Heat Shock Proteins DnaK Research Articles

Molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression in Zymomonas mobilis.

Zymomonas mobilis is a promising microorganism for industrial bioethanol production. However, ethanol produced during fermentation is toxic to Z. mobilis and affects its growth and bioethanol production. Although several reports demonstrated that the RNA-binding protein Hfq in Z. mobilis contributes to the tolerance against multiple lignocellulosic hydrolysate inhibitors, the role of Hfq on ethanol tolerance has not been investigated. In this study, hfq in Z. mobilis was either deleted or overexpressed and their effects on cell growth and ethanol tolerance were examined. Our results demonstrated that hfq overexpression improved ethanol tolerance of Z. mobilis, which is probably due to energy saving by downregulating flagellar biosynthesis and heat stress response proteins, as well as reducing the reactive oxygen species induced by ethanol stress via upregulating the sulfate assimilation and cysteine biosynthesis. To explore proteins potentially interacted with Hfq, the TEV protease mediated Yeast Endoplasmic Reticulum Sequestration Screening system (YESS) was established in Z. mobilis. YESS results suggested that Hfq may modulate the cytoplasmic heat shock response by interacting with the heat shock proteins DnaK and DnaJ to deal with the ethanol inhibition. This study thus not only revealed the underlying mechanism of enhanced ethanol tolerance by hfq overexpression, but also provided an alternative approach to investigate protein-protein interactions in Z. mobilis.

Cervimycin-Resistant Staphylococcus aureus Strains Display Vancomycin-Intermediate Resistant Phenotypes.

ABSTRACTResistance to antibiotics is an increasing problem and necessitates novel antibacterial therapies. The polyketide antibiotics cervimycin A to D are natural products of Streptomyces tendae HKI 0179 with promising activity against multidrug-resistant staphylococci and vancomycin-resistant enterococci. To initiate mode of action studies, we selected cervimycin C- and D-resistant (CmR) Staphylococcus aureus strains. Genome sequencing of CmR mutants revealed amino acid exchanges in the essential histidine kinase WalK, the Clp protease proteolytic subunit ClpP or the Clp ATPase ClpC, and the heat shock protein DnaK. Interestingly, all characterized CmR mutants harbored a combination of mutations in walK and clpP or clpC. In vitro and in vivo analyses showed that the mutations in the Clp proteins abolished ClpP or ClpC activity, and the deletion of clpP rendered S. aureus but not all Bacillus subtilis strains cervimycin-resistant. The essential gene walK was the second mutational hotspot in the CmR S. aureus strains, which decreased WalK activity in vitro and generated a vancomycin-intermediate resistant phenotype, with a thickened cell wall, a lower growth rate, and reduced cell lysis. Transcriptomic and proteomic analyses revealed massive alterations in the CmR strains compared to the parent strain S. aureus SG511, with major shifts in the heat shock regulon, the metal ion homeostasis, and the carbohydrate metabolism. Taken together, mutations in the heat shock genes clpP, clpC, and dnaK, and the walK kinase gene in CmR mutants induced a vancomycin-intermediate resistant phenotype in S. aureus, suggesting cell wall metabolism or the Clp protease system as primary target of cervimycin.IMPORTANCE Staphylococcus aureus is a frequent cause of infections in both the community and hospital setting. Resistance development of S. aureus to various antibiotics is a severe problem for the treatment of this pathogen worldwide. New powerful antimicrobial agents against Gram-positives are needed, since antibiotics like vancomycin fail to cure vancomycin-intermediate resistant S. aureus (VISA) and vancomycin-resistant enterococci (VRE) infections. One candidate substance with promising activity against these organisms is cervimycin, which is an antibiotic complex with a yet unknown mode of action. In our study, we provide first insights into the mode of action of cervimycins. By characterizing cervimycin-resistant S. aureus strains, we revealed the Clp system and the essential kinase WalK as mutational hotspots for cervimycin resistance in S. aureus. It further emerged that cervimycin-resistant S. aureus strains show a VISA phenotype, indicating a role of cervimycin in perturbing the bacterial cell envelope.

Influence of Substitutions in the Binding Motif of Proline-Rich Antimicrobial Peptide ARV-1502 on 70S Ribosome Binding and Antimicrobial Activity.

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (Kd) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.

AR-12 Has a Bactericidal Activity and a Synergistic Effect with Gentamicin against Group A Streptococcus.

Streptococcus pyogenes (group A Streptococcus (GAS) is an important human pathogen that can cause severe invasive infection, such as necrotizing fasciitis and streptococcal toxic shock syndrome. The mortality rate of streptococcal toxic shock syndrome ranges from 20% to 50% in spite of antibiotics administration. AR-12, a pyrazole derivative, has been reported to inhibit the infection of viruses, intracellular bacteria, and fungi. In this report, we evaluated the bactericidal activities and mechanisms of AR-12 on GAS infection. Our in vitro results showed that AR-12 dose-dependently reduced the GAS growth, and 2.5 μg/mL of AR-12 significantly killed GAS within 2 h. AR-12 caused a remarkable reduction in nucleic acid and protein content of GAS. The expression of heat shock protein DnaK and streptococcal exotoxins was also inhibited by AR-12. Surveys of the GAS architecture by scanning electron microscopy revealed that AR-12-treated GAS displayed incomplete septa and micro-spherical structures protruding out of cell walls. Moreover, the combination of AR-12 and gentamicin had a synergistic antibacterial activity against GAS replication for both in vitro and in vivo infection. Taken together, these novel findings obtained in this study may provide a new therapeutic strategy for invasive GAS infection.

Molecular-genetic analysis markers for identification of <i>Rhodococcus</i> bacteria species

It was established that groEL genes can be used as molecular genetic markers for species identification of Rhodococcus bacteria. A restriction analysis scheme was developed for the PCR products of groEL genes using restriction enzymes BglI, NarI, SfiI, SinI, and RseI to identify the species of natural Rhodococcus bacteria. Fifty-two genome loci determining the resistance to stressful environmental condition were annotated in genome of R. pyridinivorans strain 5Ap (i.e. 49 structural and 4 regulatory genes that determine the synthesis of 23 heat shock proteins, 9 universal stress proteins, 17 P450 cytochromes). The unique nucleotide sequences encoding the synthesis of the heat shock protein DnaK (1 plasmid gene) and P450 cytochromes (2 chromosomal and 1 plasmid) were found among these genes. Thus, they can be used for molecular typing of the biotechnological R. pyridinivorans strain 5Ap.

DETECTION OF SEROLOGICAL RESPONSE TO THE RECOMBINANT CHLAMYDIA PSITTACI PROTEIN DNAK USING DOT-ELISA TECHNIQUE

The results of dot-ELISA application for the detection of IgG specific to the recombinant Chlamydia psittaci DnaK antigen in sera of farm animals vaccinated with a commercial chlamydia vaccine are presented. It was revealed, that C. psittaci heat shock protein DnaK participates in immune response of sheep immunized with the vaccine based on the C. psittaci Rostinovo-70 strain.

Cloning, prokaryotic expression, and functional characterization of a novel 70-kDa heat shock protein (DnaK) from Bacillus persicus

The dnaK gene of Bacillus persicus strain B48(T) was cloned, sequenced, and functionally characterized in the present study. The gene was 1836 bp in length, encoding a polypeptide of 611 amino acid residues. The 3D structure of protein predicted by I-TASSER represented the similarity of the overall structures of DnaK from B. persicus strain B48(T) and human protein Hsp70 (BiP) with a homology of 89 %. Based on the results, refolding heat-denatured carbonic anhydrase increased significantly up to 80 % in the presence of the purified recombinant DnaK. In addition, salt resistance experiments demonstrated a 2.7-fold increase in the survival of recombinant E. coli BL21–DnaK in the presence of 0.4 M NaCl for 60 h compared to that of the control cells. Further, Cd2+ failed to affect DnaK refolding function, while Hg2+ ion reflected a biphasic effect (inhibiting and stimulating at lower and higher than 100 nM concentrations, respectively). Finally, DnaK from B. persicus can potentially be used for improving the functional properties of enzymes and proteins through increasing folding activity and enhancing stress tolerance.

Comparative Genome Analysis of Bacillus sporothermodurans with Its Closest Phylogenetic Neighbor, Bacillus oleronius, and Bacillus cereus and Bacillus subtilis Groups.

Bacillus sporothermodurans currently possesses one of the most highly heat-resistant spores (HRS), which can withstand ultra-high temperature (UHT) processing. Determination of multiple whole genome sequences of B. sporothermodurans provided an opportunity to perform the first comparative genome analysis between strains and with B. oleronius, B. cereus, and B. subtilis groups. In this study, five whole genome sequences of B. sporothermodurans strains, including those belonging to the HRS clone (SAD and BR12) normally isolated from UHT milk, were compared with the aforementioned Bacillus species for gene clusters responsible for heat resistance. In the phylogenomic analysis, B. sporothermodurans, with its closest phylogenetic neighbor, B. oleronius, clustered with B. thermoamylovorans and B. thermotolerans. Heat shock proteins GrpE, GroES, GroEL, and DnaK presented identical sequences for all B. sporothermodurans strains, indicating that differences in functional efficiency are not involved in the thermal resistance variations. However, comparing all species evaluated, B. sporothermodurans exhibited a different gene configuration in the chromosomal region of the heat shock protein GrpE. Furthermore, only B. sporothermodurans strains presented the stage II sporulation protein P gene located in this region. Multisequence alignment and phylogenetic analysis of the ClpB protein showed differences for HRS and non-HRS strains. The study identified ClpC, ClpE, and ClpX as the three ATPases putatively involved in protein disaggregation in B. sporothermodurans. Bacillus sporothermodurans exhibits high homology with other Bacillus species in the DnaK, DnaJ, GroEL, and GroES cluster of genes involved in heat resistance. The data presented here pave the way to select and evaluate the phenotypic effects of genes putatively involved in heat resistance.

Peptides encoded in the Streptococcus mutans RcrRPQ operon are essential for thermotolerance.

The MarR-like transcriptional regulator and two ABC transporters encoded by the rcrRPQ operon in the dental caries pathogen Streptococcus mutans have important regulatory roles related to oxidative stress tolerance, genetic competence and (p)ppGpp metabolism. A unique feature of the rcrRPQ operon, when compared to other bacteria, is the presence of two peptides, designated Pep1 and Pep2, encoded in alternative reading frames at the 3' end of rcrQ. Here, we show that the rcrRPQ operon, including Pep1 and 2, is essential for S. mutans to survive and maintain viability at elevated temperatures. No major changes in the levels of the heat shock proteins DnaK or GroEL that could account for the thermosensitivity of rcrRPQ mutants were observed. By introducing a single amino acid substitution into the comX gene that deletes an internally encoded peptide, XrpA, we found that XrpA is a contributing factor to the thermosensitive phenotype of a ΔrcrR strain. Overexpression of XrpA on a plasmid also caused a significant growth defect at 42 °C. Interestingly, loss of the gene for the RelA/SpoT homologue (RSH) enzyme, relA, restored growth of the ΔrcrR strain at 42 °C. During heat stress and when a stringent response was induced, levels of (p)ppGpp were elevated in the ΔrcrR strain. Deletion of relA in the ΔrcrR strain lowered the basal levels of (p)ppGpp to those observed in wild-type S. mutans. Thus, (p)ppGpp pools are dysregulated in ΔrcrR, which likely leads to aberrant control of transcriptional/translational processes and the thermosensitive phenotype. In summary, the genes and peptides encoded in the rcrRPQ operon are critical for thermotolerance, and in some strains these phenotypes are related to altered (p)ppGpp metabolism and increased production of the XrpA peptide.

Synergy Between Proline-Rich Antimicrobial Peptides and Small Molecule Antibiotics Against Selected Gram-Negative Pathogens in vitro and in vivo.

As monotherapy, modified proline-rich antimicrobial peptides (PrAMPs) protect animals from experimental bacteremia in a dose-dependent manner. We evaluated the in vitro synergy of a modified PrAMP, A3-APO, a dimer, previously shown to inhibit the 70 kDa bacterial heat shock protein DnaK, with imipenem or colistin against two antibiotic-resistant pathogens; a carbapenemase-expressing Klebsiella pneumoniae strain K97/09 and Acinetobacter baumannii (ATCC BAA-1605). Combining antimicrobials resulted in synergy for PrAMP/colistin combination against both K. pneumoniae and A. baumannii (ΣFIC = 0.08 both) and additive activity for the A3-APO/imipenem combination against K. pneumoniae (ΣFIC = 0.53). Chex1-Arg20, (designated as ARV-1502 in preclinical development), the single chain PrAMP monomer of A3-APO, showed synergy with meropenem against a carbapenem-resistant uropathogenic Escherichia coli strain (ΣFIC = 0.38). In a murine bacteremia model using K97/09, A3-APO at 1 mg/kg demonstrated improved survival when co-administered with standard (10 mg/kg) or subtherapeutic (1 mg/kg) doses of colistin at 36 h (p < 0.05). Surprisingly, the survival benefit of A3-APO was augmented when the A3-APO dose was decreased by 50% to 0.5 mg/kg (p < 0.02) in conjunction with a subtherapeutic colistin dose (1 mg/kg). ARV-1502, as monotherapy demonstrated prolonged (>24 h) activity in a mouse Escherichia coli infection assay. Co-treatment with ARV-1502 and subtherapeutic doses of ceftazidime (150 mg/kg) was studied in a mouse model of melioidosis. ARV-1502 provided a 50% improvement in long-term (62 days) survival, but only at the lowest of 3 administered doses; survival advantage was demonstrated at 2.5 mg/kg but not at 5 or 10 mg/kg. The mortality benefit of combination therapies was not routinely accompanied by a parallel decline in blood or tissue bacterial counts in surviving animals, suggesting that the anti-infective activity of the host defense peptides (HDP) is broader than simply bacterial eradication. In fact, the hormetic effect observed in either animal models suggest that low dose HDP treatment may change the dominant mode of action in experimental bacteremia.

Establishment of a species-specific primer pair for detecting Veillonella infantium based on the 70 kDa heat shock protein gene dnaK

Recently, Veillonella infantium was isolated from tongue biofilm of a Thai child and established as a novel Veillonella species. In this study, a species-specific primer was designed to identify V. infantium on the basis of the sequence of the 70 kDa heat shock protein (dnaK) gene of Veillonella infantium JCM 31738T (= TSD-88T). The primer pair generated a specific PCR (Polymerase Chain Reaction) product specific for V. infantium, but not for other oral Veillonella species. This specific primer pair could detect dnaK even from 1 pg of genomic DNA extracted from the V. infantium type strain. To validate the primer pair, a number of strains of Veillonella species were isolated from tongue biofilm of 3 Japanese children, DNA was isolated from each strain, and PCR was performed using species-specific primers. All oral Veillonella species except V. infantium were identified by one-step PCR method reported previously. Four kinds of Veillonella species were detected in these subjects. V. rogosae was detected in all subjects and the most predominant species with an average prevalence of 82%. However, V. infantium was detected in 2 of 3 subjects and it was the second most predominant species of oral Veillonella detected in these subjects with an average prevalence of 9.4%. V. infantium appears to coexist with other oral Veillonella species in tongue biofilm. This species-specific primer pair established in this study could be useful to detect V. infantium and support the study of Veillonella for oral health in the future.

Identification and characterization of the psychrophilic bacterium CidnaK gene in the Antarctic Chlamydomoas sp. ICE-L under freezing conditions

Heat shock protein DnaK (Hsp70) can prevent irreversible protein denaturation by chaperoning the unfolded polypeptides under extremely cold environment, and enhance cold and freezing tolerance of microorganism. From an Antarctic sea-ice green alga Chlamydomonas sp. ICE-L, eight Hsp70 genes were identified and divided into seven subfamilies: four in the cytoplasmic subfamilies, two in the chloroplast subgroups, and one in the mitochondria or endoplasmic reticulum subfamilies. Phylogenetic analyses showed that except CidnaK, each of the other Cihsp70 genes had their homologs in Chlamydomonas reinhardtii and Volvox carteri. CiDnak is a cytoplasmic protein with highly homologous to DnaK proteins from prokaryotes, and it had 100% amino acid sequence identities with the DnaK of Psychroflexus torquis ATCC 700755, a psychrophilic bacterium isolated from Antarctic sea ice. The transcription of CidnaK was significantly induced upon freezing stress in ICE-L, eminently higher than that of other Cihsp70 genes. In addition, the transformed C. reinhardtii 137c with CidnaK gene showed much higher rates of survival and growth than the wild type under freezing or low temperature conditions. Our results suggested that CidnaK was likely originated from the symbiotic bacterium in the sea-ice brine, and is an important candidate freezing inducible gene that confers low temperature tolerance to Chlamydomonas cells, and plays a crucial role in ICE-L adapting the frigid Antarctic environments.

Stress-related genes promote Edwardsiella ictaluri pathogenesis.

Edwardsiella ictaluri is a Gram-negative facultative anaerobic rod and the causative agent of enteric septicemia of channel catfish (ESC), which is one of the most prevalent diseases of catfish, causing significant economic losses in the catfish industry. E. ictaluri is resistant to complement system and macrophage killing, which results in rapid systemic septicemia. However, mechanisms of E. ictaluri stress responses under conditions of host environment are not studied well. Therefore, in this work, we report E. ictaluri stress responses during hydrogen peroxide, low pH, and catfish serum stresses as well as during catfish invasion. E. ictaluri stress responses were characterized by identifying expression of 13 universal stress protein (USP) genes (usp01-usp13) and seven USP-interacting protein genes (groEL, groES, dnaK, grpE, and clpB, grpE, relA). Data indicated that three usp genes (usp05, usp07, and usp13) were highly expressed in all stress conditions. Similarly, E. ictaluri heat shock proteins groEL, groES, dnaK, grpE, and clpB were highly expressed in oxidative stress. Also, E. ictaluri grpE and relA were highly expressed in catfish spleen and head kidney. These findings contribute to our understanding of stress response mechanisms in E. ictaluri stress response, and stress-related proteins that are essential for E. ictaluri could be potential targets for live attenuated vaccine development against ESC.

Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli.

S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial proteins in E. coli.

Identification of Api88 Binding Partners in Escherichia coli Using a Photoaffinity-Cross-Link Strategy and Label-Free Quantification.

Gene-encoded antimicrobial peptides (AMPs) kill bacteria very efficiently by either lytic mechanisms or inhibition of specific bacterial targets. Proline-rich AMPs (PrAMPs), for example, produced in insects and mammals rely on the second mechanism. They bind to the 70 kDa bacterial heat shock protein DnaK and the 60 kDa chaperonin GroEL and interfere with protein folding, but this does not explain their strong bactericidal effects. Thus, we looked for further binding partners of apidaecin 1b, originally identified in honey bees, and two rationally optimized analogues (Api88 and Api137). Because affinity chromatography using Api88 as an immobilized ligand enriched only a few proteins at low levels besides DnaK, we synthesized Api88 analogues substituting Tyr7 with p-benzoyl-phenylalanine (Bpa), which can cross-link the peptide to binding partners after UV irradiation. Escherichia coli was incubated with biotinylated Api88 Tyr7Bpa or the corresponding all-d-peptide, irradiated, and lysed. The protein extract was enriched by streptavidin, separated by SDS-PAGE, digested with trypsin, and analyzed by nanoRP-UPLC-ESI-QqTOF-MS/MS. Among the 41 proteins identified, 34 were detected only in the l-Api88 Tyr7Bpa sample, including five 70S ribosomal proteins, DNA-directed RNA polymerase, and pyruvate dehydrogenase, indicating that PrAMPs might interfere with protein translation and energy metabolism.

Ingestion of food pellets containing Escherichia coli overexpressing the heat-shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection.

Feeding aquatic animals with bacterial encapsulated heat-shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK-DnaJ-GrpE, the prokaryotic equivalents of Hsp70-Hsp40-Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g(-1) pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.

Lipopeptide biosynthesis in Pseudomonas fluorescens is regulated by the protease complex ClpAP.

BackgroundLipopeptides (LP) are structurally diverse compounds with potent surfactant and broad-spectrum antibiotic activities. In Pseudomonas and other bacterial genera, LP biosynthesis is governed by large multimodular nonribosomal peptide synthetases (NRPS). To date, relatively little is known about the regulatory genetic network of LP biosynthesis.ResultsThis study provides evidence that the chaperone ClpA, together with the serine protease ClpP, regulates the biosynthesis of the LP massetolide in Pseudomonas fluorescens SS101. Whole-genome transcriptome analyses of clpA and clpP mutants showed their involvement in the transcription of the NRPS genes massABC and the transcriptional regulator massAR. In addition, transcription of genes associated with cell wall and membrane biogenesis, energy production and conversion, amino acid transport and metabolism, and pilus assembly were altered by mutations in clpA and clpP. Proteome analysis allowed the identification of additional cellular changes associated to clpA and clpP mutations. The expression of proteins of the citrate cycle and the heat shock proteins DnaK and DnaJ were particularly affected. Combined with previous findings, these results suggest that the ClpAP complex regulates massetolide biosynthesis via the pathway-specific, LuxR-type regulator MassAR, the heat shock proteins DnaK and DnaJ, and proteins of the TCA cycle.ConclusionsCombining transcriptome and proteome analyses provided new insights into the regulation of LP biosynthesis in P. fluorescens and led to the identification of specific missing links in the regulatory pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0367-y) contains supplementary material, which is available to authorized users.

Mycoplasma heat shock proteins and their genes

Mycoplasmas (class Mollicutes) are the most simply organized prokaryotic organisms capable to self-reproduction. They are considered as a model of "minimal" cell. Systems preserved by mycoplasmas in their reductive evolution may play a fundamental role in viability of any cell. Information on the genes encoding heat shock proteins (HSP) in completely sequenced mycoplasma genomes was summarized and systematized. An attempt was made to explain the presence or absence in mycoplasmas of important bacterial chaperones and proteases. These HSP are necessary not only for cell stress resistance, but for protein homeostasis under normal conditions. The mechanisms of regulation of transcription of corresponding genes are considered. Properties and functions of the most completely characterized mycoplasmal HSP are discussed: DnaK, DnaJ-like, GroEL/GroES, ClpB, and small heat shock proteins (sHSP).

Construction of a shuttle expression vector with a promoter functioning in both halophilicArchaeaandBacteria

A shuttle expression vector, designated as pAJ, was constructed based on the Haloferax volcanii-Escherichia coli shuttle vector pSY1. This new construct contains the amyH promoter from Haloarcula hispanica and was able to confer the promoter activity in both Hfx.volcanii and E.coli. pAJ successfully expressed proteins in Hfx.volcanii or E.coli, rendering it feasible to express target proteins in corresponding domains. In addition, pAJ contains a multiple cloning site with 11 restriction sites and a 6×His tag sequence, and the vector size was decreased to 8903bp. To the best of our knowledge, pAJ is the first reported shuttle expression vector that can express proteins in both Bacteria and Archaea. Importantly, pAJ can even express the haloarchaeal heat shock protein DnaK in both domains. In conclusion, this novel vector only provides researchers with a new means to manipulate genes or express proteins in Haloarchaea but also serves as a convenient tool for the comparative study of the function of some highly conserved genes in Haloarchaea and in Bacteria.

Identification of Veillonella tobetsuensis in tongue biofilm by using a species-specific primer pair

Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, and Veillonella rogosae have been reported to be isolated from human oral cavities. The recently detected Veillonellatobetsuensis in human tongue biofilms was proposed as a novel Veillonella sp.In this study, to determine the distribution and frequency of V. tobetsuensis, we established a method for the detection and identification of V. tobetsuensis by using polymerase chain reaction (PCR) using a species-specific primer pair. The primer pair for V. tobetsuensis was designed on the basis of the nucleotide sequence of the 70-kDa heat shock protein (dnaK) gene of V. tobetsuensis JCM 17976T (=ATCC BAA-2400T). The primer pair generated a specific PCR product for V. tobetsuensis but not for other oral Veillonella spp. With the PCR procedure using the primer pair, we could detect less than 10 ng of genomic DNA extracted from V. tobetsuensis. Thus, the PCR method using this primer pair is suitable for the specific detection and identification of V. tobetsuensis. The distribution and frequency of V. tobetsuensis were investigated by PCR using this species-specific primer pair. V. tobetsuensis was detected in 5 of 27 subjects. V. tobetsuensis was recovered from 19% (5/27) of subjects with other Veillonella species. And, prevalence of V. tobetsuensis ranged from 7.6% to 20.0% in these subjects. V. tobetsuensis is likely to coexist with other Veillonella spp. in tongue biofilm.In this study, the species-specific PCR primer pair for V. tobetsuensis was designed using partial sequences of the dnaK gene. This is the first report using a species-specific primer pair for PCR to determine the distribution and frequency of V. tobetsuensis in tongue biofilm.