Effects of Phagocytosis by Rabbit Granulocytes on Macromolecular Synthesis and Degradation in Different Species of Bacteria

Phagocytosis and killing of gram-positive Bacillus megaterium and Micrococcus lysodeikticus by granulocytes in vitro is associated with almost immediate cessation of bacterial protein synthesis. By contrast, protein synthesis by Escherichia coli continues after ingestion and killing. After preincubation of E. coli with intact granulocytes for 15 min, when 95% or more of the bacteria can no longer multiply, induction of beta-galactosidase proceeds at rates about half of control values. With disrupted granulocytes, which kill E. coli as rapidly as intact cells, the rate of induction of beta-galactosidase does not fall until after 30 min of preincubation. We attribute the different effects of phagocytosis on the biochemical apparatus of these microorganisms to the different fates of their envelopes. Specifically labeled protein, ribonucleic acid, deoxyribonucleic acid, and lipid of all three species of bacteria and peptidoglycan of E. coli are apparently incompletely degraded during phagocytosis. However, the cell walls of M. lysodeikticus and B. megaterium undergo rapid and almost complete degradation. The resulting structural disintegration of these gram-positive microorganisms must cause extensive biochemical disorganization as well. Our evidence indicates that the E. coli envelope, on the other hand, retains sufficient structural organization to preserve integrated biochemical function for at least 1 h after the bacteria have lost the ability to multiply.     

Survival of bacteria and the fate of Escherichia coli DNA in amino acid deficiency

The survival rate of an E. coli polyauxotrophous strain AB1157 and the behaviour of its DNA were studied when the strain was incubated for a long time at 43 degrees C in a medium deficient in glucose, phosphates and amino acids. Under these conditions, the survival rate fell down to 10%, but no cell lysis occurred. DNA synthesis stopped within the first two hours of starvation. Neither DNA degradation, despiralization nor decrease of its molecular weight could be detected during the entire starvation. Therefore, the death of E. coli cells under these conditions was not associated with DNA damages.     

Cloning, expression, and characterization of aminopeptidase P from the hyperthermophilic archaeon Thermococcus sp. strain NA1

Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. strain NA1, revealed the presence of a 1,068-bp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the putative aminopeptidase P (APP) of Thermococcus kodakaraensis KOD1. Amino acid residues important for catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs were also conserved in the Thermococcus sp. strain NA1 APP. The archaeal APP, designated TNA1_APP (Thermococcus sp. strain NA1 APP), was cloned and expressed in Escherichia coli. The recombinant enzyme hydrolyzed the amino-terminal Xaa-Pro bond of Lys(Nepsilon-Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (Km, 0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co2+-, Mn2+-, or Zn2+-dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at pH 5 and a temperature of 100 degrees C. The APP was thermostable, with a half-life of >100 min at 80 degrees C. This study represents the first characterization of a hyperthermophilic archaeon APP.     

An oxygen dependent X-ray lesion in Escherichia coli strain B/r detected by penicillin.

Enhancement of lethal damage to E. coli B/r by penicillin was observed after X-irradiation under aerobic conditions but not after exposure to X-rays under anoxia or after U.V. (260 nm). No enhancement of damage occurred when incubation with penicillin was delayed for 2 hours after aerobic X-irradiation. This enhancing effect was only detected in this strain and not in the filamentous strain E. coli B. It is concluded that an X-ray induced lesion, sensitive to the presence of oxygen at the time of irradiation and probably located in the cell envelope, initiates filamentation in E. coli B/r, which results in lethal damage in this strain.     

Low-temperature conditional cell division mutants of Escherichia coli.

Fifteen low-temperature conditional division mutants of Escherichia coli K-12 was isolated. They grew normally at 39 degrees C but formed filaments at 30 degrees C. All exhibited a coordinated burst of cell division when the filaments were shifted to the permissive temperature (39 degrees C). None of the various agents that stimulate cell division in other mutant systems (salt, sucrose, ethanol, and chloramphenicol) was very effective in restoring colony-forming ability at 25 degrees C or in stimulating cell division in broth. One of these mutants, strain JS10, was found to have an altered cell envelope as evidenced by increased sensitivity to deoxycholate and antibiotics, as well as leakage of ribonulcease I, a periplasmic enzyme. This mutant had normal rates of DNA synthesis, RNA synthesis, and phospholipid synthesis at both the nonpermissive and permissive temperatures. However, strain JS10 required new protein synthesis in the apparent absence of new RNA synthesis for division of filaments at the permissive temperature. The division of lesion in strain JS10 is cotransducible with malA, aroB, and glpD and maps within min 72 to 75 on the E. coli chromosome.     

Use of hydrostatic pressure for inactivation of microbial contaminants in cheese

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (Escherichia coli K-12, Staphylococcus aureus ATCC 6538, and Penicillium roqueforti IMI 297987). Initially, cheese slurries inoculated with E. coli, S. aureus, and P. roqueforti were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30 degrees C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10 degrees C and by 6-log-unit cycles at temperatures of 20 and 30 degrees C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20 degrees C and >400 MPa at 30 degrees C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20 degrees C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus. The organisms were more sensitive to pressure in cheese than slurry, especially with E. coli. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by S. aureus and P. roqueforti while greatest sensitivity to HP by E. coli was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.     

Toxicity of paraquat to microorganisms

The biochemical response of the microorganisms Lipomyces starkeyi (Lod & Rij), Escherichia coli K-12 W3110, Bacillus subtilis 168 (Marburg) and Pseudomonas sp. strain TTO1 to the presence of growth-inhibitory concentrations of paraquat was studied. Paraquat was added to each culture at a concentration previously determined to reduce the culture growth rate by up to 50%. The changes in activity of a number of enzymes previously shown to be associated with the defense of the mammalian system against the action of paraquat were studied. While the response of E. coli was in agreement with that found in other studies of this microorganism and supports a commonly accepted mechanism for paraquat toxicity, the results obtained with L. starkeyi, B. subtilis, and Pseudomonas sp. strain TTO1 suggest that other mechanisms exist for protection against the toxicity of paraquat.     

Toxicity of paraquat to microorganisms.

The biochemical response of the microorganisms Lipomyces starkeyi (Lod & Rij), Escherichia coli K-12 W3110, Bacillus subtilis 168 (Marburg) and Pseudomonas sp. strain TTO1 to the presence of growth-inhibitory concentrations of paraquat was studied. Paraquat was added to each culture at a concentration previously determined to reduce the culture growth rate by up to 50%. The changes in activity of a number of enzymes previously shown to be associated with the defense of the mammalian system against the action of paraquat were studied. While the response of E. coli was in agreement with that found in other studies of this microorganism and supports a commonly accepted mechanism for paraquat toxicity, the results obtained with L. starkeyi, B. subtilis, and Pseudomonas sp. strain TTO1 suggest that other mechanisms exist for protection against the toxicity of paraquat.     

Response of an Escherichia coli-Bound Fluorescent Probe to Colicin E1

The fluorescent probe, 8-anilino-1-napthalenesulfonate (ANS) binds to Escherichia coli, showing an enhanced fluorescence. The interaction of colicin E1 with sensitive cells causes an increase of about 100% in the fluorescence of the bound ANS, and this change at equilibrium has an apparent "all-or-none" nature as a function of E1 multiplicity. Approximately 6 to 8% of the ANS is bound to the cells at equilibrium. The colicin E1-induced fluorescence increase can be attributed partly to an increase in ANS binding and partly to an increase in the fluorescence yield of the bound ANS. The kinetics of the E1-induced fluorescence increase in sensitive cells are very similar to those of the adenosine triphosphate decrease. The phosphorylation uncoupler p-trifluoromethoxy-carbonylcyanidephenylhydrazone also causes a large change in the fluorescence of bound ANS. Colicin E2 or E3 does not cause any fluorescence change, nor does colicin E1 cause fluorescence change with a colicinogenic strain. ANS appears to be a probe of structural or conformational change in the cell envelope that is closely associated with the colicin E1-induced adenosine triphosphate decrease.     

Localization of polyamine enhancement of protein synthesis to subcellular components of Escherichia coli and Pseudomonas sp. strain Kim.

At 5 mM Mg2+, spermidine stimulation of polyphenylalanine synthesis by cell-free extracts of Escherichia coli was found to be about 30 times greater than that by extracts of Pseudomonas sp. strain Kim, a unique organism which lacks detectable levels of spermidine. By means of reconstitution experiments, the target of spermidine stimulation was localized to the protein fraction of the highspeed supernatant component (S-100) of E. coli and was absent from, or deficient in, the S-100 fraction of Pseudomonas sp. strain Kim. The spermidine stimulation did not appear to be due to the presence in the E. coli S-100 fraction of ribosomal protein S1, elongation factors, or E. coli aminoacyl-tRNA synthetases. The failure to observe spermidine stimulation by the Pseudomonas sp. strain Kim S-100 fraction was also not due to a spermidine-enhanced polyuridylic acid degradation. The synthesis of polyphenylalanine by Pseudomonas sp. strain Kim extracts was stimulated by putrescine and by S-(+)-2-hydroxyputrescine to a greater degree than was synthesis by E. coli extracts. The enhancement by putrescine and by S-(+)-2-hydroxyputrescine with Pseudomonas sp. strain Kim extracts was found to be due to effects on its ribosomes.     

Cloning of the genes for degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1.

The degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-1,3,5-triazine) is associated with an indigenous plasmid in Rhodococcus sp. strain TE1. Plasmid DNA libraries of Rhodococcus sp. strain TE1 were constructed in a Rhodococcus-Escherichia coli shuttle vector, pBS305, and transferred into Rhodococcus sp. strain TE3, a derivative of Rhodococcus sp. strain TE1 lacking herbicide degradation activity, to select transformants capable of growing on EPTC as the sole source of carbon (EPTC+). Analysis of plasmids from the EPTC+ transformants indicated that the eptA gene, which codes for the enzyme required for EPTC degradation, residues on a 6.2-kb KpnI fragment. The cloned fragment also harbored the gene required for atrazine N dealkylation (atrA). The plasmid carrying the cloned fragment could be electroporated into a number of other Rhodococcus strains in which both eptA and atrA were fully expressed. No expression of the cloned genes was evident in E. coli strains. Subcloning of the 6.2-kb fragment to distinguish between EPTC- and atrazine-degrading genes was not successful.     

From PCBs to highly toxic metabolites by the biphenyl pathway

The degradation of polychlorobiphenyls (PCBs) by diverse bacteria, including Burkholderia sp. LB400, is incomplete with a concomitant accumulation of metabolic intermediates. In this study, the toxicity of diverse (chloro)biphenyls and of their biotransformation into the first two metabolic intermediates of the biphenyl pathway, were determined for the model bacterium Escherichia coli. Recombinant E. coli strains expressing different subsets of bph genes of strain LB400 accumulated metabolic intermediates from (chloro)biphenyls. During biotransformation of these compounds into metabolic intermediates, the viability and metabolic kinetics were determined. The toxicity of biotransformation of (chloro)biphenyls into different metabolic intermediates of (chloro)biphenyls varied. Dihydrodiols and dihydroxybiphenyls are very toxic metabolites for bacteria even after short incubation times, affecting the cell viability much more than (chloro)biphenyls. When bacteria transformed 2-CB into dihydrodiol or dihydroxybiphenyl, a great decrease of intact cells and abundant cell lysis was observed by transmission electronic microscopy. Cell viability of Burkholderia sp. LB400 and of E. coli exposed directly to 2,3-dihydroxybiphenyl decreased also drastically. The toxicity of metabolites generated during oxidation of PCBs may partly explain the recalcitrance to biodegradation of these pollutants. Conversion of less toxic compounds into products with increased toxicity resembles the bioactivation of xenobiotics in higher organisms.     

Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase.

An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp. strain R312. Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amdA. Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans. Moreover, amdA is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon. Our results also showed that Rhodococcus sp. strain N-774 and Brevibacterium sp. strain R312 are probably identical, or at least very similar, microorganisms. The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 mumol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide. Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30 degrees C under the control of an E. coli promoter and ribosome-binding site.     

Isolation rate of gram negative microflora and its sensitivity to antibiotics in hemoblastosis patients

A total of 67 patients with blood system diseases and infectious complications were examined. During the period of the examination 139 microorganisms were isolated. Of these gram negative microorganisms constituted 51%, gram positive microorganisms--34.8% and fungal flora--14.2%. Most frequently the following gram negative microorganisms were isolated from the patients: Pseudomonas sp. (including P. aeruginosa), Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae. All isolated microorganisms retained sensitivity to imipenem, with the exception of individual strains of Pseudomonas sp.; the latter exhibited sensitivity to amicacin and ceftazidim. Cefotaxime was active with respect to 75% of K. pneumoniae strains and all E. coli strains, ciprofloxacin was active with respect to 43% of E. coli strains, 80% of K. pneumoniae strains and 83.4% of Pseudomonas sp. strains, cefepim was active with respect to 85.7% of Pseudomonas sp. strains and all E. coli strains, ceftazidim was active with respect to all Pseudomonas sp. and E. coli strains. 75% of K. pneumoniae strains, 77.8% of Pseudomonas sp. strains and 86% of E. coli strains retained sensitivity to amicacin. 25% of K. pneumoniae strains required testing for ESBL production.     

Characterization of a psychrotrophic Arthrobacter gene and its cold-active beta-galactosidase.

Enzymes with high specific activities at low temperatures have potential uses for chemical conversions when low temperatures are required, as in the food industry. Psychrotrophic microorganisms which grow at low temperatures may be a valuable source of cold-active enzymes that have higher activities at low temperatures than enzymes found for mesophilic microorganisms. To find cold-active beta-galactosidases, we isolated and characterized several psychrotrophic microorganisms. One isolate, B7, is an Arthrobacter strain which produces beta-galactosidase when grown in lactose minimal media. Extracts have a specific activity at 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside as a substrate. Two isozymes were detected when extracts were subjected to electrophoresis in a nondenaturing polyacrylamide gel and stained for activity with 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-Gal). When chromosomal DNA was prepared and transformed into Escherichia coli, three different genes encoding beta-galactosidase activity were obtained. We have subcloned and sequenced one of these beta-galactosidase genes from the Arthrobacter isolate B7. On the basis of amino acid sequence alignment, the gene was found to have probable catalytic sites homologous to those from the E. coli lacZ gene. The gene encoded a protein of 1,016 amino acids with a predicted molecular mass of 111 kDa. The enzyme was purified and characterized. The beta-galactosidase from isolate B7 has kinetic properties similar to those of the E. coli lacZ beta-galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme.     

DNA restriction-modification systems mediate plasmid maintenance.

Two plasmid-carried restriction-modification (R-M) systems, EcoRI (from pMB1 of Escherichia coli) and Bsp6I (from pXH13 of Bacillus sp. strain RFL6), enhance plasmid segregational stability in E. coli and Bacillus subtilis, respectively. Inactivation of the endonuclease or the presence of the methylase in trans abolish the stabilizing activity of the R-M systems. We propose that R-M systems mediate plasmid segregational stability by postsegregational killing of plasmid-free cells. Plasmid-encoded methyltransferase modifies host DNA and thus prevents its digestion by the restriction endonuclease. Plasmid loss entails degradation and/or dilution of the methylase during cell growth and appearance of unmethylated sites in the chromosome. Double-strand breaks, introduced at these sites by the endonuclease, eventually cause the death of the plasmid-free cells. Contribution to plasmid stability is a previously unrecognized biological role of the R-M systems.     

A thermostable glucose isomerase having a relatively low optimum pH: study of activity and molecular cloning of the corresponding gene

A thermostable glucose isomerase from a newly isolated thermophilic Streptomyces sp. SK strain, had a wide pH range with an optimum of 6 at 60°C and 6.4 at 90°C. It was optimally active at 95°C and completely stable at 80°C for at least 5.5 h with a half-life of 5 h at 90°C. Using E.coli as a host strain and an internal fragment of xylA of S. olivochromogenes as a probe, a 6.5 kb DNA fragment from Streptomyces sp. SK was cloned. This fragment carries the entire xylA gene since the recombinant plasmid complements the E.coli xyl-5 mutant strain HB101. © Rapid Science Ltd. 1998     

Effect of soil composition, temperature, indigenous microflora, and environmental conditions on the survival of Escherichia coli O157:H7

The survival of Escherichia coli O157:H7 in replicate soil microcosms was quantified in 2 types of silty clay loam soil (high carbon and low carbon) under either sterile or nonsterile conditions. Microcosms were held at -21, 4, and 22 degrees C under constant soil moisture content. Differences existed (P < 0.05) in survival of E. coli O157:H7 in low- and high-carbon soil at all temperatures, indicating an important role of soil composition on the survival of this pathogen. The highest death rate of E. coli O157:H7 in sterile soil occurred in the low-carbon soil at 4 degrees C, whereas in nonsterile soil the highest death rate was observed in the low-carbon soil at 22 degrees C. These results suggest that the most lethal effects on E. coli O157:H7 in the sterile system occurred via the synergy of nutrient limitation and cold stress, whereas in the nonsterile system lethality was owing to inhibition by indigenous soil microorganisms and starvation. Results obtained from an in situ field survival experiment demonstrated the apparent sensitivity of E. coli O157:H7 cells to dehydration, information that may be used to reduce environmental spread of this pathogen as well as formulate appropriate waste management strategies.     

Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001

A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly (>85%) alpha-cyclomaltodextrin (alpha-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991--1997, 1999). Nucleotide sequencing of the CGTase gene (cgtA) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (cgtB), the gene encoding CGTase (cgtA), a gene homolog for a CD-binding protein (CBP) (cgtC), and a putative CBP-dependent ABC transporter involved in uptake of CDs (cgtDE). The CDase was expressed in Escherichia coli and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95 degrees C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed beta-CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to alpha-CD. However, the CBP exhibited higher thermostability in the presence of alpha-CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.