Alteromonas salexigens sp. nov., isolated from coastal seawater.

A Gram-negative, aerobic, short rod-shaped bacterium, designated ASW11-19T, was isolated from a coastal seawater sample of the Yellow Sea, PR China. Strain ASW11-19T grew optimally at 37 °C, 3.0-5.0% (w/v) NaCl and pH 7.5. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain ASW11-19T belonged to the genus Alteromonas and most closely related to Alteromonas profundi 345S023T and Alteromonas fortis 1T (98.4%, both). The draft genome was 3.55 Mb with 3150 protein-coding genes, 18 contigs, and a DNA G+C content was 44.4%. The digital DNA-DNA hybridization and average nucleotide identity values were below the species-delineating thresholds. The major fatty acids were summed feature 3 (C16:1ω7c/C16:1ω6c), summed feature 8 (C18:1ω7c/C18:1ω6c), and C16:0. The sole respiratory quinone was ubiquinone 8. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phospholipid, and two unidentified lipids. Based on these genomic data, phenotypic and chemotaxonomic properties, strain ASW11-19T is considered to represent a novel species of the genus Alteromonas. The name Alteromonas salexigens sp.nov. is proposed for ASW11-19T (=MCCC 1K07239T=KCTC 92247T).

Alteromonas Aquimaris sp. nov., Isolated from Surface Seawater.

A Gram-negative, aerobic, motile by flagellum, and rod-shaped bacterium, designated ASW11-7T, was isolated from coastal surface seawater sample collected from the Yellow Sea, PR China. Strain ASW11-7T grew optimally at 37℃, 4.0% (w/v) NaCl and pH 7.0. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain ASW11-7T belongs to the genus Alteromonas and most closely related to Alteromonas ponticola MYP5T (99.6% similarity), followed by Alteromonas confluentis DSSK2-12T (98.2%), Alteromonas lipolytica JW12T (98.2%), and Alteromonas hispanica F-32T (98.0%). The draft genome of strain ASW11-7T had a length of 3,530,922 bp with a G + C content of 44.9%, predicting 3108 coding sequences, 5 rRNA, 4 ncRNAs, 49 tRNAs genes, and 18 pseudogenes. The average nucleotide identity and digital DNA-DNA hybridization values between genomic sequences of strain ASW11-7T and closely related species of Alteromonas were in ranges of 66.9-77.8% and 18.3-27.5%, respectively. The major fatty acids of strain ASW11-7T were C16:0, summed feature 3 (C16:1ω7c/C16:1ω6c), and summed feature 8 (C18:1ω7c/C18:1ω6c). The predominant respiratory quinone was Q-8 and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. Based on the phenotypic properties, genotypic distinctiveness, and chemotaxonomic features, strain ASW11-7T is considered to represent a novel Alteromonas species, for which the name Alteromonas aquimaris sp. nov. is proposed. The type strain is ASW11-7T (= KCTC 92853T = MCCC 1K07240T).

Effects of guanidinoacetic acid supplementation on lactation performance, nutrient digestion and rumen fermentation in Holstein dairy cows.

BACKGROUND: Considering the high energy demand of lactation and the potential of guanidinoacetic acid (GAA) addition on the increase in creatine supply for cows, the present study investigated the effects of 0, 0.3, 0.6 and 0.9 g kg-1 dry matter (DM) of GAA supplementation on lactation performance, nutrient digestion and ruminal fermentation in dairy cows. The study used 40 mid-lactation multiparous Holstein cows and the study duration was 100 days. RESULTS: DM intake was not affected, but milk and milk component yields and feed efficiency increased linearly with increasing GAA addition. The total-tract digestibility of DM, organic matter, neutral detergent fibre, acid detergent fibre and non-fibre carbohydrates increased linearly and that of crude protein increased quadratically with increasing GAA addition. When the addition level of GAA increased, ruminal pH, molar percentages of butyrate, isobutyrate and isovalerate and the acetate-to-propionate ratio decreased linearly, and the total volatile fatty acids concentration and propionate molar percentage also increased linearly, whereas the acetate molar percentage and ammonia-N concentration were unaltered. The activities of fibrolytic enzymes, α-amylase and protease increased linearly. The populations of total bacteria, fungi, Ruminococcus albus, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminobacter amylophilus and Prevotella ruminicola increased linearly, whereas protozoa and methanogens decreased linearly with increasing GAA addition. As for the blood metabolites, concentrations of glucose, urea nitrogen and methionine were unchanged, total protein, albumin, creatine and homocysteine increased linearly, and folate decreased linearly with increasing GAA supply. CONCLUSION: The results of the present study indicate that supplementation of GAA improved milk performance and rumen fermentation in lactating dairy cows. © 2022 Society of Chemical Industry.

Gramella sediminis sp. nov., isolated from a tidal flat of the Yellow Sea.

A novel species of the genus Gramella, designated ASW11-100T, was isolated from a tidal flat sediment in the Yellow Sea, PR China. Phylogenetic analysis based on 16S rRNA gene sequences and single-copy orthologous clusters revealed that strain ASW11-100T belonged to the genus Gramella, and exhibited 16S rRNA gene sequence similarities of 98.9, 98.8 and 98.7 % to Gramella sabulilitoris HSMS-1T, Gramella sediminilitoris GHTF-27T and Gramella forsetii KT0803T, respectively. The genome of strain ASW11-100T harbours 2950 protein-coding genes and 105 carbohydrate-active enzymes including 38 glycoside hydrolases. Seventeen of the glycoside hydrolases are organized in five distinct polysaccharide utilization loci, which are predicted to involve in the degradation of starch, glucans, arabinoxylans, arabinomannan, arabinans and arabinogalactans. The genomic DNA G+C content was 37.3 mol%. The digital DNA-DNA hybridization and average nucleotide identity values between strain ASW11-100T and its closely related relatives were in ranges of 19.8-23.9% and 76.6-80.9 %, respectively. Cells of the isolate were Gram-negative, aerobic, non-flagellated and short rod-shaped. Carotenoid pigments were produced, but flexirubin-type pigments were absent. The major fatty acids (>10 %) were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c). The sole respiratory quinone was menaquinone-6 and the major polar lipid was phosphatidylethanolamine. Based on the above polyphasic evidence, strain ASW11-100T should be considered to represent a novel Gramella species, for which the name Gramella sediminis sp. nov. is proposed. The type strain is ASW11-100T (=KCTC 82502T=MCCC 1K05580T).

Functional analysis of the transcriptional activator XlnR of Penicillium oxalicum.

AIMS: The aim of this article is to study the functional features of Penicillium oxalicum transcriptional activator XlnR. METHODS AND RESULTS: The yeast reporter system was used to identify transcriptional activation domain of XlnR in P. oxalicum. The expression cassette was introduced into the xlnR locus of P. oxalicum by homologous recombination. In this study, several putative structural domains in P. oxalicum XlnR were predicted by bioinformatics analysis, and the transcriptional activation domain (351-694 region) was identified in XlnR relying on reporter gene system in yeast. In addition, the amino acid at XlnR 871 site (alanine) located in the regulatory region could influence the regulatory activity of XlnR directly. When the alanine at XlnR 871 site was replaced by stronger hydrophobic amino acid (e.g. valine or isoleucine), the regulatory activity will be greatly improved, especially for the regulation of hemicellulase genes expression. When alanine at XlnR 871 site was mutated to a hydrophilic amino acid (e.g. aspartic acid or arginine), the regulatory activity of XlnR will be reduced. CONCLUSIONS: The 351-694 region of P. oxalicum XlnR was identified as transcriptional activation domain, and the regulatory activity of XlnR was greatly influenced by hydrophobicity of amino acid at 871 site of XlnR in P. oxalicum. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will provide an effective target site to regulate the activity of XlnR and improve cellulase production of P. oxalicum.

Salinimicrobium sediminilitoris sp. nov., Isolated from a Tidal Flat.

A Gram-negative, facultatively anaerobic, motile, and rod-shaped bacterium, designated ASW11-47 T, was isolated from a tidal flat sediment taken from the coast of Qingdao, PR China. Phylogenetic analysis of 16S rRNA gene sequence showed that strain ASW11-47 T belongs to the genus Salinimicrobium and is most closely related to Salinimicrobium terrae YIM-C338T (98.68% similarity). The length of draft genome is 3,594,457 bp, and DNA G + C content is 40.8 mol%. The values of average nucleotide identity and digital DNA-DNA hybridization between strain ASW11-47 T and closely related strains were in ranges of 75.9-85.9 and 19.7-31.5%, respectively. The major fatty acids (> 10%) were iso-C15:0 and iso-C17:0 3-OH. The predominant respiratory quinone was menaquinone-6 and the major polar lipid was phosphatidylethanolamine. On the basis of genotypic, phenotypic, and chemotaxonomic analysis, strain ASW11-47 T represents a novel species within the genus Salinimicrobium, for which the name Salinimicrobium sediminilitoris sp. nov. is proposed. The type strain is ASW11-47 T (= KCTC 82501 T = MCCC 1K05586T).

Comparison of the microbial communities of alpacas and sheep fed diets with three different ratios of corn stalk to concentrate.

The objective of this study was to investigate the characteristics of ruminal microbial communities of alpacas (Lama pacos) and sheep (Ovis aries) fed three diets with varying ratios of roughage (corn stalk) to concentrate, 3:7 (LS), 5:5 (MS) and 7:3 (HS). Six alpacas (one-year-old and weighing 29.5 ± 7.1 kg) and six sheep (one-year-old and weighing 27.9 ± 2.7 kg) were used in this study, in a replicated 3 × 3 Latin square experiment. Total protozoa concentration was determined under the microscope; total fungi and methanogens were assessed using quantitative polymerase chain reaction and expressed as a percentage of total bacterial 16S rRNA gene copies; bacterial communities were investigated by targeted 16S rRNA gene (V3-V4 region) sequencing. The percentage of fungi was significantly higher in alpacas than in sheep under the LS diet, while the concentration of protozoa was significantly lower in alpacas under HS, MS and LS diets. The alpha diversity including Shannon, Chao l and ACE indices of bacterial communities was higher in alpacas than in sheep, under the LS diet. A total of 299 genera belonging to 22 phyla were observed in the forestomach of alpaca and sheep, with Bacteroidetes and Firmicutes dominating both animal species. Phyla Armatimonadetes and Fusobacteria, as well as 64 genera, were detected only in alpacas, whereas phyla Acidobacteria and Nitrospira, as well as 44 genera, were found only in sheep. The abundance of cellulolytic bacteria, including Butyrivibrio and Pseudobutyrivibrio, was higher in alpacas than in sheep under all three diets. These differences in the forestomach microbial communities partly explained why alpacas displayed a higher poor-quality roughage digestibility, and a lower methane production. Results also revealed that the adverse effects of high-concentrate diets (70%) were lesser in alpacas than in sheep.

Deletion of the middle region of the transcription factor ClrB in Penicillium oxalicum enables cellulase production in the presence of glucose.

Enzymes that degrade lignocellulose to simple sugars are of great interest in research and for biotechnology because of their role in converting plant biomass to fuels and chemicals. The synthesis of cellulolytic enzymes in filamentous fungi is tightly regulated at the transcriptional level, with the transcriptional activator ClrB/CLR-2 playing a critical role in many species. In Penicillium oxalicum, clrB overexpression could not relieve the dependence of cellulase expression on cellulose as an inducer, suggesting that clrB is controlled post-transcriptionally. In this study, using a reporter gene system in yeast, we identified the C-terminal region of ClrB/CLR-2 as a transcriptional activation domain. Expression of clrBID , encoding a ClrB derivative in which the DNA-binding and transcriptional activation domains are fused together to remove the middle region, led to cellulase production in the absence of cellulose in P. oxalicum Strikingly, the clrBID -expressing strain produced cellulase on carbon sources that normally repress cellulase expression, including glucose and glycerol. Results from deletion of the carbon catabolite repressor gene creA in the clrBID -expressing strain suggested that the effect of clrBID is independent of CreA's repressive function. A similar modification of clrB in Aspergillus niger resulted in the production of a mannanase in glucose medium. Taken together, these results indicate that ClrB suppression under noninducing conditions involves its middle region, suggesting a potential strategy to engineer fungal strains for improved cellulase production on commonly used carbon sources.

Introduction of heterologous transcription factors and their target genes into Penicillium oxalicum leads to increased lignocellulolytic enzyme production.

Genetic engineering of transcription factors is an efficient strategy to improve lignocellulolytic enzyme production in fungi. In this study, the xylanase transcriptional regulators of Trichoderma reesei (Xyr1) and Neurospora crassa (XLR-1), as well as their constitutively active mutants (Xyr1A824V and XLR-1A828V), were heterologously expressed in Penicillium oxalicum. The two heterologous regulators were identified to be able to activate lignocellulolytic enzyme gene expression in P. oxalicum. Particularly, expression of T. reesei Xyr1 resulted in a higher cellulase production level compared with the expression of native xylanase transcriptional regulator XlnR using the same promoter. Xyr1A824V and XLR-1A828V were found to be able to confer P. oxalicum more enhanced lignocellulolytic abilities than wild-type regulators Xyr1 and XLR-1. Furthermore, introduction of regulatory modules containing Xyr1A824V/XLR-1A828V and their target cellulase genes resulted in greater increases in cellulase production than alone expression of transcriptional regulators. Through the cumulative introduction of three regulatory modules containing regulator mutants and their corresponding target cellulase genes from P. oxalicum, T. reesei, and N. crassa, a 2.8-fold increase in cellulase production was achieved in P. oxalicum.

Identification and characterization of a long-chain fatty acid transporter in the sophorolipid-producing strain Starmerella bombicola.

The sophorolipid-producing strain Starmerella bombicola CGMCC 1576 has a remarkable ability to produce sophorolipids (SLs) under the acidic and lactonic forms with almost equal proportion. In this study, we found the gene encoding for the long-chain acyl-CoA synthetase (ALCS). This enzyme was putatively identified as a membrane-bound long-chain fatty acid transport protein and contributed to the uptake of long-chain fatty acids. Disruption of the alcs gene resulted in an impaired growth of the alcs-deleted mutant in minimal media containing different fatty acids (C12:0, C14:0, C16:0, C18:0, C22:0, and C24:0) as the sole carbon source and led to a dramatic decrease in the uptake of the fluorescent-tagged long-chain fatty acid analogue-boron dipyrromethene difluoride dodecanoic acid (BODIPY-3823). The absence of this alcs gene caused obvious phenotype changes. Compared with the wild-type strain, the yield and compositions of the SLs produced by the gene-deleted mutant of ∆alcs::six showed almost no lactonic form of SLs, and the acidic SLs were composed of medium-chain. The ALCS enzyme was heterologously expressed in Escherichia coli JM109 (DE3) with pMAL-c2x-alcs. The enzyme was purified through a maltose-binding protein (MBP) affinity chromatography column and was confirmed to be homogeneous by SDS-PAGE. The recombinant enzyme could catalyze the formation of the long-chain acyl-CoA when the long-chain fatty acids and the coenzyme A were used as substrates.