Electrophoresis and spectrometric analyses of adaptation-related proteins in thermally stressed Chromobacterium violaceum

Abstract

ABSTRACT. Chromobacterium violaceum is a Gram-negative proteobacteria found in water and soil; it is widely distributed in tropical and subtropical regions, such as the Amazon rainforest. We examined protein expression changes that occur in C. violaceum at different growth temperatures using electrophoresis and mass spectrometry. The total number of spots detected was 1985; the number ranged from 99 to 380 in each assay. The proteins that were identified spectrometrically were categorized as chaperones, proteins expressed exclusively under heat stress, enzymes involved in the respiratory and fermentation cycles, ribosomal proteins, and proteins related to transport and secretion. Controlling inverted repeat of chaperone expression and inverted repeat DNA binding sequences, as well as regions recognized by sigma factor 32, elements involved in the genetic regulation of the bacterial stress response, were identified in the promoter regions of several of the genes coding proteins, involved in the C. violaceum stress response. We found that 30°C is the optimal growth temperature for C. violaceum, whereas 25, 35, and 40°C are stressful temperatures that trigger the expression of chaperones, superoxide dismutase, a probable small heat shock protein, a probable phasing, ferrichrome-iron receptor protein, elongation factor P, and an ornithine carbamoyltransferase catabolite. This information improves our comprehension of the mechanisms involved in stress adaptation by C. violaceum.

GeLC-MS-based proteomics of Chromobacterium violaceum: comparison of proteome changes elicited by hydrogen peroxide

“…Cordeiro and colleagues analyzed how different growth temperatures influences C. violaceum metabolism while Lima et al . performed a GeLC-MS proteomics approach in the presence of high concentration of iron 14 15 . However, previous proteomic profilings disclosed at most, about 400 proteins from C. violaceum in response to an environmental stress.…”

Proteomic analysis of Chromobacterium violaceum and its adaptability to stress

“…The chaperones DnaK (heat shock protein), GroEL-2 and Phasin are both highly conserved proteins involved in biosynthesis and were both more abundant in C. violaceum cultures grown under stress conditions. DnaK, phasin, chaperonin GroEL-2 function as accessory proteins in chaperone machines [ 6 , 16 , 27 – 29 ] and increased chaperone expression was associated with stress tolerance and response to a high concentration of iron or carbon/energy or nitrogen nutrient-starvation [ 15 , 30 ]. Our data is in agreement with the role of these proteins in protection against damage caused by pH and nutrient-starvation stresses [ 31 – 34 ].…”

“…Lastly, the IPG strips were soaked in 75 mM Tris–HCl pH 6.8 for 2 min and placed on 12.5 % ( w/v ) polyacrylamide resolving gels in a SE600Rub System (GE Healthcare) under 10 mA/gel for 45 min. The polyacrylamide gels were fixed using 10 % acid acetic and 40 % methanol, followed by staining with Colloidal Coomassie Blue (8 % ammonium sulfate, 0.8 % phosphoric acid, 0.08 % Coomassie Blue G-250 and 20 % methanol) [ 16 ]. Gel images were captured by scanning (Image Scanner, GE Healthcare), and analyzed by Image Master Platinum software (Version 6).…”

“…Gel images were captured by scanning (Image Scanner, GE Healthcare), and analyzed by Image Master Platinum software (Version 6). Three reproducible gels (over 70 % in similarity) were produced to correspond with at least two independent extraction procedures from each experimental condition [ 16 ]. The spot detection parameters were determined by the ImageMaster Platinum software to detect spots in non-DIGE gels.…”

“…Laboratory studies have illustrated the remarkable adaptability of C . violaceum to changes in environmental conditions, like iron influence and stressful growth temperatures, to determine the specific enzymes and elements involved in the genetic regulation [ 15 , 16 ].…”