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US feral swine were exposed to both avian and swine influenza A viruses.
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US feral swine were exposed to both avian and swine influenza A viruses.

Appl Environ Microbiol. 2017 Jul 21;:

Authors: Martin BE, Sun H, Carrel M, Cunningham FL, Baroch JA, Hanson-Dorr KC, Young SG, Schmit B, Nolting JM, Yoon KJ, Lutman MW, Pedersen K, Lager K, Bowman AS, Slemons RD, Smith DR, DeLiberto T, Wan XF

Abstract
Influenza A viruses (IAVs) in swine can cause sporadic infections and pandemic outbreaks among humans, but how avian IAV emerges in swine is still unclear. Unlike domestic swine, feral swine are free ranging and have many opportunities for IAV exposure through contacts with various habitats and animals, including migratory waterfowl, a natural reservoir for IAVs. During 2010--2013, 8,239 serum samples were collected from feral swine across 35 US states and tested against 45 contemporary antigenic variants of avian, swine, and human IAVs; of these, 406 (4.9%) samples were IAV-antibody positive. Among 294 serum samples selected for antigenic characterization, 271 cross-reacted with ≥1 testing virus whereas the other 23 did not cross-react with any testing virus. Of the 271 IAV-positive samples, 236 cross-reacted with swine IAVs, 1 with avian IAVs, and 16 with avian and swine IAVs, indicating that feral swine were exposed to both swine and avian IAVs but predominantly to swine IAVs. Our findings suggest that feral swine could potentially be infected with both avian and swine IAVs, generating novel IAVs by hosting and reassorting IAVs from wild birds and domestic swine and facilitating adaptation of avian IAVs to other hosts, including humans, before their spillover. Continued surveillance to monitor the distribution and antigenic diversities of IAVs in feral swine is necessary to increase our understanding of the natural history of IAVs.Importance There are more than 5 million feral swine distributed across at least 35 states in the USA. In contrast to domestic swine, feral swine are free ranging and have unique opportunities for contact with wildlife, livestock and their habitats. Our serological results indicate that feral swine in the United States have been exposed to influenza A viruses (IAVs) consistent with those found in both domestic swine and wild birds, with the predominant infections consisting of swine adapted IAVs. Our findings suggest that feral swine having been infected with IAVs at low levels and could serve as hosts for the generation of novel IAVs at the interface of feral swine, wild birds, domestic swine, and humans.

PMID: 28733290 [PubMed - as supplied by publisher]




An Enterotoxin-like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity.
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An Enterotoxin-like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity.

Appl Environ Microbiol. 2017 Jul 21;:

Authors: Wei JZ, Siehl DL, Hou Z, Rosen B, Oral J, Taylor CG, Wu G

Abstract
Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated as Pp-ANP, that is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at L1 stage and killing older staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a are active when reconstituted from separate expression in Escherichia coli The binary toxin also shows strong nematicidal activity against three other free-living nematodes, Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp., but no activity was found for insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant homology to any known proteins, while Pp-ANP2a shows ∼30% homology to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates Pp-ANP2a is structurally similar to CT/LT and likely acts as a ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggests applications in controlling parasitic nematodes and opens an avenue for further research on its mechanism of action and role in bacterium-nematode interaction.IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older staged worms. It also shows strong activity on other free-living nematodes, Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp. The two subunits, Pp-ANP1a and Pp-ANP2a, can be expressed separately and reconstituted to form the active complex when mixed together. Pp-ANP shows some distinct characteristics when compared with other toxins, including Escherichia coli enterotoxin and cholera toxin. The present study indicates that Pp-ANP is a novel binary toxin and that it has potential applications in controlling parasitic nematodes and in studying toxin-host interaction.

PMID: 28733289 [PubMed - as supplied by publisher]




Bacillomycin D produced by Bacillus amyloliquefaciens is involved in the antagonistic interaction with the plant pathogenic fungus Fusarium graminearum.
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Bacillomycin D produced by Bacillus amyloliquefaciens is involved in the antagonistic interaction with the plant pathogenic fungus Fusarium graminearum.

Appl Environ Microbiol. 2017 Jul 21;:

Authors: Gu Q, Yang Y, Yuan Q, Shi G, Wu L, Lou Z, Huo R, Wu H, Borriss R, Gao X

Abstract
Fusarium graminearum (teleomorph: Ascomycota, Hypocreales, Gibberella, Gibberella zeae) is a destructive fungal pathogen that threatens the production and quality of wheat and barley worldwide. Controlling this toxin-producing pathogen is a significant challenge. In the present study, the commercially available strain Bacillus amyloliquefaciens (Bacteria, Firmicutes, Bacillales, Bacillus) FZB42 showed strong activity against F. graminearum The lipopeptide, bacillomycin D, produced by FZB42, was proven to contribute to the antifungal activity. Purified bacillomycin D showed strong activity against F. graminearum, and its 50 % effective concentration was determined as approximately 30 μg/mL. Analyses using scanning and transmission electron microscopy revealed that bacillomycin D caused morphological changes in the plasma membrane and cell wall of F. graminearum hyphae and conidia. Fluorescence microscopy combined with different dyes showed that bacillomycin D induced the accumulation of reactive oxygen species and caused cell death in F. graminearum hyphae and conidia. F. graminearum secondary metabolism also responded to bacillomycin D challenge by increased the production of deoxynivalenol. Biological control experiments demonstrated that bacillomycin D exerted good control of F. graminearum on corn silks, wheat seedlings, and wheat heads. In response to bacillomycin D, genes involved in scavenging reactive oxygen species of F. graminearum were downregulated, whereas genes involved in the synthesis of deoxynivalenol were upregulated. Phosphorylation of MGV1 and HOG1, the mitogen-activated protein kinases of F. graminearum, was increased in response to bacillomycin D. Taken together, these findings reveal the mechanism of the antifungal action of bacillomycin D.IMPORTANCE Biological control of plant disease caused by Fusarium graminearum is desirable. Bacillus amyloliquefaciens FZB42 is a representative of the biocontrol bacterial strains. In this work, the lipopeptide bacillomycin D, produced by FZB42, was proven to show strong fungicidal activity against F. graminearum Bacillomycin D caused morphological changes in the plasma membrane and cell wall of F. graminearum, induced accumulation of reactive oxygen species, and ultimately caused cell death in F. graminearum Interestingly, when challenged by bacillomycin D, deoxynivalenol production, gene expression, mitogen-activated protein kinases phosphorylation, and pathogenicity of F. graminearum were significantly altered. These findings clarified the mechanisms of bacillomycin D's activity against F. graminearum, and highlighted the potential of B. amyloliquefaciens FZB42 as biocontrol agent against F. graminearum.

PMID: 28733288 [PubMed - as supplied by publisher]




Identification of the hcb gene operon involved in catalyzing aerobic hexachlorobenzene dechlorination in Nocardioides sp. strain PD653.
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Identification of the hcb gene operon involved in catalyzing aerobic hexachlorobenzene dechlorination in Nocardioides sp. strain PD653.

Appl Environ Microbiol. 2017 Jul 21;:

Authors: Ito K, Takagi K, Iwasaki A, Tanaka N, Kanesaki Y, Martin-Laurent F, Igimi S

Abstract
Nocardioides sp. strain PD653 was the first aerobic bacterium capable of mineralizing hexachlorobenzene (HCB) that was identified. In this study, strain PD653-B2, which was unexpectedly isolated from a subculture of strain PD653, was found to lack the ability to transform HCB or pentachloronitrobenzene into pentachlorophenol. Comparative genome analysis of the two strains revealed that genetic rearrangement had occurred in strain PD653-B2, with a genomic region present in strain PD653 being deleted. In silico analysis allowed three open reading frames within this region to be identified as candidate genes involved in HCB dechlorination. Assays using recombinant Escherichia coli cells revealed that an operon is responsible for both oxidative HCB dechlorination and pentachloronitrobenzene denitration. The metabolite pentachlorophenol was detected in the cultures produced in the E. coli assays. Significantly less HCB-degrading activity occurred in assays under oxygen-limited conditions ([O2] < 0.5 mg L(-1)) than in aerobic assays, suggesting that monooxygenase is involved in the reaction. In this operon, hcbA1 was found to encode a monooxygenase involved in HCB dechlorination. this monooxygenase may form a complex with the flavin reductase encoded by hcbA3, increasing the HCB-degrading activity of PD653.Importance: The organochlorine fungicide HCB is widely distributed in the environment. Bioremediation can effectively remove HCB from contaminated sites, but HCB-degrading microorganisms have been isolated in few studies, and the genes involved in HCB degradation have not been identified. In this study, possible genes involved in the initial step of the mineralization of HCB by Nocardioides sp. strain PD653 were identified. The results improve our understanding of the protein families involved in the dechlorination of HCB to give pentachlorophenol.

PMID: 28733287 [PubMed - as supplied by publisher]




The regulator LdhR and the d-lactate dehydrogenase LdhA of Burkholderia multivorans play a role in carbon overflow and in planktonic cellular aggregates formation.
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The regulator LdhR and the d-lactate dehydrogenase LdhA of Burkholderia multivorans play a role in carbon overflow and in planktonic cellular aggregates formation.

Appl Environ Microbiol. 2017 Jul 21;:

Authors: Silva IN, Ramires MJ, Azevedo LA, Guerreiro AR, Tavares AC, Becker JD, Moreira LM

Abstract
LysR-type transcriptional regulators (LTTR) are the most commonly found regulators in Burkholderia cepacia complex, comprising opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. Despite LTTRs being global regulators of pathogenicity in several bacteria, few have been characterized in Burkholderia Here, we showed that gene ldhR of B. multivorans encoding a LTTR is co-transcribed with ldhA encoding a d-lactate dehydrogenase, and evaluate their implication in virulence traits like exopolysaccharide (EPS) synthesis and biofilm formation. Comparison of wild-type (WT) and its isogenic ΔldhR mutant grown in medium with 2% d-glucose revealed a negative impact on EPS biosynthesis and on cells' viability in the presence of LdhR. Loss of viability in WT cells was caused by intracellular acidification as consequence of cumulative organic acids secretion including d-lactate, this last one absent from the ΔldhR mutant supernatant. Furthermore, LdhR is implicated in the formation of planktonic cellular aggregates. WT cell aggregates reached 1000 μm after 24 hours in liquid cultures; in contrast to ΔldhR mutant aggregates that never grew more than 60 μm. Overexpression of d-lactate dehydrogenase LdhA in the ΔldhR mutant partially restored formed aggregates size, suggesting a role for fermentation inside aggregates. Similar results were obtained for surface-attached biofilms, with WT cells producing more biofilm. A systematic evaluation of planktonic aggregates in Burkholderia CF clinical isolates showed aggregates in 40 out of 74. As CF patients' lung environment is microaerophilic and bacteria are found as free aggregates/biofilms, LdhR and LdhA might have central roles in adaptation to this environment.IMPORTANCE Cystic fibrosis patients often suffer from chronic respiratory infections caused by several microorganisms. Among them are the Burkholderia cepacia complex bacteria which cause progressive deterioration of lung function and, in some patients, might develop into fatal necrotizing pneumoniae with bacteremia, known as "cepacia syndrome". Burkholderia pathogenesis is multifactorial since they express several virulence factors, form biofilms, and are highly resistant to antimicrobial compounds, making their eradication from the CF patients' airways very difficult. As Burkholderia is commonly found in the CF lungs in the form of cell aggregates and biofilms, the need to investigate the mechanisms of cellular aggregation is obvious. In this study we demonstrate the importance of a d-lactate dehydrogenase and a regulator, in regulating carbon overflow, cellular aggregates and surface-attached biofilm formation. This not only enhances our understanding of Burkholderia pathogenesis, but can also lead to the development of drugs against these proteins to circumvent biofilm formation.

PMID: 28733286 [PubMed - as supplied by publisher]




Transcriptional modulation of transport and metabolism associated gene clusters leading to utilization of benzoate in preference to glucose in Pseudomonas putida CSV86.
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Transcriptional modulation of transport and metabolism associated gene clusters leading to utilization of benzoate in preference to glucose in Pseudomonas putida CSV86.

Appl Environ Microbiol. 2017 Jul 21;:

Authors: Choudhary A, Modak A, Apte SK, Phale PS

Abstract
The effective elimination of xenobiotic pollutants from the environment can be achieved by efficient degradation by microorganisms even in the presence of sugars or organic acids. Pseudomonas putida CSV86 displays a unique property to utilize aromatic compounds prior to glucose. The draft genome and transcription analyses revealed that glucose uptake and benzoate transport and metabolism genes are clustered at glc and ben loci, respectively, as two distinct operons. When grown on glucose+benzoate, CSV86 displayed significantly higher expression of ben locus in the first- and glc locus in the second-log phase. Kinetics of substrate uptake and metabolism matched the transcription profiles. Inability of succinate to suppress/modulate benzoate transport and metabolism resulted in their co-utilization. When challenged with succinate or benzoate, glucose-grown cells showed rapid reduction in the glc locus transcription, glucose transport and metabolic activity, with succinate being more effective at the functional level. Benzoate and succinate failed to interact with or inhibit the activities of glucose transport components or metabolic enzymes. The data suggest that succinate as well as benzoate suppress glucose transport and metabolism at the transcription level, enabling this strain to metabolize benzoate preferentially. P. putida CSV86, thus has the potential to be an ideal host to engineer diverse metabolic pathways for efficient bioremediation.IMPORTANCEPseudomonas strains play an important role in carbon cycling in the environment and display a hierarchy in carbon utilization for organic acids followed by glucose and least for aromatic substrates. This limits their exploitation for bioremediation. This study demonstrates the substrate-dependent modulation of ben and glc operons in Pseudomonas putida CSV86, wherein benzoate suppresses the glucose transport and metabolism at the transcription level leading to preferential utilization of benzoate over glucose. Interestingly, succinate and benzoate are co-metabolized. These properties are unique as compared to other Pseudomonad and open up avenues to unravel novel regulatory processes. Strain CSV86 can serve as an ideal host to engineer and facilitate efficient removal of recalcitrant pollutants even in the presence of simpler carbon sources.

PMID: 28733285 [PubMed - as supplied by publisher]




Specific signatures of the gut microbiota and increased levels of butyrate in children treated with fermented cow's milk containing heat-killed Lactobacillus paracasei CBA L74.
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Specific signatures of the gut microbiota and increased levels of butyrate in children treated with fermented cow's milk containing heat-killed Lactobacillus paracasei CBA L74.

Appl Environ Microbiol. 2017 Jul 21;:

Authors: Berni Canani R, De Filippis F, Nocerino R, Laiola M, Paparo L, Calignano A, De Caro C, Coretti L, Chiariotti L, Gilbert JA, Ercolini D

Abstract
We recently demonstrated that cow's milk fermented with the probiotic L.paracasei CBA L74 (FM-CBAL74) reduces the incidence of respiratory and gastrointestinal tract infections in young children attending school. This effect apparently derives from a complex regulation of non-immune and immune protective mechanisms. We investigated wheter FM-CBAL74 could regulate gut microbiota composition and butyrate production.We randomly selected 20 healthy children (12-48 months) from the previous randomized controlled trial, before (t0) and after 3 months (t3) of dietary treatment with FM-CBAL74 (FM), or placebo (PL). Fecal microbiota was profiled using 16S rRNA gene amplicon sequencing and fecal butyrate concentration was also measured. Microbial alpha and beta-diversity were not significantly different between groups prior to treatment. FM-CBAL74 but not PL treatment increased the relative abundance of Lactobacillus. Individual Blautia, Roseburia and Faecalibacterium oligotypes were associated to FM-CBAL74 treatment and demonstrated correlative associations with immune biomarkers. Accordingly, PICRUSt analysis predicted an increase in the proportion of genes involved in butyrate production pathways, consistent with an increase in fecal butyrate observed only in the FM group. Dietary supplementation with FM-CBAL74 induces specific signatures in gut microbiota composition and stimulates butyrate production. These effects are associated with changes in innate and acquired immunity.Importance: The use of a fermented milk product containing the heat-killed probiotic strain L.paracasei CBAL74 induces changes in the gut microbiota, promoting the development of butyrate-producers. These changes in the gut microbiota composition correlate with increased levels of innate and acquired immunity biomarkers.

PMID: 28733284 [PubMed - as supplied by publisher]




Osmotic compounds enhance antibiotic efficacy against Acinetobacter baumannii biofilm communities.
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Osmotic compounds enhance antibiotic efficacy against Acinetobacter baumannii biofilm communities.

Appl Environ Microbiol. 2017 Jul 21;:

Authors: Falghoush A, Beyenal H, Besser TE, Omsland A, Call DR

Abstract
Biofilm-associated infections are a clinical challenge in part because a hydrated matrix protects the bacterial community from antibiotics. Herein, we evaluated how different osmotic compounds [maltodextrin, sucrose and polyethylene glycol (PEG)] enhance antibiotic efficacy against Acinetobacter baumannii biofilm communities. Established test-tube biofilms (24 h, strain ATCC 17978) were treated with osmotic compounds in the presence or absence of 10X the minimum inhibitory concentration of different antibiotics (50 μg/ml tobramycin, 20 μg/ml ciprofloxacin, 300 μg/ml chloramphenicol, 30 μg/ml nalidixic acid or 100 μg/ml erythromycin). Combining antibiotics with hypertonic concentrations of the osmotic compounds for 24 h reduced the number of biofilm bacteria by 5-7 log (P <0.05). Increasing concentrations of osmotic compounds improved the effect, but there was a tradeoff with increasing solution viscosity whereby low molecular mass compounds (sucrose, 400 Da PEG) worked better than higher mass compounds (maltodextrin, 3,350 Da PEG). Ten other A. baumannii strains were similarly treated with 400 Da PEG and tobramycin, resulting in a mean 2.7-log reduction in recoverable bacteria compared with tobramycin treatment alone. Multivariate regression models with data from different osomotic compounds and nine antibiotics demonstrated that the benefit from combining hypertonic treatments with antibiotics is a function of antibiotic mass and lipophilicity (r(2) > 0. 0.82; P <0.002), and the relationship was generalizable for biofilm formed by A. baumannii and E. coli K-12. Augmenting topical antibiotic therapies with a low-mass hypertonic treatment may enhance the efficacy of antibiotics against wound biofilms, particularly when using low-mass, hydrophilic antibiotics.Importance Biofilms form a barrier that protects bacteria from environmental insults including exposure to antibiotics. We demonstrated that multiple osmotic compounds can enhance antibiotic efficacy against Acinetobacter baumannii biofilm communities, but viscosity is a limiting factor and the most effective compounds have lower molecular mass. The synergism between osmotic compounds and antibiotics is also dependent on hydrophobicity and mass of the antibiotics. The statistical models presented herein provide a basis for predicting the optimal combination of osmotic compounds and antibiotics against surface biofilms communities.

PMID: 28733283 [PubMed - as supplied by publisher]




Differentiating botulinum-neurotoxin-producing clostridia with a simple, multiplex PCR assay.
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Differentiating botulinum-neurotoxin-producing clostridia with a simple, multiplex PCR assay.

Appl Environ Microbiol. 2017 Jul 21;:

Authors: Williamson CHD, Vazquez AJ, Hill K, Smith TJ, Nottingham R, Stone NE, Sobek CJ, Cocking JH, Fernández RA, Caballero PA, Leiser OP, Keim P, Sahl JW

Abstract
Diverse members of the genus clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum Groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This study introduces a multiplex polymerase chain reaction (PCR) assay for differentiating members of C. botulinum Group I, C. sporogenes, and two major subgroups within C. botulinum Group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha+ or orfX+). The increased availability of whole genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with bont gene clusters.Importance Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum Group I, C. sporogenes, and two major subgroups within C. botulinum Group II. Since, BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and provide information about the type (ha+ or orfX+) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

PMID: 28733282 [PubMed - as supplied by publisher]




Structural and mechanistic insights into the improvement of halotolerant ability of a marine microbial esterase by increasing intra- and inter-domain hydrophobic interactions.
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Structural and mechanistic insights into the improvement of halotolerant ability of a marine microbial esterase by increasing intra- and inter-domain hydrophobic interactions.

Appl Environ Microbiol. 2017 Jul 21;:

Authors: Li PY, Zhang Y, Xie BB, Zhang YQ, Hao J, Wang Y, Wang P, Li CY, Qin QL, Zhang XY, Su HN, Shi M, Zhang YZ, Chen XL

Abstract
Halotolerant enzymes are beneficial for industrial processes requiring high salts and low water activity. Most halophilic proteins are evolved to have reduced hydrophobic interactions on the surface and in the hydrophobic cores for their haloadaptation. However, in this study, we improved the halotolerance of a thermolablie esterase, E40, by increasing intra-protein hydrophobic interactions. E40 was quite unstable in buffers containing more than 0.3 M NaCl and its kcat and substrate affinity were both significantly reduced in 0.5 M NaCl. By introducing hydrophobic residues in the loop1 of the CAP domain and/or the α7 of the catalytic domain in E40, we obtained several mutants with improved halotolerance and the mutant M3+S202W/I203F was the most halotolerant. Then we solved the crystal structures of mutants S202W/I203F and M3+S202W/I203F to reveal the structural basis for their improved halotolerance. Structural analysis revealed that the introduction of hydrophobic residues W202 and F203 in the α7 significantly improved E40 halotolerance by strengthening intra-domain hydrophobic interactions of F203 with W202 and other residues in the catalytic domain. By further introducing hydrophobic residues in the loop1, mutant M3+S202W/I203F became more rigid and halotolerant due to the formation of additional inter-domain hydrophobic interactions between the introduced Y22 in the loop1 and W204 in the α7. These results indicate that increasing intra-protein hydrophobic interactions is also a way to improve the halotolerance of enzymes with industrial potential under high salts.Importance Esterases and lipases for industrial application are often subjected to harsh conditions such as high salts, low water activity and the presence of organic solvents. However, reports on halotolerant esterases and lipases are limited and the underlying mechanism for their halotolerance is still unclear due to the lack of structures. In this study, we focused on the improvement of the halotolerance of a salt-sensitive esterase, E40, and the underlying mechanism. The halotolerance of E40 was significantly improved by introducing hydrophobic residues. Comparative structural analysis of E40 and its halotolerant mutants revealed that increased intra-protein hydrophobic interactions make these mutants more rigid and more stable than the wild type against high salts. This study shows a new way to improve enzyme halotolerance, which is helpful for protein engineering of salt-sensitive enzymes.

PMID: 28733281 [PubMed - as supplied by publisher]




Construction and optimization of a heterologous pathway for protocatechuate catabolism in Escherichia coli enables bioconversion of model aromatic compounds.
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Construction and optimization of a heterologous pathway for protocatechuate catabolism in Escherichia coli enables bioconversion of model aromatic compounds.

Appl Environ Microbiol. 2017 Jul 21;:

Authors: Clarkson SM, Giannone RJ, Kridelbaugh DM, Elkins JG, Guss AM, Michener JK

Abstract
The production of biofuels from lignocellulose yields a substantial lignin byproduct stream that currently has few applications. Biological conversion of lignin-derived compounds into chemicals and fuels has the potential to improve the economics of lignocellulose-derived biofuels, but few microbes are able both to catabolize lignin-derived aromatic compounds and generate valuable products. While Escherichia coli has been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we have engineered E. coli to catabolize protocatechuate, a common intermediate in lignin degradation, as the sole source of carbon and energy via heterologous expression of a nine-gene pathway from Pseudomonas putida KT2440. We next used experimental evolution to select for mutations that increased growth with protocatechuate more than two-fold. Increasing the strength of a single ribosome binding site in the heterologous pathway was sufficient to recapitulate the increased growth. After optimization of the core pathway, we extended the pathway to enable catabolism of a second model compound, 4-hydroxybenzoate. These engineered strains will be useful platforms to discover, characterize, and optimize pathways for conversions of lignin-derived aromatics.Importance Lignin is a challenging substrate for microbial catabolism due to its polymeric and heterogeneous chemical structure. Therefore, engineering microbes for improved catabolism of lignin-derived aromatic compounds will require the assembly of an entire network of catabolic reactions, including pathways from genetically-intractable strains. Constructing defined pathways for aromatic compound degradation in a model host would allow rapid identification, characterization, and optimization of novel pathways. We have constructed and optimized one such pathway in E. coli to enable catabolism of a model aromatic compound, protocatechuate, and then extended the pathway to a related compound, 4-hydroxybenzoate. This optimized strain can now be used as the basis for the characterization of novel pathways.

PMID: 28733280 [PubMed - as supplied by publisher]