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Metabolites of microorganisms have long been considered as potential sources for drug discovery. In this study, five new depsidone derivatives, talaronins A-E (1–5) and three new xanthone derivatives, talaronins F–H (6–8), together with 16 known compounds (9–24), were isolated from the ethyl acetate extract of the mangrove-derived fungus Talaromyces species WHUF0362. The structures were elucidated by analysis of spectroscopic data and chemical methods including alkaline hydrolysis and Mosher’s method. Compounds 1 and 2 each attached a dimethyl acetal group at the aromatic ring. A putative biogenetic relationship of the isolated metabolites was presented and suggested that the depsidones and the xanthones probably had the same biosynthetic precursors such as chrysophanol or rheochrysidin. The antimicrobial activity assay indicated that compounds 5, 9, 10, and 14 showed potent activity against Helicobacter pylori with minimum inhibitory concentration (MIC) values in the range of 2.42–36.04 μmol/L. While secalonic acid D (19) demonstrated significant antimicrobial activity against four strains of H. pylori with MIC values in the range of 0.20 to 1.57 μmol/L. Furthermore, secalonic acid D (19) exhibited cytotoxicity against cancer cell lines Bel-7402 and HCT-116 with IC50 values of 0.15 and 0.19 μmol/L, respectively. The structure–activity relationship of depsidone derivatives revealed that the presence of the lactone ring and the hydroxyl at C-10 was crucial to the antimicrobial activity against H. pylori. The depsidone derivatives are promising leads to inhibit H. pylori and provide an avenue for further development of novel antibiotics.

Marine fungi are well known for their ability to produce a multitude of natural products and have been proved to be a particularly rich source of drug leads. Here, 20 pyrones and their analogs (1-20), including two new compounds (1 and 6), were obtained from a marine-derived fungus strain of Aspergillus sp. DM94. Their structures were determined by analyses of UV, IR, HR-ESI-MS, and NMR data. The ability to inhibit Helicobacter pylori in vitro was assessed for these isolated compounds. Results showed that the bis-naphtho-γ-pyrones exhibited potent antibacterial activity against both the standard and multidrug-resistant H. pylori strains. Structure-activity relationship (SAR) analysis suggested that the bis-naphtho[2,3-b]pyrones showed better anti-H. pylori activity than a hybrid of naphtho[2,3-b]pyrone and naphtho[1,2-b]pyrone. In addition, the free hydroxyl group of the C-8 position in the lower unit is vital for its anti-H. pylori activity. Importantly, compound 18 showed a synergistic effect in combination with amoxicillin, clarithromycin, or metronidazole, suggesting its potential use to overcome antibiotic resistance of H. pylori. This study shed light on the discovery of new anti-H. pylori agents. KEY POINTS: • New pyrones discovered from a marine-derived fungus Aspergillus sp. DM94. • Bis-naphtho-γ-pyrones showed potent anti-H. pylori activity. • The anti-H. pylori SAR analysis of bis-naphtho-γ-pyrones was discussed. • Bis-naphtho-γ-pyrone 18 showed synergistic effect with clinical antibiotics.

Unsaturated fatty acids (UFAs) are essential for functional membrane phospholipids in most bacteria. The bifunctional dehydrogenase/isomerase FabX is an essential UFA biosynthesis enzyme in the widespread human pathogen Helicobacter pylori, a bacterium etiologically related to 95% of gastric cancers. Here, we present the crystal structures of FabX alone and in complexes with an octanoyl-acyl carrier protein (ACP) substrate or with holo-ACP. FabX belongs to the nitronate monooxygenase (NMO) flavoprotein family but contains an atypical [4Fe-4S] cluster absent in all other family members characterized to date. FabX binds ACP via its positively charged α7 helix that interacts with the negatively charged α2 and α3 helices of ACP. We demonstrate that the [4Fe-4S] cluster potentiates FMN oxidation during dehydrogenase catalysis, generating superoxide from an oxygen molecule that is locked in an oxyanion hole between the FMN and the active site residue His182. Both the [4Fe-4S] and FMN cofactors are essential for UFA synthesis, and the superoxide is subsequently excreted by H. pylori as a major resource of peroxide which may contribute to its pathogenic function in the corrosion of gastric mucosa., Helicobacter pylori FabX, a dehydrogenase/isomerase flavoprotein, is required for unsaturated fatty acid synthesis. Here, the authors characterize FabX substrate recognition and catalytic mechanism, and reveal that it contains an atypical [4Fe-4S] cluster, which is essential and participates in the catalytic cycle.

Helicobacter pylori is a major global pathogen and has been implicated in gastritis, peptic ulcer, and gastric carcinoma. The efficacy of the extensive therapy of H. pylori infection with antibiotics is compromised by the development of drug resistance and toxicity toward human gut microbiota, which urgently demands novel and selective antibacterial strategies. The present study was mainly performed to assess the in vitro and in vivo effects of a natural herbal compound, dihydrotanshinone I (DHT), against standard and clinical H. pylori strains. DHT demonstrated effective antibacterial activity against H. pylori in vitro (MIC50/90, 0.25/0.5 μg/ml), with no development of resistance during continuous serial passaging. Time-kill curves showed strong time-dependent bactericidal activity for DHT. Also, DHT eliminated preformed biofilms and killed biofilm-encased H. pylori cells more efficiently than the conventional antibiotic metronidazole. In mouse models of multidrug-resistant H. pylori infection, dual therapy with DHT and omeprazole showed in vivo killing efficacy superior to that of the standard triple-therapy approach. Moreover, DHT treatment induces negligible toxicity against normal tissues and exhibits a relatively good safety index. These results suggest that DHT could be suitable for use as an anti-H. pylori agent in combination with a proton pump inhibitor to eradicate multidrug-resistant H. pylori.

Acidovorax citrulli is a seedborne pathogen that causes bacterial fruit blotch (BFB), a global threat to watermelon production. Treating watermelon seeds to eliminate A. citrulli is a critical component of BFB management, and several strategies have been evaluated to mitigate the impact of the disease. In China, watermelon seed producers routinely incubate seeds in watermelon juice (fermentation) to reduce the risk of seed infection by A. citrulli and seedling transmission of BFB. However, there has been limited effort to evaluate the efficacy of fermentation in mitigating A. citrulli seed infection. The current study showed that fermented watermelon fruit juice could inhibit A. citrulli population growth and demonstrated that the low pH conditions, not the temperature dynamic, generated during fermentation might play a major role in A. citrulli growth inhibition and could induce the viable but nonculturable (VBNC) state in A. citrulli. We developed an effective method that was based on propidium monoazide PCR to detect viable A. citrulli cells under low pH conditions or in fermented watermelon fruit juice. We also provided evidence that VBNC A. citrulli cells induced by fermented watermelon fruit juice could not be resuscitated and did not retain their virulence on watermelon seedlings. However, VBNC A. citrulli cells could be resuscitated in Luria–Bertani medium. Based on these observations, we conclude that fermentation in watermelon fruit juice may not be an effective seed treatment for BFB because it may increase the seed infection by A. citrulli.

Antibiotic resistance in Helicobacter pylori has been growing worldwide with current treatment regimens. Development of new compounds for treatment of H. pylori infections is urgently required to achieve a successful eradication therapy in the future. Armeniaspirols, a novel class of natural products isolated from Streptomyces armeniacus, have been previously identified as antibacterial agents against Gram-positive pathogens. In this study, we found that armeniaspirol A (ARM1) exhibited potent antibacterial activity against H. pylori, including multidrug-resistant strains, with MIC range values of 4-16 μg ml-1 . The underlying mechanism of action of ARM1 against H. pylori involved the disruption of bacterial cell membranes. Also, ARM1 inhibited biofilm formation, eliminated preformed biofilms and killed biofilm-encased H. pylori in a dose-dependent manner. In a mouse model of multidrug-resistant H. pylori infection, dual therapy with ARM1 and omeprazole showed efficient in vivo killing efficacy comparable to the standard triple therapy, and induced negligible toxicity against normal tissues. Moreover, at acidic pH 2.5, ARM1 exhibited a much more potent anti-H. pylori activity than metronidazole. Thus, these findings demonstrated that ARM1 is a novel potent anti-H. pylori agent, which can be developed as a promising drug lead for treatment of H. pylori infections.

The phosphopantetheinyl transferases (PPTases) catalyze the post-translational modification of carrier proteins (CPs) from fatty acid synthases (FASs) in primary metabolism and from polyketide synthases (PKSs) and non-ribosomal polypeptide synthases (NRPSs) in secondary metabolism. Based on the conserved sequence motifs and substrate specificities, two types (AcpS-type and Sfp-type) of PPTases have been identified in prokaryotes. We present here that Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, harbors merely one PPTase, namely PptP. Complementation and gene deletion experiments clearly show that PptP can replace the function of Escherichia coli AcpS and is essential for the growth of P. gingivalis. Purified PptP transfers the 4-phosphopantetheine moiety of CoA to inactive apo-acyl carrier protein (ACP) to form holo-ACP, which functions as an active carrier of the acyl intermediates of fatty acid synthesis. Moreover, PptP exhibits broad substrate specificity, modifying all ACP substrates tested and catalyzing the transfer of coenzyme A (CoA) derivatives. The lack of sequence alignment with known PPTases together with phylogenetic analyses revealed PptP as a new class of PPTases. Identification of the new PPTase gene pptP exclusive in Porphyromonas species reveals a potential target for treating P. gingivalis infections.

Helicobacter pylori is a major global pathogen and has been implicated in gastritis, peptic ulcer, and gastric carcinoma. The efficacy of the extensive therapy of H. pylori infection with antibiotics is compromised by the development of drug resistance and toxicity toward human gut microbiota, which urgently demands novel and selective antibacterial strategies. The present study was mainly performed to assess the in vitro and in vivo effects of a natural herbal compound, dihydrotanshinone I (DHT), against standard and clinical H. pylori strains. DHT demonstrated effective antibacterial activity against H. pylori in vitro (MIC(50/90), 0.25/0.5 μg/ml), with no development of resistance during continuous serial passaging. Time-kill curves showed strong time-dependent bactericidal activity for DHT. Also, DHT eliminated preformed biofilms and killed biofilm-encased H. pylori cells more efficiently than the conventional antibiotic metronidazole. In mouse models of multidrug-resistant H. pylori infection, dual therapy with DHT and omeprazole showed in vivo killing efficacy superior to that of the standard triple-therapy approach. Moreover, DHT treatment induces negligible toxicity against normal tissues and exhibits a relatively good safety index. These results suggest that DHT could be suitable for use as an anti-H. pylori agent in combination with a proton pump inhibitor to eradicate multidrug-resistant H. pylori.

The increasing complexity of environmental pollution nowadays poses a severe threat to the public health, which attracts considerable attentions in searching for nanomaterials of multiproperty. In this study, mesoporous silica of KIT-6-encapsulated bismuth oxychloride (BiOCl), an intrinsically multifunctional material exhibiting bunched structure in the composites, are facilely prepared under hydrothermal conditions. Subsequently, the produced materials of multifunctionality were applied for photocatalysis, antibacterial test, and simultaneous determination of heavy metals including lead and cadmium. A combination of physiochemical characterizations have revealed that the BiOCl-KIT-6 composites exhibit enlarged yet refined surface morphology contributing to the improved photocatalytic ability with a band gap of 3.06 eV at a molecular ratio of 8Bi-Si. Moreover, the antibacterial activities of our BiOCl-KIT-6 composites were explored, and possible antimicrobial mechanism related to the production of reactive oxygen species was discussed. Furthermore, a sensitive electrochemical determination of heavy metals of lead and cadmium using square-wave anodic stripping voltammetry was also achieved. The composites-modified glassy carbon electrode displays a linear range of calibration curve from 0.2 to 300 μg/L with a detection limit of 0.05 μg/L (Pb2+) and 0.06 μg/L (Cd2+), respectively.

Two new xanthone derivatives, spinosusones A (1) and B (2), and a new tryptoquivaline analogue, asperdiazapinone G (3), together with nine known compounds (4-12) were isolated from the EtOAc extract of the marine-derived fungus Aspergillus sp . WHUF03110. The structures of 1-3 were determined by spectroscopic analysis, and comparison with literature data. Most of these isolated compounds were evaluated for their antimicrobial activities against ten Gram-negative and seven Gram-positive bacteria, Mycobacterium smegmatis ATCC 607, Candida albicans ATCC SC5314, and C. albicans YY-1-4. Compound 10 displayed strong antibacterial activity against five Gram-positive bacteria ( Bacillus subtilis 168, Staphylococcus aureus ATCC25923, S. aureus NEWMAN, S. aureus USA300, S. aureus NRS 271) with MIC values ranging from 1.0 to 2.0 μg/mL, and displayed moderate antibacterial activity against four Gram-negative bacteria ( Helicobacter pylori 26695, H. pylori G27, H. pylori 159, H. pylori 129) and M. smegmatis ATCC 607 with a MIC value of 8.0 μg/mL.

Pimeloyl-acyl carrier protein (ACP) methyl ester esterase catalyzes the last biosynthetic step of the pimelate moiety of biotin, a key intermediate in biotin biosynthesis. The paradigm pimeloyl-ACP methyl ester esterase is the BioH protein of Escherichia coli that hydrolyses the ester bond of pimeloyl-ACP methyl ester. Biotin synthesis in E. coli also requires the function of the malonyl-ACP methyltransferase gene (bioC) to employ a methylation strategy to allow elongation of a temporarily disguised malonate moiety to a pimelate moiety by the fatty acid synthetic enzymes. However, bioinformatics analyses of the extant bacterial genomes showed that bioH is absent in many bioC-containing bacteria. The genome of the Gram-negative bacterium, Moraxella catarrhalis lacks a gene encoding a homolog of any of the six known pimeloyl-ACP methyl ester esterase isozymes suggesting that this organism encodes a novel pimeloyl-ACP methyl ester esterase isoform. We report that this is the case. The gene encoding the new isoform, called btsA, was isolated by complementation of an E. coli bioH deletion strain. The requirement of BtsA for the biotin biosynthesis in M. catarrhalis was confirmed by a biotin auxotrophic phenotype caused by deletion of btsA in vivo and a reconstituted in vitro desthiobiotin synthesis system. Purified BtsA was shown to cleave the physiological substrate pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a Ser117-His254-Asp287 catalytic triad. The lack of sequence alignment with other isozymes together with phylogenetic analyses revealed BtsA as a new class of pimeloyl-ACP methyl ester esterase. The involvement of BtsA in M. catarrhalis virulence was confirmed by the defect of bacterial invasion to lung epithelial cells and survival within macrophages in the ΔbtsA strains. Identification of the new esterase gene btsA exclusive in Moraxella species that links biotin biosynthesis to bacterial virulence, can reveal a new valuable target for development of drugs against M. catarrhalis.

A new pyrazine derivative, trypilepyrazinol (1), a new α-pyrone polyketide, (+)-neocitreoviridin (2), and a new ergostane analogue, 3β-hydroxyergosta-8,14,24(28)-trien-7-one (3), were isolated and characterized along with five known compounds from the marine-derived fungus Penicillium sp. IMB17-046. The structures of these new compounds were determined using spectroscopic data analyses (HRESIMS, 1D- and 2D-NMR), X-ray crystallography analysis, and TDDFT ECD calculation. Compounds 1 and 3 exhibited broad-spectrum antiviral activities against different types of viruses, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza A virus (IAV), with IC50 values ranging from 0.5 to 7.7 μM. Compounds 1 and 2 showed antibacterial activities against Helicobacter pylori, a causative pathogen of various gastric diseases, with minimum inhibitory concentration (MIC) values of 1–16 μg/mL.

Cyclopropane fatty acids (CFAs) are synthetized by the addition of a methylene group from S-adenosyl-l-methionine across the carbon-carbon double bonds of unsaturated fatty acid chains of membrane phospholipids. This fatty acid cyclopropanation, catalyzed by the CFA synthase (CfaS) enzyme, occurs in many bacteria, including the human pathogen Helicobacter pylori. Although the cyclopropane modification was reported to play a key role in the adaptation in response to environmental stress, its role in H. pylori remains unknown. In this study, we showed that H. pylori HP0416 encodes a functional CfaS. The enzyme was demonstrated to be required for acid resistance, antibiotic resistance, intracellular survival and mouse gastric colonization, and cell membrane integrity. Moreover, the tool compound dioctylamine, which acts as a substrate mimic, directly inhibits the CfaS function of H. pylori, resulting into sensitivity to acid stress, increased antibiotic susceptibility, and attenuated abilities to avoid macrophage killing and to colonize mouse stomachs. These results validate CfaS as a promising antibiotic target and provide new potentials for this recognized target in future anti-H. pylori drug discovery efforts. IMPORTANCE The increasing prevalence of multidrug-resistant Helicobacter pylori strains has created an urgent need for alternative therapeutic regimens that complement the current antibiotic treatment strategies for H. pylori eradication; however, this is greatly hampered due to a lack of “druggable” targets. Although the CFAs are present in H. pylori cytoplasmic membranes at high levels, their physiological role has not been established. In this report, deletion of the CFA synthase CfaS was shown to attenuate acid and drug resistance, immune escape, and gastric colonization of H. pylori. These findings were validated by inhibition of the CfaS activity with the tool compound dioctylamine. These studies identify this enzyme as an attractive target for further drug discovery efforts against H. pylori.

Helicobacter pylori is a major global pathogen, and its infection represents a key factor in the etiology of various gastric diseases, including gastritis, peptic ulcers, and gastric carcinoma. The efficacy of current standard treatment for H. pylori infection including two broad-spectrum antibiotics is compromised by toxicity toward the gut microbiota and the development of drug resistance, which will likely only be resolved through novel and selective antibacterial strategies. Here, we synthesized a small molecule, zinc linolenate (ZnLla), and investigated its therapeutic potential for the treatment of H. pylori infection. ZnLla showed effective antibacterial activity against standard strains and drug-resistant clinical isolates of H. pylori in vitro with no development of resistance during continuous serial passaging. The mechanisms of ZnLla action against H. pylori involved the disruption of bacterial cell membranes and generation of reactive oxygen species. In mouse models of multidrug-resistant H. pylori infection, ZnLla showed in vivo killing efficacy comparable and superior to the triple therapy approach when use as a monotherapy and a combined therapy with omeprazole, respectively. Moreover, ZnLla treatment induces negligible toxicity against normal tissues and causes minimal effects on both the diversity and composition of the murine gut microbiota. Thus, the high degree of selectivity of ZnLla for H. pylori provides an attractive candidate for novel targeted anti-H. pylori treatment.

Four new secondary metabolites, including one spiro[anthracenone-xanthene] derivative aspergiloxathene A (1), one penicillide analogue, Δ2′-1′-dehydropenicillide (2), and two new phthalide derivatives, 5-methyl-3-methoxyepicoccone (3) and 7-carboxy-4-hydroxy-6-methoxy-5-methylphthalide (4), together with four known compounds, yicathin C (5), dehydropenicillide (6), 3-methoxyepicoccone (7), 4-hydroxy-6-methoxy-5-methylphthalide (8), were identified from the marine-derived fungus Aspergillus sp. IMCASMF180035. Their structures were determined by spectroscopic data, including high-resolution electrospray ionization mass spectrometry (HRESIMS), 1D and 2D nuclear magnetic resonance (NMR) techniques. Compound 1 was identified as the first jointed molecule by xanthene and anthracenone moieties possessing an unprecedented carbon skeleton with spiro-ring system. All compounds were evaluated activities against Staphylococcus aureus, methicillin resistant S. aureus (MRSA), Escherichia coli, Escherichia faecium, Pseudomonas aeruginosa, and Helicobacter pylori. Compound 1 showed significant inhibitory effects against S. aureus and MRSA, with minimum inhibitory concentration (MIC) values of 5.60 and 22.40 µM. Compounds 2 and 6 exhibited potent antibacterial activities against H. pylori, with MIC values of 21.73 and 21.61 µM, respectively.

Sulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 ( apt ). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicus . IMPORTANCE Currently, the pyrEF /5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF /5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt /6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF -deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S. islandicus strains.

Algae cells were the main sources of dissolved organic nitrogen (DON) in raw water with plenty of algae, and ultrasonic pretreatment was one of the algae-controlling methods through the damage of algae cells. However, the variation of DON concentration during the ultrasonic treatment process was not confirmed. Variation of DON concentration during the processes of low frequency ultrasound treatment of Microcystis aeruginosa was investigated. In addition, the effect of sonication on the metabolite concentration, algae cellar activity and the subsequent coagulation performance were discussed. The results showed that after a long duration of ultrasonic (60 s), nearly 90% of the algal cells were damaged and the maximum concentration of DON attained more than 3 mg/L. In order to control the leakage extent of DON, the sonication time should be less than 30 s with power intensity of more than 1.0 W/cm(3). In the mean time, ultrasonic treatment could inhibit the reactivation and the proliferation of algal, keep the algae cell wall integrity and enhance coagulation effectively under the same condition. However, ultrasound frequency had little effect on DON at the frequency range used in this study (20-150 kHz).

Dissolved organic nitrogen (DON) is now considered as one of the most important precursors of nitrogenous disinfection byproducts (N-DBPs), and the algae cells were the main source of DON in eutrophic water sources. In this study, the characterizations of DON (especially proteins) in intracellular organic matter (IOM) were investigated. In addition, the DON removal by using sunlight/immobilized TiO2 was also studied. The results showed that in algae cells, about 185 kinds of proteins were found and the distribution of isoelectric points of these proteins was mostly in acidic side. Apo-α-phycocyanin and Apo-β-phycocyanin are the two main components, whose concentrations were 0.187 mg L−1 and 0.136 mg L−1, respectively. The removal rate of DON by sunlight/TiO2 system was about 29% after 7 hours irradiation, moreover, the system altered the molecular weight ranges of 30–80 kD to the range of 20–30 kD. Furthermore, proper photo-catalytic oxidation enhanced the coagulation efficiency significantly due to the direct removal and change of proteins.

In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signaling factor (DSF, 2(Z)-11-methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl-CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl-CoA ligase, Escherichia coli FadD, in the E. coli β-oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3-hydroxyacyl-acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl-ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalyzing uptake and activation of the free fatty acids to give acyl-CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl-ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.

Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the bacterial fatty acid elongation cycle. Enterococcus faecalis is unusual in that it encodes two unrelated enoyl-ACP reductases, FabI and FabK. We recently reported that deletion of the gene encoding FabI results in an unsaturated fatty acid (UFA) auxotroph despite the presence of fabK, a gene encoding a second fully functional enoyl-ACP reductase. By process of elimination, our prior report argued that poor expression was the reason that fabK failed to functionally replace FabI. We now report that FabK is indeed poorly expressed and that the expression defect is at the level of translation rather than transcription. We isolated four spontaneous mutants that allowed growth of the E. faecalis ΔfabI strain on fatty acid-free medium. Each mutational lesion (single base substitution or deletion) extended the fabK ribosome binding site. Inactivation of fabK blocked growth, indicating that the mutations acted only on fabK rather than a downstream gene. The mutations activated fabK translation to levels that supported fatty acid synthesis and hence cell growth. Furthermore, site-directed and random mutagenesis experiments showed that point mutations that resulted in increased complementarity to the 3′ end of the 16S rRNA increased FabK translation to levels sufficient to support growth, whereas mutations that decreased complementarity blocked fabK translation.

SummaryIn the classical anaerobic pathway of unsaturated fatty acid biosynthesis, that of Escherichia coli, the double bond is introduced into the growing acyl chain by the FabA dehydratase/isomerase. Another dehydratase, FabZ, functions in the chain elongation cycle. In contrast, Aerococcus viridans has only a single FabA/FabZ homolog we designate FabQ. FabQ can not only replace the function of E. coli FabZ in vivo, but it also catalyzes the isomerization required for unsaturated fatty acid biosynthesis. Most strikingly, FabQ in combination with E. coli FabB imparts the surprising ability to bypass reduction of the trans-2-acyl-ACP intermediates of classical fatty acid synthesis. FabQ allows elongation by progressive isomerization reactions to form the polyunsaturated fatty acid, 3-hydroxy-cis-5, 7-hexadecadienoic acid, both in vitro and in vivo. FabQ therefore provides a potential pathway for bacterial synthesis of polyunsaturated fatty acids.

Helicobacter pylori is a Gram-negative bacterium that inhabits human upper gastrointestinal tract and the presence of this pathogen in the gut microbiome increases the risk of peptic ulcers and stomach cancer. H. pylori depends on unsaturated fatty acid (UFA) biosynthesis for maintaining membrane structure and function. Although some of the H. pylori enzymes involved in UFA biosynthesis are functionally homologous with the enzymes found in Escherichia coli, we show here that an enzyme HP0773, now annotated as FabX, uses an unprecedented a backtracking mechanism to not only dehydrogenate decanoyl-acyl carrier protein (ACP) in a reaction that parallels that of acyl-CoA dehydrogenase, the first enzyme of the fatty acid β-oxidation cycle, but additionally isomerizes trans-2-decenoyl-ACP to cis-3-decenoyl-ACP, the key UFA synthetic intermediate. Thus, FabX reverses the normal fatty acid synthesis cycle in H. pylori at the C10 stage. Overall, this unusual FabX activity may offer a broader explanation for how many bacteria that lack the canonical pathway enzymes produce UFA-containing phospholipids.

In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was demonstrated using a reconstituted in vitro desthiobiotin synthesis system. BioV homologues seem the sole pimeloyl-ACP methyl ester esterase present in the Helicobacter species and their occurrence only in H. pylori and close relatives provide a target for development of drugs to specifically treat Helicobacter infections.

Signal molecules of the diffusible signal factor (DSF) family have been shown recently to be involved in regulation of pathogenesis and biofilm formation in diverse Gram-negative bacteria. DSF signals are reported to be active not only on their cognate bacteria, but also on unrelated bacteria and the pathogenic yeast, Candida albicans. DSFs are monounsaturated fatty acids of medium chain length containing an unusual cis-2 double bond. Although genetic analyses had identified genes involved in DSF synthesis, the pathway of DSF synthesis was unknown. The DSF of the important human pathogen Burkholderia cenocepacia (called BDSF) is cis-2-dodecenoic acid. We report that BDSF is synthesized from a fatty acid synthetic intermediate, the acyl carrier protein (ACP) thioester of 3-hydroxydodecanoic acid. This intermediate is intercepted by protein Bcam0581 and converted to cis-2-dodecenoyl-ACP. Bcam0581 is annotated as a homologue of crotonase, the first enzyme of the fatty acid degradation pathway. We demonstrated Bcam0581to be a bifunctional protein that not only catalysed dehydration of 3-hydroxydodecanoyl-ACP to cis-2-dodecenoyl-ACP, but also cleaved the thioester bond to give the free acid. Both activities required the same set of active-site residues. Although dehydratase and thioesterase activities are known activities of the crotonase superfamily, Bcam0581 is the first protein shown to have both activities.

Background FabG is the only known enzyme that catalyzes reduction of the 3-ketoacyl-ACP intermediates of bacterial fatty acid synthetic pathways. However, there are two Ralstonia solanacearum genes, RSc1052 (fabG1) and RSp0359 (fabG2), annotated as encoding putative 3-ketoacyl-ACP reductases. Both FabG homologues possess the conserved catalytic triad and the N-terminal cofactor binding sequence of the short chain dehydrogenase/reductase (SDR) family. Thus, it seems reasonable to hypothesize that RsfabG1 and RsfabG2 both encode functional 3-ketoacyl-ACP reductases and play important roles in R. solanacearum fatty acid synthesis and growth. Methods Complementation of Escherichia colifabG temperature-sensitive mutant with R. solanacearum fabGs encoded plasmids was carried out to test the function of RsfabGs in fatty acid biosynthesis. RsFabGs proteins were purified by nickel chelate chromatography and fatty acid biosynthetic reaction was reconstituted to investigate the 3-ketoacyl-ACP reductase activity of RsFabGs in vitro. Disruption of both RsfabG genes was done via DNA homologous recombination to test the function of both RsfabG in vivo. And more we also carried out pathogenicity tests on tomato plants using RsfabG mutant strains. Results We report that expression of either of the two proteins (RsFabG1 and RsFabG2) restores growth of the E. coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrate that both proteins restore fatty acid synthetic ability to extracts of the E. coli strain. The RsfabG1 gene carried on the R. solanacearum chromosome is essential for growth of the bacterium, as is the case for fabG in E. coli. In contrast, the null mutant strain with the megaplasmid-encoded RsfabG2 gene is viable but has a fatty acid composition that differs significantly from that of the wild type strain. Our study also shows that RsFabG2 plays a role in adaptation to high salt concentration and low pH, and in pathogenesis of disease in tomato plants. Conclusion R. solanacearum encodes two 3-ketoacyl-ACP reductases that both have functions in fatty acid synthesis. We supply the first evidence that, like other enzymes in the bacterial fatty acid biosynthetic pathway, one bacterium may simultaneously possess two or more 3-oxoacyl-ACP reductase isozymes. Electronic supplementary material The online version of this article (doi:10.1186/s12866-015-0554-x) contains supplementary material, which is available to authorized users.

Enoyl-acyl carrier protein (enoyl-ACP) reductase catalyzes the last step of the elongation cycle in the synthesis of bacterial fatty acids. The Enterococcus faecalis genome contains two genes annotated as enoyl-ACP reductases, a FabI-type enoyl-ACP reductase and a FabK-type enoyl-ACP reductase. We report that expression of either of the two proteins restores growth of an Escherichia coli fabI temperature-sensitive mutant strain under nonpermissive conditions. In vitro assays demonstrated that both proteins support fatty acid synthesis and are active with substrates of all fatty acid chain lengths. Although expression of E. faecalis fabK confers to E. coli high levels of resistance to the antimicrobial triclosan, deletion of fabK from the E. faecalis genome showed that FabK does not play a detectable role in the inherent triclosan resistance of E. faecalis. Indeed, FabK seems to play only a minor role in modulating fatty acid composition. Strains carrying a deletion of fabK grow normally without fatty acid supplementation, whereas fabI deletion mutants make only traces of fatty acids and are unsaturated fatty acid auxotrophs., IMPORTANCE The finding that exogenous fatty acids support growth of E. faecalis strains defective in fatty acid synthesis indicates that inhibitors of fatty acid synthesis are ineffective in countering E. faecalis infections because host serum fatty acids support growth of the bacterium.