Topographical distribution of penicillin-binding proteins in the Escherichia coli membrane.

The penicillin-binding proteins (PBPs) found in the membranes of Escherichia coli X925 minicells (primarily cell ends or septa) were compared with those found in rod-shaped cells (primarily sidewalls) in an effort to determine whether certain PBPs are unevenly distributed over the bacterial cell membrane. The seven major PBPs of E. coli were all present in minicell membranes. PBP 1B was altered in minicells, however, appearing as two bands on sodium dodecyl sulfate-polyacrylamide gels rather than the usual three. PBP 2, which is needed for longitudinal growth of the cell but not for septum formation, was significantly reduced in minicell membranes. This observation is consistent with the fact that minicells contain very little sidewall material and raises the possibility that the specialized function of PBP 2 may be determined or regulated by its uneven topographical distribution in the membrane. None of the PBPs appeared to be selectively enriched in minicell membranes.     

Changes in peptidoglycan composition and penicillin-binding proteins in slowly growing Escherichia coli.

The composition of peptidoglycan of chemostat-grown cultures of Escherichia coli was investigated as a function of growth rate. As the generation time was lengthened from 0.8 to 13.8 h, there was a decrease in the major monomer (disaccharide tetrapeptide) and dimer (bis-disaccharide tetrapeptide), while disaccharide tripeptide moieties increased to greater than 50% of the total wall. The average chain length became much shorter; lipoprotein density tripled, and the number of unusual diaminopimelyl-diaminopimelic acid crossbridges increased fivefold. As cells grew more slowly, amounts of penicillin-binding proteins (PBPs) 1a-1b complex and 4 decreased, while amounts of PBPs 3 and the 5-6 complex increased. We propose that the chemical composition of E. coli cell walls changes with growth rate in a manner consistent with alterations in the activities of PBPs and cell shape.     

Comparative susceptibility studies of clinically isolated Pseudomonas aeruginosa, Escherichia coli and Klebsiella spp to cefoperazone and cefotaxime

The in-vitro activity of cefotaxime and cefoperazone were compared using clinically isolated Escherichia coli, Klebsiella spp and Pseudomonas aeruginosa. Cefotaxime was found on a weight to weight basis, to be much more active than cefoperazone. All the three species studied show the presence of cefoperazone-resistant population which were sensitive to cefotaxime. The possible mechanisms of resistance to these antibiotics were discussed.     

Cloning and expression of Pseudomonas aeruginosa flagellin in Escherichia coli.

The flagellin gene was isolated from a Pseudomonas aeruginosa PAO1 genomic bank by conjugation into a PA103 Fla- strain. Flagellin DNA was transferred from motile recipient PA103 Fla+ cells by transformation into Escherichia coli. We show that transformed E. coli expresses flagellin protein. Export of flagellin to the E. coli cell surface was suggested by positive colony blots of unlysed cells and by isolation of flagellin protein from E. coli supernatants.     

Approaching the physiological functions of penicillin-binding proteins in Escherichia coli

A rigid shell of peptidoglycan encases and shapes bacteria and is constructed and maintained by a diverse set of enzymes, among which are the penicillin-binding proteins (PBPs). Although a great deal has been learned about how these proteins synthesize and modify peptidoglycan, the physiological functions of the multitude of bacterial PBPs remain enigmatic. We approached this problem by combining PBP mutations in a comprehensive manner and screening for effects on biochemical processes involving the passage of proteins or nucleic acids across the cell wall. The results indicate that the PBPs or their peptidoglycan product do have significant biological functions, including roles in determination of cell shape, in phage resistance, in induction of capsule synthesis, and in regulation of autolysis.     

Multiple mechanisms of membrane anchoring of Escherichia coli penicillin-binding proteins

Abstract: The major penicillin-binding proteins (PBPs) of Escherichia coli play vital roles in cell wall biosynthesis and are located in the inner membrane. The high M _r PBPs 1A, 1B, 2 and 3 are essential bifunctional transglycosylases/transpeptidases which are thought to be type II integral inner membrane proteins with their C-terminal enzymatic domains projecting into the periplasm. The low M _r PBP4 is a DD-carboxypeptidase/endopeptidase, whereas PBPs 5 and are DD-carboxypeptidases. All three low M _r , PBPs act in the modification of peptidoglycan to allow expansion of the sacculus and are thought to be periplasmic proteins attached with varying affinities to the inner membrane via C-terminal amphiphilic α-helices. It is possible that the PBPs and other inner membrane proteins form a peptidoglycan synthesizing complex to coordinate their activities.     

Secondary structure of the outer membrane proteins OmpA of Escherichia coli and OprF of Pseudomonas aeruginosa.

When purified without the use of ionic detergents, both OmpA and OprF proteins contained nearly 20% alpha-helical structures, which disappeared completely upon the addition of sodium dodecyl sulfate. This result suggests that the proteins fold in a similar manner, with an N-terminal, membrane-spanning beta-barrel domain and a C-terminal, globular, periplasmic domain.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

Cloning of Escherichia coli and Pseudomonas aeruginosa phosphomannose isomerase genes and their expression in alginate-negative mutants of Pseudomonas aeruginosa.

The phosphomannose isomerase (pmi) gene of Escherichia coli was cloned on a broad-host-range cosmid vector and expressed in Pseudomonas aeruginosa at a low level. Plasmid pAD3, which harbors the E. coli pmi gene, contains a 6.2-kilobase-pair HindIII fragment derived from the chromosome of E. coli. Subcloning produced plasmids carrying the 1.5-kilobase-pair HindIII-HpaI subfragment of pAD3 that restored alginic acid production in a nonmucoid, alginate-negative mutant of P. aeruginosa. This fragment also complemented mannose-negative, phosphomannose isomerase-negative mutants of E. coli and showed no homology by DNA-DNA hybridization to P. aeruginosa chromosomal DNA. By using a BamHI constructed cosmid clone bank of the stable alginate producing strain 8830, we have been able to isolate a recombinant plasmid of P. aeruginosa origin that also restores alginate production in the alginate-negative mutant. This new recombinant plasmid, designated pAD4, contained a 9.9-kilobase-pair EcoRI-BamHI fragment with the ability to restore alginate synthesis in the alginate-negative P. aeruginosa. This fragment showed no homology to E. coli chromosomal DNA or to plasmid pAD3. Both mucoid and nonmucoid strains of P. aeruginosa had no detectable levels of phosphomannose isomerase activity as measured by mannose 6-phosphate-to-fructose 6-phosphate conversion. However, P. aeruginosa strains harboring the cloned pmi gene of E. coli contained measurable levels of phosphomannose isomerase activity as evidenced by examining the conversion of mannose 6-phosphate to fructose 6-phosphate.     

Pseudomonas aeruginosa amidase: aggregation in recombinant Escherichia coli

The effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonas aeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of soluble amidase, relatively to other proteins, was obtained in the condition at 37°C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all the tested growth conditions. Addition of ethanol at 25°C in the culture medium improved amidase yield, which quantitatively aggregated in a biological active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37°C by FTIR analysis demonstrated a lower content of intermolecular interactions which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.     

Expression of the Pseudomonas aeruginosa PAK pilin gene in Escherichia coli.

Pseudomonas aeruginosa is a piliated opportunistic pathogen. We have recently reported the cloning of the structural gene for the pilus protein, pilin, from P. aeruginosa PAK (B. L. Pasloske, B. B. Finlay, and W. Paranchych, FEBS Lett. 183:408-412, 1985), and in this paper we present evidence that this chimera (pBP001) expresses P. aeruginosa PAK pilin in Escherichia coli independent of a vector promoter. The strength of the promoter for the PAK pilin gene was assayed, and the cellular location of the pilin protein within E. coli was examined. This protein was present mainly in the inner membrane fraction both with and without its six-amino-acid leader sequence, but it was not assembled into pili.     

Bacteriophage ecology in Escherichia coli and Pseudomonas aeruginosa mixed-biofilm communities

Phage therapy is being reexamined as a strategy for bacterial control in medical and other environments. As microorganisms often live in mixed populations, we examined the effect of Escherichia coli bacteriophage λW60 and Pseudomonas aeruginosa bacteriophage PB-1 infection on the viability of monoculture and mixed-species biofilm and planktonic cultures. In mixed-species biofilm communities, E. coli and P. aeruginosa maintained stable cell populations in the presence of one or both phages. In contrast, E. coli planktonic populations were severely depleted in coculture in the presence of λW60. Both E. coli and P. aeruginosa developed phage resistance in planktonic culture; however, reduced resistance was observed in biofilm communities. Increased phage titers and reduced resistance in biofilms suggest that phage can replicate on susceptible cells in biofilms. Infectious phage could be released from mixed-culture biofilms upon treatment with Tween 20 but not upon treatment with chloroform. Tween 20 and chloroform treatments had no effect on phage associated with planktonic cells, suggesting that planktonic phage were not cell or matrix associated. Transmission electron microscopy showed bacteriophage particles to be enmeshed in the extracellular polymeric substance component of biofilms and that this substance could be removed by Tween 20 treatment. Overall, this study demonstrates how mixed-culture biofilms can maintain a reservoir of viable phage and bacterial populations in the environment.     

Specific location of penicillin-binding proteins within the cell envelope of Escherichia coli.

This communication deals with the location of penicillin-binding proteins in the cell envelope of Escherichia coli. For this purpose, bacterial cells have been broken by various procedures and their envelopes have been fractioned. To do so, inner (cytoplasmic) and outer membranes were separated by isopycnic centrifugation in sucrose gradients. Some separation methods (Osborn et al., J. Biol. Chem. 247:3962-3972, 1972; J. Smit, Y. Kamio, and H. Nikaido, J. Bacteriol. 124:942-958, 1975) revealed that penicillin-binding proteins are not exclusively located in the inner membrane. They are also found in the outer membrane (A. Rodríguez-Tébar, J. A. Barbas, and D. Vásquez, J. Bacteriol. 161:243-248, 1985). Under the milder conditions for cell rupture used in this work, an intermembrane fraction, sedimenting between the inner and outer membrane, can be recovered from the gradients. This fraction has a high content of both penicillin-binding proteins and phospholipase B activity and may correspond to the intermembrane adhesion sites (M. H. Bayer, G. P. Costello, and M. E. Bayer, J. Bacteriol. 149:758-769, 1982). We postulate that this intermembrane fraction is a labile structure that contains a high amount of all penicillin-binding proteins which are usually found in both the inner and outer membranes when the adhesion sites are destroyed by the cell breakage and fractionation procedures.     

Daughter cell separation by penicillin-binding proteins and peptidoglycan amidases in Escherichia coli

As one of the final steps in the bacterial growth cycle, daughter cells must be released from one another by cutting the shared peptidoglycan wall that separates them. In Escherichia coli, this delicate operation is performed by several peptidoglycan hydrolases, consisting of multiple amidases, lytic transglycosylases, and endopeptidases. The interactions among these enzymes and the molecular mechanics of how separation occurs without lysis are unknown. We show here that deleting the endopeptidase PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the major peptidoglycan amidases during cell separation. Another endopeptidase, PBP 7, fulfills a secondary role. These functions may be responsible for the contributions of PBPs 4 and 7 to the generation of regular cell shape and the production of normal biofilms. In addition, we find that the E. coli peptidoglycan amidases may have different substrate preferences. When the dd-carboxypeptidase PBP 5 was deleted, thereby producing cells with higher levels of pentapeptides, mutants carrying only AmiC produced a higher percentage of cells in chains, while mutants with active AmiA or AmiB were unaffected. The results suggest that AmiC prefers to remove tetrapeptides from peptidoglycan and that AmiA and AmiB either have no preference or prefer pentapeptides. Muropeptide compositions of the mutants corroborated this latter conclusion. Unexpectedly, amidase mutants lacking PBP 5 grew in long twisted chains instead of straight filaments, indicating that overall septal morphology was also defective in these strains.     

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.     

Effect of growth rate on the penicillin-binding proteins of Escherichia coli

Abstract In an Escherichia coli strain, the levels of penicillin-binding proteins (PBPs) 1A plus 1B, both peptidoglycan transglycosylase/transpeptidases, were found to be relatively independent of the imposed growth ratw in chemostat cultures under different nutrient limitation conditions. A considerable increase in levels of PBP 6 was observed as the growth rate was reduced, whilst, in contrast, a decrease was observed in levels of the other PBPs.     

利用抗生素对大肠埃希菌和铜绿假单胞菌的影响进行菌株区分

目的为了探讨抗生素对中心碳代谢的影响,我们研究了大肠埃希菌和铜绿假单胞菌在11种不同抗生素的刺激下,三羧酸循环相关有机酸的代谢变化。方法利用毛细管电泳技术对2种菌在不同抗生素作用下细胞内的主要有机酸进行检测,然后通过多变量统计分析对数据进行处理。结果通过多变量统计分析发现,2种细菌可以通过抗生素对其胞内有机酸的影响不同而得到区分。结论胞内有机酸的变化具有菌株特异性,可以用于细菌的区分。