The <Emphasis Type="Italic">flhDC</Emphasis> gene affects motility and biofilm formation in <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

The flagella master regulatory gene flhDC of Yersinia pseudotuberculosis serotype III (YPIII) was mutated by deleting the middle region and replaced by a tetracycline resistant gene, and the subsequent mutant strain named YPIIIΔflhDC was obtained. Swimming assay showed that the swimming motility of the mutant strain was completely abolished. The promoter region of the flagella second-class regulatory gene fliA was fused with the lux box, and was conjugated with the mutant and the parent strains respectively for the first cross. LUCY assay result demonstrated that flhDC regulated the expression of fliA in YPIII as reported in E. coli. Biofilm formation of the mutant strain on abiotic and biotic surfaces was observed and quantified. The results showed that mutation of flhDC decreased biofilm formation on both abiotic and biotic surfaces, and abated the infection on Caenorhabdtis elegans. Our results suggest that mutation of the flagella master regulatory gene flhDC not only abolished the swimming motility, but also affected biofilm formation of YPIII on different surfaces. The new function of flhDC identified in this study provides a novel viewpoint for the control of bacterial biofilm formation.     

茎瘤固氮根瘤菌ORS571中motA基因的功能分析

[目的] MotA是细菌的鞭毛马达蛋白,是跨膜质子通道的重要组成结构之一,在调控鞭毛运动中具有至关重要的作用。本研究探究了Azorhizobium caulinodans ORS571中鞭毛马达基因motA对菌株表型和植物互作的影响。[方法] 通过同源重组原理和三亲接合转移方法构建突变菌株∆motA,测定野生型与突变体在菌体生长、运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力的差异。[结果] 与野生型相比,突变体菌体生长没有明显差异,但其运动能力完全丧失,固氮、胞外多糖合成、生物膜形成及根系定殖能力减弱。[结论] MotA鞭毛马达蛋白对A.caulinodans ORS571的运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力均有调控作用。     

Flagellar region 3b supports strong expression of integrated DNA and the highest chromosomal integration efficiency of the Escherichia coli flagellar regions

The Gram‐negative bacterium Escherichia coli is routinely used as the chassis for a variety of biotechnology and synthetic biology applications. Identification and analysis of reliable chromosomal integration and expression target loci is crucial for E. coli engineering. Chromosomal loci differ significantly in their ability to support integration and expression of the integrated genetic circuits. In this study, we investigate E. coli K12 MG1655 flagellar regions 2 and 3b. Integration of the genetic circuit into seven and nine highly conserved genes of the flagellar regions 2 (motA, motB, flhD, flhE, cheW, cheY and cheZ) and 3b (fliE, F, G, J, K, L, M, P, R), respectively, showed significant variation in their ability to support chromosomal integration and expression of the integrated genetic circuit. While not reducing the growth of the engineered strains, the integrations into all 16 target sites led to the loss of motility. In addition to high expression, the flagellar region 3b supports the highest efficiency of integration of all E. coli K12 MG1655 flagellar regions and is therefore potentially the most suitable for the integration of synthetic genetic circuits.     

GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli

The second messenger 3′–5′-cyclic diguanylic acid (c-di-GMP) promotes biofilm formation, and c-di-GMP is synthesized by diguanylate cyclases (characterized by a GGDEF domain) and degraded by phosphodiesterases. Here, we evaluated the effect of the 12 E. coli GGDEF-only proteins on biofilm formation and motility. Deletions of the genes encoding the GGDEF proteins YeaI, YedQ, YfiN, YeaJ, and YneF increased swimming motility as expected for strains with reduced c-di-GMP. Alanine substitution in the EGEVF motif of YeaI abolished its impact on swimming motility. In addition, extracellular DNA (eDNA) was increased as expected for the deletions of yeaI (tenfold), yedQ (1.8-fold), and yfiN (3.2-fold). As a result of the significantly enhanced motility, but contrary to current models of decreased biofilm formation with decreased diguanylate cyclase activity, early biofilm formation increased dramatically for the deletions of yeaI (30-fold), yedQ (12-fold), and yfiN (18-fold). Our results indicate that YeaI, YedQ, and YfiN are active diguanylate cyclases that reduce motility, eDNA, and early biofilm formation and contrary to the current paradigm, the results indicate that c-di-GMP levels should be reduced, not increased, for initial biofilm formation so c-di-GMP levels must be regulated in a temporal fashion in biofilms.     

Gene expression in<Emphasis Type="Italic"> Escherichia coli</Emphasis> biofilms

DNA microarrays were used to study the gene expression profile of Escherichia coli JM109 and K12 biofilms. Both glass wool in shake flasks and mild steel 1010 plates in continuous reactors were used to create the biofilms. For the biofilms grown on glass wool, 22 genes were induced significantly (p0.05) compared to suspension cells, including several genes for the stress response (hslS, hslT, hha, and soxS), type I fimbriae (fimG), metabolism (metK), and 11 genes of unknown function (ybaJ, ychM, yefM, ygfA, b1060, b1112, b2377, b3022, b1373, b1601, and b0836). The DNA microarray results were corroborated with RNA dot blotting. For the biofilm grown on mild steel plates, the DNA microarray data showed that, at a specific growth rate of 0.05/h, the mature biofilm after 5 days in the continuous reactors did not exhibit differential gene expression compared to suspension cells although genes were induced at 0.03/h. The present study suggests that biofilm gene expression is strongly associated with environmental conditions and that stress genes are involved in E. coli JM109 biofilm formation.     

Development of a Biofilm Production-Deficient Escherichia coli Strain as a Host for Biotechnological Applications

Bacteria form biofilms by adhering to biotic or abiotic surfaces. This phenomenon causes several problems, including a reduction in the transport of mass and heat, an increase in resistance to antibiotics, and a shortening of the lifetimes of modules in bioindustrial fermentors. To overcome these difficulties, we created a biofilm production-deficient Escherichia coli strain, BD123, by deleting genes involved in curli biosynthesis and assembly, Δ(csgG-csgC); colanic acid biosynthesis and assembly, Δ(wcaL-wza); and type I pilus biosynthesis, Δ(fimB-fimH). E. coli BD123 remained mostly in the form of planktonic cells under the conditions tested and became more sensitive to the antibiotics streptomycin and rifampin than the wild-type E. coli MG1655: the growth of BD123 was inhibited by one-fourth of the concentrations needed to inhibit MG1655. In addition, the transformation efficiency of BD123 was about 20 times higher than that of MG1655, and the production and secretion of recombinant proteins were ~16% and ~25% greater, respectively, with BD123 than with MG1655. These results indicate that the newly created biofilm production-deficient strain of E. coli displays several key properties that substantially enhance its utility in the biotechnology arena.     

Construction of the new <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 MG 1655 novel strain with improved growth characteristics for application in metabolic engineering

MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The “polar effect” of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of Val^S phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph ^wt from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying λattL-Cm^R-λattR and transduced with phage P1 into MG1655; later, the Cm^R marker was removed with the use of λXis/Int recombinase. Parallel to this procedure, a spontaneous Val^R mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of L-Val (50 μg/ml) was isolated. It was shown that a nucleotide deletion in the isolated Val^R strain had been generated in the region of the identified ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the Val^R phenotype in the obtained strain, was transferred to MG1655-rph ^wt using cotransduction, by analogy to the transfer of rph ^wt. Evaluation of rates of aerobically growing cells (μ, hour-1) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655,MG1655-rph ^wt, and MG1655-(rph ^wt, ilvG-15), respectively.     

Bioaugmentation of the decolorization rate of acid red GR by genetically engineered microorganism <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109 (pGEX-AZR)

The study showed that the genetically engineered microorganism (GEM) bioaugment successfully the dye wastewater biotreatment systems to enhance acid red GR (ARGR) removal. Escherichia coli JM109 (pGEX-AZR) was the GEM with higher azoreductase activity. The kinetics of the ARGR decolorization by the E. coli JM109 (pGEX-AZR) agreed with Andrews model. The kinetic parameters, r _dye,max, K _s and K _i , were found to be 42.45 mg g⁻¹ h⁻¹, 584.93 mg L⁻¹ and 556.89 mg L⁻¹, respectively. The E. coli JM109 (pGEX-AZR) was tested in anaerobic sequencing batch reactors (AnSBR) in order to enhance the ARGR decolorization. The decolorization rate of ARGR was affected by the amount of E. coli JM109 (pGEX-AZR) inoculation and the best amount of inoculation was 10%. The continuous operations of the four bioreactors with different E. coli JM109 (pGEX-AZR) immobilization supports showed that the E. coli JM109 (pGEX-AZR) could bioaugment decolorization in AnSBRs with suspended and immobilized on macroporous foam carriers. For 42 days continuous operation in the AnSBRs, both the tolerance to ARGR concentration shock and the decolorization rate in these two bioaugmented AnSBRs are higher than those of the other two systems, control system and bioaugmented AnSBRs system with the sodium-alginate immobilized cells, the decolorization rate reached 90%. Changes in microbial community were detected by ribosomal intergenic spacer analysis (RISA) and amplified ribosomal DNA restriction analysis (ARDRA), which revealed that the introduced E. coli JM109 (pGEX-AZR) was persistent in the augmented systems and maintained higher metabolic activity.     

Modulation of <Emphasis Type="Italic">Escherichia coli</Emphasis> biofilm growth by cell-free spent cultures from lactobacilli

E. coli biofilms cause serious problems in medical practice by contaminating surfaces and indwelling catheters. Due to the rapid development of antibiotic resistance, alternative approaches to biofilm suppression are needed. This study addresses whether products released by antagonistic bacteria — Lactobacillus isolates from vaginal and dairy-product samples could be useful for controlling E. coli biofilms. The effects of diluted cell-free supernatants (CFS) from late-exponential Lactobacillus cultures on the growth and biofilm production of Escherichia coli were tested. Most of the CFS applied as 10⁻² had no impact on bacterial growth, biofilm development however was influenced even by 10⁻⁴ of CFS. Initial screening by crystal violet assay showed that biofilm modulation varied between different CFS and E. coli combinations from inhibition to activation; however three of the tested CFS showed consistency in biofilm suppression. This was not due to antibacterial activity since Live/Dead fluorescence labeling showed insignificant differences in the amount of dead cells in control and treated samples. Some E. coli strain-specific mechanisms of response to the three CFS included reduction in hydrophobicity and motility. Released exoploysaccharides isolated from the three CFS stimulated sessile growth, but proteinase K reduced their inhibitory activities implying participation of protein or peptide biofilm suppression factor(s).     

Host-specific factors determine the persistence of IncP-1 plasmids

Conjugative plasmids play a very important role in bacterial adaptation through the dissemination of useful traits. Incompatibility group P-1 (IncP-1) plasmids exhibit an extreme broad-host-range among Gram-negative bacteria and known to be one of the major agents to disseminate various phenotypic traits such as antibiotic resistance and xenobiotic degradation. Although the plasmids are believed to be very stable in most Gram-negative bacteria, little is known about the factors that affect their stability in various hosts, allowing their persistence in bacterial population. Here we show that the stability of the cryptic IncP-1β plasmid pBP136 differed greatly in four different Escherichia coli K12 host backgrounds (MG1655, DH5α, EC100, and JM109), whereas the closely related plasmid pB10 was stable in all four strains. The supply of the kleF gene, which is involved in the stability of IncP-1 plasmids but absent in pBP136, did not improve the stability of the plasmid. Our findings suggest that persistence of IncP-1 plasmids in the absence of selection is affected by strain-specific factors.     

Systematic screening of Escherichia coli single-gene knockout mutants for improving recombinant whole-cell biocatalysts

Systematic screening of single-gene knockout collection of Escherichia coli BW25113 (the Keio collection) was performed to select mutants that could enhance the deethylation of 7-ethoxycoumarin catalyzed by CYP154A1. After 96-well plate high-throughput screening followed by test tube assays, four mutants (ΔcpxA, ΔgcvR, ΔglnL, and an unknown-gene-deleted one (Δuk)) were able to increase the CYP154A1 activity by approximately 1.4–1.7 times compared with that of the control strain. When new mutants were constructed by disrupting individually the cpxA, gcvR, glnL, and uk genes in E. coli BW25113, three of them (ΔcpxA, ΔgcvR, and ΔglnL) showed high levels of CYP154A1 activity. However, the uk-disruptant failed to enhance the CYP154A1 activity, suggesting that the high CYP154A1 activity of the Δuk mutant in the Keio collection was due to a spontaneous mutation in the chromosome. In-frame deletion mutants of ΔcpxA, ΔgcvR, and ΔglnL also exhibited high enzyme activity, and complementation of these mutations could decrease CYP154A1 activity. These results indicated that the enhancement of the enzyme activity was not caused by polar effects on their neighbor genes. To our knowledge, this is the first report on a genome-wide screening of the genes for deletion to improve the activity of a recombinant whole-cell biocatalyst.     

Chimeric analogs of human β-defensin 1 and θ-defensin disrupt pre-established bacterial biofilms

Antibiofilm activity of several human defensin analogs that have the ability to kill planktonic bacteria, against pre-established biofilms of Escherichia coli MG1655 and Staphylococcus aureus NCTC 8530 were examined. Linear and linear fatty acylated analogs did not show any activity while disulfide constrained analogs disrupted pre-established S. aureus biofilms. Chimeric analogs of human β-defensin 1 and θ-defensin, hBTD-1 and [d]hBTD-1 were highly active against S. aureus biofilms. Among the analogs tested, only the d-enantiomer [d]hBTD-1 showed activity against E. coli biofilm. Our study provides insights into the structural requirements for the eradication of pre-established biofilms in defensin analogs.     

ATP limitation in a pyruvate formate lyase mutant of <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655 increases glycolytic flux to <Emphasis Type="SmallCaps">d</Emphasis>-lactate

A derivative strain of Escherichia coli MG1655 for d-lactate production was constructed by deleting the pflB, adhE and frdA genes; this strain was designated “CL3.” Results show that the CL3 strain grew 44% slower than its parental strain under nonaerated (fermentative) conditions due to the inactivation of the main acetyl-CoA production pathway. In contrast to E. coli B and W3110 pflB derivatives, we found that the MG1655 pflB derivative is able to grow in mineral media with glucose as the sole carbon source under fermentative conditions. The glycolytic flux was 2.8-fold higher in CL3 when compared to the wild-type strain, and lactate yield on glucose was 95%. Although a low cell mass formed under fermentative conditions with this strain (1.2 g/L), the volumetric productivity of CL3 was 1.31 g/L h. In comparison with the parental strain, CL3 has a 22% lower ATP/ADP ratio. In contrast to wild-type E. coli, the ATP yield from glucose to lactate is 2 ATP/glucose, so CL3 has to improve its glycolytic flux in order to fulfill its ATP needs in order to grow. The aceF deletion in strains MG1655 and CL3 indicates that the pyruvate dehydrogenase (PDH) complex is functional under glucose-fermentative conditions. These results suggest that the pyruvate to acetyl-CoA flux in CL3 is dependent on PDH activity and that the decrease in the ATP/ADP ratio causes an increase in the flux of glucose to lactate.     

Identification and Validation of Novel Chromosomal Integration and Expression Loci in Escherichia coli Flagellar Region 1

Escherichia coli is used as a chassis for a number of Synthetic Biology applications. The lack of suitable chromosomal integration and expression loci is among the main hurdles of the E. coli engineering efforts. We identified and validated chromosomal integration and expression target sites within E. coli K12 MG1655 flagellar region 1. We analyzed five open reading frames of the flagellar region 1, flgA, flgF, flgG, flgI, and flgJ, that are well-conserved among commonly-used E. coli strains, such as MG1655, W3110, DH10B and BL21-DE3. The efficiency of the integration into the E. coli chromosome and the expression of the introduced genetic circuit at the investigated loci varied significantly. The integrations did not have a negative impact on growth; however, they completely abolished motility. From the investigated E. coli K12 MG1655 flagellar region 1, flgA and flgG are the most suitable chromosomal integration and expression loci.     

Inhibition of biofilm formation by the antisense peptide nucleic acids targeted at the <Emphasis Type="Italic">motA</Emphasis> gene in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 strain

Pseudomonas aeruginosa is a ubiquitous bacterium which is able to attach to many abiotic and biotic surfaces and form biofilms resulting in infections. The motA gene was an essential gene in the early phase of biofilm formation of P. aeruginosa PAO1. In this study, antisense peptide nucleic acids (PNAs) and PNAs conjugated with the peptide (KFF)₃K were used to investigate whether they could mediate gene-specific antisense effects in P. aeruginosa PAO1. We found that antisense (KFF)₃K-PNA targeted at motA gene could inhibit biofilm formation in P. aeruginosa PAO1 in a dose-dependent manner. The minimal effective concentration of this antisense agent was 1 μmol l⁻¹, and the inhibited effect could last for at least 8 h. When compared with the control group, the value of OD570 of P. aeruginosa PAO1 reduced apparently when treated with (KFF)₃K-PNA. The expression of motA was sharply reduced when treated with (KFF)₃K-PNA, but reduced slightly when treated with PNA, and had no reduction when treated with (KFF)₃K. Our results demonstrated that the cell-penetrating peptide of (KFF)₃K improved significantly the antisense inhibition effect of PNA. The (KFF)₃K-PNA conjugates might be used as antisense agent for inhibition of the biofilm formation. This provides exciting possibility for developing new tool for microbial genetic treatment.     

Significantly enhanced production of recombinant nitrilase by optimization of culture conditions and glycerol feeding

The production of a recombinant nitrilase expressed in Escherichia coli JM109/pNLE was optimized in the present work. Various culture conditions and process parameters, including medium composition, inducer, induction condition, pH and temperature, were systematically examined. The results showed that nitrilase production in E. coli JM109/pNLE was greatly affected by the pH condition and the temperature in batch culture, and the highest nitrilase production was obtained when the fermentation was carried out at 37°C, initial pH 7.0 without control and E. coli was induced with 0.2 mM isopropyl-β-d-thiogalactoside at 4.0 h. Furthermore, enzyme production could be significantly enhanced by adopting the glycerol feeding strategy with lower flow rate. The enzyme expression was also authenticated by sodium dodecyl phosphate polyacrylamide gel electrophoresis analysis. Finally, under the optimized conditions for fed-batch culture, cell growth, specific activity and nitrilase production of the recombinant E. coli were increased by 9.0-, 5.5-, and 50-fold, respectively.