Detection and characterization of the flagellar master operon in the four Shigella subgroups.

Strains in the genus Shigella are nonmotile, but they retain some cryptic flagellar operons whether functional or defective (A.Tominaga, M. A.-H. Mahmoud, T. Mukaihara, and M. Enomoto, Mol. Microbiol. 12:277-285, 1994). To disclose the cause of motility loss in shigellae, the presence or defectiveness of the flhD and flhC genes, composing the master operon whose mutation causes inactivation of the entire flagellar regulon, was examined in the four Shigella subgroups. The flhD operon cloned from Shigella boydii and Shigella sonnei can activate, though insufficiently, the regulon in the Escherichia coli flhD or flhC mutant background. The clone from Shigella dysenteriae has a functional flhD gene and nonfunctional flhC gene, and its inactivation has been caused by the IS1 element inserted in its 5' end. The operon of Shigella flexneri is nonfunctional and has suffered an IS1-insertion mutation at the 5' end of the flhD gene. Comparison of restriction maps indicates that only the central 1.8-kb region, including part of the flhC gene and its adjacent mot operon, is conserved among the four Shigella subgroups as well as in E. coli, but in Salmonella typhimurium the whole map is quite different from the others. Motility loss in shigellae is not attributable to genetic damage in the master operon of a common ancestor, but it occurs separately in respective ancestors of the four subgroups, and in both S. dysenteriae and S.flexneri IS1 insertion in the master operon might be the primary cause of motility loss.     

Motility influences biofilm architecture in Escherichia coli

Eight Escherichia coli strains were studied in minimal medium with a continuous flow system using confocal microscopy. K12 wild-type strains ATCC 25404 and MG1655 formed the best biofilms (∼43 μm thick, 21 to 34% surface coverage). JM109, DH5α, and MG1655 motA formed intermediate biofilms (∼13 μm thick, 41 to 58% surface coverage). BW25113, MG1655 qseB, and MG1655 fliA had poor biofilms (surface coverage less than 5%). The best biofilm-formers, ATCC 25404 and MG1655, displayed the highest motility, whereas the worst biofilm former, BW25113, was motility-impaired. The differences in motility were due to differences in expression of the motility loci qseB, flhD, fliA, fliC, and motA (e.g., qseB expression in MG1655 was 139-fold higher than BW25113 and 209-fold higher than JM109). Motility affected the biofilm architecture as those strains which had poor motility (E. coli JM109, E. coli MG1655 motA, and DH5α) formed flatter microcolonies compared with MG1655 and ATCC 25404, which had more dramatic vertical structures as a result of their enhanced motility. The presence of flagella was also found to be important as qseB and fliA mutants (which lack flagella) had less biofilm than the isogenic paralyzed motA strain (threefold less thickness and 15-fold less surface coverage).     

操纵子前导区的修饰及PRPP合成途径的加强对大肠杆菌积累L-组氨酸的影响

目的:基于转酮酶基因缺失菌株MG1655-ΔtktA,研究启动子替换L-组氨酸操纵子前导区及6-磷酸葡萄糖脱氢酶基因zwf、6-磷酸葡萄糖酸脱氢酶基因gnd、PRPP合成酶基因prs的过表达对大肠杆菌产L-组氨酸的影响。方法:通过Red重组系统用T5启动子替换L-组氨酸操纵子前导区;构建gnd和zwf串联表达载体gnd-zwf-pSTV28,prs表达载体prs-pQE30。通过摇瓶发酵,考察上述改造对大肠杆菌积累L-组氨酸的影响。结果:测定结果显示,改造菌株的发酵液中均能实现L-组氨酸积累,平均分别为MG1655-ΔtktA-PT5,60.12 mg/L;MG1655-ΔtktA-PT5(prs-pQE30),66.47mg/L;MG1655-ΔtktA-PT5(zwf-gnd-pSTV28),89.69 mg/L;MG1655-ΔtktA-PT5(prs-pQE30,zwf-gnd-pSTV28),111.56 mg/L。结论:L-组氨酸操纵子前导区的修饰使菌株合成L-组氨酸的能力大大增强,而氧化戊糖磷酸途径的加强和PRPP合成酶活性的提高能够进一步提高产量。     

Type 1 fimbriation and fimE mutants of Escherichia coli K-12.

We reexamined the influence of fimE, also referred to as hyp, on type 1 fimbriation in Escherichia coli K-12. We found that one strain used previously and extensively in the analysis of type 1 fimbriation, strain CSH50, is in fact a fimE mutant; the fimE gene of CSH50 contains a copy of the insertion sequence IS1. Using a recently described allelic exchange procedure, we transferred the fimE::IS1 allele from CSH50 to our present wild-type strain, MG1655. Characterization of this IS1-containing strain (AAEC137), together with another fimE mutant of MG1655 (AAEC143), led to two conclusions about the role of fimE. First, the formation of phase variant colony types, reported widely in strains of E. coli, depends on mutation of fimE, at least in K-12 strain MG1655. Here we showed that this phenomenon reflects the ability of fimE to stimulate the rapid inversion of the fim invertible element from on to off when the bacteria are grown on agar. Second, our analysis of fimE mutants, which is limited to chromosomal constructs, provided no evidence that they are hyperfimbriate. We believe that these results, which are at odds with a previous study using fim-containing multicopy plasmids, reflect differences in gene copy number.     

Aerobactin genes in clinical isolates of Escherichia coli.

The location of the aerobactin gene complex on either the chromosome or plasmid was determined in eight aerobactin-positive clinical isolates of Escherichia coli by Southern hybridization analysis, using as probes the cloned aerobactin genes from the ColV-K30 plasmid. The aerobactin genes were in two cases detected on large plasmids, whereas in the other strains the aerobactin genes are most likely located on the chromosome. Restriction mapping revealed only slight variations in the structural genes and an at least 3.4-kilobase-long upstream region conserved in all three plasmid-coded systems. A 7.7-kilobase HindIII fragment upstream and adjacent to the 16.3-kilobase HindIII fragment carrying the complete aerobactin system was cloned from the ColV-K30 plasmid. Fine-structure restriction mapping identified the left insertion sequence in the upstream region as IS1, in inverted orientation to the IS1 element downstream from the aerobactin operon. The upstream and downstream sequences of IS1 appear to have perfect homology, as indicated by S1 nuclease resistance of a 760-base-pair DNA duplex formed by both IS1 elements.     

Differentiation of Serratia liquefaciens into swarm cells is controlled by the expression of the flhD master operon.

The velocity with which a swarming colony of Serratia liquefaciens colonizes the surface of a suitable solid substratum was controlled by modulating the expression of the flhD master operon. In liquid medium, the stimulation of flhD expression resulted in filamentous, multinucleate, and hyperflagellated cells that were indistinguishable from swarm cells isolated from the edge of a swarm colony. Thus, expression of the flhD master operon appears to play a central role in the process of swarm cell differentiation.     

Identification of transposable elements which activate gene expression in Pseudomonas cepacia.

This study demonstrated that transposable elements in Pseudomonas cepacia could be inserted upstream of a poorly expressed gene and increase its expression more than 30-fold. Five elements, TnPc1, IS402, IS403, IS404, and IS405, were isolated by their ability to increase expression of the beta-lactamase gene of the broad-host-range plasmid pRP1. Increased expression resulted only from insertion of these elements, suggesting that insertional activation is an important means of elevating gene expression in this organism. Four of the elements inserted between a PstI site within the beta-lactamase gene and a BamHI site located 375 base pairs upstream of its promoter. The element IS403 inserted distal to the BamHI site within the coding region for the gene tnpR, suggesting that insertional activation can act over greater than expected distances. In addition, the element IS402 activated the beta-lactamase genes carried on plasmids pRP1 and pMR5 (temperature-sensitive pRP1) equally well in opposite orientations, demonstrating that insertional activation by this element occurs independent of its orientation.     

Spontaneous insertion of an IS2 element into the promoter region of the lac operon

A spontaneous mutation in pUC18 has revealed the insertion of a chromosomal insertion sequence (IS)2 element into the promoter region of the lac operon. The IS2 insertion site, at the pentanucleotide sequence TCGAG, is unlike previously described junctional sequences.     

Tuning of expression level of the genes of interest located in the bacterial chromosome

The new method of construction of the set of E. coli clones, differing in the promoter strength upstream the gene of interest, has been developed and tested using native E. coli MG 1655 lacZ gene as the reporter. This method includes the construction of the promoter-carrying DNA fragment obtained by PCR with consensus P(tac) as a template and the primers that lead to randomization of 4 central nucleotides in the promoter "-35"-region, linking the obtained fragments with the selective marker (Cm(R)) followed by Red-driven integration of the resulted DNA fragments directly in E. coli MG1655 chromosome instead the native lacI-gene and promoter/operator region of lac-operon. Due to direct determination of LacZ-activity in the independently obtained clones-integrants, we have found 14 new promoters (from 44 = 256 possible variants) that differ in their strength up to 100 fold (LacZ-activity in the corresponding strains smoothly varies from 10(2) for the weakest tested promoter up to 10(4) Miller U detected for the initial P(tac)). Sequencing of obtained promoters revealed that randomization of three positions in the "-35"-region is sufficient to obtain representative promoter library that would decrease the total number of potential promoter variants from 256 up to 64. It seems probable that exploiting of the developed method leading to one-step construction the library of clones with varied expression of gene/operon of interest could be useful tool in the modem metabolic engineering for optimization of genes expression.