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Archives of Microbiology (v.186, #1)

Stimulation of alkalothermophilic Aspergillus terreusxylanase by low-intensity laser radiation by Neveen S. Geweely; Salama A. Ouf; Mohamed A. Eldesoky; Amera A. Eladly (pp. 1-9).

In this study, Aspergillus terreus was irradiated by a 7.3 mW He–Ne laser in the presence of crystal violet, toluidine blue O and hematoporphyrin as photosensitizers. Xylanases recovered from non-irradiated and irradiated fungi were purified and characterized. The maximum production of xylanase (42.2 U/ml) was obtained after 5 min of laser irradiation in the absence of the photosensitizer. The irradiation of the sensitized fungus diminished the production of xylanase. On purification using G-100, the specific activity of xylanase recovered from the irradiated fungus was 292 U/mg protein representing a 37-fold purification over the crude extract compared with 95.6 U/mg protein representing the 12.8-fold for the enzyme recovered from the non-irradiated fungus. The enzyme recovered from the irradiated fungus had lower molecular weight as compared with that recovered from the non-irradiated one. Characterization of the purified enzymes revealed that the enzyme recovered from the irradiated fungus was more thermostable and had a wider range of optimum reaction temperature (60–70°C) and pH (4.0–12.0), compared to the non-irradiated one.

Keywords: Xylanase; Purification; Aspergillus terreus ; Laser radiation; Photosensitizers

Expression and processing of Vibrio anguillarum zinc-metalloprotease in Escherichia coli by Fengli Zhang; Jixiang Chen; Zhenming Chi; Long-Fei Wu (pp. 11-20).

The extracellular zinc-metalloprotease of Vibrio anguillarum is a secreted virulence factor. It is synthesized from the empA gene as a 611-residue preproprotease and processed to the active mature protease (EmpA) with concomitant secretion via the type II secretion pathway. Active EmpA has been found only in the V. anguillarum culture supernatant and the process of the activation seems to vary depending on strains analyzed. To better understand the mechanism of EmpA export and processing, the empA gene was cloned and expressed in Escherichia coli strains. Expression of empA did not have toxic effect on bacterial growth. Rupturing E. coli TOP10 cells by heating in gel-loading buffer resulted in activation of EmpA and severe proteolysis of the samples. In contrast, the same treatment of the E. coli MC4100A strain did not lead to the general proteolysis. In this strain, EmpA was exported into the periplasm via the Sec pathway. The periplasmic EmpA was detected in two active conformations. Therefore, in E. coli processing of EmpA precursor to an active enzyme did not require secretion to the media and the help of other V. anguillarum protein. Like in V. anguillarum, heterologous expression of empA in E. coli showed strain-specific activation process.

Keywords: Vibrio anguillarum ; Metalloprotease; Translocation; Activation; Processing

Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240 by Tsuyoshi Matsui; Toyokazu Yoshida; Toshihisa Hayashi; Toru Nagasawa (pp. 21-29).

We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its K _m value for 4-hydroxybenzoate was 596 μM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the K _m value was 6.80 mM. In the presence of 3 M KHCO₃ and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The K _m value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96–97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.

Keywords: 4-Hydroxybenzoate decarboxylase; 4-Hydroxybenzoate; Decarboxylation; Carboxylation; Enterobacter cloacae P240

An examination of the diversity of a novel Campylobacter reservoir by William J. Snelling; James P. McKenna; Catherine J. Hack; John E. Moore; James S. G. Dooley (pp. 31-40).

The diversity of eukaryotic populations, in particular protozoa, in the water supplies of intensively reared broilers has not been previously studied. This important food-rearing environment was screened for the molecular diversity of eukaryotes by the analysis of PCR-amplified 18S rRNA. DNA was extracted from filtered water samples that were collected from the poultry drinking water systems of five farms. The total genomic DNA was used to produce rRNA-PCR amplicons, which, with the application of TTGE, provided an overview of the eukaryotic population diversity. The rRNA-PCR amplicons were then used to generate 34 random clones that were subject to comparative sequence analysis. Twenty-five of the clones (73.5%) showed high similarity with yeasts and fungi (>92%) and 9 clones demonstrated similarity (>86%) with certain protozoan groups, including flagellates and alveolates. Further studies of the microbial diversity in the previously ignored niche of intensively reared poultry drinking water systems are required, along with subsequent in vitro co-culture assays of the detected protozoa and bacterial strains.

Keywords: Eukaryotic diversity of a Campylobacter reservoir

Plasmids for expression of heterologous proteins in Rhizopus oryzae by Jeffrey A. Mertens; Christopher D. Skory; Ashraf S. Ibrahim (pp. 41-50).

Rhizopus oryzae has long been used for enzyme production (e.g., glucoamylase and lipase), organic acid synthesis, and various fermented food applications. In this work, we describe a set of plasmid-based expression vectors that can be used for the production of heterologous proteins in R. oryzae. Three plasmid vectors have been created using either the glucoamylase A (amyA), pyruvate decarboxylase (pdcA), or phosphoglycerate kinase (pgk1) promoters to drive expression of heterologous proteins. All three plasmids use the pdcA terminator for transcription termination, the pyrG gene for restoration of uracil prototrophy, and an ampicillin resistance gene and origin of replication for maintenance in Escherichia coli. We have expressed green fluorescent protein (GFP) and compared transcription and protein accumulation for each of the expression vectors. Accumulation of GFP transcript and protein was directly correlated with the choice of promoter with pdcA > amyA > pgk1. Transcript level appears to parallel GFP protein accumulation. Plasmid copy number had little impact on transcription or protein accumulation. These vectors should be useful for overexpression of heterologous proteins and potentially, metabolic engineering of Rhizopus strains.

Keywords: Rhizopus oryzae ; Green fluorescent protein; Heterologous protein expression; Gene copy number; RT-PCR

Characterization of genes involved in fructose utilization by Lactobacillus fermentum by Miia Helanto; Johannes Aarnikunnas; Airi Palva; Matti Leisola; Antti Nyyssölä (pp. 51-59).

The genes encoding phosphoglucose isomerase (fruI) and fructokinase (fruK) of Lactobacillus fermentum NRRL-B-1932 were sequenced. They constituted an operon, which is involved in fructose metabolism of this strain by channeling intracellular fructose into the phosphoketolase pathway. A third open reading frame, unkR, upstream of the operon was identified as homologous to genes of LacI/GalR family repressors. The UnkR repressor’s role in transcriptional control of the fruIK operon could, however, not be established by electrophoretic mobility shift assay (EMSA) analysis. Sequence analysis revealed two putative catabolite responsive elements (cre) in the promoter region of fruIK suggesting that the fruIK operon is under negative regulatory control by carbon catabolite repression. Expression and enzyme activity data were compatible with the assumption that the fruIK operon is represessed by glucose. No sugar specific phosphoenolpyruvate sugar transferase system activity for the transport of fructose, glucose, sucrose or mannose could be detected in L. fermentum NRRL-B-1932 cells, which suggest that fructose is taken up by a permease system.

Keywords: Lactic acid bacteria; Lactobacillus ; Fructose metabolism; Fructokinase; Phosphoglucose isomerase; Carbon catabolite repression; Mannitol

N-Acetyltaurine dissimilated via taurine by Delftia acidovorans NAT by Jutta Mayer; Karin Denger; Theo H. M. Smits; Klaus Hollemeyer; Ulrich Groth; Alasdair M. Cook (pp. 61-67).

The naturally occurring sulfonate N-acetyltaurine was synthesized chemically and its identity was confirmed. Aerobic enrichment cultures for bacteria able to utilize N-acetyltaurine as sole source of fixed nitrogen or as sole source of carbon were successful. One representative isolate, strain NAT, which was identified as a strain of Delftia acidovorans, grew with N-acetyltaurine as carbon source and excreted stoichiometric amounts of sulfate and ammonium. Inducible enzyme activities were measured in crude extracts of this organism to elucidate the degradative pathway. Cleavage of N-acetyltaurine by a highly active amidase yielded acetate and taurine. The latter was oxidatively deaminated by taurine dehydrogenase to ammonium and sulfoacetaldehyde. This key intermediate of sulfonate catabolism was desulfonated by the known reaction of sulfoacetaldehyde acetyltransferase to sulfite and acetyl phosphate, which was further degraded to enter central metabolism. A degradative pathway including transport functions is proposed.

Keywords: Sulfoacetaldehyde acetyltransferase; Xsc; Amidase; Desulfonation

Characterization of Listeria monocytogenes protein Lmo0327 with murein hydrolase activity by Magdalena Popowska; Zdzislaw Markiewicz (pp. 69-86).

Listeria monocytogenes is an ubiquitous gram-positive, opportunistic food-borne human and animal pathogen. To date, five L. monocytogenes autolysins have been characterized: p60, p45, Ami, MurA and Auto and the preliminary results of our studies show that FlaA, a flagellar protein of L. monocytogenes, also has murein-degrading activity. In this study, a gene coding a 144 kDa protein (Lmo0327) with murein hydrolase activity was identified from a lambda Zap expression library of L. monocytogenes EGD genomic DNA, using a direct screening protocol involving the plating of infected Escherichia coli XL1-blue MRF′ cells onto medium containing Bacillus subtilis murein, a substrate for autolytic proteins. Protein Lmo0327 has a signal sequence, a N-terminal LRR domain and a C-terminal wall-anchoring LPXTG motif. In order to examine the roles of this enzyme and the putative transcription regulator coded by gene lmo0326 located upstream of lmo0327, both structural genes were insertionally inactivated by site-specific integration of a temperature-sensitive plasmid. We show that Lmo0327 is a surface protein covalently linked to murein and that the putative transcription regulator Lmo0326 can be assumed to positively regulate the expression of gene lmo0327. The enzyme, which we have shown to have murein-hydrolysing activity, plays a role in cell separation and murein turnover.

Keywords: Listeria monocytogenes ; Murein; Muramidases; Autolysin

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Archives of Microbiology (v.186, #1)