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Archives of Microbiology (v.187, #6)

Kinetics of plasmid transfer among Bacillus cereus group strains within lepidopteran larvae by Y. M. Yuan; X. M. Hu; H. Z. Liu; Bjarne Munk Hansen; J. P. Yan; Z. M. Yuan (pp. 425-431).

The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44–56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 × 10⁻⁶ CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.

Keywords: Bacillus thuringiensis ; Bacillus cereus ; Conjugation; Insect larvae; Plasmid

Regulation of the chitobiose–phosphotransferase system in Vibrio cholerae by Thorsten Berg; Stefan Schild; Joachim Reidl (pp. 433-439).

Vibrio cholerae harbours a phosphotransferase system (PTS) enabling the organism to utilise chitosan oligosaccharide, e.g. derived from deacetylated chitin. As shown recently, this utilization system is encoded by the ORFs VC1281–1283 (Meibom et al. in Proc Natl Acad Sci USA, 101:2524–2529, 2004). By using a transcriptional reporter fusion technique, we identified the regulator of the system and characterised gene expression. Furthermore, we found that gene expression of this PTS system is influenced by catabolite regulation and also by an Mlc homologue (VC2007), which in E. coli is a global regulator of sugar metabolism.

Keywords: Chitosan; Chitobiose; Vibrio cholerae ; chs operon

Denitrification in a binary culture and thiocyanate metabolism in Thiohalophilus thiocyanoxidans gen. nov. sp. nov. – a moderately halophilic chemolithoautotrophic sulfur-oxidizing Gammaproteobacterium from hypersaline lakes by Dimitry Y. Sorokin; Tatjana P. Tourova; Ekatherina Y. Bezsoudnova; Arjan Pol; Gerard Muyzer (pp. 441-450).

Anaerobic enrichment culture with thiocyanate as electron donor and nitrate as electron acceptor at 2 M NaCl inoculated with a mixture of sediments from hypersaline lakes in Kulunda Steppe (Altai, Russia) resulted in a selection of a binary consortium of moderately halophilic, obligately chemolithoautotrophic sulfur-oxidizing bacteria (SOB) capable of complete denitrification of nitrate with thiosulfate as the electron donor. One consortium member, strain HRhD 3sp, was isolated into pure culture with nitrate and thiosulfate using a density gradient. This strain was responsible for the reduction of nitrate to nitrite in the consortium, while a second strain, HRhD 2, isolated under microoxic conditions with thiosulfate as substrate, was capable of anaerobic growth with nitrite and thiosulfate. Nitrite, either as substrate or as product, was already toxic at very low concentrations for both strains. As a result, optimal growth under anaerobic conditions could only be achieved within the consortium. On the basis of phylogenetic analysis, both organisms were identified as new lineages within the Gammaproteobacteria. As well as thiosulfate, strain HRhD 2 can also use thiocyanate as electron donor, representing a first halophilic SOB capable of growth with thiocyanate at 2–4 M NaCl. Product and enzymatic analysis identified the “carbonyl sulfide (COS) pathway” of primary thiocyanate degradation in this new species. On the basis of phenotypic and genetic analysis, strain HRhD 2 is proposed to be assigned to a new genus and species Thiohalophilus thiocyanoxidans.

Keywords: Halophilic sulfur-oxidizing bacteria (SOB); Hypersaline lakes; Thiodenitrification; Thiocyanate; Thiocyanate hydrolase

N terminus determinants of MinC from Neisseria gonorrhoeae mediate interaction with FtsZ but do not affect interaction with MinD or homodimerization by V. Greco-Stewart; S. Ramirez-Arcos; M. Liao; J. R. Dillon (pp. 451-458).

While bacterial cell division has been widely studied in rod-shaped bacteria, the mechanism of cell division in round (coccal) bacteria remains largely enigmatic. In the present study, interaction between the cell division inhibitor MinC from Neisseria gonorrhoeae (MinC_Ng) and the gonococcal cell division proteins MinD_Ng and FtsZ_Ng are demonstrated. Protein truncation and site-directed mutagenic approaches determined which N-terminal residues were essential for cell division inhibition by MinC_Ng using cell morphology as an indicator of protein functionality. Truncation from or mutation at the 13th amino acid of the N terminus of MinC_Ng resulted in loss of protein function. Bioinformatic analyses predicted that point mutations of L35P and L68P would affect the α-helical conformation of the protein and we experimentally showed that these mutations alter the functionality of MinC_Ng. The bacterial two-hybrid system showed that interaction of MinC_Ng with FtsZ_Ng is abrogated upon truncation of 13 N-terminal residues while MinC_Ng-MinD_Ng interaction or MinC_Ng homodimerization is unaffected. These data confirm interactions among gonococcal cell division proteins and determine the necessity of the 13th amino acid for MinC_Ng function.

Keywords: Neisseria gonorrhoeae ; Cell division; MinC; MinD; FtsZ; Bacterial two-hybrid system

Identification of the bkdAB gene cluster, a plausible source of the starter-unit for virginiamycin M production in Streptomyces virginiae by Nattika Pulsawat; Shigeru Kitani; Hiroshi Kinoshita; Chang Kwon Lee; Takuya Nihira (pp. 459-466).

The bkdAB gene cluster, which encodes plausible E1 and E2 components of the branched-chain α-keto acid dehydrogenase (BCDH) complex, was isolated from Streptomyces virginiae in the vicinity of a regulatory island for virginiamycin production. Gene disruption of bkdA completely abolished the production of virginiamycin M (a polyketide-peptide antibiotic), while the production of virginiamycin S (a cyclodepsipeptide antibiotic) was unaffected. Complementation of the bkdA disruptant by genome-integration of intact bkdA completely restored the virginiamycin M production, indicating that the bkdAB cluster is essential for virginiamycin M biosynthesis, plausibly via the provision of isobutyryl-CoA as a primer unit. In contrast to a feature usually seen in the Streptomyces E1 component, namely, the separate encoding of the α and β subunits, S. virginiae bkdA seemed to encode the fused form of the α and β subunits, which was verified by the actual catalytic activity of the fused protein in vitro using recombinant BkdA overexpressed in Escherichia coli. Supply of an additional bkdA gene under the strong and constitutive promoter ermE* in the wild-type strain of S. virginiae resulted in enhanced production of virginiamycin M, suggesting that the supply of isobutyryl-CoA is one of the rate-limiting factors in the biosynthesis of virginiamycin M.

Keywords: Virginiamycin; Streptomyces virginiae ; Branched-chain α-keto acid dehydrogenase

Membrane cholesterol is required for activity of Vibrio vulnificus cytolysin by Hong-Nu Yu; Young-Rae Lee; Kwang-Hyun Park; So-Young Rah; Eun-Mi Noh; Eun-Kyung Song; Myung-Kwan Han; Byeong-Soo Kim; Sung-Ho Lee; Jong-Suk Kim (pp. 467-473).

Vibrio vulnificus cytolysin (VVC) forms a pore in the plasma membrane and induces cytolysis of various cells including erythrocytes, neutrophil and endothelial cells. The cytolytic activity of VVC is inhibited by exogenously added cholesterol, suggesting that membrane cholesterol might be required for VVC cytolytic activity. However, there is no direct evidence that membrane cholesterol is involved in VVC-induced cytolysis. Herein we demonstrate that membrane cholesterol is required for binding of VVC to the plasma membrane. Membrane cholesterol depletion with methyl-β-cyclodextrin inhibited VVC-induced K⁺ release, 2-deoxy glucose release and Ca²⁺ influx, which are indicators of VVC pore formation. The cholesterol depletion-induced blockage of VVC cytolysis was due to the inhibition of VVC binding to membrane. These findings suggest that interaction with cholesterol is required for activity of VVC.

Keywords: V . vulnificus cytolysin; Cholesterol; Methyl-β-cyclodextrin

A new monocupin quercetinase of Streptomyces sp. FLA: identification and heterologous expression of the queD gene and activity of the recombinant enzyme towards different flavonols by Hedda Merkens; Sonja Sielker; Karsten Rose; Susanne Fetzner (pp. 475-487).

The gene queD encoding quercetinase of Streptomyces sp. FLA, a soil isolate related to S. eurythermus ^T, was identified. Quercetinases catalyze the 2,4-dioxygenolytic cleavage of 3,5,7,3′,4′-pentahydroxyflavone to 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide. The queD gene was expressed in S. lividans and E. coli, and the recombinant hexahistidine-tagged protein (QueDHis₆) was purified. Several flavonols were converted by QueDHis₆, whereas CO formation from the 2,3-dihydroflavonol taxifolin and the flavone luteolin were not observed. In contrast to bicupin quercetinases from Aspergillus japonicus and Bacillus subtilis, and bicupin pirins showing quercetinase activity, QueD of strain FLA is a monocupin exhibiting 35.9% sequence identity to the C-terminal domain of B. subtilis quercetinase. Its native molecular mass of 63 kDa suggests a multimeric protein. A queD-specific probe hybridized with fragments of genomic DNA of four other quercetin degrading Streptomyces strains, but not with DNA of B. subtilis. Potential ORFs upstream of queD probably code for a serine protease and an endoribonuclease; two ORFs downstream of queD may encode an amidohydrolase and a carboxylesterase. This arrangement suggests that queD is not part of a catabolic gene cluster. Quercetinases might play a major role as detoxifying rather than catabolic enzymes.

Keywords: Flavonoid; Flavonol; Quercetin; Quercetinase; Dioxygenase; Monocupin; Pirin; Polyphenol; Degradation; Streptomyces

Hoc protein regulates the biological effects of T4 phage in mammals by Krystyna Dabrowska; Maria Zembala; Janusz Boratynski; Kinga Switala-Jelen; Joanna Wietrzyk; Adam Opolski; Katarzyna Szczaurska; Marek Kujawa; Joanna Godlewska; Andrzej Gorski (pp. 489-498).

We previously investigated the biological, non-antibacterial effects of bacteriophage T4 in mammals (binding to cancer cells in vitro and attenuating tumour growth and metastases in vivo); we selected the phage mutant HAP1 that was significantly more effective than T4. In this study we describe a non-sense mutation in the hoc gene that differentiates bacteriophage HAP1 and its parental strain T4. We found no substantial effects of the mutation on the mutant morphology, and its effects on electrophoretic mobility and hydrodynamic size were moderate. Only the high ionic strength of the environment resulted in a size difference of about 10 nm between T4 and HAP1. We compared the antimetastatic activity of the T2 phage, which does not express protein Hoc, with those of T4 and HAP1 (B16 melanoma lung colonies). We found that HAP1 and T2 decreased metastases with equal effect, more strongly than did T4. We also investigated concentrations of T4 and HAP1 in the murine blood, tumour (B16), spleen, liver, or muscle. We found that HAP1 was rapidly cleared from the organism, most probably by the liver. Although HAP1 was previously defined to bind cancer cells more effectively (than T4), its rapid elimination precluded its higher concentration in tumours.

Keywords: Bacteriophage T4; Bacteriophage HAP1; Hoc protein

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium Thermotoga hypogea by Xiangxian Ying; Ying Wang; Hamid R. Badiei; Vassili Karanassios; Kesen Ma (pp. 499-510).

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.

Keywords: Thermotoga hypogea ; Alcohol dehydrogenase; Iron; Extreme thermophile; Anaerobe; Thermostability

Purification, characterization and gene cloning of isoeugenol-degrading enzyme from Pseudomonas putida IE27 by Mamoru Yamada; Yukiyoshi Okada; Toyokazu Yoshida; Toru Nagasawa (pp. 511-517).

An isoeugenol-degrading enzyme was purified to homogeneity from Pseudomonas putida IE27, an isoeugenol-assimilating bacterium. The purified enzyme was a 55 kDa monomer and catalyzed the initial step of isoeugenol degradation, the oxidative cleavage of the side chain double-bond of isoeugenol, to form vanillin. Another reaction product of isoeugenol degradation besides vanillin was identified to be acetaldehyde. The values of Km and k _cat for isoeugenol were 175 μM and 5.18 s^–1, respectively. The purified enzyme catalyzed the incorporation of an oxygen atom from either molecular oxygen or water into vanillin, suggesting that the isoeugenol-degrading enzyme is a kind of monooxygenase. The gene encoding the isoeugenol-degrading enzyme and its flanking regions were isolated from P. putida IE27. The amino acid sequence of the enzyme was similar to those of lignostilbene-α,β-dioxygenases, carotenoid monooxygenases and 9-cis-epoxycarotenoid dioxygenases.

Keywords: Isoeugenol; Vanillin; Pseudomonas putida IE27; Oxygenation; Double-bond cleavage

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Archives of Microbiology (v.187, #6)