Subscribe: pubmed: 0099-2240
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pubmed: 0099-2240



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Lack of methylated hopanoids renders the cyanobacterium Nostoc punctiforme sensitive to osmotic and pH stress.

Lack of methylated hopanoids renders the cyanobacterium Nostoc punctiforme sensitive to osmotic and pH stress.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Garby TJ, Matys ED, Ongley SE, Salih A, Larkum AWD, Walter MR, Summons RE, Neilan BA

Abstract
To investigate the function of 2-methylhopanoids in modern cyanobacteria, the hpnP gene coding for the radical SAM methylase protein that acts on the C2 position of hopanoids, was deleted from the filamentous cyanobacterium Nostoc punctiforme ATCC 29133S. The resulting ΔhpnP mutant lacked all 2-methylhopanoids, but was found to produce much higher levels of two bacteriohopanepentol isomers compared to the wild type. Growth rates of ΔhpnP mutant cultures were not significantly different from those of the wild type under standard growth conditions. Akinete formation was also not impeded by the absence of 2-methylhopanoids. The relative abundances of the different hopanoid structures in akinete-dominated cultures of the wild-type and ΔhpnP mutant were similar to those of vegetative cell-dominated cultures. However, the ΔhpnP mutant was found to have decreased growth rates under both pH and osmotic stress, confirming a role for 2-methylhopanoids in stress tolerance. Evidence of elevated photosystem II yield and NAD(P)H-dependent oxidoreductase activity in the ΔhpnP mutant under stress conditions, compared to the wild type, suggested that the absence of 2-methylhopanoids increases cellular metabolic rates under stress conditions.Importance: As the first group of organisms to develop oxygenic photosynthesis, Cyanobacteria are central to the evolutionary history of life on Earth and the subsequent oxygenation of the atmosphere. To investigate the origin of cyanobacteria and emergence of oxygenic photosynthesis, geobiologists use biomarkers, the remnants of lipids produced by different organisms that are found in geologic sediments. 2-Methylhopanes have been considered indicative of cyanobacteria in some environmental settings; with the parent lipids 2-methylhopanoids being present in many contemporary cyanobacteria. We have created a Nostoc punctiforme ΔhpnP mutant strain that does not produce 2-methylhopanoids to assess the influence of 2-methylhopanoids on stress tolerance. Increased metabolic activity in the mutant under stress indicates compensatory alterations of metabolism in the absence of 2-methylhopanoids.

PMID: 28455341 [PubMed - as supplied by publisher]




Engineering Escherichia coli nicotinic acid mononucleotide adenylyltransferase for fully active amidated NAD biosynthesis.

Engineering Escherichia coli nicotinic acid mononucleotide adenylyltransferase for fully active amidated NAD biosynthesis.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Wang X, Zhou YJ, Wang L, Liu W, Liu Y, Peng C, Zhao ZK

Abstract
Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through deamidated and amidated pathway, of which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli, NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N) with high NMN preference. While in the presence of NMN both variants were sufficient to enable cell viability of the E. coli BW25113 derived NAD-auxotrophic strain YJE003, of which the last step of the deamidated pathway is blocked, the 16D8-expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering.Importance Adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively, is the key step in deamidated and amidated pathway for NAD biosynthesis. In most organisms, the canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich our understanding on NAD biosynthesis and are valuable to manipulate NAD homeostasis for metabolic engineering.

PMID: 28455340 [PubMed - as supplied by publisher]




Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-like Protein Required for Glucose-induced Endocytosis of Maltose Permease and Carbon Catabolite De-repression in Aspergillus oryzae.

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-like Protein Required for Glucose-induced Endocytosis of Maltose Permease and Carbon Catabolite De-repression in Aspergillus oryzae.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Tanaka M, Hiramoto T, Tada H, Shintani T, Gomi K

Abstract
Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the HECT ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucose-induced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by western blot, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although α-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD showed negligible effect on α-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by the disruption of the deubiquitinating enzyme-encoding creB. The α-amylase activity of the creB mutant was 1.6-fold higher than that of the wild-type, and the dephosphorylation mimic of CreD further improved the α-amylase activity by 2.6-fold. These results indicate that the combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzaeIMPORTANCE In eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucose-induced endocytosis of transporters is mediated by their ubiquitination, and arrestin-like proteins act as adaptors of transporters and ubiquitin ligases. In this study, we showed that CreD, an arrestin-like protein, was involved in glucose-induced endocytosis of maltose permease and carbon catabolite de-repression of amylase gene expression in Aspergillus oryzae Dephosphorylation of CreD was required for CCR relief triggered by the disruption of creB, which encodes a deubiquitinating enzyme; a combination of the phosphorylation-defective mutation of CreD and creB disruption dramatically improved α-amylase production. This study shows the dual function of an arrestin-like protein and provides a novel approach for improving the production of amylolytic enzymes in A. oryzae.

PMID: 28455339 [PubMed - as supplied by publisher]




The enzymes required for maltodextrin catabolism in Enterococcus faecalis exhibit several novel activities.

The enzymes required for maltodextrin catabolism in Enterococcus faecalis exhibit several novel activities.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Joyet P, Mokhtari A, Riboulet-Bisson E, Blancato VS, Espariz M, Magni C, Hartke A, Deutscher J, Sauvageot N

Abstract
Maltose and maltodextrins are formed during the degradation of starch or glycogen. Maltodextrins are composed of a mixture of maltooligosaccharides formed by α-1,4, but also some α-1,6-linked glucosyl residues. The latter are derived from branching points in the polysaccharides. In Enterococcus faecalis, maltotriose is mainly transported and phosphorylated by a phosphoenolpyruvate:carbohydrate phosphotransferase system. The formed maltotriose-6″-P is intracellularly dephosphorylated by a specific phosphatase, MapP. In contrast, maltotetraose and longer maltooligosaccharides up to maltoheptaose are taken up without phosphorylation via the ATP binding cassette transporter MdxEFG/MsmX. We show that the maltose-producing maltodextrin hydrolase MmdH (EFT41964 in strain JH2-2) catalyzes the first catabolic step of α-1,4-linked maltooligosaccharides. The purified enzyme converts even-numbered α-1,4-linked maltooligosaccharides (maltotetraose, etc.) into maltose and odd-numbered (maltotriose, etc.) into maltose and glucose. Inactivation of mmdH therefore prevents growth of E. faecalis on maltooligosaccharides ranging from maltotriose to maltoheptaose. Surprisingly, MmdH also functions as maltogenic α-1,6-glucosidase, because it converts the maltotriose isomer isopanose into maltose and glucose. E. faecalis contains in addition a glucose-producing α-1,6-specific maltodextrin hydrolase (EFT41963, renamed GmdH). This enzyme converts panose, another maltotriose isomer, into glucose and maltose. A gmdH mutant had therefore lost the capacity to grow on panose. The genes mmdH and gmdH are organized in an operon together with EFT41962 (renamed mmgT). Purified MmgT transfers glucosyl residues from one α-1,4-linked maltooligosaccharide molecule to another. For example, it catalyzes the disproportionation of maltotriose by transferring a glucosyl residue to another maltotriose molecule thereby forming maltotetraose and maltose together with a small amount of maltopentaose.IMPORTANCE The utilization of maltodextrins by Enterococcus faecalis has been shown to increase the virulence of this nosocomial pathogen. However, little is known about how this organism catabolizes maltodextrins. We therefore identified the enzymes involved in the metabolism of various α-1,4- and α-1,6-linked maltooligosaccharides. We found that one of these enzymes functions as maltose-producing α-glucosidase with relaxed linkage specificity (α-1,4 and α-1,6) and exo- and endo-glucosidase activity. A third enzyme, which resembles amylomaltases, exclusively transfers glucosyl residues from one maltooligosaccharide molecule to another. Similar enzymes are present in numerous other Firmicutes, such as streptococci and lactobacilli, suggesting that these organisms follow the same maltose degradation pathway as E. faecalis.

PMID: 28455338 [PubMed - as supplied by publisher]




Laboratory Evolution to Alternating Substrate Environments Yields Distinct Phenotypic and Genetic Adaptive Strategies.

Laboratory Evolution to Alternating Substrate Environments Yields Distinct Phenotypic and Genetic Adaptive Strategies.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Sandberg TE, Lloyd CJ, Palsson BO, Feist AM

Abstract
Adaptive Laboratory Evolution (ALE) experiments are often designed to maintain a static culturing environment to minimize confounding variables that could influence the adaptive process, but dynamic nutrient conditions occur frequently in both natural and bioprocessing settings. To study the nature of carbon substrate fitness tradeoffs, we evolved batch cultures of E. coli via serial propagation into tubes alternating between glucose and either xylose, glycerol, or acetate. Genome sequencing of evolved cultures revealed several genetic changes preferentially selected for under dynamic conditions, and different adaptation strategies depending on the substrates being switched between - in some environments a persistent "generalist" strain developed while in another, two "specialist" subpopulations arose that alternated dominance. Diauxic lag phenotype varied across the generalists and specialists, in one case being completely abolished, while gene expression data distinguished the transcriptional strategies implemented by strains in pursuit of growth optimality. Genome-scale metabolic modeling techniques were then used to help explain the inherent substrate differences giving rise to the observed distinct adaptive strategies. This study gives insight into the population dynamics of adaptation in an alternating environment, as well as the underlying metabolic and genetic mechanisms. Furthermore, ALE-generated optimized strains have phenotypes with potential industrial bioprocessing applications.Importance Evolution and natural selection inexorably lead to an organism's improved fitness in a given environment, whether in a laboratory or natural setting. However, despite the frequent natural occurrence of complex and dynamic growth environments, laboratory evolution experiments typically maintain simple, static culturing environments so as to reduce selection pressure complexity. In this study, we investigated the adaptive strategies underlying evolution to fluctuating environments by evolving E. coli to conditions of frequently switching growth substrate. Characterization of evolved strains via a number of different data types revealed the various genetic and phenotypic changes implemented in pursuit of growth optimality, and how these differed across the different growth substrates and switching protocols. This work helps to not only establish general principles of adaptation to complex environments, but also suggests strategies for experimental design to achieve desired evolutionary outcomes.

PMID: 28455337 [PubMed - as supplied by publisher]




Insights into nitrate-reducing Fe(II) oxidation mechanisms by analyzing cell-mineral associations, cell encrustation and mineralogy in the chemolithoautotrophic enrichment culture KS.

Insights into nitrate-reducing Fe(II) oxidation mechanisms by analyzing cell-mineral associations, cell encrustation and mineralogy in the chemolithoautotrophic enrichment culture KS.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Nordhoff M, Tominski C, Halama M, Byrne JM, Obst M, Kleindienst S, Behrens S, Kappler A

Abstract
Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic and depend on organic co-substrates for growth. Encrustation of cells in Fe(III)-minerals has been observed for mixotrophic NRFeOB, but not for autotrophic phototrophic and microaerophilic Fe(II)-oxidizers. So far, little is known about cell-mineral-associations in the few existing autotrophic NRFeOB. Here we investigate whether the designated autotrophic Fe(II)-oxidizing strain (closely-related to Gallionella and Sideroxydans) or the heterotrophic nitrate-reducers that are present in the autotrophic nitrate-reducing Fe(II)-oxidizing enrichment culture KS, form mineral crusts during Fe(II) oxidation under autotrophic and mixotrophic conditions. In the mixed culture, we found no significant encrustation of any of the cells both during autotrophic oxidation of 8-10 mM Fe(II) coupled to nitrate reduction and during cultivation under mixotrophic conditions with 8-10 mM Fe(II), 5 mM acetate and 4 mM nitrate, where higher numbers of heterotrophic nitrate-reducers were present. Two pure cultures of heterotrophic nitrate-reducers (Nocardioides, Rhodanobacter) isolated from culture KS were analyzed under mixotrophic growth conditions. We found green rust formation, no cell encrustation and only few mineral particles on some cell surfaces with 5 mM Fe(II), and some encrustation with 10 mM Fe(II). Our findings suggest that enzymatic, autotrophic Fe(II) oxidation coupled to nitrate reduction forms poorly-crystalline Fe(III)-oxyhydroxides and proceeds without cellular encrustation while indirect Fe(II) oxidation via heterotrophic nitrate-reduction-derived nitrite can lead to green rust as intermediate mineral and significant cell encrustation. The extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen-species depends on Fe(II) concentrations and is probably negligible at environmental conditions in most habitats.IMPORTANCE Most described nitrate-reducing Fe(II)-oxidizing bacteria (NRFeOB) are mixotrophic (their growth depends on organic co-substrates) and can become encrusted in Fe(III) minerals. Encrustation is expected to be harmful and poses a threat to cells if it also occurs at environmentally relevant conditions. Nitrite produced during heterotrophic denitrification reacts with Fe(II) abiotically and is probably the reason for encrustation in mixotrophic NRFeOB. Little is known about cell-mineral associations in autotrophic NRFeOB such as the enrichment culture KS. Here, we show that no encrustation occurs in culture KS under autotrophic and mixotrophic conditions while heterotrophic nitrate-reducing isolates from culture KS become encrusted. These findings support the hypothesis that encrustation in mixotrophic cultures is caused by the abiotic reaction of Fe(II) with nitrite and provide evidence that Fe(II) oxidation in culture KS is enzymatic. Furthermore, we show that the extent of encrustation caused by indirect Fe(II) oxidation by reactive nitrogen species depends on Fe(II) concentrations and is probably negligible in most environmental habitats.

PMID: 28455336 [PubMed - as supplied by publisher]




Yersinia pestis resists predation by Acanthamoeba castellanii and exhibits prolonged intracellular survival.

Yersinia pestis resists predation by Acanthamoeba castellanii and exhibits prolonged intracellular survival.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Benavides-Montaño JA, Vadyvaloo V

Abstract
Plague is a flea-borne rodent-associated zoonotic disease caused by Yersinia pestis The disease is characterized by epizootics with high rodent mortalities, punctuated by inter-epizootic periods when the bacterium persists in an unknown reservoir. This study investigates the interaction between Y. pestis and the ubiquitous soil free-living amoeba (FLA), Acanthamoeba castellanii, to assess if the bacterium can survive within soil amoeba and whether intracellular mechanisms are conserved between infection of mammalian macrophages and soil amoeba. The results demonstrate that during co-culture with amoeba, representative Y. pestis strains of epidemic biovars Mediaevalis, Orientalis and Antiqua are phagocytized and able to survive within amoeba for at least five days. Key Y. pestis determinants of the intracellular interaction of Y. pestis and phagocytic macrophages, PhoP and the type three secretion system (T3SS), were then tested for their roles in the Y. pestis-amoeba interaction. Consistent with a requirement for the PhoP transcriptional activator in intracellular survival of Y. pestis in macrophages, a PhoP mutant is unable to survive when co-cultured with amoeba. Additionally, induction of the T3SS blocks phagocytic uptake of Y. pestis by amoeba, similar to that which occurs during macrophage infection. Electron microscopy revealed that in A. castellanii, Y. pestis resides intact, within spacious vacuoles which were characterized using lysosomal trackers as being separated from the lysosomal compartment. This evidence for prolonged survival and subversion of intracellular digestion of Y. pestis within a FLA suggest that protozoa may serve as a protective soil reservoir for Y. pestis.ImportanceYersinia pestis is a re-emerging flea-borne zoonotic disease. Sylvatic plague cycles are characterized by an epizootic period during which the disease spreads rapidly causing high rodent mortalities, and an inter-epizootic period when the bacterium quiescently persists in an unknown reservoir. An understanding of the ecology of Y. pestis in the context of its persistence in the environment, and, reactivation to initiate a new epizootic cycle is key to implementing novel surveillance strategies to more effectively predict and prevent new disease outbreaks. Here we demonstrate prolonged survival and subversion of intracellular digestion of Y. pestis within a soil free-living amoeba. This suggests the potential role for protozoa as a protective soil reservoir for Y. pestis, which may support recrudescence of plague epizootics.

PMID: 28455335 [PubMed - as supplied by publisher]




Validation of the Hirst-type spore trap for simultaneous monitoring of prokaryotic and eukaryotic biodiversity in urban air samples by NGS.

Validation of the Hirst-type spore trap for simultaneous monitoring of prokaryotic and eukaryotic biodiversity in urban air samples by NGS.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Núñez A, Amo de Paz G, Ferencova Z, Rastrojo A, Guantes R, García AM, Alcamí A, Gutiérrez-Bustillo AM, Moreno DA

Abstract
Pollen, fungi and bacteria are the main microscopic biological entities present in the air outdoor causing allergy symptoms, disease transmission and having a significant role in atmosphere dynamics. Despite their relevance, a method for monitoring simultaneously these biological particles in metropolitan environments has not been developed yet. Here, we assessed the use of the Hirst-type spore trap to characterize the global airborne biota by high-throughput DNA sequencing, selecting regions of the genes 16S ribosomal RNA gene and internal transcribed spacer for the taxonomic assignment. We showed that aerobiological communities are well represented by this approach. The OTUs in common of two traps working synchronically compiled >87% of the total relative abundance for bacterial diversity collected in each sampler, >89% for fungi, and >97% for pollen. We found a good correspondence between traditional characterization by microscopy and genetic identification, obtaining more accurate taxonomic assignments and detecting a greater diversity using the latter. We also demonstrated that DNA sequencing accurately detects differences in biodiversity between samples. We concluded that high-throughput DNA sequencing applied to aerobiological samples obtained with Hirst spore traps provides reliable results and can be easily implemented for monitoring prokaryotic and eukaryotic entities present in the air of urban areas.Importance The detection, monitoring and characterization of the wide diversity of biological entities present in the air is a difficult task that requires time and expertise in different disciplines. We have evaluated the use of the Hirst spore trap (an equipment broadly employed in aerobiological studies) to detect and identify these organisms by DNA-based analyses. Our results showed a consistent collection of DNA and a good concordance with traditional methods for identification, suggesting that these devices can be used as a tool for continuous monitoring of the airborne biodiversity, improving the taxonomic resolution and the characterization altogether. They are also suitable for acquiring novel DNA amplicon-based information in order to gain a better understanding of the biological particles present in a scarcely known environment like the air.

PMID: 28455334 [PubMed - as supplied by publisher]




A novel quorum-quenching enzyme mediates antibiotic resistance.

A novel quorum-quenching enzyme mediates antibiotic resistance.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Kusada H, Tamaki H, Kamagata Y, Hanada S, Kimura N

Abstract
N-Acylhomoserine lactone acylase (AHL-acylase) is a well-known enzyme responsible for disrupting cell-cell communication (quorum sensing) in bacteria. Here we isolated and characterized a novel and unique AHL-acylase (designated MacQ) from a multidrug-resistant bacterium Acidovorax sp. MR-S7. The purified MacQ protein heterologously expressed in Escherichia coli degraded a wide variety of AHLs ranging from C6 to C14 side chains with or without 3-oxo substitutions. We also observed that AHL-mediated virulence factor production in a plant pathogen Pectobacterium carotovorum was dramatically attenuated by co-culturing with MacQ-overexpressing Escherichia coli, whereas E. coli with an empty vector was unable to quench the pathogenicity, which strongly indicates that MacQ can act in vivo as a quorum-quenching enzyme and interfere with the quorum-sensing system in the pathogen. In addition, this enzyme was found to be capable of degrading a wide spectrum of β-lactams (penicillin G, ampicillin, amoxicillin, carbenicillin, cephalexin and cefadroxil) by deacylation, clearly indicating that MacQ is a bifunctional enzyme that confers both quorum-quenching and antibiotic resistance on strain MR-S7. MacQ has relatively low amino acid sequence identities to any of known acylases (<39%) and has among the broadest substrate range. Our findings provide the possibility that AHL-acylase genes can be an alternative source of antibiotic resistance genes posing a threat to human health, if they migrate and transfer to pathogenic bacteria.IMPORTANCEN-Acylhomoserine lactones (AHLs) are well-known signal molecules for bacterial cell-cell communication (quorum-sensing), and AHL-acylase able to degrade AHLs has been recognized as a major target for quorum-sensing interference (quorum-quenching) in pathogens. In this work, we succeeded in isolating a novel AHL-acylase (MacQ) from a multidrug-resistant bacterium, and demonstrated that the MacQ enzyme could confer multidrug resistance as well as quorum quenching on the host organism. Indeed, the purified MacQ protein was found to be bifunctional and capable of degrading not only various AHL derivatives but also multiple β-lactam antibiotics by deacylation activities. Though quorum quenching and antibiotic resistance have been recognized to be distinct biological functions, our findings clearly link the two functions by discovering the novel bifunctional enzyme, and further provide the possibility that hitherto-overlooked antibiotic resistance mechanism mediated by quorum-quenching enzyme may exist in natural environments and perhaps in clinical settings.

PMID: 28455333 [PubMed - as supplied by publisher]




Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-hydroxybutyrate) (PHB) in Ralstonia eutropha H16.

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-hydroxybutyrate) (PHB) in Ralstonia eutropha H16.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Jüngert JR, Borisova M, Mayer C, Wolz C, Brigham CJ, Sinskey AJ, Jendrossek D

Abstract
In this study, we constructed a set of single-, double-, and triple-gene deletion strains of Ralstonia eutropha H16 in (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2) and polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) genes and determined the impact on the levels of (p)ppGpp and on accumulated PHB. Double deletion mutants in both spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were determined in strains in which the concentration of (p)ppGpp was increased by the overexpression of (p)ppGpp synthase (SpoT2) and absence of (p)ppGpp hydrolase. Determinations of (p)ppGpp levels in wild type R. eutropha under different growth conditions and induction of stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of remaining PHB in later growth phases by influencing the efficiency of the PHB mobilization system in the stationary growth. Data on a previously constructed ΔspoT2 strain (Brigham J, Speth DR, Rha C, Sinskey AJ. 2012. AEM 78:8033-44) were identified as an experimental error in strain construction and we retract the previous statement that the spoT2 gene product is essential for PHB accumulation in R. eutrophaImportance Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities over the last three decades in several laboratories have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria such as Ralstonia eutropha This implies that PHB synthase and depolymerase activities must be well-regulated to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that stringent response in Rhizobium etli and in R. eutropha is involved in regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and an HPLC-MS based detection method for the quantification of (p)ppGpp in R. eutropha This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and massive over-production of PHB. Contrary, in the absence of (p)ppGpp, mobilization of PHB was dramatically enhanced.

PMID: 28455332 [PubMed - as supplied by publisher]




Reverse Line Blot-based Detection Approaches of Microbial Pathogens in Ixodes ricinus Ticks Collected in Austria and Impact of the Chosen Method.

Reverse Line Blot-based Detection Approaches of Microbial Pathogens in Ixodes ricinus Ticks Collected in Austria and Impact of the Chosen Method.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Schötta AM, Wijnveld M, Stockinger H, Stanek G

Abstract
Ticks transmit a large number of pathogens capable of causing human disease. In this study, the PCR-reverse line blot (RLB) method was used to screen for pathogens in a total of 554 Ixodes ricinus ticks collected from all provinces of Austria. These pathogens belong to the genera Borrelia, Rickettsiae, Anaplasma/(Neo-)Ehrlichia, Babesia, and CoxiellaThe pathogens with the highest detected prevalence were spirochetes of the Borrelia burgdorferi sensu lato complex, in 142 ticks (25.6%). B. afzelii (80/142) was the most frequently detected species, followed by B. burgdorferi sensu stricto (38/142) and B. valaisiana (36/142). B. garinii/B. bavariensis, B. lusitaniae, and B. spielmanii were found in 28, five, and one ticks, respectively. Rickettsia spp. were detected in 93 ticks (16.8%): R. helvetica (39/93), R. raoultii (38/93), R. monacensis (2/93), and R. slovaca (1/93). Thirteen Rickettsia samples remain uncharacterized. Candidatus Neoehrlichia mikurensis, Babesia spp. (B. venatorum, B. divergens, B. microti), and Anaplasma phagocytophilum were found in 4.5%, 2.7%, and 0.7%, respectively. Coxiella burnetii was not detected. Multiple microorganisms were detected in 40 ticks (7.2%) and the co-occurrence of Babesia spp. and Candidatus N. mikurensis showed significant positive correlation. We also compared different PCR-RLBs for detection of Borreliaburgdorferi sensu lato and Rickettsia spp. and showed that different detection approaches provide highly diverse results, indicating that analysis of environmental samples remains challenging.Importance This study determines the wide spectrum of tick-borne bacterial and protozoal pathogens that can be encountered in Austria. Surveillance of (putative) pathogenic microorganisms occurring in the environment is of medical importance, especially when those agents can be transmitted by ticks and cause disease. The observation of significant co-infections of certain microorganisms in field-collected ticks is an initial step to an improved understanding of microbial interactions in ticks. In addition, we show that variations in molecular detection methods, such as in primer pairs and target genes, can considerably influence the final results. For instance, certain genospecies of borreliae can be better or worse detected by one or the other method; a fact of great importance for future screening studies.

PMID: 28455331 [PubMed - as supplied by publisher]




Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502.

Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Fredrick CM, Lin G, Johnson EA

Abstract
Botulinum neurotoxin (BoNT) produced by neurotoxigenic clostridia is the most potent biological toxin known and the causative agent of the paralytic disease botulism. The nutritional, environmental, and genetic regulation of BoNT synthesis, activation, stability, and toxin complex (TC) formation are not well studied. Previous studies indicated that growth and BoNT formation was affected by arginine and glucose in C. botulinum types A and B. In the present study, C. botulinum ATCC 3502 was grown in toxin production medium (TPM) with different levels of arginine, glucose and three products of arginine metabolism, citrulline, proline and ornithine. Cultures were analyzed for growth (OD600), spore formation, BoNT and TC formation by Western blots and immunoprecipitation, and BoNT activity by mouse bioassay. A high level of arginine (20 g/l) repressed BoNT production approximately 1000-fold, enhanced growth, slowed lysis, and reduced endospore production by greater than 1000-fold. Similar effects on toxin production were seen with equivalent levels of citrulline, but not ornithine or proline. In TPM lacking glucose, formation of BoNT/A1 and TC were significantly decreased, and extracellular BoNT and TC proteins were partially inactivated after the first day of culture. An understanding of the regulation of C. botulinum growth and BoNT and TC formation should be valuable in defining requirements for BoNT formation in foods and clinical samples, improving the quality of BoNT for pharmaceutical preparations, and elucidating the biological functions of BoNTs for the bacterium.IMPORTANCE Botulinum neurotoxin (BoNT) is a major food safety and bioterrorism concern, and also an important pharmaceutical, yet the regulation of its synthesis, activation, and stability in culture media, foods, and clinical samples is not well understood. This paper provides insights into the effects of critical nutrients on growth, lysis, spore formation, BoNT and TC production, and stability of BoNTs of C. botulinum We show that for C. botulinum ATCC 3502 cultured in a complex medium, a high level of arginine repressed BoNT expression by ca. 1000-fold, and also strongly reduced sporulation. Arginine stimulated growth and compensated for a lack of glucose. BoNT and toxin complex proteins were partially inactivated in a complex medium lacking glucose. This work should aid in optimizing BoNT production for pharmaceutical uses, and furthermore, an understanding of the nutritional regulation on growth and BoNT formation may provide insights into growth and BoNT formation in foods and clinical samples, and the enigmatic function of BoNTs in nature.

PMID: 28455330 [PubMed - as supplied by publisher]




Rational engineering of a cold-adapted α-amylase from the Antarctic ciliate Euplotes focardii for simultaneous improvement of thermostability and catalytic activity.

Rational engineering of a cold-adapted α-amylase from the Antarctic ciliate Euplotes focardii for simultaneous improvement of thermostability and catalytic activity.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Yang G, Yao H, Mozzicafreddo M, Ballarini P, Pucciarelli S, Miceli C

Abstract
The α-amylases are endo-acting enzyme which hydrolyze starch by randomly cleaving the 1,4-α-D-glucosidic linkages between the adjacent glucose units in linear amylose chain. It has significant advantages in a wide range of applications, in particular in food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, differently from most of the α-amylases characterized so far. Furthermore, EfAmy shows the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants: in one mutant we introduced Pro residues on the A and B domains in surface loops. In the second mutant we changed Val into Thr residues close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for changes in the biochemical properties are discussed by analyzing the three-dimensional structural model.IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered the novel EfAmy with improved thermostability, substrate binding affinity and catalytic efficiency to various extents, without impact on its pH preference. These characteristics can be considered an important property to be used in food, detergents, textiles and other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications.

PMID: 28455329 [PubMed - as supplied by publisher]




Coxiella burnetii circulation in a naturally infected flock of sheep: individual follow-up of antibodies in serum and milk.

Coxiella burnetii circulation in a naturally infected flock of sheep: individual follow-up of antibodies in serum and milk.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Joulié A, Rousset E, Gasqui P, Lepetitcolin E, Leblond A, Sidi-Boumedine K, Jourdain E

Abstract
The control of Q fever, a zoonotic disease caused by the Coxiella burnetii bacterium, remains a scientific challenge. Domestic ruminants are considered the main reservoir, shedding C. burnetii essentially through parturition products during abortion or birth. Sheep are particularly frequently associated with human outbreaks, but there are insufficient field data to fully understand disease dynamics and instigate efficient control measures.A longitudinal follow-up study of a naturally infected sheep flock was performed in order to: (a) investigate relationships between seropositivity and bacterial shedding in the vaginal mucus, (b) describe the kinetics of antibodies, including responses to vaccination, (c) monitor maternal antibodies in ewe lambs, and (d) compare serological results in milk and serum samples.For eight months, we collected blood samples every three weeks from 11 aborting and 26 non-aborting dairy ewes, 20 non-aborting suckler ewes and nine ewe lambs. Individual milk samples were also obtained from lactating females. All serum and milk samples were tested by ELISA whereas vaginal swabs were tested by quantitative PCR.We found that some dairy females did not seroconvert although they shed C. burnetii in their vaginal mucus or despite vaccination. Overall, antibody levels in adult females were found to remain stable over time, with exceptions during the mating and lambing periods. Maternal antibodies decreased during the first month after birth. Interestingly, antibody levels in milk were correlated with those in serum.This study provides valuable field data that will help improve Q fever surveillance and within-flock management measures.Importance Field data are necessary to improve the surveillance, diagnosis and sanitary management of Q fever in livestock. Here, we provide extensive serological data obtained from serum and milk samples of infected and vaccinated ewes belonging to a naturally infected flock of sheep. We show that antibody levels are stable over time and that seropositivity and vaginal shedding are not clearly correlated, whereas antibody levels in milk are strongly correlated with those in serum. Accordingly, we find that antibody levels in bulk tank milk are consistent with the variations observed in the serum of dairy females over time. For the first time, we report the existence of maternal antibody transmission to ewe lambs and we show that the presence of maternal antibodies at birth does not impact the serological response to vaccination at the age of 4 months. Finally, we report that adult ewes generally seroconvert after vaccination, including during pregnancy.

PMID: 28455328 [PubMed - as supplied by publisher]




Novel method for the reliable identification of Siccibacter and Franconibacter strains: From 'pseudo-Cronobacter' to new Enterobacteriaceae genera.

Novel method for the reliable identification of Siccibacter and Franconibacter strains: From 'pseudo-Cronobacter' to new Enterobacteriaceae genera.

Appl Environ Microbiol. 2017 Apr 28;:

Authors: Svobodová B, Vlach J, Junková P, Karamonová L, Blažková M, Fukal L

Abstract
In the last decade, strains of the genera Franconibacter and Siccibacter have been misclassified as first Enterobacter and later Cronobacter Because Cronobacter is a serious food-borne pathogen that affects premature neonates and elderly individuals, such misidentification may not only falsify epidemiological statistics, but also lead to tests of powdered infant formula or other foods giving false results. Currently, the main ways of identifying Franconibacter and Siccibacter strains are by biochemical testing or by sequencing of the fusA gene as part of Cronobacter MLST, but in relation to these strains the former is generally highly difficult and unreliable while the latter remains expensive. To address this, we developed a fast, simple and, most importantly, reliable method for Franconibacter and Siccibacter identification based on intact cell MALDI-TOF MS. Our method integrates the following steps: data preprocessing using mMass software; principal component analysis (PCA) for the selection of mass spectra fingerprints of Franconibacter and Siccibacter strains; optimization of the Biotyper database settings for the creation of Main Spectra Projections (MSPs). This methodology enabled us to create an in-house MALDI MS database that extends the current MALDI Biotyper database with Franconibacter and Siccibacter strains. Finally, we verified our approach using seven previously unclassified strains, all of which were correctly identified, thereby validating our method.IMPORTANCE We show that the majority of methods currently used for the identification of Franconibacter and Siccibacter bacteria are not able to properly distinguish these strains from those of Cronobacter While sequencing of the fusA gene as part of Cronobacter MLST remains the most reliable such method, it is highly expensive and time consuming. Here, we demonstrate a cost-effective and reliable alternative that correctly distinguishes between Franconibacter, Siccibacter, and Cronobacter bacteria, and identifies Franconibacter and Siccibacter at the species level. Using intact cell MALDI-TOF MS, we extend the current MALDI Biotyper database with 11 Franconibacter and Siccibacter MSPs. In addition, the use of our approach is likely to lead to more reliable identification scheme for Franconibacter and Siccibacter strains and, consequently, more trustworthy epidemiological picture of their involvement in disease.

PMID: 28455327 [PubMed - as supplied by publisher]