Identification of Conserved, RpoS-Dependent Stationary-Phase Genes of Escherichia coli

During entry into stationary phase, many free-living, gram-negative bacteria express genes that impart cellular resistance to environmental stresses, such as oxidative stress and osmotic stress. Many genes that are required for stationary-phase adaptation are controlled by RpoS, a conserved alternative sigma factor, whose expression is, in turn, controlled by many factors. To better understand the numbers and types of genes dependent upon RpoS, we employed a genetic screen to isolate more than 100 independent RpoS-dependent gene fusions from a bank of several thousand mutants harboring random, independent promoter-lacZ operon fusion mutations. Dependence on RpoS varied from 2-fold to over 100-fold. The expression of all fusion mutations was normal in an rpoS/rpoS⁺ merodiploid (rpoS background transformed with an rpoS-containing plasmid). Surprisingly, the expression of many RpoS-dependent genes was growth phase dependent, albeit at lower levels, even in an rpoS background, suggesting that other growth-phase-dependent regulatory mechanisms, in addition to RpoS, may control postexponential gene expression. These results are consistent with the idea that many growth-phase-regulated functions in Escherichia coli do not require RpoS for expression. The identities of the 10 most highly RpoS-dependent fusions identified in this study were determined by DNA sequence analysis. Three of the mutations mapped to otsA, katE, ecnB, and osmY—genes that have been previously shown by others to be highly RpoS dependent. The six remaining highly-RpoS-dependent fusion mutations were located in other genes, namely, gabP, yhiUV, o371, o381, f186, and o215.     

Isolation, Characterization, and Genetic Analysis of Mutator Genes in Escherichia coli B and K-12

Twenty-one Mut mutants were obtained from Escherichia coli B (B/UV) and K-12 (JC355) after treatment with mutagens. These Mut strains are characterized by rates of mutation to streptomycin resistance and T-phase resistance which are significantly higher than the parental (Mut(+)) rates. Mutator genes in 12 strains have been mapped at three locations on the E. coli chromosome: one close to the leu locus; five close to the purA locus; and six close to cysC. In addition, eight mutator strains derived from E. coli B/UV are still unmapped. Some effort was made to deduce the mode of action of the mutator genes. These isolates have been examined for possible defects in deoxyribonucleic acid repair mechanisms (dark repair of ultraviolet damage, host-cell reactivation, recombination ability, repair of mitomycin C damage). By using transductional analysis, it was found that the ultraviolet sensitivity of NTG119 and its mutator property results from two separate but closely linked mutations. PurA(+) transductants that receive mut from NTG119 or NTG35 are all more sensitive to mitomycin C than is the PurA recipient. Unless transduction selects for sensitivity, a probable interpretation is that defective repair of mitomycin C-induced damage is related to the mode of action of mut in these transductants and the donor. Abnormal purine synthesis may be involved in the mutability of some strains with cotransduction of the mutator properly and purA (100% cotransduction for NTG119). Three mutators are recombination-deficient and may have a defective step in recombination repair. One maps near three rec genes close to cysC.     

Porin activity in the osmotic shock fluid of Escherichia coli.

Osmotic shock fluid of Escherichia coli exhibited pore-forming activity. This activity could be followed by an in vitro assay based on the conductivity increase for ions due to the presence of pores in black lipid membranes. The histogram (the distribution of conductivity increments in a single pore experiment) obtained with osmotic shock fluid from E. coli was identical to the histogram obtained by detergent-solubilized porin isolated from the outer membrane. The osmotic shock fluid from porin-negative mutants also exhibited pore activity, although the histogram and ion specificity were different from those of porin. Antibodies raised against detergent-solubilized porin were able to form precipitin lines by the Ouchterlony immunodiffusion technique when shock fluids, but not detergent-solubilized porin, were used. These antibodies prevented the formation of pores when shock fluids contained porin but not when shock fluids obtained from porin-negative mutants were used. Macroscopic membrane conductivity of shock fluids due to porin exhibited a concentration dependence, in contrast to detergent-solubilized porin. These results indicate that the hydrodynamic properties of periplasmic or "soluble" porin are different from those of the detergent-solubilized porin of the outer membrane. Periplasmic porin comprises about 0.7% of total protein in the osmotic shock fluid.     

Two Rep Genes in Small Cryptic Plasmid pKST21 of Escherichia coli

The complete nucleotide sequence of a small cryptic plasmid pKST21 from Escherichia coli was determined. This plasmid is 1,460 bp long with an overall GC content of 51 %. Based on sequence analysis, the presence of two segments with different average GC density was observed. The segment with higher GC content revealed 98–90 % similarity to several small plasmids of E. coli and to pCR1 from Gram-positive Corynebacterium renale. Plasmid pKST21 possesses two conversely oriented open reading frames encoding proteins with a high degree of amino acid identity to Rep proteins involved in replication. ORF1 encodes replication protein similar to RepA protein of Bartonella tribocorum or Bacillus cereus plasmids or to the putative plasmid Rep protein from ecologically close Selenomonas ruminantium. ORF2 similarly encodes a replication protein, which shares 97 % homology with Rep protein from C. renale. Genetic diversity observed in plasmid pKST21 indicates a mosaic structure of the plasmid with different segments acquired from different sources. Deletion analysis showed that both fragments carrying the repA and repB genes are necessary for the replication of pKST21 in E. coli. The presence of plasmid with the same gene composition was revealed in 14 % of tested E. coli isolates from the rumen of sheep. All these strains produced identical ERIC-PCR profiles indicating isogenic origin of the strain and lack of horizontal gene transfer of pKST21 plasmid.     

Identification and Characterization of a New Lipoprotein, NlpI, in Escherichia coli K-12

We report here the identification of a new lipoprotein, NlpI, in Escherichia coli K-12. The NlpI structural gene (nlpI) is located between the genes pnp (polynucleotide phosphorylase) and deaD (RNA helicase) at 71 min on the E. coli chromosome. The nlpI gene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. An nlpI::cm mutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation in nlpI, since it was complemented by the wild-type nlpI gene alone. Overexpression of the unaltered nlpI gene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.     

Porin channels in Escherichia coli: studies with beta-lactams in intact cells

Wild-type Escherichia coli K-12 produces two porins, OmpF (protein 1a) and OmpC (protein 1b). In mutants deficient in both of these "normal" porins, secondary mutants that produce a "new" porin, protein PhoE (protein E), are selected for. We determined the properties of the channels produced by each of these porins by measuring the rates of diffusion of various cephalosporins through the outer membrane in strains producing only one porin species. We found that all porin channels retarded the diffusion of more hydrophobic cephalosporins and that with monoanionic cephalosporins a 10-fold increase in the octanol-water partition coefficient of the solute produced a 5- to 6-fold decrease in the rate of penetration. Electrical charges of the solutes had different effects on different channels. Thus, with the normal porins (i.e., OmpF and OmpC proteins) additional negative charge drastically reduced the penetration rate through the channels, whereas additional positive charge significantly accelerated the penetration. In contrast, diffusion through the PhoE channel was unaffected by the presence of an additional negative charge. We hypothesize that the relative exclusion of hydrophobic and negatively charged solutes by normal porin channels is of ecological advantage to E. coli, which must exclude hydrophobic and anionic bile salts in its natural habitat. The properties of the PhoE porin are also consistent with the recent finding (M. Argast and W. Boos, J. Bacteriol. 143:142-150, 1980; J. Tommassen and B. Lugtenberg, J. Bacteriol. 143:151-157, 1980) that its biosynthesis is derepressed by phosphate starvation; the channel may thus act as an emergency pore primarily for the uptake of phosphate and phosphorylated compounds.     

Evidence for Two Genes Specifically Involved in Unsaturated Fatty Acid Biosynthesis in Escherichia coli

Unsaturated fatty acid auxotrophs of Eschericha coli have been divided into two distinct cistrons by extract complementation and genetic complementation based on abortive transduction. Lesions in one cistron result in the loss of the beta-hydroxydecanoyl thioester dehydrase which produces the first biosynthetic intermediate in unsaturated fatty acid formation. Evidence is presented which indicates that lesions in the second cistron result in the lack of a second enzyme specifically involved in the biosynthesis of unsaturated fatty acids.     

Identification of Two New Proteins in Spermidine Nucleoids Isolated from Escherichia coli

The Escherichia coli nucleoid contains DNA in a condensed but functional form. Analysis of proteins released from isolated spermidine nucleoids after treatment with DNase I reveals significant amounts of two proteins not previously detected in wild-type E. coli. Partial amino-terminal sequencing has identified them as the products of rdgC and yejK. These proteins are strongly conserved in gram-negative bacteria, suggesting that they have important cellular roles.     

Identification of Two Essential Arginine Residues in UhpT, the Sugar Phosphate Antiporter of Escherichia coli

Three lines of evidence indicate that arginine-46 (R46) and arginine-275 (R275) are essential to the function of UhpT, the Pi-linked antiport protein of Escherichia coli. A role for arginine was initially suggested by the sensitivity of UhpT to inhibition by 2,3-butanedione, an arginine-directed probe. Since the presence of substrate protected against this inhibition, this work further suggested that arginine(s) may lie at or near the UhpT active site. In other work, each UhpT arginine was examined individually by using site-directed mutagenesis to generate a cysteine or a lysine derivative. With two exceptions (R46, R275), all arginines could be replaced by either cysteine (10 of 14 residues) or lysine (12 of 14) without loss of function, implicating R46 and R275 as essential to UhpT function. This idea was strengthened by examining a multiple alignment of the eleven known UhpT-related proteins (≥30% identity). That alignment showed R46 and R275 were two of the only three arginines strongly conserved in this group of proteins. Considered together, these different approaches lead us to conclude that UhpT and its relatives have only two arginine residues (R46, R275) whose presence is essential to function. Prior biochemical work had placed R275 at the external entrance to the translocation pathway, and a symmetry argument emerging from the multiple alignment suggests a similar position for R46. Accordingly, by virtue of their locations at the entrance to this pathway, we speculate that R46 and R275 function in establishing substrate specificity. Received: 29 January 1998/Revised: 13 April 1998     

Identification,Source, and Metabolism of N-Ethylglutamate in Escherichia coli

N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ∼4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 μM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. l-[ethyl-²H₄]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.During work on developing methods for the analysis of the amino acids generated by recombinant archaeal mutases, I developed procedures for the recovery and analysis of the free amino acids present in cell extracts of Escherichia coli. When these methods were applied to analysis of E. coli grown on LB broth, I always found a large amount of an unknown amino acid. Here I report on the identification of this amino acid as N-ethylglutamate (NEG). NEG has never been reported as a natural product. I demonstrate that NEG is readily taken up by E. coli and can serve as the sole source of nitrogen when the cells are grown on M9 glucose medium.     

大肠杆菌trpBA和serA基因的串联表达

大肠杆菌trpBA基因编码的色氨酸合成酶(tryptophan synthetase, TSase)是色氨酸合成的关键酶; serA基因编码的磷酸甘油酸脱氢酶(D-3-phosphoglycerate-dehydrogenase, PGDH)为L-丝氨酸合成(色氨酸合成的底物)的关键酶。为了通过基因工程手段来增加色氨酸的产量, 在利用高效的原核表达载体pET22b(+)分别对trpBA和serA基因克隆表达的基础上, 采用PCR方法扩增了抗反馈抑制的serA和trpBA基因, 将两基因串联于pET22b(+)载体上, 共构建了4种方式的串联质粒, 实现了2种蛋白酶在大肠杆菌中的共表达。聚丙烯酰胺电泳分析显示, ABA-Ⅰ重组菌株在37 kD (PGDH)、29 kD(色氨酸合成酶的α亚基)、44 kD(β亚基)处均有明显的蛋白表达带。4种串联表达质粒重组菌的TSase酶活性, 分别比含空载体菌相应酶的活性提高2~4倍, PGDH酶活性分别提高约2.1~3.6倍。经摇瓶发酵实验表明酶活性较高的ABA-I菌株色氨酸合成量亦最高, 约为对照菌株的20.2倍。     

Identification of microRNA-Size, Small RNAs in Escherichia coli

Noncoding small regulatory RNA molecules control gene expression and microRNAs provide one of the best examples in eukaryotes. However, bacterial RNAs of comparable size to eukaryotic microRNAs have received little attention. Here, we demonstrate the existence of microRNA-size, small RNAs (msRNAs) in the model bacterium Escherichia coli. We examined the small RNAs in E. coli using a deep sequencing approach, and analyzed 33.2 million small RNA clone reads after size fractionation. Bioinformatic analysis of the whole set revealed more than 400 individual msRNA species. The cellular contents of selected highly expressed msRNAs were verified by quantitative RT-PCR and northern blotting. Although, the functional significance of these RNAs is unclear, their high abundance suggests that they may play specialized roles in bacteria, analogous to miRNAs in eukaryotes.     

大肠杆菌O150 O-抗原基因簇的破译和dTDP-鼠李糖合成酶基因的鉴定

利用鸟枪法对大肠杆菌O150 O-抗原基因簇进行测序,序列全长13551bp,用生物信息学的方法进行序列分析,共发现11个基因,分别为鼠李糖合成酶基因(rmlB、rmlD、rmlA、rmlC)糖基转移酶基因(3个)、O-抗原转运酶基因(wzx)和O-抗原聚合酶基因(wzy),另外还有两个基因功能未知。用PCR的方法筛选出了针对大肠杆菌O150的特异基因,可以用于基因芯片或PCR方法对大肠杆菌O150的快速检测。另外,通过进化分析发现大肠杆菌O150的O-抗原基因簇中携带有典型的大肠杆菌鼠李糖合成酶基因,并且这些基因参与了O-抗原基因簇间的重组以形成新的基因簇的过程。     

Genes involved in copper homeostasis in Escherichia coli

Recently, genes for two copper-responsive regulatory systems were identified in the Escherichia coli chromosome. In this report, data are presented that support a hypothesis that the putative multicopper oxidase CueO and the transenvelope transporter CusCFBA are involved in copper tolerance in E. coli.