Proteome analysis of Azotobacter vinelandii ∆arrF mutant that overproduces poly-ß-hydroxybutyrate polymer.

Azotobacter vinelandii ArrF is an iron-responsive small RNA that is under negative control of Ferric uptake regulator protein. A. vinelandii ∆arrF mutant that had a deletion of the entire arrF gene was known to overproduce poly-ß-hydroxybutyrate (PHB). Proteins differentially expressed in the mutant were identified by gel-based proteomics and confirmed by real-time RT-PCR. 6-Phosphogluconolactonase and E(1) component of pyruvate dehydrogenase complex, which leads to the production of NADPH and acetyl-CoA, were upregulated, while proteins in the tricarboxylic acid cycle that consumes acetyl-CoA were downregulated. Heat-shock proteins such as HSP20 and GroEL were highly overexpressed in the mutant. Antioxidant proteins such as Fe-containing superoxide dismutase (FeSOD), a putative oxidoreductase, alkyl hydroperoxide reductase, flavorprotein WrbA, and cysteine synthase were also overexpressed in the ∆arrF mutant, indicating that the PHB accumulation is stressful to the cells. Upregulated in the ∆arrF mutant were acetyl-CoA carboxylase, flagellin, and adenylate kinase, though the reasons for their overexpression are unclear. Among genes upregulated in the mutant, sodB coding for FeSOD and phbF encoding PHB synthesis regulator PhbF were negatively regulated by small RNA ArrF probably in an antisense mechanism. The deletion of arrF gene, therefore, would increase PhbF and FeSOD levels, which favors PHB synthesis in the mutant. On the other hand, glutamate synthetase, elongation factor-Tu, iron ABC transporter, and major outer membrane porin OprF were downregulated in the ∆arrF mutant. Based on the results, it is concluded that multiple factors including the direct effect of small RNA ArrF might be responsible for the PHB overproduction in the mutant.

Incidence and persistence of Listeria monocytogenes in the catfish processing environment and fresh fillets.

Incidence of Listeria spp. in whole raw catfish, catfish fillets, and processing environments from two catfish processing facilities was determined in August 2008 and August 2009. Thirty-nine (18.4%) of 212 samples collected in August 2008 were positive for Listeria monocytogenes. Prevalences of Listeria species L. innocua and L. seeligeri-L. welshimeri-L. ivanovii were 11.3 and 23.6%, respectively. Of 209 samples collected in August 2009, 12.4% were positive for L. monocytogenes, 11% for L. innocua, and 19.6% for L. seeligeri-L. welshimeri-L. ivanovii. No Listeria grayi was detected in any of the samples. L. monocytogenes was not found in catfish skins and intestines, but was detected in catfish fillets, on food contact surfaces, and on non-food contact surfaces with frequencies of 45.0, 12.0, and 11.1%, respectively. In August 2008 isolates, serotypes 1/2b (62.2%) and 3b (15.6%) were frequently isolated, whereas the majority of the August 2009 isolates (92.3%) were serotype 1/2b. Genotyping analyses revealed that some genotypes of L. monocytogenes isolates were detected in one facility even after a year, but no persistence of L. monocytogenes was observed in the other facility. In addition, some L. monocytogenes isolates from fresh fillets showed genotypes that were either identical, or more than 90% similar, to those of L. monocytogenes isolates from food contact surfaces in the processing lines. The results of this study suggest that processing environment rather than whole raw catfish is an important source of L. monocytogenes contamination in the catfish fillets. These results should assist the catfish industry to develop better control and prevention strategies for L. monocytogenes.

Prevalence and contamination patterns of Listeria monocytogenes in catfish processing environment and fresh fillets.

Catfish skins, intestines, fresh fillets, processing surfaces at different production stages, chiller water and non-food contact surfaces were sampled for Listeria monocytogenes and other Listeria species. Among 315 samples, prevalence of L. monocytogenes, Listeria innocua and a group of Listeria seeligeri-Listeria welshimeri-Listeria ivanovii was 21.6, 13.0 and 29.5%, respectively. No Listeria grayi was detected in this survey. While no L. monocytogenes strains were isolated from catfish skins and intestines, the strains were found with a frequency of 76.7% in chilled fresh catfish fillets and 43.3% in unchilled fillets. L. monocytogenes and Listeria spp. were also detected in fish contact surfaces such as deheading machine, trimming board, chiller water, conveyor belts at different stages, and fillet weighing table. Among L. monocytogenes, 1/2b (47.0%), 3b (16.0%) and 4c (14%) were the predominant serotypes isolated, whereas 4b, 4e, 1/2c and 1/2a were detected at much lower frequencies. Genotype analyses of L. monocytogenes isolates using serotyping, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed that chiller water represented an important contamination source of L. monocytogenes in the chilled catfish fillets of two processing facilities, whereas fillet weighing table significantly contributed to the catfish fillet contamination of the third facility. This study suggests that L. monocytogenes contamination in the processed catfish fillets originates from the processing environment, rather than directly from catfish. Results from this study can aid the catfish industry to develop a plant-specific proper cleaning and sanitation procedure for equipment and the processing environment designed to specifically target L. monocytogenes contamination.

Overproduction of poly-beta-hydroxybutyrate in the Azotobacter vinelandii mutant that does not express small RNA ArrF.

Azotobacter vinelandii contains an iron-regulatory small RNA ArrF whose expression is dependent upon the levels of iron and ferric uptake regulator. The deletion of this ArrF-encoding gene resulted in a 300-fold increase in the production of poly-beta-hydroxybutyrate (PHB), a polymer of industrial importance. This arrF mutant exhibited wild-type growth and growth-associated PHB production. Limited iron and aeration elevated the PHB production in the mutant as well as wild type. Real-time RT-PCR revealed that phbB, phbA, and phbC were upregulated approximately 61-, 18-, and eightfold, respectively, in the mutant. The phbR transcript of the activator PhbR for this operon was also approximately 11 times more abundant. The analysis of phbR transcript predicted a region of complementarity near its Shine-Dalgarno sequence that could potentially basepair with the conserved region of ArrF. These results suggest that ArrF represses the expression of PhbR in an antisense manner and derepression of this activator in the mutant elevates the expression of phbB, phbA, and phbC, resulting in the PHB overproduction.

Small RNA ArrF regulates the expression of sodB and feSII genes in Azotobacter vinelandii.

Azotobacter vinelandii contains a prrF-like sequence in a noncoding region of the chromosome. Like the Pseudomonas aeruginosa PrrF small RNA-encoding genes, the expression of the sequence, herein named arrF (Azotobacter regulatory RNA involving Fe), was increased 100-fold in wild-type cells in response to iron depletion. The level of ArrF was also increased to the same degree in the iron-replete fur mutant, but down back to a wild-type level when this fur mutant was complemented with the wild-type fur gene. These results, with the location of arrF gene in a noncoding region, suggest that this gene encodes an iron-responsive small RNA whose expression is negatively regulated by the Fur-Fe(2+) complex. Disruption of this arrF gene upregulated the expression of iron-containing superoxide dismutase and FeSII protein, whereas fur mutation or iron depletion decreased the level of their transcript. A short region in the 5'-untranslated region of each transcript was predicted to be quite complementary to the core sequence of ArrF, assuming that ArrF represses the expression of the genes under Fur control by an antisense RNA mechanism. However, unlike the P. aeruginosa PrrF that has extensive targets in the tricarboxylic acid cycle and glyoxylate cycle, ArrF had little effect on those genes. The findings that there is a poor overlap between ArrF and PrrF targets and that the FeSII gene, which is present only in the chromosome of nitrogen-fixing bacterial species, is controlled by ArrF suggest that ArrF might have unique targets, some of which are involved in N(2) fixation.

E1 component of pyruvate dehydrogenase complex does not regulate the expression of NADPH-ferredoxin reductase in Azotobacter vinelandii.

In Azotobacter vinelandii, the E1 component of pyruvate dehydrogenase complex (PDHE1) is proposed to be a key regulatory protein in an oxidative stress management system that responds to superoxide. This proposal was tested by constructing an A. vinelandii mutant that had a disruption of aceE gene encoding PDHE1. This mutant exhibited wild-type exponential growth and a normal response to oxidative stress induced by paraquat. Electrophoretic mobility-shift assays revealed that a protein previously shown to bind to a paraquat-activatable DNA promoter was still present in the extract prepared from the mutant, implying that the protein cannot be PDHE1. These observations strongly contradict the previous claim that PDHE1 is a DNA-binding protein that is directly involved in the A. vinelandii oxidative stress-regulatory system.

Optimization of hot water treatment for removing microbial colonies on fresh blueberry surface.

UNLABELLED: Blueberries for the frozen market are washed but this process sometimes is not effective or further contaminates the berries. This study was designed to optimize conditions for hot water treatment (temperature, time, and antimicrobial concentration) to remove biofilm and decrease microbial load on blueberries. Scanning electron microscopy (SEM) image showed a well-developed microbial biofilm on blueberries dipped in room temperature water. The biofilm consisted of yeast and bacterial cells attached to the berry surface in the form of microcolonies, which produced exopolymer substances between or upon the cells. Berry exposure to 75 and 90 °C showed little to no microorganisms on the blueberry surface; however, the sensory quality (wax/bloom) of berries at those temperatures was unacceptable. Response surface plots showed that increasing temperature was a significant factor on reduction of aerobic plate counts (APCs) and yeast/mold counts (YMCs) while adding Boxyl® did not have significant effect on APC. Overlaid contour plots showed that treatments of 65 to 70 °C for 10 to 15 s showed maximum reductions of 1.5 and 2.0 log CFU/g on APCs and YMCs, respectively; with acceptable level of bloom/wax score on fresh blueberries. This study showed that SEM, response surface, and overlaid contour plots proved successful in arriving at optima to reduce microbial counts while maintaining bloom/wax on the surface of the blueberries. PRACTICAL APPLICATION: Since chemical sanitizing treatments such as chlorine showed ineffectiveness to reduce microorganisms loaded on berry surface (Beuchat and others 2001, Sapers 2001), hot water treatment on fresh blueberries could maximize microbial reduction with acceptable quality of fresh blueberries.

Enhanced antimicrobial activity of starch-based film impregnated with thermally processed tannic acid, a strong antioxidant.

Starch-based films impregnated with fresh tannic acid (FTA/starch film) and thermally processed tannic acid (PTA/starch film) were assessed for inhibition of Escherichia coli O157:H7 and Listeria monocytogenes. Disc-diffusion assay revealed that the PTA/starch film showed larger clear zone around the film on the bacterial lawn than the FTA/starch film at the same tannic acid concentrations (0.45 to 4.5mg per disc). Viable cell count assays in tryptic soy broth showed that the PTA/starch film also had a stronger antimicrobial activity on these foodborne pathogens than the FTA/starch film. L. monocytogenes did not replicate in trypic soy broth containing the FTA/starch film for the first 8h but multiplied up to 9.22 log CFU/ml at 48 h of incubation. The PTA/starch film caused a 2.72-log decrease in L. monocytogenes cells over the same time period. While 5-log E. coli O157:H7 cells were inactivated by the FTA/starch film within 48 h, more than 7-log E. coli O157:H7 cells were killed by the PTA/starch film over the same period. The antimicrobial activity of FTA/starch and PTA/starch film was primarily pH independent. HPLC measurement of the FTA or PTA release from starch film in water revealed that their release kinetic curves were in well match with their inactivation curves for E. coli O157:H7 and L. monocytogenes in 0.1% peptone water. In addition to antimicrobial activity, FTA showed antioxidant activity on soybean oil by doubling the induction time of oil oxidation. PTA further enhanced the oxidative stability of the oil by 17%. These results suggested that the use of processed tannic acid in starch films could improve the safety and quality of foods.

Mechanisms of redox-coupled proton transfer in proteins: role of the proximal proline in reactions of the [3Fe-4S] cluster in Azotobacter vinelandii ferredoxin I.

The 7Fe ferredoxin from Azotobacter vinelandii (AvFdI) contains a [3Fe-4S](+/0) cluster that binds a single proton in its reduced level. Although the cluster is buried, and therefore inaccessible to solvent, proton transfer from solvent to the cluster is fast. The kinetics and energetics of the coupled electron-proton transfer reaction at the cluster have been analyzed in detail by protein-film voltammetry, to reveal that proton transfer is mediated by the mobile carboxylate of an adjacent surface residue, aspartate-15, the pK of which is sensitive to the charge on the cluster. This paper examines the role of a nearby proline residue, proline-50, in proton transfer and its coupling to electron transfer. In the P50A and P50G mutants, a water molecule has entered the cluster binding region; it is hydrogen bonded to the backbone amide of residue-50 and to the Asp-15 carboxylate, and it is approximately 4 A from the closest sulfur atom of the cluster. Despite the water molecule linking the cluster more directly to the solvent, proton transfer is not accelerated. A detailed analysis reveals that Asp-15 remains a central part of the mechanism. However, the electrostatic coupling between cluster and carboxylate is almost completely quenched, so that cluster reduction no longer induces such a favorable shift in the carboxylate pK, and protonation of the base no longer induces a significant shift in the pK of the cluster. The electrostatic coupling is crucial for maintaining the efficiency of proton transfer both to and from the cluster, over a range of pH values.

Azotobacter vinelandii ferredoxin I: a sequence and structure comparison approach to alteration of [4Fe-4S]2+/+ reduction potential.

The reduction potential (E(0)') of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (AvFdI) and related ferredoxins is approximately 200 mV more negative than the corresponding clusters of Peptostreptococcus asaccharolyticus ferredoxin and related ferredoxins. Previous studies have shown that these differences in E(0)' do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the [4Fe-4S](2+/+) clusters. Recently, a third, quite distinct class of ferredoxins (represented by the structurally characterized Chromatium vinosum ferredoxin) was shown to have a [4Fe-4S](2+/+) cluster with a very negative E(0)' similar to that of AvFdI. The observation that the sequences and structures surrounding the very negative E(0)' clusters in quite dissimilar proteins were almost identical inspired the construction of three additional mutations in the region of the [4Fe-4S](2+/+) cluster of AvFdI. The three mutations, V19E, P47S, and L44S, that incorporated residues found in the higher E(0)' P. asaccharolyticus ferredoxin all led to increases in E(0)' for a total of 130 mV with a 94-mV increase in the case of L44S. The results are interpreted in terms of x-ray structures of the FdI variants and show that the major determinant for the large increase in L44S is the introduction of an OH-S bond between the introduced Ser side chain and the Sgamma atom of Cys ligand 42 and an accompanying movement of water.

Crystal structures of ferredoxin variants exhibiting large changes in [Fe-S] reduction potential.

Elucidating how proteins control the reduction potentials (E0') of [Fe--S] clusters is a longstanding fundamental problem in bioinorganic chemistry. Two site-directed variants of Azotobacter vinelandii ferredoxin I (FdI) that show large shifts in [Fe--S] cluster E0' (100--200 mV versus standard hydrogen electrode (SHE)) have been characterized. High resolution X-ray structures of F2H and F25H variants in their oxidized forms, and circular dichroism (CD) and electron paramagnetic resonance (EPR) of the reduced forms indicate that the overall structure is not affected by the mutations and reveal that there is no increase in solvent accessibility nor any reorientation of backbone amide dipoles or NH--S bonds. The structures, combined with detailed investigation of the variation of E0' with pH and temperature, show that the largest increases in E0' result from the introduction of positive charge due to protonation of the introduced His residues. The smaller (50--100 mV) increases observed for the neutral form are proposed to occur by directing a Hdelta+--Ndelta- dipole toward the reduced form of the cluster.