four operons which are regulated by the AgmR protein: the exaA operon encodes the pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the exaBC operon encodes a soluble cytochrome c 550 and an aldehyde dehydrogenase, the pqqABCDE operon carries the PQQ biosynthetic genes, and operon exaDE encodes a two-component regulatory system which controls transcription of the exaA operon. Transcription of exaA was restored by transformation of NG3 with a pUCP20T derivative carrying the exaDE genes under lac-promoter control. These data indicate that the AgmR response regulator and the exaDE two-component regulatory system are organized in a hierarchical manner. Gene PA1977, which appears to form an operon with the agmR gene, was found to be non-essential for growth on ethanol.

PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors

1986 ◽  
Vol 6 (7) ◽  
pp. 2490-2499
Author(s):  
G Ammerer ◽  
C P Hunter ◽  
J H Rothman ◽  
G C Saari ◽  
L A Valls ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Structural Gene ◽  
Yeast Cells ◽  
Linkage Data ◽  
Predicted Amino Acid Sequence ◽  
Multicopy Plasmid ◽  
Proteinase A ◽  
Pep4 Gene ◽  
Cloned Gene

The proteinase A structural gene of Saccharomyces cerevisiae was cloned by using an immunological screening procedure that allows detection of yeast cells which are aberrantly secreting vacuolar proteins (J. H. Rothman, C. P. Hunter, L. A. Valls, and T. H. Stevens, Proc. Natl. Acad. Sci. USA, 83:3248-3252, 1986). A second cloned gene was obtained on a multicopy plasmid by complementation of a pep4-3 mutation. The nucleotide sequences of these two genes were determined independently and were found to be identical. The predicted amino acid sequence of the cloned gene suggests that proteinase A is synthesized as a 405-amino-acid precursor which is proteolytically converted to the 329-amino-acid mature enzyme. Proteinase A shows substantial homology to mammalian aspartyl proteases, such as pepsin, renin, and cathepsin D. The similarities may reflect not only analogous functions but also similar processing and intracellular targeting mechanisms for the two proteins. The cloned proteinase A structural gene, even when it is carried on a single-copy plasmid, complements the deficiency in several vacuolar hydrolase activities that is observed in a pep4 mutant. A strain carrying a deletion in the genomic copy of the gene fails to complement a pep4 mutant of the opposite mating type. Genetic linkage data demonstrate that integrated copies of the cloned proteinase A structural gene map to the PEP4 locus. Thus, the PEP4 gene encodes a vacuolar aspartyl protease, proteinase A, that is required for the in vivo processing of a number of vacuolar zymogens.

scholarly journals Prior Exposure to Live Mycobacterium bovis BCG Decreases Cryptococcus neoformans-Induced Lung Eosinophilia in a Gamma Interferon-Dependent Manner

2003 ◽  
Vol 71 (6) ◽  
pp. 3384-3391 ◽  
Author(s):  
Gerhard Walzl ◽  
Ian R. Humphreys ◽  
Ben G. Marshall ◽  
Lorna Edwards ◽  
Peter J. M. Openshaw ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Mycobacterium Bovis ◽  
Mycobacterial Infection ◽  
Gamma Interferon ◽  
Delayed Type Hypersensitivity ◽  
Infection Model ◽  
Dependent Manner ◽  
Interleukin 5 ◽  
Lung Eosinophilia ◽  
Mycobacterial Antigens

ABSTRACT Some common childhood infections appear to prevent the development of atopy and asthma. In some Mycobacterium bovis BCG-vaccinated populations, strong delayed-type hypersensitivity responses to mycobacterial antigens are associated with a reduced risk of atopy. Although BCG exposure decreases allergen-induced lung eosinophilia in animal models, little attention has been given to the effect of immunity to BCG on responses against live pathogens. We used the murine Cryptococcus neoformans infection model to investigate whether prior BCG infection can alter such responses. The present study shows that persistent pulmonary BCG infection of C57BL/6 mice induced an increase in gamma interferon, a reduction in interleukin-5, and a decrease in lung eosinophilia during subsequent Cryptococcus infection. This effect was long lasting, depended on the presence of live bacteria, and required persistence of mycobacterial infection in the lung. Reduction of eosinophilia was less prominent after infection with a mutant BCG strain (ΔhspR), which was rapidly cleared from the lungs. These observations have important implications for the development of vaccines designed to prevent Th2-mediated disease and indicate that prior lung BCG vaccination can alter the pattern of subsequent host inflammation.

scholarly journals Escherichia coli Mutants Lacking All Possible Combinations of Eight Penicillin Binding Proteins: Viability, Characteristics, and Implications for Peptidoglycan Synthesis

1999 ◽  
Vol 181 (13) ◽  
pp. 3981-3993 ◽  
Author(s):  
Sylvia A. Denome ◽  
Pamela K. Elf ◽  
Thomas A. Henderson ◽  
David E. Nelson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Binding Proteins ◽  
Kanamycin Resistance ◽  
Open Reading Frames ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Kanamycin Resistance Cassette ◽  
Genes Encoding

ABSTRACT The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology ofEscherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two ressites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via λ phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bpres site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, thedacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the β-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.

scholarly journals Lactobacillus rhamnosus Ameliorates Multi-Drug-Resistant Bacillus cereus-Induced Cell Damage through Inhibition of NLRP3 Inflammasomes and Apoptosis in Bovine Endometritis

2022 ◽  
Vol 10 (1) ◽  
pp. 137
Author(s):  
Ning Liu ◽  
Xue Wang ◽  
Qiang Shan ◽  
Le Xu ◽  
Yanan Li ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Cell Damage ◽  
Membrane Surface ◽  
Lactobacillus Rhamnosus ◽  
Animal Husbandry ◽  
Infection Model ◽  
Drug Resistant ◽  
Cell Infection ◽  
Endometrial Epithelial Cell ◽  
Food Borne

Bacillus cereus, considered a worldwide human food-borne pathogen, has brought serious health risks to humans and animals and huge losses to animal husbandry. The plethora of diverse toxins and drug resistance are the focus for B. cereus. As an alternative treatment to antibiotics, probiotics can effectively alleviate the hazards of super bacteria, food safety, and antibiotic resistance. This study aimed to investigate the frequency and distribution of B. cereus in dairy cows and to evaluate the effects of Lactobacillus rhamnosus in a model of endometritis induced by multi-drug-resistant B. cereus. A strong poisonous strain with a variety of drug resistances was used to establish an endometrial epithelial cell infection model. B. cereus was shown to cause damage to the internal structure, impair the integrity of cells, and activate the inflammatory response, while L. rhamnosus could inhibit cell apoptosis and alleviate this damage. This study indicates that the B. cereus-induced activation of the NLRP3 signal pathway involves K+ efflux. We conclude that LGR-1 may relieve cell destruction by reducing K+ efflux to the extracellular caused by the perforation of the toxins secreted by B. cereus on the cell membrane surface.

scholarly journals Cloning and Characterization of α-Methylacyl Coenzyme A Racemase from Gordonia polyisoprenivorans VH2

2008 ◽  
Vol 74 (22) ◽  
pp. 7085-7089 ◽  
Author(s):  
Quyen Arenskötter ◽  
Jens Heller ◽  
David Dietz ◽  
Matthias Arenskötter ◽  
Alexander Steinbüchel
Keyword(s):  
Cytochrome P450 ◽  
Reverse Transcription ◽  
Kanamycin Resistance ◽  
P450 Monooxygenase ◽  
Transcription Analysis ◽  
Kanamycin Resistance Cassette ◽  
Short Chain Dehydrogenase ◽  
Gordonia Polyisoprenivorans ◽  
Rubber Degradation

ABSTRACT The mcr gene of Gordonia polyisoprenivorans VH2 is not clustered with genes required for rubber degradation. Its disruption by insertion of a kanamycin resistance cassette impaired growth on methyl-branched isoprenoids but not on linear hydrocarbons. Intact mcr from this bacterium or from Nocardia farcinica IFM 10152 complemented the mutant. Reverse transcription analysis showed similar mcr VH2 expression results during cultivation with poly(cis-1,4-isoprene) and propionate. Additional genes coding for a putative cytochrome P450 monooxygenase and a short-chain dehydrogenase/reductase involved in β-oxidation and poly(cis-1,4-isoprene) degradation were also characterized.

scholarly journals Staphylococcus aureus internalization impairs osteoblastic activity and early differentiation process

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
W. Mouton ◽  
J. Josse ◽  
C. Jacqueline ◽  
L. Abad ◽  
S. Trouillet-Assant ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Post Infection ◽  
Infection Model ◽  
Dependent Manner ◽  
Differentiation Process ◽  
Osteoblastic Activity ◽  
Bone Parameters ◽  
Early Differentiation

AbstractStaphylococcus aureus is the most frequent aetiology of bone and joint infections (BJI) and can cause relapsing and chronic infections. One of the main factors involved in the chronicization of staphylococcal BJIs is the internalization of S. aureus into osteoblasts, the bone-forming cells. Previous studies have shown that S. aureus triggers an impairment of osteoblasts function that could contribute to bone loss. However, these studies focused mainly on the extracellular effects of S. aureus. Our study aimed at understanding the intracellular effects of S. aureus on the early osteoblast differentiation process. In our in vitro model of osteoblast lineage infection, we first observed that internalized S. aureus 8325-4 (a reference lab strain) significantly impacted RUNX2 and COL1A1 expression compared to its non-internalized counterpart 8325-4∆fnbAB (with deletion of fnbA and fnbB). Then, in a murine model of osteomyelitis, we reported no significant effect for S. aureus 8325-4 and 8325-4∆fnbAB on bone parameters at 7 days post-infection whereas S. aureus 8325-4 significantly decreased trabecular bone thickness at 14 days post-infection compared to 8325-4∆fnbAB. When challenged with two clinical isogenic strains isolated from initial and relapse phase of the same BJI, significant impairments of bone parameters were observed for both initial and relapse strain, without differences between the two strains. Finally, in our in vitro osteoblast infection model, both clinical strains impacted alkaline phosphatase activity whereas the expression of bone differentiation genes was significantly decreased only after infection with the relapse strain. Globally, we highlighted that S. aureus internalization into osteoblasts is responsible for an impairment of the early differentiation in vitro and that S. aureus impaired bone parameters in vivo in a strain-dependent manner.

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