Cloning and transcription analysis of the ndh(A-I-G-E) gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803

1992 ◽  
Vol 20 (6) ◽  
pp. 1097-1110 ◽  
Author(s):  
Ulrike Ellersiek ◽  
Klaus Steinm�ller
Keyword(s):  
Gene Cluster ◽  
Transcription Analysis ◽  
E Gene ◽  
Synechocystis Sp

scholarly journals Transcription Analysis of the Major Antigenic Protein 1 Multigene Family of Three In Vitro-Cultured Ehrlichia ruminantium Isolates

2005 ◽  
Vol 187 (14) ◽  
pp. 4782-4791 ◽  
Author(s):  
Cornelis P. J. Bekker ◽  
Milagros Postigo ◽  
Amar Taoufik ◽  
Lesley Bell-Sakyi ◽  
Conchita Ferraz ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Cell Lines ◽  
Multigene Family ◽  
Vaccine Development ◽  
Tick Cell ◽  
Transcription Analysis ◽  
Antigenic Protein ◽  
Ehrlichia Ruminantium ◽  
Reverse Transcription Pcr

ABSTRACT Ehrlichia ruminantium, an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, causes heartwater disease in ruminants. The gene coding for the major antigenic protein MAP1 is part of a multigene family consisting of a cluster containing 16 paralogs. In the search for differentially regulated genes between E. ruminantium grown in endothelial and tick cell lines that could be used in vaccine development and to determine if differences in the map1 gene cluster exist between different isolates of E. ruminantium, we analyzed the map1 gene cluster of the Senegal and Gardel isolates of E. ruminantium. Both isolates contained the same number of genes, and the same organization as found in the genome sequence of the Welgevonden isolate (H. Van Heerden, N. E. Collins, K. A. Brayton, C. Rademeyer, and B. A. Allsopp, Gene 330:159-168, 2004). However, comparison of two subpopulations of the Gardel isolate maintained in different laboratories demonstrated that recombination between map1-3 and map1-2 had occurred in one subpopulation with deletion of one entire gene. Reverse transcription-PCR on E. ruminantium derived mRNA from infected cells using gene-specific primers revealed that all 16 map1 paralogs were transcribed in endothelial cells. In one vector (Amblyomma variegatum) and several nonvector tick cell lines infected with E. ruminantium, transcripts were found for between 4 and 11 paralogs. In all these cases the transcript for the map1-1 gene was detected and was predominant. Our results indicate that the map1 gene cluster is relatively conserved but can be subject to recombination, and differences in the transcription of map1 multigenes in host and vector cell environments exist.

scholarly journals Analysis of promoter regions in the saxitoxin gene cluster identifies potential bottlenecks in heterologous biosynthesis

2019 ◽  
Author(s):  
Paul Michael D'Agostino ◽  
Bakir Al-Sinawi ◽  
Rabia Mazmouz ◽  
Julia Muenchhoff ◽  
Brett Anthony Neilan ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Local Anaesthetics ◽  
Bidirectional Promoter ◽  
Biosynthetic Gene Cluster ◽  
Reporter System ◽  
Promoter Regions ◽  
E Coli ◽  
Synechocystis Sp ◽  
Pcc 6803

Abstract Background: Dolichospermum circinale is a filamentous bloom-forming cyanobacterium responsible for biosynthesis of the paralytic shellfish toxins (PST), including saxitoxin. PSTs are neurotoxins and in their purified form are important analytical standards for monitoring the quality of water and seafood and biomedical research tools for studying neuronal sodium channels. More recently, PSTs have been recognised for their utility as local anaesthetics. Characterisation of the transcriptional elements within the saxitoxin ( sxt ) biosynthetic gene cluster (BGC) is a first step towards accessing these molecules for biotechnology. Results: In D. circinale AWQC131C the sxt BGC is transcribed from two bidirectional promoter regions encoding five individual promoters. These promoters were identified experimentally using 5ʹ RACE and their activity assessed via coupling to a lux reporter system in E. coli and Synechocystis sp. PCC 6803. Transcription of the predicted drug/metabolite transporter (DMT) encoded by sxtPER was found to initiate from two promoters, P sxtPER and P sxtPER2 . In E. coli , strong expression of lux from P sxtP , P sxtD and P sxtPER was observed while expression from P orf24 and P sxtPER2 was remarkably weaker. In contrast, heterologous expression in Synechocystis sp. PCC 6803 showed that expression of lux from P sxtP , P sxtPER , and P orf24 promoters was statistically higher compared to the non-promoter control, while P sxtD showed poor activity under the described conditions. Conclusions: Both of the heterologous hosts investigated in this study exhibited high expression levels from three of the five sxt promoters. These results indicate that future experiments to clone the complete sxt BGC into either heterologous host should result in the transcription of the complete pathway following engineering of the least active promoters. Therefore, heterologous expression of the sxt BGC in either E. coli or Synechocystis could be a viable option for producing PSTs for industrial or biomedical purposes.

scholarly journals A GC-Rich Prophage-Like Genomic Region of Mycoplasma bovirhinis HAZ141_2 Carries a Gene Cluster Encoding Resistance to Kanamycin and Neomycin

2020 ◽  
Author(s):  
Inna Lysnyansky ◽  
Ilya Borovok
Keyword(s):  
Gene Cluster ◽  
Field Isolate ◽  
Gc Content ◽  
Bioinformatic Analysis ◽  
Genomic Region ◽  
Transcription Analysis ◽  
E Coli ◽  
Functional Defect ◽  
Rapid Amplification ◽  
Carboxy Terminal

Recently, a complete genome of Mycoplasma bovirhinis HAZ141_2 has been published showing presence of 54-kB prophage-like region. Bioinformatic analysis revealed that this region has a more than 40-% GC content and a chimeric organization with three structural elements – a prophage continuous region, a restriction-modification cassette, and a highly transmittable aadE-sat4-aphA-3 gene cluster found in both Gram-positive and Gram-negative bacteria. It is known that aadE confers resistance to streptomycin, sat4 - resistance to streptothricin/nourseothricin, and aphA-3 - resistance to kanamycin and structurally related antibiotics. An aadE-like (aadE*) gene of strain HAZ141_2 encodes a 228 amino acid (aa) polypeptide whose carboxy-terminal domain (44-206) is almost identical to that of a functional 302 aa AadE (140-302). Transcription analysis of the aadE*-sat4-aphA-3 genes showed their co-transcription in M. bovirhinis HAZ141_2. Moreover, a common promoter for aadE*-sat4-aphA-3 was mapped upstream of the aadE* using 5′ rapid amplification of cDNA ends analysis. Determination of MICs to aminoglycosides and nourseothricin revealed that M. bovirhinis HAZ141_2 is highly resistant to kanamycin and neomycin (≥512 μg/ml). However, MICs to streptomycin (64 μg/ml) and nourseothricin (16-32 μg/ml) were similar to these identified in the prophageless M. bovirhinis type strain PG43 and Israeli field isolate 316981. We cloned the aadE*-sat4-aphA-3 genes into a low-copy number vector and transferred them into antibiotic-sensitive Escherichia coli cells. While the obtained E. coli transformants were highly resistant to kanamycin, neomycin as well as to nourseothricin (MICs ≥256 μg/ml), there were no changes in MICs to streptomycin suggesting a functional defect of the aadE*.

scholarly journals A Gene Cluster Involved in Metal Homeostasis in the Cyanobacterium Synechocystis sp. Strain PCC 6803

2000 ◽  
Vol 182 (6) ◽  
pp. 1507-1514 ◽  
Author(s):  
Mario García-Domínguez ◽  
Luis Lopez-Maury ◽  
Francisco J. Florencio ◽  
José C. Reyes
Keyword(s):  
Gene Cluster ◽  
Metal Binding ◽  
Open Reading Frames ◽  
Response System ◽  
Metal Binding Domain ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Reading Frames ◽  
Synechocystis Sp ◽  
Pcc 6803

ABSTRACT A gene cluster composed of nine open reading frames (ORFs) involved in Ni2+, Co2+, and Zn2+ sensing and tolerance in the cyanobacterium Synechocystis sp. strain PCC 6803 has been identified. The cluster includes an Ni2+response operon and a Co2+ response system, as well as a Zn2+ response system previously described. Expression of the Ni2+ response operon (nrs) was induced in the presence of Ni2+ and Co2+. Reduced Ni2+ tolerance was observed following disruption of two ORFs of the operon (nrsA and nrsD). We also show that the nrsD gene encodes a putative Ni2+ permease whose carboxy-terminal region is a metal binding domain. The Co2+ response system is composed of two divergently transcribed genes, corR and corT, mutants of which showed decreased Co2+ tolerance. Additionally, corR mutants showed an absence of Co2+-dependent induction of corT, indicating that CorR is a transcriptional activator of corT. To our knowledge, CorR is the first Co2+-sensing transcription factor described. Our data suggest that this region of theSynechocystis sp. strain PCC 6803 genome is involved in sensing and homeostasis of Ni2+, Co2+, and Zn2+.

scholarly journals Identification of promoter elements in the Dolichospermum circinale AWQC131C saxitoxin gene cluster and the experimental analysis of their use for heterologous expression.

2020 ◽  
Author(s):  
Paul Michael D'Agostino ◽  
Bakir Al-Sinawi ◽  
Rabia Mazmouz ◽  
Julia Muenchhoff ◽  
Brett Anthony Neilan ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Local Anaesthetics ◽  
Bidirectional Promoter ◽  
Biosynthetic Gene Cluster ◽  
Reporter System ◽  
Promoter Regions ◽  
E Coli ◽  
Paralytic Shellfish Toxins ◽  
Synechocystis Sp

Abstract Background: Dolichospermum circinale is a filamentous bloom-forming cyanobacterium responsible for biosynthesis of the paralytic shellfish toxins (PST), including saxitoxin. PSTs are neurotoxins and in their purified form are important analytical standards for monitoring the quality of water and seafood and biomedical research tools for studying neuronal sodium channels. More recently, PSTs have been recognised for their utility as local anaesthetics. Characterisation of the transcriptional elements within the saxitoxin ( sxt ) biosynthetic gene cluster (BGC) is a first step towards accessing these molecules for biotechnology. Results: In D. circinale AWQC131C the sxt BGC is transcribed from two bidirectional promoter regions encoding five individual promoters. These promoters were identified experimentally using 5ʹ RACE and their activity assessed via coupling to a lux reporter system in E. coli and Synechocystis sp. PCC 6803. Transcription of the predicted drug/metabolite transporter (DMT) encoded by sxtPER was found to initiate from two promoters, P sxtPER1 and P sxtPER2 . In E. coli, strong expression of lux from P sxtP , P sxtD and sxtPER1 was observed while expression from P orf24 and P sxtPER2 was remarkably weaker. In contrast, heterologous expression in Synechocystis sp. PCC6803 showed that expression of lux from P sxtP , P sxtPER , and P orf24 promoters was statistically higher compared to the non-promoter control, while P sxtD showed poor activity under the described conditions. Conclusions: Both of the heterologous hosts investigated in this study exhibited high expression levels from three of the five sxt promoters. These results indicate that the majority of the native sxt promoters appear active in different heterologous hosts, simplifying initial cloning efforts. Therefore, heterologous expression of the sxt BGC in either E. coli or Synechocystis could be a viable first option for producing PSTs for industrial or biomedical purposes.

MOLECULAR STRUCTURE AND EVOLUTION OF THE CHLOROPLAST atpB/E GENE CLUSTER IN THE DIATOM ODONTELLA SINENSIS1

1995 ◽  
Vol 31 (6) ◽  
pp. 962-969 ◽  
Author(s):  
Peter G. Kroth-Pancic ◽  
Ulrich Freier ◽  
Heinrich Strotmann ◽  
Klaus V. Kowallik
Keyword(s):  
Molecular Structure ◽  
Gene Cluster ◽  
E Gene

scholarly journals Sequencing the Botulinum Neurotoxin Gene and Related Genes in Clostridium botulinum Type E Strains Reveals orfx3 and a Novel Type E Neurotoxin Subtype

2007 ◽  
Vol 189 (23) ◽  
pp. 8643-8650 ◽  
Author(s):  
Ying Chen ◽  
Hannu Korkeala ◽  
Johannes Aarnikunnas ◽  
Miia Lindström
Keyword(s):  
Amino Acid ◽  
Gene Cluster ◽  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
The Other ◽  
E Gene ◽  
Botulinum Type ◽  
Clostridium Botulinum Type E ◽  
First Time ◽  
Distinct Subtype

ABSTRACT Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (≤3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.

scholarly journals Phenol Degradation in the Strictly Anaerobic Iron-Reducing Bacterium Geobacter metallireducens GS-15

2009 ◽  
Vol 75 (12) ◽  
pp. 3912-3919 ◽  
Author(s):  
Kathleen M. Schleinitz ◽  
Sirko Schmeling ◽  
Nico Jehmlich ◽  
Martin von Bergen ◽  
Hauke Harms ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Posttranscriptional Regulation ◽  
Phenol Degradation ◽  
Regulation Mechanism ◽  
Strictly Anaerobic ◽  
Carboxylase Activity ◽  
Transcription Analysis ◽  
Geobacter Metallireducens ◽  
Isotope Exchange Reaction ◽  
Cell Extracts

ABSTRACT Information on anaerobic phenol metabolism by physiological groups of bacteria other than nitrate reducers is scarce. We investigated phenol degradation in the strictly anaerobic iron-reducing deltaproteobacterium Geobacter metallireducens GS-15 using metabolite, transcriptome, proteome, and enzyme analyses. The results showed that the initial steps of phenol degradation are accomplished by phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) as known from Thauera aromatica, but they also revealed some distinct differences. The pps-ppc gene cluster identified in the genome is functional, as shown by transcription analysis. In contrast to T. aromatica, transcription of the pps- and ppc-like genes was induced not only during growth on phenol, but also during growth on benzoate. In contrast, proteins were detected only during growth on phenol, suggesting the existence of a posttranscriptional regulation mechanism for these initial steps. Phenylphosphate synthase and phenylphosphate carboxylase activities were detected in cell extracts. The carboxylase does not catalyze an isotope exchange reaction between 14CO2 and 4-hydroxybenzoate, which is characteristic of the T. aromatica enzyme. Whereas the enzyme of T. aromatica is encoded by ppcABCD, the pps-ppc gene cluster of G. metallireducens contains only a ppcB homologue. Nearby, but oriented in the opposite direction, is a ppcD homologue that is transcribed during growth on phenol. Genome analysis did not reveal obvious homologues of ppcA and ppcC, leaving open the question of whether these genes are dispensable for phenylphosphate carboxylase activity in G. metallireducens or are quite different from the Thauera counterparts and located elsewhere in the genome.

Overexpression of the putative extracytoplasmic function sigma (σ) factor FujE enhances FK506 production in Streptomyces sp. strain KCCM 11116P

2014 ◽  
Vol 60 (6) ◽  
pp. 363-369 ◽  
Author(s):  
Sung-Kwon Lee ◽  
Seung Hwan Yang ◽  
Choong-Min Kang ◽  
SangJoon Mo ◽  
Joo-Won Suh
Keyword(s):  
Gene Cluster ◽  
Morphological Differentiation ◽  
Polymerase Chain Reaction Analysis ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Transcription Analysis ◽  
Streptomyces Sp ◽  
Σ Factor ◽  
Integrative Vectors

The role of the putative extracytoplasmic function sigma (σ) factor FujE, which has not been characterized as a member of the FK506 biosynthetic gene cluster, on FK506 production was identified by gene deletion, overexpression, and transcription analysis experiments in Streptomyces sp. strain KCCM 11116P. Inactivation of fujE had no effect on FK506 production, growth, or morphological differentiation. Overexpression of fujE with integrative vectors increased FK506 production by 2.87-fold (24.5 ± 1.4 mg·L–1) compared with the wild type (8.5 ± 0.5 mg·L–1). Semiquantitative reverse transcription – polymerase chain reaction analysis indicated that the overexpression of fujE stimulates the transcription of the FK506 biosynthetic genes. These results demonstrated that fujE is a new member of the FK506 biosynthetic gene cluster.

Sign in / Sign up

Export Citation Format

Share Document