The potential of cold-shock promoters for the expression of recombinant proteins in microbes and mammalian cells.

BACKGROUND: Low-temperature expression of recombinant proteins may be advantageous to support their proper folding and preserve bioactivity. The generation of expression vectors regulated under cold conditions can improve the expression of some target proteins that are difficult to express in different expression systems. The cspA encodes the major cold-shock protein from Escherichia coli (CspA). The promoter of cspA has been widely used to develop cold shock-inducible expression platforms in E. coli. Moreover, it is often necessary to employ expression systems other than bacteria, particularly when recombinant proteins require complex post-translational modifications. Currently, there are no commercial platforms available for expressing target genes by cold shock in eukaryotic cells. Consequently, genetic elements that respond to cold shock offer the possibility of developing novel cold-inducible expression platforms, particularly suitable for yeasts, and mammalian cells. CONCLUSIONS: This review covers the importance of the cellular response to low temperatures and the prospective use of cold-sensitive promoters to direct the expression of recombinant proteins. This concept may contribute to renewing interest in applying white technologies to produce recombinant proteins that are difficult to express.

Correction to: Autolysis of Pichia pastoris induced by cold.

The original version of this article (Bartolo-Aguilar et al. 2017) was written and published including the first construction strategy of pLGC09, but not the final one. This error was pointed out by a reader and an analysis of sequences of parts of the plasmid corroborated this. The final construction strategy was reanalysed and confirmed the error. This error affected the text, Table 2, Fig. 1 and Additional files, but did not affect the results and conclusions stated in the paper. The authors regret that this error occurred in the original publication of the article. The corrected text, Table 2 and Fig. 1, and Additional files (Additional file 1. Construction strategy of pLGC09 and Additional file 2. Plasmid pLGC09) are given in this correction.

Investigation on the Evolutionary Relation of Diverse Polyhydroxyalkanoate Gene Clusters in Betaproteobacteria.

Products of numerous genes (phaC, phaA, phaB, phaP, phaR, and phaZ) are involved in the synthesis and degradation processes of the ubiquitous prokaryotic polyhydroxyalkanoate (PHA) intracellular reserve storage system. In this study, we performed a bioinformatics analysis to identify PHA-related genes and proteins in the genome of 66 selected organisms (class: Betaproteobacteria) that occur in various habitats; besides, evolutionary trajectories of the PHA system are reported here. The identified PHA-related genes were organized into clusters, and the gene arrangement was highly diverse. The occurrence and distribution of PHA-related clusters revealed that a single cluster was primarily segmented into small gene groups among various genomes, which were further reorganized as novel clusters based on various functional genes. The individual phylogenies of gene and protein sequences supported that the clusters were assembled through the relocation of native orthologous genes that underwent insertion, deletion, and elongation events. Furthermore, the neighboring genes provided valuable evolutionary and functional cues regarding the conservation and maintenance of PHA-related genes in the genome. Overall, the aforementioned results strongly indicate the influence of horizontal gene transfer on the organization of PHA-related gene clusters. Therefore, our results reveal new insights into the organization, evolutionary history, and cluster conservation of the PHA-related gene inventories among Betaproteobacterial organisms.

Phylogenetic diversification and developmental implications of poly-(R)-3-hydroxyalkanoate gene cluster assembly in prokaryotes.

Many polyhydroxyalkanoates (PHAs) system genes, such as phaC, phaA, phaB, phaR, phaP and phaZ, are often found to be organised in the form of operon-like clusters. In this study, a genome survey was performed to identify such clustered PHA systems among 256 prokaryotic organisms. These data were then used to generate a comprehensive 16S rRNA species tree depicting the phylogenetic distribution of the observed clusters with diverse gene arrangements. In addition, the gene occurrences and physical linkages between PHA system genes were quantitatively estimated. From this, we identified a centrally connected hub gene, i.e. the phaC gene of PHA. Furthermore, a comparative investigation was performed between the clusters of PHA and glycogen, which decoded the role of the hub gene in the cluster organisation of both systems. Together, these findings suggest that the highly connected hub gene might contribute substantively towards the organisation and maintenance of the gene network connectivity in the clusters, particularly in the storage reserve systems.

Autolysis of Pichia pastoris induced by cold.

The production of recombinant biopharmaceutical proteins is a multi-billion dollar market. Protein recovery represents a major part of the production costs. Pichia pastoris is one of the microbial systems most used for the production of heterologous proteins. The use of a cold-induced promoter to express lytic enzymes in the yeast after the growth stage could reduce protein recovery costs. This study shows that a cold-shock can be applied to induce lysis of the yeast cells. A strain of P. pastoris was constructed in which the endogenous eng gene encoding a putative endo-ß-1,3-glucanase was overexpressed using the cold-shock induced promoter of the cctα gene from Saccharomyces cerevisiae. In the transgenic P. pastoris, the expression of eng increased 3.6-fold after chilling the cells from 30 to 4 °C (cold-shock stage) followed by incubation for 6 h (eng expression stage). The culture was heated to 30 °C for 6 h (ENG synthesis stage) and kept at 37 °C for 24 h (lysis stage). After this procedure the cell morphology changed, spheroplasts were obtained and cellular lysis was observed. Thus, a clone of P. pastoris was obtained, which undergoes autolysis after a cold-shock.

Bioavailability and dissipation of anthracene from soil with different alkalinity and salinity.

Bioavailability of contaminants, such as anthracene (Anthra), a polycyclic aromatic hydrocarbon (PAHs), and their removal from soil has been related to their extractability with non-exhaustive techniques, such as hydroxypropyl-beta-cyclodextrin (HPCD) or n-butanol. Anthra was extracted with HPCD, n-butanol and by exhaustive ultrasonic extraction method from sterilized and unsterilized alkaline soil of the former lake Texcoco, having pH ranging from pH 8.2 to 10.1 and electrolytic conductivity varying from 1.2 dS m(-1) to 95.2 dS m(-1), respectively. About 24.4 and 37.6% of Anthra was removed biologically from soil as estimated by exhaustive technique after 56 days. The percentage of Anthra that was removed from soil by exhaustive technique was not related to the amount thatwas extractable with HPCD or n-butanol. It was found that the Anthra extractable with n-butanol or HPCD did not correlate well with the removal of the contaminant from soil. In this study, the removal of Anthra from soil could not be predicted by the amount of Anthra that was extracted with n-butanol or HPCD .

Characterization of the hrpZ gene from Pseudomonas syringae pv. maculicola M2.

Pseudomonas syringae pv. maculicola is a natural pathogen of members of the Brassicaceae plant family. Using a transposon-based mutagenesis strategy in Pseudomonas syringaepv. maculicola M2 (PsmM2), we conducted a genetic screen to identify mutants that were capable of growing in M9 medium supplemented with a crude extract from the leaves of Arabidopsis thaliana. A mutant containing a transposon insertion in the hrpZ gene (PsmMut8) was unable to infect adult plants from Arabidopsis thaliana or Brassica oleracea, suggesting a loss of pathogenicity. The promotorless cat reporter present in the gene trap was expressed if PsmMut8 was grown in minimal medium (M9) supplemented with the leaf extract but not if grown in normal rich medium (KB). We conducted phylogenetic analysis using hrpAZB genes, showing the classical 5-clade distribution, and nucleotide diversity analysis, showing the putative position for selective pressure in this operon. Our results indicate that the hrpAZB operon from Pseudomonas syringaepv. maculicola M2 is necessary for its pathogenicity and that its diversity would be under host-mediated diversifying selection.

Characterization of the hrpZ gene from Pseudomonas syringae pv. maculicolaM2

Pseudomonas syringae pv. maculicola is a natural pathogen of members of the Brassicaceae plant family. Using a transposon-based mutagenesis strategy in Pseudomonas syringaepv. maculicola M2 (PsmM2), we conducted a genetic screen to identify mutants that were capable of growing in M9 medium supplemented with a crude extract from the leaves of Arabidopsis thaliana. A mutant containing a transposon insertion in the hrpZ gene (PsmMut8) was unable to infect adult plants from Arabidopsis thaliana or Brassica oleracea, suggesting a loss of pathogenicity. The promotorless cat reporter present in the gene trap was expressed if PsmMut8 was grown in minimal medium (M9) supplemented with the leaf extract but not if grown in normal rich medium (KB). We conducted phylogenetic analysis using hrpAZB genes, showing the classical 5-clade distribution, and nucleotide diversity analysis, showing the putative position for selective pressure in this operon. Our results indicate that the hrpAZB operon from Pseudomonas syringaepv. maculicola M2 is necessary for its pathogenicity and that its diversity would be under host-mediated diversifying selection.

Characterization of the hrpZ gene from Pseudomonas syringae pv. maculicolaM2

Pseudomonas syringae pv. maculicola is a natural pathogen of members of the Brassicaceae plant family. Using a transposon-based mutagenesis strategy in Pseudomonas syringaepv. maculicola M2 (PsmM2), we conducted a genetic screen to identify mutants that were capable of growing in M9 medium supplemented with a crude extract from the leaves of Arabidopsis thaliana. A mutant containing a transposon insertion in the hrpZ gene (PsmMut8) was unable to infect adult plants from Arabidopsis thaliana or Brassica oleracea, suggesting a loss of pathogenicity. The promotorless cat reporter present in the gene trap was expressed if PsmMut8 was grown in minimal medium (M9) supplemented with the leaf extract but not if grown in normal rich medium (KB). We conducted phylogenetic analysis using hrpAZB genes, showing the classical 5-clade distribution, and nucleotide diversity analysis, showing the putative position for selective pressure in this operon. Our results indicate that the hrpAZB operon from Pseudomonas syringaepv. maculicola M2 is necessary for its pathogenicity and that its diversity would be under host-mediated diversifying selection.

Molecular cloning and characterization of the ATP citrate lyase from carotenogenic yeast Phaffia rhodozyma.

ATP citrate lyase (ACL), is a key cytosolic source of acetyl-CoA for fatty acid and sterol biosynthesis and appear to be involved in carotenoid biosynthesis in yeasts. Three homologous DNA sequences encoding ACLs in Phaffia rhodozyma were isolated i.e two genes and one cDNA. The two genes were multi-intronic, with 3450-bp-coding sequences and both genes, as the cDNA, encoded identical 120.1-kDa polypeptides. Full-length amino acid sequences of these ACLs showed the two multidomains, PLN02235 and PLN02522, which are necessary for activity. The ACLs showed 82-87% similarity to putative ACLs from other basidiomycetes and 71% similarity to human ACL. The acl cDNA was used to express the heterologous ACL 6XHis-tagged which was identified using MALDI-TOF-MS. The sequenced peptides with 42.2% coverage showed 100% identity to the amino acid sequence generated in silico. The recombinant ACL purified to homogeneity showed an activity of 2 U. This is the first study to characterize a recombinant ACL from a carotenogenic yeast. The present study provides a key foundation for future studies to assess (a) the possible occurrence of alternative splicing, (b) identify the promoter(s) sequence(s) and (c) the involvement of ACL in the differential regulation of fatty acid and carotenoid biosynthesis in yeasts.

Archaeal Communities in a Heterogeneous Hypersaline-Alkaline Soil.

In this study the archaeal communities in extreme saline-alkaline soils of the former lake Texcoco, Mexico, with electrolytic conductivities (EC) ranging from 0.7 to 157.2 dS/m and pH from 8.5 to 10.5 were explored. Archaeal communities in the 0.7 dS/m pH 8.5 soil had the lowest alpha diversity values and were dominated by a limited number of phylotypes belonging to the mesophilic Candidatus Nitrososphaera. Diversity and species richness were higher in the soils with EC between 9.0 and 157.2 dS/m. The majority of OTUs detected in the hypersaline soil were members of the Halobacteriaceae family. Novel phylogenetic branches in the Halobacteriales class were detected in the soil, and more abundantly in soil with the higher pH (10.5), indicating that unknown and uncharacterized Archaea can be found in this soil. Thirteen different genera of the Halobacteriaceae family were identified and were distributed differently between the soils. Halobiforma, Halostagnicola, Haloterrigena, and Natronomonas were found in all soil samples. Methanogenic archaea were found only in soil with pH between 10.0 and 10.3. Retrieved methanogenic archaea belonged to the Methanosarcinales and Methanomicrobiales orders. The comparison of the archaeal community structures considering phylogenetic information (UniFrac distances) clearly clustered the communities by pH.

Changes in methane oxidation activity and methanotrophic community composition in saline alkaline soils.

The soil of the former Lake Texcoco is a saline alkaline environment where anthropogenic drainage in some areas has reduced salt content and pH. Potential methane (CH4) consumption rates were measured in three soils of the former Lake Texcoco with different electrolytic conductivity (EC) and pH, i.e. Tex-S1 a >18 years drained soil (EC 0.7 dS m(-1), pH 8.5), Tex-S2 drained for ~10 years (EC 9.0 dS m(-1), pH 10.3) and the undrained Tex-S3 (EC 84.8 dS m(-1), pH 10.3). An arable soil from Alcholoya (EC 0.7 dS m(-1), pH 6.7), located nearby Lake Texcoco was used as control. Methane oxidation in the soil Tex-S1 (lowest EC and pH) was similar to that in the arable soil from Alcholoya (32.5 and 34.7 mg CH4 kg(-1) dry soil day(-1), respectively). Meanwhile, in soils Tex-S2 and Tex-S3, the potential CH4 oxidation rates were only 15.0 and 12.8 mg CH4 kg(-1) dry soil day(-1), respectively. Differences in CH4 oxidation were also related to changes in the methane-oxidizing communities in these soils. Sequence analysis of pmoA gene showed that soils differed in the identity and number of methanotrophic phylotypes. The Alcholoya soil and Tex-S1 contained phylotypes grouped within the upland soil cluster gamma and the Jasper Ridge, California JR-2 clade. In soil Tex-S3, a phylotype related to Methylomicrobium alcaliphilum was detected.

Isolation and phylogenic identification of soil haloalkaliphilic strains in the former Texcoco Lake.

A wide diversity of organisms exists in soil. Well-adapted groups can be found in extreme environments. A great economic and metabolic potential for extremozymes produced by organisms living at extreme environments has been reported. Extreme characteristics such as high salt content and high pH level make the soil of the former Texcoco Lake a unique place which has not been exploited. Therefore, in this study, 66 strains from soil of the former Texcoco Lake were isolated and phylogenetically analyzed using universal oligonucleotide primers. Different genera such as Kocuria, Micrococcus, Nesterenkonia, Halomonas, Salinicoccus, Kurthia, Gracilibacillus, and Bacillus were found. However, only 22 from all isolated strains were identified at specie level.

The bacterial community structure in an alkaline saline soil spiked with anthracene

Background: The application of polycyclic aromatic hydrocarbons (PAHs) will affect the bacterial community structure as some groups will be favoured and others not. An alkaline saline soil with electrolytic conductivity (EC) 56 dS m-1 was spiked with anthracene and acetone while their effect on bacterial community structure was investigated. Results: The percentages of Acidobacteria and Actinobacteria decreased over time, while the percentage of Proteobacteria, mostly Xanthomonadales, increased. The percentage of the phylotypes belonging to the Nocardioides, Rhodococcus and Streptomyces, known degraders of PAHs, was larger in the anthracene-amended soil than in the acetone-amended and unamended soil at day 14. The phylotypes belonging to the genera Sphingomonas, also a known degrader of PAHs, however, was lower. Weighted and unweighted PCoA with UniFrac indicated that phylotypes were similar in the different treatments at day 0, but changed at day 1. After 14 days, phylotypes in the unamended and acetone-amended soil were similar, but different from those in the anthracene-spiked soil. Conclusions: It was found that incubating the soil and contaminating it with anthracene changed the bacterial community structure, but spiking the soil with acetone had little or no effect on the bacterial community structure compared to the unamended soil.

Texcoconibacillus texcoconensis gen. nov., sp. nov., alkalophilic and halotolerant bacteria isolated from soil of the former lake Texcoco (Mexico).

A novel Gram-positive, rod-shaped, spore-forming bacterium, designated 13CC(T) was isolated from soil of the former lake Texcoco. The strain was aerobic, catalase-positive and oxidase-negative. It grew at salinities of 0-26% (w/v) NaCl with an optimum at 9-16% (w/v) NaCl. The cells contain peptidoglycan type A1γ, A1γ' with glycine instead of l-alanine and three variations of peptidoglycan type A4γ. The only quinone detected was MK-7. The major fatty acid was anteiso-C(15:0). The polar lipids fraction consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and three different phospholipids. The DNA G+C content was 37.5 mol%. Maximum-likelihood phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 13CC(T) was closely related to members of the genus Bacillus and shared 92.35% similarity with Bacillus agaradhaerens, 92.28% with Bacillus neizhouensis and 92.21% with Bacillus locisalis. It is proposed based on the phenotypic, genotypic and phylogenetic analyses that the novel isolate should be classified as a representative of a new genus and novel species, for which the name Texcoconibacillus texcoconensis gen. nov., sp. nov. is proposed. The type strain of Texcoconibacillus texcoconensis is 13CC(T) ( =JCM 17654(T) =DSM 24696(T)).

The ability of soil bacteria to receive the conjugative IncP1 plasmid, pKJK10, is different in a mixed community compared to single strains.

Horizontal gene transfer by conjugation is common among bacterial populations in soil. It is well known that the host range of plasmids depends on several factors, including the identity of the plasmid host cell. In the present study, however, we demonstrate that the composition of the recipient community is also determining for the dissemination of a conjugative plasmid. We isolated 15 different bacterial strains from soil and assessed the conjugation frequencies of the IncP1 plasmid, pKJK10, by flow cytometry, from two different donors, Escherichia coli and Pseudomonas putida, to either 15 different bacterial strains or to the mixed community composed of all the 15 strains. We detected transfer of pKJK10 from P. putida to Stenotrophomonas rhizophila in a diparental mating, but no transfer was observed to the mixed community. In contrast, for E. coli, transfer was observed only to the mixed community, where Ochrobactrum rhizosphaerae was identified as the dominating plasmid recipient. Our results indicate that the presence of a bacterial community impacts the plasmid permissiveness by affecting the ability of strains to receive the conjugative plasmid.

Natronorubrum texcoconense sp. nov., a haloalkaliphilic archaeon isolated from soil of the former lake Texcoco (Mexico).

A new haloalkaliphilic archaeon, strain B4(T), was isolated from the former lake Texcoco in Mexico. The cells were Gram-negative, pleomorphic-shaped, pink to red pigmented and aerobic. Strain B4(T) required at least 2.5 M NaCl for growth, with optimum growth at 3.4 M NaCl. It was able to grow over a pH range of 7.5-10.0 and temperature of 25-50 °C, with optimal growth at pH 9 and 37 °C. Cells are lysed in hypotonic treatment with less than 1.3 M NaCl. The major polar lipids of strain B4(T) were phosphatidylglycerol and methyl-phosphatidylglycerophosphate. Phospholipids were detected, but not glycolipids. The nucleotide sequence of the 16S rRNA gene revealed that the strain B4(T) was phylogenetically related to members of the genus Natronorubrum. Sequence similarity with Natronorubrum tibetense was 96.28 %, with Natronorubrum sulfidifaciens 95.06 % and Natronorubrum sediminis 94.98 %. The G+C content of the DNA was 63.3 mol%. The name of Natronorubrum texcoconense sp. nov. is proposed. The type strain is B4(T) (=CECT 8067(T) = JCM 17497(T)).

Enhanced dissipation of polycyclic aromatic hydrocarbons in the rhizosphere of the Athel tamarisk (Tamarix aphylla L. Karst.) grown in saline-alkaline soils of the former lake Texcoco.

Remediation of polycyclic aromatic hydrocarbons (PAHs) contaminated alkaline saline soil with phreatophyte or "water loving plants" was investigated by spiking soil from the former lake Texcoco with 100 mg phenanthrene (Phen) kg(-1) soil, 120 mg anthracene (Ant)kg(-1) soil and 45 mg benzo(a)pyrene (BaP) kg(-1) soil and vegetating it with Athel tamarisk (Tamarix aphylla L Karst.). The growth of the Athel tamarisk was not affected by the PAHs. In soil cultivated with Athel tamarisk, the leaching of PAHs to the 32-34 cm layer decreased 2-fold compared to the uncultivated soil. The BaP concentration decreased to 39% of the initial concentration at a distance smaller than 3 cm from the roots and to 45% at a distance larger than 3cm, but 59% remained in unvegetated soil after 240 days. Dissipation of Ant and Phen decreased with depth, but not BaP. The biodegradation of PAHs was affected by their chemical properties and increased in the presence of T. aphylla, but decreased with depth.

Greenhouse gas emissions under conservation agriculture compared to traditional cultivation of maize in the central highlands of Mexico.

In 1991, the 'International Maize and Wheat Improvement Center' (CIMMYT) started a field experiment in the rain fed Mexican highlands to investigate conservation agriculture (CA) as a sustainable alternative for conventional maize production practices (CT). CT techniques, characterized by deep tillage, monoculture and crop residue removal, have deteriorated soil fertility and reduced yields. CA, which combines minimum tillage, crop rotations and residue retention, restores soil fertility and increases yields. Soil organic matter increases in CA compared to CT, but increases in greenhouse gas emissions (GHG) in CA might offset the gains obtained to mitigate global warming. Therefore, CO(2), CH(4) and N(2)O emissions, soil temperature, C and water content were monitored in CA and CT treatments in 2010-2011. The cumulative GHG emitted were similar for CA and CT in both years, but the C content in the 0-60 cm layer was higher in CA (117.7 Mg C ha(-1)) than in CT (69.7 Mg C ha(-1)). The net global warming potential (GWP) of CA (considering soil C sequestration, GHG emissions, fuel use, and fertilizer and seeds production) was -7729 kg CO(2) ha(-1) y(-1) in 2008-2009 and -7892 kg CO(2) ha(-1) y(-1) in 2010-2011, whereas that of CT was 1327 and 1156 kg CO(2) ha(-1) y(-1). It was found that the contribution of CA to GWP was small compared to that of CT.

Molecular phylogeny and paclitaxel screening of fungal endophytes from Taxus globosa.

We studied the endophytic mycoflora associated with Taxus globosa, the Mexican yew. The study localities; Las Avispas (LA), San Gaspar (SG), and La Mina (LM) were three segments of cloud forest within the range of Sierra Gorda Biosphere Reserve, México. Overall, 245 endophytes were isolated and 105 representative Ascomycota (morphotaxons) were chosen for phylogenetic and genotypic characterization. Maximum likelihood analyses of large subunit of ribosomal RNA (LSU) rDNA showed well-supported clades of Dothideomycetes, Eurotiomycetes, Leotiomycetes, Pezizomycetes, and Sordariomycetes. Analyses of ITS rDNA groups showed 57 genotypes (95% sequence similarity), in general consistent with the phylogenetically delimitated taxa based on LSU rDNA sequences. The endophyte diversity measured by Fisher's α, Shanonn, and Simpson indices was ca. three-fold and ca. two-fold greater in LM than in LA and SG respectively. A screening for paclitaxel using a competitive inhibition enzyme immunoassay showed 16 positive isolates producing between 65 and 250 ng l(-1). The isolates included Acremonium, Botryosphaeria, Fusarium, Gyromitra, Nigrospora, Penicillium, three novel Pleosporales, and Xylaria.