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Archives of Microbiology (v.186, #6)

Cascade of sigma factors in streptomycetes: identification of a new extracytoplasmic function sigma factor σ^J that is under the control of the stress-response sigma factor σ^H in Streptomyces coelicolor A3(2) by Vladislava Mazurakova; Beatrica Sevcikova; Bronislava Rezuchova; Jan Kormanec (pp. 435-446).

By using the previously established Escherichia coli two-plasmid system, we identified a promoter recognized by the Streptomyces coelicolor A3(2) stress-response sigma factor σ^H. The promoter directed expression of the sigJ gene encoding an extracytoplasmic function (ECF) sigma factor. S1-nuclease mapping using RNA prepared from E. coli containing the two-plasmid system, and S. coelicolor A3(2) from various developmental stages identified an identical transcription start point in both strains, corresponding to the sigJp promoter. The sigJp promoter was induced during sporulation of aerial hyphae. The level of the transcript from sigJp was dramatically reduced in a S. coelicolor A3(2) sigH mutant and unaffected in a sigF mutant. The S. coelicolor A3(2) core RNA polymerase, after complementation with σ^H, was able to recognize the sigJp promoter in vitro. A sigJ mutation had no obvious effect on growth, stress response, differentiation, and production of antibiotics. The results suggested that the S. coelicolor A3(2) sigJ gene is under the control of stress-response σ^H, thus indicating a cascade of sigma factors in Streptomyces stress response and development. Considering the expression of sigJ and its direct dependence upon developmentally-regulated σ^H, we assume that σ^J may play a role in the later stages of development of S. coelicolor A3(2).

Keywords: Differentiation; Gene disruption; Recombinant DNA; RNA polymerase; Sigma factor; Stress response; Streptomyces coelicolor A3(2)

Investigation of the functional properties and regulation of three glutamine synthetase-like genes in Streptomyces coelicolor A3(2) by H. U. Rexer; T. Schäberle; W. Wohlleben; A. Engels (pp. 447-458).

Streptomyces coelicolor A3(2) has three additional glnA-type genes besides the glutamine synthetase genes glnA (encoding GSI) and glnII (encoding GSII). The aim of this work was to characterize their functional properties and regulation. Sequence analyses revealed that GlnA2, GlnA3, and GlnA4 are dissimilar to S. coelicolor GSI and lack highly conserved amino acid residues involved in catalysis. In heterologous expression experiments, glnA2, glnA3, and glnA4, in contrast to glnA and glnII, were not capable of complementing the l-glutamine auxotrophy of an Escherichia coli glnA mutant. The lack of a conserved sequence motif reflecting adenylylation control of enzyme activity suggests that GlnA2, GlnA3, and GlnA4 are not regulated via adenylyltransferase-mediated modification. In DNA-binding assays, the OmpR-like regulator of nitrogen metabolism GlnRII, which interacts with the glnA and glnII promoters, did not bind to the upstream regions of glnA2, glnA3, and glnA4. These findings support the conclusion that glnA2, glnA3, and glnA4 are not directly involved in l-glutamine synthesis and nitrogen assimilation and are not subject to nitrogen control in S. coelicolor. The glnA3 gene product is similar to FluG, which is required for asexual sporulation in Aspergillus nidulans. However, inactivation of glnA3 does not block morphological differentiation in S. coelicolor.

Keywords: Streptomyces coelicolor ; Nitrogen metabolism; Glutamine synthetase

Tetracycline-inducible gene expression in mycobacteria within an animal host using modified Streptomyces tcp830 regulatory elements by S. Moises Hernandez-Abanto; Samuel C. Woolwine; Sanjay K. Jain; W. R. Bishai (pp. 459-464).

Inducible expression systems are powerful tools for studying gene function. Though several inducible expression systems are now available for mycobacteria, none have been used to modulate bacterial gene expression during an animal infection. A tetracycline-inducible expression system from Streptomyces coelicolor was successfully adapted for use in mycobacteria. To prevent baseline expression without induction, S. coelicolor tetR gene was overexpressed using the acetamidase promoter and regulatory gene block. Target gene expression was controlled by the S. coelicolor tcp830 promoter and operator allele. The −10 promoter consensus sequence of the tcp830 promoter was modified to better resemble known strong mycobacterial promoters. Using this system, induction of tetR fully repressed tcp830-dependent expression of green fluorescent protein (GFP) to baseline levels. Addition of anhydrotetracycline led to a 62-fold induction of GFP expression in vitro and 15-fold induction in a mouse mycobacterial peritonitis model in the presence of maximal tetR expression. Chemically regulatable gene expression during animal infection may be a useful tool in studying mycobacterial pathogenesis.

Keywords: Anhydrotetracycline; Acetamide; Mycobacteria

Pheromone-induced expression of recombinant proteins in Streptococcus thermophilus by Trinelise Blomqvist; Hilde Steinmoen; Leiv Sigve Håvarstein (pp. 465-473).

A locus encoding proteins with high homology to the pneumococcal BlpABCHR quorum-sensing system was identified in Streptococcus thermophilus LMG 18311. The BlpABCHR system regulates bacteriocin production in Streptococcus pneumoniae by monitoring the extracellular concentration of a peptide-pheromone encoded by blpC. The homologous system in S. thermophilus, termed StbABCHR, contains a corresponding gene (stbC) encoding a possible peptide-pheromone (STP) that presumably controls bacteriocin production in S. thermophilus. We synthesized this peptide and found that it activates transcription of a gusA reporter gene placed behind the promoter of the bacteriocin-like gene stbD. Furthermore, deletion mapping and mutational analysis of the stbD promoter region were used to identify a degenerated direct repeat motif required for STP induced GusA expression. Our findings provide strong evidence that STP regulates bacteriocin production in S. thermophilus LMG 18311, and show that the StbABCHR quorum-sensing system can be exploited for inducible expression of recombinant proteins in this bacterial species.

Keywords: Streptococcus thermophilus ; Inducible expression of proteins; Peptide pheromone; Bacteriocin

The lipopeptides mycosubtilin and surfactin enhance spreading of Bacillus subtilis strains by their surface-active properties by Valérie Leclère; Romain Marti; Max Béchet; Patrick Fickers; Philippe Jacques (pp. 475-483).

The colonizing behaviour and the pellicle formation of Bacillus subtilis strains producing different families of lipopeptides were evaluated under several cultural conditions. The pattern of lipopeptides produced determined the architecture of the colony on a swarming medium as well as the flotation and the thickness of the pellicle formed at the air/liquid interface. The overproduction of mycosubtilin, a lipopeptide of the iturin family, led to increased spreading but had no effect on pellicle formation. A physico-chemical approach was developed to gain an insight into the mode of action of the biosurfactants facilitating the colonization. A relationship between surface tension of the culture medium and spreading of a lipopeptide non-producing strain, B. subtilis 168, was established. Goniometry was used to highlight the modification of the in situ wettability in the area where spreading was enhanced. On a solid medium, co-cultures of a surfactin producing with other strains showed a diffusion ring of the surfactin around the colony. This ring characterized by a higher wettability favoured the propagation of other colonies.

Keywords: Bacillus subtilis ; Spreading; Swarming; Surfactin; Mycosubtilin; Surface tension; Wettability

Relationships among the biosyntheses of ubiquinone, carotene, sterols, and triacylglycerols in Zygomycetes by Vera Kuzina; Carlos Domenech; Enrique Cerdá-Olmedo (pp. 485-493).

The Zygomycetes Phycomyces blakesleeanus and Blakeslea trispora are actual or potential sources of β-carotene, ergosterol, ubiquinone, edible oil, and other compounds. By feeding [¹⁴C]acetyl-CoA, L-[¹⁴C]leucine, or R-[¹⁴C]mevalonate in the presence of excess unlabeled glucose, we found that ubiquinone (the terpenoid moiety), β-carotene, and triacylglycerols were made from separate pools of all their common intermediates; the pools for ubiquinone and ergosterol were indistinguishable. Fatty acids were not labeled from mevalonate, showing the absence in these fungi of a shunt pathway that would recycle carbon from mevalonate and its products back to central metabolism. The overproduction of carotene in a Phycomyces mutant and in sexually mated cultures of Blakeslea modified the relative use of labeled and unlabeled carbon sources in the production of carotene, but not of the other compounds. We concluded that carotene, ubiquinone, and triacylglycerols are synthesized in separate subcellular compartments, while sterols and ubiquinone are synthesized in the same compartments or in compartments that exchange precursors. Carotene biosynthesis was regulated specifically and not by flow diversion in a branched pathway.

Keywords: Terpenoid pathways; Subcellular compartments; Ubiquinone; Carotene; Ergosterol; Triacylglycerols; Phycomyces ; Blakeslea

Identification of glycine betaine as compatible solute in Synechococcus sp. WH8102 and characterization of its N-methyltransferase genes involved in betaine synthesis by Wei-Dong Lu; Zhen-Ming Chi; Chuan-Dong Su (pp. 495-506).

Biosynthesis of glycine betaine from simple carbon sources as compatible solute is rare among aerobic heterotrophic eubacteria, and appears to be almost exclusive to the non-halophilic and slightly halophilic phototrophic cyanobacteria. Although Synechococcus sp. WH8102 (CCMP2370), a unicellular marine cyanobacterium, could grow up to additional 2.5% (w/v) NaCl in SN medium, natural abundance ¹³C nuclear magnetic resonance spectroscopy identified glycine betaine as its major compatible solute. Intracellular glycine betaine concentrations were dependent on the osmolarity of the growth medium over the range up to additional 2% NaCl in SN medium, increasing from 6.8 ± 1.5 to 62.3 ± 5.5 mg/g dw. The ORFs SYNW1914 and SYNW1913 from Synechococcus sp. WH8102 were found as the homologous genes coding for glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase, heterologously over-expressed respectively as soluble fraction in Escherichia coli BL21(DE3)pLysS and purified by Ni-NTA His•bind resins. Their substrate specificities and the values of the kinetic parameters were determined by TLC and ¹H NMR spectroscopy. RT-PCR analysis revealed that the two ORFs were both transcribed in cells of Synechococcus sp. WH8102 growing in SN medium without additional NaCl, which confirmed the pathway of de novo synthesizing betaine from glycine existing in these marine cyanobacteria.

Keywords: Synechococcus sp.; WH8102; Glycine betaine; Glycine sarcosine N-methyltransferase; Dimethylglycine N-methyltransferase

Evaluation of TatABC overproduction on Tat- and Sec-dependent protein secretion in Streptomyces lividans by Sophie De Keersmaeker; Kristof Vrancken; Lieve Van Mellaert; Elke Lammertyn; Jozef Anné; Nick Geukens (pp. 507-512).

The majority of bacterial proteins are exported across the cytoplasmic membrane via the Sec pathway, but also the more recently discovered twin-arginine translocation (Tat) route seems to play an important role for protein secretion in Streptomyces lividans in whose genome tatA, tatB and tatC have been identified. In the present work we showed that simultaneous overproduction of TatABC improved the Tat-dependent secretion capacity as could be concluded from the increased amount of secreted xylanase C, an exclusive Tat-dependent substrate. This result demonstrates that next to the availability of energy to drive secretion, also the number of translocases can be rate-limiting for Tat-dependent secretion. On the other hand, tatABC overexpression was found to diminish secretion of the Sec-dependent proteins xylanase B and subtilisin inhibitor in S. lividans. These results reveal cross-talk between both pathways in S. lividans.

Keywords: Streptomyces lividans ; Twin-arginine translocation; Tat

The ntp operon encoding the Na⁺ V-ATPase of the thermophile Caloramator fervidus by Trees Ubbink-Kok; Jeroen Nijland; Dirk-Jan Slotboom; Juke S. Lolkema (pp. 513-517).

The V-type ATPase of the thermophile Caloramator fervidus is an ATP-driven Na⁺ pump. The nucleotide sequence of the ntpFIKECGABD operon containing the structural genes coding for the nine subunits of the enzyme complex was determined. The identity of the proteins in two pairs of subunits (D, E and F, G) that have very similar mobilities on SDS-PAGE of the purified complex (24.3 and 22.7 kDa, and 12.3 and 11.6 kDa) was established by tryptic digestion of the protein bands followed by mass spectrometric analysis of the peptides.

Keywords: ntp Operon; V-ATPase; Thermophile; Caloramator fervidus ; Molecular motor; Central stalk

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Archives of Microbiology (v.186, #6)