Immune Response to the Recombinant Apa Protein from Mycobacterium tuberculosis Expressed in Streptomyces lividans After Intranasal Administration in Mice. Induction of Protective Response to Tubercle Bacillus Aerosols Exposure.

Identifying and evaluating potential vaccine candidates has become one of the main objectives to combat tuberculosis. Among them, mannosylated Apa antigen from Mycobacterium tuberculosis and the non-mannosylated protein expressed in Escherichia coli, have been studied. Although both proteins can induce a protective response in mice, it has been considered that native protein can be dispensed. In this work, we study the protective response induced by Apa expressed in E. coli and in Streptomyces lividans. The latter, like native is secreted as a double band of 45/47 kDa, however, only its 47 kDa band is mannosylated. Both antigens and BCG were intranasal administrated in mice, and animals were then challenged by aerosol with M. tuberculosis H37Rv. The results showed that both, Apa from S. lividans and E. coli conferred statistically significantly protection to animals compared to controls. The cytokine immune response was studied by an immunoassay after animals' immunization, revealing that Apa from S. lividans induced a statistically significant proliferation of T cell, as well as the expression of IFN-γ, IL-1ß, IL-17 and IL-10. In contrast, non-proliferation was obtained with non-mannosylated protein, but induction of IL-12 and IL-17 was observed. Together, these results demonstrate that both proteins were able to modulate a specific immune response against M. tuberculosis, that could be driven by different mechanisms possibly associated with the presence or not of mannosylation. Furthermore, stimulation of cells from BCG-vaccinated animals with the proteins could be an important tool, to help define the use of a given subunit-vaccine after BCG vaccination.

Pseudomonas aeruginosa strains belonging to phylogroup 3 frequently exhibit an atypical quorum sensing response: the case of MAZ105, a tomato rhizosphere isolate.

Pseudomonas aeruginosa is a widespread γ-proteobacterium and an important opportunistic pathogen. The genetically diverse P. aeruginosa phylogroup 3 strains are characterized by producing the pore-forming ExlA toxin and by their lack of a type III secretion system. However, like all strains of this species, they produce several virulence-associated traits, such as elastase, rhamnolipids and pyocyanin, which are regulated by quorum sensing (QS). The P. aeruginosa QS response comprises three systems (Las, Rhl and Pqs, respectively) that hierarchically regulate these virulence factors. The Pqs QS system is composed of the PqsR transcriptional factor, which, coupled with the alkyl-quinolones HHQ or PQS, activates the transcription of the pqsABCDE operon. The products of the first four genes of this operon produce HHQ, which is then converted to PQS by PqsH, while PqsE forms a complex with RhlR and stabilizes it. In this study we report that mutations affecting the Pqs system are particularly common in phylogroup 3 strains. To better understand QS in phylogroup 3 strains we studied strain MAZ105 isolated from tomato rhizosphere and showed that it contains mutations in the central QS transcriptional regulator, LasR, and in the gene encoding the PqsA enzyme involved in the synthesis of PQS. However, it can still produce QS-regulated virulence factors and is virulent in Galleria mellonella and mildly pathogenic in the mouse abscess/necrosis model; our results show that this may be due to the expression of pqsE from a different PqsR-independent promoter than the pqsA promoter. Our results indicate that using anti-virulence therapy based on targeting the PQS system will not be effective against infections by P. aeruginosa phylogroup 3 strains.

PqsR-independent quorum-sensing response of Pseudomonas aeruginosa ATCC 9027 outlier-strain reveals new insights on the PqsE effect on RhlR activity.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen that represents an important health hazard. The quorum-sensing response regulates the expression of several virulence factors and involves three regulons: Las, Rhl, and Pqs. The P. aeruginosa ATCC 9027 strain, which belongs to the genetically diverse PA7 clade, contains a frame-shift mutation in the pqsR gene that encodes a transcriptional activator necessary for pyocyanin (PYO) synthesis in type strains PAO1 and PA14. Here we characterize the PqsE-dependent production of PYO in strain ATCC 9027. We show that this strain expresses pqsE independently of PqsR and in the absence of quinolone production, and that PqsE promotes the RhlR-dependent production of PYO, yet this production is not strictly dependent on PqsE. In addition, we show that in both strains ATCC 9027 and PAO1, PqsE overexpression causes an increased concentration of RhlR and enhances PYO production but does not affect rhamnolipids (RL) production in the same way. These results suggest that PqsE interaction with RhlR preferentially modifies its ability to activate transcription of genes involved in PYO production and provide new evidence about PqsE-dependent RhlR activation, highlighting the variability of the QS response among different P. aeruginosa clades and strains. HIGHLIGHTS: Pseudomonas aeruginosa ATCC 9027 is able to produce pyocyanin in phosphate limiting conditions, even in the absence of a functional PqsR. This strain does not produce alkyl quinolones like PQS and HHQ, but expresses pqsE. Synthesis of pyocyanin by ATCC 9027 is only partially dependent on pqsE. The overexpression of pqsE in the ATCC 9027 and PAO1 strains causes pyocyanin overproduction. The overexpression of pqsE in these strains causes an increased RhlR concentration without affecting rhlR transcription or translation. Rhamnolipids production is not affected to the same extent as pyocyanin by overexpression of pqsE in these strains.

The Rhl Quorum-Sensing System Is at the Top of the Regulatory Hierarchy under Phosphate-Limiting Conditions in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is a major nosocomial pathogen that presents high-level resistance to antibiotics. Its ability to cause infections relies on the production of multiple virulence factors. Quorum sensing (QS) regulates the expression of many of these virulence factors through three QS systems: Las, Rhl, and PQS. The Las system positively regulates the other two systems, so it is at the top of a hierarchized regulation. Nevertheless, clinical and environmental strains that lack a functional Las system have been isolated, and, surprisingly, some of them still have the ability to produce virulence factors and infect animal models, so it has been suggested that the hierarchy is flexible under some conditions or with atypical strains. Here, we analyze the PAO1 type strain and its ΔlasR-derived mutant and report, for the first time, a growth condition (phosphate limitation) where LasR absence has no effect either on virulence factor production or on the gene expression profile, in contrast to a condition of phosphate repletion where the LasR hierarchy is maintained. This work provides evidence on how the QS hierarchy can change from being a strictly LasR-dependent to a LasR-independent RhlR-based hierarchy under phosphate limitation even in the PAO1 type strain.IMPORTANCEPseudomonas aeruginosa is an important pathogen, considered a priority for the development of new therapeutic strategies. An important approach to fight its infections relies on blocking quorum sensing. The Las system is the main regulator of the quorum-sensing response, so many research efforts aim to block this system to suppress the entire response. In this work, we show that LasR is dispensable in a phosphate-limited environment in the PAO1 type strain, which has been used to define the quorum-sensing response hierarchy, and that under this condition RhlR is at the top of the regulation hierarchy. These results are highly significant, since phosphate limitation represents a similar environment to the one that P. aeruginosa faces when establishing infections.

Evolution of bacteria seen through their essential genes: the case of Pseudomonas aeruginosa and Azotobacter vinelandii.

Pseudomonas aeruginosa is a metabolically versatile bacterium and also an important opportunistic pathogen. It has a remarkable genomic structure since the genetic information encoding its pathogenicity-related traits belongs to its core-genome while both environmental and clinical isolates are part of the same population with a highly conserved genomic sequence. Unexpectedly, considering the high level of sequence identity and homologue gene number shared between different P. aeruginosa isolates, the presence of specific essential genes of the two type strains PAO1 and PA14 has been reported to be highly variable. Here we report the detailed bioinformatics analysis of the essential genes of P. aeruginosa PAO1 and PA14 that have been previously experimentally identified and show that the reported gene variability was owed to sequencing and annotation inconsistencies, but that in fact they are highly conserved. This bioinformatics analysis led us to the definition of 348 P. aeruginosa general essential genes. In addition we show that 342 of these 348 essential genes are conserved in Azotobacter vinelandii, a nitrogen-fixing, cyst-forming, soil bacterium. These results support the hypothesis of A. vinelandii having a polyphyletic origin with a Pseudomonads genomic backbone, and are a challenge to the accepted theory of bacterial evolution.

Inactivation of the quorum-sensing transcriptional regulators LasR or RhlR does not suppress the expression of virulence factors and the virulence of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an environmental bacterium but is also an opportunistic pathogen. The aim of this work is to evaluate the contribution of P. aeruginosa LasR and RhlR transcriptional regulators of the quorum-sensing response (QSR) to the production of virulence factors, and to its virulence in a mouse abscess model. The QSR is a complex regulatory network that modulates the expression of several virulence factors, including elastase, pyocyanin and rhamnolipids. LasR, when complexed with the auto-inducer 3-oxo-dodecanoyl lactone (3O-C12-HSL), produced by LasI, is at the top of the QSR regulatory cascade since it activates transcription of some genes encoding virulence factors (such as the gene coding for elastase, lasB) and also transcription of both rhlR and rhlI, encoding the synthase of the auto-inducer butanoyl-homoserine lactone (C4-HSL). In turn RhlR, coupled with C4-HSL, activates the transcription of genes encoding for the enzymes involved in pyocyanin and rhamnolipid production. Several efforts have been made to obtain inhibitors of LasR activity that would suppress the QSR. However, these attempts have used chemical compounds that might not be specific for LasR inactivation. In this work we show that individual inactivation of either lasR or rhlR did not block the QSR, nor did it impair P. aeruginosa virulence, and that even a lasR rhlR double mutant still presented residual virulence, even lacking the production of virulence factors. These results show that the inhibition of either lasR or rhlR is not a straightforward approach to blocking P. aeruginosa virulence, due to the great complexity of the QSR.

Role of ß-oxidation and de novo fatty acid synthesis in the production of rhamnolipids and polyhydroxyalkanoates by Pseudomonas aeruginosa.

Pseudomonas aeruginosa are ubiquitous γ-proteobacteria capable of producing the biosurfactant rhamnolipids (RL) and the polymer polyhydroxyalkanoate (PHA). RL are glycolipids with high biotechnological potential, whereas PHA is used for the production of biodegradable plastics. It has been proposed that the ß-oxidation pathway provides intermediates for RL biosynthesis, even when using a non-fatty acid carbon source for growth, while an intermediate of de novo fatty acid biosynthesis (FASII) pathway [(R)-3-hydroxyacyl-ACP] is used for PHA biosynthesis. The aim of this work is to study the inter-relationship of the RL and PHA biosynthetic pathways in a culture medium with a non-fatty acid carbon source, focusing on the role of FASII and ß-oxidation in supplying the substrates for the first step in RL and PHA synthesis, carried out by the RhlA and PhaG enzymes, respectively. The PHA synthases (PhaC1 and PhaC2) are only able to use CoA-linked 3-hydroxy acids and the PhaG enzyme catalyzes the conversion of (R)-3-hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA, the substrate of PhaC1 and PhaC2. RhlA in turn catalyzes the synthesis of the RL precursor 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAA) by the dimerization of two 3-hydroxyalkanoic acid molecules (that have been shown to be also (R)-3-hydroxyacyl-ACP). In this work, we show that RhlA can produce both RL and PHA precursors (presumably CoA-linked HAA), that the blockage of carbon flux through ß-oxidation pathway does not decrease RL titer, and that the enoyl-CoA hydratase RhlY and enoyl-CoA hydratase/isomerase RhlZ produce the main fatty acids precursor of RL using as substrate also a FASII intermediate (presumably (S)-3-hydroxyacyl-CoA).

5p and 3p Strands of miR-34 Family Members Have Differential Effects in Cell Proliferation, Migration, and Invasion in Cervical Cancer Cells.

The micro RNA (miR)-34 family is composed of 5p and 3p strands of miR-34a, miR-34b, and miR-34c. The 5p strand's expression and function is studied in cervical cancer. The 3p strand's function and regulation remain to be elucidated. To study the function of the passenger strands of miR-34 family members, we overexpressed 5p and 3p strands using a synthetic miRNA in cervical cell lines. Cell proliferation was evaluated using crystal violet. Migration and invasion were tested using transwell assays, Western blot, and zymography. Possible specific targets and cell signaling were investigated for each strand. We found that miR-34a-5p inhibited proliferation, migration, and cell invasion accompanied by matrix metalloproteinase 9 (MMP9) activity and microtubule-associated protein 2 (MAP2) protein reduction. We also found that miR-34b-5p and miR-34c-5p inhibit proliferation and migration, but not invasion. In contrast, miR-34c-5p inhibits MMP9 activity and MAP2 protein, while miR-34b-5p has no effect on these genes. Furthermore, miR-34a-3p and miR-34b-3p inhibit proliferation and migration, but not invasion, despite the later reducing MMP2 activity, while miR-34c-3p inhibit proliferation, migration, and cell invasion accompanied by MMP9 activity and MAP2 protein inhibition. The difference in cellular processes, MMP2 and MMP9 activity, and MAP2 protein inhibition by miR-34 family members suggests the participation of other regulated genes. This study provides insights into the roles of passenger strands (strand*) of the miR-34 family in cervical cancer.

Overproduction of rhamnolipids in Pseudomonas aeruginosa PA14 by redirection of the carbon flux from polyhydroxyalkanoate synthesis and overexpression of the rhlAB-R operon.

OBJECTIVE: To construct Pseudomonas aeruginosa PA14 derivatives that overproduce rhamnolipids (RL) by blocking the synthesis of the carbon-storage polymer polyhydroxyalkanoates (PHA) and by overexpressing the rhlAB-R operon that encodes for enzymes of RL synthesis and the RhlR transcriptional regulator. RESULTS: In contrast to previous results showing that overexpression of rhlAB-R genes in two P. aeruginosa strains (PAO1 and ATCC 9027) is sufficient to overproduce RL, we show that a PA14 derivative overexpressing the rhlAB-R operon did not increase the synthesis of these biosurfactants. In addition, PA14 mutants deficient in PHA production did not overproduce RL either. However, if the rhlAB-R genes were expressed in a mutant that is completely impaired in PHA synthesis, a significant increase in RL production was observed (59%). These results show that RL production in PA14 is limited both by the availability of fatty acid precursors and by the levels of the RhlA and RhlB enzymes that are involved in the synthesis of mono-RL. CONCLUSIONS: The limitation of RL production by P. aeruginosa PA14 is multifactorial and diverse from the results obtained with other strains. Thus, the factors that limit RL production are particular to each P. aeruginosa strain, so strain-specific strategies should be developed to increase their production.

Regulation of Pseudomonas aeruginosa virulence factors by two novel RNA thermometers.

In a number of bacterial pathogens, the production of virulence factors is induced at 37 °C; this effect is often regulated by mRNA structures formed in the 5' untranslated region (UTR) that block translation initiation of genes at environmental temperatures. At 37 °C, the RNA structures become unstable and ribosomes gain access to their binding sites in the mRNAs. Pseudomonas aeruginosa is an important opportunistic pathogen and the expression of many of its virulence-associated traits is regulated by the quorum-sensing (QS) response, but the effect of temperature on virulence-factor expression is not well-understood. The aim of this work is the characterization of the molecular mechanism involved in thermoregulation of QS-dependent virulence-factor production. We demonstrate that traits that are dependent on the QS transcriptional regulator RhlR have a higher expression at 37 °C, correlating with a higher RhlR concentration as measured by Western blot. We also determined, using gene fusions and point mutations, that RhlR thermoregulation is a posttranscriptional effect dependent on an RNA thermometer of the ROSE (Repression Of heat-Shock gene Expression) family. This RNA element regulates the expression of the rhlAB operon, involved in rhamnolipid production, and of the downstream rhlR gene. We also identified a second functional thermometer in the 5' UTR of the lasI gene. We confirmed that these RNA thermometers are the main mechanism of thermoregulation of QS-dependent gene expression in P. aeruginosa using quantitative real-time PCR. This is the first description, to our knowledge, of a ROSE element regulating the expression of virulence traits and of an RNA thermometer controlling multiple genes in an operon through a polar effect.

Use of Mature miRNA Strand Selection in miRNAs Families in Cervical Cancer Development.

Aberrant miRNA expression is well recognized as a cancer hallmark, nevertheless miRNA function and expression does not always correlate in patients tissues and cell lines studies. In addition to this issue, miRNA strand usage conduces to increased cell signaling pathways modulation diversifying cellular processes regulation. In cervical cancer, 20 miRNA families are involved in carcinogenesis induction and development to this moment. These families have 5p and 3p strands with different nucleotide (nt) chain sizes. In general, mature 5p strands are larger: two miRNAs of 24 nt, 24 miRNAs of 23 nt, 35 miRNAs of 22 nt and three miRNAs of 21 nt. On the other hand, the 3p strands lengths observed are: seven miRNAs of 23 nt, 50 miRNAs of 22 nt, six miRNAs of 21 nt and four miRNAs of 20 nt. Based on the analysis of the 20 miRNA families associated with cervical cancer, 67 3p strands and 65 5p strands are selected suggesting selectivity and specificity mechanisms regulating cell processes like proliferation, apoptosis, migration, invasion, metabolism and Warburg effect. The insight reviewed here could be used in the miRNA based therapy, diagnosis and prognosis approaches.

Pseudomonas aeruginosa ATCC 9027 is a non-virulent strain suitable for mono-rhamnolipids production.

Rhamnolipids produced by Pseudomonas aeruginosa are biosurfactants with a high biotechnological potential, but their extensive commercialization is limited by the potential virulence of P. aeruginosa and by restrictions in producing these surfactants in heterologous hosts. In this work, we report the characterization of P. aeruginosa strain ATCC 9027 in terms of its genome-sequence, virulence, antibiotic resistance, and its ability to produce mono-rhamnolipids when carrying plasmids with different cloned genes from the type strain PAO1. The genes that were expressed from the plasmids are those coding for enzymes involved in the synthesis of this biosurfactant (rhlA and rhlB), as well as the gene that codes for the RhlR transcriptional regulator. We confirm that strain ATCC 9027 forms part of the PA7 clade, but contrary to strain PA7, it is sensitive to antibiotics and is completely avirulent in a mouse model. We also report that strain ATCC 9027 mono-rhamnolipid synthesis is limited by the expression of the rhlAB-R operon. Thus, this strain carrying the rhlAB-R operon produces similar rhamnolipids levels as PAO1 strain. We determined that strain ATCC 9027 with rhlAB-R operon was not virulent to mice. These results show that strain ATCC 9027, expressing PAO1 rhlAB-R operon, has a high biotechnological potential for industrial mono-rhamnolipid production.

Families of microRNAs Expressed in Clusters Regulate Cell Signaling in Cervical Cancer.

Tumor cells have developed advantages to acquire hallmarks of cancer like apoptosis resistance, increased proliferation, migration, and invasion through cell signaling pathway misregulation. The sequential activation of genes in a pathway is regulated by miRNAs. Loss or gain of miRNA expression could activate or repress a particular cell axis. It is well known that aberrant miRNA expression is well recognized as an important step in the development of cancer. Individual miRNA expression is reported without considering that miRNAs are grouped in clusters and may have similar functions, such as the case of clusters with anti-oncomiRs (23b~27b~24-1, miR-29a~29b-1, miR-29b-2~29c, miR-99a~125b-2, miR-99b~125a, miR-100~125b-1, miR-199a-2~214, and miR-302s) or oncomiRs activity (miR-1-1~133a-2, miR-1-2~133a-1, miR-133b~206, miR-17~92, miR-106a~363, miR183~96~182, miR-181a-1~181b-1, and miR-181a-2~181b-2), which regulated mitogen-activated protein kinases (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), NOTCH, proteasome-culling rings, and apoptosis cell signaling. In this work we point out the pathways regulated by families of miRNAs grouped in 20 clusters involved in cervical cancer. Reviewing how miRNA families expressed in cluster-regulated cell path signaling will increase the knowledge of cervical cancer progression, providing important information for therapeutic, diagnostic, and prognostic methodology design.

Pseudomonas aeruginosa clinical and environmental isolates constitute a single population with high phenotypic diversity.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen with a high incidence of hospital infections that represents a threat to immune compromised patients. Genomic studies have shown that, in contrast to other pathogenic bacteria, clinical and environmental isolates do not show particular genomic differences. In addition, genetic variability of all the P. aeruginosa strains whose genomes have been sequenced is extremely low. This low genomic variability might be explained if clinical strains constitute a subpopulation of this bacterial species present in environments that are close to human populations, which preferentially produce virulence associated traits. RESULTS: In this work, we sequenced the genomes and performed phenotypic descriptions for four non-human P. aeruginosa isolates collected from a plant, the ocean, a water-spring, and from dolphin stomach. We show that the four strains are phenotypically diverse and that this is not reflected in genomic variability, since their genomes are almost identical. Furthermore, we performed a detailed comparative genomic analysis of the four strains studied in this work with the thirteen previously reported P. aeruginosa genomes by means of describing their core and pan-genomes. CONCLUSIONS: Contrary to what has been described for other bacteria we have found that the P. aeruginosa core genome is constituted by a high proportion of genes and that its pan-genome is thus relatively small. Considering the high degree of genomic conservation between isolates of P. aeruginosa from diverse environments, including human tissues, some implications for the treatment of infections are discussed. This work also represents a methodological contribution for the genomic study of P. aeruginosa, since we provide a database of the comparison of all the proteins encoded by the seventeen strains analyzed.

Strong seed-bank effects in bacterial evolution.

Bacterial genomes are mosaics with fragments showing distinct phylogenetic origins or even being unrelated to any other genetic information (ORFan genes). Thus the analysis of bacterial population genetics is in large part a collection of explanations for anomalies in relation to classical population genetic models such as the Wright-Fisher model and the Kingman coalescent that do not adequately describe bacterial population genetics, genomics or evolution. The concept of "species" as an evolutionary coherent biological group that is genetically isolated and shares genetic information through recombination among its members cannot be applied to any bacterial group. Recently, a simple probabilistic model considering the role of strong seed-bank effects in population genetics has been proposed by Blath et al. This model suggests the existence of a genetic pool with high diversity that is not subject to classical selection and extinction. We reason that certain bacterial population genetics anomalies could be explained by the prevalence of strong seed-bank effects among bacteria. To address this possibility we analyzed the genome of the bacterium Azotobacter vinelandii and show that genes that code for functions that are essential for the bacterium biology do not have a relation of ancestry with closely related bacteria, or are ORFan genes. The existence of essential genes that are not inherited from the most recent ancestor cannot be explained by classical population genetics models and is irreconcilable with the current view of genes acquired by horizontal transfer as being accessory or adaptive.

Scale-up from shake flasks to bioreactor, based on power input and Streptomyces lividans morphology, for the production of recombinant APA (45/47 kDa protein) from Mycobacterium tuberculosis.

Culture conditions in shake flasks affect filamentous Streptomyces lividans morphology, as well the productivity and O-mannosylation of recombinant Ala-Pro-rich O-glycoprotein (known as the 45/47 kDa or APA antigen) from Mycobacterium tuberculosis. In order to scale up from previous reported shake flasks to bioreactor, data from the literature on the effect of agitation on morphology of Streptomyces strains were used to obtain gassed volumetric power input values that can be used to obtain a morphology of S. lividans in bioreactor similar to the morphology previously reported in coiled/baffled shake flasks by our group. Morphology of S. lividans was successfully scaled-up, obtaining similar mycelial sizes in both scales with diameters of 0.21 ± 0.09 mm in baffled and coiled shake flasks, and 0.15 ± 0.01 mm in the bioreactor. Moreover, the specific growth rate was successfully scaled up (0.09 ± 0.02 and 0.12 ± 0.01 h(-1), for bioreactors and flasks, respectively), and the recombinant protein productivity measured by densitometry, as well. More interestingly, the quality of the recombinant glycoprotein measured as the amount of mannoses attached to the C-terminal of APA was also scaled- up; with up to five mannose residues in cultures carried out in shake flasks; and six in the bioreactor. However, final biomass concentration was not similar, indicating that although the process can be scaled-up using the power input, others factors like oxygen transfer rate, tip speed or energy dissipation/circulation function can be an influence on bacterial metabolism.

Possible involvement of the sco2127 gene product in glucose repression of actinorhodin production in Streptomyces coelicolor.

Streptomyces coelicolor mutants resistant to 2-deoxyglucose are insensitive to carbon catabolite repression (CCR). Total reversion to CCR sensitivity is observed by mutant complementation with a DNA region harboring both glucose kinase glkA gene and the sco2127 gene. The sco2127 is located upstream of glkA and encodes a putative protein of 20.1 kDa. In S. coelicolor, actinorhodin production is subject to glucose repression. To explore the possible involvement of both SCO2127 and glucose kinase (Glk) in the glucose sensitivity of actinorhodin production, this effect was evaluated in a wild-type S. coelicolor A3(2) M145 strain and a sco2127 null mutant (Δsco2127) derived from this wild-type strain. In comparison with strain M145, actinorhodin production by the mutant was insensitive to glucose repression. Under repressive conditions, only minor differences were observed in glucose utilization and Glk production between these strains. SCO2127 was detected mainly during the first 36 h of fermentation, just before the onset of antibiotic production, and its synthesis was not related to a particular carbon source. The glucose sensitivity of antibiotic production was restored to wild-type phenotype by transformation with an integrative plasmid containing sco2127. Our results support the hypothesis that SCO2127 is a negative regulator of actinorhodin production and suggest that the effect is independent of Glk.

Transcriptional regulation of Pseudomonas aeruginosa rhlR: role of the CRP orthologue Vfr (virulence factor regulator) and quorum-sensing regulators LasR and RhlR.

The production of many virulence factors by Pseudomonas aeruginosa is regulated by the quorum-sensing (QS) response. In this regulatory network LasR and RhlR, bound to their corresponding autoinducers, play a central role. The QS response has a hierarchical structure: LasR/3O-C12-HSL activates the transcription of rhlR, and RhlR/C4-HSL activates the transcription of several genes, including the rhlAB operon, which encodes the enzymes responsible for rhamnolipid synthesis. The rhlAB operon is located immediately upstream of the rhlR gene. rhlR has four transcription start sites, two of which are located in the rhlB coding region. Vfr directly activates transcription of lasR, and has been reported to be also involved in rhlR expression. The aim of this work was to characterize the details of the mechanism of rhlR transcriptional regulation. We show that Vfr directly regulates rhlR transcription through its binding to several Vfr-binding sites (VBSs) present in the rhlR promoter region, one of which has a negative effect on transcription. Two of the VBSs overlap with las boxes where LasR/3O-C12-HSL binds to activate rhlR transcription. We also show that rhlR transcription is subject to positive-feedback autoregulation through RhlR/C4-HSL activation of the rhlA promoter. This positive autoregulation plays a major role in rhlR expression.

The O-mannosylation and production of recombinant APA (45/47 KDa) protein from Mycobacterium tuberculosis in Streptomyces lividans is affected by culture conditions in shake flasks.

BACKGROUND: The Ala-Pro-rich O-glycoprotein known as the 45/47 kDa or APA antigen from Mycobacterium tuberculosis is an immunodominant adhesin restricted to mycobacterium genus and has been proposed as an alternative candidate to generate a new vaccine against tuberculosis or for diagnosis kits. In this work, the recombinant O-glycoprotein APA was produced by the non-pathogenic filamentous bacteria Streptomyces lividans, evaluating three different culture conditions. This strain is known for its ability to produce heterologous proteins in a shorter time compared to M. tuberculosis. RESULTS: Three different shake flask geometries were used to provide different shear and oxygenation conditions; and the impact of those conditions on the morphology of S. lividans and the production of rAPA was characterized and evaluated. Small unbranched free filaments and mycelial clumps were found in baffled and coiled shake flasks, but one order of magnitude larger pellets were found in conventional shake flasks. The production of rAPA is around 3 times higher in small mycelia than in larger pellets, most probably due to difficulties in mass transfer inside pellets. Moreover, there are four putative sites of O-mannosylation in native APA, one of which is located at the carboxy-terminal region. The carbohydrate composition of this site was determined for rAPA by mass spectrometry analysis, and was found to contain different glycoforms depending on culture conditions. Up to two mannoses residues were found in cultures carried out in conventional shake flasks, and up to five mannoses residues were determined in coiled and baffled shake flasks. CONCLUSIONS: The shear and/or oxygenation parameters determine the bacterial morphology, the productivity, and the O-mannosylation of rAPA in S. lividans. As demonstrated here, culture conditions have to be carefully controlled in order to obtain recombinant O-glycosylated proteins with similar "quality" in bacteria, particularly, if the protein activity depends on the glycosylation pattern. Furthermore, it will be an interesting exercise to determine the effect of shear and oxygen in shake flasks, to obtain evidences that may be useful in scaling-up these processes to bioreactors. Another approach will be using lab-scale bioreactors under well-controlled conditions, and study the impact of those on rAPA productivity and quality.

Interaction of SCO2127 with BldKB and its possible connection to carbon catabolite regulation of morphological differentiation in Streptomyces coelicolor.

In Streptomyces coelicolor, the sco2127 gene is located upstream of the gene encoding for glucose kinase. This region restores sensitivity to carbon catabolite repression (CCR) of Streptomyces peucetius var. caesius mutants, resistant to 2-deoxyglucose (Dog(R)). In order to search for the possible mechanisms behind this effect, sco2127 was overexpressed and purified for protein-protein interaction studies. SCO2127 was detected during the late growth phase of S. coelicolor grown in a complex media supplemented with 100 mM glucose. Pull-down assays using crude extracts from S. coelicolor grown in the same media, followed by far-western blotting, allowed detection of two proteins bound to SCO2127. The proteins were identified by MALDI-TOF mass spectrometry as SCO5113 and SCO2582. SCO5113 (BldKB) is a lipoprotein ABC-type permease (∼66 kDa) involved in mycelium differentiation by allowing the transport of the morphogenic oligopeptide Bld261. SCO2582, is a putative membrane metalloendopeptidase (∼44 kDa) of unknown function. In agreement with the possible role of SCO2127 in mycelium differentiation, delayed aerial mycelium septation and sporulation was observed when S. coelicolor A3(2) was grown in the presence of elevated glucose concentrations (100 mM), an effect not seen in a Δ-sco2127 mutant derived from it. We speculate that SCO2127 might represent a key factor in CCR of mycelium differentiation by interacting with BldKB.