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Archives of Microbiology (v.182, #1)

Lipoprotein trafficking in Escherichia coli by Shin-ichiro Narita; Shin-ichi Matsuyama; Hajime Tokuda (pp. 1-6).

Bacterial lipoproteins comprise a subset of membrane proteins that are covalently modified with lipids at the amino-terminal Cys. Lipoproteins are involved in a wide variety of functions in bacterial envelopes. Escherichia coli has more than 90 species of lipoproteins, most of which are located on the periplasmic surface of the outer membrane, while others are located on that of the inner membrane. In order to elucidate the mechanisms by which outer-membrane-specific lipoproteins are sorted to the outer membrane, biochemical, molecular biological and crystallographic approaches have been taken. Localization of lipoproteins on the outer membrane was found to require a lipoprotein-specific sorting machinery, the Lol system, which is composed of five proteins (LolABCDE). The crystal structures of LolA and LolB, the periplasmic chaperone and outer-membrane receptor for lipoproteins, respectively, were determined. On the basis of the data, we discuss here the mechanism underlying lipoprotein transfer from the inner to the outer membrane through Lol proteins. We also discuss why inner membrane-specific lipoproteins remain on the inner membrane.

Keywords: Lipoprotein; Lol system; Outer membrane; Sorting signal; ABC transporter

Pseudomonas aeruginosa dihydroorotases: a tale of three pyrCs by Dayna M. Brichta; Kamran N. Azad; Pooja Ralli; Gerard A. O’Donovan (pp. 7-17).

Pseudomonas aeruginosa PAO1 was shown to contain three pyrC sequences. Two of these genes, designated pyrC (PA3527) and pyrC2 (PA5541), encode polypeptides with dihydroorotase (DHOase) activity, while the third, pyrC′ (PA0401), encodes a DHOase-like polypeptide that lacks DHOase activity, but is necessary for the structure and function of ATCase. Both pyrC and pyrC2 were cloned and complemented an Escherichia coli pyrC mutant. In addition, pyrC and pyrC2 were individually inactivated in P. aeruginosa by homologous exchange with a mutated allele of each. The resulting mutant strains were prototrophic. A pyrC, pyrC2 double mutant was also constructed, and this strain had an absolute requirement for pyrimidines. The transcriptional activity of pyrC and pyrC2 was measured using lacZ promoter fusions. While pyrC was found to be constitutively expressed, pyrC2 was expressed only in the pyrC mutant background. An in vitro transcriptional/translational system was used to estimate the size of the pyrC2 gene product. The expressed polypeptide was approximately 47 kDa, which is in keeping with the theoretical molecular mass of 48 kDa, making it the largest prokaryotic DHOase polypeptide identified to date. To our knowledge, this is the first report of a true DHOase mutant in P. aeruginosa and also the first confirmation that pyrC2 encodes a polypeptide with DHOase activity.

Characterization and expression of genes from the RubisCO gene cluster of the chemoautotrophic symbiont of Solemya velum: cbbLSQO by Julie Schwedock; Tara L. Harmer; Kathleen M. Scott; Harm J. Hektor; Angelica P. Seitz; Matthew C. Fontana; Daniel L. Distel; Colleen M. Cavanaugh (pp. 18-29).

Chemoautotrophic endosymbionts residing in Solemya velum gills provide this shallow water clam with most of its nutritional requirements. The cbb gene cluster of the S. velum symbiont, including cbbL and cbbS, which encode the large and small subunits of the carbon-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), was cloned and expressed in Escherichia coli. The recombinant RubisCO had a high specific activity, ∼3 μmol min⁻¹ mg protein ⁻¹, and a K _CO2 of 40.3 μM. Based on sequence identity and phylogenetic analyses, these genes encode a form IA RubisCO, both subunits of which are closely related to those of the symbiont of the deep-sea hydrothermal vent gastropod Alviniconcha hessleri and the photosynthetic bacterium Allochromatium vinosum. In the cbb gene cluster of the S. velum symbiont, the cbbLS genes were followed by cbbQ and cbbO, which are found in some but not all cbb gene clusters and whose products are implicated in enhancing RubisCO activity post-translationally. cbbQ shares sequence similarity with nirQ and norQ, found in denitrification clusters of Pseudomonas stutzeri and Paracoccus denitrificans. The 3′ region of cbbO from the S. velum symbiont, like that of the three other known cbbO genes, shares similarity to the 3′ region of norD in the denitrification cluster. This is the first study to explore the cbb gene structure for a chemoautotrophic endosymbiont, which is critical both as an initial step in evaluating cbb operon structure in chemoautotrophic endosymbionts and in understanding the patterns and forces governing RubisCO evolution and physiology.

Keywords: RubisCO; Symbiosis; Chemosynthesis; Solemya velum ; Ribulose 1,5-bisphosphate carboxylase/oxygenase; K _CO2

Genetic diversity in Rhizopus oryzae strains as revealed by the sequence of lactate dehydrogenase genes by Katsuichi Saito; Akane Saito; Masao Ohnishi; Yuji Oda (pp. 30-36).

Twenty-seven strains of Rhizopus oryzae accumulating predominantly lactic acid were shown to possess two ldh genes, ldhA and ldhB, encoding NAD-dependent lactate dehydrogenases. Variation in nucleotide sequence was identified for each gene from different strains, and similar phylogenetic trees were obtained based on the nucleotide sequences of both genes. The other 21 strains of R. oryzae accumulating predominantly fumaric and malic acids contained a single ORF of ldhB. Compared to the strains accumulating predominantly lactic acid, a lower degree of sequence divergence was found in ldhB, resulting in a separate cluster in the phylogenetic tree. The high similarity (>90%) spanning the ORF and adjacent regions demonstrates that ldhA and ldhB are derived from the same ancestor gene. The strains accumulating predominantly fumaric and malic acids lack functional ldhA, which plays a role in lactic acid synthesis and may form a lineage separated from the strains accumulating predominantly lactic acid in the genus Rhizopus.

Keywords: Rhizopus oryzaeLactate dehydrogenase; Phylogeny; Polymorphisms

Expression of cspH upon nutrient up-shift in Salmonella enterica serovar Typhimurium by Bae Hoon Kim; Hyeon Guk Kim; Gun Ick Bae; Iel Soo Bang; Sung Ho Bang; Jong Hak Choi; Yong Keun Park (pp. 37-43).

The gene cspH , which encodes one of the cold-shock proteins in Salmonella enterica serovar Typhimurium, has previously been reported to be induced during early exponential phase at 37°C. In the present study, the expression of cspH upon nutrient up-shift at 37°C was investigated and found to be affected by DNA gyrase and DNA-binding protein Fis. When cells at stationary phase were subcultured into a rich medium, the mRNA level of cspH increased dramatically prior to the first cell division. However, when the cells were treated with DNA gyrase inhibitors, cspH mRNA was not induced upon nutrient up-shift. The low level of DNA superhelical density at the cspH promoter in part affected the expression of cspH mRNA in vitro. In addition, a fis-deficient strain had a lower level of cspH mRNA than the wild-type upon nutrient up-shift. Finally, a cspH–lacZ construct, in which the putative binding region for Fis was deleted in the cspH promoter, expressed a low level of LacZ, in contrast to the native cspH–lacZ construct.

Keywords: cspH ; DNA gyrase; Nutrient up-shift; Fis

Cloning and characterization of a gene encoding the γ-butyrolactone autoregulator receptor from Streptomyces clavuligerus by Hyun Soo Kim; Yong Jik Lee; Chang Kwon Lee; Sun Uk Choi; Soo-Hwan Yeo; Yong Il Hwang; Tae Shick Yu; Hiroshi Kinoshita; Takuya Nihira (pp. 44-50).

With primers designed for the conserved region of the γ-butyrolactone autoregulator receptor proteins from Streptomyces species, PCR using the Streptomyces clavuligerus genome DNA as a template gave a clear band of 100 bp, the sequence of which revealed high similarity to the expected region of a receptor gene. By Southern blot and colony hybridization with the 100-bp insert as a probe, plasmid pSCA, harboring a 4.2 kb-SalI fragment, was obtained. Sequence analysis on the insert revealed a 702-bp ORF encoding a protein with a moderate similarity (identity, 33–43%; similarity, 51–62%) to known γ-butyrolactone autoregulator receptor proteins from Streptomyces sp. The ORF was named scaR (S. clavuligerus autoregulator receptor). The scaR/pET-3d plasmid was constructed for overexpression of the recombinant ScaR protein (rScaR) in Escherichia coli, and the rScaR protein was purified to homogeneity by DEAE-ion-exchange HPLC. The molecular mass of the purified rScaR protein was determined to be 27 kDa as determined by SDS-PAGE, and 54 kDa by gel filtration HPLC under nondenatured conditions at a low protein concentration, indicating that the majority of the native ScaR is present in the form of a dimer, although rScaR tended to aggregate into a higher molecular form of 230 kDa at a high protein concentration. A binding assay with tritium-labeled autoregulators indicated that IM-2 type compounds with a long C2 side chain were the most effective ligands for rScaR, demonstrating for the first time that the β-lactam producer S. clavuligerus contains a gene for the γ-butyrolactone autoregulator receptor.

Keywords: Streptomyces clavuligerusγ-butyrolactone; Autoregulator; Receptor; Purification

Oxidation and ring cleavage of dibenzofuran by the filamentous fungus Paecilomyces lilacinus by Manuela Gesell; Elke Hammer; Annett Mikolasch; Frieder Schauer (pp. 51-59).

The ability of the imperfect soil fungus Paecilomyces lilacinus to transform the environmental pollutant dibenzofuran was investigated. Transformation of dibenzofuran and related derivatives lead to 14 products, which were identified by UV spectroscopy, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Biotransformation was initiated by two separate hydroxylation steps, leading to the accumulation of 4-monohydroxylated and 4-dihydroxylateddibenzofurans. Hydroxylation at both aromatic rings produced 2,7-dihydroxydibenzofuran, 3,7-dihydroxydibenzofuran, and 2,8-dihydroxydibenzofuran. Further oxidation yields ring cleavage of dibenzofuran, which has not been described before for filamentous fungi. The ring fission products were identified as benzo[b]furo[3,2-d]-2-pyrone-6-carboxylic acid and [2-(1-carboxy-methylidene)-benzofuran-3-ylidene]-hydroxy-acetic acid and its derivatives hydroxylated at carbon 7 and 8 at the non-cleaved ring. Other metabolites were riboside-conjugates of 2-hydroxydibenzofuran and 3-hydroxydibenzofuran. The results showed that P. lilacinus transforms the hydrophobic compound dibenzofuran by phase I/phase II reactions to produce hydroxylated products and excretable sugar conjugates.

Keywords: Dibenzofuran; Biotransformation; Hydroxylation; Ring cleavage; Filamentous fungiPaecilomyces lilacinusConjugates

Ribosome modulation factor protects Escherichia coli during heat stress, but this may not be dependent on ribosome dimerisation by Gordon W. Niven (pp. 60-66).

The role of ribosome modulation factor (RMF) in protecting heat-stressed Escherichia coli cells was identified by the observation that cultures of a mutant strain lacking functional RMF (HMY15) were highly heat sensitive in stationary phase compared to those of the parent strain (W3110). No difference in heat sensitivity was observed between these strains in exponential phase, during which RMF is not synthesised. Studies by differential scanning calorimetry demonstrated that the ribosomes of stationary-phase cultures of the mutant strain had lower thermal stability than those of the parent strain in stationary phase, or exponential-phase ribosomes. More rapid breakdown of ribosomes in the mutant strain during heating was confirmed by rRNA analysis and sucrose density gradient centrifugation. Analyses of ribosome composition showed that the 100S dimers dissociated more rapidly during heating than 70S particles. While ribosome dimerisation is a consequence of the conformational changes caused by RMF binding, it may not therefore be essential for RMF-mediated ribosome stabilisation.

Keywords: Ribosome stability; Ribosome modulation factor; Heat stress; Ribosome dimerisation

Distribution and sub-cellular localization of the aflatoxin enzyme versicolorin B synthase in time-fractionated colonies of Aspergillus parasiticus by Ching-Hsun Chiou; Li-Wei Lee; Shirley A. Owens; Joanne H. Whallon; Karen L. Klomparens; Craig A. Townsend; John E. Linz (pp. 67-79).

Aflatoxins are highly toxic and carcinogenic fungal secondary metabolites. At least 18 enzyme activities are required for aflatoxin biosynthesis in the filamentous fungus Aspergillus parasiticus. One of these enzymes, versicolorin B synthase (VBS), catalyzes bisfuran ring closure in versiconal hemiacetal (a reaction near the middle of the pathway) to form versicolorin B. This reaction is required for the subsequent activation to aflatoxin B₁-8,9 epoxide, a highly reactive and toxic aflatoxin metabolite, and is important for aflatoxin toxicity. We analyzed the localization of VBS in the aflatoxin-producing strain A. parasiticus SU-1 grown on solid media using a colony fractionation technique developed previously. A highly specific polyclonal antibody, raised against a maltose-binding protein–VBS fusion protein synthesized in Escherichia coli, was used to detect VBS in SU-1 grown on a rich solid medium via immunofluorescence confocal laser scanning microscopy (CLSM) and immunogold transmission electron microscopy (TEM). VBS was detected in both vegetative hyphae and in asexual developmental structures, called conidiophores. Western blot and CLSM analyses demonstrated the highest abundance of VBS in colony fraction S2 consisting of cells that had grown for 24–48 h; this fraction also contained the highest levels of newly developed conidiophores and the highest abundance of aflatoxin B₁, consistent with VBS abundance. At the subcellular level, CLSM and TEM detected VBS distributed throughout the cytoplasm and concentrated in ring-like structures surrounding nuclei. It is uncertain whether enzymatically active VBS is present in either or both locations.

Keywords: Confocal microscopy; Transmission electron microscopy; Aflatoxin biosynthesis; Fungal development; Secondary metabolism

Purification and characterization of ferredoxin-NADP⁺ reductase encoded by Bacillus subtilis yumC by Daisuke Seo; Kei Kamino; Kazuhito Inoue; Hidehiro Sakurai (pp. 80-89).

From Bacillus subtilis cell extracts, ferredoxin-NADP⁺ reductase (FNR) was purified to homogeneity and found to be the yumC gene product by N-terminal amino acid sequencing. YumC is a ∼94-kDa homodimeric protein with one molecule of non-covalently bound FAD per subunit. In a diaphorase assay with 2,6-dichlorophenol-indophenol as electron acceptor, the affinity for NADPH was much higher than that for NADH, with K_m values of 0.57 μM vs >200 μM. K_cat values of YumC with NADPH were 22.7 s⁻¹ and 35.4 s⁻¹ in diaphorase and in a ferredoxin-dependent NADPH-cytochrome c reduction assay, respectively. The cell extracts contained another diaphorase-active enzyme, the yfkO gene product, but its affinity for ferredoxin was very low. The deduced YumC amino acid sequence has high identity to that of the recently identified Chlorobium tepidum FNR. A genomic database search indicated that there are more than 20 genes encoding proteins that share a high level of amino acid sequence identity with YumC and which have been annotated variously as NADH oxidase, thioredoxin reductase, thioredoxin reductase-like protein, etc. These genes are found notably in gram-positive bacteria, except Clostridia, and less frequently in archaea and proteobacteria. We propose that YumC and C. tepidum FNR constitute a new group of FNR that should be added to the already established plant-type, bacteria-type, and mitochondria-type FNR groups.

Keywords: Ferredoxin; Ferredoxin-NADP⁺ reductase; NADPH oxidase; NADPH-thioredoxin reductaseBacillus subtilisyumC

Anaerobiosis and low specific growth rates enhance hemolysin BL production by Bacillus cereus F4430/73 by Catherine Duport; Séverine Thomassin; Gérald Bourel; Philippe Schmitt (pp. 90-95).

Bacillus cereus F4430/73 produced the highest levels of hemolysin BL (HBL) when grown under anaerobiosis in MOD medium. Anaerobic cells grown in a chemostat at low specific growth rate (0.1–0.2 h⁻¹) expressed up to sevenfold more HBL than did cells held at a faster growth rate. At 0.2 h⁻¹, the presence of 90 mM glucose resulted in inhibition of HBL production. Glucose was found to repress HBL induction at the mRNA level, indicating the potential involvement of catabolite repression in the regulation of HBL. Based on these data, it is suggested that growth rate could be an effector of catabolite regulation of HBL.

Keywords: Chemostat; RT-PCR; Bacillus cereus ; Enterotoxin; Anaerobiosis; Growth rate; Glucose repression

DpsA protects the human pathogen Burkholderia pseudomallei against organic hydroperoxide by Suvit Loprasert; Wirongrong Whangsuk; Ratiboot Sallabhan; Skorn Mongkolsuk (pp. 96-101).

The human pathogen, Burkholderia pseudomalle, is able to survive and multiply in hostile environments such as within macrophages. In an attempt to understand its strategy to cope with oxidative stress, the physiological role and gene regulation of a nonspecific DNA-binding protein (DpsA) was investigated. Expression of dpsA increases in response to oxidative stress through increased transcription from the upstream katG (catalase–peroxidase) promoter, which is OxyR dependent. dpsA is also transcribed from its own promoter, which is activated by osmotic stress in an OxyR-independent manner. DpsA-deficient mutants are hypersensitive to tert-butyl hydroperoxide, while overexpression of DpsA leads to increased resistance to organic oxidants. B. pseudomallei DpsA can also protect Escherichia coli against organic hydroperoxide toxicity. The mechanism of DpsA-mediated resistance to organic hydroperoxides was shown to differ from that of alkyl hydroperoxide reductase.

Keywords: Melioidosis; Oxidative stress; Nonspecific DNA-binding protein; Organic hydroperoxide

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Archives of Microbiology (v.182, #1)