Antimicrobial susceptibility profile, Adherence and invasion to mammalian cells of Brucella melitensis isolates

Susceptibilities of 66 Brucella isolates were tested in vitro. All isolates were susceptible to doxycycline, gentamic in and streptomycin. In addition, propyl paraben, cresol and benzalkoniumchloride were found to be the most powerful tested preservative, disinfectant and antiseptic, respectively. All isolates adhered to and invaded into Vero cells by variable degrees. Adherence and invasion of most isolates were significantly reduced by: [1] pretreatment of test isolates with trypsin and sodium metaperiodate; [2] pretreatment of Vero cells with lipase, neuraminidase and sodium metaperiodate; [3] presence of Ca++, Mg++ and 200mM mannose in the assay medium and [4] growth of test isolates in half MICs of different antimicrobial agents. On the other hand, pretreatment of Vero cells with trypsin increased the adherence and invasion of most test isolates. No significant change in adhesion and invasion by changing the temperature from 27degree C to 42 degree Cor the pH from 6 to 8. Log phase cultures showed higher adherence and invasion than stationary phase cultures

Physiological factors affecting the level of soluble form of enzyme ii glucose in Escherichia coli

The membrane integrated enzyme II glucose [EIICB[Glc]], the product of ptsG gene, was found to be also present as soluble form. This form appeared as a second activity peak when the 2 h high speed supernatant [2 HSS] of crude cell lysate of E. coli is gel filtered. While, the first activity peak produced represents the membranous form of the enzyme. The relative percentage of peak 2 to peak 1 [P2/P1] was not constant in the presence of various sugar concentrations in different E. coli strains. With E. coli strain 301, in Luria Bertani [LB] alone, the relative percentage P2/P1 was about 52% while in M9 medium containing 10 mM glucose, this ratio was about 4% . Incorporation of non-phosphotransferase [PTS] sugars [lactose, maltose and melibiose] but not the PTS sugar glucose, in LB at 1 mM, caused an increase in P2/P1 ratio and this increase was more pronounced in case of lactose. While, both glucose and the non PTS sugars tested at 10 mM caused a decrease [about 40-50% in case of glucose and 85% or more for other sugars] in the P2/P1 ratio. In case of E. coli MG1655, and under pH buffered growth, P2/P1 ratio in LB was about 30% while the incorporation of glucose, lactose, maltose or melibiose at 11 mM concentration decreased P2/P1 ratios to values ranged between 6 and 16% . In case of E. coli strains with point mutations in the ptsG gene the P2/P1 ratios were different among different mutants. Comparing to the wild type strain BW3214, the point mutations in strains BW3256, BW3259, BW3260 and BW3261 resulted in higher P2/P1 ratios. On the other hand, comparing to the wild type strain BW3407, the point mutations in strains BW3406 and BW3408 resulted in lower P2/P1 ratios. In conclusion, the soluble form of EIICB[Glc] [P2] is present in different E. coli strains and its level relative to the membranous form [P1] in the 2 HSS is not constant. Such level differs from one strain to another and is affected by the sugar type in the growth medium and its concentration, culture pH and point mutations in ptsG gene that encodes the enzyme. High sugar concentration either of PTS or non PTS sugars greatly reduced the EIICB[Glc] soluble form. This gives another evidence for the existence of such soluble form of enzyme II glucose [EIICB[Glc]]

Cloning and expression of the Escherichia coli ptsG gene using an expression system enabling fusion to maltose binding protein

The ptsG gene carried onto the recombinant vector pET19b-ptsG-H4-ampr was amplified by PCR and separated by gel electrophoresis. The isolated ptsG gene was cloned downstream the Escherichia coli malE gene which carried onto the expression vector pMALE-ampr. Seven recombinant plasmid preparations were obtained and all of them gave the expected restriction map using EcoRI and BamHI restriction enzymes. Also PvuII restriction map for each of these recombinant plasmid preparations had the characteristic restriction pattern of the proper direction of ptsG insertion. This pattern showed five bands of molecular sizes 3179, 1689, 1676, 769 and 674 bp, three separated bands and two overlapping bands. Upon sequencing the inserted gene of four recombinant plasmid preparations, all of them showed the sequence of ptsG gene. One of these recombinant plasmid preparations [pMAL-ptsG-ampr] was electroporated into E. coli BL21DE3 to give the recombinant strain BL21DE3 [pMALE-ptsG-ampr]. The enzyme II glucose was expressed as a fused protein with maltose binding protein which encoded by malE gene. The produced enzyme was detected by transphosphorylation catalytic activity and western blot. The results showed that maltose binding protein can be used as a fusion legend for enzyme II glucose without abolishing its catalytic activity