Late Log Phase Of Growth Research Articles

Methanooceanicella nereidis gen. nov., sp. nov., the first oceanic Methanocellaceae methanogen, isolated from potential methane hydrate bearing area offshore southwestern Taiwan.

A novel mesophilic, hydrogenotrophic methanogen, strain CWC-04T, was obtained from a sediment sample extracted from a gravity core retrieved at station 22 within the KP-9 area off the southwestern coast of Taiwan during the ORIII-1368 cruise in 2009. Cells of strain CWC-04T were rod-shaped, 1.4-2.9 µm long by 0.5-0.6 µm wide, and occurred singly. Strain CWC-04Tutilized formate, H2/CO2, 2-propanol/CO2 or 2-butanol/CO2 as catabolic substrates. The optimal growth conditions were 42 °C, 0.17 M NaCl and pH 5.35. The genomic DNA G+C content calculated from the genome sequence of strain CWC-04T was 46.19 mol%. Phylogenetic analysis of 16S rRNA gene revealed that strain CWC-04T is affiliated with the genus Methanocella. The 16S rRNA gene sequences similarities within strains Methanocella arvoryzae MRE50T, Methanocella paludicola SANAET and Methanocella conradii HZ254T were 93.7, 93.0 and 91.3 %, respectively. In addition, the optical density of CWC-04T culture dropped abruptly upon entering the late-log growth phase, with virus-like particles (150 nm in diameter) being observed on and around the cells. This observation suggests that strain CWC-04T harbours a lytic virus. Based on these phenotypic, phylogenetic and genomic results, we propose that strain CWC-04T represents a novel species of a novel genus in the family Methanocellaceae, for which the name Methanooceanicella nereidis gen. nov., sp. nov. is proposed. The type strain is CWC-04T (=BCRC AR10050T=NBRC 113165T).

Improved lipid production and component of mycosporine-like amino acids by co-overexpression of amt1 and aroB genes in Synechocystis sp. PCC6803

Implementing homologous overexpression of the amt1 (A) and aroB (B) genes involved in ammonium transporter and the synthesis of mycosporine-like amino acids (MAAs) and aromatic amino acids, respectively, we created three engineered Synechocystis sp. PCC6803 strains, including Ox-A, Ox-B, and Ox-AB, to study the utilization of carbon and nitrogen in cyanobacteria for the production of valuable products. With respect to amt1 overexpression, the Ox-A and Ox-AB strains had a greater growth rate under (NH4)2SO4 supplemented condition. Both the higher level of intracellular accumulation of lipids in Ox-A and Ox-AB as well as the increased secretion of free fatty acids from the Ox-A strain were impacted by the late-log phase of cell growth. It is noteworthy that among all strains, the Ox-B strain undoubtedly spotted a substantial accumulation of glycogen as a consequence of aroB overexpression. Additionally, the ammonium condition drove the potent antioxidant activity in Ox strains with a late-log phase, particularly in the Ox-B and Ox-AB strains. This was probably related to the altered MAA component inside the cells. The higher proportion of P4-fraction was induced by the ammonium condition in both Ox-B and Ox-AB, while the noted increase of the P1 component was found in the Ox-A strain.

Microbial thermogenesis is dependent on ATP concentrations and the protein kinases ArcB, GlnL, and YccC.

Organisms necessarily release heat energy in their pursuit of survival. This process is known as cellular thermogenesis and is implicated in many processes from cancer metabolism to spontaneous farm fires. However, the molecular basis for this fundamental phenomenon is yet to be elucidated. Here, we show that the major players involved in the cellular thermogenesis of Escherichia coli are the protein kinases ArcB, GlnL, and YccC. We also reveal the substrate-level control of adenosine triphosphate (ATP)-driven autophosphorylation that governs cellular thermogenesis. Specifically, through live cell microcalorimetry, we find these regulatory proteins, when knocked out in a model E. coli strain, dysregulate cellular thermogenesis. This dysregulation can be seen in an average 25% or greater increase in heat output by these cells. We also discover that both heat output and intracellular ATP levels are maximal during the late log phase of growth. Additionally, we show that microbial thermogenesis can be engineered through overexpressing glnL. Our results demonstrate a correlation between ATP concentrations in the cell and a cell's ability to generate excess heat. We expect this work to be the foundation for engineering thermogenically tuned organisms for a variety of applications.

Fluoride and gallein inhibit polyphosphate accumulation by oral pathogen Rothia dentocariosa.

The uptake and storage of extracellular orthophosphate (Pi) by polyphosphate (polyP) accumulating bacteria may contribute to mineral dissolution in the oral cavity. To test the effect of potential inhibitors of polyP kinases on Rothia dentocariosa, gallein (0, 25, 50, and 100 μM) and fluoride (0, 50, and 100 ppm) were added to R. dentocariosa cultures grown in brain-heart infusion broth. At a late log growth phase (8 h), extracellular Pi was measured using an ascorbic acid assay, and polyP was isolated from bacterial cells treated with RNA/DNAases using a neutral phenol/chloroform extraction. Extracts were hydrolyzed and quantified as above. Gallein and fluoride had minor effects on bacterial growth with NaF having a direct effect on media pH. Gallein (≥25 μM) and fluoride (≥50 ppm) attenuated the bacterial drawdown of extracellular Pi by 56.7% (P<0.05) and 37.3% (P<0.01). There was a corresponding polyP synthesis decrease of 73.2% (P<0.0001) from gallein and 83.1% (P<0.0001) from fluoride. Attenuated total reflectance-Fourier-transform infrared spectroscopy validated the presence of polyP and its reduced concentration in R. dentocariosa bacterial cells following gallein and fluoride treatment. Rothia dentocariosa can directly change extracellular Pi and accumulate intracellular polyP, but the mechanism is attenuated by gallein and NaF.

Metabolism of Scenedesmus obliquus cultivated with raw plant substrates.

The potential benefits of adding raw, non-food, lignocellulosic plant material as a carbon source for mixotrophic growth of microalgae have previously been demonstrated. This approach has advantages over using traditional carbon sources like glucose or acetate due to wide-spread plant biomass availability and substrate recalcitrance to bacterial contamination. Here, we report the overall growth characteristics and explore the metabolic patterns of Scenedesmus obliquus cultured in the presence raw plant substrate. An initial screen of plant substrate candidates showed an increase in specific growth rate and biomass accumulation when S. obliquus was cultured in the presence of switchgrass or yard waste compared to media alone. We observed a near doubling of microalgal dry weight when S. obliquus was grown with 0.2% (w/v) switchgrass under ambient CO2. Scanning electron microscopy (SEM) of corn stem after S. obliquus cultivation exhibited substantial phloem degradation. Transcriptomic analyses of S. obliquus during mid- and late-log phase growth revealed a dynamic metabolic landscape within many KEGG pathways. Notably, differential expression was observed for several potential glycosyl hydrolases. We also investigated the influence of switchgrass on the growth of S. obliquus at 50 L volume in mini raceway ponds to determine the scalability of this approach.

In vitro activity of imipenem/relebactam plus aztreonam against metallo-β-lactamase-producing, OprD-deficient Pseudomonas aeruginosa with varying levels of Pseudomonas-derived cephalosporinase production

BackgroundTreatment options for metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa infections are limited. Imipenem/relebactam (I/R) plus aztreonam (ATM) may be an option. MethodsTen OprD(-) P. aeruginosa isolates (3 parent strains; 7 MBL-producers) were evaluated using checkerboard methodology and Fractional Inhibitory Concentration Index (FICI). Isolates exhibiting synergy in checkerboard studies (FICI ≤0.5) were evaluated using 24-h static concentration time-kill. Bacteria in late log-phase growth were diluted to 1 × 106 cfu/mL and incubated at 37°C for 24 h. Samples were drawn at 0, 2, 4, 6 and 24 h. Physiological fCmax, fCss,avg and fCmin of imipenem (26.7, 5.6, 0.5 mg/L), relebactam (REL; 13.1, 4, 0.8 mg/L) and ATM (62, 29, 8 mg/L) were used. Synergy in time-kill studies was defined as >2 log10 cfu/mL reduction compared with the most active individual agent. ResultsSynergy was observed in five isolates in checkerboard studies, including three of seven MBL-producing isolates. Isolates that were OprD(-) and harbored inducible Pseudomonas-derived cephalosporinases (PDCs) did not show synergy as defined by FICI; however, ATM minimum inhibitory concentrations (MICs) were significantly reduced with the combination. In time-kill studies, ATM alone was as active as combination regimens for MBL-producing isolates with deleted or inducible PDC production. For strains exhibiting constitutive PDC production, I/R plus ATM was synergistic at fCss,avg concentrations but exhibited similar activity to ATM at fCmin and fCmax concentrations. ConclusionsI/R plus ATM appears to exhibit synergy for some MBL-producing P. aeruginosa at physiological concentrations. Further study of the effect of dynamic concentrations is needed to fully understand the utility of this combination.

Assessment of β-D-glucosidase activity and bgl gene expression of Oenococcus oeni SD-2a.

Glycosidases enhance flavor during wine-making by mediating the enzymatic release of aroma molecules. In order to better understand the aroma enhancement potential of Oenococcus oeni SD-2a, β-D-glucosidase (βG) activities in the culture supernatant, whole cells, and disrupted cell lysate were assessed at mid log, late log and stationary growth phase. The enzymatic activity was also compared further from cell cultures with 5 different carbon sources (glucose, cellobiose, arbutin, glucose and cellobiose, glucose and arbutin) at late log phase. Correspondingly, expression levels of 3 bgl genes, OEOE-0224, OEOE-1210, and OEOE-1569 were investigated from cell cultures of the 3 growth phases, and the 5 cell cultures with different carbon sources. Finally, the volatile aroma compounds released by O. oeni SD-2a in synthetic wines with natural glycosides were evaluated by GC-MS. Results showed βG of O. oeni SD-2a was not extracellular enzyme, and the location of it didn’t change with the change of growth phase and carbon source studied. βG activities in the whole cells and disrupted cell lysate were similar and constant at the 3 growth phases. As for the carbon sources, βG activities of whole cells and disrupted lysate were positively affected by cellobiose. While arbutin displayed positive and negative effect on βG activity of whole cells and disrupted lysate, respectively. It is probably that bgl genes OEOE-0224 and OEOE-1210 were related to βG activity of SD-2a whole cells, while OEOE-1569 was responsible for βG activity of disrupted lysate. More kinds of volatile compounds and higher total concentration were released by SD-2a in synthetic wine compared with control. Thus, SD-2a showed a great potential for flavor enhancement under wine-like conditions. This study provides more information for further study of βG activity from O. oeni SD-2a.

Novel Genes and Metabolite Trends in Bifidobacterium longum subsp. infantis Bi-26 Metabolism of Human Milk Oligosaccharide 2\u2032-fucosyllactose

Human milk oligosaccharides (HMOs) function as prebiotics for beneficial bacteria in the developing gut, often dominated by Bifidobacterium spp. To understand the relationship between bifidobacteria utilizing HMOs and how the metabolites that are produced could affect the host, we analyzed the metabolism of HMO 2′-fucosyllactose (2′-FL) in Bifidobacterium longum subsp. infantis Bi-26. RNA-seq and metabolite analysis (NMR/GCMS) was performed on samples at early (A600 = 0.25), mid-log (0.5–0.7) and late-log phases (1.0–2.0) of growth. Transcriptomic analysis revealed many gene clusters including three novel ABC-type sugar transport clusters to be upregulated in Bi-26 involved in processing of 2′-FL along with metabolism of its monomers glucose, fucose and galactose. Metabolite data confirmed the production of formate, acetate, 1,2-propanediol, lactate and cleaving of fucose from 2′-FL. The formation of acetate, formate, and lactate showed how the cell uses metabolites during fermentation to produce higher levels of ATP (mid-log compared to other stages) or generate cofactors to balance redox. We concluded that 2′-FL metabolism is a complex process involving multiple gene clusters, that produce a more diverse metabolite profile compared to lactose. These results provide valuable insight on the mode-of-action of 2′-FL utilization by Bifidobacterium longum subsp. infantis Bi-26.

Non-targeted metabolomics unravels a media-dependent prodiginines production pathway in Streptomyces coelicolor A3(2).

The genus Streptomyces is the best-known source of therapeutic secondary metabolites, especially antibiotics with pharmaceutical applications. Here, we performed a comparative study based on the time-resolved metabolic disparity in S. coelicolor A3(2) subjected to fermentative cultivation in two different types of media (R2YE and RSM3) in order to investigate secondary metabolite production pathways. The relative abundance of secondary metabolites, such as prodiginines, indoles, germicidins, and selected diketopiperazines, was increased in S. coelicolor A3(2) cultivated in R2YE medium compared to that in RSM3 medium, variably at the late-log and stationary phases of fermentative growth. Correlation analysis indicated that “antibiotic prodiginines” contributed maximally to the absorption maxima (A530) of culture supernatants, indicating their optimal production at 96 hours in R2YE medium. A higher abundance of L-proline (48–72 hours) followed by prodiginines (96 hours) was evident, substantiating the intertwined links between precursor and activated prodiginines pathway. Similarly, the higher abundance of indoles was concurrent with tryptophan levels in the shikimate pathway, whereas diketopiperazines were synchronously abundant along with the levels of phenylalanine, leucine, and proline. Additionally, acetyl-CoA induced the acetate pathway, resulting in the production of germicidins. Thus, our results demonstrate that S. coelicolor A3(2) produces specific secondary metabolites by enhancing the dedicated metabolic pathway responsible for their production. In conclusion, our results from this study provide insight into the metabolic pathways of S. coelicolor A3(2), and can be applied to further optimize the production of prodiginines.

Pleiotropic control of antibiotic biosynthesis, flagellar operon expression, biofilm formation, and carbon source utilization by RpoN in Pseudomonas protegens H78.

The rhizobacterium Pseudomonas protegens H78 biosynthesizes a number of antibiotic compounds, including pyoluteorin, 2,4-diacetylphloroglucinol, and pyrrolnitrin. Here, we investigated the global regulatory function of the nitrogen metabolism-related sigma factor RpoN in P. protegens H78 through RNA-seq and phenotypic analysis. During the mid- to late-log growth phase, transcriptomic profiling revealed that 562 genes were significantly upregulated, and 502 genes were downregulated by at least twofold at the RNA level in the rpoN deletion mutant in comparison with the wild-type strain H78. With respect to antibiotics, Plt biosynthesis and the expression of its operon were positively regulated, while Prn biosynthesis and the expression of its operon were negatively regulated by RpoN. RpoN is responsible for the global activation of operons involved in flagellar biogenesis and assembly, biofilm formation, and bacterial mobility. In contrast, RpoN was shown to negatively control a number of secretion system operons including one type VI secretion system operon (H1-T6SS), two pilus biogenesis operons (Flp/Tad-T4b pili and Csu-T1 pili), and one polysaccharide biosynthetic operon (psl). In addition, two operons that are involved in mannitol and inositol utilization are under the positive regulation of RpoN. Consistent with this result, the ability of H78 to utilize mannitol or inositol as a sole carbon source is positively influenced by RpoN. Taken together, the RpoN-mediated global regulation is mainly involved in flagellar biogenesis and assembly, bacterial mobility, biofilm formation, antibiotic biosynthesis, secretion systems, and carbon utilization in P. protegens H78.

Quantitative multiplexed profiling of Penicillium funiculosum secretome grown on polymeric cellulase inducers and glucose

Filamentous fungi respond to the need to secure utilisable carbon from their growth milieu by secreting unique extracellular proteins depending upon the types of polymeric substrates. We have here profiled the variations in the secretome pattern of a non-model hypercellulolytic fungus – Penicillium funiculosum, grown in minimal media containing four different polymeric cellulase inducers, i.e., Avicel, wheat bran, ammonium-pretreated wheat straw and Avicel & wheat bran, and glucose over its early and late log phases of growth. Of the 137 secreted proteins validated at 1% FDR, we identified the quantified proteins in three clusters as early, persistently or lately expressed. The type of carbon substrate present in the culture media significantly affected the levels of cellulolytic enzymes expression by the fungus. The top abundant proteins quantified in the secretome for Avicel and wheat bran were cellobiohydrolaseI [GH7-CBM1], cellobiohydrolaseII [GH6-CBM1], β-glucosidase [GH3], arabinofuranosidase [GH51] and β-xylosidase [GH3], with bicupin being highest in case of wheat straw. Our results further suggested that the fungus secreted the extracellular proteins in waves, such that the initial responders act to hydrolyse the composite substrates in the culture environment before the second wave of proteins which tend to be more tailored to the specific substrate in the cultivating media. Biological significanceIn this article, we have comprehensively examined the dynamics of the secretome of a non-model hypercellulolytic fungus produced in response to model and composite cellulase inducers. Our study has provided additional insights into how the fungus enzyme machinery responds to the presence of different polymeric cellulase inducers over the two different growth phases (early growth and late growth phase). The comprehensive typing and quantification of the different proteins present in the secretomes of the cellulolytic fungal strains in response to diverse nutrient sources hold many prospects in understanding the fungus unique enzyme machinery and dynamics for the downstream biotechnological applications.

Metabolic dependent and independent pH-drop shuts down VirSR quorum sensing in Clostridium perfringens

Clostridium perfringens produces various exotoxins and enzymes that cause food poisoning and gas gangrene. The genes involved in virulence are regulated by the agr-like quorum sensing (QS) system, which consists of a QS signal synthesis system and a VirSR two-component regulatory system (VirSR TCS) which is a global regulatory system composed of signal sensor kinase (VirS) and response regulator (VirR). We found that the perfringolysin O gene (pfoA) was transiently expressed during mid-log phase of bacterial growth; its expression was rapidly shut down thereafter, suggesting the existence of a self-quorum quenching (sQQ) system. The sQQ system was induced by the addition of stationary phase culture supernatant (SPCS). Activity of the sQQ system was heat stable, and was present following filtration through the ultrafiltration membrane, suggesting that small molecules acted as sQQ agents. In addition, sQQ was also induced by pure acetic and butyric acids at concentrations equivalent to those in the stationary phase culture, suggesting that organic acids produced by C.perfringens were involved in sQQ. In pH-controlled batch culture, sQQ was greatly diminished; expression level of pfoA extended to late-log growth phase, and was eventually increased by one order of magnitude. Furthermore, hydrochloric acid induced sQQ at the same pH as was used in organic acids. SPCS also suppressed the expression of genes regulated by VirSR TCS. Overall, the expression of virulence factors of C.perfringens was downregulated by the sQQ system, which was mediated by primary acidic metabolites and acidic environments. This suggested the possibility of pH-controlled anti-virulence strategies.

Preliminary Characterization of a Nisin Z Bacteriocin with Activity Against the Fish Pathogen Streptococcus iniae

This is a preliminary characterisation of a bacteriocin, BacL49 produced by Lactococcus lactis ssp. lactis. This bacteriocin is significant due to its activity against Streptococcus iniae, a bacterial pathogen causing severe economic losses in the global aquaculture of various fish. Spot-on-lawn and microtitre plate assays were used to test antagonistic activity of the bacteriocin. BacL49 is heat and pH stable (100 °C for 60min, pH 2.5-9.5), and sensitive to proteinase K, α-chymotrypsin, trypsin and papain. BacL49 has a bactericidal mode of action and is produced during late log phase growth. BacL49 exhibits a broad activity spectrum against S. iniae, antagonising 93.75% (45/48) of S. iniae isolates collected from a variety of hosts and environments. The apparent molecular masses of the active protein components determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis are 5 and 54kDa. Molecular analyses were performed to locate the genetic determinants of BacL49. PCR of chromosomal DNA successfully amplified the structural gene encoding the precursor of nisin. Subsequent analysis of nucleotide sequences of the PCR products revealed it to be identical to the nis Z structural gene of nisin Z. There is a paucity of reports examining the inhibition of S. iniae by a lactococcal bacteriocin or even L. lactis as an aquacultural probiotic. This is one of the first studies to identify nisin genes in a strain of L. lactis exhibiting activity against S. iniae. BacL49 is a candidate biocontrol agent for mitigation of this important fish pathogen.

The protein and neutral lipid composition of lipid droplets isolated from the fission yeast, Schizosaccharomyces pombe.

Lipid droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer with bound proteins. Much of the information on lipid droplet function comes from proteomic and lipodomic studies that identify the components of droplets isolated from organisms throughout the phylogenetic tree. Here, we add to that important inventory by reporting lipid droplet factors from the fission yeast, Schizosaccharomyces pombe. Unique to this study was the fact that cells were cultured in three different environments: 1) late log growth phase in glucose-based media, 2) stationary phase in glucosebased media, and 3) late log growth phase in media containing oleic acid. We confirmed colocalization of major factors with lipid droplets using live-cell fluorescent microscopy. We also analyzed droplets from each of the three conditions for sterol ester (SE) and triacylglycerol (TAG) content, along with their respective fatty acid compositions. We identified a previously undiscovered lipid droplet protein, Vip1p, which affects droplet size distribution. The results provide further insight into the workings of these ubiquitous organelles.

Heat shock at higher cell densities improves measles hemagglutinin translocation and human GRP78/BiP secretion in Saccharomyces cerevisiae

The yield of heterologous proteins is often limited by several bottlenecks in the secretory pathway of yeast Saccharomyces cerevisiae. It was shown earlier that synthesis of measles virus hemagglutinin (MeH) is inefficient mostly due to a bottleneck in the translocation of viral protein precursors into the endoplasmic reticulum (ER) of yeast cells. Here we report that heat shock with subsequent induction of MeH expression at 37°C improved translocation of MeH precursors when applied at higher cell densities. The amount of MeH glycoprotein increased by about 3-fold after heat shock in the late-log phases of both glucose and ethanol growth. The same temperature conditions increased both secretion titer and yield of another heterologous protein human GRP78/BiP by about 50%. Furthermore, heat shock at the late-log glucose growth phase also improved endogenous invertase yield by approximately 2.7-fold. In contrast, a transfer of yeast culture to lower temperature at diauxic shift followed by protein expression at 20°C almost totally inhibited translocation of MeH precursors. The difference in amounts of MeH glycoprotein under expression at 37°C and 20°C was about 80-fold, while amounts of unglycosylated MeH polypeptides were similar under both conditions. Comparative proteomic analysis revealed that besides over-expressed ER-resident chaperone Kar2, an increased expression of several cytosolic proteins (such as Hsp104, Hsp90 and eEF1A) may contribute to improved translocation of MeH.

Fatty acid hydrolysis of acyl marinobactin siderophores by Marinobacter acylases.

The marine bacteria Marinobacter sp. DS40M6 and Marinobacter nanhaiticus D15-8W produce a suite of acyl peptidic marinobactin siderophores to acquire iron under iron-limiting conditions. During late-log phase growth, the marinobactins are hydrolyzed to form the marinobactin headgroup with release of the corresponding fatty acid tail. The bntA gene, a homologue of the Pseudomonas aeruginosa pyoverdine acylase gene, pvdQ, was identified from Marinobacter sp. DS40M6. A bntA knockout mutant of Marinobacter sp. DS40M6 produced the suite of acyl marinobactins A-E, without the usual formation of the marinobactin headgroup. Another marinobactin-producing species, M. nanhaiticus D15-8W, is predicted to have two pvdQ homologues, mhtA and mhtB. MhtA and MhtB have 67% identical amino acid sequences. MhtA catalyzes hydrolysis of the apo-marinobactin siderophores as well as the quorum sensing signaling molecule, dodecanoyl-homoserine lactone. In contrast to hydrolysis of the suite of apo-marinobactins by MhtA, hydrolysis of the iron(III)-bound marinobactins was not observed.

Microbial tailoring of acyl peptidic siderophores.

Marine bacteria produce an abundance of suites of acylated siderophores characterized by a unique, species-dependent headgroup that binds iron(III) and one of a series of fatty acid appendages. Marinobacter sp. DS40M6 produces a suite of seven acylated marinobactins, with fatty acids ranging from saturated and unsaturated C12–C18 fatty acids. In the present study, we report that in the late log phase of growth, the fatty acids are hydrolyzed by an amide hydrolase producing the peptidic marinobactin headgroup. Halomonas aquamarina str. DS40M3, another marine bacterium isolated originally from the same sample of open ocean water as Marinobacter sp. DS40M6, produces the acyl aquachelins, also as a suite composed of a peptidic headgroup distinct from that of the marinobactins. In contrast to the acyl marinobactins, hydrolysis of the suite of acyl aquachelins is not detected, even when H. aquamarina str. DS40M3 is grown into the stationary phase. The Marinobacter cell-free extract containing the acyl amide hydrolase is active toward exogenous acyl-peptidic siderophores (e.g., aquachelin C, loihichelin C, as well as octanoyl homoserine lactone used in quorum sensing). Further, when H. aquamarina str. DS40M3 is cultured together with Marinobacter sp. DS40M6, the fatty acids of both suites of siderophores are hydrolyzed, and the aquachelin headgroup is also produced. The present study demonstrates that coculturing bacteria leads to metabolically tailored metabolites compared to growth in a single pure culture, which is interesting given the importance of siderophore-mediated iron acquisition for bacterial growth and that Marinobacter sp. DS40M6 and H. aquamarina str. DS40M3 were isolated from the same sample of seawater.

English

Repeated use of L-asparaginase II enzyme, in the treatment of acute lymphoblastic leukaemia, is commonly needed because of the enzyme&rsquo;s instability and relatively short half-life which leads to more serious side effects on patients. In the present study, we report on the cloning and expression of L-asparaginase from a thermotolerant strain of Escherichia coli (KH027) which was isolated from camel manure and could grow at 45&deg;C. Expression of recombinant asparaginase was conducted by fusion asparaginase gene to pelB leader sequence and 6His residues at the C-terminus under the inducible T7 promoter in DH5&alpha; cells. Induction of the cells with 0.1 mM isopropyl-&beta;-D-thiogalactopyranoside (IPTG) at late log phase of growth resulted in 0.6-fold (2111 UI) higher to that obtained in early log phase induction (1319 UI) and 0.3-fold compared with mid log phase induction (1623 UI). The recombinant asparaginase protein was purified from the culture supernatant through nickel affinity chromatography. The apparent molecular weight of the tetramer enzyme was found to be ~141 kDa. Overall yield (87 mg/L) of the purified recombinant asparaginase was achieved at the shake flask level. The purified protein showed optimum activities at a temperature of 43&deg;C and pH 6. The Km and Kcat parameters were 3.8 mM-1 and 2.92 &times; 103s&minus;1, respectively. The enzyme retained around 57 and 30% of its initial activity after 30 and 60 min of incubation at 50&deg;C, respectively. Recombinant L-asparaginase was evaluated for its antiproliferative effect in the leukemia cell lines of RS4; 11 and HL60 after 96 and 72 h of incubation. The doses of 100 &mu;g/mL and time-response effect of 96 h caused a reduction value of 50% in cell viability of RS4. However, cell viability of 50% in the leukemic cells HL-60 was noticed with a concentration of 200 &mu;g/mL with an incubation period of 72 h. In vitro antiproliferative results in the leukemia cell lines encourage for making in vivo investigation to increase the possibility of using this thermostable enzyme in leukaemia therapy. &nbsp; Key words: L-Asparaginase II, leukemia cell, recombinant asparaginase.

Microarray Analysis of the Ler Regulon in Enteropathogenic and Enterohaemorrhagic Escherichia coli Strains

The type III protein secretion system is an important pathogenicity factor of enteropathogenic and enterohaemorrhagic Escherichia coli pathotypes. The genes encoding this apparatus are located on a pathogenicity island (the locus of enterocyte effacement) and are transcriptionally activated by the master regulator Ler. In each pathotype Ler is also known to regulate genes located elsewhere on the chromosome, but the full extent of the Ler regulon is unclear, especially for enteropathogenic E. coli. The Ler regulon was defined for two strains of E. coli: E2348/69 (enteropathogenic) and EDL933 (enterohaemorrhagic) in mid and late log phases of growth by DNA microarray analysis of the transcriptomes of wild-type and ler mutant versions of each strain. In both strains the Ler regulon is focused on the locus of enterocyte effacement – all major transcriptional units of which are activated by Ler, with the sole exception of the LEE1 operon during mid-log phase growth in E2348/69. However, the Ler regulon does extend more widely and also includes unlinked pathogenicity genes: in E2348/69 more than 50 genes outside of this locus were regulated, including a number of known or potential pathogenicity determinants; in EDL933 only 4 extra-LEE genes, again including known pathogenicity factors, were activated. In E2348/69, where the Ler regulon is clearly growth phase dependent, a number of genes including the plasmid-encoded regulator operon perABC, were found to be negatively regulated by Ler. Negative regulation by Ler of PerC, itself a positive regulator of the ler promoter, suggests a negative feedback loop involving these proteins.

The two-component system CBO2306/CBO2307 is important for cold adaptation of Clostridium botulinum ATCC 3502

Clostridium botulinum is a notorious foodborne pathogen. Its ability to adapt to and grow at low temperatures is of interest for food safety. Two-component systems (TCSs) have been reported to be involved in cold-shock and growth at low temperatures. Here we show the importance of TCS CBO2306/CBO2307 in the cold-shock response of C. botulinum ATCC 3502. The relative expression levels of the cbo2306 and cbo2307 were up to 4.4-fold induced in the cold-shocked cultures but negatively regulated in the late-log and stationary growth phase in relation to early logarithmic growth phase in non-shocked cultures. Importance of the CBO2306/CBO2307 in the cold stress was further demonstrated by impaired growth of insertional cbo2306 or cbo2307 knockout mutants in relation to the wild-type strain ATCC 3502. The results suggest that the TCS CBO2306/CBO2307 is important for cold-shock response and adaptation of C. botulinum ATCC 3502 to low temperature.