groESL Operon Research Articles

Genome-wide transcription response of Staphylococcus epidermidis to heat shock and medically relevant glucose levels.

Skin serves as both barrier and interface between body and environment. Skin microbes are intermediaries evolved to respond, transduce, or act in response to changing environmental or physiological conditions. We quantified genome-wide changes in gene expression levels for one abundant skin commensal, Staphylococcus epidermidis, in response to an internal physiological signal, glucose levels, and an external environmental signal, temperature. We found 85 of 2,354 genes change up to ~34-fold in response to medically relevant changes in glucose concentration (0-17 mM; adj p ≤0.05). We observed carbon catabolite repression in response to a range of glucose spikes, as well as upregulation of genes involved in glucose utilization in response to persistent glucose. We observed 366 differentially expressed genes in response to a physiologically relevant change in temperature (37-45°C; adj p ≤ 0.05) and an S. epidermidis heat-shock response that mostly resembles the heat-shock response of related staphylococcal species. DNA motif analysis revealed CtsR and CIRCE operator sequences arranged in tandem upstream of dnaK and groESL operons. We identified and curated 38 glucose-responsive genes as candidate ON or OFF switches for use in controlling synthetic genetic systems. Such systems might be used to instrument the in-situ skin microbiome or help control microbes bioengineered to serve as embedded diagnostics, monitoring, or treatment platforms.

Multilocus genetic analysis indicates taxonomic status of “Candidatus Rickettsia mendelii” as a separate basal group

To date, the phylogeny of Rickettsia spp. from basal groups is based on the small number of identified species. Thus, the finding of “Candidatus Rickettsia mendelii” in 2016 is of great interest. In this study, “Ca. R. mendelii” was first identified in the Asian region in a new carrier, Ixodes pavlovskyi. “Candidatus R. mendelii”, along with “Candidatus Rickettsia tarasevichiae”, were found in Ixodes ticks collected on Russky Island (the Far East), where I. pavlovskyi coexists with I. persulcatus. To establish the taxonomic position of “Ca. R. mendelii”, a detailed genetic study was carried out. “Candidatus R. mendelii” was genotyped by five genetic fragments (16S rRNA, gltA, and ompB genes, groESL operon, and 23S–5S IGS region); among them, the ompB gene, groESL operon and 23S–5S IGS region were sequenced for the first time. In addition, “Ca. R. tarasevichiae” was genetically characterized by eight genetic loci (16S rRNA, gltA, ompA, ompB, sca4, htrA genes, groESL operon, and 23S–5S IGS region), of which the sca4 gene was first determined. Phylogenetic analysis indicated that regardless of analyzed genetic loci, “Ca. R. mendelii” formed a separate well-supported cluster on each phylogenetic tree. Phylogenetic analysis based on concatenated sequences of gltA, ompB, and groEL gene fragments (total length of 3191 bp) demonstrated that “Ca. R. mendelii”, like Rickettsia bellii, is a basal group of Rickettsia.

Molecular detection and identification of piroplasms (Babesia spp. and Theileria spp.) and Anaplasma phagocytophilum in questing ticks from northwest Spain.

Anaplasma phagocytophilum and some piroplasm species are pathogens mainly transmitted by Ixodes ricinus. Considering that this tick species is predominant in north-western Spain, individual specimens (652 nymphs, 202 females and 202 males) and 23 larval pools were processed to determine the prevalence of these pathogens in questing I. ricinus from that region. Additionally, Dermacentor marginatus, Dermacentor reticulatus, Ixodes frontalis and Ixodes acuminatus were individually analysed. The groESL operon as well as the 16S rRNA and msp2 genes of Anaplasma were analysed. Similarly, piroplasms were identified at the 18S rRNA gene and the ITS1 of Babesia spp. and Theileria spp. Babesia venatorum (1.5%), A. phagocytophilum (0.7%), Babesia microti (0.3%) and Theileria sp. OT3 (0.2%) were detected in I. ricinus. A single I. frontalis (8.3%) tested positive to A. phagocytophilum. Although a low percentage of I. ricinus were infected with A. phagocytophilum and piroplasms, a potentially human pathogenic variant of A. phagocytophilum was detected, and both Babesia species found were zoonotic. Since the vector of Theileria sp. OT3 remains unknown, further investigations are needed to unravel the role of I. ricinus in the transmission of this piroplasm.

Long-term persistence of Anaplasma phagocytophilum and Ehrlichia muris in wild rodents

Wild animals are reservoir hosts for a number of tick-transmitted agents, and long-term persistence of the agents is a key factor for their effective transmission from animal hosts to ticks. To study the persistence of Anaplasmataceae in rodents, 59 adult Myodes spp. voles (M. rutilus, M. rufocanus, and M. glareolus) were captured in Omsk Province, Asian Russia, freed from all ectoparasites, and kept in individual cages. Their blood samples were regularly analyzed for the presence of DNA of Anaplasma phagocytophilum, Ehrlichia muris, and “Candidatus Neoehrlichia mikurensis”. Anaplasma phagocytophilum, E. muris, and mixed infections were found in 29 (49 %), 17 (29 %), and 14 (24 %) voles, respectively. DNA of “Candidatus N. mikurensis” was not identified. Long-term persistence of A. phagocytophilum and E. muris in naturally infected Myodes spp. was shown for the first time. Thus, 12 animals with A. phagocytophilum were found infected for more than three months and four of them carried the bacterium throughout 22–55 weeks of observation. Four voles were infected with E. muris for 26–40 weeks. Fragments of groESL operon were sequenced for genotyping A. phagocytophilum and E. muris. In voles with A. phagocytophilum, the determined sequences belonged to two phylogenetic clusters. Most sequences were identical or closely related to those found in small mammals and Ixodes trianguliceps ticks from Western Siberia and European countries; one sequence was previously identified in rodents and Ixodes persulcatus and Ixodes pavlovskyi ticks from Asian Russia. Sequences of groESL fragments from E. muris were close to those determined previously in small mammals and I. persulcatus ticks from Siberia. In addition, a new groESL sequence of E. muris was identified in one vole and the sequence substantially differed from all known corresponding E. muris sequences (≥ 14 mismatches).

Detection of Ehrlichia spp. and Theileria spp. in Hyalomma anatolicum ticks collected in Tajikistan.

The objectives of our study were to survey the prevalence of genetic markers for Rickettsia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Theileria spp. in Hyalomma anatolicum ticks collected in southwestern Tajikistan and to perform sequencing and phylogenetic analysis of fragments of the 16S rRNA gene and groESL operon from Ehrlichia spp. and fragments of the 18S rRNA gene of Theileria spp. detected in H. anatolicum ticks. Hyalomma anatolicum ticks collected in the Tursunzade and Rudaki districts of Tajikistan were tested for DNA of Rickettsia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Theileria spp. by PCR with specific primers. The amplified fragments were sequenced and analyzed. DNA of Ehrlichia spp. (3.3 %) and Theileria spp. (3.3 %) was detected only in H. anatolicum ticks collected from the Rudaki district, and DNA of Ehrlichia spp. (0.7 %) was found in H. anatolicum ticks from the Tursunzade district. Sequence analysis of fragments of the 16S rRNA gene and groESL operon from Ehrlichia spp. revealed high similarity to Ehrlichia spp. The Tajik isolates of Theileria spp. were genotyped as Theileria annulata based on the analysis of 18S rRNA gene sequences. The phylogenetic analysis demonstrates that Ehrlichia spp. isolates are highly similar to Ehrlichia spp. circulating in China and Brazil. The isolate Tajikistan-5 is closely related to the putative novel species Ehrlichia mineirensis. The Tajik isolates of Theileria spp. were clustered with T. annulata isolates from Turkey, Iran, Pakistan, and China by phylogenetic analyses.

Regulation of the groESL1 transcription by the HrcA repressor and a novel transcription factor Orf7.5 in the cyanobacterium Synechococcus elongatus PCC7942.

The CIRCE/HrcA system is highly conserved in cyanobacterial genomes. We have shown that heat-shock induction of the groESL1 operon in the cyanobacterium Synechocystis sp. PCC6803 is negatively regulated by the CIRCE/HrcA system. In Synechococcus elongatus PCC7942, a novel heat shock protein, Orf7.5, is involved in positive regulation of the groESL1 transcription. However, Orf7.5 is not conserved in some cyanobacteria, including Synechocystis sp. PCC6803. The purpose of this study is to evaluate the functional conservation of the CIRCE/HrcA system in S. elongatus PCC7942 and to understand the interplay between the CIRCE/HrcA system and the Orf7.5 regulatory system. We constructed single and double mutants of S. elongatus orf7.5, hrcA and orf7.5/hrcA and heat induction of the groESL1 transcription in these mutants was analyzed. Unexpectedly, derepression of the groESL1 transcription in an hrcA mutant was not observed. In all these mutants, the transcription was greatly suppressed under both normal and heat stress conditions, indicating that both HrcA and Orf7.5 are involved in regulation of the groESL1 transcription in a positive way. Consistent with the decrease in the groESL1 mRNA level, all the single and double mutants showed a great loss of acquired thermotolerance. Heat induction of the orf7.5 promoter activity was totally diminished in the orf7.5 mutant, indicating that Orf7.5 activates its own transcription. Yeast two hybrid analysis showed that the principle sigma factor RpoD1 interacts with Orf7.5. These results indicate that Orf7.5 enhances the transcription of groESL1 and orf7.5 by interacting with RpoD1.

Computational selection of minimum length groESL operon required for Anaplasma species attribution and strain diversity analysis

Anaplasmosis is a tick-borne rickettsial disease caused by Anaplasma marginale, A. centrale, A. phagocytophilum, A. bovis, A. ovis and A. platys. Understanding the phylogenetic relations among these species is fundamental to perform an accurate identification and an informative intra-specific analysis. Heat shock groESL operon is frequently employed in phylogenetic analysis of Anaplasma species and, for the most cases, the use of partial sequences of this operon is randomly done without knowing the most appropriate regions to be used either in species attribution or in intra-specific diversity analysis. In this study, on the basis of all fully and nearly complete groESL sequences available in the GenBank, we firstly selected a minimum partial length sequence which allows species delineation and gives a similar topology to that found by analyzing the complete sequence. By using other in silico analyses, we obtained two minimal partial sequences that are the most interesting to describe intra-specific diversity within A. ovis and A. centrale. Our results raise concern on the use of randomly selected partial sequences of groESL operon employed for the detection and the characterization of Anaplasma species and provide additional background about minimum length groESL operon required for Anaplasma species attribution and strains diversity analysis.

Different roles of two groEL homologues in methylotrophic utiliser of dichloromethane Methylorubrum extorquens DM4.

The genome of methylotrophic bacteria Methylorubrum extorquens DM4 contains two homologous groESL operons encoding the 60-kDa and 10-kDa subunits of GroE heat shock chaperones with highly similar amino acid sequences. To test a possible functional redundancy of corresponding GroEL proteins we attempted to disrupt the groEL1 and groEL2 genes. Despite the large number of recombinants analysed and the gentle culture conditions the groEL1-lacking mutant was not constructed suggesting that the loss of GroEL1 was lethal for cells. At the same time the ∆groEL2 strain was viable and varied from the wild-type by increased sensitivity to acid, salt and desiccation stresses as well as by the impaired growth with a toxic halogenated compound-dichloromethane (DCM). The evaluation of activity of putative PgroE1 and PgroE2 promoters using the reporter gene of green fluorescent protein (GFP) showed that the expression of groESL1 operon greatly prevails (about two orders of magnitude) over those of groESL2 under all tested conditions. However the above promoters demonstrated differential regulation in response to stresses. The expression from PgroE1 was heat-inducible, while the activity of PgroE2 was upregulated upon acid shock and cultivation with DCM. Based on these results we conclude that the highly conservative groESL1 operon (old locus tags METDI5839-5840) encodes the housekeeping chaperone essential for fundamental cellular processes. On the contrary the second pair of paralogues (METDI4129-4130) is dispensable, but corresponding GroE2 chaperone promotes the tolerance to acid and salt stresses, in particular, during the growth with DCM.

Detection and genotyping of Anaplasma phagocytophilum in I. persulcatus and D. reticulatus ticks collected in Tomsk (Western Siberia) in 2015–2016

Introduction. The detection of the first cases of tick-borne human granulocytic anaplasmosis in Russia, discovery of genetic markers for Anaplasma spp. in ixodid ticks and reporting of a significant number of cases of tick-borne infections in the southern part of Western Siberia give reason to suppose that causative agents of tick-borne anaplasmosis may be transmitted in Tomsk and its suburbs. Objective. To study the distribution and species biodiversity of A. phagocytophilum in ixodid ticks in Tomsk Region. Materials and methods. The analysis of 690 individual ixodid ticks (larvae and adults) was carried out for Ixodes persulcatus (n = 530) and Dermacentor reticulatus (n = 160) ticks collected in 2015–2016 on the territory of urban and suburban biotopes of Tomsk. Primary screening of ticks for the presence of genetic material of A. phagocytophilum was conducted using two-round PCR with species-specific primers for the 16S rRNA gene. The amplification (1,220 kB) of the groESL fragment of the heat shock protein operon was performed for positive isolates with subsequent determination of the nucleotide sequence in the gene fragment for phylogenetic analysis. Results. The number of A. phagocytophilum positive samples for I. persulcatus (larvae) was 1.2 ± 0.6%, I. persulcatus (adult) was 1.8 ± 0.7%; and D. reticulatus (adult) was 0.6 ± 0.3%. Analysis of the nucleotide sequence of the gene fragments in groESL operon for nine isolates confirmed that the genetic material of the granulocytic anaplasmosis was detected. Phylogenetic analysis showed that all the isolates belonged to the first group of the “new cluster” of A. phagocytophilum. Conclusion. The causative agent of human granulocytic anaplasmosis has been newly detected in I. persulcatus ticks collected in urban and suburban biotopes of Tomsk and in D. reticulatus from urban foci.

Species and Genetic Diversity of Representatives of the <i>Anaplasmataceae</i> Family Found in the Sympatry Zone of the <i>Ixodes, Dermacentor</i> and <i>Haemaphysalis</i> Genera Ticks

Introduction.On the territory of the Ekhirit-Bulagatsky district of the Irkutsk region zones of sympatry of four Ixodes ticks species are found, where the species and genetic diversity of infectious agents transmitted through tick bites may be more pronounced than in foci with a mono-dominant type of ticks’ population. In this connection, the study of the species and genetic diversity of representatives of the Anaplasmataceae family in the sympatry zone of the Ixodes ticks of closely related species was of scientific interest.Objective: To study the species and genetic diversity of members of the Anaplasmataceae family in the zones of sympatry of Ixodes ticks Ixodes persulcatus, Dermacentor silvarum, D. nuttalli and Haemaphysalis concinna, to identify the main carriers and potential reservoir hosts of ehrlichia and anaplasma.Methods. In the course of the study, 1106 specimens of adult ticks and 49 samples of small mammalian livers from the Ekhirit-Bulagatsky area were analyzed. Anaplasma and ehrlichia DNA were detected by two-round PCR in the presence of genus- and species-specific primers from the 16S rRNA gene region. The nucleotide sequences of the 16S rRNA gene and the fragment of the groESL operon were identified in some samples. Sequencing was carried out according to the Sanger method. Comparative analysis was performed using the BLASTN program and ClustalW method. Epidemiological data analysis was performed using parametric methods of statistical processing of the material.Results. The DNA of Ehrlichia muris and Anaplasma phagocytophilum were detected in all studied species of ticks in their sympatry area. However, the rate of infection of taiga ticks was significantly higher than that of H. concinna and Dermacentor spp. Potential reservoir hosts of the Anaplasmataceae family members can be classified as Microtus oeconomus, M. gregalis, Myodes rutilus and Sorex spp. When analyzing the nucleotide sequences of the 16S rRNA gene, three genetic variants of anaplasma were detected. The nucleotide sequences of the A. phagocytophilum groESL operon belonged to two genetic groups.

Lineage structure of Streptococcus pneumoniae may be driven by immune selection on the groEL heat-shock protein

Populations of Streptococcus pneumoniae (SP) are typically structured into groups of closely related organisms or lineages, but it is not clear whether they are maintained by selection or neutral processes. Here, we attempt to address this question by applying a machine learning technique to SP whole genomes. Our results indicate that lineages evolved through immune selection on the groEL chaperone protein. The groEL protein is part of the groESL operon and enables a large range of proteins to fold correctly within the physical environment of the nasopharynx, thereby explaining why lineage structure is so stable within SP despite high levels of genetic transfer. SP is also antigenically diverse, exhibiting a variety of distinct capsular serotypes. Associations exist between lineage and capsular serotype but these can be easily perturbed, such as by vaccination. Overall, our analyses indicate that the evolution of SP can be conceptualized as the rearrangement of modular functional units occurring on several different timescales under different pressures: some patterns have locked in early (such as the epistatic interactions between groESL and a constellation of other genes) and preserve the differentiation of lineages, while others (such as the associations between capsular serotype and lineage) remain in continuous flux.

Myxococcus xanthus DK1622 Coordinates Expressions of the Duplicate groEL and Single groES Genes for Synergistic Functions of GroELs and GroES.

Chaperonin GroEL (Cpn60) requires cofactor GroES (Cpn10) for protein refolding in bacteria that possess single groEL and groES genes in a bicistronic groESL operon. Among 4,861 completely-sequenced prokaryotic genomes, 884 possess duplicate groEL genes and 770 possess groEL genes with no neighboring groES. It is unclear whether stand-alone groEL requires groES in order to function and, if required, how duplicate groEL genes and unequal groES genes balance their expressions. In Myxococcus xanthus DK1622, we determined that, while duplicate groELs were alternatively deletable, the single groES that clusters with groEL1 was essential for cell survival. Either GroEL1 or GroEL2 required interactions with GroES for in vitro and in vivo functions. Deletion of groEL1 or groEL2 resulted in decreased expressions of both groEL and groES; and ectopic complementation of groEL recovered not only the groEL but also groES expressions. The addition of an extra groES gene upstream groEL2 to form a bicistronic operon had almost no influence on groES expression and the cell survival rate, whereas over-expression of groES using a self-replicating plasmid simultaneously increased the groEL expressions. The results indicated that M. xanthus DK1622 cells coordinate expressions of the duplicate groEL and single groES genes for synergistic functions of GroELs and GroES. We proposed a potential regulation mechanism for the expression coordination.

High-Efficiency Secretion of β-Mannanase in Bacillus subtilis through Protein Synthesis and Secretion Optimization.

The manno endo-1,4-mannosidase (β-mannanase, EC. 3.2.1.78) catalyzes the random hydrolysis of internal (1 → 4)-β-mannosidic linkages in the mannan polymers. A codon optimized β-mannanase gene from Bacillus licheniformis DSM13 was expressed in Bacillus subtilis. When four Sec-dependent and two Tat-dependent signal peptide sequences cloned from B. subtilis were placed upstream of the target gene, the highest activity of β-mannanase was observed using SPlipA as a signal peptide. Then a 1.25-fold activity of β-mannanase was obtained when another copy of groESL operon was inserted into the genome of host strain. Finally, five different promoters were separately used to enhance the synthesis of the target protein. The results showed that promoter Pmglv, a modified maltose-inducible promoter, significantly elevated the production of β-mannanase. After 72 h of flask fermentation, the enzyme activity of β-mannanase in the supernatant when using locust bean gum as substrate reached 2207 U/mL. This work provided a promising β-mannanase production strain in industrial application.

The alr-groEL1 operon in Mycobacterium tuberculosis: an interplay of multiple regulatory elements

Threonylcarbamoyladenosine is a universally conserved essential modification of tRNA that ensures translational fidelity in cellular milieu. TsaD, TsaB and TsaE are identified as tRNA-A37-threonylcarbamoyl (t6A)-transferase enzymes that have been reconstituted in vitro, in few bacteria recently. However, transcriptional organization and regulation of these genes are not known in any of these organisms. This study describes the intricate architecture of a complex multicistronic alr-groEL1 operon, harboring essential genes, namely tsaD, tsaB, tsaE, groES, groEL1, and alr (required for cell wall synthesis), and rimI encoding an N-α- acetyltransferase in Mycobacterium tuberculosis. Using northern blotting, RT-PCR and in vivo fluorescence assays, genes alr to groEL1 were found to constitute an ~6.3 kb heptacistronic operon with multiple internal promoters and an I-shaped intrinsic hairpin-like cis-regulatory element. A strong promoter PtsaD within the coding sequence of rimI gene is identified in M. tuberculosis, in addition. The study further proposes an amendment in the known bicistronic groESL1 operon annotation by providing evidence that groESL1 is co-transcribed as sub-operon of alr-groEL1 operon. The architecture of alr-groEL1 operon, conservation of the genetic context and a mosaic transcriptional profile displayed under various stress conditions convincingly suggest the involvement of this operon in stress adaptation in M. tuberculosis.

Anaplasma platys in dogs from Uruguay

Anaplasmataceae family members include vector-borne bacteria of veterinary importance that may also affect humans. Ehrlichia canis and Anaplasma platys are the main members of this family detected in dogs worldwide. In Uruguay there are not many published studies on tick-borne pathogens affecting dogs, the only haemoparasite molecularly confirmed in dogs, is the piroplasm Rangelia vitalii. The aim of the present work was to detect the presence of A. platys and E. canis in dogs and dogs-associated ticks of two localities in Northwestern Uruguay. Blood samples from dogs with and without clinical signs associated with vector-borne diseases, and Rhipicephalus sanguineus obtained from these dogs were analyzed by PCR for Anaplasmataceae. Positive dogs were further analyzed by PCR for Ehrlichia spp. and A. platys. All the ticks were found negative. No dog was detected infected with E. canis, while eight dogs (4.2%) were found to be infected with A. platys. Phylogenetic analysis of groESL operon sequence for A. platys revealed no differences with sequences described for A. platys in neighbor countries and from other regions of the world. This is the first report of the presence of A. platys in Uruguay.

Real-time PCR detection and phylogenetic relationships of Neorickettsia spp. in digeneans from Egypt, Philippines, Thailand, Vietnam and the United States

Neorickettsia (Rickettsiales, Anaplasmataceae) is a genus of obligate intracellular bacterial endosymbionts of digeneans (Platyhelminthes, Digenea). Some Neorickettsia are able to invade cells of the digenean's vertebrate host and are known to cause diseases of domestic animals, wildlife, and humans. In this study we report the results of screening digenean samples for Neorickettsia collected from bats in Egypt and Mindoro Island, Philippines, snails and fishes from Thailand, and fishes from Vietnam and the USA. Neorickettsia were detected using a real-time PCR protocol targeting a 152bp fragment of the heat shock protein coding gene, GroEL, and verified with nested PCR and sequencing of a 1853bp long region of the GroESL operon and a 1371bp long region of 16S rRNA. Eight unique genotypes of Neorickettsia were obtained from digenean samples. Neorickettsia sp. 8 obtained from Lecithodendrium sp. from Egypt; Neorickettsia sp. 9 and 10 obtained from two species of Paralecithodendrium from Mindoro, Philippines; Neorickettsia sp. 11 from Lecithodendrium sp. and Neorickettsia sp. 4 (previously identified from Saccocoelioides lizae, from China) from Thailand; Neorickettsia sp. 12 from Dicrogaster sp. Florida, USA; Neorickettsia sp. 13 and SF agent from Vietnam. Sequence comparison and phylogenetic analysis demonstrated that the forms, provisionally named Neorickettsia sp. 8–13, represent new genotypes. We have for the first time detected Neorickettsia in a digenean from Egypt (and the African continent as a whole), the Philippines, Thailand and Vietnam based on PCR and sequencing evidence. Our findings suggest that further surveys from the African continent, SE Asia, and island countries are likely to reveal new Neorickettsia lineages as well as new digenean host associations.

GroEL of the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31 exhibits GroES and ATP-independent refolding activity.

The nitrogen-fixing cyanobacterium, Anabaena L-31 has two Hsp60 proteins, 59 kDa GroEL coded by the second gene of groESL operon and 61 kDa Cpn60 coded by cpn60 gene. Anabaena GroEL formed stable higher oligomer (>12-mer) in the presence of K(+) and prevented thermal aggregation of malate dehydrogenase (MDH). Using three protein substrates (MDH, All1541 and green fluorescent protein), it was found that the refolding activity of Anabaena GroEL was lower than that of Escherichia coli GroEL, but independent of both GroES and ATP. This correlated with in vivo data. GroEL exhibited ATPase activity which was enhanced in the presence of GroES and absence of a denatured protein, contrary to that observed for bacterial GroEL. However, a significant role for ATP could not be ascertained during in vitro folding assays. The monomeric Cpn60 exhibited much lower refolding activity than GroEL, unaffected by GroES and ATP. In vitro studies revealed inhibition of the refolding activity of Anabaena GroEL by Cpn60, which could be due to their different oligomeric status. The role of GroES and ATP may have been added during the course of evolution from the ancient cyanobacteria to modern day bacteria enhancing the refolding ability and ensuring wider scope of substrates for GroEL.

Analysis of Clostridium beijerinckii NCIMB 8052's transcriptional response to ferulic acid and its application to enhance the strain tolerance.

BackgroundPlant-based cellulose presents the best source of renewable sugars for biofuel production. However, the lignin associated with plant cellulose presents a hurdle as hydrolysis of this component leads to the production of inhibitory compounds, such as ferulic acid.ResultsThe impacts of ferulic acid, a phenolic compound commonly found in lignin hydrolysates, on the growth, solvent production, and transcriptional responses of Clostridium beijerinckii NCIMB 8052 were determined. Addition of ferulic acid to growing cultures resulted in a decrease in the growth and solvent production by 30% and 25%, respectively, when compared to the control cultures. To better understand the toxicity of this compound, microarray analyses were performed using samples taken from these cultures at three different growth states. Several gene ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified showing significant change at each status, including ATP-binding cassette (ABC) transporters, two component system, and oxidoreductase activity. Moreover, genes related with efflux systems and heat shock proteins were also strongly up-regulated. Among these, expression of the groESL operon was induced by more than fourfold and was consequently selected to improve C. beijerinckii tolerance to ferulic acid. Real-time quantitative PCR (RT-qPCR) analysis confirmed that C. beijerinckii harboring the plasmid, pSAAT-ptb_Gro, had a two- to fivefold increased groESL operon expression during growth of these cultures. Moreover, this strain was more tolerant to ferulic acid as the growth of this recombinant strain and its bioconversion of glucose into solvents were both improved.ConclusionsUsing transcriptomics, we identified numerous genes that are differentially expressed when C. beijerinckii cultures were exposed to ferulic acid for varying amounts of time. The operon expressing groESL was consistently up-regulated, suggesting that this gene cluster may contribute to strain tolerance. This was confirmed as recombinant cultures showed both an enhanced growth and solvent yield in the presence of 0.5 g/L ferulic acid.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0252-9) contains supplementary material, which is available to authorized users.

Cloning of some heat shock proteins genes for further transcriptional study of Planktothrix agardhii exposed to abiotic stress.

Planktothrix agardhii is one of the freshwater cyanobacteria that can produce the hepatotoxin microcystins (MC)-a real threat to human and animal health. Knowledge of the biological role of MC in producing organisms is highly desired to understand the driving force of MC production. Recently, emerging evidences have suggested that MC may have protective role in cells facing environmental stress. If this is true, one should expect differences in the cellular protective mechanisms between MC-containing and MC-deficient mutant strains. To test this hypothesis, it would be essential to investigate the consequences of the loss of MC in Planktothrix in the transcriptional responses of its heat shock proteins (Hsps) to abiotic stresses-an important component of cellular stress response. However, a crucial first step is prerequisite for the isolation of hsp genes here, as the genome of Planktothrix has not been fully published. Therefore, we have successfully isolated four hsp genes including clpC (hsp100), htpG (hsp90), groEL (hsp60), and groES (hsp10) from Planktothrix agardhii PCC 7805 using ramped annealing PCR (RAN-PCR) with consensus-degenerate hybrid oligonucleotide primers (CODEHOP) and annealing control primer (ACP) system. In addition, some putative regulatory sequences found in the upstream region of groESL operon of Planktothrix agardhii were also discussed.

Genetic variability of Anaplasma phagocytophilum in ticks and voles from Ixodes persulcatus/Ixodes trianguliceps sympatric areas from Western Siberia, Russia

Anaplasma phagocytophilum is a causative agent of granulocytic anaplasmosis in different mammals. The presence of A. phagocytophilum was assayed in Ixodes persulcatus, Ixodes trianguliceps ticks and Myodes spp. voles from two I. persulcatus/I. trianguliceps sympatric areas in the Omsk region (Western Siberia, Russia). In total, A. phagocytophilum was found in 42/108 (38.9%) of vole blood samples, 13/34 (38.2%) of I. trianguliceps ticks removed from voles, 1/12 (8.3%) of I. persulcatus removed from voles, and 18/279 (7.2%) of questing I. persulcatus. GroESL operon sequence analysis of positive samples revealed three distinct A. phagocytophilum genetic groups previously identified in ticks and mammals in Russia. Genetic group 1 was found in 6/36 (16.7%) of sequenced positive blood samples; this group was previously revealed in I. persulcatus and Myodes spp. voles in different regions of Russia. Genetic group 2 was found in 30/36 (83.3%) of sequenced positive blood samples and all positive I. trianguliceps; this group was previously revealed only in Myodes spp. voles and common shrews (Sorex araneus) in I. persulcatus/I. trianguliceps sympatric areas in the Northern Ural. Genetic group 3 was found in all positive questing I. persulcatus and one blood sample; this group was previously revealed in I. persulcatus and Siberian chipmunks (Tamias sibiricus). We suppose that I. trianguliceps is the most probable vector for A. phagocytophilum of group 2. Analysis of the msp4 gene, intergenic region DOV1, and some other genetic loci has shown that isolates from different genetic groups significantly differ in all studied loci and that A. phagocytophilum of group 2 is closely related to A. phagocytophilum isolates revealed in voles and I. trianguliceps in Europe. A. phagocytophilum of groups 1 and 2 are the most similar to each other, while A. phagocytophilum of group 3 clusters with European A. phagocytophilum isolates from I. ricinus and various mammalian species.