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Archives of Microbiology (v.182, #6)

Different molecular rearrangements in the integron of the IncP-1β resistance plasmid pB10 isolated from a wastewater treatment plant result in elevated β-lactam resistance levels by Rafael Szczepanowski; Irene Krahn; Alfred Pühler; Andreas Schlüter (pp. 429-435).

The multiresistance IncP-1β plasmid pB10 conferring resistance to ampicillin, streptomycin, sulfonamides, tetracycline and mercury ions was previously obtained from activated sludge bacteria by applying the exogenous isolation method with Pseudomonas sp. strain GFP2 as recipient. A pB10 derivative, designated pB10-1, occurred spontaneously and displays an extended NotI restriction fragment. From the pB10 nucleotide sequence, it is known that the corresponding NotI fragment of this plasmid contains a complete class 1 integron with an oxa2 and an orfE-like gene cassette. Sequencing of the integron-specific variable region present on pB10-1 revealed that a second copy of the oxa2 gene cassette has inserted downstream of the orfE-like cassette. Sequences flanking the second oxa2 cassette indicate that this cassette was excised from pB10 and reinserted at a new site in an integrase-catalyzed manner. Duplication of the oxa2 cassette is associated with a higher level of ampicillin resistance. Another pB10 derivative, designated pB10-2, conferring higher resistance to ampicillin, was shown to carry an IS10 insertion upstream of the oxa2 cassette. Since IS10 possesses a promoter-out activity, it can be assumed that the elevated ampicillin resistance level is due to enhanced transcription of the β-lactamase gene.

Keywords: β-Lactam resistance; Broad-host-range plasmid; Conjugative plasmid; Mobile genetic element; Site-specific recombination

Complementation studies of the DnaK–DnaJ–GrpE chaperone machineries from Vibrio harveyi and Escherichia coli, both in vivo and in vitro by Michał A. Żmijewski; Joanna M. Kwiatkowska; Barbara Lipińska (pp. 436-449).

The marine bacterium Vibrio harveyi is a potential indicator organism for evaluating marine environmental pollution. The DnaK–DnaJ–GrpE chaperone machinery of V. harveyi has been studied as a model of response to stress conditions and compared to the Escherichia coli DnaK system. The genes encoding DnaK, DnaJ and GrpE of V. harveyi were cloned into expression vectors and grpE was sequenced. It was found that V. harveyi possesses a unique organization of the hsp gene cluster (grpE–gltP–dnaK–dnaJ), which is present exclusively in marine Vibrio species. In vivo experiments showed that suppression of the E. coli dnaK mutation by V. harveyi DnaK protein was weak or absent, while suppression of the dnaJ and grpE mutations by V. harveyi DnaJ and GrpE proteins was efficient. These results suggest higher species-specificity of the DnaK chaperone than the GrpE and DnaJ cochaperones. Proteins of the DnaK chaperone machinery of V. harveyi were purified to homogeneity and their efficient cooperation with the E. coli chaperones in the luciferase refolding reaction and in stimulation of DnaK ATPase activity was demonstrated. Compared to the E. coli system, the purified DnaK–DnaJ–GrpE system of V. harveyi exhibited about 20% lower chaperoning activity in the luciferase reactivation assay. ATPase activity of V. harveyi DnaK protein was at least twofold higher than that of the E. coli model DnaK but its stimulation by the cochaperones DnaJ and GrpE was significantly (10 times) weaker. These results indicate that, despite their high structural identity (approximately 80%) and similar mechanisms of action, the DnaK chaperones of closely related V. harveyi and E.coli bacteria differ functionally.

Keywords: Vibrio harveyiDnaK chaperone system; DnaJ; GrpE; gltP; Luciferase refolding; DnaK ATPase activity ; Chaperone species-specificity

Identification of a region involved in the pheromone receptor function of the histidine kinase PlnB by Ola Johnsborg; Linda H. Godager; Ingolf F. Nes (pp. 450-457).

Bacteriocin biosynthesis in Lactobacillus plantarum is an inducible process, triggered by the secreted inducer peptide pheromone IP-C11. The environmental concentration of IP-C11 is monitored by the membrane-bound histidine protein kinase PlnB, which is part of a two-component signal transduction pathway. Upon interaction with IP-C11, PlnB phosphorylates the cognate response regulator PlnC. This regulator subsequently activates transcription of the bacteriocin genes. PlnB belongs to the HPK₁₀ subfamily of peptide-pheromone-activated histidine kinases. All members of this subfamily have an unusual polytopic membrane domain that previously has been shown to contain the peptide pheromone receptor. Employing an in vivo reporter assay, the present work investigated the receptor functionality of various mutagenized PlnB membrane domains. The results indicated that important determinants for receptor function locate to the most N-terminal extracytoplasmatic loop of the membrane domain. In addition, this region appears to be involved in the peptide pheromone interaction of ComD, another member of the HPK₁₀ subfamily.

Keywords: Histidine protein kinase; Peptide pheromone; Receptor

Relations and functions of dye-linked formaldehyde dehydrogenase from Hyphomicrobium zavarzinii revealed by sequence determination and analysis by Arnold C. Schwartz; Gaby Gockel; Julia Groß; Bernd Moritz; Helmut E. Meyer (pp. 458-466).

faoA, the gene of the dye-linked NAD(P)-independent quinone-containing formaldehyde dehydrogenase of methylamine-grown Hyphomicrobium zavarzinii strain ZV 580 was sequenced and analyzed together with an apparent promoter region and adjoining genes in a 7.2-kb fragment of hyphomicrobial DNA. The formaldehyde dehydrogenase, identified as a periplasmic enzyme by its signal sequence, is distantly related to the soluble pyrroloquinoline-quinone-dependent glucose dehydrogenase of Acinetobacter calcoaceticus and to other predicted glucose dehydrogenase sequences. The promoter region, containing about 400 nucleotides upstream of faoA, comprised potential binding sites identical or highly similar to known consensus sequences of the sigma factors σ⁷⁰ (housekeeping), σ^H (heat shock), σ^F (flagellar) and σ^N (nitrogen). The complex regulation of the transcription of faoA, which is suggested by this setting and emphasized by a possible heat-shock promoter, supports a hypothesis proposing an auxiliary role of the enzyme in lowering detrimental elevated concentrations of formaldehyde, which might arise in the course of stress or regulatory transitions disturbing balanced C₁ metabolism.

Keywords: C₁ metabolism; Dye-linked formaldehyde dehydrogenase; Dye-linked formaldehyde dehydrogenase-encoding genefaoAFormaldehyde toxicity; Glucose dehydrogenaseHyphomicrobium zavarziniiHyphomicrobium DNA; Promoter consensus sequences; Stress

Global transcription profiles and intracellular pH regulation measured in Bacillus licheniformis upon external pH upshifts by Tina Hornbæk; Mogens Jakobsen; Jens Dynesen; Allan K. Nielsen (pp. 467-474).

For optimization of propagation conditions for an industrially used Bacillus licheniformis, this study examines the effect of transferring cells at the early-stationary growth phase (pH 5.3) to fresh growth medium at pH 5.0–8.0. Intracellular pH (pH_i) was measured on a single-cell level, using fluorescence ratio imaging microscopy after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester. Transcription profiles were determined using a genome DNA microarray. The optimum extracellular pH (pH_ex) value for growth of B. licheniformis was found to be pH 7.0, resulting in the shortest lag phase, highest maximum specific growth rate and maximum biomass formation. An average pH gradient (ΔpH = pH_i − pH_ex) of approx. 1.0 was found in B. licheniformis 15 min after transfer to pH_ex 5.0–8.0. Up-regulation of genes involved in sucrose uptake at pH 7.0 could be related to the optimum growth observed. Transcription profiles indicated that the organism was experiencing phosphate starvation upon transfer to pH 7.0 and pH 8.0. Mechanisms involved in pH_i regulation appeared to include changes in fatty acid synthesis to yield a more rigid cell membrane structure at low pH_ex values and conversion of pyruvate to acetoin instead of acetate for neutralization of low pH_ex values.

The kgmB gene, encoding ribosomal RNA methylase from Streptomyces tenebrarius, is autogenously regulated by Sandra Vajic; Natasa Anastasov; Branka Vasiljevic (pp. 475-481).

The KgmB methylase (the kanamycin–gentamicin resistance methylase from Streptomyces tenebrarius) acts at G-1405 of 16S rRNA within the sequence CGUCA that is also found 6 bp in front of ribosomal binding site of the kgmB gene. The kgmB∷lacZ gene and operon fusions were used in order to test for translational autoregulation of kgmB gene. Overexpression of kgmB either in cis or in trans drastically decreased the level of expression of the fusion protein. However, mutagenesis eliminated any role for the CGUCA sequence in translational autoregulation. Hence, the role of second putative regulatory sequence (CGCCC) that was shown to be involved in regulation of another methylase, Sgm (sisomicin–gentamicin methylase gene from Micromonospora zionensis) was examined. It was shown that the Sgm methylase can also decrease the level of expression of the kgmB∷lacZ fusion protein.

Keywords: rRNA methylaseStreptomyces tenebrariuskgmB autoregulationlacZ gene fusions

Propanol as an end product of threonine fermentation by Peter H. Janssen (pp. 482-486).

Clostridium sp. strain 17cr1 was able to ferment l-threonine to propionate and propanol. Electrons arising in the oxidation of 2-oxobutyrate to propionyl-CoA were apparently used in reductive pathway leading to propanol formation. Part of the propionyl-CoA was used to form propionate in an ATP-forming pathway via a propionate kinase, so that the final ATP yield was 0.5 mol per mol of l-threonine metabolised. Other growth substrates were fermented mainly to acetate and butyrate, and the reductive formation of butyrate, from 2 mol of acetyl-CoA or from crotonate or 3-hydroxybutyrate, was the main route for recycling reduced electron carriers arising during oxidative pathways for most substrates.

Keywords: AnaerobeClostridiumFermentation; Threonine; Propanol

Comparative analysis of the hspA mutant and wild-type Synechocystis sp. strain PCC 6803 under salt stress: evaluation of the role of hspA in salt-stress management by Asadulghani; Koji Nitta; Yasuko Kaneko; Kouji Kojima; Hideya Fukuzawa; Hideo Kosaka; Hitoshi Nakamoto (pp. 487-497).

DNA microarray analysis has previously revealed that hspA, which encodes a small heat-shock protein, is the second most highly expressed gene under salt stress in Synechocystis sp. strain PCC 6803. Consequently, an hspA deletion mutant was studied under various salt stresses in order to identify a potential role of HspA in salt stress management. The mutant had a growth disadvantage under moderate salt stress. It lost the ability to develop tolerance to a lethal salt treatment by a moderate salt pre-treatment when the tolerance was evaluated by cell survival and the level of major soluble proteins, phycocyanins, while the wild-type acquired tolerance. Under various salt stresses, the mutant failed to undergo the ultrastructural changes characteristic of wild-type cells. The mutant, which showed higher survival than the wild-type after a direct shift to lethal salt conditions, accumulated higher levels of groESL1 and groEL2 transcripts and the corresponding proteins, GroES, GroEL1, and GroEL2, suggesting a role for these heat-shock proteins in conferring basal salt tolerance. Under salt stress, heat-shock genes, such as hspA, groEL2, and dnaK2, were transcriptionally induced and greatly stabilized, indicating a transcriptional and post-transcriptional mechanism of acclimation to salt stress involving these heat-shock genes.

Keywords: Synechocystis sp. strain PCC 6803; Cyanobacterium; Heat-shock protein; GroEL; GroES; HspA; DnaK; Salt tolerance

Studies on hydrogenase activity and chlorobenzene respiration in Dehalococcoides sp. strain CBDB1 by Gopalakrishnan Jayachandran; Helmut Görisch; Lorenz Adrian (pp. 498-504).

Hydrogen oxidation and electron transport were studied in the chlorobenzene-utilizing anaerobe Dehalococcoides sp. strain CBDB1. While Cu²⁺ and Hg²⁺ ions irreversibly inhibited hydrogenase activity in intact cells, Ni²⁺ ions inhibited reversibly. About 80% of the initial hydrogenase activity was inactivated within 30 s when the cells were exposed to air. In contrast, hydrogenase was active at a redox potential of +10 mV when this redox potential was established anoxically with a redox indicator. Viologen dyes served both as electron acceptor for hydrogenase and electron donor for the dehalogenase. A menaquinone analogue, 2,3-dimethyl 1,4-naphthoquinone, served neither as electron acceptor for the hydrogenase nor as electron donor for the dehalogenase. In addition, the menaquinone antagonist 2-n-heptyl-4-hydroxyquinoline-N-oxide had no effect on dechlorination catalyzed by cell suspensions or isolated membranes with hydrogen as electron donor, lending further support to the notion that menaquinone is not involved in electron transport. The ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenylhydrazone did not inhibit dechlorination by cell suspensions, indicating that strain CBDB1 does not require reverse electron transport. The ATP-synthase inhibitor N,N′-dicyclohexylcarbodiimide inhibited the dechlorination reaction with cell suspensions; however, the latter effect was partially relieved by the addition of tetrachlorosalicylanilide. 1,2,3,4-Tetrachlorobenzene strongly inhibited dechlorination of other chlorobenzenes by cell suspensions with hydrogen as electron donor, but it did not interfere with either hydrogenase or dehalogenase activity.

Keywords: Dehalococcoides; Chlorobenzenes; Reductive dechlorination; Dehalorespiration; Hydrogenase

Rhizobium leguminosarum methyl-accepting chemotaxis protein genes are down-regulated in the pea nodule by Christopher K. Yost; Kate L. Del Bel; Jürgen Quandt; Michael F. Hynes (pp. 505-513).

Regulation of methyl-accepting chemotaxis protein (MCP) genes of Rhizobium leguminosarum was studied under symbiotic conditions. Transcriptional fusions using both β-galactosidase and β-glucuronidase genes within two different mcp genes demonstrated that mcp expression decreased significantly during nodulation. Immunoblots using an anti-MCP antibody detected MCPs in free-living cells but not in bacteroids. Down-regulation during nodulation was not dependent upon known regulatory proteins involved in induction of expression of genes involved in nitrogen fixation. Environmental conditions found in the bacteroid that may trigger down-regulation were investigated by growing free-living cultures under a variety of growth conditions. Growth under low oxygen concentration or using succinate as a sole carbon source did not lower expression of the mcp gene fusions.

Keywords: Bacteroid; Methyl-accepting chemotaxis proteins; Nodulation; Rhizobia; Chemotaxis; Chemoreceptors; Gene regulation

Degradation pathway of 2-chloroethanol in Pseudomonas stutzeri strain JJ under denitrifying conditions by John A. Dijk; Jan Gerritse; Gosse Schraa; Alfons J. M. Stams (pp. 514-519).

The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected.

Keywords: Pseudomonas stutzeri strain JJ; Chloroethanol; Chloroethanol dehydrogenase; Degradation; Pathway; Denitrifying bacterium; PQQ

Heterologous overexpression of quorum-sensing regulators to study cell-density-dependent phenotypes in a symbiotic plant bacterium Mesorhizobium huakuii by Hui Wang; Zengtao Zhong; Tao Cai; Shunpeng Li; Jun Zhu (pp. 520-525).

Quorum-sensing is widespread among many prokaryotic lineages. In order to investigate quorum regulation in the plant bacterium Mesorhizobium huakuii which produces an N-acyl homoserine lactone (AHL) quorum signal, the Agrobacterium quorum-sensing regulator TraR was heterologously expressed in this bacterium. The resulting strains showed reduced AHL production in the supernatant compared to wild-type, but similar intracellular levels of AHLs were detected, suggesting that M. huakuii AHLs can be bound to intracellular TraR proteins and thus become unavailable for its own quorum systems. M. huakuii overexpressing TraR formed thinner biofilms than the wild-type, suggesting a role played by quorum-sensing in biofilm formation.

Keywords: Quorum-sensingMesorhizobium huakuiiN-acyl homoserine lactones; Biofilm formation

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Archives of Microbiology (v.182, #6)