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Archives of Microbiology (v.180, #6)

Type IV pili-related natural transformation systems: DNA transport in mesophilic and thermophilic bacteria by Beate Averhoff; Alexandra Friedrich (pp. 385-393).

Horizontal gene flow is a driving force for bacterial adaptation. Among the three distinct mechanisms of gene transfer in bacteria, conjugation, transduction, and transformation, the latter, which includes competence induction, DNA binding, and DNA uptake, is perhaps the most versatile mechanism and allows the incorporation of free DNA from diverse bacterial species. Here we review DNA transport machineries mediating uptake of naked DNA in gram-positive and gram-negative bacteria. Different putative models of transformation machineries comprising components similar to proteins of type IV pili are presented. Emphasis is placed on a comparative discussion of the underlying mechanisms of DNA transfer in mesophilic and extremely thermophilic bacteria, highlighting conserved and distinctive features of these transformation machineries.

Keywords: Natural transformation; Competence proteins; Type IV pili

Purification and properties of the formate dehydrogenase and characterization of the fdhA gene of Sulfurospirillum multivorans by Roland P. H. Schmitz; Gabriele Diekert (pp. 394-401).

The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene (fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe₄/S₄ cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.

Keywords: Dehalorespiration; Dehalospirillum multivorans ; Sulfurospirillum multivorans ; Formate dehydrogenase; fdhA ; Tetrachloroethene reductive dehalogenase; Selenocysteine; SECIS; Iron-sulfur protein

PCR-based identification of microcystin-producing genotypes of different cyanobacterial genera by Michael Hisbergues; Guntram Christiansen; Leo Rouhiainen; Kaarina Sivonen; Thomas Börner (pp. 402-410).

Microcystins are harmful hepatotoxins produced by many, but not all strains of the cyanobacterial genera Anabaena, Microcystis, Anabaena, Planktothrix, and Nostoc. Waterbodies have to be monitored for the mass development of toxic cyanobacteria; however, because of the close genetic relationship of microcystin-producing and non-producing strains within a genus, identification of microcystin-producers by morphological criteria is not possible. The genomes of microcystin-producing cells contain mcy genes coding for the microcystin synthetase complex. Based on the sequence information of mcy genes from Microcystis and Planktothrix, a primer pair for PCR amplification of a mcyA gene fragment was designed. PCR with this primer pair is a powerful means to identify microcystin-producing strains of the genera Anabaena, Microcystis, and Planktothrix. Moreover, subsequent RFLP analysis of the PCR products generated genus-specific fragments and allowed the genus of the toxin producer to be identified. The assay can be used with DNA from field samples.

Keywords: Anabaena ; Cyanobacteria; Microcystin; Microcystis ; Nostoc ; Nodularia ; Peptide synthetase; Planktothrix ; Toxin

Dehalorespiration with hexachlorobenzene and pentachlorobenzene by Dehalococcoides sp. strain CBDB1 by Gopalakrishnan Jayachandran; Helmut Görisch; Lorenz Adrian (pp. 411-416).

The chlororespiring anaerobe Dehalococcoides sp. strain CBDB1 used hexachlorobenzene and pentachlorobenzene as electron acceptors in an energy-conserving process with hydrogen as electron donor. Previous attempts to grow Dehalococcoides sp. strain CBDB1 with hexachlorobenzene or pentachlorobenzene as electron acceptors failed if these compounds were provided as solutions in hexadecane. However, Dehalococcoides sp. strain CBDB1 was able to grow with hexachlorobenzene or pentachlorobenzene when added in crystalline form directly to cultures. Growth of Dehalococcoides sp. strain CBDB1 by dehalorespiration resulted in a growth yield (Y) of 2.1±0.24 g protein/mol Cl⁻ released with hexachlorobenzene as electron acceptor; with pentachlorobenzene, the growth yield was 2.9±0.15 g/mol Cl⁻. Hexachlorobenzene was reductively dechlorinated to pentachlorobenzene, which was converted to a mixture of 1,2,3,5- and 1,2,4,5-tetrachlorobenzene. Formation of 1,2,3,4-tetrachlorobenzene was not detected. The final end-products of hexachlorobenzene and pentachlorobenzene dechlorination were 1,3,5-trichlorobenzene, 1,3- and 1,4-dichlorobenzene, which were formed in a ratio of about 3:2:5. As reported previously, Dehalococcoides sp. strain CBDB1 converted 1,2,3,5-tetrachlorobenzene exclusively to 1,3,5-trichlorobenzene, and 1,2,4,5-tetrachlorobenzene exclusively to 1,2,4-trichlorobenzene. The organism therefore catalyzes two different pathways to dechlorinate highly chlorinated benzenes. In the route leading to 1,3,5-trichlorobenzene, only doubly flanked chlorine substituents were removed, while in the route leading to 1,3-and 1,4-dichlorobenzene via 1,2,4-trichlorobenzene singly flanked chlorine substituents were also removed. Reductive dehalogenase activity measurements using whole cells pregrown with different chlorobenzene congeners as electron acceptors indicated that different reductive dehalogenases might be induced by the different electron acceptors. To our knowledge, this is the first report describing reductive dechlorination of hexachlorobenzene and pentachlorobenzene via dehalorespiration by a pure bacterial culture.

Keywords: Dehalococcoides ; Hexachlorobenzene; Pentachlorobenzene; Chlorobenzene; Reductive dechlorination; Dehalorespiration.

Characterization of the chlorosome antenna of the filamentous anoxygenic phototrophic bacterium Chloronema sp. strain UdG9001 by Frederic Gich; Ruth L. Airs; Marianne Danielsen; Brendan J. Keely; Carles A. Abella; Jesús Garcia-Gil; Mette Miller; Carles M. Borrego (pp. 417-426).

The absorption and fluorescence properties of chlorosomes of the filamentous anoxygenic phototrophic bacterium Chloronema sp. strain UdG9001 were analyzed. The chlorosome antenna of Chloronema consists of bacteriochlorophyll (BChl) d and BChl c together with γ-carotene as the main carotenoid. HPLC analysis combined with APCI LC-MS/MS showed that the chlorosomal BChls comprise a highly diverse array of homologues that differ in both the degree of alkylation of the macrocycle at C-8 and/or C-12 and the alcohol moiety esterified to the propionic acid group at C-17. BChl c and BChl d from Chloronema were mainly esterified with geranylgeraniol (33% of the total), heptadecanol (24%), octadecenol (19%), octadecanol (14%), and hexadecenol (9%). Despite this pigment heterogeneity, fluorescence emission of the chlorosomes showed a single peak centered at 765 nm upon excitation at wavelengths ranging from 710 to 740 nm. This single emission, assigned to BChl c, indicates an energy transfer from BChl d to BChl c within the same chlorosome. Likewise, incubation of chlorosomes under reducing conditions caused a weak increase in fluorescence emission, which indicates a small redox-dependent fluorescence. Finally, protein analysis of Chloronema chlorosomes using SDS-PAGE and MALDI-TOF-MS revealed the presence of a chlorosomal polypeptide with a molecular mass of 5.7 kDa, resembling the CsmA protein found in Chloroflexus aurantiacus and Chlorobium tepidum chlorosomes. Several minor polypeptides were also detected but not identified. These results indicate that, compared with other members of filamentous anoxygenic phototrophic bacteria and green sulfur bacteria, Chloronema possesses an antenna system with novel features that may be of interest for further investigations.

Keywords: Antenna pigments; Bacteriochlorophyll homologues; Chloronema ; Chlorosomes; Filamentous anoxygenic phototrophic bacteria; Green sulfur bacteria

Analysis of phenomena involved in the apical growth of Phycomyces blakesleeanus by José Ruiz-Herrera; Guadalupe Martínez-Cadena; Flavia Loarca; Rubén Salcedo-Hernández (pp. 427-433).

The effects of the Ca²⁺/H⁺ exchanger A23187 and the K⁺/H⁺ exchanger nigericin, the electrogenic membrane-potential depleters valinomycin and CCCP, and the calcium channel blockers ruthenium red, nifedipine, and nitrendipine on the apical growth of Phycomyces blakesleeanus were analyzed. While all of the compounds inhibited the growth of germlings in liquid medium, the Ca²⁺ channel blockers were the least effective. Chitin synthesis in vivo was also sensitive to the inhibitors; here again, the calcium channel blockers were less efficient, and their effect occurred after a lag phase, in contrast to the electroneutral ionophores whose effects were immediate. The ionophores rapidly inhibited protein secretion, and reduced the number of secretory vesicles and chitosomes in the hyphal apex of P. blakesleeanus. The results suggest that not only tip-to-base calcium gradients but also transmembrane ionic gradients and membrane potential have a role in the apical growth of P. blakesleeanus. They are probably involved in the formation, migration, and/or fusion with the plasmalemma of secretory vesicles and chitosomes.

Keywords: Apical growth; Ionophores; Calcium channels; Cell wall; Apical vesicles; Phycomyces blakesleeanus

Biochemical and genetic characterization of ChiA, the major enzyme component for the solubilization of chitin by Cellulomonas uda by Gemma Reguera; Susan B. Leschine (pp. 434-443).

Cellulomonas uda efficiently solubilized chitinous substrates with a simple chitinase system composed of an endochitinase, designated ChiA, which hydrolyzed insoluble substrates into long-chain chitooligosaccharides, and an as yet uncharacterized exochitinase activity. ChiA, isolated from culture supernatant fluids, was found to be a glycosylated endochitinase with an apparent molecular mass of approximately 70 kDa and pI of 8.5. The gene encoding ChiA was cloned in Escherichia coli and sequenced, revealing an open reading frame of 1,716 bp encoding a 571-amino-acid protein with a predicted molecular mass of 59.2 kDa. The region upstream of chiA included a conserved −35 hexamer flanked by two direct repeats analogous to those found in many Streptomyces chitinase promoters, and thought to function as binding sequences for regulatory proteins. Analysis of the deduced amino acid sequence showed a modular protein consisting of a signal peptide at its N terminus, a family 2 carbohydrate-binding module (CBM2) that was closely related to the substrate-binding domains of glycosyl hydrolases from distantly related bacteria, and a family 18 glycosyl hydrolase catalytic module related to Streptomyces chitinases. In contrast to the fibronectin type III domains of Streptomyces chitinases, the linker region between modules in ChiA consisted of a long proline- and threonine-rich module, thought to contribute to the glycosylation and flexibility of the mature protein.

Keywords: Chitinase; ChiA; Cellulomonas uda ; Chitin; Chitinolysis

Diverse Mesorhizobium plurifarium populations native to Mexican soils by En Tao Wang; Feng Ling Kan; Zhi Yuan Tan; Ivonne Toledo; Wen Xin Chen; Esperanza Martínez-Romero (pp. 444-454).

Forty-six Mesorhizobium strains associated with the leguminous plants Leucaena leucocephala and Sesbania herbacea in an uncultivated Mexican field were characterized using a polyphasic approach. The strains were identified as Mesorhizobium plurifarium based upon the close relationships with the reference strains for this species in PCR-based restriction fragment length polymorphism analyses, sequencing of 16S rRNA genes, multilocus enzyme electrophoresis, and DNA-DNA hybridization. Although the strains isolated from both plants formed the same group in multilocus enzyme electrophoresis and cross-nodulations were observed in the laboratory, different electrophoretic types were obtained from the two plants grown in natural soils, indicating the existence of a preferable association between the plants and the rhizobia. The M. plurifarium strains from Mexico and the reference strains from Africa and Brazil formed different phenotypic clusters in a numerical taxonomy. The Mexican strains did not grow at 37 °C and were sensitive to salty-alkaline conditions, while the reference strains from Africa and Brazil grew at 42 °C and were more resistant to salty-alkaline conditions. These results demonstrate that both the plants and environmental factors affected the evolution of rhizobia and that the Mexican strains had adapted to the neutral soils and the cool climate where they were isolated.

Keywords: Mesorhizobium plurifarium ; Leucaena ; Sesbania ; Phylogeny; Diversity

Identification of the 7,8-didemethyl-8-hydroxy-5-deazariboflavin synthase required for coenzyme F₄₂₀ biosynthesis by David E. Graham; Huimin Xu; Robert H. White (pp. 455-464).

The hydride carrier coenzyme F₄₂₀ contains the unusual chromophore 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Microbes that generate F₄₂₀ produce this FO moiety using a pyrimidine intermediate from riboflavin biosynthesis and the 4-hydroxyphenylpyruvate precursor of tyrosine. The fbiC gene, cloned from Mycobacterium smegmatis, encodes the bifunctional FO synthase. Expression of this protein in Escherichia coli caused the host cells to produce FO during growth, and activated cell-free extracts catalyze FO biosynthesis in vitro. FO synthase in the methanogenic euryarchaeon Methanocaldococcus jannaschii comprises two proteins encoded by cofG (MJ0446) and cofH (MJ1431). Both subunits were required for FO biosynthesis in vivo and in vitro. Cyanobacterial genomes encode homologs of both genes, which are used to produce the coenzyme for FO-dependent DNA photolyases. A molecular phylogeny of the paralogous cofG and cofH genes is consistent with the genes being vertically inherited within the euryarchaeal, cyanobacterial, and actinomycetal lineages. Ancestors of the cyanobacteria and actinomycetes must have acquired the two genes, which subsequently fused in actinomycetes. Both CofG and CofH have putative radical S-adenosylmethionine binding motifs, and pre-incubation with S-adenosylmethionine, Fe²⁺, sulfide, and dithionite stimulates FO production. Therefore a radical reaction mechanism is proposed for the biosynthesis of FO.

Keywords: Methanocaldococcus jannaschii ; Mycobacterium smegmatis ; Coenzyme F₄₂₀ ; 7,8-Didemethyl-8-hydroxy-5-deazariboflavin; S-adenosylmethionine; 4-Hydroxyphenylpyruvate; Riboflavin; FO synthase; Radical reaction

Significance of phosphoglucose isomerase for the shift between heterolactic and mannitol fermentation of fructose by Oenococcus oeni by Hanno Richter; Albert A. De Graaf; Inka Hamann; Gottfried Unden (pp. 465-470).

The bacterium Oenococcus oeni employs the heterolactic fermentation pathway (products lactate, ethanol, CO₂) during growth on fructose as a substrate, and the mannitol pathway when using fructose as an electron acceptor. In this study, [U-¹³C]glucose, [U-¹³C]fructose, HPLC, NMR spectroscopy, and enzyme analysis were applied to elucidate the use of both pathways by the hexoses. In the presence of glucose or pyruvate, fructose was metabolized either by the mannitol or the phosphoketolase pathways, respectively. Phosphoglucose isomerase, which is required for channeling fructose into the phosphoketolase pathways, was inhibited by a mixed-type inhibition composed of competitive (K _i=180 μM) and uncompetitive (K′_i=350 μM) inhibition by 6-phosphogluconate. Erythrose 4-phosphate inhibited phosphoglucose isomerase competitively (K _i=1.3 μM) with a low contribution of uncompetitive inhibition (K′_i=13 μM). The cellular 6-phosphogluconate content during growth on fructose plus pyruvate (<75 μM) was significantly lower than during growth on fructose alone or fructose plus glucose (550 and 480 μM). We conclude that competitive inhibition of phosphoglucose isomerase by 6-phosphogluconate (and possibly erythrose 4-phosphate) is responsible for exclusion of fructose from the phosphoketolase pathway during growth on fructose plus glucose, but not during growth on fructose plus pyruvate.

Keywords: Oenococcus oeni ; Phosphoglucose isomerase; Phosphoketolase pathway; Heterolactic fermentation; Mannitol formation; 6-phosphogluconate; Erythrose 4-phosphate

Comparative analysis of idiA and isiA transcription under iron starvation and oxidative stress in Synechococcus elongatus PCC 7942 wild-type and selected mutants by Naeima Yousef; Elfriede K. Pistorius; Klaus-Peter Michel (pp. 471-483).

In the mesophilic cyanobacterium Synechococcus elongatus PCC 7942, iron starvation induces the expression of a number of proteins, including IdiA and IsiA. Whereas IdiA protects photosystem (PS) II under mild iron limitation against oxidative stress in a yet unknown way, prolonged iron starvation leads to the formation of the PS I–IsiA supercomplex. Transcription of idiA is positively regulated by IdiB under iron starvation, and Fur represses transcription of isiAB under iron-sufficient growth conditions. In this report, data are presented suggesting a strong interrelationship between iron homeostasis and oxidative stress in S. elongatus PCC 7942, and showing that transcription of major iron-regulated genes, such as isiA, isiAB, idiA, idiB, mapA, and irpA, is induced by oxidative stress within a few minutes by treatment of cells with hydrogen peroxide or methylviologen. The overall results suggest that isiA/isiAB as well as idiB transcription in response to oxidative stress might be controlled by a transcriptional repressor possibly of the PerR-type. This fact also explains the observed cross-talk between IdiB- and Fur-mediated transcriptional regulation of gene expression and for the role of H₂O₂ as a superior trigger coordinating expression of iron-regulated genes under iron starvation and oxidative stress. Measuring 77 K chlorophyll a fluorescence, it is shown that hydrogen peroxide treatment causes a transient short-term modification of PS II and PS I most likely leading to increased cyclic electron transport around PS I. In this context, the intriguing observation was made that idiB is transcribed as part of an operon together with a gene encoding a potential [2Fe–2S]-protein. This protein has similarity to [Fe–S]-proteins involved in the electron transport activity of the NDH I complex in eubacteria. Since the NDH I complex is involved in cyclic electron transport activity around PS I in cyanobacteria and both adaptation to iron starvation and adaptation to oxidative stress lead to an enhanced cyclic electron transport activity around PS I, this potential [Fe–S]-protein might participate in the overall adaptational response to iron starvation and/or oxidative stress in Synechococcus.

Keywords: Synechococcus elongatus PCC 7942; Iron-responsive gene expression; Oxidative stress; Cyclic electron flow around photosystem I

Expression of the trxA gene for thioredoxin 1 in Rhodobacter sphaeroides during oxidative stress by Kuanyu Li; Cécile Pasternak; Gabriele Klug (pp. 484-489).

Expression of the thioredoxin (trxA) gene of Rhodobacter sphaeroides is regulated by oxidative stress at the transcriptional and post-transcriptional levels. All oxidative stress agents tested resulted in a moderate or strong increase of trxA mRNA levels, which was not due to increased mRNA stability. While the kinetics of increased trxA mRNA and of sodB mRNA, encoding superoxide dismutase, were similar after addition of tert-butyl hydroperoxide (t-BOOH) or hydrogen peroxide (H₂O₂), different kinetics were observed after addition of diamide or paraquat, indicating the involvement of different stress responses in the regulation of these genes. The level of TrxA did not increase to the same extent as trxA mRNA levels. Furthermore, the addition of H₂O₂ or t-BOOH led to increased turnover of the protein. Apparently, increased txA transcription compensated, at least in part, for the reduced stability of the protein. A strain expressing lower levels of thioredoxin 1 showed decreased resistance to diamide and H₂O₂ but increased resistance to paraquat and t-BOOH compared to the wild-type. These data implicate the involvement of various systems in the response to different types of oxidative stress and the participation of thioredoxin 1 in the defense against oxidative stress caused by diamide or H₂O₂.

Keywords: Rhodobacter ; Oxidative stress; Thioredoxin; Gene expression

Purification and characterization of a lactonase from Erwinia cypripedii 314B that hydrolyzes (S)-5-oxo-2-tetrahydrofurancarboxylic acid by Kazuya Mochizuki (pp. 490-493).

A bacterium, strain 314B, able to assimilate (S)-5-oxo-2-tetrahydrofurancarboxylic acid was isolated from soil and identified as Erwinia cypripedii. A lactonase hydrolyzing (S)-5-oxo-2-tetrahydrofurancarboxylic acid to l-α-hydroxyglutaric acid was purified 63-fold with 2% recovery from crude extracts of this bacterium to homogeneity as judged by SDS-PAGE. The molecular masses estimated by SDS-PAGE and gel filtration were 41 kDa and 79 kDa, respectively. The maximum activity was observed at pH 6.5–7.5 and 35–45 °C. The enzyme showed lower activity toward dl-2-oxotetrahydrofuran-4,5-dicarboxylic acid, but did not act on (R)-5-oxo-2-tetrahydrofurancarboxylic acid and other natural and synthetic lactones tested.

Keywords: (S)-5-oxo-2-tetrahydrofurancarboxylic acid; Lactonase; Erwinia cypripedii 314B

Chemical identification of N-acyl homoserine lactone quorum-sensing signals produced by Sinorhizobium meliloti strains in defined medium by Max Teplitski; Anatol Eberhard; Matthew R. Gronquist; Mengsheng Gao; Jayne B. Robinson; Wolfgang D. Bauer (pp. 494-497).

The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.

Keywords: Quorum sensing; Sinorhizobium ; N-acyl homoserine lactones; Mass spectrometry

Compensatory increase in ahpC gene expression and its role in protecting Burkholderia pseudomallei against reactive nitrogen intermediates by Suvit Loprasert; Ratiboot Sallabhan; Wirongrong Whangsuk; Skorn Mongkolsuk (pp. 498-502).

In the human pathogen Burkholderia pseudomallei, katG encodes the antioxidant defense enzyme catalase-peroxidase. Interestingly, a B. pseudomallei mutant, disrupted in katG, is hyperresistant to organic hydroperoxide. This hyperresistance is due to the compensatory expression of the alkyl hydroperoxide reductase gene (ahpC) and depends on a global regulator OxyR. The KatG-deficient mutant is also highly resistant to reactive nitrogen intermediates (RNI). When overproduced, the B. pseudomallei AhpC protein, protected cells against killing by RNI. The levels of resistance to both organic peroxide and RNI returned to those of the wild-type when the katG mutant was complemented with katG. These studies establish the partially overlapping defensive activities of KatG and AhpC.

Keywords: Melioidosis; Alkyl hydroperoxide reductase; Catalase-peroxidase

Different glycolytic pathways for glucose and fructose in the halophilic archaeon Halococcus saccharolyticus by Ulrike Johnsen; Martina Selig; Karina B. Xavier; Helena Santos; Peter Schönheit (pp. 503-503).

Mechanisms of acetate formation and acetate activation in halophilic archaea by Christopher Bräsen; Peter Schönheit (pp. 504-504).

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Archives of Microbiology (v.180, #6)