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Archives of Microbiology (v.192, #9)

The small RNA Aar in Acinetobacter baylyi: a putative regulator of amino acid metabolism by Dominik Schilling; Sven Findeiß; Andreas S. Richter; Jennifer A. Taylor; Ulrike Gerischer (pp. 691-702).

Small non-coding RNAs (sRNAs) are key players in prokaryotic metabolic circuits, allowing the cell to adapt to changing environmental conditions. Regulatory interference by sRNAs in cellular metabolism is often facilitated by the Sm-like protein Hfq. A search for novel sRNAs in A. baylyi intergenic regions was performed by a biocomputational screening. One candidate, Aar, encoded between trpS and sucD showed Hfq dependency in Northern blot analysis. Aar was expressed strongly during stationary growth phase in minimal medium; in contrast, in complex medium, strongest expression was in the exponential growth phase. Whereas over-expression of Aar in trans did not affect bacterial growth, seven mRNA targets predicted by two in silico approaches were upregulated in stationary growth phase. All seven mRNAs are involved in A. baylyi amino acid metabolism. A putative binding site for Lrp, the global regulator of branched-chain amino acids in E. coli, was observed within the aar gene. Both facts imply an Aar participation in amino acid metabolism.

Keywords: A. baylyi ; Hfq; IntaRNA; RNAup; sRNAs

The quinohaemoprotein alcohol dehydrogenase from Gluconacetobacter xylinus: molecular and catalytic properties by J. L. Chávez-Pacheco; M. Contreras-Zentella; J. Membrillo-Hernández; R. Arreguín-Espinoza; G. Mendoza-Hernández; S. Gómez-Manzo; J. E. Escamilla (pp. 703-713).

Gluconacetobacter xylinus possesses a constitutive membrane-bound oxidase system for the use of ethanol. Its alcohol dehydrogenase complex (ADH) was purified to homogeneity and characterized. It is a 119-kDa heterodimer (68 and 41 kDa subunits). The peroxidase reaction confirmed the presence of haem C in both subunits. Four cytochromes c per enzyme were determined by pyridine hemochrome spectroscopy. Redox titrations of the purified ADH revealed the presence of four haem c redox centers, with apparent mid-point potential values (Em₇) of −33, +55, +132 and +310 mV, respectively. The ADH complex contains one mol of pyrroloquinoline quinone as determined by HPLC. The enzyme was purified in full reduced state; oxidation was induced by potassium ferricyanide and substrate restores full reduction. Activity responses to pH were sharp, showing two distinct optimal pH values (i.e. pH 5.5 and 6.5) depending on the electron acceptor used. Purified ADH oxidizes primary alcohols (C2–C6) but not methanol. Noteworthy, aliphatic aldehydes (C1–C4) were also good substrates. Myxothiazol and antymicin A were powerful inhibitors of the purified ADH complex, most likely acting at the ubiquinone acceptor site in subunit II.

Keywords: Alcohol dehydrogenase; Acetic acid bacteria; Gluconacetobacter xylinus

Environmental and genetic factors that contribute to Escherichia coli K-12 biofilm formation by Birgit M. Prüß; Karan Verma; Priyankar Samanta; Preeti Sule; Sunil Kumar; Jianfei Wu; David Christianson; Shelley M. Horne; Shane J. Stafslien; Alan J. Wolfe; Anne Denton (pp. 715-728).

Biofilms are communities of bacteria whose formation on surfaces requires a large portion of the bacteria’s transcriptional network. To identify environmental conditions and transcriptional regulators that contribute to sensing these conditions, we used a high-throughput approach to monitor biofilm biomass produced by an isogenic set of Escherichia coli K-12 strains grown under combinations of environmental conditions. Of the environmental combinations, growth in tryptic soy broth at 37°C supported the most biofilm production. To analyze the complex relationships between the diverse cell-surface organelles, transcriptional regulators, and metabolic enzymes represented by the tested mutant set, we used a novel vector-item pattern-mining algorithm. The algorithm related biofilm amounts to the functional annotations of each mutated protein. The pattern with the best statistical significance was the gene ontology ‘pyruvate catabolic process,’ which is associated with enzymes of acetate metabolism. Phenotype microarray experiments illustrated that carbon sources that are metabolized to acetyl-coenzyme A, acetyl phosphate, and acetate are particularly supportive of biofilm formation. Scanning electron microscopy revealed structural differences between mutants that lack acetate metabolism enzymes and their parent and confirmed the quantitative differences. We conclude that acetate metabolism functions as a metabolic sensor, transmitting changes in environmental conditions to biofilm biomass and structure.

Keywords: Biofilm formation; Environmental conditions; Genetic factors; High-throughput experimentation; Vector-item pattern-mining algorithm; Acetate metabolism; Two-component signaling

Differential regulation of groESL operon expression in response to heat and light in Anabaena by Hema Rajaram; Shree Kumar Apte (pp. 729-738).

The HrcA protein is known to bind the cis-element CIRCE and repress expression of hsp60 in certain bacteria. However, recent data from cyanobacteria have seriously questioned the HrcA/CIRCE interaction paradigm. A hrcA null mutant showed constitutive expression of Hsp60 proteins [GroEL/Cpn60(GroEL2)], and an unexpected further increase in GroEL during temperature upshift, suggesting involvement of regulatory mechanisms other than HrcA in groESL expression in Anabaena. The negative regulation of both hsp60 genes [groEL and cpn60 (groEL2)] at CIRCE element was established by: (1) constitutive expression of Green Fluorescent Protein gene, tagged to Anabaena hsp60 promoters, in E. coli, and its repression upon co-expression of Anabaena HrcA and (2) specific binding of Anabaena HrcA to the CIRCE element. Deletion analysis of other cis-elements further distinguished (a) a photo-regulation by the K-box and (b) thermoregulation from a novel H-box, over and above the negative regulation by HrcA at CIRCE.

Keywords: Heat-regulation; HrcA; CIRCE; groESL ; cpn60 ; Anabaena

Identification and biochemical characterization of a unique Mn²⁺-dependent UMP kinase from Helicobacter pylori by Mon-Juan Lee; Liu Chien-Liang; Ju-Ying Tsai; Wae-Ting Sue; Wan-Shun Hsia; Haimei Huang (pp. 739-746).

Uridine monophosphate (UMP) kinase converts UMP to the corresponding UDP in the presence of metal ions and ATP and is allosterically regulated by nucleotides such as UTP and GTP. Although the UMP kinase reported to date is Mg²⁺-dependent, we found in this study that the UMP kinase of Helicobacter pylori had a preference for Mn²⁺ over Mg²⁺, which may be related to a conformational difference between the Mn²⁺-bound and Mg²⁺-bound UMP kinase. Similar to previous findings, the UMP kinase activity of H. pylori UMP kinase was inhibited by UTP and activated by GTP. However, a relatively low GTP concentration (0.125 mM) was required to activate H. pylori UMP kinase to a level similar to other bacterial UMP kinases using a higher GTP concentration (0.5 mM). In addition, depending on the presence of either Mg²⁺ or Mn²⁺, a significant difference in the level of GTP activation was observed. It is therefore hypothesized that the Mg²⁺-bound and Mn²⁺-bound H. pylori UMP kinase may be activated by GTP through different mechanisms.

Keywords: Helicobacter pylori ; Uridine monophosphate; UMP kinase; Manganese preference

CspC and CspD are essential for Caulobacter crescentus stationary phase survival by Heloise Balhesteros; Ricardo R. Mazzon; Carolina A. P. T. da Silva; Elza A. S. Lang; Marilis V. Marques (pp. 747-758).

The cold shock response in bacteria involves the expression of low-molecular weight cold shock proteins (CSPs) containing a nucleic acid-binding cold shock domain (CSD), which are known to destabilize secondary structures on mRNAs, facilitating translation at low temperatures. Caulobacter crescentus cspA and cspB are induced upon cold shock, while cspC and cspD are induced during stationary phase. In this work, we determined a new coding sequence for the cspC gene, revealing that it encodes a protein containing two CSDs. The phenotypes of C. crescentus csp mutants were analyzed, and we found that cspC is important for cells to maintain viability during extended periods in stationary phase. Also, cspC and cspCD strains presented altered morphology, with frequent non-viable filamentous cells, and cspCD also showed a pronounced cell death at late stationary phase. In contrast, the cspAB mutant presented increased viability in this phase, which is accompanied by an altered expression of both cspC and cspD, but the triple cspABD mutant loses this characteristic. Taken together, our results suggest that there is a hierarchy of importance among the csp genes regarding stationary phase viability, which is probably achieved by a fine tune balance of the levels of these proteins.

Keywords: Stationary phase; Cold shock proteins; Cold shock domain; Caulobacter crescentus

First description of a laccase-like enzyme in soil algae by Benjamin Otto; Dietmar Schlosser; Werner Reisser (pp. 759-768).

Laccases (EC 1.10.3.2) are versatile multi-copper oxidases so far found in higher plants, fungi, insects, prokaryotes and lichens. In the present study, the production of an extracellular laccase-like enzyme by the coccoid green soil alga Tetracystis aeria was investigated and the enzyme was partly characterized, thereby providing the first description of a laccase-like enzyme in soil algae. Enzyme production in algae cultures was considerably increased by addition of the fungal laccase inducer copper sulphate. Maximal enzyme production was observed during the stationary growth phase. Peroxidase or tyrosinase activity was not detected. The native enzyme exhibits an apparent molecular mass of about 212 kDa as observed with size exclusion chromatography and about 210–260 kDa as estimated by zymograms. The enzyme efficiently oxidizes 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), syringaldazine (SGZ) and the anthraquinone dye Acid Blue 62, while guaiacol and Remazol Brilliant Blue R are only poorly oxidized. The apparent kinetic parameters obtained for ABTS, 2,6-DMP and SGZ oxidation are within the range reported for fungal laccases. Oxidation of the phenolic substrate 2,6-DMP displays a remarkably high pH optimum (pH 8.0–8.5), which is interesting with respect to potential biotechnological applications.

Keywords: Algae; Laccase; Laccase-like enzyme; Oxidase; Soil algae; Tetracystis

Heterologous expression of secreted biologically active human interleukin-10 in Bifidobacterium breve by E. V. Khokhlova; B. A. Efimov; L. I. Kafarskaia; A. N. Shkoporov (pp. 769-774).

Construction of Bifidobacterium breve capable of production of secreted biologically active human interleukin-10 (hIL-10) is described. ORF coding for full-length mature human interleukin-10 was cloned into a series of expression vectors. This resulted in generation of translational fusions between hIL-10 and signal peptides sequences derived from Bifidobacterium breve genes sec2, apuB and B. adolescentis gene amyB under the control of constitutively active bifidobacterial promoter. We have shown that fusion to amyB signal peptide resulted in highest expression level of hIL-10 at the mRNA and protein level. Secreted hIL-10 was highly unstable in bifidobacterial culture supernatants in standard growth conditions. However, incubation of stationary cultures in buffered tissue culture medium resulted in production of stable biologically active hIL-10, albeit in low amounts (1.9 ng/ml).

Keywords: Bifidobacterium ; Inflammatory bowel disease; Interleukin-10; Vector

A direct comparison between extracted tooth and filter-membrane biofilm models of endodontic irrigation using Enterococcus faecalis by Christopher K. Hope; Stuart G. Garton; Qian Wang; Girvan Burnside; Peter J. Farrelly (pp. 775-781).

Endodontic restorations often fail due to inadequate disinfection of the root canal even though the antimicrobial irrigants used have been shown to be capable of killing the bacterium frequently implicated in this complication, Enterococcus faecalis (Ef). Extracted human teeth were root-prepared and filled with a liquid culture of Ef. Following incubation, the root canals were irrigated with 1% sodium hypochlorite (NaOCl), electrochemically activated water or saline control. Irrigation was modelled using an electronic pipette to deliver the solutions at a reproducible flow velocity. A series of parallel experiments employed a membrane biofilm model that was directly immersed into irrigant. Experimental conditions where contiguous between the extracted tooth model and biofilm model wherever possible. After 60 s of exposure, 1% NaOCl effectively sterilised the biofilm model, whereas log 3.36 viable Ef where recoverable from the analogous extracted tooth model, the other irrigants proved ineffective. Biofilms of Ef were susceptible to concentrations of irrigant that proved ineffective in the tooth model. NaOCl was the most effective biocide in either case. This suggests that the biofilm modality of bacterial growth may not be the most important factor for the recalcitrance of root canal infections during endodontic irrigation; it is more likely due to the inability of the irrigant to access the infection.

Keywords: Enterococcus faecalis ; In vitro modelling; Hypochlorous acid; Sodium hypochlorite

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Archives of Microbiology (v.192, #9)

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Archives of Microbiology (v.192, #9)