Distinct microbiota composition and fermentation products indicate functional compartmentalization in the hindgut of a marine herbivorous fish.

Many marine herbivorous fishes harbour diverse microbial communities in the hindgut that can play important roles in host health and nutrition. Kyphosus sydneyanus is a temperate marine herbivorous fish that feeds predominantly on brown seaweeds. We employed 16S rRNA gene amplicon sequencing and gas chromatography to characterize microbial communities and their metabolites in different hindgut regions of six K. sydneyanus. Measurements were confined to three distal sections of the intestine, labelled III, IV and V from anterior to posterior. A total of 625 operational taxonomic units from 20 phyla and 123 genera were obtained. Bacteroidota, Firmicutes and Proteobacteria were the major phyla in mean relative abundance, which varied along the gut. Firmicutes (76%) was the most dominant group in section III, whereas Bacteroidota (69.3%) dominated section V. Total short-chain fatty acid (SCFA) concentration was highest in sections IV and V, confirming active fermentation in these two most distal sections. The abundance of Bacteroidota correlated with propionate concentration in section V, while Firmicutes positively correlated with formate in sections III and IV. Acetate levels were highest in sections IV and V, which correlated with abundance of Bacteroidota. Despite differences in gut microbial community composition, SCFA profiles were consistent between individual fish in the different hindgut regions of K. sydneyanus, although proportions of SCFAs differed among gut sections. These findings demonstrate functional compartmentalization of the hindgut microbial community, highlighting the need for regional sampling when interpreting overall microbiome function. These results support previous work suggesting that hindgut microbiota in marine herbivorous fish are important to nutrition in some host species by converting dietary carbohydrates into metabolically useful SCFAs.

The Pseudoenzyme PDX1.2 Sustains Vitamin B6 Biosynthesis as a Function of Heat Stress.

Plants sense temperature changes and respond by altering growth and metabolic activity to acclimate to the altered environmental conditions. The B vitamins give rise to vital coenzymes that are indispensable for growth and development but their inherent reactive nature renders them prone to destruction especially under stress conditions. Therefore, plant survival strategies would be expected to include mechanisms to sustain B vitamin supply under demanding circumstances. Here, using the example of vitamin B6, we investigate the regulation of biosynthesis across eudicot and monocot species under heat stress. Most eudicots carry a pseudoenzyme PDX1.2 that is a noncatalytic homolog of the PDX1 subunit of the vitamin B6 biosynthesis protein machinery, PYRIDOXINE BIOSYNTHESIS PROTEIN1. Using Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) as models, we show that PDX12 is transcriptionally regulated by the HSFA1 transcription factor family. Monocots only carry catalytic PDX1 homologs that do not respond to heat stress as demonstrated for rice (Oryza sativa) and maize (Zea mays), suggesting fundamental differences in the regulation of vitamin B6 biosynthesis across the two lineages. Investigation of the molecular mechanism of PDX12 transcription reveals two alternative transcriptional start sites, one of which is exclusive to heat stress. Further data suggest that PDX1.2 leads to stabilization of the catalytic PDX1s under heat stress conditions, which would serve to maintain vitamin B6 homeostasis in times of need in eudicots that carry this gene. Our analyses indicate an important abiotic stress tolerance strategy in several eudicots, which has not been evolutionarily adapted (or is not required) by monocots such as grasses.

Consequences of a deficit in vitamin B6 biosynthesis de novo for hormone homeostasis and root development in Arabidopsis.

Vitamin B(6) (pyridoxal 5'-phosphate) is an essential cofactor of many metabolic enzymes. Plants biosynthesize the vitamin de novo employing two enzymes, pyridoxine synthase1 (PDX1) and PDX2. In Arabidopsis (Arabidopsis thaliana), there are two catalytically active paralogs of PDX1 (PDX1.1 and PDX1.3) producing the vitamin at comparable rates. Since single mutants are viable but the pdx1.1 pdx1.3 double mutant is lethal, the corresponding enzymes seem redundant. However, the single mutants exhibit substantial phenotypic differences, particularly at the level of root development, with pdx1.3 being more impaired than pdx1.1. Here, we investigate the differential regulation of PDX1.1 and PDX1.3 by identifying factors involved in their disparate phenotypes. Swapped-promoter experiments clarify the presence of distinct regulatory elements in the upstream regions of both genes. Exogenous sucrose (Suc) triggers impaired ethylene production in both mutants but is more severe in pdx1.3 than in pdx1.1. Interestingly, Suc specifically represses PDX1.1 expression, accounting for the stronger vitamin B6 deficit in pdx1.3 compared with pdx1.1. Surprisingly, Suc enhances auxin levels in pdx1.1, whereas the levels are diminished in pdx1.3. In the case of pdx1.3, the previously reported reduced meristem activity combined with the impaired ethylene and auxin levels manifest the specific root developmental defects. Moreover, it is the deficit in ethylene production and/or signaling that triggers this outcome. On the other hand, we hypothesize that it is the increased auxin content of pdx1.1 that is responsible for the root developmental defects observed therein. We conclude that PDX1.1 and PDX1.3 play partially nonredundant roles and are differentially regulated as manifested in disparate root growth impairment morphologies.

The pseudoenzyme PDX1.2 boosts vitamin B6 biosynthesis under heat and oxidative stress in Arabidopsis.

Vitamin B6 is an indispensable compound for survival, well known as a cofactor for numerous central metabolic enzymes and more recently for playing a role in several stress responses, particularly in association with oxidative stress. Regulatory aspects for the use of the vitamin in these roles are not known. Here we show that certain plants carry a pseudoenzyme (PDX1.2), which is involved in regulating vitamin B6 biosynthesis de novo under stress conditions. Specifically, we demonstrate that Arabidopsis PDX1.2 enhances the activity of its catalytic paralogs by forming a heterododecameric complex. PDX1.2 is strongly induced by heat as well as singlet oxygen stress, concomitant with an enhancement of vitamin B6 production. Analysis of pdx1.2 knockdown lines demonstrates that boosting vitamin B6 content is dependent on PDX1.2, revealing that this pseudoenzyme acts as a positive regulator of vitamin B6 biosynthesis during such stress conditions in plants.

Enhanced levels of vitamin B(6) increase aerial organ size and positively affect stress tolerance in Arabidopsis.

Vitamin B6 is an essential nutrient in the human diet derived primarily from plant sources. While it is well established as a cofactor for numerous metabolic enzymes, more recently, vitamin B6 has been implicated as a potent antioxidant. The de novo vitamin B6 biosynthesis pathway in plants has recently been unraveled and involves only two proteins, PDX1 and PDX2. To provide more insight into the effect of the compound on plant development and its role as an antioxidant, we have overexpressed the PDX proteins in Arabidopsis, generating lines with considerably higher levels of the vitamin in comparison with other recent attempts to achieve this goal. Interestingly, it was possible to increase the level of only one of the two catalytically active PDX1 proteins at the protein level, providing insight into the mechanism of vitamin B6 homeostasis in planta. Vitamin B6 enhanced lines have considerably larger vegetative and floral organs and although delayed in pre-reproductive development, do not have an altered overall morphology. The vitamin was observed to accumulate in seeds and the enhancement of its levels was correlated with an increase in their size and weight. This phenotype is predominantly a consequence of embryo enlargement as reflected by larger cells. Furthermore, plants that overaccumulate the vitamin have an increased tolerance to oxidative stress providing in vivo evidence for the antioxidant functionality of vitamin B6. In particular, the plants show an increased resistance to paraquat and photoinhibition, and they attenuate the cell death response observed in the conditional flu mutant.

High-throughput Method for Novel Medium Development for Culture of Anaerobic Gut Bacteria.

Gut microbiota play important roles in the health of their host and detailed investigation of these organisms requires in vitro culture. Culturing strictly anaerobic bacteria can be a challenge as the gut environment they inhabit is nutritionally complex. Use of complex media containing nutritionally rich but undefined gut fluid reduces the accuracy of physiological and metabolomic studies. Here we present a high-throughput protocol for comparing growth rates of fastidiously anaerobic bacteria on different media. These protocols can be used to develop a solid medium made up of commercially sourced ingredients, providing replicable growth conditions for previously uncultured anaerobic bacteria. As many fastidious bacteria grow poorly in a liquid broth, these protocols measure bacterial growth rate on solid media. These protocols speed up and simplify the growth rate measurement process by using a multiwell format and equations in place of physical McFarland standards to calculate approximate cell density. Bacterial strains belonging to the families Erysipelotrichaceae and Lachnospiraceae (phylum Firmicutes) isolated from the hindgut of Kyphosus sydneyanus were used to demonstrate the efficacy of these protocols. Bacterial growth rates were compared between a nutritionally rich medium with gut fluid versus a novel replicable medium with mannitol. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of solid YCFA growth medium Basic Protocol 2: Collection of fish gut samples and plating to single isolates Basic Protocol 3: Genetic identification of single isolates with colony PCR and 16S rRNA gene sequencing Basic Protocol 4: Measurement of bacterial growth rates on solid media.

Activation of the cytochrome c peroxidase of Pseudomonas aeruginosa. The role of a heme-linked protein loop: a mutagenesis study.

Mutagenesis studies have been used to investigate the role of a heme ligand containing protein loop (67-79) in the activation of di-heme peroxidases. Two mutant forms of the cytochrome c peroxidase of Pseudomonas aeruginosa have been produced. One mutant (loop mutant) is devoid of the protein loop and the other (H71G) contains a non-ligating Gly at the normal histidine ligand site. Spectroscopic data show that in both mutants the distal histidine ligand of the peroxidatic heme in the un-activated enzyme is lost or is exchangeable. The un-activated H71G and loop mutants show, respectively, 75% and 10% of turnover activity of the wild-type enzyme in the activated form, in the presence of hydrogen peroxide and the physiological electron donor cytochrome c(551). Both mutant proteins show the presence of constitutive reactivity with peroxide in the normally inactive, fully oxidised, form of the enzyme and produce a radical intermediate. The radical product of the constitutive peroxide reaction appears to be located at different sites in the two mutant proteins. These results show that the loss of the histidine ligand from the peroxidatic heme is, in itself, sufficient to produce peroxidatic activity by providing a peroxide binding site and that the formation of radical intermediates is very sensitive to changes in protein structure. Overall, these data are consistent with a major role for the protein loop 67-79 in the activation of di-heme peroxidases and suggest a "charge hopping" mechanism may be operative in the process of intra-molecular electron transfer.

The rise of operon-like gene clusters in plants.

Gene clusters are common features of prokaryotic genomes also present in eukaryotes. Most clustered genes known are involved in the biosynthesis of secondary metabolites. Although horizontal gene transfer is a primary source of prokaryotic gene cluster (operon) formation and has been reported to occur in eukaryotes, the predominant source of cluster formation in eukaryotes appears to arise de novo or through gene duplication followed by neo- and sub-functionalization or translocation. Here we aim to provide an overview of the current knowledge and open questions related to plant gene cluster functioning, assembly, and regulation. We also present potential research approaches and point out the benefits of a better understanding of gene clusters in plants for both fundamental and applied plant science.

Strategies for vitamin B6 biofortification of plants: a dual role as a micronutrient and a stress protectant.

Vitamin B6 has an essential role in cells as a cofactor for several metabolic enzymes. It has also been shown to function as a potent antioxidant molecule. The recent elucidation of the vitamin B6 biosynthesis pathways in plants provides opportunities for characterizing their importance during developmental processes and exposure to stress. Humans and animals must acquire vitamin B6 with their diet, with plants being a major source, because they cannot biosynthesize it de novo. However, the abundance of the vitamin in the edible portions of the most commonly consumed plants is not sufficient to meet daily requirements. Genetic engineering has proven successful in increasing the vitamin B6 content in the model plant Arabidopsis. The added benefits associated with the enhanced vitamin B6 content, such as higher biomass and resistance to abiotic stress, suggest that increasing this essential micronutrient could be a valuable option to improve the nutritional quality and stress tolerance of crop plants. This review summarizes current achievements in vitamin B6 biofortification and considers strategies for increasing vitamin B6 levels in crop plants for human health and nutrition.

Redox-linked structural changes associated with the formation of a catalytically competent form of the diheme cytochrome c peroxidase from Pseudomonas aeruginosa.

A recombinant form of the prototypic diheme bacterial cytochrome c peroxidase (BCCP) from Pseudomonas aeruginosa (PsaCCP) has been expressed in Escherichia coli and purified to homogeneity. This material was used to carry out the first integrated biochemical, spectroscopic and structural investigation of the factors leading to reductive activation of this class of enzymes. A single, tightly bound, Ca2+ ion (K = 3 x 10(10) M-1) found at the domain interface of both the fully oxidized and mixed-valence forms of the enzyme is absolutely required for catalytic activity. Reduction of the electron-transferring (high-potential) heme in the presence of Ca2+ ions triggers substantial structural rearrangements around the active-site (low-potential) heme to allow substrate binding and catalysis. The enzyme also forms a mixed-valence state in the absence of Ca2+ ions, but a combination of electronic absorption, and EPR spectroscopies suggests that under these circumstances the low potential heme remains six-coordinate, unable to bind substrate and therefore catalytically inactive. Our observations strongly suggest that the two mixed-valence forms of native PsaCCP reported previously by Foote and colleagues (Foote, N., Peterson, J., Gadsby, P., Greenwood, C., and Thomson, A. (1985) Biochem. J. 230, 227-237) correspond to the Ca2+-loaded and -depleted forms of the enzyme.

Intramolecular electron transfer in the dihaem cytochrome c peroxidase of Pseudomonas aeruginosa.

Mutant forms of the enzyme cytochrome c peroxidase from Pseudomonas aeruginosa, in which the peroxidatic haem ligand (H71) and putative haem-bridging amino acid (W94) have been mutated, were produced in an E. coli expression system as a means of investigating possible mechanisms of intramolecular electron transfer within the enzyme. EPR spectroscopy indicated the presence of a high-spin, presumably five-coordinate, peroxidatic haem site in the H71G and H71G/W94A mutants, whilst the W94A mutant apparently retained the normal six-coordinate haem structures. In turnover experiments, these mutants show 55, 4, and <1% activity, respectively, as compared to the wild-type enzyme. The W94A mutant shows essentially no activity in turnover experiments. Circular dichroism spectroscopy indicates no measurable difference in the secondary structure of the H71G mutant from that of the native enzyme, whilst some small differences are observed for the double mutant. Treatment of the oxidised mutant proteins with hydrogen peroxide, in the absence of preactivation or exogenous reductants, yields products that suggest the formation of a tryptophan radical species in the case of the H71 mutant and the production of a porphyrin radical in the case of the double mutant. These results are discussed in terms of the intramolecular electron transfer in this enzyme.

Codon pairs in the genome of Escherichia coli.

MOTIVATION: The effect of two neighboring codons (codon pairs) on gene expression is mediated via the interaction of their cognate tRNAs occupying the two functional ribosomal sites during the translation elongation step. For steric reasons it is reasonable to assume that not all combinations of codons and therefore of tRNAs are equally favorable when situated on the ribosome surface. Aiming of identifying preferential and rare codon pairs, we have determined the frequency of occurrence of all possible combinations of codon pairs in the entire genome of Escherichia coli (E.coli). RESULTS: The frequency of occurrence of the 3904 codon pairs comprising both sense:sense and sense:stop codon pairs in the full set of E.coli 4289 ORFs was found to vary from zero to 4913 times. For most of the pairs we have observed a significant difference between the real and statistically predicted frequency of occurrence. The analysis of 334 highly expressed and 303 poorly expressed E.coli genes showed that codon pair usage is different for the two gene categories. Using an especially defined criterion (Delta(REG)), the codon pairs are classified as 'hypothetically attenuating' (HAP) and 'hypothetically non-attenuating' (HNAP) and their possible effect on translation is discussed. AVAILABILITY: The program used in this study is available at http://www.bio21.bas.bg/codonpairs/

Effect of 3' terminal codon pairs with different frequency of occurrence on the expression of cat gene in Escherichia coli.

In a previous study, we have identified four types of 3' terminal codon pairs depending on their frequency of occurrence in the Escherichia coli genome: overrepresented, moderately represented, underrepresented, and missing. In this study, the influence of eight codon pairs belonging to these four groups on the efficiency of chloramphenicol acetyltransferase ( cat) gene expression in E. coli is examined. Our results show that the missing codon pairs CCU:UAG (Pro:Stop) and CCC:UAG (Pro:Stop) had decreasing effect, whereas another missing pair CCU:AGG (Pro:Arg) had an opposite effect on the yield of CAT protein in comparison with the wild-type cat gene.