scholarly journals Transcriptional Analysis of thetet(P) Operon from Clostridium perfringens

2001 ◽  
Vol 183 (24) ◽  
pp. 7110-7119 ◽  
Author(s):  
Priscilla A. Johanesen ◽  
Dena Lyras ◽  
Trudi L. Bannam ◽  
Julian I. Rood
Keyword(s):  
Clostridium Perfringens ◽  
Gene Deletion ◽  
Transmembrane Protein ◽  
Tetracycline Resistance ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Control Element ◽  
Overlapping Genes ◽  
Reporter Construct ◽  
P Gene

ABSTRACT The Clostridium perfringens tetracycline resistance determinant from the 47-kb conjugative R-plasmid pCW3 is unique in that it consists of two overlapping genes, tetA(P) andtetB(P), which mediate resistance by different mechanisms. Detailed transcriptional analysis has shown that the inducible tetA(P) and tetB(P) genes comprise an operon that is transcribed from a single promoter, P3, located 529 bp upstream of the tetA(P) start codon. Deletion of P3 or alteration of the spacing between the −35 and −10 regions significantly reduced the level of transcription in a reporter construct. Induction was shown to be mediated at the level of transcription. Unexpectedly, a factor-independent terminator, T1, was detected downstream of P3 but before the start of thetetA(P) gene. Deletion or mutation of this terminator led to increased read-through transcription in the reporter construct. It is postulated that the T1 terminator is an intrinsic control element of the tet(P) operon and that it acts to prevent the overexpression of the TetA(P) transmembrane protein, even in the presence of tetracycline.

scholarly journals Genetic organization and distribution of tetracycline resistance determinants in Clostridium perfringens.

1996 ◽  
Vol 40 (11) ◽  
pp. 2500-2504 ◽  
Author(s):  
D Lyras ◽  
J I Rood
Keyword(s):  
Clostridium Perfringens ◽  
Transmembrane Protein ◽  
Tetracycline Resistance ◽  
Wide Distribution ◽  
Pcr Analysis ◽  
Nucleotide Sequence Analysis ◽  
Significant Similarity ◽  
Active Efflux ◽  
P Gene ◽  
Carboxy Terminal

The Tet P determinant from the conjugative Clostridium perfringens R plasmid pCW3 two functional overlapping tetracycline resistance genes, tetA(P) and tetB(P). The tetA(P) gene encodes a putative 46-kDa transmembrane protein which mediates active efflux of tetracycline from the cell, while tetB(P) encodes a putative 72.6-kDa protein which has significant similarity to Tet M-like tetracycline resistance proteins (J. Sloan, L.M. McMurry, D. Lyras, S. B. Levy, and J. I. Rood, Mol. Microbiol. 11:403-415, 1994). In the present study, hybridization and PCR analysis of 81 tetracycline-resistant isolates of C. perfringens showed that they all carried the tetA(P) gene. Most of these isolates (93%) carried a second tetracycline resistance gene, with 53% carrying tetB(P) and 40% carrying a tet(M)-like gene. Despite the wide distribution of the tetB(P) and tet(M) genes, no isolate which carried both of these determinants was detected. In isolates that carried both tetA(P) and tetB(P) these genes overlapped, as in pCW3. Isolates carrying this combination of genes originated from diverse geographical locations and environmental sources. The single Clostridium paraputrificum isolate examined carried tetA(P), indicating that this gene is not confined to C.perfringens. However, neither tetA(P) nor tetB(P) was detected in the nine Clostridium difficile isolates tested. Nucleotide sequence analysis of isolates lacking tetB(P) revealed that they contained the tetA408(P) gene, which lacked the codons for the 12 carboxy-terminal amino acids of the TetA(P) protein.

scholarly journals Transcriptional Analysis of the Adeno-Associated Virus Integration Site

2009 ◽  
Vol 83 (23) ◽  
pp. 12512-12525 ◽  
Author(s):  
Nathalie Dutheil ◽  
Els Henckaerts ◽  
Erik Kohlbrenner ◽  
R. Michael Linden
Keyword(s):  
Transcriptional Activity ◽  
Integration Site ◽  
Regulatory Elements ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Complex Interactions ◽  
Site Specific Integration ◽  
Virus Integration

ABSTRACT The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the human MBS85 gene, which is embedded in AAVS1 on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon of MBS85 suggests that the transcriptional activity of MBS85 might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the human MBS85 promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate that AAVS1 is defined by a complex transcriptional environment and that the MBS85 promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectional MBS85 promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5′ untranslated region of the gene and play a posttranscriptional role in the regulation of MBS85 expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.

scholarly journals New Insights into the Genetic Organization of the FK228 Biosynthetic Gene Cluster inChromobacterium violaceumNo. 968

2010 ◽  
Vol 77 (4) ◽  
pp. 1508-1511 ◽  
Author(s):  
Vishwakanth Y. Potharla ◽  
Shane R. Wesener ◽  
Yi-Qiang Cheng
Keyword(s):  
Natural Product ◽  
Gene Cluster ◽  
Gene Deletion ◽  
Regulatory Gene ◽  
Biosynthetic Gene Cluster ◽  
Transcriptional Analysis ◽  
Biosynthetic Gene ◽  
Genetic Organization

ABSTRACTThe biosynthetic gene cluster of FK228, an FDA-approved anticancer natural product, was identified and sequenced previously. The genetic organization of this gene cluster has now been delineated through systematic gene deletion and transcriptional analysis. As a result, the gene cluster is redefined to contain 12 genes:depAthroughdepJ,depM, and a newly identified pathway regulatory gene,depR.

scholarly journals Analysis of Cytadherence-Deficient, GapA-NegativeMycoplasma gallisepticum Strain R

2000 ◽  
Vol 68 (12) ◽  
pp. 6643-6649 ◽  
Author(s):  
L. Papazisi ◽  
K. E. Troy ◽  
T. S. Gorton ◽  
X. Liao ◽  
S. J. Geary
Keyword(s):  
Shuttle Vector ◽  
Phenotypic Expression ◽  
Mycoplasma Genitalium ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Wild Type ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Low Passage ◽  
Translational Start

ABSTRACT Comparison of the phenotypic expression of Mycoplasma gallisepticum strain R low (passage 15) to that of strain R high (passage 164) revealed that three proteins, i.e., the cytadhesin molecule GapA, a 116-kDa protein (p116), and a 45-kDa protein (p45), are missing in strain R high. Sequence analysis confirmed that the insertion of an adenine 105 bp downstream of the gapAtranslational start codon resulted in premature termination of translation in R high. A second adenine insertion had also occurred at position 907. Restoration of expression of wild-type gapAin R high (clone designated GT5) allowed us to evaluate the extent to which the diminished cytadherence capacity could be attributed to GapA alone. The results indicated that GT5 attached to the same limited extent as the parental R high, from which it was derived. The cytadherence capability of the parental R high was not restored solely by gapA complementation alone, indicating that either p116 or p45 or both may play a role in the overall cytadherence process. The gene encoding p116 was found to be immediately downstream ofgapA in the same operon and was designatedcrmA. This gene exhibited striking homology to genes encoding molecules with cytadhesin-related functions in bothMycoplasma pneumoniae and Mycoplasma genitalium. Transcriptional analysis revealed thatcrmA is not transcribed in R high. We are currently constructing a shuttle vector containing both the wild-typegapA and crmA for transformation into R high to assess the role of CrmA in the cytadherence process.

scholarly journals Necrotic Enteritis-Derived Clostridium perfringens Strain with Three Closely Related Independently Conjugative Toxin and Antibiotic Resistance Plasmids

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Trudi L. Bannam ◽  
Xu-Xia Yan ◽  
Paul F. Harrison ◽  
Torsten Seemann ◽  
Anthony L. Keyburn ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Clostridium Perfringens ◽  
Tetracycline Resistance ◽  
Necrotic Enteritis ◽  
Sequencing Analysis ◽  
Content Type ◽  
Conjugative Plasmids ◽  
Resistance Plasmid ◽  
Large Plasmids ◽  
Beta2 Toxin

ABSTRACTThe pathogenesis of avian necrotic enteritis involves NetB, a pore-forming toxin produced by virulent avian isolates ofClostridium perfringenstype A. To determine the location and mobility of thenetBstructural gene, we examined a derivative of the tetracycline-resistant necrotic enteritis strain EHE-NE18, in whichnetBwas insertionally inactivated by the chloramphenicol and thiamphenicol resistance genecatP. Both tetracycline and thiamphenicol resistance could be transferred either together or separately to a recipient strain in plate matings. The separate transconjugants could act as donors in subsequent matings, which demonstrated that the tetracycline resistance determinant and thenetBgene were present on different conjugative elements. Large plasmids were isolated from the transconjugants and analyzed by high-throughput sequencing. Analysis of the resultant data indicated that there were actually three large conjugative plasmids present in the original strain, each with its own toxin or antibiotic resistance locus. Each plasmid contained a highly conserved 40-kb region that included plasmid replication and transfer regions that were closely related to the 47-kb conjugative tetracycline resistance plasmid pCW3 fromC. perfringens. The plasmids were as follows: (i) a conjugative 49-kb tetracycline resistance plasmid that was very similar to pCW3, (ii) a conjugative 82-kb plasmid that contained thenetBgene and other potential virulence genes, and (iii) a 70-kb plasmid that carried thecpb2gene, which encodes a different pore-forming toxin, beta2 toxin.IMPORTANCEThe anaerobic bacteriumClostridium perfringenscan cause an avian gastrointestinal disease known as necrotic enteritis. Disease pathogenesis is not well understood, although the plasmid-encoded pore-forming toxin NetB, is an important virulence factor. In this work, we have shown that the plasmid that carries thenetBgene is conjugative and has a 40-kb region that is very similar to replication and transfer regions found within each of the sequenced conjugative plasmids fromC. perfringens. We also showed that this strain contained two additional large plasmids that were also conjugative and carried a similar 40-kb region. One of these plasmids encoded beta2 toxin, and the other encoded tetracycline resistance. To our knowledge, this is the first report of a bacterial strain that carries three closely related but different independently conjugative plasmids. These results have significant implications for our understanding of the transmission of virulence and antibiotic resistance genes in pathogenic bacteria.

scholarly journals Characterization of a Second Rhodococcus erythropolis SQ1 3-Ketosteroid 9α-Hydroxylase Activity Comprising a Terminal Oxygenase Homologue, KshA2, Active with Oxygenase-Reductase Component KshB

2008 ◽  
Vol 74 (23) ◽  
pp. 7197-7203 ◽  
Author(s):  
R. van der Geize ◽  
G. I. Hessels ◽  
M. Nienhuis-Kuiper ◽  
L. Dijkhuizen
Keyword(s):  
Mutant Strain ◽  
Parent Strain ◽  
Gene Deletion ◽  
Amino Acid Level ◽  
Rhodococcus Erythropolis ◽  
Pcr Primers ◽  
Transcriptional Analysis ◽  
Reporter Protein ◽  
Steroid Substrate ◽  
Key Enzyme

ABSTRACT Previously we have characterized 3-ketosteroid 9α-hydroxylase (KSH), a key enzyme in microbial steroid degradation in Rhodococcus erythropolis strain SQ1, as a two-component iron-sulfur monooxygenase, comprised of the terminal oxygenase component KshA1 and the oxygenase-reductase component KshB. Deletion of the kshA1 gene resulted in the loss of the ability of mutant strain RG2 to grow on the steroid substrate 4-androstene-3,17-dione (AD). Here we report characteristics of a close KshA1 homologue, KshA2 of strain SQ1, sharing 60% identity at the amino acid level. Expression of the kshA2 gene in mutant strain RG2 restored growth on AD and ADD, indicating that kshA2 also encodes KSH activity. The functional complementation was shown to be dependent on the presence of kshB. Transcriptional analysis showed that expression of kshA2 is induced in parent strain R. erythropolis SQ1 in the presence of AD. However, promoter activity studies, using β-lactamase of Escherichia coli as a convenient transcription reporter protein for Rhodococcus, revealed that the kshA2 promoter in fact is highly induced in the presence of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) or a metabolite thereof. Inactivation of kshA2 in parent strain SQ1 by unmarked gene deletion did not affect growth on 9OHAD, cholesterol, or cholic acid. We speculate that KshA2 plays a role in preventing accumulation of toxic intracellular concentrations of ADD during steroid catabolism. A third kshA homologue was additionally identified in a kshA1 kshA2 double gene deletion mutant strain of R. erythropolis SQ1. The developed degenerate PCR primers for kshA may be useful for isolation of kshA homologues from other (actino) bacteria.

scholarly journals Molecular Characterization and Transcriptional Analysis of adhE2, the Gene Encoding the NADH-Dependent Aldehyde/Alcohol Dehydrogenase Responsible for Butanol Production in Alcohologenic Cultures of Clostridium acetobutylicum ATCC 824

2002 ◽  
Vol 184 (3) ◽  
pp. 821-830 ◽  
Author(s):  
Lisa Fontaine ◽  
Isabelle Meynial-Salles ◽  
Laurence Girbal ◽  
Xinghong Yang ◽  
Christian Croux ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Clostridium Acetobutylicum ◽  
Transcriptional Analysis ◽  
Start Codon ◽  
Butanol Production ◽  
Gene Encoding ◽  
E Coli ◽  
Butanol Dehydrogenase ◽  
Transcriptional Start Sites ◽  
Extension Analysis

ABSTRACT The adhE2 gene of Clostridium acetobutylicum ATCC 824, coding for an aldehyde/alcohol dehydrogenase (AADH), was characterized from molecular and biochemical points of view. The 2,577-bp adhE2 codes for a 94.4-kDa protein. adhE2 is expressed, as a monocistronic operon, in alcohologenic cultures and not in solventogenic cultures. Primer extension analysis identified two transcriptional start sites 160 and 215 bp upstream of the adhE2 start codon. The expression of adhE2 from a plasmid in the DG1 mutant of C. acetobutylicum, a mutant cured of the pSOL1 megaplasmid, restored butanol production and provided elevated activities of NADH-dependent butyraldehyde and butanol dehydrogenases. The recombinant AdhE2 protein expressed in E. coli as a Strep-tag fusion protein and purified to homogeneity also demonstrated NADH-dependent butyraldehyde and butanol dehydrogenase activities. This is the second AADH identified in C. acetobutylicum ATCC 824, and to our knowledge this is the first example of a bacterium with two AADHs. It is noteworthy that the two corresponding genes, adhE and adhE2, are carried by the pSOL1 megaplasmid of C. acetobutylicum ATCC 824.

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