Incompatibility of outer membrane proteins OmpA and OmpF of Escherichia coli with secretion in Bacillus subtilis: Fusions with secretable peptides

The secretion of the outer membrane proteins OmpA and OmpF of Escherichia coli has previously been found to be blocked at an early intracellular step, when these proteins were fused to a bacillar signal sequence and expressed in Bacillus subtilis. We have now fused these proteins to long secretable polypeptides, the amino-terminal portions of alpha-amylase or beta-lactamase. In spite of this, no secretion of the fusion proteins was detected in B. subtilis. With the exception of a small fraction of the beta-lactamase fusion, the proteins were cell-bound with uncleaved signal sequences. Protease accessibility indicated that the fusion proteins were not even partially exposed on the outer surface of the cytoplasmic membrane. Thus there was no change of the location compared to the OmpA or OmpF fused to the signal sequence only. We conclude that, like OmpA and OmpF, the fusion proteins fold into an export-incompatible conformation in B. subtilis before the start of translocation, which we postulate to be a late post-translational event.     

Conformation of Escherichia coli outer membrane protein OmpA produced in Bacillus subtilis: Influence of lipopolysaccharide

Abstract The conformation of the outer membrane protein OmpA of Escherichia coli produced in Bacillus subtilis and solubilized in Sarkosyl was studied by measuring its ability to bind OmpA-specific phage K3 and to inhibit F-mediated conjugation. The partially purified protein was inactive in both these assays. Refolding of the protein in the presence of lipopolysaccharide resulted in preparations with full phage-binding and conjugation-inhibiting capacity, indicating the formation of surface-exposed loops of OmpA of native conformation. The finding is of importance for the potential use of outer membrane proteins of Gram-negative bacteria as vaccines.     

Export and sorting of theEscherichia coli outer membrane protein OmpA

Results of studies, mostly using the outer membrane, 325 residue protein OmpA, are reviewed which concern its translocation across the plasma membrane and incorporation into the outer membrane ofEscherichia coli. For translocation, neither a unique export signal, acting in a positive fashion within the mature part of the precursor, nor a unique conformation of the precursor is required. Rather, the mature part of a secretory protein has to be export-compatible. Export-incompatibility can be caused by a stretch of 16 (but not 8 or 12) hydrophobic residues, too low a size of the polypeptide (smaller than 75 residue precursors), net positive charge at the N-terminus, or lack of a turn potential at the same site. It is not yet clear whether binding sites for chaperonins (SecB, trigger factor, GroEL) within OmpA are importantin vivo. The mechanism of sorting of outer membrane proteins is not yet understood. The membrane part of OmpA, encompassing residues 1 to about 170, it thought to traverse the membrane eight times in antiparallel -sheet conformation. At least the structure of the last -strand (residues 160–170) is of crucial importance for membrane assembly. It must be amphiphilic or hydrophobic, these properties must extend over at least nine residues, and it must not contain a proline residue at or near its center. Membrane incorporation of OmpA involves a conformational change of the protein and it could be that the last -strand initiates folding and assembly in the outer membrane.     

Cyclic AMP stimulates the protein tyrosine kinase activity of Acinetobacter calcoaceticus

The protein tyrosine kinase activity of Acinetobacter calcoaceticus was analyzed in vitro through the specific phosphorylation of an endogenous protein which is modified exclusively at tyrosine residues. A strong stimulation of this activity by cyclic AMP was observed. This finding represents the first example of a protein tyrosine kinase, in prokaryotes as well as in eukaryotes, whose functioning is cyclic nucleotide-dependent.     

Characterization of the gltF gene product of Escherichia coli

The glt operon of Escherichia coli comprises the structural genes for the glutamate synthase subunits (gltB and gltD) and gltF, whose product was previously suggested to have regulatory functions. The A/T-rich region between gltD and gltF contains a weak promoter and a translation initiation site for gltF. The GltF protein is preceded by a signal peptide, which is cleaved off during export into the periplasmic space. A gltF::Km(R) insertion mutant was constructed and shown here to have no detectable phenotype with respect to amino acid utilization or ammonium transport. Thus, GltF is apparently not involved in regulation of nitrogen catabolism.     

Cloning of the ethidium efflux gene from Escherichia coli

The gene specifying the ethidium efflux system of Escherichia coli has been cloned on a 3.2 kbp HindIII fragment and located on a 1.2 kbp fragment within this. Cross-resistance studies indicate that the system has a broad specificity for monovalent cations and the gene shows no hybridisation with similar genes found in Staphylococci.     

Nucleotide sequence of the promoter region of the melibiose operon of Escherichia coli

The nucleotide sequence of the promoter region of the melibiose operon of E. coli was determined. Consensus sequences for the -35 region, the Pribnow box and the binding site for cyclic AMP receptor protein were found in this region. The possible secondary structure of this DNA region was very similar to that of the promoter region of the lactose operon. A possible initiation ATG preceded by a Shine-Dalgarno sequence with proper spacing was present just downstream of the promoter region. The possible sequence of 52 amino acid residues in the NH2 terminus of the alpha-galactosidase were determined.     

Cyclic AMP transport across membrane vesicles of ultra-violet light irradiatedEscherichia coli

Transport of cyclic AMP acrossEscherichia coli membrane was studied using membrane vesicles. Uptake of cyclic AMP was measured using normally oriented vesicles, whereas uptake in everted vesicles was taken as a measure of the efflux of cyclic AMP. Ultra-violet irradiation of the cells led to an inhibition of both uptake and efflux of cyclic AMP across the membrane. The presence of cyclic AMP in the growth medium prior to ultra-violet irradiation caused an enhancement of the uptake and efflux. The uptake and efflux of cyclic AMP were less in vesicles from glucose grown cells as compared to the uptake and efflux by the vesicles prepared from glycerol grown cells. Similarly both uptake and efflux of cyclic AMP were more in vesicles prepared from cells grown on glycerol or glucose in the presence of cyclic AMP than in vesicles from cells grown in absence of cyclic AMP. It is suggested that the number of cyclic AMP carrier molecules were reduced in cells under catabolite repression by glucose as well as by ultra-violet irradiation     

Cyclic AMP and higher plants

Despite the evidence in support, the extent of which is outlined in this review, the occurrence of cyclic AMP in tissues of higher plants has been doubted by a number of previous reviewers. Recent MS and other evidence vindicates earlier identification of an adenosine nucleotide from plant tissues as adenosine 3′:5′-cyclic monophosphate. The additional demonstration of 3′: 5′-cyclic nucleotide phosphodiesterases in higher plants, together with adenylate cyclase, a specific cyclic AMP binding protein, and calmodulin, means that plants possess all the necessary components for a functional cyclic AMP-regulated system. Whether such a system does function in plants is considered as are also the reported physiological effects of exogenously supplied cyclic AMP on plant tissues.     

Cyclic AMP independence of Escherichia coli protein phosphorylation

The effect of cyclic AMP on protein phosphorylation was analyzed comparatively in two strains of E.coli differing in their capacity to synthesize this nucleotide, one of them lacking the adenylate cyclase activity. The results obtained from both in vivo and in vitro experiments concurred in showing that the bacterial protein kinase activity is cAMP-independent.     

大肠埃希菌蛋白指纹图谱的建立

目的建立大肠埃希菌（Escherichia coli,E．coli）蛋白指纹图谱,为Ecoli感染快速诊断奠定基础。方法收集临床分离E.coli88株,提取细菌DNA,PCR检测Ecoli 16S rRNA。蛋白提取液提取细菌蛋白,干化学法测蛋白浓度,应用表面增强激光解析电离飞行时间质谱技术（SELDI-TOF-MS）检测Ecoli蛋白,采用Ciphergen Pro-teinchip软件自动采集数据。重复测定20次Ecoli混合标本,评价SELDI检测Ecoli蛋白分子量的重复性。结果E．coil标准菌株ATCC 25922和临床分离株均可检出16S rRNA。AU芯片能捕获近30个E．coli蛋白峰,其中19个蛋白峰构成E．coli特征性蛋白指纹图谱,各蛋白峰在临床分离E．coli间分子量变异系数≤0．2％。SELDI重复检测20次E．coli混合标本显示同一蛋白峰的分子量变异系数≤0．05％。结论E．coli在分子量3～20kD范围内具有特征性蛋白指纹图谱,为快速诊断E.coli感染提供了新思路。     

Cyclic AMP binding protein from Jerusalem artichoke rhizome tissues

A cyclic AMP binding protein has been purified to electrophoretic homogeneity from Jerusalem artichoke rhizome tissues. Its MW is ca. 240 000 and the apparent constant of cyclic AMP binding to the protein is 2.3 × 10^?7 M. When tested using Millipore filter assay, cyclic AMP binding activity was enhanced by protamine and histone, but not by casein and phosvitin. Of several purine derivatives tested, only 5′-AMP and adenosine inhibited significantly the binding of cyclic AMP by the protein. The protein also binds adenosine and this binding is not affected by cyclic AMP or by other purine derivatives. The apparent binding constant for adenosine is 1.0 × 10^?6 M. The binding protein did not show protein kinase activity. In addition, it did not affect the chromatin-bound DNA dependent RNA polymerase of homologous origin, either in the presence or absence of cyclic AMP. The binding protein is devoid of the following activities: cyclic AMP phosphodiesterase, 5′-nucleotidase, adenosine deaminase and ATPase.     

Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay

DNA binding properties of the proteins required for induction of the Escherichia coli L-arabinose operon were measured using a polyacrylamide gel electrophoresis assay. The mechanisms of induction and repression were studied by observing the multiple interactions of RNA polymerase, cyclic AMP receptor protein and araC protein with short DNA fragments containing either the araC or araBAD promoter regions. These studies show that binding of araC protein to the operator site, araO1, directly blocks RNA polymerase binding at the araC promoter, pC. We find that cyclic AMP receptor protein and araC protein do not bind co-operatively at their respective sites to linear DNA fragments containing the pBAD promoter. Nevertheless, both these positive effectors must be present on the DNA to stimulate binding of RNA polymerase. Additionally, binding of the proteins to the DNA is not sufficient; araC protein must also be in the inducing state, for RNA polymerase to bind. Equilibrium binding constraints and kinetics were determined for araC protein binding to the araI and the araO1 sites. In the presence of inducer, L-arabinose, araC protein binds with equal affinity to DNA fragments containing either of these sites. In the presence of anti-inducer, D-fucose, the affinity for both sites is reduced 40-fold. The apparent equilibrium binding constants for both states of the protein vary in parallel with the buffer salt concentration. This result suggests that the inducing and repressing forms of araC protein displace a similar number of cations upon binding DNA.