Microalgal-bacterial treatment of ice-cream wastewater to remove organic waste and harvest oil-rich biomass.

The diversity of microalgae and bacteria allows them to form beneficial consortia for efficient wastewater treatment and nutrient recovery. This study aimed to evaluate the feasibility of a new microalgal-bacterial combination in the treatment of ice cream wastewater for biomass harvest. The bacterium Novosphingobium sp. ICW1 was natively isolated from ice cream wastewater and the microalga Vischeria sp. WL1 was a terrestrial oil-producing strain of Eustigmatophyceae. The ice cream wastewater was diluted 4 folds for co-cultivation, which was relatively less inhibitory for the growth of Vischeria sp. WL1. Four initial algal-bacterial combinations (v:v) of 150:0 (single algal cultivation), 150:1, 150:2, and 150:4 were assessed. During 24 days of co-cultivation, algal pigmentation was dynamically changed, particularly at the algal-bacterial combination of 150:4. Algal growth (in terms of cell number) was slightly promoted during the late phase of co-cultivation at the combinations of 150:2 and 150:4, while in the former the cellular oil yield was obviously elevated. Treated by these algal-bacterial combinations, total carbon was reduced by 67.5 ~ 74.5% and chemical oxygen demand was reduced by 55.0 ~ 60.4%. Although single bacterial treatment was still effective for removing organic nutrients, the removal efficiency was obviously enhanced at the algal-bacterial combination of 150:4. In addition, the harvested oils contained 87.1 ~ 88.3% monounsaturated fatty acids. In general, this study enriches the biotechnological solutions for the sustainable treatment of organic matter-rich food wastewater.

Exploitation of inland salt lake water by dilution and nutrient enrichment to cultivate Vischeria sp. WL1 (Eustigmatophyceae) for biomass and oil production.

Salt lakes are significant components of global inland waters. Salt lake (SL) water can provide precious mineral resource for microbial growth. The prospect of utilizing diluted SL water for cultivation of a terrestrial oil-producing microalga Vischeria sp. WL1 was evaluated under laboratory conditions. Based on the detected mineral element composition, the water from Gouchi Salt Lake was diluted 2, 4, 6 and 8 folds and used with supplementation of additional nitrogen, phosphorus and iron (SL+ water). It was found that 4 folds diluted SL+ water was most favorable for biomass and oil production. When cultivated in this condition, Vischeria sp. WL1 gained a biomass yield of 0.82 g L-1 and an oil yield of 0.56 g L-1 after 24 days of cultivation, which is comparable to the optimum productivity we previously established. In addition, total monounsaturated fatty acid contents (64.4∼68.1 %) of the oils resulted from cultures in diluted SL+waters were higher than that in the control (55.5 %). It was also noteworthy that in all these cultures the oil contents (652.0∼681.0 mg g-1) accounted for the most of the biomass, which are far more than the protein and starch contents. This study demonstrates the feasibility of using SL water as a cost-effective mineral resource to cultivate microalgae for biomass and oil production.

Comparative genomic analysis of Halomonas campaniensis wild-type and ultraviolet radiation-mutated strains reveal genomic differences associated with increased ectoine production / El análisis genómico comparativo de cepas mutadas por radiación ultravioleta y de tipo salvaje de Halomonas campaniensis revela diferencias genómicas asociadas con una mayor producción de ectoína

Ectoine is a natural amino acid derivative and one of the most widely used compatible solutes produced by Halomonas species that affects both cellular growth and osmotic equilibrium. The positive effects of UV mutagenesis on both biomass and ectoine content production in ectoine-producing strains have yet to be reported. In this study, the wild-type H. campaniensis strain XH26 (CCTCCM2019776) was subjected to UV mutagenesis to increase ectoine production. Eight rounds of mutagenesis were used to generate mutated XH26 strains with different UV-irradiation exposure times. Ectoine extract concentrations were then evaluated among all strains using high-performance liquid chromatography analysis, alongside whole genome sequencing with the PacBio RS II platform and comparison of the wild-type strain XH26 and the mutant strain G8-52 genomes. The mutant strain G8-52 (CCTCCM2019777) exhibited the highest cell growth rate and ectoine yields among mutated strains in comparison with strain XH26. Further, ectoine levels in the aforementioned strain significantly increased to 1.51 ± 0.01 g L−1 (0.65 g g−1 of cell dry weight), representing a twofold increase compared to wild-type cells (0.51 ± 0.01 g L−1) when grown in culture medium for ectoine accumulation. Concomitantly, electron microscopy revealed that mutated strain G8-52 cells were obviously shorter than wild-type strain XH26 cells. Moreover, strain G8-52 produced a relatively stable ectoine yield (1.50 g L−1) after 40 days of continuous subculture. Comparative genomics analysis suggested that strain XH26 harbored 24 mutations, including 10 nucleotide insertions, 10 nucleotide deletions, and unique single nucleotide polymorphisms. Notably, the genes orf00723 and orf02403 (lipA) of the wild-type strain mutated to davT and gabD in strain G8-52 that encoded for 4-aminobutyrate-2-oxoglutarate transaminase and NAD-dependent succinate-semialdehyde dehydrogenase, respectively. Consequently, these genes may be involved in increased ectoine yields. These results suggest that continuous multiple rounds of UV mutation represent a successful strategy for increasing ectoine production, and that the mutant strain G8-52 is suitable for large-scale fermentation applications.(AU)

Metabolic engineering of Halomonas campaniensis strain XH26 to remove competing pathways to enhance ectoine production.

Ectoine has gained considerable attention as a high-value chemical with significant application potential and market demand. This study aimed to increase ectoine yields by blocking the metabolic shunt pathway of L-aspartate-4-semialdehyde, the precursor substrate in ectoine synthesis. The homoserine dehydrogenase encoded by hom in H. campaniensis strain XH26 is responsible for the metabolic shunt of L-aspartate-4-semialdehyde to glycine. CRISPR/Cas9 technology was used to seamlessly knockout hom, blocking the metabolic shunt pathway to increase ectoine yields. The ectoine yield of XH26/Δhom was 351.13 mg (g CDW)-1 after 48 h of incubation in 500 mL shake flasks using optimal medium with 1.5 mol L-1 NaCl, which was significantly higher than the 239.18 mg (g CDW)-1 of the wild-type strain. Additionally, the absence of the ectoine metabolic shunt pathway affects betaine synthesis, and thus the betaine yields of XH26/Δhom was 19.98 mg (g CDW)-1, considerably lower than the 69.58 mg (g CDW)-1 of the wild-type strain. Batch fermentation parameters were optimized, and the wild-type strain and XH26/Δhom were fermented in 3 L fermenters, resulting in a high ectoine yield of 587.09 mg (g CDW)-1 for the defective strain, which was significantly greater than the ectoine yield of 385.03 mg (g CDW)-1 of the wild-type strain. This study showed that blocking the metabolic shunt of synthetic substrates effectively increases ectoine production, and a reduction in the competitively compatible solute betaine appears to promote increased ectoine synthesis.

Comparative genomic analysis of Halomonas campaniensis wild-type and ultraviolet radiation-mutated strains reveal genomic differences associated with increased ectoine production.

Ectoine is a natural amino acid derivative and one of the most widely used compatible solutes produced by Halomonas species that affects both cellular growth and osmotic equilibrium. The positive effects of UV mutagenesis on both biomass and ectoine content production in ectoine-producing strains have yet to be reported. In this study, the wild-type H. campaniensis strain XH26 (CCTCCM2019776) was subjected to UV mutagenesis to increase ectoine production. Eight rounds of mutagenesis were used to generate mutated XH26 strains with different UV-irradiation exposure times. Ectoine extract concentrations were then evaluated among all strains using high-performance liquid chromatography analysis, alongside whole genome sequencing with the PacBio RS II platform and comparison of the wild-type strain XH26 and the mutant strain G8-52 genomes. The mutant strain G8-52 (CCTCCM2019777) exhibited the highest cell growth rate and ectoine yields among mutated strains in comparison with strain XH26. Further, ectoine levels in the aforementioned strain significantly increased to 1.51 ± 0.01 g L-1 (0.65 g g-1 of cell dry weight), representing a twofold increase compared to wild-type cells (0.51 ± 0.01 g L-1) when grown in culture medium for ectoine accumulation. Concomitantly, electron microscopy revealed that mutated strain G8-52 cells were obviously shorter than wild-type strain XH26 cells. Moreover, strain G8-52 produced a relatively stable ectoine yield (1.50 g L-1) after 40 days of continuous subculture. Comparative genomics analysis suggested that strain XH26 harbored 24 mutations, including 10 nucleotide insertions, 10 nucleotide deletions, and unique single nucleotide polymorphisms. Notably, the genes orf00723 and orf02403 (lipA) of the wild-type strain mutated to davT and gabD in strain G8-52 that encoded for 4-aminobutyrate-2-oxoglutarate transaminase and NAD-dependent succinate-semialdehyde dehydrogenase, respectively. Consequently, these genes may be involved in increased ectoine yields. These results suggest that continuous multiple rounds of UV mutation represent a successful strategy for increasing ectoine production, and that the mutant strain G8-52 is suitable for large-scale fermentation applications.

[Advances in the biomedical application research of halophilic microorganisms].

In recent years, antibiotic resistance has become increasingly serious, and the number of cancer patients keeps increasing. There is an urgent need to develop new drugs with antibacterial and antitumor effects. Halophilic microorganisms are a special group of microorganisms living in extreme environment. They have the characteristics of metabolic diversity, low nutritional requirements and adaptability to harsh conditions, thus can serve as promising candidates for new drug discovery. To date, researchers have isolated a variety of metabolites and enzymes with antibacterial and/or antitumor activities from halophilic microorganisms. This review summarized the functions and potential biomedical applications of halophilic microorganisms and their related products, such as antibacterial, anti-inflammatory, antitumor, antioxidant, biomedical materials and drug carriers. In particular, novel antibacterial and antitumor substances recently discovered in halophilic microorganisms, as well as the biomedical applications of ectoine, a unique metabolite found in halophilic microorganisms, were introduced. Finally, future development and utilization of halophilic microorganisms in biomedical and industrial fields were prospected.

Study of osmoadaptation mechanisms of halophilic Halomonas alkaliphila XH26 under salt stress by transcriptome and ectoine analysis.

Halophilic bacteria such as the genus Halomonas are promising candidates in diverse industrial, agricultural and biomedical applications. Here, we successfully isolated a halophilic Halomonas alkaliphila strain XH26 from Xiaochaidan Salt Lake, and studied its osmoadaptation strategies using transcriptome and ectoine analysis. Divergent mechanisms were involved in osmoadaptation at different salinities in H. alkaliphila XH26. At moderate salinity (6% NaCl), increased transcriptions of ABC transporters related to iron (III), phosphate, phosphonate, monosaccharide and oligosaccharide import were observed. At high salinity (15% NaCl), transcriptions of flagellum assembly and cell motility were significantly inhibited. The transcriptional levels of ABC transporter genes related to iron (III) and iron3+-hydroxamate import, glycine betaine and putrescine uptake, and cytochrome biogenesis and assembly were significantly up-regulated. Ectoine synthesis and accumulation was significantly increased under salt stress, and the increased transcriptional expressions of ectoine synthesis genes ectB and ectC may play a key role in high salinity induced osmoadaptation. At extreme high salinity (18% NaCl), 5-hydroxyectoine and ectoine worked together to maintain cell survival. Together these results give valuable insights into the osmoadaptation mechanisms of H. alkaliphila XH26, and provide useful information for further engineering this specific strain for increased ectoine synthesis and related applications.

Whole genome sequencing of the halophilic Halomonas qaidamensis XH36, a novel species strain with high ectoine production.

Here, we report the whole genome of a novel halophilic Halomonas species strain XH36 with high ectoine production potential. The genome was 3,818,310 bp in size with a GC content of 51.97%, and contained 3533 genes, 61 tRNAs and 18 rRNAs. The phylogenetic analysis using the 16s rRNA genes, the UBCGs and the TYGS database indicated that XH36 belongs to a novel Halomonas species, which we named as Halomonas qaidamensis. Osmoadaptation related genes including Na(+) and K(+) transport and compatible solute accumulation were both present in the XH36 genome, the latter of which mainly contained ectoine, 5-hydroxyectoine and betaine. HPLC validation studies showed that H. qaidamensis XH36 accumulated ectoine to cope with salt stress, and the content of ectoine could be as high as 315 mg/g CDW under 3 mol/l NaCl. Our results show that XH36 is a new promising industrial strain for ectoine production, and the genomic analysis will guide us to better understand its salt-induced osmoadaptation mechanisms, and provide theoretical references for future application research of ectoine.

[Advances in the microbial production of the compatible solute ectoine: a review].

Ectoine is an amino acid derivative and an important natural product in halophilic microorganisms. It plays an important role in protecting cells and stabilizing biological macromolecules, and can be widely used in biomedical fields such as drug preparation adjuvants, organ transplantation and preservation, skin wound repair and cosmetics. Due to the medical value and commercial market demand of ectoine, this article summarized the recent advances in the microbial production of ectoine, including the mutation and breeding of hyper-producing strains, construction of genetically and metabolically engineered strains, optimization of fermentation processes, and extraction and purification processes. The application of multi-omics technologies and computational biology to develop an ectoine producing cell factory was prospected, with the aim to provide a reference for ectoine overproduction.

Distinctive distributions of halophilic Archaea across hypersaline environments within the Qaidam Basin of China.

Halophilic Archaea are widely distributed globally in hypersaline environments. However, little is known of how dominant halophilic archaeal genera are distributed across environments and how they may co-associate across ecosystems. Here, the archaeal community composition and diversity from hypersaline environments (> 300 g/L salinity; total of 33 samples) in the Qaidam Basin of China were investigated using high-throughput Illumina sequencing of 16S rRNA genes. The archaeal communities (total of 3,419 OTUs) were dominated by the class Halobacteria (31.7-99.6% relative abundances) within the phylum Euryarchaeota (90.8-99.9%). Five predominant taxa, including Halorubrum, Halobacterium, Halopenitus, Methanothrix, and Halomicrobium, were observed across most samples. However, several distinct genera were associated with individual samples and were inconsistently distributed across samples, which contrast with previous studies of hypersaline archaeal communities. Additionally, co-occurrence network analysis indicated that five network clusters were present and potentially reflective of interspecies interactions among the environments, including three clusters (clusters II, III, and IV) comprising halophilic archaeal taxa within the Halobacteriaceae and Haloferacaceae families. In addition, two other clusters (clusters I and V) were identified that comprised methanogens. Finally, salinity comprising ionic concentrations (in the order of Na+ > Ca2+ > Mg2+) and pH were most correlated with taxonomic distributions across sample sites.

Genetic variants in GHR and PLCE1 genes are associated with susceptibility to esophageal cancer.

BACKGROUND: Esophageal cancer (EC) is the leading cause of cancer-related mortality worldwide. The underlying genetic risk factors remain unclear. The association between gene growth hormone receptor (GHR) and phospholipase C epsilon 1 (PLCE1) polymorphisms and the EC risk were identified in this study. METHODS: A total of 506 EC cases and 507 controls were included in this research. Two SNPs (rs6898743 of GHR and rs2274223 of PLCE1) were selected and genotyped. The associations between gene polymorphisms and the EC risk were assessed by logistic regression analysis. The databases RegulomeDB, GTEx, and UALCAN were used for functional annotations. RESULTS: In the allelic frequencies analysis, the rs6898743 of GHR was associated with decreased susceptibility of EC (OR = 0.83, 95% CI: 0.70-1.00, p = 0.049), while rs2274223 of PLCE1 was associated with increased 0.25-fold EC risk (OR = 1.25, 95% CI: 1.02-1.53, p = 0.037). The "GC" genotype of rs6898743 was associated with a 0.24-fold decreased risk of EC under co-dominant model (OR = 0.76, 95% CI: 0.58-0.99, p = 0.046), and the "GA" genotype of rs2274223 was associated with increased EC risk under co-dominant model (OR = 1.36, 95% CI: 1.04-1.77, p = 0.023). Using GTEx database, rs2274223 was found to be significant associated with increased PLCE1 expression (p = 4.1 × 10-7 ) in esophagus muscularis. The UALCAN database demonstrated that the GHR gene was under-expressed in esophageal cancer tissues (p = 0.017). CONCLUSION: The gene GHR and PLCE1 polymorphisms are associated with EC in the general population and the results need to be verified in future.

Bacterial and Archaeal Communities within an Ultraoligotrophic, High-altitude Lake in the Pre-Himalayas of the Qinghai-Tibet Plateau.

Puma Yumco Lake (PYL) is an ultraoligotrophic freshwater lake that sits an altitude of 5030 m within the Qinghai-Tibet Plateau of China. The bacterial and archaeal diversity of the lake remains poorly understood, despite their potential to inform on biogeochemical cycling and environment-microbial associations in these unique environments. Here, the bacterial and archaeal communities of PYL were investigated using high-throughput sequencing analysis of community 16S rRNA gene sequences. Further, the relationships among dominant taxa and environmental factors were comprehensively evaluated. Bacterial diversity comprised 31 phyla and 371 genera (10,645 operational taxonomic units [OTUs], Shannon index values of 5.21-6.16) and was significantly higher than that of Archaea (five phyla and 24 genera comprising 1141 OTUs and Shannon index values of 1.18-3.28). The bacterial communities were dominated by Proteobacteria (48.42-59.97% relative abundances), followed by Bacteroidetes (12.5-32.51%), Acidobacteria (2.07-11.56%), Firmicutes (0.65-6.32%), Planctomycetes (0.99-3.56%), Gemmatimonadetes (0.38-3.57%), Actinobacteria (1.67-3.52%), Verrucomicrobia (0.87-2.01%), and Chloroflexi (0.5-1.17%). In addition, archaeal communities were dominated by Thaumarchaeota (33.22-93.00%), followed by Euryarchaeota (2.89-35.47%), Woesearchaeota (0.99-31.04%), and Pacearchaeota (0.01-1.14%). The most abundant bacterial genus was Rhodoferax (5.73-26.62%) and the most abundant archaeal genus was the ammonia-oxidizing Nitrososphaera (29.18-91.46%). These results suggest that the Rhodoferax and Nitrososphaera are likely to participate in biogeochemical cycles in these environments through photoheterotrophy and nitrification, respectively. Taken together, these results provide valuable data for better understanding microbial interactions with each other and with these unique environments.

An evaluation of the core bacterial communities associated with hypersaline environments in the Qaidam Basin, China.

Hypersaline lakes and saltern areas are important industrial and biodiversity resources in the Qaidam Basin of China that reside at > 2600 m asl. Most hypersaline environments in this area are characterized by saturated salinity (~ 300 g/L salinity), nearly neutral pH, intense ultraviolet radiation, and extremely variable temperature fluctuations. The core bacterial communities associated with these stressful environments have nevertheless remained uninvestigated. 16S rRNA gene Illumina sequencing analyses revealed that the bacterial communities were dominated by core lineages including the Proteobacteria (39.4-64.6%) and the Firmicutes (17.0-42.7%). However, the relative abundances of common lineages, and especially the five most abundant taxa of Pseudomonas, Lactococcus, Anoxybacillus, Acinetobacter, and Brevundimonas, were highly variable across communities and closely associated with hypersaline characteristics in the samples. Network analysis revealed the presence of co-occurrence high relative abundance taxa (cluster I) that were highly correlated across all hypersaline samples. Additionally, temperature, total organic carbon, K+, and Mg2+ correlated highest with taxonomic distributions across communities. These results highlight the potential mechanisms that could underlie survival and adaptation to these extreme hypersaline ecosystems.

The effect of temperature fluctuation on the microbial diversity and community structure of rural household biogas digesters at Qinghai Plateau.

Seasonal temperature-fluctuation has been regarded as a key environmental factor affecting rural biogas fermentation yields. The present study investigated the impact of seasonal temperature-fluctuation on operating-temperatures and biogas production in rural household digesters at Qinghai Plateau and revealed the related changes in microbial diversity and community structure by 16S rRNA gene high-throughput sequencing (HTS) analysis. Our results showed closely positive correlation between operating-temperatures and biogas production. HTS analysis indicated the highest diversity for bacteria community in autumn (at highest operating-temperatures) and late winter (at lowest operating-temperatures) and for archaea community only in autumn. HTS analysis classified bacteria into 21 phyla and 346 genera with the most predominant phyla Firmicutes, Bacteroidetes and Proteobacteria (> 72.4% in total) and the most predominant genera Proteiniphilum, Clostridium sensustricto 1, Petrimonas, Pseudomonas and Fastidiosipila (37.09-38.61% in total). HTS analysis also revealed two main archaea orders (Methanomicrobiales and Methanobacteriales) and one predominant genus Methanogenium to support plateau biogas fermentation. Especially, a remarkable impact of temperature on the community abundances of bacteria phyla Synergistetes and archaea genera Methanogenium and Thermogymnomonas was observed, and such microbial community structure changes were positively consistent with the biogas production. The present work provided the first set of evidences to link temperature-controlled modulation of microbial community structure with rural household biogas production at Qinghai Plateau.

Evaluating Nanoparticles in Preclinical Research Using Microfluidic Systems.

Nanoparticles (NPs) have found a wide range of applications in clinical therapeutic and diagnostic fields. However, currently most NPs are still in the preclinical evaluation phase with few approved for clinical use. Microfluidic systems can simulate dynamic fluid flows, chemical gradients, partitioning of multi-organs as well as local microenvironment controls, offering an efficient and cost-effective opportunity to fast screen NPs in physiologically relevant conditions. Here, in this review, we are focusing on summarizing key microfluidic platforms promising to mimic in vivo situations and test the performance of fabricated nanoparticles. Firstly, we summarize the key evaluation parameters of NPs which can affect their delivery efficacy, followed by highlighting the importance of microfluidic-based NP evaluation. Next, we will summarize main microfluidic systems effective in evaluating NP haemocompatibility, transport, uptake and toxicity, targeted accumulation and general efficacy respectively, and discuss the future directions for NP evaluation in microfluidic systems. The combination of nanoparticles and microfluidic technologies could greatly facilitate the development of drug delivery strategies and provide novel treatments and diagnostic techniques for clinically challenging diseases.

PCR-DGGE Analysis on Microbial Community Structure of Rural Household Biogas Digesters in Qinghai Plateau.

To investigate contribution of environmental factor(s) to microbial community structure(s) involved in rural household biogas fermentation at Qinghai Plateau, we collected slurry samples from 15 digesters, with low-temperature working conditions (11.1-15.7 °C) and evenly distributed at three counties (Datong, Huangyuan, and Ledu) with cold plateau climate, to perform polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and further sequencing. The bacterial communities in the total 15 digesters were classified into 38 genera with Mangroviflexus (12.1%) as the first dominant, and the archaeal communities into ten genera with Methanogenium (38.5%) as the most dominant. For each county, the digesters with higher biogas production, designated as HP digesters, exclusively had 1.6-3.1 °C higher fermentation temperature and the unique bacterial structure composition related, i.e., unclassified Clostridiales for all the HP digesters and unclassified Marinilabiliaceae and Proteiniclasticum for Ledu HP digesters. Regarding archaeal structure composition, Methanogenium exhibited significantly higher abundances at all the HP digesters and Thermogymnomonas was the unique species only identified at Ledu HP digesters with higher-temperature conditions. Redundancy analysis also confirmed the most important contribution of temperature to the microbial community structures investigated. This report emphasized the correlation between temperature and specific microbial community structure(s) that would benefit biogas production of rural household digesters at Qinghai Plateau.

Microbial community structure and diversity within hypersaline Keke Salt Lake environments.

Keke Salt Lake is located in the Qaidamu Basin of China. It is a unique magnesium sulfate-subtype hypersaline lake that exhibits a halite domain ecosystem, yet its microbial diversity has remained unstudied. Here, the microbial community structure and diversity was investigated via high-throughput sequencing of the V3-V5 regions of 16S rRNA genes. A high diversity of operational taxonomic units was detected for Bacteria and Archaea (734 and 747, respectively), comprising 21 phyla, 43 classes, and 201 genera of Bacteria and 4 phyla, 4 classes, and 39 genera of Archaea. Salt-saturated samples were dominated by the bacterial genera Bacillus (51.52%-58.35% relative abundance), Lactococcus (9.52%-10.51%), and Oceanobacillus (8.82%-9.88%) within the Firmicutes phylum (74.81%-80.99%), contrasting with other hypersaline lakes. The dominant Archaea belonged to the Halobacteriaceae family, and in particular, the genera (with an abundance of >10% of communities) Halonotius, Halorubellus, Halapricum, Halorubrum, and Natronomonas. Additionally, we report the presence of Nanohaloarchaeota and Woesearchaeota in Qinghai-Tibet Plateau lakes, which has not been previously documented. Total salinity (especially Mg2+, Cl-, Na+, and K+) mostly correlated with taxonomic distribution across samples. These results expand our understanding of microbial resource utilization within hypersaline lakes and the potential adaptations of dominant microorganisms that allow them to inhabit such environments.

Identification and characterization of ectoine biosynthesis genes and heterologous expression of the ectABC gene cluster from Halomonas sp. QHL1, a moderately halophilic bacterium isolated from Qinghai Lake.

The moderately halophilic bacterium Halomonas sp. QHL1 was identified as a member of the genus Halomonas by 16S rRNA gene sequencing. HPLC analysis showed that strain QHL1 synthesizes ectoine in its cytoplasm. The genes involved in the ectoine biosynthesis pathway were identified on the chromosome in the order ectABC. Subsequently, the ectB gene from this strain was amplified by PCR, and the entire ectABC gene cluster (3,580 bp) was cloned using genome walking. Analysis showed that the ectA (579 bp), ectB (1269 bp), and ectC (390 bp) genes were organized in a single transcriptional unit and were predicted to encode three peptides of 21.2 kDa, 46.4 kDa, and 14.7 kDa, respectively. Two putative promoters, a δ(70)-dependent promoter and a δ(38)-controlled promoter, as well as several conserved motifs with unknown function were identified. Individual ectA, ectB, and ectC genes, and the entire ectABC gene cluster were inserted into the expression plasmid pET-28a(+) to generate the recombinant plasmids pET-28a(+)-ectA, pET-28a(+)-ectB, pET-28a(+)-ectC and pET-28a(+)-ectABC, respectively. Heterologous expression of these proteins in Escherichia coli BL21 (DE3) was confirmed by SDS-PAGE. The recombinant E. coli strain BL21 (pET-28a (+)-ectABC) displayed a higher salt tolerance than native E. coli cells but produced far less ectoine than the wild-type QHL1 strain.