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Archives of Microbiology (v.191, #7)

Transcriptional regulation and structural modeling of the FutC subunit of an ABC-type iron transporter in Synechocystis sp. strain PCC 6803 by Anna-Maria Brandt; Wuttinun Raksajit; Paula Mulo; Aran Incharoensakdi; Tiina A. Salminen; Pirkko Mäenpää (pp. 561-570).

The futC gene encodes a subunit of an ATP-binding cassette (ABC)-type iron transporter in Synechocystis sp. strain PCC 6803. In the present study, we have focused on the environmental regulation of futC transcription in the model organism Synechocystis sp. strain PCC 6803 and, moreover, studied the transcriptional regulation of the other transporter subunits, futA1, futA2 and futB. The steady-state amounts of the futA1, futA2, futB and futC transcripts were regulated under several conditions studied including darkness, temperature, alternative nitrogen source, salt and osmotic stresses and iron deficiency. Transcription of all subunits of the FutABC-iron transporter seems to be under similar regulation, which, according to our results, may also apply to genes encoding subunits of other transporters in Synechocystis. The sequence alignment, including sequences from six different organisms, revealed the conserved nature of FutC. Based on the sequence alignment and the structural model of FutC, the monomer consists of a nucleotide-binding domain (NBD) and a regulatory domain. The NBD is well conserved indicating completely functional ATP binding.

Keywords: futC ; Iron transport; Transcriptional regulation; Homology model; Synechocystis sp. strain PCC 6803

Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants by M. Victoria Delpino; Diego J. Comerci; Mary Ann Wagner; Michel Eschenbrenner; Cesar V. Mujer; Rodolfo A. Ugalde; Carlos A. Fossati; Pablo C. Baldi; Vito G. DelVecchio (pp. 571-581).

The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis–trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.

Keywords: Brucella ; Extracellular proteins; Type IV secretion system

Deletion analysis of the C-terminal region of a molecular chaperone DnaK from Bacillus licheniformis by Wan-Chi Liang; Min-Guan Lin; Meng-Chun Chi; Hui-Yu Hu; Huei-Fen Lo; Hui-Ping Chang; Long-Liu Lin (pp. 583-593).

Bacillus licheniformis DnaK (BlDnaK) is predicted to consist of a 45-kDa N-terminal ATPase domain and a 25-kDa C-terminal substrate-binding domain. In this study, the full-length BlDnaK and its T86W and three C-terminally truncated mutants were constructed to evaluate the role of up to C-terminal 255 amino acids of the protein. The steady-state ATPase activity for BlDnaK, T86W, T86W/ΔC120, T86W/ΔC249, and T86W/ΔC255 was 65.68, 53.21, 116.04, 321.38, and 90.59 nmol Pi/min per mg, respectively. In vivo, BldnaK, T86W and T86W/ΔC120 genes allowed an E. coli dnaK756-ts mutant to grow at 44°C. Except for T86W/ΔC255, simultaneous addition of B. licheniformis DnaJ and GrpE, and NR-peptide synergistically stimulated the ATPase activity of BlDnaK, T86W, T86W/ΔC120, and T86W/ΔC249 by 16.9-, 13.9-, 33.9-, 9.9-fold, respectively. Measurement of intrinsic tryptophan fluorescence revealed significant alterations of microenvironment of aromatic amino acids in the C-terminally truncated mutants. The temperature-dependent signal in the far-UV region for T86W was consistent with that of BlDnaK, but the C-terminally truncated mutant proteins showed a higher sensitivity toward temperature-induced denaturation. These results suggest that C-terminal truncations alter the ATPase activity and thermal stability of BlDnaK and induce the conformation change of the ATPase domain.

Keywords: Bacillus licheniformis ; DnaK; ATPase activity; Truncational mutants; DnaJ; GrpE

Involvement of digalactosyldiacylglycerol in cellular thermotolerance in Synechocystis sp. PCC 6803 by Naoki Mizusawa; Shinya Sakata; Isamu Sakurai; Naoki Sato; Hajime Wada (pp. 595-601).

The effects of digalactosyldiacylglycerol (DGDG) deficiency on photosynthesis at high temperatures were examined using a dgdA mutant of Synechocystis sp. PCC 6803 incapable of DGDG biosynthesis. The dgdA mutant cells showed significant growth retardation when the temperature was increased from 30 to 38°C, although wild-type cells grew normally. The degree of growth retardation was enhanced by increasing light intensity. In addition, dgdA mutant cells showed increased sensitivity to the photoinhibition of photosynthesis when illuminated at 38°C. Analysis of photosynthesis in intact cells suggested that the inhibition of repair processes and accelerated photodamage resulted in growth retardation in dgdA mutant cells at high temperatures.

Keywords: Digalactosyldiacylglycerol; High-temperature stress; Photoinhibition; Photosynthesis; Photosystem II

Functional analysis of a putative regulatory gene, tadR, involved in aniline degradation in Delftia tsuruhatensis AD9 by Lizhao Geng; Ming Chen; Quanfeng Liang; Wei Liu; Wei Zhang; Shuzhen Ping; Wei Lu; Yongliang Yan; Weiwei Wang; Masahiro Takeo; Min Lin (pp. 603-614).

Delftia tsuruhatensis AD9 contains the chromosomally encoded tad gene cluster responsible for the complete metabolism of aniline to TCA cycle intermediates. The tadQTA1A2B genes encode a multi-component aniline dioxygenase, the first enzyme of aniline metabolism, and the tadR gene directly downstream of this gene cluster encodes a putative LysR-type regulatory protein. Inactivation of tadR resulted in the inability to degrade aniline and to grow on aniline. Transcriptional assays using a tadQ promoter (P tadQ)–lacZ fusion revealed that the transcriptional activation of tadQ from P tadQ was dependent on the presence of tadR and aniline, suggesting that tadR encodes a positive regulatory protein for the expression of at least six genes. Induction experiments using the same P tadQ–lacZ fusion showed that, of the 22 chemical compounds, aniline and monochloroanilines activated transcription from P tadQ in wild-type AD9. Sequential deletions of a 1,003-bp region just upstream of tadQ showed that a 148-bp segment upstream of the transcription start site of tadQ, containing one inverted repeat named IR6, was essential for the transcriptional activation of tadQ. Moreover, gel shift assay confirmed the binding of the gene product to the tadQ promoter region. These results clarified the outline of the regulatory mechanism for aniline degradation in AD9.

Keywords: Aniline; Gene regulation; Delftia ; LysR-type regulator; Promoter

Gene-specific disruption in the filamentous fungus Cercospora nicotianae using a split-marker approach by Bang-Jau You; Miin-Huey Lee; Kuang-Ren Chung (pp. 615-622).

To determine if DNA configuration, gene locus, and flanking sequences will affect homologous recombination in the phytopathogenic fungus Cercospora nicotianae, we evaluated and compared disruption efficiency targeting four cercosporin toxin biosynthetic genes encoding a polyketide synthase (CTB1), a monooxygenase/O-methyltransferase (CTB3), a NADPH-dependent oxidoreductase (CTB5), and a FAD/FMN-dependent oxidoreductase (CTB7). Transformation of C. nicotianae using a circular plasmid resulted in low disruption frequency. The use of endonucleases or a selectable marker DNA fragment flanked by homologous sequence either at one end or at both ends in the transformation procedures, increased disruption efficiency in some but not all CTB genes. A split-marker approach, using two DNA fragments overlapping within the selectable marker, increased the frequency of targeted gene disruption and homologous integration as high as 50%, depending on the target gene and on the length of homologous DNA sequence flanking the selectable marker. The results indicate that the split-marker approach favorably decreased ectopic integration and thus, greatly facilitated targeted gene disruption in this important fungal pathogen.

Keywords: Gene replacement; Filamentous fungi; Pathogenicity; Plant pathogen; Recombination; Split marker; Toxins; Virulence

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Archives of Microbiology (v.191, #7)

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Archives of Microbiology (v.191, #7)