scholarly journals A Type Ib ParB Protein Involved in Plasmid Partitioning in a Gram-Positive Bacterium

2006 ◽  
Vol 188 (23) ◽  
pp. 8103-8108 ◽  
Author(s):  
Ping Yin ◽  
Tai-Yuan Li ◽  
Mao-Hua Xie ◽  
Lina Jiang ◽  
Yi Zhang
Keyword(s):  
Dna Sequences ◽  
Plasmid Stability ◽  
Direct Repeat ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Reading Frame ◽  
Plasmid Partition ◽  
Orf4 Protein ◽  
Bacterial Plasmid

ABSTRACT Our current understanding of segregation of prokaryotic plasmids has been derived mainly from the study of the gram-negative bacterial plasmids. We previously reported a replicon of the cryptic plasmid from a gram-positive bacterium, Leifsonia xyli subsp. cynodontis. The replicon contains a putative plasmid partition cassette including a Walker-type ATPase followed by open reading frame 4 without sequence homologue. Here we reported that the orf4 gene was essential for maintaining the plasmid stability in L. xyli subsp. cynodontis. Furthermore, the purified orf4 protein specifically and cooperatively bound to direct repeat sequences located upstream of the parA gene in vitro, indicating that orf4 is a parB gene and that the direct repeat DNA sequences constitute a partition site, parS. The location of parS and the features of ParA and ParB proteins suggest that this plasmid partition cassette belongs to type Ib, representing the first type Ib cassette identified from a gram-positive bacterial plasmid.

Sequence of a Rhodococcus gene cluster encoding the subunits of ethanolamine ammonia-lyase and an APC-like permease

1994 ◽  
Vol 40 (5) ◽  
pp. 403-407 ◽  
Author(s):  
René De Mot ◽  
Istvan Nagy ◽  
Geert Schoofs ◽  
Jos Vanderleyden
Keyword(s):  
Open Reading Frame ◽  
Genomic Fragment ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gene Encoding ◽  
Reading Frame ◽  
Transporter Family ◽  
Rhodococcus Species ◽  
Rhodococcus Sp

Sequence analysis of a 5173-bp genomic fragment from the nocardioform actinomycete Rhodococcus sp. strain NI86/21 revealed the presence of two genes, eutB and eutC, encoding the putative homologues of the large and small subunits of the ethanolamine ammonia-lyase, respectively, from Salmonella typhimurium. This is the first report of the characterization of these genes in a Gram-positive species. Immediately upstream of eutB, a gene encoding a putative permease of the APC (amino acids, polyamines, choline) transporter family was located. At present, no other Gram-positive members of this permease family are known. The translational coupling of these eut genes suggests an operon-like organization of the ethanolamine genes in Rhodococcus species. A truncated open reading frame downstream of eutC contained an N-terminal motif characteristic of membrane-anchored lipoproteins.Key words: nocardioform actinomycete, cobalamin, APC transporter, membrane-anchored lipoprotein, Gram-positive bacterium.

The topology of plasmid-monomerizing Xer site-specific recombination

2013 ◽  
Vol 41 (2) ◽  
pp. 589-594 ◽  
Author(s):  
Sean D. Colloms
Keyword(s):  
Dna Sequences ◽  
Direct Repeat ◽  
Accessory Proteins ◽  
Site Specific ◽  
Circular Dna ◽  
Site Specific Recombination ◽  
Daughter Cells ◽  
The Core ◽  
Bacterial Plasmid ◽  
Negative Supercoils

Xer site-specific recombination at cer and psi converts bacterial plasmid multimers into monomers so that they can be efficiently segregated to both daughter cells at cell division. Recombination is catalysed by the XerC and XerD recombinases acting at ~30 bp core sites, and is regulated by the action of accessory proteins bound to accessory DNA sequences adjacent to the core sites. Recombination normally occurs only between sites in direct repeat in a negatively supercoiled circular DNA molecule, and yields two circular products linked together in a right-handed four-node catenane with antiparallel sites. These and other topological results are explained by a model in which the accessory DNA sequences are interwrapped around the accessory proteins, trapping three negative supercoils so that strand exchange by the XerC and XerD yields the observed four-node catenane.

scholarly journals Mapping of the interactions between partition proteins Delta and Omega of plasmid pSM19035 from Streptococcus pyogenes

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1009-1020 ◽  
Author(s):  
Michal Dmowski ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Streptococcus Pyogenes ◽  
Dna Sequences ◽  
Copy Number ◽  
Terminal Part ◽  
Partition System ◽  
Plasmid Partition ◽  
Nucleoprotein Complex ◽  
Interaction Domains

Formation of the segrosome, a nucleoprotein complex crucial for proper functioning of plasmid partition systems, involves interactions between specific partition proteins (ParA-like and ParB-like), ATP and specific DNA sequences (the centromeric sites). Although partition systems have been studied for many years, details of the segrosome formation are not yet clear. Organization of the pSM19035-encoded partition system is unique; in contrast with other known par systems, here, the δ and ω genes do not constitute an operon. Moreover, Omega [a ParB-like protein which has a Ribbon-Helix-Helix (RHH) structure] recognizes multiple centromeric sequences located in the promoters of δ, ω and copS (copy-number control gene). The ParA-like protein Delta is a Walker-type ATPase. In this work, we identify the interaction domains and requirements for dimerization and hetero-interactions of the Delta and Omega proteins of pSM19035 plasmid. The RHH structures are involved in Omega dimerization in vivo and its N-terminal unstructured part is indispensable for association with Delta, both in vivo and in vitro. Omega does not need to form dimers to interact with Delta. ATP binding is not required for Delta dimerization but is important for interaction with Omega in vivo. The in vitro interaction between Delta and Omega depends on ATP but does not require the presence of specific DNA segments (the centromere) recognized by Omega. The C-terminal part of the Delta protein (aa 198–284) is indispensable for interaction with Omega. Delta most probably interacts with Omega as a dimer since two amino acid substitutions in a conserved region between the A′ and B motifs abolish both the dimerization of Delta and its interaction with Omega.

scholarly journals Codon-Optimized Fluorescent Proteins Designed for Expression in Low-GC Gram-Positive Bacteria

2009 ◽  
Vol 75 (7) ◽  
pp. 2099-2110 ◽  
Author(s):  
Inka Sastalla ◽  
Kannie Chim ◽  
Gordon Y. C. Cheung ◽  
Andrei P. Pomerantsev ◽  
Stephen H. Leppla
Keyword(s):  
Codon Usage ◽  
Dna Sequences ◽  
Fluorescent Protein ◽  
Protective Antigen ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Gc Content ◽  
Virulence Plasmid ◽  
Positive Bacterium ◽  
Gram Positive

ABSTRACT Fluorescent proteins have wide applications in biology. However, not all of these proteins are properly expressed in bacteria, especially if the codon usage and genomic GC content of the host organism are not ideal for high expression. In this study, we analyzed the DNA sequences of multiple fluorescent protein genes with respect to codons and GC content and compared them to a low-GC gram-positive bacterium, Bacillus anthracis. We found high discrepancies for cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and the photoactivatable green fluorescent protein (PAGFP), but not GFP, with regard to GC content and codon usage. Concomitantly, when the proteins were expressed in B. anthracis, CFP- and YFP-derived fluorescence was undetectable microscopically, a phenomenon caused not by lack of gene transcription or degradation of the proteins but by lack of protein expression. To improve expression in bacteria with low genomic GC contents, we synthesized a codon-optimized gfp and constructed optimized photoactivatable pagfp, cfp, and yfp, which were in contrast to nonoptimized genes highly expressed in B. anthracis and in another low-GC gram-positive bacterium, Staphylococcus aureus. Using optimized GFP as a reporter, we were able to monitor the activity of the protective antigen promoter of B. anthracis and confirm its dependence on bicarbonate and regulators present on virulence plasmid pXO1.

scholarly journals Structure and expression of the human immunoglobulin lambda genes.

1990 ◽  
Vol 172 (2) ◽  
pp. 609-620 ◽  
Author(s):  
T J Vasicek ◽  
P Leder
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Rna Splicing ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Enhancer Activity ◽  
Initiation Point ◽  
Reading Frame ◽  
Duplication Events

We determined the DNA sequence of two large regions of chromosome 22: 33.7 kb containing the C lambda complex; and 5.2 kb 5' of the functionally rearranged lambda gene from the human myeloma, U266. Analysis of these sequences reveals the complete structure of the human C lambda complex and a previously undescribed seventh C lambda region that may encode the Ke+Oz- lambda protein. The seven constant regions are organized in a tandem array, and each is preceded by a single J lambda region. lambda 1, lambda 2, lambda 3, and lambda 7 are apparently active genes, while lambda 4, lambda 5, and lambda 6 are pseudogenes. There are no other J lambda or C lambda regions within a 60-kb region surrounding the C lambda complex; however, there are at least four other lambda-like genes and lambda pseudogenes in the human genome. The lambda genes appear to have evolved via a series of gene duplication events resulting from unequal crossing over or gene conversion between the highly conserved C lambda regions on mispaired chromosomes. The lack of Alu sequences in this large segment of DNA suggests that the C lambda complex resulted from a recent amplification of a smaller Alu-free segment of DNA. Illegitimate recombination between repeated sequences containing lambda 2 and lambda 3 may be responsible for variable amplification of the lambda genes. We also found a 1,377-bp open reading frame (ORF) located on the opposite strand in the region containing lambda 7. While this ORF is flanked by potential RNA splicing signals, we have no evidence that it is part of a functional gene. We also discovered a V lambda pseudogene, called psi V lambda 1, 3 kb upstream of the U266 lambda gene. Using primer extension analysis to map the transcription start in the human lambda gene, we have identified its initiation point 41 bp upstream of the initiation codon. Analysis of the lambda promoter reveals that it contains a TATAA box at position -29 relative to the transcription initiation site and an octamer sequence at -67. Computer analysis of 40 kb of DNA sequences surrounding the human lambda locus has revealed no sequences resembling the kappa or IgH transcriptional enhancers, nor have in vitro analyses for function revealed enhancer activity. A comparison of these results with those obtained in separate studies with transgenic mice point to a complex, developmentally linked mechanism of transcriptional activation.

Identification of Erythroid Cell Stimulating Factor as a Unique Protein, Quiescin Q6: Role in Erythropoiesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4270-4270
Author(s):  
Kodetthoor B. Udupa ◽  
Chhanda Bose
Keyword(s):  
Dna Sequences ◽  
Antigenic Property ◽  
Erythroid Cell ◽  
Mouse Serum ◽  
Restriction Enzyme Digestion ◽  
Reading Frame ◽  
Unique Protein ◽  
Stimulating Factor

Abstract We demonstrated earlier the presence of an erythroid cell stimulating factor (ESF) in mouse serum that enhanced the erythropoietin-dependent erythroid cell proliferation in vitro. Employing a highly specific monoclonal antibody generated in our laboratory, we showed further that ESF had an essential role in normal erythropoiesis in vivo [Blood98, 296a, 2001, Exp Hematol.31, Supp. 1, 165, 2003]. In order to clone and express this protein, ESF was purified from mouse serum and on Western blotting using the monoclonal anti-ESF antibody the purified protein was stained as bands of ~62/64 kDa. These bands were analyzed by Mass Spectrometry. The data were processed using Protein Lynx 2.1 (Waters) and the resulting peak list was submitted for database searching on Mascot. One of the peptide sequences had a highest similarity with Quiescin Q6 (Q6). This protein was then BLAST searched for mouse DNA sequences in the NCBI database for nucleotides corresponding to the mouse Q6. We designed the primers to reclone Q6 ORF (Open reading frame) having restriction sites NCO1 and ECOR1. Amplified product was sequenced to confirm DNA fragment. Fragment was cloned in pGEMT vector and subsequently re-cloned in expression vector pET30a(+). After each cloning step, the orientation of the insert was analyzed using restriction enzyme digestion. His-tagged Q6 protein was over-expressed in E.coli BL21 Codon Plus (DE3)-RP cells. Western blot analysis using anti-ESF antibody identified 62/64 kDa-bands of Q6 in the soluble fraction of the bacterial extract. Further characteristics of this recombinant protein and its CFU-E enhancing properties are being investigated and will be reported. Thus we have identified ESF to be a unique protein, quiescin Q6 and successfully cloned and expressed it. Expressed protein is similar to ESF as regards its antigenic property.

Incorporation of Fluorinated Pyridine in the Side Chain of 4-Aminoquinolines: Synthesis, Characterization and Antibacterial Activity

Drug Research ◽  
2017 ◽  
Vol 68 (01) ◽  
pp. 17-22 ◽  
Author(s):  
Reza Ranjbar-Karimi ◽  
Alireza Poorfreidoni
Keyword(s):  
Antibacterial Activity ◽  
Chemical Structure ◽  
Side Chain ◽  
Gram Negative Bacteria ◽  
Pyridine Derivatives ◽  
Positive Bacterium ◽  
Gram Positive ◽  
In Vitro Antibacterial Activity ◽  
Ft Ir

AbstractA series of hybrid of 4-aminoquinoline and fluorinated pyridine derivatives were synthesized and their chemical structure were confirmed by 19F-NMR, 1H-NMR, 13C-NMR and FT-IR. All compounds were tested against one Gram-positive and one Gram-negative bacteria to assess their in vitro antibacterial activity. Compounds 10a, 10b, 11a and 12b showed moderate antibacterial activity against Gram-positive bacterium, Staphylococcus aureus.

scholarly journals Gene Targeting in the Gram-Positive Bacterium Lactococcus lactis, Using Various Delta Ribozymes

2006 ◽  
Vol 72 (1) ◽  
pp. 869-879 ◽  
Author(s):  
Karine Fiola ◽  
Jean-Pierre Perreault ◽  
Benoit Cousineau
Keyword(s):  
Gene Targeting ◽  
Lactococcus Lactis ◽  
Half Life ◽  
Bacterial Cells ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Mammalian Cell Lines ◽  
Delta Ribozyme

ABSTRACT The trans-acting antigenomic delta ribozyme, isolated from the human hepatitis delta virus, was shown to be highly stable and active in vitro, as well as in mammalian cell lines. However, the stability and gene-targeting competence of this small ribozyme have not been studied previously in bacterial cells. In this paper we describe the use of two variants of the trans-acting antigenomic delta ribozyme targeting the abundant EF-Tu mRNA in the industrially important gram-positive bacterium Lactococcus lactis. These two delta ribozyme variants were expressed at significant levels and were shown to be highly stable in vivo. The half-life of the EF-Tu mRNA was slightly but consistently reduced in the presence of the classical delta ribozymes (7 to 13%). In contrast, delta ribozymes harboring a specific on/off riboswitch (SOFA-delta ribozymes) targeting the same sites on the EF-Tu mRNA considerably reduced the half-life of this mRNA (22 to 47%). The rates of catalysis of the SOFA-delta ribozymes in L. lactis were similar to the rates determined in vitro, showing that this new generation of delta ribozymes was highly efficient in these bacterial cells. Clearly, SOFA-delta ribozymes appear to be an ideal means for development of gene inactivation systems in bacteria.

scholarly journals Identification of a Streptogramin A Acetyltransferase Gene in the Chromosome of Yersinia enterocolitica

2000 ◽  
Vol 44 (4) ◽  
pp. 905-909 ◽  
Author(s):  
Asunción Seoane ◽  
Juan M. García Lobo
Keyword(s):  
Yersinia Enterocolitica ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Gram Negative Bacteria ◽  
Gene Products ◽  
Gram Positive ◽  
Reading Frame ◽  
Gram Positive Bacteria ◽  
Polypeptide Antibiotics

ABSTRACT Streptogramins are polypeptide antibiotics inhibiting protein synthesis by the prokaryotic ribosome. Gram-positive organisms are susceptible to streptogramins, while most gram-negative bacteria are intrinsically resistant. We have found a genomic fragment from aYersinia enterocolitica isolate with an open reading frame coding for a polypeptide similar to the virginiamycin acetyltransferases found in various plasmids from gram-positive bacteria. The susceptible Escherichia coli strain DB10 was transformed to resistance to the type A streptogramins and to mixed (A + B) streptogramins upon introduction of a plasmid containing that gene. In addition, we showed streptogramin acetylating activity in vitro dependent on the presence of the Y. enterocolitica sat gene. Southern blot hybridization experiments showed that thesat gene was present in all the Y. enterocolitica isolates examined. These data together show that the gene in the Y. enterocolitica chromosome encoded an active streptogramin acetyltransferase. The deduced sequence of theY. enterocolitica Sat protein was close to those ofsat gene products found in gram-positive bacteria and cyanobacteria, suggesting a common evolutionary origin.

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