Crystal structure and acetylation of BioQ suggests a novel regulatory switch for biotin biosynthesis in Mycobacterium smegmatis.

Biotin (vitamin B7), a sulfur-containing fatty acid derivative, is a nutritional virulence factor in certain mycobacterial species. Tight regulation of biotin biosynthesis is important because production of biotin is an energetically expensive process requiring 15-20 equivalents of ATP. The Escherichia coli bifunctional BirA is a prototypical biotin regulatory system. In contrast, mycobacterial BirA is an unusual biotin protein ligase without DNA-binding domain. Recently, we established a novel two-protein paradigm of BioQ-BirA. However, structural and molecular mechanism for BioQ is poorly understood. Here, we report crystal structure of the M. smegmatis BioQ at 1.9 Å resolution. Structure-guided functional mapping defined a seven residues-requiring motif for DNA-binding activity. Western blot and MALDI-TOF MS allowed us to unexpectedly discover that the K47 acetylation activates crosstalking of BioQ to its cognate DNA. More intriguingly, excess of biotin augments the acetylation status of BioQ in M. smegmatis. It seems likely that BioQ acetylation proceeds via a non-enzymatic mechanism. Mutation of this acetylation site K47 in BioQ significantly impairs its regulatory role in vivo. This explains in part (if not all) why BioQ has no detectable requirement of the presumable bio-5'-AMP effecter, which is a well-known ligand for the paradigm E. coli BirA regulator system. Unlike the scenario seen with E. coli carrying a single biotinylated protein, AccB, genome-wide search and Streptavidin blot revealed that no less than seven proteins require the rare post-translational modification, biotinylation in M. smegmatis, validating its physiological demand for biotin at relatively high level. Taken together, our finding defines a novel biotin regulatory machinery by BioQ, posing a possibility that development of new antibiotics targets biotin, the limited nutritional virulence factor in certain pathogenic mycobacterial species.

Dissemination and Mechanism for the MCR-1 Colistin Resistance.

Polymyxins are the last line of defense against lethal infections caused by multidrug resistant Gram-negative pathogens. Very recently, the use of polymyxins has been greatly challenged by the emergence of the plasmid-borne mobile colistin resistance gene (mcr-1). However, the mechanistic aspects of the MCR-1 colistin resistance are still poorly understood. Here we report the comparative genomics of two new mcr-1-harbouring plasmids isolated from the human gut microbiota, highlighting the diversity in plasmid transfer of the mcr-1 gene. Further genetic dissection delineated that both the trans-membrane region and a substrate-binding motif are required for the MCR-1-mediated colistin resistance. The soluble form of the membrane protein MCR-1 was successfully prepared and verified. Phylogenetic analyses revealed that MCR-1 is highly homologous to its counterpart PEA lipid A transferase in Paenibacili, a known producer of polymyxins. The fact that the plasmid-borne MCR-1 is placed in a subclade neighboring the chromosome-encoded colistin-resistant Neisseria LptA (EptA) potentially implies parallel evolutionary paths for the two genes. In conclusion, our finding provids a first glimpse of mechanism for the MCR-1-mediated colistin resistance.

Screening and Characterization of a Non-cyp51A Mutation in an Aspergillus fumigatus cox10 Strain Conferring Azole Resistance.

The rapid and global emergence of azole resistance in the human pathogen Aspergillus fumigatus has drawn attention. Thus, a thorough understanding of its mechanisms of drug resistance requires extensive exploration. In this study, we found that the loss of the putative calcium-dependent protein-encoding gene algA causes an increased frequency of azole-resistant A. fumigatus isolates. In contrast to previously identified azole-resistant isolates related to cyp51A mutations, only one isolate carries a point mutation in cyp51A (F219L mutation) among 105 independent stable azole-resistant isolates. Through next-generation sequencing (NGS), we successfully identified a new mutation (R243Q substitution) conferring azole resistance in the putative A. fumigatus farnesyltransferase Cox10 (AfCox10) (AFUB_065450). High-performance liquid chromatography (HPLC) analysis verified that the decreased absorption of itraconazole in related Afcox10 mutants is the primary reason for itraconazole resistance. Moreover, a complementation experiment by reengineering the mutation in a parental wild-type background strain demonstrated that both the F219L and R243Q mutations contribute to itraconazole resistance in an algA-independent manner. These data collectively suggest that the loss of algA results in an increased frequency of azole-resistant isolates with a non-cyp51A mutation. Our findings indicate that there are many unexplored non-cyp51A mutations conferring azole resistance in A. fumigatus and that algA defects make it possible to isolate drug-resistant alleles. In addition, our study suggests that genome-wide sequencing combined with alignment comparison analysis is an efficient approach to identify the contribution of single nucleotide polymorphism (SNP) diversity to drug resistance.

Transcriptional Repression of the VC2105 Protein by Vibrio FadR Suggests that It Is a New Auxiliary Member of the fad Regulon.

UNLABELLED: Recently, our group along with others reported that the Vibrio FadR regulatory protein is unusual in that, unlike the prototypical fadR product of Escherichia coli, which has only one ligand-binding site, Vibrio FadR has two ligand-binding sites and represents a new mechanism for fatty acid sensing. The promoter region of the vc2105 gene, encoding a putative thioesterase, was mapped, and a putative FadR-binding site (AA CTG GTA AGA GCA CTT) was proposed. Different versions of the FadR regulatory proteins were prepared and purified to homogeneity. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) determined the direct interaction of the vc2105 gene with FadR proteins of various origins. Further, EMSAs illustrated that the addition of long-chain acyl-coenzyme A (CoA) species efficiently dissociates the vc2105 promoter from the FadR regulator. The expression level of the Vibrio cholerae vc2105 gene was elevated 2- to 3-fold in a fadR null mutant strain, validating that FadR is a repressor for the vc2105 gene. The ß-galactosidase activity of a vc2105-lacZ transcriptional fusion was increased over 2-fold upon supplementation of growth medium with oleic acid. Unlike the fadD gene, a member of the Vibrio fad regulon, the VC2105 protein played no role in bacterial growth and virulence-associated gene expression of ctxAB (cholera toxin A/B) and tcpA (toxin coregulated pilus A). Given that the transcriptional regulation of vc2105 fits the criteria for fatty acid degradation (fad) genes, we suggested that it is a new member of the Vibrio fad regulon. IMPORTANCE: The Vibrio FadR regulator is unusual in that it has two ligand-binding sites. Different versions of the FadR regulatory proteins were prepared and characterized in vitro and in vivo. An auxiliary fad gene (vc2105) from Vibrio was proposed that encodes a putative thioesterase and has a predicted FadR-binding site (AAC TGG TA A GAG CAC TT). The function of this putative binding site was proved using both EMSA and SPR. Further in vitro and in vivo experiments revealed that the Vibrio FadR is a repressor for the vc2105 gene. Unlike fadD, a member of the Vibrio fad regulon, VC2105 played no role in bacterial growth and expression of the two virulence-associated genes (ctxAB and tcpA). Therefore, since transcriptional regulation of vc2105 fits the criteria for fad genes, it seems likely that vc2105 acts as a new auxiliary member of the Vibrio fad regulon.

Mycobacterium smegmatis†BioQ defines a new regulatory network for biotin metabolism.

Biotin (vitamin H), the sulfur-containing enzyme cofactor, is an essential micronutrient for three domains of life. Given the fact that biotin is an energetically expensive molecule whose de novo biosynthesis demands 20 ATP equivalents each, it is reasonable that bacteria have evolved diversified mechanisms in various microorganisms to tightly control biotin metabolism. Unlike the Escherichia coli BirA, the prototypical bi-functional version of biotin protein ligase (BPL) in that it acts as a repressor for biotin biosynthesis pathway, the BirA protein of Mycobacterium smegmatis (M. smegmatis), a closely relative of the tuberculosis-causing pathogen, Mycobacterium tuberculosis, lacked the DNA-binding activity. It raised a possibility that an alternative new regulator might be present to compensate the loss of regulatory function. Here we report that this is the case. Genomic context analyses of M. smegmatis detected a newly identified BioQ homolog classified into the TetR family of transcription factor and its recognizable palindromes. The M. smegmatis BioQ protein was overexpressed and purified to homogeneity. Size-exclusion chromatography combined with chemical cross-linking studies demonstrated that the BioQ protein had a propensity to dimerize. The promoters of bioFD and bioQ/B were mapped using 5'-RACE. Electrophoretic mobility shift assays revealed that BioQ binds specifically to the promoter regions of bioFD and bioQ/B. Further DNase I foot-printing elucidated the BioQ-binding palindromes. Site-directed mutagenesis suggested the important residues critical for BioQ/DNA binding. The isogenic mutant of bioQ (ΔbioQ) was generated using the approach of homologous recombination. The in vivo data from the real-time qPCR combined with the lacZ transcriptional fusion experiments proved that removal of bioQ gave significant increment with expression of bio operons. Also, expression of bio operons were repressed by exogenous addition of biotin, and this repression seemed to depend on the presence of BioQ protein. Thereby, we believed that M. smegmatis BioQ is not only a negative auto-regulator but also a repressor for bioFD and bioB operons involved in the biotin biosynthesis pathway. Collectively, this finding defined the two-protein paradigm of BirA and BioQ, representing a new mechanism for bacterial biotin metabolism.

Calcium signaling mediates antifungal activity of triazole drugs in the Aspergilli.

Azoles are widely applied and largely effective as antifungals; however, the increasing prevalence of clinically resistant isolates has yet to be matched by approaches to improve the efficacy of antimicrobial therapy. In this study, using the model fungus Aspergillus nidulans and one of the most common human pathogen Aspergillus fumigatus as research materials, we present the evidence that calcium signaling is involved in the azole-antifungals-induced stress-response reactions. In normal media, antifungal-itraconazole (ITZ) is able to induce the [Ca(2+)]c increased sharply but the addition of calcium chelator-EGTA or BAPTA almost blocks the calcium influx responses, resulted in the dramatically decreasing of [Ca(2+)]c transient. Real-time PCR analysis verified that six-tested Ca(2+)-inducible genes-two calcium channels (cchA/midA), a calmodulin-dependent phosphatase-calcineurin (cnaA), a transcription factor-crzA, and two calcium transporters (pmrA/pmcA)-could be transiently up-regulated by adding ITZ, indicating these components are involved in the azole stress-response reaction. Defect of cnaA or crzA caused more susceptibility to azole antifungals than did single mutants or double deletions of midA and cchA. Notably, EGTA may influence Rh123 accumulation as an azole-mimicking substrate through the process of the drug absorption. In vivo studies of a Galleria mellonella model identified that the calcium chelator works as an adjunct antifungal agent with azoles for invasive aspergillosis. Most importantly, combination of ITZ and EGTA or ITZ with calcium signaling inhibitor-FK506 greatly enhances the ITZ efficacy. Thus, our study provides potential clues that specific inhibitors of calcium signaling could be clinically useful adjuncts to conventional azole antifungals in the Aspergilli.

A pericentrin-related protein homolog in Aspergillus nidulans plays important roles in nucleus positioning and cell polarity by affecting microtubule organization.

Pericentrin is a large coiled-coil protein in mammalian centrosomes that serves as a multifunctional scaffold for anchoring numerous proteins. Recent studies have linked numerous human disorders with mutated or elevated levels of pericentrin, suggesting unrecognized contributions of pericentrin-related proteins to the development of these disorders. In this study, we characterized AnPcpA, a putative homolog of pericentrin-related protein in the model filamentous fungus Aspergillus nidulans, and found that it is essential for conidial germination and hyphal development. Compared to the hyphal apex localization pattern of calmodulin (CaM), which has been identified as an interactive partner of the pericentrin homolog, GFP-AnPcpA fluorescence dots are associated mainly with nuclei, while the accumulation of CaM at the hyphal apex depends on the function of AnPcpA. In addition, the depletion of AnPcpA by an inducible alcA promoter repression results in severe growth defects and abnormal nuclear segregation. Most interestingly, in mature hyphal cells, knockdown of pericentrin was able to significantly induce changes in cell shape and cytoskeletal remodeling; it resulted in some enlarged compartments with condensed nuclei and anucleate small compartments as well. Moreover, defects in AnPcpA significantly disrupted the microtubule organization and nucleation, suggesting that AnPcpA may affect nucleus positioning by influencing microtubule organization.

Gut microbes exacerbate systemic inflammation and behavior disorders in neurologic disease CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a cerebral small vessel disease that carries mutations in NOTCH3. The clinical manifestations are influenced by genetic and environmental factors that may include gut microbiome. RESULTS: We investigated the fecal metagenome, fecal metabolome, serum metabolome, neurotransmitters, and cytokines in a cohort of 24 CADASIL patients with 28 healthy household controls. The integrated-omics study showed CADASIL patients harbored an altered microbiota composition and functions. The abundance of bacterial coenzyme A, thiamin, and flavin-synthesizing pathways was depleted in patients. Neurotransmitter balance, represented by the glutamate/GABA (4-aminobutanoate) ratio, was disrupted in patients, which was consistent with the increased abundance of two major GABA-consuming bacteria, Megasphaera elsdenii and Eubacterium siraeum. Essential inflammatory cytokines were significantly elevated in patients, accompanied by an increased abundance of bacterial virulence gene homologs. The abundance of patient-enriched Fusobacterium varium positively correlated with the levels of IL-1ß and IL-6. Random forest classification based on gut microbial species, serum cytokines, and neurotransmitters showed high predictivity for CADASIL with AUC = 0.89. Targeted culturomics and mechanisms study further showed that patient-derived F. varium infection caused systemic inflammation and behavior disorder in Notch3R170C/+ mice potentially via induction of caspase-8-dependent noncanonical inflammasome activation in macrophages. CONCLUSION: These findings suggested the potential linkage among the brain-gut-microbe axis in CADASIL. Video Abstract.

Quasispecies of SARS-CoV-2 revealed by single nucleotide polymorphisms (SNPs) analysis.

New SARS-CoV-2 mutants have been continuously indentified with enhanced transmission ever since its outbreak in early 2020. As an RNA virus, SARS-CoV-2 has a high mutation rate due to the low fidelity of RNA polymerase. To study the single nucleotide polymorphisms (SNPs) dynamics of SARS-CoV-2, 158 SNPs with high confidence were identified by deep meta-transcriptomic sequencing, and the most common SNP type was C > T. Analyses of intra-host population diversity revealed that intra-host quasispecies' composition varies with time during the early onset of symptoms, which implicates viral evolution during infection. Network analysis of co-occurring SNPs revealed the most abundant non-synonymous SNP 22,638 in the S glycoprotein RBD region and 28,144 in the ORF8 region. Furthermore, SARS-CoV-2 variations differ in an individual's respiratory tissue (nose, throat, BALF, or sputum), suggesting independent compartmentalization of SARS-CoV-2 populations in patients. The positive selection analysis of the SARS-CoV-2 genome uncovered the positive selected amino acid G251V on ORF3a. Alternative allele frequency spectrum (AAFS) of all variants revealed that ORF8 could bear alternate alleles with high frequency. Overall, the results show the quasispecies' profile of SARS-CoV-2 in the respiratory tract in the first two months after the outbreak.

Initial whole-genome sequencing and analysis of the host genetic contribution to COVID-19 severity and susceptibility.

The COVID-19 pandemic has accounted for millions of infections and hundreds of thousand deaths worldwide in a short-time period. The patients demonstrate a great diversity in clinical and laboratory manifestations and disease severity. Nonetheless, little is known about the host genetic contribution to the observed interindividual phenotypic variability. Here, we report the first host genetic study in the Chinese population by deeply sequencing and analyzing 332 COVID-19 patients categorized by varying levels of severity from the Shenzhen Third People's Hospital. Upon a total of 22.2 million genetic variants, we conducted both single-variant and gene-based association tests among five severity groups including asymptomatic, mild, moderate, severe, and critical ill patients after the correction of potential confounding factors. Pedigree analysis suggested a potential monogenic effect of loss of function variants in GOLGA3 and DPP7 for critically ill and asymptomatic disease demonstration. Genome-wide association study suggests the most significant gene locus associated with severity were located in TMEM189-UBE2V1 that involved in the IL-1 signaling pathway. The p.Val197Met missense variant that affects the stability of the TMPRSS2 protein displays a decreasing allele frequency among the severe patients compared to the mild and the general population. We identified that the HLA-A*11:01, B*51:01, and C*14:02 alleles significantly predispose the worst outcome of the patients. This initial genomic study of Chinese patients provides genetic insights into the phenotypic difference among the COVID-19 patient groups and highlighted genes and variants that may help guide targeted efforts in containing the outbreak. Limitations and advantages of the study were also reviewed to guide future international efforts on elucidating the genetic architecture of host-pathogen interaction for COVID-19 and other infectious and complex diseases.

A single regulator NrtR controls bacterial NAD+ homeostasis via its acetylation.

Nicotinamide adenine dinucleotide (NAD+) is an indispensable cofactor in all domains of life, and its homeostasis must be regulated tightly. Here we report that a Nudix-related transcriptional factor, designated MsNrtR (MSMEG_3198), controls the de novo pathway of NAD+biosynthesis in M. smegmatis, a non-tuberculosis Mycobacterium. The integrated evidence in vitro and in vivo confirms that MsNrtR is an auto-repressor, which negatively controls the de novo NAD+biosynthetic pathway. Binding of MsNrtR cognate DNA is finely mapped, and can be disrupted by an ADP-ribose intermediate. Unexpectedly, we discover that the acetylation of MsNrtR at Lysine 134 participates in the homeostasis of intra-cellular NAD+ level in M. smegmatis. Furthermore, we demonstrate that NrtR acetylation proceeds via the non-enzymatic acetyl-phosphate (AcP) route rather than by the enzymatic Pat/CobB pathway. In addition, the acetylation also occurs on the paralogs of NrtR in the Gram-positive bacterium Streptococcus and the Gram-negative bacterium Vibrio, suggesting that these proteins have a common mechanism of post-translational modification in the context of NAD+ homeostasis. Together, these findings provide a first paradigm for the recruitment of acetylated NrtR to regulate bacterial central NAD+ metabolism.

Deciphering MCR-2 Colistin Resistance.

Antibiotic resistance is a prevalent problem in public health worldwide. In general, the carbapenem ß-lactam antibiotics are considered a final resort against lethal infections by multidrug-resistant bacteria. Colistin is a cationic polypeptide antibiotic and acts as the last line of defense for treatment of carbapenem-resistant bacteria. Very recently, a new plasmid-borne colistin resistance gene, mcr-2, was revealed soon after the discovery of the paradigm gene mcr-1, which has disseminated globally. However, the molecular mechanisms for MCR-2 colistin resistance are poorly understood. Here we show a unique transposon unit that facilitates the acquisition and transfer of mcr-2 Evolutionary analyses suggested that both MCR-2 and MCR-1 might be traced to their cousin phosphoethanolamine (PEA) lipid A transferase from a known polymyxin producer, Paenibacillus Transcriptional analyses showed that the level of mcr-2 transcripts is relatively higher than that of mcr-1 Genetic deletions revealed that the transmembrane regions (TM1 and TM2) of both MCR-1 and MCR-2 are critical for their location and function in bacterial periplasm, and domain swapping indicated that the TM2 is more efficient than TM1. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) confirmed that all four MCR proteins (MCR-1, MCR-2, and two chimeric versions [TM1-MCR-2 and TM2-MCR-1]) can catalyze chemical modification of lipid A moiety anchored on lipopolysaccharide (LPS) with the addition of phosphoethanolamine to the phosphate group at the 4' position of the sugar. Structure-guided site-directed mutagenesis defined an essential 6-residue-requiring zinc-binding/catalytic motif for MCR-2 colistin resistance. The results further our mechanistic understanding of transferable colistin resistance, providing clues to improve clinical therapeutics targeting severe infections by MCR-2-containing pathogens.IMPORTANCE Carbapenem and colistin are the last line of refuge in fighting multidrug-resistant Gram-negative pathogens. MCR-2 is a newly emerging variant of the mobilized colistin resistance protein MCR-1, posing a potential challenge to public health. Here we report transfer of the mcr-2 gene by a unique transposal event and its possible origin. Distribution of MCR-2 in bacterial periplasm is proposed to be a prerequisite for its role in the context of biochemistry and the colistin resistance. We also define the genetic requirement of a zinc-binding/catalytic motif for MCR-2 colistin resistance. This represents a glimpse of transferable colistin resistance by MCR-2.

Structural and Functional Characterization of the FadR Regulatory Protein from Vibrio alginolyticus.

The structure of Vibrio cholerae FadR (VcFadR) complexed with the ligand oleoyl-CoA suggests an additional ligand-binding site. However, the fatty acid metabolism and its regulation is poorly addressed in Vibrio alginolyticus, a species closely-related to V. cholerae. Here, we show crystal structures of V. alginolyticus FadR (ValFadR) alone and its complex with the palmitoyl-CoA, a long-chain fatty acyl ligand different from the oleoyl-CoA occupied by VcFadR. Structural comparison indicates that both VcFadR and ValFadR consistently have an additional ligand-binding site (called site 2), which leads to more dramatic conformational-change of DNA-binding domain than that of the E. coli FadR (EcFadR). Isothermal titration calorimetry (ITC) analyses defines that the ligand-binding pattern of ValFadR (2:1) is distinct from that of EcFadR (1:1). Together with surface plasmon resonance (SPR), electrophoresis mobility shift assay (EMSA) demonstrates that ValFadR binds fabA, an important gene of unsaturated fatty acid (UFA) synthesis. The removal of fadR from V. cholerae attenuates fabA transcription and results in the unbalance of UFA/SFA incorporated into membrane phospholipids. Genetic complementation of the mutant version of fadR (Δ42, 136-177) lacking site 2 cannot restore the defective phenotypes of ΔfadR while the wild-type fadR gene and addition of exogenous oleate can restore them. Mice experiments reveals that VcFadR and its site 2 have roles in bacterial colonizing. Together, the results might represent an additional example that illustrates the Vibrio FadR-mediated lipid regulation and its role in pathogenesis.

Diversified mcr-1-Harbouring Plasmid Reservoirs Confer Resistance to Colistin in Human Gut Microbiota.

UNLABELLED: Colistin is an ultimate line of refuge against multidrug-resistant Gram-negative pathogens. Very recently, the emergence of plasmid-mediatedmcr-1colistin resistance has become a great challenge to global public health, raising the possibility that dissemination of themcr-1gene is underestimated and diversified. Here, we report three cases of plasmid-carried MCR-1 colistin resistance in isolates from gut microbiota of diarrhea patients. Structural and functional analyses determined that the colistin resistance is conferred purely by the singlemcr-1gene. Genetic and sequence mapping revealed thatmcr-1-harbouring plasmid reservoirs are present in diversity. Together, the data represent the first evidence of diversity inmcr-1-harbouring plasmid reservoirs of human gut microbiota. IMPORTANCE: The plasmid-mediated mobile colistin resistance gene (mcr-1) challenged greatly the conventional idea mentioned above that colistin is an ultimate line of refuge against lethal infections by multidrug-resistant Gram-negative pathogens. It is a possibility that diversified dissemination of themcr-1gene might be greatly underestimated. We report three cases of plasmid-carried MCR-1 colistin resistance in isolates from gut microbiota of diarrhea patients and functionally define the colistin resistance conferred purely by the singlemcr-1gene. Genetic and sequence mapping revealed unexpected diversity among themcr-1-harbouring plasmid reservoirs of human gut microbiota.

Binding of Shewanella FadR to the fabA fatty acid biosynthetic gene: implications for contraction of the fad regulon.

The Escherichia coli fadR protein product, a paradigm/prototypical FadR regulator, positively regulates fabA and fabB, the two critical genes for unsaturated fatty acid (UFA) biosynthesis. However the scenario in the other Ɣ-proteobacteria, such as Shewanella with the marine origin, is unusual in that Rodionov and coworkers predicted that only fabA (not fabB) has a binding site for FadR protein. It raised the possibility of fad regulon contraction. Here we report that this is the case. Sequence alignment of the FadR homologs revealed that the N-terminal DNA-binding domain exhibited remarkable similarity, whereas the ligand-accepting motif at C-terminus is relatively-less conserved. The FadR homologue of S. oneidensis (referred to FadR_she) was over-expressed and purified to homogeneity. Integrative evidence obtained by FPLC (fast protein liquid chromatography) and chemical cross-linking analyses elucidated that FadR_she protein can dimerize in solution, whose identity was determined by MALDI-TOF-MS. In vitro data from electrophoretic mobility shift assays suggested that FadR_she is almost functionally-exchangeable/equivalent to E. coli FadR (FadR_ec) in the ability of binding the E. coli fabA (and fabB) promoters. In an agreement with that of E. coli fabA, S. oneidensis fabA promoter bound both FadR_she and FadR_ec, and was disassociated specifically with the FadR regulatory protein upon the addition of long-chain acyl-CoA thioesters. To monitor in vivo effect exerted by FadR on Shewanella fabA expression, the native promoter of S. oneidensis fabA was fused to a LacZ reporter gene to engineer a chromosome fabA-lacZ transcriptional fusion in E. coli. As anticipated, the removal of fadR gene gave about 2-fold decrement of Shewanella fabA expression by ß-gal activity, which is almost identical to the inhibitory level by the addition of oleate. Therefore, we concluded that fabA is contracted to be the only one member of fad regulon in the context of fatty acid synthesis in the marine bacteria Shewanella genus.

The Atypical Occurrence of Two Biotin Protein Ligases in Francisella novicida Is Due to Distinct Roles in Virulence and Biotin Metabolism.

UNLABELLED: The physiological function of biotin requires biotin protein ligase activity in order to attach the coenzyme to its cognate proteins, which are enzymes involved in central metabolism. The model intracellular pathogen Francisella novicida is unusual in that it encodes two putative biotin protein ligases rather than the usual single enzyme. F. novicida BirA has a ligase domain as well as an N-terminal DNA-binding regulatory domain, similar to the prototypical BirA protein in E. coli. However, the second ligase, which we name BplA, lacks the N-terminal DNA binding motif. It has been unclear why a bacterium would encode these two disparate biotin protein ligases, since F. novicida contains only a single biotinylated protein. In vivo complementation and enzyme assays demonstrated that BirA and BplA are both functional biotin protein ligases, but BplA is a much more efficient enzyme. BirA, but not BplA, regulated transcription of the biotin synthetic operon. Expression of bplA (but not birA) increased significantly during F. novicida infection of macrophages. BplA (but not BirA) was required for bacterial replication within macrophages as well as in mice. These data demonstrate that F. novicida has evolved two distinct enzymes with specific roles; BplA possesses the major ligase activity, whereas BirA acts to regulate and thereby likely prevent wasteful synthesis of biotin. During infection BplA seems primarily employed to maximize the efficiency of biotin utilization without limiting the expression of biotin biosynthetic genes, representing a novel adaptation strategy that may also be used by other intracellular pathogens. IMPORTANCE: Our findings show that Francisella novicida has evolved two functional biotin protein ligases, BplA and BirA. BplA is a much more efficient enzyme than BirA, and its expression is significantly induced upon infection of macrophages. Only BplA is required for F. novicida pathogenicity, whereas BirA prevents wasteful biotin synthesis. These data demonstrate that the atypical occurrence of two biotin protein ligases in F. novicida is linked to distinct roles in virulence and biotin metabolism.

A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein.

Escherichia coli (E. coli) FadR regulator plays dual roles in fatty acid metabolism, which not only represses the fatty acid degradation (fad) system, but also activates the unsaturated fatty acid synthesis pathway. Earlier structural and biochemical studies of FadR protein have provided insights into interplay between FadR protein with its DNA target and/or ligand, while the missing knowledge gap (esp. residues with indirect roles in DNA binding) remains unclear. Here we report this case through deep mapping of old E. coli fadR mutants accumulated. Molecular dissection of E. coli K113 strain, a fadR mutant that can grow on decanoic acid (C10) as sole carbon sources unexpectedly revealed a single point mutation of T178G in fadR locus (W60G in FadRk113). We also observed that a single genetically-recessive mutation of W60G in FadR regulatory protein can lead to loss of its DNA-binding activity, and thereby impair all the regulatory roles in fatty acid metabolisms. Structural analyses of FadR protein indicated that the hydrophobic interaction amongst the three amino acids (W60, F74 and W75) is critical for its DNA-binding ability by maintaining the configuration of its neighboring two ß-sheets. Further site-directed mutagenesis analyses demonstrated that the FadR mutants (F74G and/or W75G) do not exhibit the detected DNA-binding activity, validating above structural reasoning.