Overexpression of serine alkaline protease encoding gene in Bacillus species: performance analyses

Bacillus species carrying subC gene encoding serine alkaline protease (SAP) enzyme were developed in order to increase the yield and selectivity in the bioprocess for SAP production. For this aim, subC gene was cloned into pHV1431 Escherichia coli–Bacillus shuttle vector, and transferred into nine host Bacillus species, i.e. B. alvei, B. amyloliquefaciens, B. badius, B. cereus, B. coagulans, B. firmus, B. licheniformis, B. sphaericus and B. subtilis. The influence of the host Bacillus species on SAP production on a defined medium with glucose was investigated in bioreactor systems. For each of the recombinant (r-) Bacillus species, effects of initial glucose concentration on cell growth and SAP production were investigated; and, physiological differences and similarities between the wild-type and r-Bacillus species are discussed. The highest biomass concentration was obtained with r-B. coagulans as 3.8 kg m⁻³ at the initial glucose concentration of C_Go=20 kg m⁻³ and the highest volumetric SAP activity was obtained with r-B. amyloliquefaciens as 1650 U cm⁻³ at C_Go=20 kg m⁻³. Overall SAP activity per amount of substrate consumed was the highest for r-B. sphaericus (137 U g⁻¹ cm⁻³) and r-B. licheniformis (130 U g⁻¹ cm⁻³). Among the r-Bacillus species the highest activity increase compared to the wild types was obtained with r-B. sphaericus while the lowest increase was obtained with r-B. amyloliquefaciens and r-B. licheniformis due to high SAP production potential of the wild-type strains. During storage of the host microorganisms, r-B. alvei and r-B. amyloliquefaciens were not able to bear the recombinant plasmid, probably, due to the restriction enzymes synthesized. Due to the highest stable volumetric activities r-B. licheniformis (950 U cm⁻³) and r-B. sphaericus (820 U cm⁻³) appear to be the favorable hosts for the production of SAP. All the r-Bacillus species excreted organic acids oxaloacetic and succinic acids, but, none excreted the amino acid valine. The variations in by-product distributions with each recombinant organism were also discussed.     

Gordonia lacunae sp. nov., isolated from an estuary

An actinomycete, strain BS2^T, was isolated from a sand sample collected from an estuary in Plettenberg Bay, Western Cape Province, South Africa. Based on 16S rRNA gene sequence similarities and chemotaxonomy, strain BS2^T was identified as a member of the genus Gordonia. It exhibited weak antibiosis against Mycobacterium aurum A+ and Mycobacterium smegmatis LR222. Phenotypic and phylogenetic results allowed for the differentiation of strain BS2^T from other species within the genus Gordonia. DNA–DNA hybridization further differentiated strain BS2^T from its nearest phylogenetic neighbour, Gordonia terrae NRRL B-16283^T (57.5±4.4% DNA relatedness). Strain BS2^T is therefore a novel species within the genus Gordonia, for which the name Gordonia lacunae is proposed, with the type strain being BS2^T (=DSM 45085^T=NRRL B-24551^T).     

台湾地区芽胞杆菌物种多样性

了解芽胞杆菌资源多样性, 可为芽胞杆菌功能资源挖掘和菌剂开发提供基础。从台湾地区8个市(县)采集土壤样本, 从20份土壤样品中分离获得了136株芽胞杆菌, 采用16S rRNA基因同源性将其鉴定为芽胞杆菌科的2个属、20个种。分别属于芽胞杆菌属(Bacillus)的16个种和赖氨酸芽胞杆菌属(Lysinibacillus)的4个种。根据分离频度分析得知, 台湾地区土壤中的芽胞杆菌优势菌群为阿氏芽胞杆菌(Bacillus aryabhattai)、苏云金芽胞杆菌(B. thuringiensis)和蜡样芽胞杆菌(B. cereus), 其他种类分布极其不均匀。芽胞杆菌Shannon多样性指数为1.2925-2.5850, 最高的为台中市和嘉义市(2.5850), 最低的为桃园县(1.2925)。根据分离频度对芽胞杆菌种类的聚类分析显示, 当欧式距离λ = 20时, 芽胞杆菌种类可分为高频度分布类型如阿氏芽胞杆菌(B. aryabhattai), 低频度分布类型如简单芽胞杆菌(B. simplex)。依据分离频度对8个采样点间的聚类分析未发现采样点间的芽胞杆菌种类分布的相关性。本研究认为台湾地区土壤中蕴藏着丰富芽胞杆菌种类多样性高, 具有很大的开发潜力。     

Rhodococcus phenolicus sp. nov., a novel bioprocessor isolated actinomycete with the ability to degrade chlorobenzene, dichlorobenzene and phenol as sole carbon sources

The aerobic degradation of phenol, chlorobenzene and dichlorobenzene as a sole carbon source has been observed in bacterial Gram-positive strain G2P^T isolated from a wastewater bioprocessor. Cells display branching mycelia fragmenting into rod and coccoid elements when grown on TSA. Aerial hyphae formation occurs when grown on phenol and chlorinated aromatics as the sole carbon source. Growth was observed at up to 0.75% phenol as a sole carbon source, indicating a strong tolerance for the compound. The 16S rRNA gene sequence shares the greatest similarity with members of the Rhodococcus genus, with the closest shared nucleotide identity of 98% with the aromatic toxin degrading bacteria Rhodococcus zopfii DSM 44108^T. Neighbor-joining and parsimony analysis of Corynebacterineae 16S rRNA gene sequences consistently places strain G2P^T in a clade shared with R. zopfii within the Rhodococcus rhodochrous subclade. Based on a unique polyphasic profile involving phenotypic, ribosomal DNA sequence analysis, DNA–DNA hybridization, mol% DNA G+C content and fatty acid composition, G2P^T is proposed to represent a previously uncharacterized, novel species in the genus Rhodococcus. The name Rhodococcus phenolicus is proposed for the isolate with the type strain G2P^T (=DSM 44812) (=NRL B-24343).     

Production and partial characterization of bacteriocin-like pepitdes by Bacillus licheniformis ZJU12

Bacillus licheniformis ZJU12, which was isolated from soil, could produce bacteriocin-like peptides that exhibited a broad spectrum of antagonistic activity against various species of Gram-positive bacteria and fungal pathogens, but not against Gram-negative bacteria tested except Xanthomonas oryzae pv. oryzae, a rice pathogen. The bacteriocin-like peptides were sensitive to proteinase K and trypsin. The activity was stable during temperature exposure up to 100 °C for 30 min, but lost completely at 121 °C for 15 min. The cell-free supernatant of B. licheniformis ZJU12 was shown to retain the activity within the pH range of 2–9, and the optimum pH for the activity was about 6.5. No adverse effect of the antagonistic compound to mice was observed in acute toxicity tests with the dose of 0.8 mg/20 g.     

Hydrogen and polyhydroxybutyrate producing abilities of microbes from diverse habitats by dark fermentative process

Thirty five bacterial isolates from diverse environmental sources such as contaminated food, nitrogen rich soil, activated sludges from pesticide and oil refineries effluent treatment plants were found to belong to Bacillus, Bordetella, Enterobacter, Proteus, and Pseudomonas sp. on the basis of 16S rRNA gene sequence analysis. Under dark fermentative conditions, maximum hydrogen (H₂) yields (mol/mol of glucose added) were recorded to be 0.68 with Enterobacter aerogenes EGU16 followed by 0.63 with Bacillus cereus EGU43 and Bacillus thuringiensis EGU45. H₂ constituted 63–69% of the total biogas evolved. Out of these 35 microbes, 18 isolates had the ability to produce polyhydroxybutyrate (PHB), which varied up to 500 mg/l of medium, equivalent to a yield of 66.6%. The highest PHB yield was recorded with B. cereus strain EGU3. Nine strains had high hydrolytic activities (zone of hydrolysis): lipase (34–38 mm) – Bacillus sphaericus strains EGU385, EGU399 and EGU542; protease (56–62 mm) – Bacillus sp. strains EGU444, EGU447 and EGU445; amylase (23 mm) – B. thuringiensis EGU378, marine bacterium strain EGU409 and Pseudomonas sp. strain EGU448. These strains with high hydrolytic activities had relatively low H₂ producing abilities in the range of 0.26–0.42 mol/mol of glucose added and only B. thuringiensis strain EGU378 had the ability to produce PHB. This is the first report among the non-photosynthetic microbes, where the same organism(s) – B. cereus strain EGU43 and B. thuringiensis strain EGU45, have been shown to produce H₂ – 0.63 mol/mol of glucose added and PHB – 420–435 mg/l medium.     

Sustained expression of keratinase gene under PxylA and PamyL promoters in the recombinant Bacillus megaterium MS941

The ker gene encoding pre-pro keratinase of Bacillus licheniformis MKU3 was cloned with xylose inducible promoter (PxylA) or -amylase promoter (PamyL) or both in Escherichia coli–Bacillus shuttle vector, pWH1520 generating recombinant plasmids pWHK3, pWAK3 and pWXAK3 respectively. Compared with Bacillius megaterium MS941 (pWXAK3) expressing ker gene with PxylA–PamyL promoters, B. megaterium MS941 (pWAK3) with PamyL displayed higher keratinase yield (168.6 U/ml) and specific activity (14.59 U/mg) after 36 h of growth in LB medium, however the keratinase yield decreased in the culture grown in LB medium supplemented with starch or xylose or both. A maximum yield of 186.3 U/ml with specific activity of 17.25 U/mg was obtained from xylose induced keratinase expression in B. megaterium MS941 (pWHK3) grown for 24 h. The recombinant plasmids were stably maintained with sustained expression of keratinase for about 60 generations in B. megaterium MS941 rather than in B. megaterium 14945.     

WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line

Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2–3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P = 0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44–52 h. A positive correlation (R² = 0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption.     

武夷山自然保护区土壤可培养芽胞杆菌 的物种多样性及分布

为了解武夷山自然保护区土壤中可培养芽胞杆菌的分布状况, 2012年6月从该保护区的黄岗山顶部、中部、底部和桐木关、挂墩、大竹岚等6个地点采集土样75份。用80℃水浴加热、稀释平板法进行芽胞杆菌的分离, 并根据16S rRNA基因序列分析对菌株进行初步鉴定。从土样中分离出芽胞杆菌418株, 鉴定为8个属42个种, 其中Bacillus属的种数最多, 有20种, Paenibacillus属和Lysinibacillus属分别有8种和7种。不同地点分离到的芽胞杆菌在种类、数量上存在差异: 从大竹岚土壤中分离到的芽胞杆菌种类最多, 从黄岗山中部和底部分离到的种类数则较少; 挂墩、大竹岚土壤中芽胞杆菌的数量较大, 达3.6×10⁶ cfu/g以上, 而黄岗山顶部和中部土壤中的数量则少于4.9×10⁵ cfu/g。Bacillus cereus、B. mycoides、B. thuringiensis和Lysinibacillus xylanilyticus等4个种在6个地点的土样中均有分离到, 其中B. thuringiensis、L. xylanilyticus是该保护区土壤中的优势种。桐木关土壤中芽胞杆菌的种类多样性和均匀度指数都比其他5个地点的高, 而挂墩土壤中芽胞杆菌的Shannon-Wiener多样性、均匀度和优势度指数都最低。B. mycoides和B. thuringiensis的数量与海拔显著相关, 相关系数分别为0.852和-0.834, B. cereus、B. mycoides、B. thuringiensis的分离频度与海拔的相关性极显著, 相关系数分别为0.960、0.952和-0.931。研究结果表明, 武夷山自然保护区土壤中可培养芽胞杆菌的种类丰富、数量较大, 具有较高的多样性。     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Potential for control of seedling blight of wheat caused by Fusarium graminearum and related species using the bacterial endophyte Bacillus mojavensis

Fusarium-infected wheat seed decreases germination, seedling emergence, and causes post emergence seedling death, and can contribute to wheat scab and ear rot of maize, with consequent production of mycotoxins such as deoxynivalenol and zearalenone. Current seed treatments have proved ineffective in controlling seedling blight and scab. A patented endophytic bacterial strain, Bacillus mojavensis RRC 101, and several other strains of this species were studied to determine in vitro antagonism to some Fusarium species and to assess the potential of this bacterium to serve as an endophytic biocontrol for seedling blight of wheat produced by species within the F. graminearum complex, as well as other species of Fusarium. Seedling emergence and seed germination were two tests used as indicators of seedling blight. These tests were conducted in growth rooms with two wheat cultivars highly susceptible to scab, Norm and Pioneer 2552, and other cultivars with varying resistance to scab. The results indicated that all strains of this bacterium were antagonistic in vitro to the strains of F. graminearum and its seven related species, as well as four strains of F. pseudograminearum and the two strains of F. verticillioides. Germination of the highly scab susceptible cultivar 2552 was increased from 77 to 97% when planted in soil containing a mixed inoculum of F. graminearum and related species. Seedling emergence in the very susceptible wheat cultivar Norm increased from 20 to 82% when treated with the bacterium. The data indicated that inoculating wheat kernels with B. mojavensis reduced seedling blight of wheat produced by F. graminearum and related Fusarium species indicating the potential for this bacterium as a biocontrol under field condition.     

Cloning and production of Xylanase B from Paenibacillus barcinonensis in Bacillus subtilis hosts

Xylanase B from Paenibacillus barcinonensis was cloned in shuttle vectors for Escherichia coli and Bacillus subtilis, and expressed in Bacillus hosts. Several recombinant strains were constructed, among which B. subtilis MW15/pRBSPOX20 showed the highest production. This recombinant strain consists of a protease double mutant host containing P. barcinonensis xynB gene under the control of a phage SPO2 strong promoter. Maximum production was found when the strain was cultured in nutrient broth supplemented with xylans. Analysis of xylanase B location in B. subtilis MW15/pRBSPOX20 showed that the enzyme remained cell-associated in young cultures, consistent with its intracellular location in its original host, P. barcinonensis, and the lack of a signal peptide. However, when cultures reached the stationary phase, xylanase B was released to the external medium as a result of cell lysis. The amount of enzyme located in the supernatants of old cultures could account for 50% of total xylanase activity. Analysis by SDS-PAGE showed that xylanase B is an abundant protein found in the culture medium in late stationary phase cultures.     

Development of a rapid and sensitive immunoassay for detection and subsequent recovery of Bacillus anthracis spores in environmental samples

Bacillus anthracis is considered a major threat as an agent of bioterrorism. B. anthracis spores are readily dispersed as aerosols, are very persistent, and are resistant to normal disinfection treatments. Immunoassays have been developed to rapidly detect B. anthracis spores at high concentrations. However, detection of B. anthracis spores at lower concentrations is problematic due to the fact that closely related Bacillus species (e.g., B. thuringiensis) can cross-react with anti-B. anthracis antibodies, resulting in false positive detections. Subsequent polymerase chain reaction (PCR) analysis is required to differentiate virulent strains. We report here on a protocol for the rapid, sensitive detection of B. anthracis spore using the Integrating Waveguide Biosensor followed by a method for the rapid release and germination of immunocaptured spores. A detection limit of ca. 10³ spores was achieved by incubating spores simultaneously with capture and detection antibodies (“liquid-phase” assay) prior to capture on capillary tubes/waveguides. Subsequent incubation with BHI broth directly in capillary tubes allowed for rapid germination, outgrowth, and release of spores, resulting in vegetative cells for PCR analysis.     

Biotransformation of isoeugenol to vanillin by a newly isolated Bacillus pumilus strain: identification of major metabolites

A bacterial strain S-1 capable of transforming isoeugenol to vanillin was isolated. The strain was identified as Bacillus pumilus based on biochemical tests, cellular fatty acid composition, riboprint pattern and 16S rRNA gene sequence analyses. In the biotransformation of isoeugenol, vanillin was the main product. With the growing culture of B. pumilus S-1, 10 g l⁻¹ isoeugenol was converted to 3.75 g l⁻¹ vanillin in 150 h, with a molar yield of 40.5% that is the highest up to now. Dehydrodiisoeugenol, a dimer of isoeugenol, was separated by preparative thin layer chromatography and identified by gas chromatography–mass spectrometry. Based on the accurate masses obtained from gas chromatography–high resolution mass spectrometry, two key intermediates, isoeugenol-epoxide (IE) and isoeugenol-diol (ID), were identified by mass spectra interpretations. The biotransformation with resting cells showed that vanillin was oxidized to vanillic acid and then to protocatechuic acid before the aromatic ring was broken. These findings suggest that isoeugenol is degraded through an epoxide-diol pathway.     

Improved high thermal stability of pullulanase from a newly isolated thermophilic Bacillus sp. AN-7

A thermophilic Bacillus sp. strain AN-7, isolated from a soil in India, produced an extracellular pullulanase upon growth on starch–peptone medium. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The optimum temperature and pH for activity was 90 °C and 6.0. With half-life time longer than one day at 80 °C the enzyme proves to be thermostable in the pH range 4.5–7.0. The pullulanase from Bacillus strain lost activity rapidly when incubated at temperature higher than 105 °C or at pH lower than 4.5. Pullulanase was completely inhibited by the Hg²⁺ ions. Ca²⁺, dithiothreitol, and Mn²⁺ stimulated the pullulanase activity. Kinetic experiments at 80 °C and pH 6.0 gave V_max and K_m values of 154 U mg⁻¹ and 1.3 mg ml⁻¹. The products of pullulan were maltotriose and maltose. This proved that the purified pullulanase (pullulan-6-glucanohydrolase, EC 3.2.1.41) from Bacillus sp. AN-7 is classified under pullulanase type I. To our knowledge, this Bacillus pullulanase is the most highly thermostable type I pullulanase known to date.     

Characterization of Desulfomicrobium salsuginis sp. nov. and Desulfomicrobium aestuarii sp. nov., two new sulfate-reducing bacteria isolated from the Adour estuary (French Atlantic coast) with specific mercury methylation potentials

Three strains of sulfate-reducing bacteria (ADR21, ADR26 and ADR28) were isolated from Adour estuary sediments (French South Atlantic coast). Cells of these isolates were rod-shaped, motile and stained Gram-negative. The 16S rRNA and dsrAB genes sequence analyses indicated that these three strains belonged to the genus Desulfomicrobium within the delta Proteobacteria, with Desulfomicrobium escambiense strain DSM10707^T as their closest relative. According to phenotypic characteristics, strains ADR21 and ADR28 could be considered as members of the same species. The relatedness values, based on DNA–DNA hybridization studies, between strains ADR21/DSM10707^T, ADR26/DSM10707^T and ADR21/ADR26 ranged between 30.6–40.8%, 45.2–43.0% and 19.0–26.4%, respectively. Strains ADR21 and ADR28 grew well on lactate, fumarate, malate, formate, ethanol and H₂/acetate in the presence of sulfate as an electron acceptor. Thiosulfate, nitrate, fumarate and DMSO were alternative electron acceptors. Malate was well fermented but pyruvate and fumarate only poorly. Strain ADR26 could not grow on ethanol or fumarate and was unable to use DMSO or fumarate as electron acceptors. The three new strains exhibited differences compared to the type strain of D. escambiense, such as temperature optima, substrate utilization and mercury methylation capacities. On the basis of both genetic and phenotypic evidences, strain ADR21 is proposed as the type strain of the species Desulfomicrobium salsuginis sp. nov., and strain ADR26 as the type strain of the species Desulfomicrobium aestuarii sp. nov.     

Meta-pathway degradation of phenolics by thermophilic Bacilli

Thermophilic bacteria capable of degrading phenol as the sole carbon source were isolated from sewage effluent. The isolates were aerobic, sporulating, motile rod-shaped bacteria characterized as Bacillus species with growth temperature optima of 50–60°C. The enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol-2,3-dioxygenase, was detected in all isolates grown in the presence of phenol. One strain, designated Bacillus strain Cro3.2, was capable of degrading phenol, o-, m-, and p-cresol via the meta-pathway and tolerated phenol at concentrations up to 0.1% (w/v) without apparent inhibition of growth. Phenol degradation activities in strain Cro3.2 were induced 3–5 h after supplementation by phenol, orcinol, and the cresols but not by halo- or nitro-substituted phenols. Maximal rates of phenol degradation in stirred bioreactors (10 μmol/min⁻¹/g⁻¹ cells) were achieved at an O₂ delivery rate of 1.0 vvm and temperatures of 45–60°C; however, catechol-2,3-dioxygenase (but not 2-hydroxymuconic semialdehyde dehydrogenase) was rapidly inactivated at high oxygen concentrations. Whole cells of Bacillus strain Cro3.2 entrapped in calcium alginate, polyacrylamide, and agarose gels showed widely different rates of phenol degradation. In calcium alginate gels, rapid loss of phenol-degrading activity was attributed to calcium-induced inactivation of catechol-2,3-dioxygenase. No stabilization with respect to oxygen-induced inactivation was observed under any of the immobilization conditions. It is concluded that the counteractive effects of oxygen limitation at low dO₂ and inactivation of catechol-2,3-dioxygenase at high dO₂ levels pose a significant impediment to the use of resting thermophile cells in the treatment of phenolic waste streams.     

Burkholderia phenoliruptrix sp. nov., to accommodate the 2,4,5-trichlorophenoxyacetic acid and halophenol-degrading strain AC1100

Strain AC1100 is well-known for its ability to degrade a variety of recalcitrant xenobiotics, including 2,4,5-trichlorophenoxyacetic acid. We performed a polyphasic-taxonomic study to determine its taxonomic position. The G+C content of strain AC1100 was 62.6 mol%. On the basis of 16S rRNA gene sequence similarity, strain AC1100 belonged to the b-Proteobacteria and was most closely related to Burkholderia fungorum (98.3% similarity). DNA-DNA hybridisations, comparison of protein profiles, cellular fatty acid analysis and biochemical tests allowed genotypic and phenotypic differentiation of strain AC1100 from other Burkholderia species. Our data show that strain AC1100 represents a novel species for which the name Burkholderia phenoliruptrix sp. nov. is proposed. The type strain is AC1100^T (= LMG 22037^T = CCUG 48558^T).     

yhcZ基因在苏云金芽胞杆菌生长中的功能研究

在枯草芽胞杆菌和蜡样芽胞杆菌中,yhcZ基因和yhcY基因组成双组分系统调控细菌生长,但yhcZ基因在苏云金芽胞杆菌中发挥的生物学功能尚未明确。本研究通过基因功能注释、上下游基因排列分析和氨基酸序列比对,证实苏云金芽胞杆菌库斯塔克亚种HD73中HD73_5824基因为yhcZ基因,推测其与HD73_5825基因(yhcY基因)共同组成双组份系统调控细菌生长。利用同源重组技术敲除HD73菌株中的yhcZ基因获得缺失突变体HD (ΔyhcZ),其在LB和SSM培养基中生长均慢于野生型HD73,而互补菌株HD(ΔyhcZ::yhcZ)菌株则能够部分恢复生长,表明yhcZ基因的缺失影响了该菌株细胞的生长。在以0.4%葡萄糖为唯一碳源的M9培养基中,HD (ΔyhcZ)生长速度快于HD73,表明yhcZ基因在该菌株吸收利用葡萄糖的过程中发挥重要作用。Biolog实验显示HD (ΔyhcZ)的单孔颜色变化率低于HD73,且对D/L-丝氨酸、甲酸、D-葡糖酸、L-组胺,D-乳酸甲酯以及柠檬酸等的吸收利用能力低于HD73,表明yhcZ基因能显著影响HD73菌株对碳源的利用。同时,HD(ΔyhcZ)对8% NaCl的耐受能力弱于HD73,表明该基因可能参与细菌细胞应力响应相关基因的表达与调控。以上结果表明yhcZ基因在HD73菌株生长过程中对葡萄糖及其他碳源的利用具有重要的促进作用。本研究结果为解析yhcZ基因调控葡萄糖及碳源利用的分子机制奠定基础,且为进一步研究细菌生长及发酵提供参考。