Regulation of quorum sensing activities by the stringent response gene rsh in sphingomonads is species-specific and culture condition dependent.

Stringent response and quorum sensing (QS) are two essential mechanisms that control bacterial global metabolism for better survival. Sphingomonads are a clade of bacteria that survive successfully in diverse ecosystems. In silico survey indicated that 36 out of 79 investigated sphingomonads strains contained more than one luxI homolog, the gene responsible for the biosynthesis of QS signal acyl homoserine lactones (AHLs). Investigation of the regulatory effects of the stringent response gene rsh on QS related bioactivities were carried out using rsh mutants of Sphingobium japonicum UT26 and Sphingobium sp. SYK-6, both had three luxI homologs. Results indicated that deletion of rsh upregulated the overall production of AHLs and extracellular polymeric substances (EPS) in both UT26 and SYK-6 in rich medium, but affected expressions of these luxI/luxR homologs in different ways. In the poor medium (1% LB), rsh mutant of SYK-6 significantly lost AHLs production in broth cultivation but not in biofilm cultivation. The regulatory effects of rsh on QS activities were growth phase dependent in UT26 and culture condition dependent in SYK-6. Our results demonstrated the negative regulatory effect of rsh on QS activities in sphingomonads, which were very different from the positive effect found in sphingomonads containing only one luxI/R circuit. This study extends the current knowledge on the intricate networks between stringent response and QS system in sphingomonads, which would help to understand their survival advantage.

Regulation of the transcription factor CdnL promotes adaptation to nutrient stress in Caulobacter.

In response to nutrient deprivation, bacteria activate a conserved stress response pathway called the stringent response (SR). During SR activation in Caulobacter crescentus, SpoT synthesizes the secondary messengers guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate (collectively known as (p)ppGpp), which affect transcription by binding RNA polymerase (RNAP) to down-regulate anabolic genes. (p)ppGpp also impacts the expression of anabolic genes by controlling the levels and activities of their transcriptional regulators. In Caulobacter, a major regulator of anabolic genes is the transcription factor CdnL. If and how CdnL is controlled during the SR and why that might be functionally important are unclear. In this study, we show that CdnL is down-regulated posttranslationally during starvation in a manner dependent on SpoT and the ClpXP protease. Artificial stabilization of CdnL during starvation causes misregulation of ribosomal and metabolic genes. Functionally, we demonstrate that the combined action of SR transcriptional regulators and CdnL clearance allows for rapid adaptation to nutrient repletion. Moreover, cells that are unable to clear CdnL during starvation are outcompeted by wild-type cells when subjected to nutrient fluctuations. We hypothesize that clearance of CdnL during the SR, in conjunction with direct binding of (p)ppGpp and DksA to RNAP, is critical for altering the transcriptome in order to permit cell survival during nutrient stress.

Unusual and Unconsidered Mechanisms of Bacterial Resilience and Resistance to Quinolones.

Quinolone resistance has been largely related to the presence of specific point mutations in chromosomal targets, with an accessory role of impaired uptake and enhanced pump-out. Meanwhile the relevance of transferable mechanisms of resistance able to protect the target of pump-out or inactivate quinolones has been increasingly reported since 1998. Nevertheless, bacteria have other strategies and mechanisms allowing them to survive and even proliferate in the presence of quinolones, which might be qualified as resistance or resilience mechanisms. These include decreasing levels of quinolone target production, transient amoeba protection, benthonic lifestyle, nutrient-independent slow growth, activation of stringent response, inactivation or degradation of quinolones as well as apparently unrelated or forgotten chromosomal mutations. These mechanisms have been largely overlooked, either because of the use of classical approaches to antibiotic resistance determination or due to the low increase in final minimum inhibitory concentration levels. This article is devoted to a review of a series of these mechanisms.

Breaking bad nucleotides: understanding the regulatory mechanisms of bacterial small alarmone hydrolases.

Guanosine tetra- and pentaphosphate nucleotides, (p)ppGpp, function as central secondary messengers and alarmones in bacterial cell biology, signalling a range of stress conditions, including nutrient starvation and exposure to cell-wall-targeting antibiotics, and are critical for survival. While activation of the stringent response and alarmone synthesis on starved ribosomes by members of the RSH (Rel) class of proteins is well understood, much less is known about how single-domain small alarmone synthetases (SASs) and their corresponding alarmone hydrolases, the small alarmone hydrolases (SAHs), are regulated and contribute to (p)ppGpp homeostasis. The substrate spectrum of these enzymes has recently been expanded to include hyperphosphorylated adenosine nucleotides, suggesting that they take part in a highly complex and interconnected signalling network. In this review, we provide an overview of our understanding of the SAHs and discuss their structure, function, regulation, and phylogeny.

Molecular regulation of virulence in Legionella pneumophila.

Legionella pneumophila is a gram-negative bacteria found in natural and anthropogenic aquatic environments such as evaporative cooling towers, where it reproduces as an intracellular parasite of cohabiting protozoa. If L. pneumophila is aerosolized and inhaled by a susceptible person, bacteria may colonize their alveolar macrophages causing the opportunistic pneumonia Legionnaires' disease. L. pneumophila utilizes an elaborate regulatory network to control virulence processes such as the Dot/Icm Type IV secretion system and effector repertoire, responding to changing nutritional cues as their host becomes depleted. The bacteria subsequently differentiate to a transmissive state that can survive in the environment until a replacement host is encountered and colonized. In this review, we discuss the lifecycle of L. pneumophila and the molecular regulatory network that senses nutritional depletion via the stringent response, a link to stationary phase-like metabolic changes via alternative sigma factors, and two-component systems that are homologous to stress sensors in other pathogens, to regulate differentiation between the intracellular replicative phase and more transmissible states. Together, we highlight how this prototypic intracellular pathogen offers enormous potential in understanding how molecular mechanisms enable intracellular parasitism and pathogenicity.

The stringent response is strongly activated in the antibiotic producing strain, Streptomyces coelicolor.

S. lividans and S. coelicolor are phylogenetically closely related strains with different abilities to produce the same specialized metabolites. Previous studies revealed that the strong antibiotic producer, S. coelicolor, had a lower ability to assimilate nitrogen and phosphate than the weak producer, Streptomyces lividans, and this resulted into a lower growth rate. A comparative proteomic dataset was used to establish the consequences of these nutritional stresses on the abundance of proteins of the translational apparatus of these strains, grown in low and high phosphate availability. Our study revealed that most proteins of the translational apparatus were less abundant in S. coelicolor than in S. lividans whereas it was the opposite for ET-Tu 3 and a TrmA-like methyltransferase. The expression of the latter being known to be under the positive control of the stringent response whereas that of the other ribosomal proteins is under its negative control, this indicated the occurrence of a strong activation of the stringent response in S. coelicolor. Furthermore, in S. lividans, ribosomal proteins were more abundant in phosphate proficiency than in phosphate limitation suggesting that a limitation in phosphate, that was also shown to trigger RelA expression, contributes to the induction of the stringent response.

Gut microbes modulate (p)ppGpp during a time-restricted feeding regimen.

IMPORTANCE: Mammals do not eat continuously, instead concentrating their feeding to a restricted portion of the day. This behavior presents the mammalian gut microbiota with a fluctuating environment with consequences for host-microbiome interaction, infection risk, immune response, drug metabolism, and other aspects of health. We demonstrate that in mice, gut microbes elevate levels of an intracellular signaling molecule, (p)ppGpp, during the fasting phase of a time-restricted feeding regimen. Disabling this response in a representative human gut commensal species significantly reduces colonization during this host-fasting phase. This response appears to be general across species and conserved across mammalian gut communities, highlighting a pathway that allows healthy gut microbiomes to maintain stability in an unstable environment.

Complete loss of RelA and SpoT homologs in Arabidopsis reveals the importance of the plastidial stringent response in the interplay between chloroplast metabolism and plant defense response.

The highly phosphorylated nucleotide, guanosine tetraphosphate (ppGpp), functions as a secondary messenger in bacteria and chloroplasts. The accumulation of ppGpp alters plastidial gene expression and metabolism, which are required for proper photosynthetic regulation and robust plant growth. However, because four plastid-localized ppGpp synthases/hydrolases function redundantly, the impact of the loss of ppGpp-dependent stringent response on plant physiology remains unclear. We used the CRISPR/Cas9 technology to generate an Arabidopsis thaliana mutant lacking all four ppGpp synthases/hydrolases, and characterized its phenotype. The mutant showed over 20-fold less ppGpp levels than the wild type (WT) under normal growth conditions, and exhibited leaf chlorosis and increased expression of defense-related genes as well as salicylic acid and jasmonate levels upon transition to nitrogen-starvation conditions. These results demonstrate that proper levels of ppGpp in plastids are required for controlling not only plastid metabolism but also phytohormone signaling, which is essential for plant defense.

The YmgB-SpoT interaction triggers the stringent response in Escherichia coli.

Virtually all bacterial species synthesize (p)ppGpp (guanosine penta- or tetraphosphate), a pleiotropic regulator of the so-called stringent response, which controls many aspects of cellular physiology and metabolism. In Escherichia coli, (p)ppGpp levels are controlled by two homologous enzymes: the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified several protein candidates that can modulate (p)ppGpp levels in E. coli. In this work, we show that the putative two-component system connector protein YmgB can promote SpoT-dependent accumulation of ppGpp in E. coli. Importantly, we determined that the control of SpoT activities by YmgB is independent of its proposed role in the two-component Rcs system, and these two functions can be uncoupled. Using genetic and structure-function analysis, we show that the regulation of SpoT activities by YmgB occurs by functional and direct binding in vivo and in vitro to the TGS and Helical domains of SpoT. These results further support the role of these domains in controlling the reciprocal enzymatic states.

Trichoderma viride Colonizes the Roots of Brassica napus L., Alters the Expression of Stress-Responsive Genes, and Increases the Yield of Canola under Field Conditions during Drought.

In this work, we present the results of the inoculation of canola seeds (Brassica napus L.) with Trichoderma viride strains that promote the growth of plants. Seven morphologically different strains of T. viride (TvI-VII) were shown to be capable of synthesizing auxins and exhibited cellulolytic and pectinolytic activities. To gain a deeper insight into the molecular mechanisms underlying canola-T. viride interactions, we analyzed the canola stress genes metallothioneins (BnMT1-3) and stringent response genes (BnRSH1-3 and BnCRSH). We demonstrated the presence of cis-regulatory elements responsive to fungal elicitors in the promoter regions of B. napus MT and RSH genes and observed changes in the levels of the transcripts of the above-mentioned genes in response to root colonization by the tested fungal strains. Of the seven tested strains, under laboratory conditions, T. viride VII stimulated the formation of roots and the growth of canola seedlings to the greatest extent. An experiment conducted under field conditions during drought showed that the inoculation of canola seeds with a suspension of T. viride VII spores increased yield by 16.7%. There was also a positive effect of the fungus on the height and branching of the plants, the number of siliques, and the mass of a thousand seeds. We suggest that the T. viride strain TvVII can be used in modern sustainable agriculture as a bioinoculant and seed coating to protect B. napus from drought.

The transcription factor DksA exerts opposing effects on cell division depending on the presence of ppGpp.

Bacterial cell size is a multifactorial trait that is influenced by variables including nutritional availability and the timing of cell division. Prior work revealed a negative correlation between concentration of the alarmone (p)ppGpp (ppGpp) and cell length in Escherichia coli, suggesting that ppGpp may promote assembly of the division machinery (divisome) and cytokinesis in this organism. To clarify this counterintuitive connection between a starvation-induced stress response effector and cell proliferation, we undertook a systematic analysis of growth and division in E. coli cells defective in ppGpp synthesis and/or engineered to overproduce the alarmone. Our data indicate that ppGpp acts indirectly on divisome assembly through its role as a global mediator of transcription. Loss of either ppGpp (ppGpp0) or the ppGpp-associated transcription factor DksA led to increased average length, with ppGpp0 mutants also exhibiting a high frequency of extremely long filamentous cells. Using heat-sensitive division mutants and fluorescently labeled division proteins, we confirmed that ppGpp and DksA are cell division activators. We found that ppGpp and DksA regulate division through their effects on transcription, although the lack of known division genes or regulators in available transcriptomics data strongly suggests that this regulation is indirect. We also found that DksA inhibits division in ppGpp0 cells, contrary to its role in a wild-type background. We propose that the ability of ppGpp to switch DksA from a division inhibitor to a division activator helps tune cell length across different concentrations of ppGpp. IMPORTANCE Cell division is a key step in the bacterial lifecycle that must be appropriately regulated to ensure survival. This work identifies the alarmone (p)ppGpp (ppGpp) as a general regulator of cell division, extending our understanding of the role of ppGpp beyond a signal for starvation and other stress. Even in nutrient-replete conditions, basal levels of ppGpp are essential for division to occur appropriately and for cell size to be maintained. This study establishes ppGpp as a "switch" that controls whether the transcription factor DksA behaves as a division activator or inhibitor. This unexpected finding enhances our understanding of the complex regulatory mechanisms employed by bacteria to coordinate division with diverse aspects of cell growth and stress response. Because division is an essential process, a better understanding of the mechanisms governing the assembly and activation of the division machinery could contribute to the development of novel therapeutics to treat bacterial infections.

Stringent Response of Cyanobacteria and Other Bacterioplankton during Different Stages of a Harmful Cyanobacterial Bloom.

We conducted a field study to investigate the role of stringent response in cyanobacteria and coexisting bacterioplankton during nutrient-deprived periods at various stages of bloom in a freshwater lake (Utah Lake) for the first time. Using metagenomics and metatranscriptomics analyses, we examined the cyanobacterial ecology and expression of important functional genes related to stringent response, N and P metabolism, and regulation. Our findings mark a significant advancement in understanding the mechanisms by which toxic cyanobacteria survive and proliferate during nitrogen (N) and phosphorus (P) limitations. We successfully identified and analyzed the metagenome-assembled genomes (MAGs) of the dominant bloom-forming cyanobacteria, namely, Dolichospermum circinale, Aphanizomenon flos-aquae UKL13-PB, Planktothrix agardhii, and Microcystis aeruginosa. By mapping RNA-seq data to the coding sequences of the MAGs, we observed that these four prevalent cyanobacteria species activated multiple functions to adapt to the depletion of inorganic nutrients. During and after the blooms, the four dominant cyanobacteria species expressed high levels of transcripts related to toxin production, such as microcystins (mcy), anatoxins (ana), and cylindrospermopsins (cyr). Additionally, genes associated with polyphosphate (poly-P) storage and the stringent response alarmone (p)ppGpp synthesis/hydrolysis, including ppk, relA, and spoT, were highly activated in both cyanobacteria and bacterioplankton. Under N deficiency, the main N pathways shifted from denitrification and dissimilatory nitrate reduction in bacterioplankton toward N2-fixing and assimilatory nitrate reduction in certain cyanobacteria with a corresponding shift in the community composition. P deprivation triggered a stringent response mediated by spoT-dependent (p)ppGpp accumulation and activation of the Pho regulon in both cyanobacteria and bacterioplankton, facilitating inorganic and organic P uptake. The dominant cyanobacterial MAGs exhibited the presence of multiple alkaline phosphatase (APase) transcripts (e.g., phoA in Dolichospermum, phoX in Planktothrix, and Microcystis), suggesting their ability to synthesize and release APase enzymes to convert ambient organic P into bioavailable forms. Conversely, transcripts associated with bacterioplankton-dominated pathways like denitrification were low and did not align with the occurrence of intense cyanoHABs. The strong correlations observed among N, P, stringent response metabolisms and the succession of blooms caused by dominant cyanobacterial species provide evidence that the stringent response, induced by nutrient limitation, may activate unique N and P functions in toxin-producing cyanobacteria, thereby sustaining cyanoHABs.

Clinical rel mutations in Staphylococcus aureus prime pathogen expansion under nutrient stress.

Persistent infection by Staphylococcus aureus has been linked to the bacterial stringent response (SR), a conserved stress response pathway regulated by the Rel protein. Rel synthesizes (p)ppGpp "alarmones" in response to amino acid starvation, which enables adaptation to stress by modulating bacterial growth and virulence. We previously identified five novel protein-altering mutations in rel that arose in patients with persistent methicillin-resistant S. aureus bacteremia. The mutations mapped to both the enzymatic and regulatory protein domains of Rel. Here, we set out to characterize the phenotype of these mutations to understand how they may have been selected in vivo. After introducing each mutation into S. aureus strain JE2, we analyzed growth, fitness, and antibiotic profiles. Despite being located in different protein domains, we found that all of the mutations converged on the same phenotype. Each shortened the time of lag phase growth and imparted a fitness advantage in nutritionally depleted conditions. Through quantification of intracellular (p)ppGpp, we link this phenotype to increased SR activation, specifically during the stationary phase of growth. In contrast to two previously identified clinical rel mutations, we find that our rel mutations do not cause antibiotic tolerance. Instead, our findings suggest that in vivo selection was due to an augmented SR that primes cells for growth in nutrient-poor conditions, which may be a strategy for evading host-imposed nutritional immunity. Importance Host and pathogen compete for available nutrition during infection. For bacteria, the stringent response (SR) regulator Rel responds to amino acid deprivation by signaling the cell to modulate its growth rate, metabolism, and virulence. In this report, we characterize five rel mutations that arose during cases of persistent methicillin-resistant Staphylococcus aureus bacteremia. We find that all of the mutations augmented SR signaling specifically under nutrient-poor conditions, enabling the cell to more readily grow and survive. Our findings reveal a strategy used by bacterial pathogens to evade the nutritional immunity imposed by host tissues during infection.

Coping with stress: How bacteria fine-tune protein synthesis and protein transport.

Maintaining a functional proteome under different environmental conditions is challenging for every organism, in particular for unicellular organisms, such as bacteria. In order to cope with changing environments and stress conditions, bacteria depend on strictly coordinated proteostasis networks that control protein production, folding, trafficking, and degradation. Regulation of ribosome biogenesis and protein synthesis are cornerstones of this cellular adaptation in all domains of life, which is rationalized by the high energy demand of both processes and the increased resistance of translationally silent cells against internal or external poisons. Reduced protein synthesis ultimately also reduces the substrate load for protein transport systems, which are required for maintaining the periplasmic, inner, and outer membrane subproteomes. Consequences of impaired protein transport have been analyzed in several studies and generally induce a multifaceted response that includes the upregulation of chaperones and proteases and the simultaneous downregulation of protein synthesis. In contrast, generally less is known on how bacteria adjust the protein targeting and transport machineries to reduced protein synthesis, e.g., when cells encounter stress conditions or face nutrient deprivation. In the current review, which is mainly focused on studies using Escherichia coli as a model organism, we summarize basic concepts on how ribosome biogenesis and activity are regulated under stress conditions. In addition, we highlight some recent developments on how stress conditions directly impair protein targeting to the bacterial membrane. Finally, we describe mechanisms that allow bacteria to maintain the transport of stress-responsive proteins under conditions when the canonical protein targeting pathways are impaired.

Photodynamic Activity of Chlorophyllin and Polyethylenimine on Pseudomonas aeruginosa Planktonic, Biofilm and Persister Cells.

Antimicrobial photodynamic inactivation is considered a promising antimicrobial approach that may not develop resistance in the near future. Here, we investigate the influence of the photosensitizer chlorophyllin (CHL) and the cationic permeabilizer polyethylenimine (PEI), exposed to a red light-emitting diode, on the human pathogen Pseudomonas aeruginosa free-living planktonic cells, the sessile biofilm and persister cells. The broth microdilution checkerboard method was used to test antimicrobial susceptibility. As a substrate for biofilms, the Calgary biofilm device was used, and the quantification of the biofilm biomass was carried out using a crystal violet assay. Serine hydroxamate was used for the induction of persisters. Our findings reveal that PEI ameliorates the antimicrobial activity of CHL against P. aeruginosa planktonic and biofilm states, and the concentration required to eradicate the bacteria in the biofilm is more than fourfold that is required to eradicate planktonic cells. Interestingly, the persister cells are more susceptible to CHL/PEI (31.25/100 µg mL-1) than the growing cells by 1.7 ± 0.12 and 0.4 ± 0.1 log10 reduction, respectively, after 15 min of illumination. These data demonstrate that CHL excited with red light together with PEI is promising for the eradication of P. aeruginosa, and the susceptibility of P. aeruginosa to CHL/PEI is influenced by the concentrations and the exposure time.

(p)ppGpp and moonlighting RNases influence the first step of lipopolysaccharide biosynthesis in Escherichia coli.

The outer membrane (OM) protects Gram-negative bacteria from harsh environmental conditions and provides intrinsic resistance to many antimicrobial compounds. The asymmetric OM is characterized by phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. Previous reports suggested an involvement of the signaling nucleotide ppGpp in cell envelope homeostasis in Escherichia coli. Here, we investigated the effect of ppGpp on OM biosynthesis. We found that ppGpp inhibits the activity of LpxA, the first enzyme of LPS biosynthesis, in a fluorometric in vitro assay. Moreover, overproduction of LpxA resulted in elongated cells and shedding of outer membrane vesicles (OMVs) with altered LPS content. These effects were markedly stronger in a ppGpp-deficient background. We further show that RnhB, an RNase H isoenzyme, binds ppGpp, interacts with LpxA, and modulates its activity. Overall, our study uncovered new regulatory players in the early steps of LPS biosynthesis, an essential process with many implications in the physiology and susceptibility to antibiotics of Gram-negative commensals and pathogens.

Ribose 5-phosphate: the key metabolite bridging the metabolisms of nucleotides and amino acids during stringent response in Escherichia coli?

The bacterial stringent response and its effector alarmone guanosine penta- or tetra - phosphates (p)ppGpp are vital for bacterial tolerance and survival of various stresses in environments (including antibiotics) and host cells (virulence). (p)ppGpp does so by binding to its numerous target proteins and reprograming bacterial transcriptome to tune down the synthesis of nucleotides and rRNA/tRNA, and up-regulate amino acid biosynthesis genes. Recent identification of more novel (p)ppGpp direct binding proteins in Escherichia coli and their deep studies have unveiled unprecedented details of how (p)ppGpp coordinates the nucleotide and amino acid metabolic pathways upon stringent response; however, the mechanistic link between nucleotide and amino acid metabolisms remains still incompletely understood. Here we propose the metabolite ribose 5'-phosphate as the key link between nucleotide and amino acid metabolisms and a working model integrating both the transcriptional and metabolic effects of (p)ppGpp on E. coli physiological adaptation during the stringent response.

New seed coating containing Trichoderma viride with anti-pathogenic properties.

Background: To ensure food security in the face of climate change and the growing world population, multi-pronged measures should be taken. One promising approach uses plant growth-promoting fungi (PGPF), such as Trichoderma, to reduce the usage of agrochemicals and increase plant yield, stress tolerance, and nutritional value. However, large-scale applications of PGPF have been hampered by several constraints, and, consequently, usage on a large scale is still limited. Seed coating, a process that consists of covering seeds with low quantities of exogenous materials, is gaining attention as an efficient and feasible delivery system for PGPF. Methods: We have designed a new seed coating composed of chitin, methylcellulose, and Trichoderma viride spores and assessed its effect on canola (Brassica napus L.) growth and development. For this purpose, we analyzed the antifungal activity of T. viride against common canola pathogenic fungi (Botrytis cinerea, Fusarium culmorum, and Colletotrichum sp.). Moreover, the effect of seed coating on germination ratio and seedling growth was evaluated. To verify the effect of seed coating on plant metabolism, we determined superoxide dismutase (SOD) activity and expression of the stress-related RSH (RelA/SpoT homologs). Results: Our results showed that the T. viride strains used for seed coating significantly restricted the growth of all three pathogens, especially F. culmorum, for which the growth was inhibited by over 40%. Additionally, the new seed coating did not negatively affect the ability of the seeds to complete germination, increased seedling growth, and did not induce the plant stress response. To summarize, we have successfully developed a cost-effective and environmentally responsible seed coating, which will also be easy to exploit on an industrial scale.

Persistence of obligate intracellular pathogens: alternative strategies to overcome host-specific stresses.

In adapting to the intracellular niche, obligate intracellular bacteria usually undergo a reduction of genome size by eliminating genes not needed for intracellular survival. These losses can include, for example, genes involved in nutrient anabolic pathways or in stress response. Living inside a host cell offers a stable environment where intracellular bacteria can limit their exposure to extracellular effectors of the immune system and modulate or outright inhibit intracellular defense mechanisms. However, highlighting an area of vulnerability, these pathogens are dependent on the host cell for nutrients and are very sensitive to conditions that limit nutrient availability. Persistence is a common response shared by evolutionarily divergent bacteria to survive adverse conditions like nutrient deprivation. Development of persistence usually compromises successful antibiotic therapy of bacterial infections and is associated with chronic infections and long-term sequelae for the patients. During persistence, obligate intracellular pathogens are viable but not growing inside their host cell. They can survive for a long period of time such that, when the inducing stress is removed, reactivation of their growth cycles resumes. Given their reduced coding capacity, intracellular bacteria have adapted different response mechanisms. This review gives an overview of the strategies used by the obligate intracellular bacteria, where known, which, unlike model organisms such as E. coli, often lack toxin-antitoxin systems and the stringent response that have been linked to a persister phenotype and amino acid starvation states, respectively.

Quantitative Physiology and Proteome Adaptations of Bifidobacterium breve NRBB57 at Near-Zero Growth Rates.

In natural environments, nutrients are usually scarce, causing microorganisms to grow slowly while staying metabolically active. These natural conditions can be simulated using retentostat cultivations. The present study describes the physiological and proteome adaptations of the probiotic Bifidobacterium breve NRBB57 from high (0.4 h-1) to near-zero growth rates. Lactose-limited retentostat cultivations were carried out for 21 days in which the bacterial growth rate progressively reduced to 0.00092 h-1, leading to a 3.4-fold reduction of the maintenance energy requirement. Lactose was mainly converted into acetate, formate, and ethanol at high growth rates, while in the retentostat, lactate production increased. Interestingly, the consumption of several amino acids (serine, aspartic acid, and glutamine/arginine) and glycerol increased over time in the retentostat. Morphological changes and viable but nonculturable cells were also observed in the retentostat. Proteomes were compared for all growth rates, revealing a downregulation of ribosomal proteins at near-zero growth rates and an upregulation of proteins involved in the catabolism of alternative energy sources. Finally, we observed induction of the stringent response and stress defense systems. Retentostat cultivations were proven useful to study the physiology of B. breve, mimicking the nutrient scarcity of its complex habitat, the human gut. IMPORTANCE In natural environments, nutrients are usually scarce, causing microorganisms to grow slowly while staying metabolically active. In this study we used retentostat cultivation to investigate how the probiotic Bifidobacterium breve adapts its physiology and proteome under severe nutrient limitation resulting in near-zero growth rates (<0.001 h-1). We showed that the nutrient limitation induced a multifaceted response including stress defense and stringent response, metabolic shifts, and the activation of novel alternative energy-producing pathways.