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CT554 is expressed throughout the entire chlamydial developmental cycle. HeLa cells were infected using an MOI of 1, and total RNA was prepared at 2, 8, 16, 24, and 44 h p.i. using Trizol for analysis by quantitative real-time RT-PCR. The expression of CT554 was normalized to chlamydial 16S rRNA, and the average mRNA levels relative to the level at 24 h p.i. are shown; the error bars indicate standard errors. Experiments were done in duplicate.
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Chlamydiaceae are obligate intracellular bacterial pathogens that strictly depend on host metabolites, such as nucleotides, lipids, and amino acids. Depletion of amino acids in cell culture media results in abnormal chlamydial development in vitro. Surprisingly, enrichment of certain amino acids also retards chlamydial growth. Our experiments revea...
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Membrane proteins comprise an important class of molecules whose study is largely frustrated by several intrinsic constraints, such as their hydrophobicity and added requirements for correct folding. Additionally, the complexity of the cellular mechanisms that are required to insert membrane proteins functionally in the membrane and to monitor thei...
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... Finally, sip2 was sensitive to high concentrations of isoleucine. To assess if amino acid metabolism is altered in sip2, we modified an approach that was used previously to identify substrates of a chlamydial BCAA transporter, BrnQ (24). Braun noted that high concentrations of isoleucine, leucine, methionine, and phenylalanine block chlamydial development because they antagonize BrnQ-mediated import of valine, an amino acid Chlamydia spp. ...
Many obligate intracellular bacteria, including members of the genus Chlamydia, cannot synthesize a variety of amino acids de novo and acquire these from host cells via largely unknown mechanisms. Previously, we determined that a missense mutation in ctl0225, a conserved Chlamydia open reading frame of unknown function, mediated sensitivity to interferon gamma. Here, we show evidence that CTL0225 is a member of the SnatA family of neutral amino acid transporters that contributes to the import of several amino acids into Chlamydia cells. Further, we show that CTL0225 orthologs from two other distantly related obligate intracellular pathogens (Coxiella burnetii and Buchnera aphidicola) are sufficient to import valine into Escherichia coli. We also show that chlamydia infection and interferon exposure have opposing effects on amino acid metabolism, potentially explaining the relationship between CTL0225 and interferon sensitivity. Overall, we show that phylogenetically diverse intracellular pathogens use an ancient family of amino acid transporters to acquire host amino acids and provide another example of how nutritional virulence and immune evasion can be linked in obligate intracellular pathogens.
... Earlier studies on brnQ revealed its main role in leucine, isoleucine, and valine uptake in E. coli and other organisms (Saier et al., 2016). Further, it was reported that valine uptake by E. coli brnQ was competitively inhibited by threonine (Braun et al., 2008), indicating that threonine is a possible substrate. The results provided here support this hypothesis due to the impaired and the rapid decline in dye accumulation in the wild type and RE-KITd mutant, respectively. ...
Membrane transport proteins are potential targets for medical and biotechnological applications. However, more than 30% of reported membrane transporter families are either poorly characterized or lack adequate functional annotation. Here, adaptive laboratory evolution was leveraged to identify membrane transporters for a set of four amino acids as well as specific mutations that modulate the activities of these transporters. Specifically, Escherichia coli was adaptively evolved under increasing concentrations of L–histidine, L–phenylalanine, L–threonine, and L–methionine separately with multiple replicate evolutions. Evolved populations and isolated clones displayed growth rates comparable to the unstressed ancestral strain at elevated concentrations (four-to six-fold increases) of the targeted amino acids. Whole genome sequencing of the evolved strains revealed a diverse number of key mutations, including SNPs, small deletions, and copy number variants targeting the transporters leuE for histidine, yddG for phenylalanine, yedA for methionine, and brnQ and rhtC for threonine. Reverse engineering of the mutations in the ancestral strain established mutation causality of the specific mutations for the tolerant phenotypes. The functional roles of yedA and brnQ in the transport of methionine and threonine, respectively, are novel assignments and their functional roles were validated using a flow cytometry cellular accumulation assay. To demonstrate how the identified transporters can be leveraged for production, an L–phenylalanine overproduction strain was shown to be a superior producer when the identified yddG exporter was overexpressed. Overall, the results revealed the striking efficiency of laboratory evolution to identify transporters and specific mutational mechanisms to modulate their activities, thereby demonstrating promising applicability in transporter discovery efforts and strain engineering.
... The inhibition of microbial growth by some amino acids has been reported for several strains, such as Escherichia coli [13,24], Thiobacillus neapolitanus [27], Caulobacter crescentus [16], Neisseria gonorrhoeae [5], Chlamydia sp. [7], and even for mammalian cells [15]. ...
Piscirickettsia salmonis is a facultative Gram-negative intracellular bacterium that produces piscirickettsiosis, disease that causes a high negative impact in salmonid cultures. The so-far-unidentified nutritional requirements have hindered its axenic culture at laboratory and industrial scales for the formulation of vaccines. The present study describes the development of a defined culture medium for P. salmonis. The culture medium was formulated through rational design involving auxotrophy test and statistical designs of experiments, considering the genome-scale metabolic reconstruction of P. salmonis reported by our group. The whole optimization process allowed for a twofold increase in biomass and a reduction of about 50% of the amino acids added to the culture medium. The final culture medium contains twelve amino acids, where glutamic acid, threonine and arginine were the main carbon and energy sources, supporting 1.65 g/L of biomass using 6.5 g/L of amino acids in the formulation. These results will contribute significantly to the development of new operational strategies to culture this bacterium for the production of vaccines.
... One possible explanation for these concentration-dependent effects may be that growth medium formulations applied at high concentrations contain large amounts of inhibitory substances-substances that are reduced to noninhibitory levels in dilute medium formulations. For example, a key amino acid transporter in Chlamydia trachomatis can be blocked by nonessential amino acids, preventing the transport of required amino acids, resulting in growth inhibition (51). A similar phenomenon was demonstrated in the extreme marine oligotroph "Candidatus Pelagibacter ubique," where alanine was conditionally required for cell division but abolished growth at higher concentrations (52). ...
Isolating environmental microbes and studying their physiology under controlled conditions are essential aspects of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities—the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes.
... The publication of the first C. trachomatis genome was reported in 1998, and showed an organism with a reduced genome (ca. 1 million bp) that encoded the major metabolic pathways (glycolysis, DNA synthesis, RNA synthesis, protein synthesis, and phospholipid synthesis) found in free-living bacteria, but lacked many genes necessary to synthesize the precursors to these pathways (Stephens et al., 1998). Instead, C. trachomatis encoded a variety of pumps to acquire these precursors such as amino acids, nucleotides, and cofactors from the host (Braun et al., 2008;Bastidas et al., 2013). The genome sequence of C. trachomatis paints the picture of a free-living bacteria that adapted to intracellular growth by stealing metabolic precursors instead of synthesizing them endogenously, but retained the major metabolic pathways. ...
Chlamydia trachomatis is an obligate intracellular pathogen with a reduced genome reflecting its host cell dependent life style. However, C. trachomatis has retained all of the genes required for fatty acid and phospholipid synthesis that are present in free-living bacteria. C. trachomatis assembles its cellular membrane using its own biosynthetic machinery utilizing glucose, isoleucine, and serine. This pathway produces disaturated phospholipid molecular species containing a branched-chain 15-carbon fatty acid in the 2-position, which are distinct from the structures of host phospholipids. The enoyl reductase step (FabI) is a target for antimicrobial drug discovery, and the developmental candidate, AFN-1252, blocks the activity of CtFabI. The x-ray crystal structure of the CtFabI•NADH•AFN-1252 ternary complex reveals the interactions between the drug, protein, and cofactor. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infection cycle reduces infectious titers, and treatment at the time of infection prevents the first cell division. Fatty acid synthesis is essential for C. trachomatis proliferation within its eukaryotic host, and CtFabI is a validated therapeutic target against C. trachomatis.
... In our experiments, the methionine analog AHA was efficiently taken up by EBs in the inclusion; thus, it is likely that at least the BrnQ branched-chain amino acid permease is functional in the EB. BrnQ transports valine, leucine, isoleucine, methionine, and phenylalanine (40). In addition, it has been reported that phenylalanine is taken up by the EB (11). ...
Bacteria of the genus Chlamydia include the significant human pathogens Chlamydia trachomatis and C. pneumoniae. All chlamydiae are obligate intracellular parasites that depend on infection of a host cell and transition through a biphasic developmental cycle. Following host cell invasion by the infectious elementary body (EB), the pathogen transitions to the replicative but noninfectious reticulate body (RB). Differentiation of the RB back to the EB is essential to generate infectious progeny. While the EB form has historically been regarded as metabolically inert, maintenance of infectivity during incubation with specific nutrients has revealed active maintenance of the infectious phenotype. Using transcriptome sequencing, we show that the transcriptome of extracellular EBs incubated under metabolically stimulating conditions does not cluster with germinating EBs but rather with the transcriptome of EBs isolated directly from infected cells. In addition, the transcriptional profile of the extracellular metabolizing EBs more closely resembled that of EB production than germination. Maintenance of infectivity of extracellular EBs was achieved by metabolizing chemically diverse compounds, including glucose 6-phosphate, ATP, and amino acids, all of which can be found in extracellular environments, including mucosal secretions. We further show that the EB cell type actively maintains infectivity in the inclusion after terminal differentiation. Overall, these findings contribute to the emerging understanding that the EB cell form is actively maintained through metabolic processes after terminal differentiation to facilitate prolonged infectivity within the inclusion and under host cell free conditions, for example, following deposition at mucosal surfaces.
IMPORTANCE
Chlamydiae are obligate intracellular Gram-negative bacteria that are responsible for a wide range of diseases in both animal and human hosts. According to the Centers for Disease Control and Prevention, C. trachomatis is the most frequently reported sexually transmitted infection in the United States, costing the American health care system nearly $2.4 billion annually. Every year, there are over 4 million new cases of Chlamydia infections in the United States and an estimated 100 million cases worldwide. To cause disease, Chlamydia must successfully complete its complex biphasic developmental cycle, alternating between an infectious cell form (EB) specialized for initiating entry into target cells and a replicative form (RB) specialized for creating and maintaining the intracellular replication niche. The EB cell form has historically been considered metabolically quiescent, a passive entity simply waiting for contact with a host cell to initiate the next round of infection. Recent studies and data presented here demonstrate that the EB maintains its infectious phenotype by actively metabolizing a variety of nutrients. Therefore, the EB appears to have an active role in chlamydial biology, possibly within multiple environments, such as mucosal surfaces, fomites, and inside the host cell after formation.
... All of the eight prokarya-specific secondary transporter families (Additional file 2: Data S1) were energy-dependent, i.e., dependent on a proton or sodium gradient, or utilizing a combination of electro-and chemical potential of the membrane [46][47][48][49][50][51]. A surprising insight here was related to the six evolutionarily younger secondary transporter families found only in eukaryotes (See Additional file 2: Data S1): four of these, about which we have experimental information, were energy-independent and low-energy-demanding transporter families for bile acid, choline, silicate, and vitamin A. The last two belong to the 4 TMS multidrug endosomal transporter and chloroplast maltose exporter families. ...
Background: Transporter proteins mediate the translocation of substances across the membranes of living cells. Many transport processes are energetically expensive and the cells use 20 to 60% of their energy to power the transportomes. We hypothesized that there may be an evolutionary selection pressure for lower energy transporters.
Results: We performed a genome-wide analysis of the compositional reshaping of the transportomes across the kingdoms of bacteria, archaea, and eukarya. We found that the share of ABC transporters is much higher in bacteria and archaea (ca. 27% of the transportome) than in primitive eukaryotes (13%), algae and plants (10%) and in fungi and animals (5-6%). This decrease is compensated by an increased occurrence of secondary transporters and ion channels. The share of ion channels is particularly high in animals (ca. 30% of the transportome) and algae and plants with (ca. 13%), when compared to bacteria and archaea with only 6-7%. Therefore, our results show a move to a preference for the low-energy-demanding transporters (ion channels and carriers) over the more energy-costly transporter classes (ATP-dependent families, and ABCs in particular) as part of the transition from prokaryotes to eukaryotes. The transportome analysis also indicated seven bacterial species, including Neorickettsia risticii and Neorickettsia sennetsu, as likely origins of the mitochondrion in eukaryotes, based on the phylogenetically restricted presence therein of clear homologues of modern mitochondrial solute carriers.
Conclusions: The results indicate that the transportomes of eukaryotes evolved strongly towards a higher energetic efficiency, as ATP-dependent transporters diminished and secondary transporters and ion channels proliferated. These changes have likely been important in the development of tissues performing energetically costly cellular functions.
... While the biological relevance of trpBA induction during iron starvation is unclear, we reason that Chlamydia could in fact prepare for further immune insult (e.g., IFN-␥ induction of indoleamine 2,3-dioxygenase expression) by increasing intracellular Trp levels. Taking the data together, iron starvation may increase levels of serine, aspartate, glutamate, branched-chain amino acids, and tryptophan, many of which are essential for normal development (56)(57)(58)(59)(60). Amino acid biosynthetic genes were significantly overrepresented (4.38-fold; P value ϭ 0.0464) in the set of differentially expressed midcycle genes as determined by the PANTHER overexpression test (61). ...
Iron is essential for growth and development of Chlamydia. Its long-term starvation in cultured mammalian cells leads to production of aberrant non-infectious chlamydial forms, also known as persistence. Immediate transcriptional responses to iron limitation have not been characterized, leaving a knowledge gap of how Chlamydia regulates its response to changes in iron availability. We used the fast-chelating agent 2,2-Bipyridyl (BPDL) to homogeneously starve Chlamydia trachomatis serovar L2 of iron, starting at 6 or 12h post-infection. Immediate transcriptional responses were monitored after only 3 or 6h of BPDL-treatment, well before formation of aberrant Chlamydia. The first genome-wide transcriptional response of C. trachomatis to iron-starvation was subsequently determined utilizing RNA-sequencing. Only 7% and 8% of the genome was differentially expressed in response to iron-starvation at early and mid-stages of development, respectively. Biological pathway analysis revealed an overarching theme. Synthesis of macromolecular precursors (deoxynucleotides, amino acids, charged tRNAs, and acetyl-coA) was up-regulated, while energy-expensive processes (ABC transport and translation) were down-regulated. A large fraction of differentially down-regulated genes are involved in translation, including ribosome assembly, initiation and termination factors, which could be analogous to the translation down-regulation triggered by stress in other prokaryotes during stringent responses. Additionally, transcriptional up-regulation of DNA repair, oxidative stress, and tryptophan salvage genes reveals a possible coordination of responses to multiple antimicrobial and immunological insults. These responses of replicative-phase Chlamydia to iron-starvation indicate a prioritization of survival over replication, enabling the pathogen to stock the pantry with ingredients needed for rapid growth once optimal iron levels are restored.
... Bacteria acquire BCAAs via dedicated active transporters, including BrnQ (Gram-negative and-positive bacteria), BcaP (Gram-positive bacteria), and the high affinity ATP-Binding Cassette (ABC) transporter LIV-I (Gram-negative bacteria) [23][24][25][26][27][28][29][30][31][32][33][34][35][36]. S. aureus encodes three BrnQ homologs (BrnQ1, BrnQ2, BrnQ3), and BcaP. ...
Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed S. aureus uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that S. aureus can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a trans-acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a cis-acting leucine-responsive attenuator, uncovering how S. aureus regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in S. aureus, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on S. aureus environmental adaptation and virulence via CodY.
... All of the eight prokarya-specific secondary transporter families (Additional file 2: Data S1) were energy-dependent, i.e., dependent on a proton or sodium gradient, or utilizing a combination of electro-and chemical potential of the membrane [46][47][48][49][50][51]. A surprising insight here was related to the six evolutionarily younger secondary transporter families found only in eukaryotes (See Additional file 2: Data S1): four of these, about which we have experimental information, were energy-independent and low-energy-demanding transporter families for bile acid, choline, silicate, and vitamin A. The last two belong to the 4 TMS multidrug endosomal transporter and chloroplast maltose exporter families. ...
Transporter proteins mediate the translocation of substances across the membranes of living cells. We performed a genome-wide analysis of the compositional reshaping of cellular transporters (the transportome) across the kingdoms of bacteria, archaea, and eukarya. We show that the transportomes of eukaryotes evolved strongly towards a higher energetic efficiency, as ATP-dependent transporters diminished and secondary transporters and ion channels proliferated. This change has likely been important in the development of tissues performing energetically costly cellular functions. The transportome analysis also indicated seven bacterial species, including Neorickettsia risticii and Neorickettsia sennetsu , as likely origins of the mitochondrion in eukaryotes, due to the restricted presence therein of clear homologues of modern mitochondrial solute carriers.
