Synthetic redesign of Escherichia coli W for faster metabolism of sugarcane molasses.

BACKGROUND: Sugarcane molasses, rich in sucrose, glucose, and fructose, offers a promising carbon source for industrial fermentation due to its abundance and low cost. However, challenges arise from the simultaneous utilization of multiple sugars and carbon catabolite repression (CCR). Despite its nutritional content, sucrose metabolism in Escherichia coli, except for W strain, remains poorly understood, hindering its use in microbial fermentation. In this study, E. coli W was engineered to enhance sugar consumption rates and overcome CCR. This was achieved through the integration of a synthetically designed csc operon and the optimization of glucose and fructose co-utilization pathways. These advancements facilitate efficient utilization of sugarcane molasses for the production of 3-hydroxypropionic acid (3-HP), contributing to sustainable biochemical production processes. RESULTS: In this study, we addressed challenges associated with sugar metabolism in E. coli W, focusing on enhancing sucrose consumption and improving glucose-fructose co-utilization. Through targeted engineering of the sucrose utilization system, we achieved accelerated sucrose consumption rates by modulating the expression of the csc operon components, cscB, cscK, cscA, and cscR. Our findings revealed that monocistronic expression of the csc genes with the deletion of cscR, led to optimal sucrose utilization without significant growth burden. Furthermore, we successfully alleviated fructose catabolite repression by modulating the binding dynamics of FruR with the fructose PTS regulon, enabling near-equivalent co-utilization of glucose and fructose. To validate the industrial applicability of our engineered strain, we pursued 3-HP production from sugarcane molasses. By integrating heterologous genes and optimizing metabolic pathways, we achieved improvements in 3-HP titers compared to previous studies. Additionally, glyceraldehyde-3-phosphate dehydrogenase (gapA) repression aids in carbon flux redistribution, enhancing molasses conversion to 3-HP. CONCLUSIONS: Despite limitations in sucrose metabolism, the redesigned E. coli W strain, adept at utilizing sugarcane molasses, is a valuable asset for industrial fermentation. Its synthetic csc operon enhances sucrose consumption, while mitigating CCR improves glucose-fructose co-utilization. These enhancements, coupled with repression of gapA, aim to efficiently convert sugarcane molasses into 3-HP, addressing limitations in sucrose and fructose metabolism for industrial applications.

Exploring the transcription start sites and other genomic features facilitates the accurate identification and annotation of small RNAs across multiple stress conditions in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) is a pathogen that is known for its ability to persist in harsh environments and cause chronic infections. Understanding the regulatory networks of MTB is crucial for developing effective treatments. Small regulatory RNAs (sRNAs) play important roles in gene expression regulation in all kingdoms of life, and their classification based solely on genomic location can be imprecise due to the computational-based prediction of protein-coding genes in bacteria, which often neglects segments of mRNA such as 5'UTRs, 3'UTRs, and intercistronic regions of operons. To address this issue, our study simultaneously discovered genomic features such as TSSs, UTRs, and operons together with sRNAs in the M. tuberculosis H37Rv strain (ATCC 27294) across multiple stress conditions. Our analysis identified 1,376 sRNA candidates and 8,173 TSSs in MTB, providing valuable insights into its complex regulatory landscape. TSS mapping enabled us to classify these sRNAs into more specific categories, including promoter-associated sRNAs, 5'UTR-derived sRNAs, 3'UTR-derived sRNAs, true intergenic sRNAs, and antisense sRNAs. Three of these sRNA candidates were experimentally validated using 3'-RACE-PCR: predictedRNA_0240, predictedRNA_0325, and predictedRNA_0578. Future characterization and validation are necessary to fully elucidate the functions and roles of these sRNAs in MTB. Our study is the first to simultaneously unravel TSSs and sRNAs in MTB and demonstrate that the identification of other genomic features, such as TSSs, UTRs, and operons, allows for more accurate and specific classification of sRNAs.

Enzymatic promiscuity and underground reactions accounted for the capability of Escherichia coli to use the non-natural chemical synthon 2,4-dihydroxybutyric acid as a carbon source for growth.

2,4-dihydroxybutyric acid (DHB) and 2-keto-4-hydroxybutyrate (OHB) are non-natural molecules obtained through synthetic pathways from renewable carbon source. As they are structurally similar to lactate and pyruvate respectively, they could possibly interfere with the metabolic network of Escherichia coli. In fact, we showed that DHB can be easily oxidized by the membrane associated L and D-lactate dehydrogenases encoded by lldD, dld and ykgF into OHB, and the latter being cleaved into pyruvate and formaldehyde by several pyruvate-dependent aldolases, with YagE being the most effective. While formaldehyde was readily detoxified into formate, Escherichia coli K12 MG1655 strain failed to grow on DHB despite of the production of pyruvate. To find out the reason for this failure, we constructed a mutant strain whose growth was rendered dependent on DHB and subjected this strain to adaptive evolution. Genome sequencing of the adapted strain revealed an essential role for ygbI encoding a transcriptional repressor of the threonate operon in this DHB-dependent growth. This critical function was attributed to the derepression of ygbN encoding a putative threonate transporter, which was found to exclusively transport the D form of DHB. A subsequent laboratory evolution was carried out with E. coli K12 MG1655 deleted for ΔygbI to adapt for growth on DHB as sole carbon source. Remarkably, only two additional mutations were disclosed in the adapted strain, which were demonstrated by reverse engineering to be necessary and sufficient for robust growth on DHB. One mutation was in nanR encoding the transcription repressor of sialic acid metabolic genes, causing 140-fold increase in expression of nanA encoding N-acetyl neuraminic acid lyase, a pyruvate-dependent aldolase, and the other was in the promoter of dld leading to 14-fold increase in D-lactate dehydrogenase activity on DHB. Taken together, this work illustrates the importance of promiscuous enzymes in underground metabolism and moreover, in the frame of synthetic pathways aiming at producing non-natural products, these underground reactions could potentially penalize yield and title of these bio-based products.

CyAbrB2 is a nucleoid-associated protein in Synechocystis controlling hydrogenase expression during fermentation.

The hox operon in Synechocystis sp. PCC 6803, encoding bidirectional hydrogenase responsible for H2 production, is transcriptionally upregulated under microoxic conditions. Although several regulators for hox transcription have been identified, their dynamics and higher-order DNA structure of hox region in microoxic conditions remain elusive. We focused on key regulators for the hox operon: cyAbrB2, a conserved regulator in cyanobacteria, and SigE, an alternative sigma factor. Chromatin immunoprecipitation sequencing revealed that cyAbrB2 binds to the hox promoter region under aerobic conditions, with its binding being flattened in microoxic conditions. Concurrently, SigE exhibited increased localization to the hox promoter under microoxic conditions. Genome-wide analysis revealed that cyAbrB2 binds broadly to AT-rich genome regions and represses gene expression. Moreover, we demonstrated the physical interactions of the hox promoter region with its distal genomic loci. Both the transition to microoxic conditions and the absence of cyAbrB2 influenced the chromosomal interaction. From these results, we propose that cyAbrB2 is a cyanobacterial nucleoid-associated protein (NAP), modulating chromosomal conformation, which blocks RNA polymerase from the hox promoter in aerobic conditions. We further infer that cyAbrB2, with altered localization pattern upon microoxic conditions, modifies chromosomal conformation in microoxic conditions, which allows SigE-containing RNA polymerase to access the hox promoter. The coordinated actions of this NAP and the alternative sigma factor are crucial for the proper hox expression in microoxic conditions. Our results highlight the impact of cyanobacterial chromosome conformation and NAPs on transcription, which have been insufficiently investigated.

A 3'UTR-derived small RNA represses pneumolysin synthesis and facilitates pneumococcal brain invasion.

Pneumolysin (Ply) of Streptococcus pneumoniae (pneumococcus) at relatively high and low levels facilitates pneumococcal invasion into the lung and brain, respectively; however, the regulatory mechanisms of Ply expression are poorly understood. Here, we find that a small RNA plyT, processed from the 3'UTR of the ply operon, is expressed higher in anaerobically- than in statically-cultured pneumococcus D39. Using bioinformatic, biochemical and genetic approaches, we reveal that PlyT inhibits Ply synthesis and hemolytic activities by pairing with an RBS-embedded intergenic region of the ply operon. The RNA-binding protein SPD_1558 facilitates the pairing. Importantly, PlyT inhibition of Ply synthesis is stronger in anaerobic culture and leads to lower Ply abundance. Deletion of plyT decreases the number of pneumococci in the infected mouse brain and reduces the virulence, demonstrating that PlyT-regulated lower Ply in oxygen-void microenvironments, such as the blood, is important for pneumococcus to cross the blood-brain barrier and invade the brain. PlyT-mediated repression of Ply synthesis at anoxic niches is also verified in pneumococcal serotype 4 and 14 strains; moreover, the ply operon with a 3'UTR-embedded plyT, and the pairing sequences of IGR and plyT are highly conserved among pneumococcal strains, implying PlyT-regulated Ply synthesis might be widely employed by pneumococcus.

Metalloproteomics Reveals Multi-Level Stress Response in Escherichia coli When Exposed to Arsenite.

The arsRBC operon encodes a three-protein arsenic resistance system. ArsR regulates the transcription of the operon, while ArsB and ArsC are involved in exporting trivalent arsenic and reducing pentavalent arsenic, respectively. Previous research into Agrobacterium tumefaciens 5A has demonstrated that ArsR has regulatory control over a wide range of metal-related proteins and metabolic pathways. We hypothesized that ArsR has broad regulatory control in other Gram-negative bacteria and set out to test this. Here, we use differential proteomics to investigate changes caused by the presence of the arsR gene in human microbiome-relevant Escherichia coli during arsenite (AsIII) exposure. We show that ArsR has broad-ranging impacts such as the expression of TCA cycle enzymes during AsIII stress. Additionally, we found that the Isc [Fe-S] cluster and molybdenum cofactor assembly proteins are upregulated regardless of the presence of ArsR under these same conditions. An important finding from this differential proteomics analysis was the identification of response mechanisms that were strain-, ArsR-, and arsenic-specific, providing new clarity to this complex regulon. Given the widespread occurrence of the arsRBC operon, these findings should have broad applicability across microbial genera, including sensitive environments such as the human gastrointestinal tract.

CyuR is a dual regulator for L-cysteine dependent antimicrobial resistance in Escherichia coli.

Hydrogen sulfide (H2S), mainly produced from L-cysteine (Cys), renders bacteria highly resistant to oxidative stress and potentially increases antimicrobial resistance (AMR). CyuR is a Cys-dependent transcription regulator, responsible for the activation of the cyuPA operon and generation of H2S. Despite its potential importance, its regulatory network remains poorly understood. In this study, we investigate the roles of the CyuR regulon in a Cys-dependent AMR mechanism in E. coli strains. We show: (1) Generation of H2S from Cys affects the sensitivities to growth inhibitors; (2) Cys supplementation decreases stress responses; (3) CyuR negatively controls the expression of mdlAB encoding a potential transporter for antibiotics; (4) CyuR binds to a DNA sequence motif 'GAAwAAATTGTxGxxATTTsyCC' in the absence of Cys; and (5) CyuR may regulate 25 additional genes which were not reported previously. Collectively, our findings expand the understanding of the biological roles of CyuR relevant to antibiotic resistance associated with Cys.

Tad pili contribute to the virulence and biofilm formation of virulent Aeromonas hydrophila.

Type IV pili (T4P) are versatile proteinaceous protrusions that mediate diverse bacterial processes, including adhesion, motility, and biofilm formation. Aeromonas hydrophila, a Gram-negative facultative anaerobe, causes disease in a wide range of hosts. Previously, we reported the presence of a unique Type IV class C pilus, known as tight adherence (Tad), in virulent Aeromonas hydrophila (vAh). In the present study, we sought to functionalize the role of Tad pili in the pathogenicity of A. hydrophila ML09-119. Through a comprehensive comparative genomics analysis of 170 A. hydrophila genomes, the conserved presence of the Tad operon in vAh isolates was confirmed, suggesting its potential contribution to pathogenicity. Herein, the entire Tad operon was knocked out from A. hydrophila ML09-119 to elucidate its specific role in A. hydrophila virulence. The absence of the Tad operon did not affect growth kinetics but significantly reduced virulence in catfish fingerlings, highlighting the essential role of the Tad operon during infection. Biofilm formation of A. hydrophila ML09-119 was significantly decreased in the Tad operon deletant. Absence of the Tad operon had no effect on sensitivity to other environmental stressors, including hydrogen peroxide, osmolarity, alkalinity, and temperature; however, it was more sensitive to low pH conditions. Scanning electron microscopy revealed that the Tad mutant had a rougher surface structure during log phase growth than the wildtype strain, indicating the absence of Tad impacts the outer surface of vAh during cell division, of which the biological consequences are unknown. These findings highlight the role of Tad in vAh pathogenesis and biofilm formation, signifying the importance of T4P in bacterial infections.

Pseudomonas aeruginosa mucinous phenotypes and algUmucABD operon mutant characteristics obtained from inpatients with bronchiectasis and their correlation with acute aggravation.

Objective: Although the mechanism is unclear, Pseudomonas aeruginosa (PA) infection directly affects the frequency of acute exacerbations in patients with bronchiectasis. The aims of this article are to analyze the genetic mutation characteristics of the algUmucABD operon in PA, isolated from hospitalized patients with bronchiectasis, and to explore independent risk factors for frequent acute exacerbations of bronchiectasis. Methods: Based on the number of acute exacerbations that occurred in the past year, these patients with bronchiectasis were divided into those with frequent acute exacerbations (Group A) and those with non-frequent acute exacerbations (Group B). We identified the distribution of mucoid phenotypes (MPs) and alginate morphotypes (AMs) in PA, and classified them into I-IV categories based on their different AMs; otherwise, the gene mutation types (GMTs) of the algUmucABD operon were tested. Subsequently, the relationship between GMT, MP, and AM and the independent risk factors for frequent acute exacerbations in patients with bronchiectasis were explored. Results: A total of 93 patients and 75 PA strains, from January 2019 to August 2023, were included in this study. The MP and AM distributions of PA were as follows: 64 strains (85.33%) of mucoid (the AMs were 38 strains of type I, 3 strains of type II, and 23 strains of type IV) and 11 strains of non-mucoid (the AM was type III only). Mucoid PA with algU, mucA, mucB, and mucD mutations accounted for 19.61%, 74.51%, 31.37%, and 50.98%, respectively. GMT was divided into the following: mucA mutations only, mucA combined with other gene mutations, other gene mutations without mucA mutations, and without gene mutations. In 91.7% of PA with type I of AM, only mucA mutations occurred, and in both separate MP and AM, the GMT differences were statistically significant. Lastly, the number of lung lobes with bronchiectasis and the number of PA with mucA mutations only were the independent risk factors for frequent acute exacerbations. Conclusion: The mucA mutation was primarily responsible for the mucoid of MP and type I of AM in PA, and it was also an independent risk factor for frequent exacerbations of bronchiectasis.

Role of the polyamine transporter PotABCD during biofilm formation by Streptococcus pneumoniae.

Streptococcus pneumoniae is a bacterium of great global importance, responsible for more than one million deaths per year. This bacterium is commonly acquired in the first years of life and colonizes the upper respiratory tract asymptomatically by forming biofilms that persist for extended times in the nasopharynx. However, under conditions that alter the bacterial environment, such as viral infections, pneumococci can escape from the biofilm and invade other niches, causing local and systemic disease of varying severity. The polyamine transporter PotABCD is required for optimal survival of the organism in the host. Immunization of mice with recombinant PotD can reduce subsequent bacterial colonization. PotD has also been suggested to be involved in pneumococcal biofilm development. Therefore, in this study we aimed to elucidate the role of PotABCD and polyamines in pneumococcal biofilm formation. First, the formation of biofilms was evaluated in the presence of exogenous polyamines-the substrate transported by PotABCD-added to culture medium. Next, a potABCD-negative strain was used to determine biofilm formation in different model systems using diverse levels of complexity from abiotic surface to cell substrate to in vivo animal models and was compared with its wild-type strain. The results showed that adding more polyamines to the medium stimulated biofilm formation, suggesting a direct correlation between polyamines and biofilm formation. Also, deletion of potABCD operon impaired biofilm formation in all models tested. Interestingly, more differences between wild-type and mutant strains were observed in the more complex model, which emphasizes the significance of employing more physiological models in studying biofilm formation.

Strain-dependent induction of primary bile acid 7-dehydroxylation by cholic acid.

BACKGROUND: Bile acids (BAs) are steroid-derived molecules with important roles in digestion, the maintenance of host metabolism, and immunomodulation. Primary BAs are synthesized by the host, while secondary BAs are produced by the gut microbiome through transformation of the former. The regulation of microbial production of secondary BAs is not well understood, particularly the production of 7-dehydroxylated BAs, which are the most potent agonists for host BA receptors. The 7-dehydroxylation of cholic acid (CA) is well established and is linked to the expression of a bile acid-inducible (bai) operon responsible for this process. However, little to no 7-dehydroxylation has been reported for other host-derived BAs (e.g., chenodeoxycholic acid, CDCA or ursodeoxycholic acid, UDCA). RESULTS: Here, we demonstrate that the 7-dehydroxylation of CDCA and UDCA by the human isolate Clostridium scindens is induced when CA is present, suggesting that CA-dependent transcriptional regulation is required for substantial 7-dehydroxylation of these primary BAs. This is supported by the finding that UDCA alone does not promote expression of bai genes. CDCA upregulates expression of the bai genes but the expression is greater when CA is present. In contrast, the murine isolate Extibacter muris exhibits a distinct response; CA did not induce significant 7-dehydroxylation of primary BAs, whereas BA 7-dehydroxylation was promoted upon addition of germ-free mouse cecal content in vitro. However, E. muris was found to 7-dehydroxylate in vivo. CONCLUSIONS: The distinct expression responses amongst strains indicate that bai genes are regulated differently. CA promoted bai operon gene expression and the 7-dehydroxylating activity in C. scindens strains. Conversely, the in vitro activity of E. muris was promoted only after the addition of cecal content and the isolate did not alter bai gene expression in response to CA. The accessory gene baiJ was only upregulated in the C. scindens ATCC 35704 strain, implying mechanistic differences amongst isolates. Interestingly, the human-derived C. scindens strains were also capable of 7-dehydroxylating murine bile acids (muricholic acids) to a limited extent. This study shows novel 7-dehydroxylation activity in vitro resulting from the presence of CA and suggests distinct bai gene expression across bacterial species.

Induction of bacterial expression at the mRNA level by light.

Vital organismal processes, including development, differentiation and adaptation, involve altered gene expression. Although expression is frequently controlled at the transcriptional stage, various regulation mechanisms operate at downstream levels. Here, we leverage the photoreceptor NmPAL to optogenetically induce RNA refolding and the translation of bacterial mRNAs. Blue-light-triggered NmPAL binding disrupts a cis-repressed mRNA state, thereby relieves obstruction of translation initiation, and upregulates gene expression. Iterative probing and optimization of the circuit, dubbed riboptoregulator, enhanced induction to 30-fold. Given action at the mRNA level, the riboptoregulator can differentially regulate individual structural genes within polycistronic operons. Moreover, it is orthogonal to and can be wed with other gene-regulatory circuits for nuanced and more stringent gene-expression control. We thus advance the pAurora2 circuit that combines transcriptional and translational mechanisms to optogenetically increase bacterial gene expression by >1000-fold. The riboptoregulator strategy stands to upgrade numerous regulatory circuits and widely applies to expression control in microbial biotechnology, synthetic biology and materials science.

Type 1 fimbria and P pili: regulatory mechanisms of the prototypical members of the chaperone-usher fimbrial family.

Adherence to both cellular and abiotic surfaces is a crucial step in the interaction of bacterial pathogens and commensals with their hosts. Bacterial surface structures known as fimbriae or pili play a fundamental role in the early colonization stages by providing specificity or tropism. Among the various fimbrial families, the chaperone-usher family has been extensively studied due to its ubiquity, diversity, and abundance. This family is named after the components that facilitate their biogenesis. Type 1 fimbria and P pilus, two chaperone-usher fimbriae associated with urinary tract infections, have been thoroughly investigated and serve as prototypes that have laid the foundations for understanding the biogenesis of this fimbrial family. Additionally, the study of the mechanisms regulating their expression has also been a subject of great interest, revealing that the regulation of the expression of the genes encoding these structures is a complex and diverse process, involving both common global regulators and those specific to each operon.

Identification and characterization of a novel type II toxin-antitoxin system in Aeromonas veronii.

The bacterial type II toxin-antitoxin (TA) system is a rich genetic element that participates in various physiological processes. Aeromonas veronii is the main bacterial pathogen threatening the freshwater aquaculture industry. However, the distribution of type II TA system in A. veronii was seldom documented and its roles in the life activities of A. veronii were still unexplored. In this study, a novel type II TA system AvtA-AvtT was predicted in a fish pathogen Aeromonas veronii biovar sobria with multi-drug resistance using TADB 2.0. Through an Escherichia coli host killing and rescue assay, we demonstrated that AvtA and AvtT worked as a genuine TA system, and the predicted toxin AvtT actually functioned as an antitoxin while the predicted antitoxin AvtA actually functioned as a toxin. The binding ability of AvtA with AvtT proteins were confirmed by dot blotting analysis and co-immunoprecipitation assay. Furthermore, we found that the toxin and antitoxin labelled with fluorescent proteins were co-localized. In addition, it was found that the transcription of AvtAT bicistronic operon was repressed by the AvtAT protein complex. Deletion of avtA gene and avtT gene had no obvious effect on the drug susceptibility. This study provides first characterization of type II TA system AvtA-AvtT in aquatic pathogen A. veronii.

Transcriptional regulation of the anaerobic 3-hydroxybenzoate degradation pathway in Aromatoleum sp. CIB.

Phenolic compounds are commonly found in anoxic environments, where they serve as both carbon and energy sources for certain anaerobic bacteria. The anaerobic breakdown of m-cresol, catechol, and certain lignin-derived compounds yields the central intermediate 3-hydroxybenzoate/3-hydroxybenzoyl-CoA. In this study, we have characterized the transcription and regulation of the hbd genes responsible for the anaerobic degradation of 3-hydroxybenzoate in the ß-proteobacterium Aromatoleum sp. CIB. The hbd cluster is organized in three catabolic operons and a regulatory hbdR gene that encodes a dimeric transcriptional regulator belonging to the TetR family. HbdR suppresses the activity of the three catabolic promoters (PhbdN, PhbdE and PhbdH) by binding to a conserved palindromic operator box (ATGAATGAN4TCATTCAT). 3-Hydroxybenzoyl-CoA, the initial intermediate of the 3-hydroxybenzoate degradation pathway, along with benzoyl-CoA, serve as effector molecules that bind to HbdR inducing the expression of the hbd genes. Moreover, the hbd genes are subject to additional regulation influenced by the presence of non-aromatic carbon sources (carbon catabolite repression), and their expression is induced in oxygen-deprived conditions by the AcpR transcriptional activator. The prevalence of the hbd cluster among members of the Aromatoleum/Thauera bacterial group, coupled with its association with mobile genetic elements, suggests acquisition through horizontal gene transfer. These findings significantly enhance our understanding of the regulatory mechanisms governing the hbd gene cluster in bacteria, paving the way for further exploration into the anaerobic utilization/valorization of phenolic compounds derived from lignin.

D-galactonate metabolism in enteric bacteria: a molecular and physiological perspective.

D-galactonate, a widely prevalent sugar acid, was first reported as a nutrient source for enteric bacteria in the 1970s. Since then, decades of research enabled a description of the modified Entner-Doudoroff pathway involved in its degradation and reported the structural and biochemical features of its metabolic enzymes, primarily in Escherichia coli K-12. However, only in the last few years, the D-galactonate transporter has been characterized, and the regulation of the dgo operon, encoding the structural genes for the transporter and enzymes of D-galactonate metabolism, has been detailed. Notably, in recent years, multiple evolutionary studies have identified the dgo operon as a dominant target for adaptation of E. coli in the mammalian gut. Despite considerable research on dgo operon, numerous fundamental questions remain to be addressed. The emerging relevance of the dgo operon in host-bacterial interactions further necessitates the study of D-galactonate metabolism in other enterobacterial strains.

Dual Glycosyltransferases from Campylobacter concisus Diverge from the Canonical Campylobacter N-Linked Glycan Assembly Pathway.

Species within the Campylobacter genus are recognized as emerging human pathogens. Common to all known members of the genus is the presence of an asparagine-linked glycosylation pathway encoded by the pgl operon. Campylobacter species are divided into two major groups, Group I and Group II. To date, most biochemical studies have focused on the Group I species including Campylobacter jejuni. We recently reported that the Group II Campylobacter concisus pathway deviates from that of Group I by the inclusion of a C-6″-oxidized GalNAc (GalNAcA) at the third position installed by PglJ. Herein, we investigate the diversification of the PglH enzymes that act subsequent to installation of GalNAcA. The majority of pgl operons from Group II species, including C. concisus, encode two GT-B fold glycosyltransferases (GTs), PglH1 and PglH2. As the functions of these GTs were not clear by simple comparison of their sequences to that of C. jejuni PglH, further analyses were required. We show that subsequent to the action of PglJ, PglH2 installs the next HexNAc followed by PglH1 adding a single sugar. These steps diverge from the C. jejuni pathway not only in the identity of the sugar donors (UDP-GlcNAc) but also in installing single sugars rather than acting processively. These biochemical studies were extended via bioinformatics to identify sequence signatures that provide predictive capabilities for unraveling the prokaryotic glycan landscape. Phylogenetic analysis showed early divergence between the C. jejuni PglH orthologs and C. concisus PglH1/PglH2 orthologs, leading to diversification of the final glycan.

Enhanced accumulation of γ-aminobutyric acid by deletion of aminotransferase genes involved in γ-aminobutyric acid catabolism in engineered Halomonas elongata.

Halomonas elongata OUT30018 is a moderately halophilic bacterium that synthesizes and accumulates ectoine as an osmolyte by activities of the enzymes encoded by the high salinity-inducible ectABC operon. Previously, we engineered a γ-aminobutyric acid (GABA)-producing H. elongata GOP-Gad (ΔectABC::mCherry-HopGadmut) from an ectoine-deficient mutant of this strain due to its ability to use high-salinity biomass waste as substrate. Here, to further increase GABA accumulation, we deleted gabT, which encodes GABA aminotransferase (GABA-AT) that catalyzes the first step of the GABA catabolic pathway, from the H. elongata GOP-Gad genome. The resulting strain H. elongata ZN3 (ΔectABC::mCherry-HopGadmut ΔgabT) accumulated 291 µmol/g cell dry weight (CDW) of GABA in the cells, which is a 1.5-fold increase from H. elongata GOP-Gad's 190 µmol/g CDW. This result has confirmed the role of GABA-AT in the GABA catabolic pathway. However, redundancy in endogenous GABA-AT activity was detected in a growth test, where a gabT-deletion mutant of H. elongata OUT30018 was cultured in a medium containing GABA as the sole carbon and nitrogen sources. Because L-2,4-diaminobutyric acid aminotransferase (DABA-AT), encoded by an ectB gene of the ectABC operon, shares sequence similarity with GABA-AT, a complementation analysis of the gabT and the ectB genes was performed in the H. elongata ZN3 genetic background to test the involvement of DABA-AT in the redundancy of GABA-AT activity. Our results indicate that the expression of DABA-AT can restore GABA-AT activity in H. elongata ZN3 and establish DABA-AT's aminotransferase activity toward GABA in vivo. IMPORTANCE: In this study, we were able to increase the yield of GABA by 1.5 times in the GABA-producing H. elongata ZN3 strain by deleting the gabT gene, which encodes GABA-AT, the initial enzyme of the GABA catabolic pathway. We also report the first in vivo evidence for GABA aminotransferase activity of an ectB-encoded DABA-AT, confirming a longstanding speculation based on the reported in vitro GABA-AT activity of DABA-AT. According to our findings, the DABA-AT enzyme can catalyze the initial step of GABA catabolism, in addition to its known function in ectoine biosynthesis. This creates a cycle that promotes adequate substrate flow between the two pathways, particularly during the early stages of high-salinity stress response when the expression of the ectB gene is upregulated.

Tail assembly interference is a common strategy in bacterial antiviral defenses.

Many bacterial immune systems recognize phage structural components to activate antiviral responses, without inhibiting the function of the phage component. These systems can be encoded in specific chromosomal loci, known as defense islands, and in mobile genetic elements such as prophages and phage-inducible chromosomal islands (PICIs). Here, we identify a family of bacterial immune systems, named Tai (for 'tail assembly inhibition'), that is prevalent in PICIs, prophages and P4-like phage satellites. Tai systems protect their bacterial host population from other phages by blocking the tail assembly step, leading to the release of tailless phages incapable of infecting new hosts. To prevent autoimmunity, some Tai-positive phages have an associated counter-defense mechanism that is expressed during the phage lytic cycle and allows for tail formation. Interestingly, the Tai defense and counter-defense genes are organized in a non-contiguous operon, enabling their coordinated expression.

Evolution of a bistable genetic system in fluctuating and nonfluctuating environments.

Epigenetic mechanisms can generate bacterial lineages capable of spontaneously switching between distinct phenotypes. Currently, mathematical models and simulations propose epigenetic switches as a mechanism of adaptation to deal with fluctuating environments. However, bacterial evolution experiments for testing these predictions are lacking. Here, we exploit an epigenetic switch in Salmonella enterica, the opvAB operon, to show clear evidence that OpvAB bistability persists in changing environments but not in stable conditions. Epigenetic control of transcription in the opvAB operon produces OpvABOFF (phage-sensitive) and OpvABON (phage-resistant) cells in a reversible manner and may be interpreted as an example of bet-hedging to preadapt Salmonella populations to the encounter with phages. Our experimental observations and computational simulations illustrate the adaptive value of epigenetic variation as an evolutionary strategy for mutation avoidance in fluctuating environments. In addition, our study provides experimental support to game theory models predicting that phenotypic heterogeneity is advantageous in changing and unpredictable environments.