Metabolic engineering of Serratia marcescens with the bacterial hemoglobin gene: alterations in fermentation pathways

Serratia marcescens was transformed with plasmid vector pUC8 or pUC8 containing the bacterial (Vitreoscilla) hemoglobin gene (vgb) on either a 2.3-kb fragment (pUC8:15) or 1.4-kb fragment (pUC8:16) of Vitreoscilla DNA. The vgb-bearing strains were compared with the pUC8 transformant and untransformed S. marcescens with respect to growth in Luria-Bertani (LB) broth supplemented with glucose or casein acid hydrolysate. Growth (on a viable cell basis) was similar to that in unsupplemented LB. Total acid excretion (as estimated by medium pH) was similar for all strains in both LB plus 2% casein acid hydrolysate and LB without additions. Acid excretion in LB plus 2% glucose was somewhat greater at up to 10 h in culture for the two vgb-bearing strains; from 10 to 26 h in culture, the pHs of these cultures continued to decrease (to 4.1-4.2), whereas those of the non-vgb-bearing strains returned to near the starting pH (7.4-7.8). Concomitantly, after 26 h of culture in LB plus 2% glucose, the non-vgb-bearing strains had produced about 15 times as much acetoin and about three to four times as much 2,3-butanediol as the vgb-bearing strains. In general, for all strains, much more acetoin and 2,3-butanediol were produced in LB plus 2% glucose than in unsupplemented LB. The exception was acetoin production by the strain bearing vgb on plasmid pUC8:15; after 26 h of culture in LB without supplementation it was between three and four times that of the other strains, and about 50% higher than its level in LB plus 2% glucose. When grown with the 2% casein acid hydrolysate supplement, the strain bearing vgb on plasmid pUC8:15 produced much more acetoin and 2,3-butanediol than the other strains after 26 hours in culture. The results confirm that vgb can significantly alter carbon metabolism and suggest that the use of vgb technology for directed metabolic engineering may be a complicated process, depending in part on medium composition.     

Effect of Salt Concentration in the Recovery Medium on Heat-injured Streptococcus faecalis

Properties relating to the recovery of three heat-injured strains of Streptococcus faecalis were studied. All strains were cultured in all purpose plus Tween broth (APT) at 30 C for 24 hr before being subjected to heat in fresh APT broth. APT recovery medium containing various added amounts of NaCl, KCl, MgCl(2), or KCl and MgCl(2) was used to assess the effect of salts on the recovery of thermally injured S. faecalis. It was evident that, upon exposure to heat, S. faecalis cells became sensitive to increased salt concentrations. Analyses to determine the ribonucleic acid (RNA) content of heated cells showed a reduction of cellular RNA, but the per cent reduction was not directly proportional to the per cent reduction of the viable cells.     

Culturing enterohemorrhagic Escherichia coli in the presence and absence of glucose as a simple means of evaluating the acid tolerance of stationary-phase cells.

Prior growth of seven enterohemorrhagic and one nonenterohemorrhagic strains of Escherichia coli in tryptic soy broth with (TSB+G) and without (TSB-G) 1% glucose was evaluated for its effect on acid tolerance. The final pHs of 18-h TSB+G and TSB-G cultures were 4.6 to 5.2 and 6.9 to 7.0, respectively. Cells were then transferred to brain heart infusion broth adjusted to pH 2.5 or 3.0 with HCl, incubated at 37 degrees C for up to 7 h, and assayed periodically for viable populations with brain heart infusion and MacConkey agars. All enterohemorrhagic strains were acid resistant (< 0.5 log decline after 7 h) when initially cultured in TSB+G, but substantial differences in acid tolerance were observed among strains cultured in TSB-G (log declines ranged from < 0.3 to > 3.8). The results indicated that prior growth in a medium with and without a fermentable carbohydrate is a convenient way to studying the induction of acid tolerance, that acid inactivation is preceded by a period of acid injury, and that pH-independent and pH-dependent stationary-phase acid tolerance phenotypes may exist among strains of enterohemorrhagic E. coli.     

Enhancing the antimicrobial effects of bovine lactoferrin against Escherichia coli O157:H7 by cation chelation, NaCl and temperature

AIM: To evaluate the effect of NaCl, growth medium and temperature on the antimicrobial activity of bovine lactoferrin (LF) against Escherichia coli O157:H7 in the presence of different chelating agents. METHODS AND RESULTS: LF (32 mg ml(-1)) was tested against E. coli O157:H7 strain 3081 in Luria broth (LB) and All Purpose Tween (APT) broth with metal ion chelators sodium bicarbonate (SB), sodium lactate (SL), sodium hexametaphosphate (SHMP), ethylene diamine tetraacetic acid (EDTA) or quercetin at 0.5 and 2.5% NaCl at 10 and 37 degrees C. LF and the chelators were tested against four other E. coli O157:H7 strains in LB at 2.5% NaCl and 10 degrees C. LF alone was bacteriostatic against strains 3081 and LCDC 7283 but other strains grew. Antimicrobial effectiveness of LF was reduced in APT broth but enhanced by SB at 2.5% NaCl and 10 degrees C where 4.0 log(10) CFU ml(-1) inoculated cells were killed. EDTA enhanced antimicrobial action of the LF-SB combination. SL alone was effective against E. coli O157:H7 but a reduction in its activity at 2.5% NaCl and 10 degrees C was reversed by LF. The combinations LF-SHMP and LF-quercetin were more effective at 37 degrees C and NaCl effects varied. CONCLUSIONS: LF plus SB or SL were bactericidal toward the same 3/5 E. coli O157:H7 strains and inhibited growth of the others at 2.5% NaCl and 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of LF with either SL or SB shows potential for reducing viability of E. coli O157:H7 in food systems containing NaCl at reduced, but growth permissive temperature.     

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37°C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 10⁶ to 10⁸ cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.Download video file.^{(109M, mp4)}     

应用DNA芯片技术对禽致病性大肠杆菌可能致病基因体外表达水平的研究

应用DNA芯片研究禽致病性大肠杆菌可能致病基因的表达.构建禽致病性大肠杆菌毒力基因、潜在毒力基因的DNA芯片,应用基因芯片技术对同属O2血清型的禽高致病性大肠杆菌E058株和低致病性大肠杆菌E526株在体外LB培养基和鸡血清培养状态下进行差异表达分析.结果:在体外LB静置培养状态下,低致病株E526与高致病株E058相比共有16个差异基因,均为下调基因.在鸡血清静置培养中,E526与E058相比共有15个差异基因,均为下调基因.应用基因芯片成功筛选了禽致病性大肠杆菌在体外不同条件下的毒力基因及可能毒力基因中差异表达基因,表明一些铁摄取系统相关基因对APEC的毒力较重要,同时也筛选出了一些新的可能致病基因aes-1,aes-2,aes-3,aes-4,aes-6,aes-8,aes-10,aes-13,aes-15,aes-31等.     

Lipid composition of Staphylococcus aureus and its derived L-forms.

Two strains of Staphylococcus aureus (Newman and Tazaki) and their derived L-forms were cultured in serum-containing broth and the differences in their lipid compositions were analyzed. Cardiolipin accounted for more than 50% of the total phospholipid phosphorus in L-forms, but for less than 25% in parent bacteria. The cardiolipin content of L-forms was very high through all growth phases, although it increased gradually as growth proceeded. Significant amounts of cholesterol and its esters were present in parent strains and L-forms, all of which incorporated serum cholesterol into the cell membrane. On the other hand, they could be detected in the L-forms but not in the parent strains when they were cultured in serum-free broth. To examine the ability of L-forms to synthesize cholesterol, the cholesterol content of L-forms cultured in serum-free broth was compared with that of the medium. The results indicated that staphylococcal L-forms could synthesize cholesterol and its esters. These differences in lipid composition suggested that modification of membrane lipids may occur as an adaptational change in response to the disappearance of the cell wall.     

The effect of chlorogenic, gallic and quinic acids on the growth of spoilage strains of Lactobacillus collinoides and Lactobacillus brevis

The naturally occurring complex organic acids, chlorogenic acid, gallic acid and quinic acid, at concentrations of 100, 500 and 1000 mg l^-1 were evaluated for effects on the growth of three spoilage strains of Lactobacillus collinoides and one of Lact. brevis in acid tomato broth containing 5% (v/v) ethanol at pH 4.8. During early stages of growth, all the complex acids at each concentration stimulated growth of Lact. collinoides but not of Lact. brevis. During stationary phase, chlorogenic and gallic acids produced greater cell densities of all strains, whereas quinic acid generally had less effect. The presence of these complex acids in fruit products may increase the requirement for added preservative in order to prevent spoilage by certain strains of lactic acid bacteria.     

Ecological insights into the underlying evolutionary patterns of biofilm formation from biological wastewater treatment systems: Red or Black Queen Hypothesis?

Interspecies interactions and phylogenetic distances were studied to reveal the underlying evolutionary adaptations of biofilms sourced from wastewater treatment processes. Based on 380 pairwise cocultures of 40 strains from two microbial aggregates (surface-attached and mobile aggregates [flocs]) at two substrate concentrations (LB broth and 0.1× LB broth), interspecies interactions were explored using biofilm classification schemes. There was a strong source-dependence of biofilm development formed by the monocultures, that is, a higher biofilm formation potential for strains from attached aggregates than for those from sludge flocs at both substrate concentrations. Interestingly, the results showed that total biofilm reduction was dominant in the dual-species biofilm sourced from flocs in both LB broth (67.37%) and 0.1× LB broth (64.21%), indicating high interspecific competition in mobile aggregates and the independence of substrate concentrations. However, biofilm reduction was higher (33.68%) than induction (19.37%) for the biofilms formed by surface-attached aggregates in LB broth, while the opposite trend was apparent in 0.1× LB broth, suggesting the occurrence of indeterministic processes for biofilm formation and important roles of substrate concentrations. In addition, the more closely related phylogenetic relationships of cocultures from mobile aggregates were consistent with higher competition compared with those from surface-attached aggregates. Overall, the underlying evolutionary patterns of biofilms formed from mobile aggregates consistently followed the essence of the “Red Queen Hypothesis,” while biofilms developed from surface-attached aggregates were not deterministic. This study advanced our understanding of biofilm-related treatment processes using the principles of microbial ecology.     

Preservation of Haemophilus influenzae and Haemophilus parainfluenzae at -70 degrees C.

We have studied the viability of Haemophilus spp. preserved for 5 to 12 months at -70 degrees C. The following media were used: Laboratoire de Santé Publique du Québec (LSPQ) preservation medium, trypticase soy broth with 10 degrees C (vol/vol) glycerol and 40 degrees C (vol/vol) horse serum (TSBG), and Levinthal's broth (LB) medium. Three clinical isolates of both H. influenzae and H. parainfluenzae were used. After 5 months no differences in viability were observed between strains preserved in TSBG and strains preserved in LB, but a significant loss of viability was observed in strains preserved in LSPQ medium. No significant changes in antimicrobial susceptibility were observed after 5-month storage in any medium. After 12 months, TSBG appeared to be the most suitable cryopreservation medium for the six strains tested. We conclude that TSBG represents a good medium for the maintenance of Haemophilus spp. at -70 degrees C for up to 1 year.     

Resuscitation of acid-injured Salmonella in enrichment broth, in apple juice and on the surfaces of fresh-cut cucumber and apple

AIMS: To investigate the resuscitation of acid-injured Salmonella enterica in selected enrichment broths, in apple juice and on cut surfaces of apple and cucumber slices. METHODS AND RESULTS: Following exposure to 2.4% acetic acid for 7 min, S. enterica (serovars Mbandaka, Chester and Newport) cells were used to inoculate enrichment broths, phosphate-buffered saline (PBS), apple juice and fruit slices. Injured Salmonella cells resuscitated and regained the ability to form colonies on selective agar (Xylose-Lysine-Tergitol 4) if they were incubated in lactose broth (LB), universal pre-enrichment broth (UPB) or buffered peptone water (BPW), but not in tetrathionate broth, PBS or apple juice. The resuscitation occurred at a significantly (P > 0.05) faster rate in UPB than in LB or BPW. The resuscitation also occurred on the surfaces of fresh-cut cucumber at 20 degrees C, but not at 4 degrees C. CONCLUSIONS: Acid-injured Salmonella cells resuscitated in nonselective enrichment broths at different rates, but not in selective enrichment broth, apple juice, PBS or on fresh-cut apple. SIGNIFICANCE AND IMPACT OF THE STUDY: Pre-enrichment of food samples in UPB prior to selective enrichment is recommended. Injured Salmonella cells have the ability to resuscitate on fresh-cut surfaces of cucumber when stored at abusive temperatures.     

Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli

Plasmid-host cell interactions have been investigated experimentally using Escherichia coli HB101, plasmid RSF1050 which contains the origin of replication of pMB1, and four other closely related copy number mutant plasmids. Growth characteristics of these recombinant strains and beta-lactamase activity expressed from a plasmid gene were investigated in Luria broth (LB) and in minimal medium (M9) containing in some cases casamino acids or different concentrations of alpha-methylglucoside, a competitive inhibitor of glucose transport. Maximum specific growth rates in LB and minimal media were reduced for increasing plasmid content per cell. Plasmid copy number increased when specific growth rate was reduced by changing medium composition. Growth rates of high copy number strains were less sensitive to alpha-methylglucoside than lower copy number strains and the plasmidfree host. The overall efficiency of plasmid gene expression, measured as the ratio of beta-lactamase specific activity to plasmid content, decreased significantly with increasing plasmid content in LB medium.     

酸性土壤中耐铝细菌的筛选鉴定及其耐铝能力分析

以含有1mmol／LAl3＋的s—LB培养基作为筛选培养基,从酸性土壤中分离到13株耐铝的细菌菌株,选取其中6株进行形态学分析,结果观察到这些菌株的菌体均呈杆状,其中1株为革兰阳性反应,其余5株为革兰阴性反应。以细菌通用引物扩增这些菌株的16SrDNA并测序,将得到的序列与GenBank中的序列进行BLAST比对,利用MEGA4．0软件,按照Neighbor-joining法构建系统进化树,这6个菌株分别与Enterobacter endosymbiont,Serratia marcescens ,Pantoea agglomerans ,Enterobacter aerogenes .Bacillus subtilis 和 Enterobacter asburiae的亲缘关系最近。将这些菌株接种到加有2mmol／LAl3＋、pH4．5的s—LB固体培养基上培养时,它们都能生长,说明这些菌株具有较好的耐铝能力,这些菌株为进一步研究细菌的耐铝机制提供了极好的材料。     

Isolation,identification and characterization of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> LB44 and <Emphasis Type="Italic">Weissella confusa</Emphasis> LM85 for the presence of bacteriocin-like inhibitory substances (BLIS)

Lactic acid bacteria (LAB) were isolated from different soil samples such as dairy, garden, rhizospheric soil and sediments of lake. Thirty nine strains of bacteria were isolated using direct spreading method but only one showed the characteristics of LAB. Whereas, using accumulation with incubation method, fifty seven strains were found to be LAB out of sixty eight strains of bacteria isolated. Therefore, accumulation with incubation method was found to be more effective and faster. On the basis of higher growth and antimicrobial activity, two potential isolates LB44 and LM85 were selected for the further characterization. Strain LB44 and LM85 were identified using 16S rDNA amplification and sequencing as Pediococcus pentosaceus LB44 and Weissella confusa LM85, respectively. Cell free supernatant (CFS) of these strains showed antimicrobial activity, stability at higher temperatures, acidic pH and in the presence of organic solvents, detergents and surfactants. Antimicrobial activity was not affected by treatment with catalase and lipase but reduced after treatment with proteinase K reveals the presence of bacteriocin-like inhibitory substance in CFS. CFS inhibited the growth of other Gram-positive and Gram-negative bacteria such as Micrococcus luteus, Lactobacillus delbrueckii, L. curvatus, L. plantarum, L. acidophilus, Enterobacter cloaceae, Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, Bacillus subtilis, B. licheniformis, Salmonella typhi and Escherichia coli.     

Expression of beta-lactamase by recombinant Escherichia coli strains containing plasmids of different sizes--effects of pH, phosphate, and dissolved oxygen

The characteristics of growth and synthesis of plasmid-encoded protein were studied for strains of recombinant E. coli JM103 which carried the beta-lactamase gene on plasmids of different sizes. The plasmids used included the vector pUC8 and its recombinant derivatives containing varying-sized inserts of Drosophila DNA (not expressed in E. coli). Luria broth (LB) and a minimal medium (M9) supplemented in some cases with additional inorganic phosphate were used as growth media. There was no evidence of segregational instability in these experiments, where no antibiotic selection pressure was employed. Responses of the recombinant strains to variations in environmental parameters including pH, phosphate concentration in the medium, and aeration rate were examined. While the cell growth rate in LB decreased with pH in the range 7.0-8.0, the bulk beta-lactamase activity was maximized at an intermediate pH. The recombinant cell growth rate decreases with increasing plasmid size in the minimal medium, while such decrease is not significant when a rich medium such as LB is used. There is an intermediate plasmid size in the range studied (2.7-8.7 kb), at which beta-lactamase activity is maximum. While reduction in aeration rate (which determines the dissolved oxygen level) is detrimental for cell growth, it is beneficial for beta-lactamase synthesis. The bulk beta-lactamase activity therefore exhibits a maximum with respect to aeration rate. Cell growth and beta-lactamase production are affected in a similar manner by phosphate concentration in the minimal medium and therefore both are maximized at the same phosphate concentration. This investigation demonstrates clearly how the production of a recombinant plasmid-encoded protein can be maximized by proper manipulation of culture conditions and how it is affected by plasmid size.     

Fats and fatty acids as growth factors for Lactobacillus delbrueckii

The effects of various fats and fatty acids on the growth of Lactobacillus delbrueckii strains have been studied using modified MRS broth without Tween 80 as a basic growth medium. Among the six L. delbrueckii strains studied all except one strain required Tween 80 or Tween 20 as a fatty acid supplement for the growth. Tween 40 and Tween 60, which contain solely medium and long chain saturated fatty acids, inhibited the growth of all L. delbrueckii strains when present as a sole fat supplement in MRS broth. Free oleic acid but not free lauric acid could substitute Tween 80 or Tween 20 supplement suggesting that unsaturated fatty acids are essential growth factors for most L. delbrueckii strains. Among the natural food oils tested, the oils containing the lowest amounts of saturated long chain fatty acids promoted the growth of L. delbrueckii most effectively. Especially cellular C18:1 and C19 cyclopropane fatty acid contents of L. delbrueckii were strongly affected by exogenous fatty acid composition and by strain suggesting genetic diversity and polymorphism among the genes encoding and/or regulating cyclopropane synthase. In addition obviously most if not all L. delbrueckii strains lack particular synthase, desaturase and/or dehydrase activities required for de novo synthesis of long chain unsaturated fatty acids. These biochemical features could be used as informative chemotaxonomic characteristics for L. delbrueckii starter strain identification and selection.     

L-天冬酰胺酶工程菌株培养条件及稳定性

L-天冬酰胺酶工程菌株的酶活和表达水平受菌体生物量和诱导时间的影响。在生物量Ａ６００03×１０左右,热诱导4h酶活力和表达水平可达到较高水平。葡萄糖对酶的生成有阻遏作用,当葡萄糖浓度大于025%时,对工程菌酶的合成造成阻遏。确定了工程菌培养的培养基、pH值、接种量等因素。重组质粒pASN在\%E.coli\% JM105,TG1和AS1357等宿主菌中具有很好的稳定性,工程菌培养50代以上重组质粒保留90%以上,在LB和M\|3培养基中也较稳定。     

Modulation of the immune response to an Aeromonas hydrophila aroA live vaccine in rainbow trout: effect of culture media on the humoral immune response and complement consumption

The Aeromonas hydrophila aroA is an attenuated strain that has been assessed as a live vaccine in rainbow trout, Oncorhynchus mykiss. In this study the effects of different culture media used to grow the strain on its survival after in vitro exposure to rainbow trout serum, and on its immunogenicity in rainbow trout were compared. Four culture media were tested: Luria broth (LB), Luria broth with 0.25% glucose, trypticase soy broth (TSB), and brain-heart infusion broth (BHIB). Bacteria grown in culture media with glucose (TSB, BHIB and LB with 0.25% glucose) showed reduced complement consumption and a lower serum susceptibility. O. mykiss vaccinated with inocula prepared with BHIB- and LB-grown aroA cells resuspended in phosphate-buffered saline (PBS) showed higher and longer-lasting serum agglutinating antibody titres than those vaccinated with TSB-grown bacteria. Thus, a direct relationship between serum resistance and immunogenicity could not be established, but BHIB and LB culture media were the most effective in increasing the immunogenicity of the A. hydrophila aroA vaccine.     

松材线虫携带一株荧光假单胞菌分泌毒素的初步研究

本文研究了离体情况下松材线虫携带的致病菌一株荧光假单胞菌(Pseudomonas fluorescens GcM5-1A)在LB、NB和PD三种培养基中的毒性, 以及产生的毒素对黑松(Pinus thunbergii)切根苗和悬浮细胞的效应。结果显示, 菌体在LB和NB 培养液的毒性较高, 其中 LB培养液的毒性最高, 且培养液的pH值为7时比pH值为5时毒性高, 而该菌在PD培养基中几乎不产毒。细菌培养液经硫酸铵分级沉淀, 得到了主要含有50 kDa蛋白的蛋白组分, 该蛋白组分对黑松悬浮细胞和切根苗均有较高的毒性, 并能改变黑松悬浮细胞细胞膜的透性, 导致胞内可溶性糖和游离氨基酸外渗。