The Sinorhizobium meliloti LpxXL and AcpXL Proteins Play Important Roles in Bacteroid Development within Alfalfa

Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of S. meliloti bacteroid lipid A with VLCFAs.Sinorhizobium meliloti and Brucella abortus form chronic intracellular infections within legumes and mammalian hosts, respectively (3, 20), and their BacA proteins play essential roles in these processes (8, 12). The precise function(s) of the BacA proteins has not been resolved, but free-living S. meliloti and B. abortus mutants lacking BacA have increased resistance to the glycopeptide bleomycin (9, 12) and there are ∼50% decreases in their lipid A very-long-chain fatty acid (VLCFA) contents (4, 7). It has also been determined that the increased resistance of an S. meliloti bacA null mutant to bleomycin and a truncated eukaryotic peptide, Bac7_1-16, is independent of its lipid A VLCFA alteration (6, 15). Together, these findings support a model in which BacA could have multiple nonoverlapping functions which lead to lipid A VLCFA modification and peptide uptake. The fact that two symbiotically defective S. meliloti BacA site-directed mutants (Q193G and R389G) (13) show defects in BacA-mediated lipid A VLCFA modification (4) but are still capable of peptide uptake (15) suggests that the S. meliloti lipid A VLCFA modification could play a key role in the symbiosis of this organism with alfalfa.Since the mechanism by which BacA leads to the lipid A VLCFA modification has not been resolved (4), S. meliloti mutants were constructed with mutations in the lpxXL and acpXL genes, which encode a lipid A VLCFA acyl transferase and a VLCFA acyl carrier protein directly involved in the biosynthesis of VLCFA-modified lipid A (5, 23). The S. meliloti lpxXL and acpXL mutants completely lack the lipid A VLCFA modification in their free-living states, but, unlike the S. meliloti bacA null mutant, these mutants can still form a successful symbiosis with alfalfa (5, 8, 23). However, the fact that the S. meliloti acpXL and lpxXL mutants are substantially less competitive in the alfalfa symbiosis than the parent strain (5, 23) indicates that the AcpXL and LpxXL proteins play important roles in at least one of the stages of the alfalfa symbiosis. Although the free-living S. meliloti acpXL and lpxXL mutants completely lack the lipid A VLCFA, they produce different species of lipid A (5). For example, in the absence of AcpXL, S. meliloti is able to modify lipid A with either C16:0 or C18:0 in the position normally modified with the VLCFA in the parent strain lipid A. This process is LpxXL dependent, as it does not occur in either an S. meliloti lpxXL single mutant or an S. meliloti acpXL lpxXL double mutant. In addition, since a Rhizobium leguminosarum acpXL mutant completely lacks the lipid A VLCFA modification in its free-living state but its lipid A is partially modified with the VLCFA to ∼58% of the amount in the parent strain lipid A during passage through peas (25), it is also possible that the S. meliloti acpXL mutant and possibly the S. meliloti lpxXL mutant undergo further lipid A changes during the interaction with alfalfa.In this study, we found that LpxXL and AcpXL play important but distinct roles in S. meliloti bacteroid development during alfalfa symbiosis. Additionally, we demonstrated that there is a minor host-induced AcpXL-independent mechanism by which S. meliloti bacteroid lipopolysaccharide (LPS) can be modified with the VLCFA. In contrast, we found that the LpxXL protein plays an essential role in the modification of S. meliloti bacteroids with VLCFAs.     

Functional and Structural Study of the Dimeric Inner Membrane Protein SbmA

SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability.     

Functional Characterization of SbmA,a Bacterial Inner Membrane Transporter Required for Importing the Antimicrobial Peptide Bac7(1-35)

SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated K_m value of 6.95 ± 0.89 μM peptide and a V_max of 53.91 ± 3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein.     

An acpXL Mutant of Rhizobium leguminosarum bv. phaseoli Lacks 27-Hydroxyoctacosanoic Acid in Its Lipid A and Is Developmentally Delayed during Symbiotic Infection of the Determinate Nodulating Host Plant Phaseolus vulgaris

Rhizobium leguminosarum is a Gram-negative bacterium that forms nitrogen-fixing symbioses with compatible leguminous plants via intracellular invasion and establishes a persistent infection within host membrane-derived subcellular compartments. Notably, an unusual very-long-chain fatty acid (VLCFA) is found in the lipid A of R. leguminosarum as well as in the lipid A of the medically relevant pathogens Brucella abortus, Brucella melitensis, Bartonella henselae, and Legionella pneumophila, which are also able to persist within intracellular host-derived membranes. These bacterial symbionts and pathogens each contain a homologous gene region necessary for the synthesis and transfer of the VLCFA to the lipid A. Within this region lies a gene that encodes the specialized acyl carrier protein AcpXL, on which the VLCFA is built. This study describes the biochemical and infection phenotypes of an acpXL mutant which lacks the VLCFA. The mutation was created in R. leguminosarum bv. phaseoli strain 8002, which forms symbiosis with Phaseolus vulgaris, a determinate nodulating legume. Structural analysis using gas chromatography and mass spectrometry revealed that the mutant lipid A lacked the VLCFA. Compared to the parent strain, the mutant was more sensitive to the detergents deoxycholate and dodecyl sulfate and the antimicrobial peptide polymyxin B, suggesting a compromise to membrane stability. In addition, the mutant was more sensitive to higher salt concentrations. Passage through the plant restored salt tolerance. Electron microscopic examination showed that the mutant was developmentally delayed during symbiotic infection of the host plant Phaseolus vulgaris and produced abnormal symbiosome structures.     

BacA Is Essential for Bacteroid Development in Nodules of Galegoid,but not Phaseoloid,Legumes

BacA is an integral membrane protein, the mutation of which leads to increased resistance to the antimicrobial peptides bleomycin and Bac7_1-35 and a greater sensitivity to SDS and vancomycin in Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. phaseoli, and Rhizobium etli. The growth of Rhizobium strains on dicarboxylates as a sole carbon source was impaired in bacA mutants but was overcome by elevating the calcium level. While bacA mutants elicited indeterminate nodule formation on peas, which belong to the galegoid tribe of legumes, bacteria lysed after release from infection threads and mature bacteroids were not formed. Microarray analysis revealed almost no change in a bacA mutant of R. leguminosarum bv. viciae in free-living culture. In contrast, 45 genes were more-than 3-fold upregulated in a bacA mutant isolated from pea nodules. Almost half of these genes code for cell membrane components, suggesting that BacA is crucial to alterations that occur in the cell envelope during bacteroid development. In stark contrast, bacA mutants of R. leguminosarum bv. phaseoli and R. etli elicited the formation of normal determinate nodules on their bean host, which belongs to the phaseoloid tribe of legumes. Bacteroids from these nodules were indistinguishable from the wild type in morphology and nitrogen fixation. Thus, while bacA mutants of bacteria that infect galegoid or phaseoloid legumes have similar phenotypes in free-living culture, BacA is essential only for bacteroid development in indeterminate galegoid nodules.Bacteria of the family Rhizobiaceae are alphaproteobacteria, which form a species-specific symbiotic relationship with leguminous plants. Plants release flavonoids that typically induce the synthesis of lipochitooligosaccharides by rhizobia, which in turn initiate a signaling cascade in the plant, leading to nodule formation (34). Rhizobia become trapped by curling root hairs, which they enter via infection threads that grow and ramify into the root cortex, where newly induced meristematic cells form the nodule (34). Bacteria are released from infection threads and engulfed by a plant-derived symbiosome membrane. In galegoid legumes (a clade in the subfamily Papilionoideae, such as Medicago, Pisum, or Vicia), which form indeterminate nodules that have a persistent meristem, bacteria undergo the endoreduplication of their chromosome, resulting in dramatic increases in size, shape, and DNA content to become terminally differentiated bacteroids (32). However, in phaseoloid legumes (e.g., lotus, bean, and soybean), which form determinate nodules with a transient meristem, bacteria do not undergo endoreduplication and therefore do not enlarge substantially. These bacteroids retain a normal DNA content and can regrow after isolation from nodules (32). The endoreduplication of bacteroids is controlled by the plant, and it is believed that nodule-specific cysteine-rich (NCR) peptides, which are made in indeterminate, but not in determinate, nodules, may be responsible for inducing and maintaining bacteroid development (31, 32). Finally, mature bacteroids receive dicarboxylic acids from the plant, which they use as a carbon, reductant, and energy source for the reduction of N₂ to ammonia (38). The ammonia is secreted to the plant, where it is assimilated into amino acids or ureides, depending on the legume, for export to the shoot.Sinorhizobium meliloti BacA protein was the first bacterial factor identified to be essential for bacteroid development (15). More recently, it also has been shown to be essential for the Mesorhizobium-Astragalus symbiosis (42). S. meliloti elicits the formation of indeterminate nodules on alfalfa, and while S. meliloti bacA null mutants induce nodule formation, bacteria lyse soon after endocytosis but prior to bacteroid differentiation (15, 20). BacA is a cytoplasmic membrane protein that shares 64% identity with SbmA from Escherichia coli (15, 25). SbmA/BacA proteins belong to the ATP binding cassette (ABC) superfamily and share sequence similarity with a family of eukaryotic peroxisomal membrane proteins, including the human adrenoleukodystrophy protein, which is required for the efficient transport of very-long-chain fatty acids (VLCFAs) out of the cytoplasm (9). Consistent with this, S. meliloti BacA is required for the complete modification of lipid A with VLCFAs (9). However, since S. meliloti mutants, which are directly involved in the biosynthesis of VLCFA-modified lipid A, show bacteroid abnormalities but still can form a successful alfalfa symbiosis, the effect of BacA on lipid A VLCFA modification does not fully account for its essential role in bacteroid development (10, 11, 16). Strains mutated in bacA also have an increased resistance to the glycopeptide bleomycin, a low-level resistance to aminoglycoside antibiotics, and an increased sensitivity to ethanol, sodium dodecyl sulfate (SDS), and deoxycholate relative to the sensitivities of the parent strain (12, 18, 25). More recently it has been shown that an S. meliloti bacA null mutant has an increased resistance to a truncated form of a eukaryotic proline-rich peptide, Bac7_1-16, and was unable to accumulate a fluorescently labeled form of this peptide (28). This finding, combined with the increased resistance of an S. meliloti bacA null mutant to bleomycin, led to the hypothesis that BacA is itself a putative peptide transporter (BacA mediated) or able to alter the activity of such a transporter (BacA influenced) (11, 15, 18, 28).As the increased resistance of the S. meliloti bacA null mutant to bleomycin and Bac7_1-16 appears to be independent of the VLCFA modification of lipid A (11, 28), this suggested that either BacA-mediated or BacA-influenced peptide uptake into S. meliloti plays a role in bacteroid development. Since indeterminate galegoid nodules contain hundreds of NCR peptides, whereas determinate phaseoloid nodules lack these host peptides (31), we considered it important to assess the role of BacA in bacteroid development during the formation of both nodule types.Here, we show that bacA mutants of Rhizobium leguminosarum bv. viciae strains 3841 and A34 failed to develop bacteroids and did not fix nitrogen in indeterminate pea (Pisum sativum) nodules. However, bacA mutants of both R. leguminosarum bv. phaseoli 4292 and Rhizobium etli CE3 formed normal bacteroids and fixed nitrogen at wild-type rates in determinate bean (Phaseolus vulgaris) nodules. This is consistent with BacA being a key component of bacteroid development in indeterminate galegoid nodules that is not required for functional bacteroid formation in determinate phaseoloid nodules.     

流产布氏杆菌烯脂酰ACP还原酶的鉴定

烯脂酰ACP还原酶是细菌脂肪酸合成的关键酶之一．流产布氏杆菌基因组有2个注释为烯脂酰ACP还原酶基因fabI的同源基因：fabI1和fabI2．由这2个fabI同源基因编码的蛋白质分别与大肠杆菌FabI有50%和51%的同源性,且都拥有与大肠杆菌FabI一样的催化中心Tyr-(Xaa)₆-Lys序列．分别用携带这2个同源基因的质粒载体转化大肠杆菌fabI温度敏感突变菌株JP1111．转化子能在42℃生长,表明这2个基因均能遗传互补大肠杆菌fabI突变,并使此菌株恢复脂肪酸的合成．另外,体外酶学分析显示,由这2个同源基因编码的蛋白质都拥有烯脂酰ACP还原酶活性,均能参与细菌脂肪酸合成．上述结果证实,流产布氏杆菌同时拥有2个同种类型的烯脂酰ACP还原酶,是一种新的烯脂酰ACP多样性的表现．     

Erratum

Escherichia coli cells (unsaturated fatty acid auxotroph) have been adapted to grow on branched-chain fatty acids. Membrane vesicles were isolated from cells grown on a mixture of branched-chain fatty acids isolated from the lipids of Bacillus subtilis (E. coli (B. subtilis) membranes) and on a pure synthetic anti-isononadecanoic acid (E. coli (aC19) membranes).We have shown, using wide-angle X-ray diffraction and differential scanning calorimetry, that the ordered state of the lipids is perturbed in the case of E. coli (aC19) membranes. The perturbation leads to the presence of a large wide-angle X-ray diffraction at 4.25–4.3 Å, as opposed to the presence of a sharp 4.2 Å reflection in unperturbed systems.We have shown, using freeze-fracture electron microscopy, that a protein segregation exists in the case of E. coli (aC19) membranes (at low temperature the integral membrane proteins aggregate in the membrane domains containing the disordered lipids); we do not observe such segregation in the case of E. coli (B. subtilis) membranes. We conclude that in cases where the branching of the fatty acids introduces a perturbation of the lipid order, the integral membrane proteins can still be accommodated in membrane domains containing the ‘perturbed’ ordered lipids.Finally, we have determined the rate of β-galactoside transport in E. coli (aC19) and E. coli (B. subtilis) membranes as a function of temperature. We have shown that, in both cases, the Arrhenius representations display an increased slope in the region of the disorder-to-order transition. We conclude that such an increased slope may have different origins. In the case of E. coli (aC19) membranes, it is the result of the aggregation of the β-galactoside carriers together with other integral membrane proteins which may lead to the inactivation of the carriers; in the case of E. coli (B. subtilis) membranes, it is the result of the partial immobilisation of the carriers embedded in a lipid environment, of which the fluidity, despite the perturbation of its lipid order, is still much less than that associated with lipids in a totally disordered state.     

Genetic modification of the Streptomyces cholesterol oxidase gene for expression in Escherichia coli and development of promoter-probe vectors for use in enteric bacteria

Streptomyces cholesterol oxidase was produced in Escherichia coli by a modification of the cholesterol oxidase gene (choA′) in which the native codons for the precursor NH₂-terminal region and the ribosome binding site were substituted for those favored by E. coli. The choA′ gene was expressed under the control of the lac or tac promoter in a multiple copy plasmid vector, although no expression of the native choA gene from Streptomyces was observed in E. coli. E. coli cells carrying the plasmid, pCo117, produced 2-fold more cholesterol oxidase intracellularly during 18-h culture than did the producing strain of Streptomyces sp. SA-COO cultured for 4 d. The NH₂-terminal amino acid sequence of cholesterol oxidase produced by E. coli appeared to be processed between Ala²⁰ and Ala²¹ of the precursor enzyme, while the Streptomyces enzyme was processed between Ala⁴² and Asp⁴³. Based on the facts that the cholesterol oxidase was stable, could be assayed rapidly, and no endogenous cholesterol oxidase activity was found in any enteric bacteria, we developed two widely applicable, new promoter-probe vectors posessing the choA′ gene, multiple cloning sites, and either a low or high copy number plasmid. Since these plasmids can replicate in enteric bacteria, the new plasmid vectors have a great potential for use in enteric bacteria without the isolation of Cho⁻ mutants.     

Incorporation of Substrate Cell Lipid A Components into the Lipopolysaccharide of Intraperiplasmically Grown Bdellovibrio bacteriovorus

The composition of Bdellovibrio bacteriovorus lipopolysaccharide (LPS) was determined for cells grown axenically and intraperiplasmically on Escherichia coli or Pseudomonas putida. The LPS of axenically grown bdellovibrios contained glucose and fucosamine as the only detectable neutral sugar and amino sugar, and nonadecenoic acid (19:1) as the predominant fatty acid. Additional fatty acids, heptose, ketodeoxyoctoic acid, and phosphate were also detected. LPS from bdellovibrios grown intraperiplasmically contained components characteristic of both axenically grown bdellovibrios and the substrate cells. Substrate cell-derived LPS fatty acids made up the majority of the bdellovibrio LPS fatty acids and were present in about the same proportions as in the substrate cell LPS. Glucosamine derived from E. coli LPS amounted to about one-third of the hexosamine residues in intraperiplasmically grown bdellovibrio LPS. However, galactose, characteristic of the E. coli outer core and O antigen, was not detected in the bdellovibrio LPS, suggesting that only lipid A components of the substrate cell were incorporated. Substrate cell-derived and bdellovibrio-synthesized LPS materials were conserved in the B. bacteriovorus outer membrane for at least two cycles of intraperiplasmic growth. When bdellovibrios were grown on two different substrate cells successively, lipid A components were taken up from the second while the components incorporated from the lipid A of the first were conserved in the bdellovibrio LPS. The data show that substrate cell lipid A components were incorporated into B. bacteriovorus lipid A during intraperiplasmic growth with little or no change, and that these components, fatty acids and hexosamines, comprised a substantial portion of bdellovibrio lipid A.     

Mechanistic Basis of Plasmid-Specific DNA Binding of the F Plasmid Regulatory Protein,TraM

The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon–helix–helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.     

Brucella abortus induces apoptosis of human T lymphocytes

Immune evasion is essential for Brucella abortus to survive in the face of robust adaptive CD4+ T cell response. We have previously demonstrated that B. abortus can indirectly inhibit CD4+ T cells by down-regulating MHC-II expression and antigen presentation on macrophages. However, whether B. abortus is able to directly interfere with T lymphocytes is not known. We report here that B. abortus induces apoptosis of human T lymphocytes, even though invasion of T lymphocytes was low and non-replicative. The ability of heat-killed B. abortus to reproduce the same phenomenon suggested that there was a bacterial structural component involved. We demonstrated that a prototypical B. abortus outer membrane lipoprotein (l-Omp19), but not its unlipidated form, induced T lymphocyte apoptosis. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also induced an increase in T lymphocyte cell death, indicating that the structural component implicated in the phenomenon could be any B. abortus lipoprotein. B. abortus-induced T lymphocyte apoptosis was dependent on the secretion of TNF-α since pre-incubation of T lymphocytes with anti-TNF-α mAb inhibited the apoptosis of the cells. Overall, these results represent a new mechanism whereby B. abortus by directly inhibiting T cell-mediated responses may evade adaptive immune responses.     

pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis

Two novel Enterococcus faecalis-Escherichia coli shuttle vectors that utilize the promoter and ribosome binding site of bacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli and E. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in E. coli and E. faecalis at levels of 10³ and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.     

苜蓿中华根瘤菌fabA和fabB基因功能的鉴定

在大肠杆菌(Escherichia coli)脂肪酸合成酶体系中,fabA基因编码有双功能的3-羟基脂酰ACP脱水异构酶,其异构产物能被fabB基因编码的3-酮基脂酰ACP合成酶Ⅰ延伸,合成不饱和脂肪酸,该FabA-FabB途径被认为是缺氧条件下不饱和脂肪酸合成的经典途径．生物信息学分析发现,苜蓿中华根瘤菌(Sinorhizobium meliloti)的SmFabA与EcFabA相似性达到60.6%,具有相同的保守活性位点和两个保守的α螺旋结构;SmFabB与EcFabB相似性达到61.1%,具有相同的Cys-His-His活性中心．用携带SmfabA和SmfabB的质粒载体遗传互补大肠杆菌温度敏感突变株CY57和CY242,在添加三氯森(TCL)抑制烯脂酰ACP还原酶活性的条件下,转化子能在42℃恢复生长,且放射性薄层层析能检测到转化子中不饱和脂肪酸棕榈油酸(Δ9C16:1)和十八碳烯酸(Δ11C18:1)的合成．体外重建脂肪酸合成反应表明,SmFabA能催化羟脂酰ACP的脱水反应且能够使反-2-癸烯酰ACP异构化,SmFabB能催化不同链长的脂酰ACP和丙二酸单酰ACP的聚合反应．另外,未得到SmFabA和SmFabB的突变株,表明SmFabA和SmFabB可能是苜蓿中华根瘤菌脂肪酸合成酶系中必不可少的关键蛋白．上述结果证实了苜蓿中华根瘤菌fabA和fabB两个基因在不饱和脂肪酸合成中的功能．     

The Rhizobium etli FixL protein differs in structure from other known FixL proteins

The central heme-binding domain in the FixL proteins of Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium leguminosarum biovar viciae and Azorhizobium caulinodans, is highly conserved. The similarity with the corresponding domain in the Rhizobium etli FixL protein is considerably less. This observation prompted us to analyze the heme-binding capacities of the R. etli FixL protein. The R. etlifixL gene was overexpressed in Escherichia coli. In the presence of S. meliloti FixJ, the overexpressed R. etli FixL protein was able to enhance FixJ-mediated activation of an S. meliloti pnifA-lacZ fusion, indicating that the R.?etli FixL protein possesses an active conformation in E. coli. Subsequently, using a non-denaturing gel assay for heme, we analyzed the heme-binding capacity of the R.?etli FixL protein expressed in E. coli, taking the S.?meliloti FixL protein as a positive control. The R. etli FixL protein expressed in E. coli does not contain a heme group, in contrast to the S. meliloti FixL protein. Therefore we conclude that the R. etli FixL is a non-heme protein in the nif regulatory cascade.     

A microglial cell model for acyl-CoA oxidase 1 deficiency

Acyl-CoA oxidase 1 (ACOX1) deficiency is a rare and severe peroxisomal leukodystrophy associated with a very long-chain fatty acid (VLCFA) β–oxidation defect. This neurodegenerative disease lacks relevant cell models to further decipher the pathomechanisms in order to identify novel therapeutic targets. Since peroxisomal defects in microglia appear to be a key component of peroxisomal leukodystrophies, we targeted the Acox1 gene in the murine microglial BV-2 cell line. Using CRISPR/Cas9 gene editing, we generated an Acox1-deficient cell line and validated the allelic mutations, which lead to the absence of ACOX1 protein and enzymatic activity. The activity of catalase, the enzyme degrading H₂O₂, was increased, likely in response to the alteration of redox homeostasis. The mutant cell line grew more slowly than control cells without obvious morphological changes. However, ultrastructural analysis revealed an increased number of peroxisomes and mitochondria associated with size reduction of mitochondria. Changes in the distribution of lipid droplets containing neutral lipids have been observed in mutant cells; lipid analysis revealed the accumulation of saturated and monounsaturated VLCFA. Besides, expression levels of genes encoding interleukin-1 beta and 6 (IL-1β and IL-6), as well as triggering receptor expressed on myeloid cells 2 (Trem2) were found modified in the mutant cells suggesting modification of microglial polarization and phagocytosis ability. In summary, this Acox1-deficient cell line presents the main biochemical characteristics of the human disease and will serve as a promising model to further investigate the consequences of a specific microglial peroxisomal β–oxidation defect on oxidative stress, inflammation and cellular functions.     

Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production

Here we report recombinant expression and activity of several type I fatty acid synthases that can function in parallel with the native Escherichia coli fatty acid synthase. Corynebacterium glutamicum FAS1A was the most active in E. coli and this fatty acid synthase was leveraged to produce oleochemicals including fatty alcohols and methyl ketones. Coexpression of FAS1A with the ACP/CoA-reductase Maqu2220 from Marinobacter aquaeolei shifted the chain length distribution of fatty alcohols produced. Coexpression of FAS1A with FadM, FadB, and an acyl-CoA-oxidase from Micrococcus luteus resulted in the production of methyl ketones, although at a lower level than cells using the native FAS. This work, to our knowledge, is the first example of in vivo function of a heterologous fatty acid synthase in E. coli. Using FAS1 enzymes for oleochemical production have several potential advantages, and further optimization of this system could lead to strains with more efficient conversion to desired products. Finally, functional expression of these large enzyme complexes in E. coli will enable their study without culturing the native organisms.     

Characteristics of the response of natural and recombinant luminescent microorganisms in the presence of Fe2+ ions

It was found that divalent iron ions have alternative effects on the bioluminescence of the natural marine microorganism Photobacterium phosphoreum and the recombinant Escherichia coli strain with a cloned lux operon of P. leiognathi. In the presence of 0.25–5.0 mM FeSO₄, the bioluminescence intensity of the former and the latter increased and decreased, respectively. To establish the causes of these differences, we studied the characteristics of the fatty acid composition of the compared microorganisms. The fatty acid profile of E. coli was characterized by a high proportion of unsaturated 11-octadecenoic (vaccenic) acid. A study of this acid in a cell-free enzyme system used for bioluminescence generation showed that it is a potent inhibitor of bacterial bioluminescence. It was found that such effects are enhanced if 11-octadecenoid acid is preincubated with Fe²⁺.     

Characterization of the lipopolysaccharide of <Emphasis Type="Italic">Escherichia coli</Emphasis> 126

The lipopolysaccharide (LPS) of Escherichia coli 126 was isolated and studied. The lipid A fatty acid composition of the investigated LPS was similar to that of other members of the family Enterobacteriaceae. The E. coli 126 LPS was more toxic than the LPSs of previously studied E. coli strains and of other members of the Enterobacteriaceae (Budvicia aquatica and Pragia fontium), and was less pyrogenic than pyrogenal. SDS-PAG electrophoresis showed a bimodal distribution typical of S-form LPSs. The LPS of E. coli 126 decreased the adhesive index indicating a possible competition between LPS molecules of E. coli 126 and adhesins of E. coli F-50 on rabbit erythrocytes. The LPS of E. coli 126 in a homologous system showed antigenic activity in the reactions of double immunodiffusion in agar by Ouchterlony. No serological cross-reaction of the LPS of other E. coli strains, as well as of that of the B. aquatica type strain, with the antiserum to E. coli 126 was observed. The structural components of the lipopolysaccharide obtained by mild acid hydrolysis were lipid A, the core oligosaccharide, and the O-specific polysaccharide. Based on the data of monosaccharide analysis and ¹H and ¹³C NMR spectroscopy it was found that the O-specific polysaccharide had the structure characteristic of the representatives of E. coli serogroup O15.