Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12

The various [35S]DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M). Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column. A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively. The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent. The polypeptides could be divided into two main groups according to pI values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63). The ratio between acidic and basic polypeptides decreased with increasing salt concentrations in the eluates. The majority of the basic polypeptides had molecular weights in the range 10 000-30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.     

Properties of the penicillin-binding proteins of Escherichia coli K12,.

Benzyl[14C]penicillin binds to six proteins with molecular weights between 40000 and 91000 in the inner membrane of Escherichia coli. Two additional binding proteins with molecular weights of 29000 and 32000 were sometimes detected. All proteins were accessible to benzyl[14C]penicillin in whole cells. Proteins 5 and 6 released bound benzyl[14C]penicillin with half times of 5 and 19 min at 30 degrees C but the other binding proteins showed less than 50% release during a 60-min period at 30 degrees C. The rate of release of bound penicillin from some of the proteins was greatly stimulated by 2-mercaptoethanol and neutral hydroxylamine. Release of benzyl[14C]penicillin did not occur if the binding proteins were denatured in anionic detergent and so was probably enzymic. No additional binding proteins were detected with two [14C]cephalosporins. These beta-lactams bound to either all or some of those proteins to which benzyl[14C]penicillin bound. No binding proteins have been detected in the outer membrane of E coli with any beta-[14C]lactam. The binding of a range of unlabelled penicillins and cephalosporins were studied by measuring their competition for the binding of benzyl[14C]penicillin to the six penicillin-binding proteins. These results, together with those obtained by direct binding experiments with beta-[14C]lactams, showed that penicillins bind to all six proteins but that at least some cephalosporins fail to bind, or bind very slowly, to proteins 2, 5 and 6, although they bind to the other proteins. Since these cephalosporins inhibited cell division and caused cell lysis at concentrations where we could detect no binding to proteins 2, 5 and 6, we believe that these latter proteins are not the target at which beta-lactams bind to elicit the above physiological responses. The binding properties of proteins 1, 3, and 4 correlate reasonably well with those expected for the above killing targets.     

TolB protein of Escherichia coli K-12 interacts with the outer membrane peptidoglycan-associated proteins Pal, Lpp and OmpA

The Tol–Pal proteins of Escherichia coli are involved in maintaining outer membrane integrity. Transmembrane domains of TolQ, TolR and TolA interact in the cytoplasmic membrane, while TolB and Pal form a complex near the outer membrane. TolB and the central domain of TolA interact in vitro with the outer membrane porins. In this study, both genetic and biochemical analyses were carried out to analyse the links between TolB, Pal and other components of the cell envelope. It was shown that TolB could be cross-linked in vivo with Pal, OmpA and Lpp, while Pal was associated with TolB and OmpA. The isolation of pal and tolB mutants disrupting some interactions between these proteins represents a first approach to characterizing the residues contributing to the interactions. We propose that TolB and Pal are part of a multiprotein complex that links the peptidoglycan to the outer membrane. The Tol–Pal proteins might form transenvelope complexes that bring the two membranes into close proximity and help some outer membrane components to reach their final destination.     

Sulphate permease of Escherichia coli K12.

Some properties of the sulphate transport system and the isolation of sulphate permease mutants in E. coli K12 are described. The gene coding for sulphate permease is located in the same region as the cysA gene in Salmonella typhimurium.     

Homoserine kinase from Escherichia coli K12.

Homoserine kinase was purified to apparent homogeneity from a derepressed strain of Escherichia coli K12, using standard fractionation techniques. It is a dimer (Mr = 60000) composed of apparently identical polypeptide chains (Mr = 29000). Its amino acid composition and N-terminal sequence have been determined. L-Threonine is a competitive inhibitor of the substrate L-homoserine; this inhibition is straighforward and shows no sign of co-operativity. Evidence is presented that homoserine and threonine bind to the same site of this non-allosteric enzyme. The binding of homoserine and threonine can also be studied by difference spectroscopy; the latter studies reveal an unexpected effect of magnesium ions, which might be the basis for the unusual high Mg2+ requirement for optimal enzyme reaction.     

Biochemical Genetics of the Cryptic Gene System for Cellobiose Utilization in Escherichia coli K12

The cellobiose catabolic system of Escherichia coli K12 is being used to study the role of cryptic genes in microbial evolution. Wild-type E. coli K12 do not utilize the beta-glucoside sugars, arbutin, salicin and cellobiose. A Cel+ (cellobiose utilizing) mutant which grows on cellobiose, arbutin, and salicin was isolated previously from wild-type E. coli K12. Biochemical assays indicate that a cel structural gene (celT) specifies a single transport protein that is a beta-glucoside specific enzyme of the phosphoenolpyruvate-dependent phosphotransferase system. The transport protein phosphorylates beta-glucosides at the expense of phosphoenolpyruvate. A single phosphoglucosidase, specified by celH, hydrolyzes phosphorylated cellobiose, arbutin, and salicin. The genes of the cel system are expressed constitutively in the Cel+ mutant, whereas they are not expressed at a detectable level in the wild-type strain. The transport and hydrolase genes are simultaneously silenced or simultaneously expressed and thus constitute an operon. Cel+ strains which fail to utilize one or more beta-glucosides express the transport system at a lower level than do Cel+ strains which grow on all three beta-glucosides. Other strains inducibly express a gene which specifies transport of arbutin but not the other beta-glucosides. The arbutin transport gene, arbT, maps outside of the cel locus.     

Characterization of proteins in different subcellular localizations for Escherichia coli K12

Knowing the comprehensive knowledge about the protein subcellular localization is an important step to understand the function of the proteins. Recent advances in system biology have allowed us to develop more accurate methods for characterizing the proteins at subcellular localization level. In this study, the analysis method was developed to characterize the topological properties and biological properties of the cytoplasmic proteins, inner membrane proteins, outer membrane proteins and periplasmic proteins in Escherichia coli (E. coli). Statistical significant differences were found in all topological properties and biological properties among proteins in different subcellular localizations. In addition, investigation was carried out to analyze the differences in 20 amino acid compositions for four protein categories. We also found that there were significant differences in all of the 20 amino acid compositions. These findings may be helpful for understanding the comprehensive relationship between protein subcellular localization and biological function     

A specific polyadenylase from Escherichia coli K12.

A polyadenylase, degrading specifically poly(A) sequences was isolated from Escherichia coli K12. The enzyme was purified about 850 times to practically electrophoretic homogeneity. It was free of poly(A) polymerase activity, as well as of the well known E. coli RNAases I and II. It is stimulated by bivalent cations like Mg2+ and Mn2+ and splits poly(A) to 3'-AMP and therefore it can be considered as an exonuclease. The enzyme does not degrade any other ribohomopolymer or RNA.     

Pores in the outer membrane of Escherichia coli K12: involvement of proteins b and e in the functioning of pores for nucleotides.

Summary Mutants lacking outer membrane proteins were studied in order to investigate the role of these proteins in the functioning of aqueous pores through the outer membrane. Protein b is involved in the functioning of pores through which low concentrations of adenosine-monophosphate (AMP), guanosine-monophosphate (GMP), bis(paranitrophenyl)-phosphate (bis-PNPP) and, although less pronounced, also cytidine-monophosphate (CMP) enter the cell. In the absence of the receptor protein of phage T6, which facilitates the permeation of various nucleosides (Hantke, 1976), protein b plays a major role in the uptake of adenosine. Proteins c and d and the receptor proteins of phages lambda and T6 do not have a pore function for AMP, GMP, CMP and bis-PNPP. However, a newly discovered peptidoglycan-associated protein, protein e, can also mediate the permeation of the latter four components, but not of adenosine, through the outer membrane.Protein b does not play a major role in the uptake of higher concentrations of AMP. Obviously mainly other pores are used under those conditions. The results strongly suggest that at low concentrations of nucleoside monophosphates (and possibly of other nutrients) protein b functions as a specific pore.     

Isolation of haemin-requiring mutants of Escherichia coli K12.

Fifty-five haemin-requiring mutants were isolated from haemin-permeable mutants. According to their growth responses to haem precursors and their patterns of porphyrin accumulation, the 55 mutants fell into three groups which were judged to have defects in 5-aminolaevulinate dehydratase, ferrochelatase, and uroporphyrinogen III cosynthase or uroporphyrinogen decarboxylase. In mutants of the group deficient in 5-aminolaevulinate dehydratase, the mutations were adjacent to lac, and evidence is presented that the mutations were in hemB and were commonly deletions extending into proC.