ABSTRACT The last step of the initiation phase of fatty acid biosynthesis in most bacteria is catalyzed by the 3-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). Pseudomonas syringae pv. syringae strain B728a encodes two FabH homologs, Psyr_3467 and Psyr_3830, which we designated PssFabH1 and PssFabH2, respectively. Here, we explored the roles of these two 3-ketoacyl-ACP synthase (KAS) III proteins. We found that PssFabH1 is similar to the Escherichia coli FabH in using acetyl-acetyl-coenzyme A (CoA ) as a substrate in vitro, whereas PssFabH2 uses acyl-CoAs (C 4 –C 10 ) or acyl-ACPs (C 6 –C 10 ). Mutant analysis showed that neither KAS III protein is essential for the de novo fatty acid synthesis and cell growth. Loss of PssFabH1 reduced the production of an acyl homoserine lactone (AHL) quorum-sensing signal, and this production was partially restored by overexpressing FabH homologs from other bacteria. AHL production was also restored by inhibiting fatty acid elongation and providing exogenous butyric acid. Deletion of PssFabH1 supports the redirection of acyl-ACP toward biosurfactant synthesis, which in turn enhances swarming motility. Our study revealed that PssFabH1 is an atypical KAS III protein that represents a new KAS III clade that functions in providing a critical fatty acid precursor, butyryl-ACP, for AHL synthesis. IMPORTANCE Acyl homoserine lactones (AHLs) are important quorum-sensing compounds in Gram-negative bacteria. Although their formation requires acylated acyl carrier proteins (ACPs), how the acylated intermediate is shunted from cellular fatty acid synthesis to AHL synthesis is not known. Here, we provide in vivo evidence that Pseudomonas syringae strain B728a uses the enzyme PssFabH1 to provide the critical fatty acid precursor butyryl-ACP for AHL synthesis. Loss of PssFabH1 reduces the diversion of butyryl-ACP to AHL, enabling the accumulation of acyl-ACP for synthesis of biosurfactants that contribute to bacterial swarming motility. We report that PssFabH1 and PssFabH2 each encode a 3-ketoacyl-acyl carrier protein synthase (KAS) III in P. syringae B728a. Whereas PssFabH2 is able to function in redirecting intermediates from β-oxidation to fatty acid synthesis, PssFabH1 is an atypical KAS III protein that represents a new KAS III clade based on its sequence, non-involvement in cell growth, and novel role in AHL synthesis.

Biotin is an essential micro-nutrient across the three domains of life. The paradigm earlier step of biotin synthesis denotes “BioC-BioH” pathway in Escherichia coli . Here we report that BioZ bypasses the canonical route to begin biotin synthesis. In addition to its origin of Rhizobiales , protein phylogeny infers that BioZ is domesticated to gain an atypical role of β-ketoacyl-ACP synthase III. Genetic and biochemical characterization demonstrates that BioZ catalyzes the condensation of glutaryl-CoA (or ACP) with malonyl-ACP to give 5’-keto-pimeloyl ACP. This intermediate proceeds via type II fatty acid synthesis (FAS II) pathway, to initiate the formation of pimeloyl-ACP, a precursor of biotin synthesis. To further explore molecular basis of BioZ activity, we determine the crystal structure of Agrobacterium tumefaciens BioZ at 1.99 Å, of which the catalytic triad and the substrate-loading tunnel are functionally defined. In particular, we localize that three residues (S84, R147, and S287) at the distant bottom of the tunnel might neutralize the charge of free C-carboxyl group of the primer glutaryl-CoA. Taken together, this study provides molecular insights into the BioZ biotin synthesis pathway.

The Pseudomonas putida F1 genome contains five genes annotated as encoding 3-ketoacyl-acyl carrier protein (ACP) synthases. Four are annotated as encoding FabF (3-ketoacylACP synthase II) proteins, and the fifth is annotated as encoding a FabB (3-ketoacyl-ACP synthase I) protein. Expression of one of the FabF proteins, FabF2, is cryptic in the native host and becomes physiologically important only when the repressor controlling fabF2 transcription is inactivated. When derepressed, FabF2 can functionally replace FabB, and when expressed from a foreign promoter, had weak FabF activity. Complementation of Escherichia coli fabB and fabF mutant strains with high expression showed that P. putida fabF1 restored E. coli fabF function, whereas fabB restored E. coli fabB function and fabF2 restored the functions of both E. coli fabF and fabB. The P. putida AfabF1 deletion strain was almost entirely defective in synthesis of cis-vaccenic acid, whereas the AfabB strain is an unsaturated fatty acid (UFA) auxotroph that accumulated high levels of spontaneous suppressors in the absence of UFA supplementation. This was due to increased expression of fabF2 that bypasses loss of fabB because of the inactivation of the regulator, Pput_2425, encoded in the same operon as fabF2. Spontaneous suppressor accumulation was decreased by high levels of UFA supplementation, whereas competition by the P. putida n-oxidation pathway gave increased accumulation. The AfabB Delta fabF2 strain is a stable UFA auxotroph indicating that suppressor accumulation requires FabF2 function. However, at low concentrations of UFA supplementation, the AfabF2 APput_2425 double mutant strain still accumulated suppressors at low UFA concentrations.

Abstract Homologous regulatory factors are widely present in bacteria, but whether homologous regulators synergistically or differentially regulate different biological functions remains mostly unknown. Here, we report that the homologous regulators RpoN1 and RpoN2 of the plant pathogen Xanthomonas campestris pv. campestris (Xcc) play different regulatory roles with respect to virulence traits, flagellar biosynthesis, and basal metabolism. RpoN2 directly regulated Xcc fliC and fliQ to modulate flagellar synthesis in X. campestris, thus affecting the swimming motility of X. campestris. Mutation of rpoN2 resulted in reduced production of biofilms and extracellular polysaccharides in Xcc. These defects may together cause reduced virulence of the rpoN2 mutant against the host plant. Moreover, we demonstrated that RpoN1 could regulate branched‐chain fatty acid production and modulate the synthesis of diffusible signal factor family quorum sensing signals. Although RpoN1 and RpoN2 are homologues, the regulatory roles and biological functions of these proteins were not interchangeable. Overall, our report provides new insights into the two different molecular roles that form the basis for the transcriptional specialization of RpoN homologues.

Bacterial 3-oxoacyl-ACP reductase (OAR) catalyzes the 3-oxoacyl-ACP reduction step in the fatty acid synthesis pathway. At least 12 genes in the Pseudomonas aeruginosa genome are annotated as OAR-encoding genes. In this study, we characterized the functions of these genes with biochemical and genetic techniques. With the exception of PA2967, which encodes FabG, an essential protein in fatty acid synthesis, only the PA4389 and PA4786 gene products had OAR activity, and the single deletion of these two genes reduced the ability of P. aeruginosa to produce several specific quorum-sensing (QS) signals. However, PA4389 and PA4786 do not have key roles in fatty acid synthesis. Moreover, although most OAR homologs had no OAR activity, some may function in carbon utilization. The PA3128 product may play a role in the TCA cycle, and PA0182 and PA1470 seem to be required for the utilization of several amino acids. The rest of the OAR homologs have no roles in carbon utilization, but the deletion of one of these genes might affect the production of virulence factors by P. aeruginosa. We conclude that most OAR homolog genes do not encode OAR enzymes, and that these proteins do not function in fatty acid synthesis.IMPORTANCE:We report that although all P. aeruginosa OAR homologs have similar structures and the conserved catalytic triad of the bacterial OAR enzymes, only a few OAR homologs have OAR activity.

To deregulate the purine operon of the purine biosynthetic pathway and optimize energy generation of the respiratory chain to improve the yield of guanosine in Bacillus amyloliquefaciens XH7.

To construct a promoter probe vector, pBE-bgaB, to screen strong promoters from Bacillus amyloliquefaciens.

解淀粉芽孢杆菌(Bacillus amyloliquefaciens)是一类重要的工业微生物,在农业、医药和食品等领域有广泛用途.为了开发高效的基因操作技术,在解淀粉芽孢杆菌中引入单链核苷酸(ssDNA)介导的同源重组方法.首先,通过敲除mutS基因干扰解淀粉芽孢杆菌的错配修复系统,然后导入单链结合蛋白(Beta)表达质粒,构建了宿主菌B.amyloliquefaciens XH7(mutS-,bet+).其次,通过设计88 bp的ssDNA电转化导入以上构建的宿主菌中实现了以rpoB基因为靶点的有效同源重组,转化株产生利福平抗性.实验优化了ssDNA介导同源重组的参数:75μg的ssDNA,电转细胞复苏时间为6～12h.本研究首次成功实现了ssDNA同源重组技术在解淀粉芽孢杆菌的应用,对解淀粉芽孢杆菌和其它难转化的芽孢杆菌属进行有效的遗传操作手段提供新的思路.

Bacillus amyloliquefaciens is an important industrial microbe for the production of many industrial enzymes and primary metabolites. Although the complete genome sequence of B. amyloliquefaciens has been now published, transcript structures of B. amyloliquefaciens remain poorly defined. In this study, high-throughput RNA sequencing (RNA-seq) technology was applied to dissect the transcriptome of B. amyloliquefaciens strain XH7. In total, 3936 out of a total of 4204 B. amyloliquefaciens genes (93.6%) were transcribed under the selected growth condition. Transcriptional start sites (TSS) of 1064 annotated genes and 749 operons were identified. To screen for strong promoters, a beta-galactoside reporter was fused to eight candidate promoters from 288 genes with higher expression levels (RPKM values) than the control gene P43-bgaB. The results illustrated that the candidate promoter Pr2 (promoter for the sigW gene) displayed the strongest beta-galactosidase specific activity during the post-log phase, suggesting that it could be used effectively for heterologous gene expression. The presented data will contribute to the further study of the B. amyloliquefaciens transcriptome by identifying useful promoters for industrial uses.

In the phosphotransferase system of Bacillus amyloliquefaciens, glucose is transported into the cell mainly through enzymes EI, HPr and EIIGlc encoded by the ptsGHI operon. In this investigation, first, about 1 kbp upstream and downstream DNA fragments of ptsG and ptsHI genes were amplified and were ligated into one fragment by using fusion PCR. Then, the fused fragment was inserted into the temperature-sensitive plasmid pKS2 and transformed by electroporation into Bacillus amyloliquefaciens XH7, thus constructing ptsG-and ptsHI-deficient strains. Experimental results show that (1) in LB medium, there is no obvious difference in the growth characteristics between the deficient strains and the wild-type strains; (2) in LB medium supplemented with 2% glucose, the ptsG gene-deficient strain shows a maximal cell density that is 28% higher than the wild-type one as well as a longer steady period than the control, while the ptsHI gene-deficient strain shows a maximal cell density that is about 32% lower than the wild-type one, and its growth accords well with that in LB medium; and (3) in guanosine fermentation experiments, the guanosine production yield of the ptsG gene-deficient strain is about 24% higher than that of the wild-type one, while that of the ptsHI gene-deficient strain is about 82% lower These results demonstrate that ptsG gene-deficient strains are of good ability in growth and guanosine biosynthesis.

ABSTRACT Here we report the draft annotated genome sequence of Escherichia coli XH140A, which is used to produce l -threonine in industry. The genome sequence will allow the characterization of the molecular mechanisms underlying its beneficial properties.

ABSTRACT l -Threonine has been widely used as a supplement in the food, pharmaceutical, and cosmetic industries. Here, we present a high-quality draft annotated genome sequence of Escherichia coli XH001, a producer of l -threonine in industry. Its genome and plasmid sequence will provide clues about the molecular mechanisms underlying its beneficial properties.

Here, we report the complete annotated genome sequence of Bacillus amyloliquefaciens XH7, which is used to produce purine nucleosides in industry. The genome sequence will allow for the characterization of the molecular mechanisms underlying its beneficial properties.

Corynebacterium acetoacidophilum was used as the original strain for stepwise mutation by whole genome fragmentation mutation(UV and NaNO2)and subsequent sulfaguanidine resistance screening.A L-proline over-producing mutant was obtained.The optimum contents of glucose,biotin and thiamin in shake-flask fermentation were 16%,300μg/L and 400μg/L respectively,and pH is 6.8~7.0,where 25ml medium was cultured in 500ml shake-flask.After 72h by flask-shaking batch fermentation,the production of L-proline reached 75.6g/L,5% higher than with the control.To investigate the effect of cell growth on the L-proline production,50L fed-batch culture was performed at the specific growth rate(μ)of 0.06/h,0.08/h and 0.1/h.The results showed that the specific L-proline production rate reached 0.091g/(g.h)and the highest production was 82.1g/L,increased by 14% compared with the control when the specific growth rate(μ)was 0.08/h.

Through genetic engineering method,the Vitreoscilla hemoglobin gene(vhb) was inserted into the Escherichia coli-Bacillus subtilis shuttle vector pBEP43-DFE.The recombinant plasmid pBEP43-vhb was constructed and chemically transformed into Bacillus subtilis WB800.Molecular analysis of the recombinant was carried by enzyme digestion and PCR.The Vitreoscilla hemoglobin gene was demonstrated by carbon monoxide binding analysis,and the result show that active Vitreoscilla hemoglobin was expressed in Bacillus subtilis WB800.According to limited oxygen experiment,Vitreoscilla hemoglobin enhanced cell growth under the low dissolved oxygen concentrations.

Objective To invesgate the effects of zwf2 gene knock-in and disruption on the specific activity of glucose-6-phosphate dehydrogenase(G6PDH),cell growth and oxytetracycline(OTC) biosynthesis in Streptomy-ces rimosus M4018.Methods Taking parent strain S.rimosus M4018 as control,the changes of the specific acitivity of G6PDH,cell growth and OTC level in the media were measured after the mutants M4018-Δzwf 2 and M4018-(zwf2) respectively obtained through gene disruption and knock-in were cultured in shaking flask.Results The specific acitivity of G6PDH in mutant M4018-Δzwf 2 was only half of that in parent strain,while the specific acitivity of G6PDH in mutant M4018-(zwf2) was a bit higher than that in parent strain over the fermentation period.However,the OTC level in mutant M4018-Δzwf 2 was 27 % and 40 % higher than that in parent strain and mutant M4018-(zwf2),respectively.As for the cell growth,the dry cell weight of mutant M4018-(zwf2) was the highest and the difference between mutant M4018-Δzwf 2 and parent strain was not distinct.Conclusion The zwf2 gene disruption benefits the OTC biosynthesis,on the contrary,the reinforced zwf2 gene was better for cell growth.