Catabolic Network of the Fermentative Gut Bacterium Phocaeicola vulgatus (Phylum Bacteroidota) from a Physiologic-Proteomic Perspective.

INTRODUCTION: Phocaeicola vulgatus (formerly Bacteroides vulgatus) is a prevalent member of human and animal guts, where it influences by its dietary-fiber-fueled, fermentative metabolism the microbial community as well as the host health. Moreover, the fermentative metabolism of P. vulgatus bears potential for a sustainable production of bulk chemicals. The aim of the present study was to refine the current understanding of the P. vulgatus physiology. METHODS: P. vulgatus was adapted to anaerobic growth with 14 different carbohydrates, ranging from hexoses, pentoses, hemicellulose, via an uronic acid to deoxy sugars. These substrate-adapted cells formed the basis to define the growth stoichiometries by quantifying growth/fermentation parameters and to reconstruct the catabolic network by applying differential proteomics. RESULTS: The determination of growth performance revealed, e.g., doubling times (h) from 1.39 (arabinose) to 14.26 (glucuronate), biomass yields (gCDW/mmolS) from 0.01 (fucose) to 0.27 (α-cyclodextrin), and ATP yields (mMATP/mMC) from 0.21 (rhamnose) to 0.60 (glucuronate/xylan). Furthermore, fermentation product spectra were determined, ranging from broad and balanced (with xylan: acetate, succinate, formate, and propanoate) to rather one sided (with rhamnose or fucose: mainly propane-1,2-diol). The fermentation network serving all tested compounds is composed of 56 proteins (all identified), with several peripheral reaction sequences formed with high substrate specificity (e.g., conversion of arabinose to d-xylulose-3-phosphate) implicating a fine-tuned regulation. By contrast, central modules (e.g., glycolysis or the reaction sequence from PEP to succinate) were constitutively formed. Extensive formation of propane-1,2-diol from rhamnose and fucose involves rhamnulokinase (RhaB), rhamnulose-1-phosphate kinase (RhaD), and lactaldehyde reductase (FucO). Furthermore, Sus-like systems are apparently the most relevant uptake systems and a complex array of transmembrane electron-transfer systems (e.g., Na+-pumping Rnf and Nqr complexes, fumarate reductase) as well as F- and V-type ATP-synthases were detected. CONCLUSIONS: The present study provides insights into the potential contribution of P. vulgatus to the gut metabolome and into the strain's biotechnological potential for sustainable production of short-chain fatty acids and alcohols.

Systems Biology of Aromatic Compound Catabolism in Facultative Anaerobic Aromatoleum aromaticum EbN1T.

Members of the genus Aromatoleum thrive in diverse habitats and use a broad range of recalcitrant organic molecules coupled to denitrification or O2 respiration. To gain a holistic understanding of the model organism A. aromaticum EbN1T, we studied its catabolic network dynamics in response to 3-(4-hydroxyphenyl)propanoate, phenylalanine, 3-hydroxybenzoate, benzoate, and acetate utilized under nitrate-reducing versus oxic conditions. Integrated multi-omics (transcriptome, proteome, and metabolome) covered most of the catabolic network (199 genes) and allowed for the refining of knowledge of the degradation modules studied. Their substrate-dependent regulation showed differing degrees of specificity, ranging from high with 3-(4-hydroxyphenyl)propanoate to mostly relaxed with benzoate. For benzoate, the transcript and protein formation were essentially constitutive, contrasted by that of anoxia-specific versus oxia-specific metabolite profiles. The matrix factorization of transcriptomic data revealed that the anaerobic modules accounted for most of the variance across the degradation network. The respiration network appeared to be constitutive, both on the transcript and protein levels, except for nitrate reductase (with narGHI expression occurring only under nitrate-reducing conditions). The anoxia/nitrate-dependent transcription of denitrification genes is apparently controlled by three FNR-type regulators as well as by NarXL (all constitutively formed). The resequencing and functional reannotation of the genome fostered a genome-scale metabolic model, which is comprised of 655 enzyme-catalyzed reactions and 731 distinct metabolites. The model predictions for growth rates and biomass yields agreed well with experimental stoichiometric data, except for 3-(4-hydroxyphenyl)propanoate, with which 4-hydroxybenzoate was exported. Taken together, the combination of multi-omics, growth physiology, and a metabolic model advanced our knowledge of an environmentally relevant microorganism that differs significantly from other bacterial model strains. IMPORTANCE Aromatic compounds are abundant constituents not only of natural organic matter but also of bulk industrial chemicals and fuel components of environmental concern. Considering the widespread occurrence of redox gradients in the biosphere, facultative anaerobic degradation specialists can be assumed to play a prominent role in the natural mineralization of organic matter and in bioremediation at contaminated sites. Surprisingly, differential multi-omics profiling of the A. aromaticum EbN1T studied here revealed relaxed regulatory stringency across its four main physiological modi operandi (i.e., O2-independent and O2-dependent degradation reactions versus denitrification and O2 respiration). Combining multi-omics analyses with a genome-scale metabolic model aligned with measured growth performances establishes A. aromaticum EbN1T as a systems-biology model organism and provides unprecedented insights into how this bacterium functions on a holistic level. Moreover, this experimental platform invites future studies on eco-systems and synthetic biology of the environmentally relevant betaproteobacterial Aromatoleum/Azoarcus/Thauera cluster.

Effects of antibiotics on bacterial cell morphology and their physiological origins.

Characterizing the physiological response of bacterial cells to antibiotic treatment is crucial for the design of antibacterial therapies and for understanding the mechanisms of antibiotic resistance. While the effects of antibiotics are commonly characterized by their minimum inhibitory concentrations or the minimum bactericidal concentrations, the effects of antibiotics on cell morphology and physiology are less well characterized. Recent technological advances in single-cell studies of bacterial physiology have revealed how different antibiotic drugs affect the physiological state of the cell, including growth rate, cell size and shape, and macromolecular composition. Here, we review recent quantitative studies on bacterial physiology that characterize the effects of antibiotics on bacterial cell morphology and physiological parameters. In particular, we present quantitative data on how different antibiotic targets modulate cellular shape metrics including surface area, volume, surface-to-volume ratio, and the aspect ratio. Using recently developed quantitative models, we relate cell shape changes to alterations in the physiological state of the cell, characterized by changes in the rates of cell growth, protein synthesis and proteome composition. Our analysis suggests that antibiotics induce distinct morphological changes depending on their cellular targets, which may have important implications for the regulation of cellular fitness under stress.

Coordination of gene expression with cell size enables Escherichia coli to efficiently maintain motility across conditions.

To swim and navigate, motile bacteria synthesize a complex motility machinery involving flagella, motors, and a sensory system. A myriad of studies has elucidated the molecular processes involved, but less is known about the coordination of motility expression with cellular physiology: In Escherichia coli, motility genes are strongly up-regulated in nutrient-poor conditions compared to nutrient-replete conditions; yet a quantitative link to cellular motility has not been developed. Here, we systematically investigated gene expression, swimming behavior, cell growth, and available proteomics data across a broad spectrum of exponential growth conditions. Our results suggest that cells up-regulate the expression of motility genes at slow growth to compensate for reduction in cell size, such that the number of flagella per cell is maintained across conditions. The observed four or five flagella per cell is the minimum number needed to keep the majority of cells motile. This simple regulatory objective allows E. coli cells to remain motile across a broad range of growth conditions, while keeping the biosynthetic and energetic demands to establish and drive the motility machinery at the minimum needed. Given the strong reduction in flagella synthesis resulting from cell size increases at fast growth, our findings also provide a different physiological perspective on bacterial cell size control: A larger cell size at fast growth is an efficient strategy to increase the allocation of cellular resources to the synthesis of those proteins required for biomass synthesis and growth, while maintaining processes such as motility that are only needed on a per-cell basis.

Antibiotic Resistance via Bacterial Cell Shape-Shifting.

Bacteria have evolved to develop multiple strategies for antibiotic resistance by effectively reducing intracellular antibiotic concentrations or antibiotic binding affinities, but the role of cell morphology in antibiotic resistance remains poorly understood. By analyzing cell morphological data for different bacterial species under antibiotic stress, we find that bacteria increase or decrease the cell surface-to-volume ratio depending on the antibiotic target. Using quantitative modeling, we show that by reducing the surface-to-volume ratio, bacteria can effectively reduce the intracellular antibiotic concentration by decreasing antibiotic influx. The model further predicts that bacteria can increase the surface-to-volume ratio to induce the dilution of membrane-targeting antibiotics, in agreement with experimental data. Using a whole-cell model for the regulation of cell shape and growth by antibiotics, we predict shape transformations that bacteria can utilize to increase their fitness in the presence of antibiotics. We conclude by discussing additional pathways for antibiotic resistance that may act in synergy with shape-induced resistance.

Cellular resource allocation strategies for cell size and shape control in bacteria.

Bacteria are highly adaptive microorganisms that thrive in a wide range of growth conditions via changes in cell morphologies and macromolecular composition. How bacterial morphologies are regulated in diverse environmental conditions is a long-standing question. Regulation of cell size and shape implies control mechanisms that couple the growth and division of bacteria to their cellular environment and macromolecular composition. In the past decade, simple quantitative laws have emerged that connect cell growth to proteomic composition and the nutrient availability. However, the relationships between cell size, shape, and growth physiology remain challenging to disentangle and unifying models are lacking. In this review, we focus on regulatory models of cell size control that reveal the connections between bacterial cell morphology and growth physiology. In particular, we discuss how changes in nutrient conditions and translational perturbations regulate the cell size, growth rate, and proteome composition. Integrating quantitative models with experimental data, we identify the physiological principles of bacterial size regulation, and discuss the optimization strategies of cellular resource allocation for size control.

Integrative growth physiology and transcriptome profiling of probiotic Limosilactobacillus reuteri KUB-AC5.

Limosilactobacillus reuteri KUB-AC5 has been widely used as probiotic in chicken for Salmonella reduction. However, a preferable carbon source and growth phase is poorly characterized underlying metabolic responses on growth and inhibition effects of L. reuteri KUB-AC5. This study therefore aimed to investigate transcriptome profiling of L. reuteri KUB-AC5 revealing global metabolic responses when alteration of carbon sources and growth phases. Interestingly, L. reuteri KUB-AC5 grown under sucrose culture showed to be the best for fast growth and inhibition effects against Salmonella Enteritidis S003 growth. Towards the transcriptome profiling and reporter proteins/metabolites analysis, the results showed that amino acid transport via ABC systems as well as sucrose metabolism and transport are key metabolic responses at Logarithmic (L)-phase of L. reuteri KUB-AC5 growth. Considering the Stationary (S)-phase, we found the potential reporter proteins/metabolites involved in carbohydrate metabolism e.g., levansucrase and levan. Promisingly, levansucrase and levan were revealed to be candidates in relation to inhibition effects of L. reuteri KUB-AC5. Throughout this study, L. reuteri KUB-AC5 had a metabolic control in acclimatization to sucrose and energy pools through transcriptional co-regulation, which supported the cell growth and inhibition potentials. This study offers a perspective in optimizing fermentation condition through either genetic or physiological approaches for enhancing probiotic L. reuteri KUB-AC5 properties.

Global Response of Phaeobacter inhibens DSM 17395 to Deletion of Its 262-kb Chromid Encoding Antibiotic Synthesis.

The marine alphaproteobacterium Phaeobacter inhibens DSM 17395, a member of the Roseobacter group, was recently shown to markedly enhance growth upon deletion of its 262-kb chromid encoding biosynthesis of tropodithietic acid (TDA). To scrutinize the metabolic/regulatory adaptations that underlie enhanced growth of the Δ262 mutant, its transcriptome and proteome compared to the wild type were investigated in process-controlled bioreactors with Casamino Acids as growth substrate. Genome resequencing revealed only few additional genetic changes (a heterogenic insertion, prophage activation, and several point mutations) between wild type and Δ262 mutant, albeit with no conceivable effect on the studied growth physiology. The abundances of the vast majority of transcripts and proteins involved in the catabolic network for complete substrate oxidation to CO2 were found to be unchanged, suggesting that the enhanced amino acid utilization of the Δ262 mutant did not require elevated synthesis of most enzymes of the catabolic network. Similarly, constituents of genetic information processing and cellular processes remained mostly unchanged. In contrast, 426 genes displayed differential expression, of which 410 were localized on the 3.2-Mb chromosome, 5 on the 65-kb chromid, and 11 on the 78-kb chromid. Notably, the branched-chain amino transferase IlvE acting on rapidly utilized Val, Ile, and Leu was upregulated. Moreover, the transportome was reconfigured, as evidenced from increased abundances of transcripts and proteins of several uptake systems for amino acids and inorganic nutrients (e.g., phosphate). Some components of the respiratory chain were also upregulated, which correlates with the higher respiration rates of the Δ262 mutant. Furthermore, chromosomally encoded transcripts and proteins that are peripherally related to TDA biosynthesis (e.g., the serine acyl transferase CysE) were strongly downregulated in the Δ262 mutant. Taken together, these observations reflect adaptations to enhanced growth as well as the functional interconnectivity of the replicons of P. inhibens DSM 17395.

Characterization of a New Rhamnolipid Biosurfactant Complex from Pseudomonas Isolate DYNA270.

The chemical and physical properties of extracellular rhamnolipid synthesized by a nonfluorescent Pseudomonas species soil isolate, identified as DYNA270, is described, along with characteristics of rhamnolipid production under varying growth conditions and substrates. The biosurfactant is determined to be an anionic, extracellular glycolipid consisting of two major components, the rhamnopyranoside ß-1-3-hydroxydecanoyl-3-hydroxydecanoic acid (GU-6) and rhamnopyranosyl ß→ß2-rhamnopyranoside-ß1-3-hydroxydecanoyl-3-hydroxydecanoic acid (GL-2), of molecular weight 504 and 649 daltons, respectively. These glycolipids are produced in a stoichiometric ratio of 1:3, respectively. The purified rhamnolipid mixture exhibits a critical micelle concentration of 20 mg/L, minimum surface (air/water interface) tension of 22 mN/m, and minimum interfacial tension values of 0.005 mN/m (against hexane). The pH optimum, critical micelle concentration, and effective alkane carbon number were established for Pseudomonas species DYNA270 and compared to those of rhamnolipid produced by Pseudomonas aeruginosa PG201. Significant differences are documented in the physical properties of extracellular rhamnolipids derived from these two microorganisms. The surface properties of this rhamnolipid are unique in that ultra-low surface and interfacial tension values are present in both purified rhamnolipid and culture broth containing the rhamnolipid complex (GU6 and GL2). We are not aware of prior studies reporting surface activity values this low for rhamnolipids. An exception is noted for an extracellular trehalose glycolipid produced by Rhodococcus species H13-A, which measured 0.00005 mN/m in the presence of the co-agent pentanol (Singer et al. 1990). Similar CMC values of 20 mg/L have been reported for rhamnolipids, a few being recorded as 5-10 mg/L for Pseudomonas species DSM2874 (Lang et al. 1984).

Surface-to-volume scaling and aspect ratio preservation in rod-shaped bacteria.

Rod-shaped bacterial cells can readily adapt their lengths and widths in response to environmental changes. While many recent studies have focused on the mechanisms underlying bacterial cell size control, it remains largely unknown how the coupling between cell length and width results in robust control of rod-like bacterial shapes. In this study we uncover a conserved surface-to-volume scaling relation in Escherichia coli and other rod-shaped bacteria, resulting from the preservation of cell aspect ratio. To explain the mechanistic origin of aspect-ratio control, we propose a quantitative model for the coupling between bacterial cell elongation and the accumulation of an essential division protein, FtsZ. This model reveals a mechanism for why bacterial aspect ratio is independent of cell size and growth conditions, and predicts cell morphological changes in response to nutrient perturbations, antibiotics, MreB or FtsZ depletion, in quantitative agreement with experimental data.

Non-Redfield, nutrient synergy and flexible internal elemental stoichiometry in a marine bacterium.

The stoichiometric constraints of algal growth are well understood, whereas there is less knowledge for heterotrophic bacterioplankton. Growth of the marine bacterium Phaeobacter inhibens DSM 17395, belonging to the globally distributed Roseobacter group, was studied across a wide concentration range of NH4+ and PO43-. The unique dataset covers 415 different concentration pairs, corresponding to 207 different molar N:P ratios (from 10-2 to 105). Maximal growth (by growth rate and biomass yield) was observed within a restricted concentration range at N:P ratios (∼50-120) markedly above Redfield. Experimentally determined growth parameters deviated to a large part from model predictions based on Liebig's law of the minimum, thus implicating synergistic co-limitation due to biochemical dependence of resources. Internal elemental ratios of P. inhibens varied with external nutrient supply within physiological constraints, thus adding to the growing evidence that aquatic bacteria can be flexible in their internal elemental composition. Taken together, the findings reported here revealed that P. inhibens is well adapted to fluctuating availability of inorganic N and P, expected to occur in its natural habitat (e.g. colonized algae, coastal areas). Moreover, this study suggests that elemental variability in bacterioplankton needs to be considered in the ecological stoichiometry of the oceans.

Quantification of oxidative stress phenotypes based on high-throughput growth profiling of protein kinase and phosphatase knockouts.

Cellular responses to oxidative stress are important for restoring redox balance and ensuring cell survival. Genetic defects in response factors can lead to impaired response to oxidative damage and contribute to disease and aging. In single cell organisms, such as yeasts, the integrity of the oxidative stress response can be observed through its influences on growth characteristics. In this study, we investigated the time-dependent batch growth effects as a function of oxidative stress levels in protein kinase and phosphatase deletion backgrounds of Saccharomyces cerevisiae. In total, 41 different protein kinases and phosphatase mutants were selected for their known activities in oxidative stress or other stress response pathways and were investigated for their dosage-dependent response to hydrogen peroxide. Detailed growth profiles were analyzed after the induction of stress for growth rate, lag time duration and growth efficiency, and by a novel method to identify stress-induced diauxic shift delay. This approach extracts more phenotypic information than traditional plate-based methods due to the assessment of time dynamics in the time scale of minutes. With this approach, we were able to identify surprisingly diverse sensitivity and resistance patterns as a function of gene knockout.

A brief history of bacterial growth physiology.

Arguably, microbial physiology started when Leeuwenhoek became fascinated by observing a Vorticella beating its cilia, my point being that almost any observation of microbes has a physiological component. With the advent of modern microbiology in the mid-19th century, the field became recognizably distinctive with such discoveries as anaerobiosis, fermentation as a biological phenomenon, and the nutritional requirements of microbes. Soon came the discoveries of Winogradsky and his followers of the chemical changes in the environment that result from microbial activities. Later, during the first half of the 20th century, microbial physiology became the basis for much of the elucidation of central metabolism. Bacterial physiology then became a handmaiden of molecular biology and was greatly influenced by the discovery of cellular regulatory mechanisms. Microbial growth, which had come of age with the early work of Hershey, Monod, and others, was later pursued by studies on a whole cell level by what became known as the "Copenhagen School." During this time, the exploration of physiological activities became coupled to modern inquiries into the structure of the bacterial cell. Recent years have seen the development of a further phase in microbial physiology, one seeking a deeper quantitative understanding of phenomena on a whole cell level. This pursuit is exemplified by the emergence of systems biology, which is made possible by the development of technologies that permit the gathering of information in huge amounts. As has been true through history, the research into microbial physiology continues to be guided by the development of new methods of analysis. Some of these developments may well afford the possibility of making stunning breakthroughs.

Quantitative proteomic analysis reveals a simple strategy of global resource allocation in bacteria.

A central aim of cell biology was to understand the strategy of gene expression in response to the environment. Here, we study gene expression response to metabolic challenges in exponentially growing Escherichia coli using mass spectrometry. Despite enormous complexity in the details of the underlying regulatory network, we find that the proteome partitions into several coarse-grained sectors, with each sector's total mass abundance exhibiting positive or negative linear relations with the growth rate. The growth rate-dependent components of the proteome fractions comprise about half of the proteome by mass, and their mutual dependencies can be characterized by a simple flux model involving only two effective parameters. The success and apparent generality of this model arises from tight coordination between proteome partition and metabolism, suggesting a principle for resource allocation in proteome economy of the cell. This strategy of global gene regulation should serve as a basis for future studies on gene expression and constructing synthetic biological circuits. Coarse graining may be an effective approach to derive predictive phenomenological models for other 'omics' studies.

Effects of exogenous nitric oxide on physiology of seed germination and seedling growth of Silybum marianum under NaCl stress / 中草药

Objective: To investigate the effects of exogenous nitric oxide (NO) on the physiology of the seed germination and seedling growth of Silybum marianum under NaCl stress. Methods: The seeds of S. marianum were treated by sodium nitro prusside (SNP) at the concentration of 0.05-0.60 mmol/L under 0.7% NaCl stress. Some physiological indexes were measured, such as germination energy, germination rate, germination index, and vigor index of the seeds, and contents of malondialdehyde (MDA), photosynthetic pigment, osmosis substances, and the activities of the protective enzymes in leaves. Results: The seed germination and seedling growth of S. marianum were obviously inhibited under NaCl stress. Soaking seeds with 0.05-0.60 mmol/L SNP could alleviate the damage of NaCl stress. Under this treatment, the contents of photosynthetic pigment (including chlorophyll a, chlorophyll b, total chlorophyll, and carotinoid) and osmosis substances (including soluble sugar, soluble protein, and proline), and the activities of protective enzymes (including SOD, POD, and CAT) in the leaves were significantly increased, while the MDA content in the leaves was decreased. Conclusion: Soaking seeds with 0.05-0.60 mmol/L SNP could promote the salt resistance of the seeds and seedlings of S. marianum. The different cultivars of S. marianum differ in the sensitivity to SNP. The optimal concentration of SNP for the seed soaking of S. marianum with white and black skins is 0.10 and 0.40 mmol/L, respectively.

O crescimento de crianças alimentadas com leite materno exclusivo nos primeiros 6 meses de vida

Resumo Objetivo: avaliar a evolução do crescimento de crianças alimentadas exclusivamente com leite materno durante os primeiros 6 meses de vida. Métodos: estudo longitudinal de 184 crianças que receberam atenção primária em instituição pública (Programa de Incentivo ao Aleitamento Materno Exclusivo) de Belém (PA), entre fevereiro de 2000 e janeiro de 2001. Finalizaram o estudo 102 lactentes nascidos a termo, com peso igual ou superior a 2.500 g, sem intercorrências no período neonatal e alimentados exclusivamente com leite materno sob livre demanda desde o nascimento. Acompanhados mensalmente, foram avaliados os registros de peso e comprimento ao nascer, no primeiro, quarto e sexto meses de vida. Avaliou-se o crescimento através das médias de peso e estatura para cada idade, comparando-se os resultados com o percentil 50° dos gráficos de crescimento do National Center for Health Statistics (NCHS) e com outros estudos anteriores com crianças brasileiras em aleitamento materno exclusivo. Resultados: as crianças tiveram crescimento adequado, dobrando de peso antes do quarto mês de vida, com desaceleração do ganho pôndero-estatural após o quarto mês, porém chegando aos 6 meses com médias de peso superiores aos padrões utilizados para comparação. Conclusões: os resultados mostraram que crianças em aleitamento materno exclusivo chegaram aos 6 meses com peso médio superior ao percentil 50° do NCHS, confirmando as vantagens nutricionais do leite materno, principalmente quando as mães recebem orientação sobre a técnica adequada para amamentar.

Summary Objective: to evaluate the growth of exclusively breastfed infants from birth to 6 months of life. Methods: this was a longitudinal study of 184 children who received primary care at a public institution (Exclusive Breastfeeding Stimulation Program, PROAME) in Belém, state of Pará, Brazil, between February 2000 and January 2001. A total of 102 children completed the study. All were born at full term with a birth weight greater than or equal to 2,500 g. No events were recorded during the neonatal period and the infants were exclusively breastfed, on demand, since their birth. The infants were followed up monthly, and the weight and length measured at birth and at the end of the first, fourth and sixth months of life were compared to the 50th percentile line of the NCHS growth charts and to other previous studies of Brazilian children who were exclusively breastfed. Results: growth was adequate for all the children, who doubled their weight before the fourth month of life. Despite a slow-down in weight and height gain after the fourth month, the weight averages at 6 months were greater than the standards used for comparison. Conclusions: the average weight of these exclusively breastfed children was above the 50th percentile of the NCHS curve at 6 months, thus confirming the nutritional advantages of breast milk, especially when the mothers receive guidance regarding the appropriate techniques for breastfeeding.