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The eutT Gene of Salmonella enterica Encodes an Oxygen-Labile, Metal-Containing ATP:Corrinoid Adenosyltransferase Enzyme. Nicole R. Buan, 2004.The eutT gene of Salmonella enterica was cloned and overexpressed, and the function of its product was established in vivo and in vitro . The EutT protein has an oxygen-labile, metal-containing ATP:co(I)rrinoid adenosyltransferase activity associated with it . Functional redundancy between EutT and the housekeeping ATP:co(I)rrinoid adenosyltransferase CobA enzyme was demonstrated through phenotypic analyses of mutant strains . Lack of CobA and EutT blocked ethanolamine utilization . EutT was necessary and sufficient for growth of an S . enterica cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source . A eutT+ gene provided in trans corrected the adenosylcobalamin-dependent transcription of a eut-lacZ operon fusion in a cobA strain . Cell extracts enriched for EutT protein contained strong, readily detectable ATP:co(I)rrinoid adenosyltransferase activity . The activity was only detected in extracts maintained under anoxic conditions, with complete loss of activity upon exposure to air or treatment with the Fe2+ ion chelator bathophenanthroline . While the involvement of another metal ion cannot be ruled out, the observed sensitivity to air and bathophenanthroline suggests involvement of Fe2+ . We propose that the EutT protein is a unique metal-containing ATP:co(I)rrinoid adenosyltransferase . It is unclear whether the metal ion plays a structural or catalytic role . Improved Stress Tolerance of GroESL-Overproducing Lactococcus lactis and Probiotic Lactobacillus paracasei NFBC 338. C. Desmond, 2004.The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes . Two-dimensional polyacrylamide gel electrophoresis revealed that GroEL expression in probiotic Lactobacillus paracasei NFBC 338 was increased under heat adaptation conditions (52°C for 15 min) . Subsequently, the groESL operon of L . paracasei NFBC 338 was PCR amplified, and by using the nisin-inducible expression system, two plasmids, pGRO1 and pGRO2, were constructed on the basis of vectors pNZ8048 and pMSP3535, respectively . These vectors were transferred into Lactococcus lactis(pGRO1) and L . paracasei(pGRO2), and after induction with nisin, overexpressed GroEL represented 15 and 20% of the total cellular protein in each strain, respectively . Following heat shock treatment of lactococci (at 54°C) and lactobacilli (at 60°C), the heat-adapted cultures maintained the highest level of viability (5-log-unit increase, approximately) in each case, while it was found that the GroESL-overproducing strains performed only moderately better (1-log-unit increase) than the controls . On the other hand, the salt tolerance of both GroESL-overproducing strains (in 5 M NaCl) was similar to that of the parent cultures . Interestingly, both strains overproducing GroESL exhibited increased solvent tolerance, most notably, the ability to grow in the presence of butanol (0.5% [vol/vol]) for 5 h, while the viability of the parent strain declined . These results confirm the integral role of GroESL in solvent tolerance, and to a lesser extent, thermotolerance of lactic acid bacteria . Furthermore, this study demonstrates that technologically sensitive cultures, including certain probiotic lactobacilli, can potentially be manipulated to become more robust for survival under harsh conditions, such as food product development and gastrointestinal transit . DapE Can Function as an Aspartyl Peptidase in the Presence of Mn2+. Daniel H. Broder, 2003.Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on Mn2+ . Purification of this activity followed by N-terminal sequencing of the protein suggested that the Mn2+-dependent peptidase is DapE (N-succinyl-L,L-diaminopimelate desuccinylase) . A dapE chromosomal disruption was constructed and transduced into a multiply peptidase-deficient (MPD) strain . Crude extracts of this strain showed no aspartyl peptidase activity, and the strain failed to utilize Asp-Leu as a leucine source . The dapE gene was cloned into expression vectors in order to overproduce either the native protein (DapE) or a hexahistidine fusion protein (DapE-His6) . Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of Mn2+-dependent aspartyl peptidase activity relative to the MPD dapE+ strain . In addition, purified DapE-His6 exhibited Mn2+-dependent peptidase activity toward aspartyl dipeptides . Growth of the MPD strain carrying a single genomic copy of dapE on Asp-Leu as a Leu source was slow but detectable . Overproduction of DapE in the MPD dapE strain allowed growth on Asp-Leu at a much faster rate . DapE was found to be specific for N-terminal aspartyl dipeptides: no N-terminal Glu, Met, or Leu peptides were hydrolyzed, nor were any peptides containing more than two amino acids . DapE is known to bind two divalent cations: one with high affinity and the other with lower affinity . Our data indicate that the form of DapE active as a peptidase contains Zn2+ in the high-affinity site and Mn2+ in the low-affinity site . Depth Distribution of Microbial Diversity in Mono Lake, a Meromictic Soda Lake in California. Shaheen B. Humayoun, 2003.We analyzed the variation with depth in the composition of members of the domain Bacteria in samples from alkaline, hypersaline, and currently meromictic Mono Lake in California . DNA samples were collected from the mixolimnion (2 m), the base of the oxycline (17.5 m), the upper chemocline (23 m), and the monimolimnion (35 m) . Composition was assessed by sequencing randomly selected cloned fragments of 16S rRNA genes retrieved from the DNA samples . Most of the 212 sequences retrieved from the samples fell into five major lineages of the domain Bacteria:  - and
 -Proteobacteria (6 and 10%, respectively), Cytophaga-Flexibacter-Bacteroides (19%), high-G+C-content gram-positive organisms (Actinobacteria; 25%), and low-G+C-content gram-positive organisms (Bacillus and Clostridium; 19%) . Twelve percent were identified as chloroplasts . The remaining 9% represented ß- and
 -Proteobacteria, Verrucomicrobiales, and candidate divisions . Mixolimnion and oxycline samples had low microbial diversity, with only 9 and 12 distinct phylotypes, respectively, whereas chemocline and monimolimnion samples were more diverse, containing 27 and 25 phylotypes, respectively . The compositions of microbial assemblages from the mixolimnion and oxycline were not significantly different from each other (P = 0.314 and 0.877), but they were significantly different from those of chemocline and monimolimnion assemblages (P < 0.001), and the compositions of chemocline and monimolimnion assemblages were not significantly different from each other (P = 0.006 and 0.124) . The populations of sequences retrieved from the mixolimnion and oxycline samples were dominated by sequences related to high-G+C-content gram-positive bacteria (49 and 63%, respectively) distributed in only three distinct phylotypes, while the population of sequences retrieved from the monimolimnion sample was dominated (52%) by sequences related to low-G+C-content gram-positive bacteria distributed in 12 distinct phylotypes . Twelve and 28% of the sequences retrieved from the chemocline sample were also found in the mixolimnion and monimolimnion samples, respectively . None of the sequences retrieved from the monimolimnion sample were found in the mixolimnion or oxycline samples . Elevated diversity in anoxic bottom water samples relative to oxic surface water samples suggests a greater opportunity for niche differentiation in bottom versus surface waters of this lake .
Methicillin (Oxacillin)-Resistant Staphylococcus aureus Strains Isolated from Major Food Animals and Their Potential Transmission to Humans. John Hwa Lee, 2003.From May 2001 to April 2003, various types of specimens from cattle, pigs, and chickens were collected and examined for the presence of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA) . S . aureus was isolated and positively identified by using Gram staining, colony morphology, tests for coagulase and urease activities, and an API Staph Ident system . Among 1,913 specimens collected from the animals, 421 contained S . aureus; of these, 28 contained S . aureus resistant to concentrations of oxacillin higher than 2 µg/ml . Isolates from 15 of the 28 specimens were positive by PCR for the mecA gene . Of the 15 mecA-positive MRSA isolates, 12 were from dairy cows and 3 were from chickens . Antimicrobial susceptibility tests of mecA-positive MRSA strains were performed by the disk diffusion method . All isolates were resistant to members of the penicillin family, such as ampicillin, oxacillin, and penicillin . All isolates were also susceptible to amikacin, vancomycin, and trimethoprim-sulfamethoxazole . To determine molecular epidemiological relatedness of these 15 animal MRSA isolates to isolates from humans, random amplified polymorphic DNA (RAPD) patterns were generated by arbitrarily primed PCR . The RAPD patterns of six of the isolates from animals were identical to the patterns of certain isolates from humans . The antibiotypes of the six animal isolates revealed types similar to those of the human isolates . These data suggested that the genomes of the six animal MRSA isolates were very closely related to those of some human MRSA isolates and were a possible source of human infections caused by consuming contaminated food products made from these animals .
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