Light Response of Pseudomonas putida KT2440 Mediated by Class II LitR, a Photosensor Homolog.

Pseudomonas putida KT2440 retains three homologs (PplR1 to PplR3) of the LitR/CarH family, an adenosyl B12-dependent light-sensitive MerR family transcriptional regulator. Transcriptome analysis revealed the existence of a number of photoinducible genes, including pplR1, phrB (encoding DNA photolyase), ufaM (furan-containing fatty acid synthase), folE (GTP cyclohydrolase I), cryB (cryptochrome-like protein), and multiple genes without annotated/known function. Transcriptional analysis by quantitative reverse transcription-PCR with knockout mutants of pplR1 to pplR3 showed that a triple knockout completely abolished the light-inducible transcription in P. putida, which indicates the occurrence of ternary regulation of PplR proteins. A DNase I footprint assay showed that PplR1 protein specifically binds to the promoter regions of light-inducible genes, suggesting a consensus PplR1-binding direct repeat, 5'-T(G/A)TACAN12TGTA(C/T)A-3'. The disruption of B12 biosynthesis cluster did not affect the light-inducible transcription; however, disruption of ppSB1-LOV (where LOV indicates "light, oxygen, or voltage") and ppSB2-LOV, encoding blue light photoreceptors adjacently located to pplR3 and pplR2, respectively, led to the complete loss of light-inducible transcription. Overall, the results suggest that the three PplRs and two PpSB-LOVs cooperatively regulate the light-inducible gene expression. The wide distribution of the pplR/ppSB-LOV cognate pair homologs in Pseudomonas spp. and related bacteria suggests that the response and adaptation to light are similarly regulated in the group of nonphototrophic bacteria.IMPORTANCE The LitR/CarH family is a new group of photosensor homologous to MerR-type transcriptional regulators. Proteins of this family are distributed to various nonphototrophic bacteria and grouped into at least five classes (I to V). Pseudomonas putida retaining three class II LitR proteins exhibited a genome-wide response to light. All three paralogs were functional and mediated photodependent activation of promoters directing the transcription of light-induced genes or operons. Two LOV (light, oxygen, or voltage) domain proteins, adjacently encoded by two litR genes, were also essential for the photodependent transcriptional control. Despite the difference in light-sensing mechanisms, the DNA binding consensus of class II LitR [T(G/A)TA(C/T)A] was the same as that of class I. This is the first study showing the actual involvement of class II LitR in light-induced transcription.

Comparative Biochemical Analysis of Cellulosomes Isolated from Clostridium clariflavum DSM 19732 and Clostridium thermocellum ATCC 27405 Grown on Plant Biomass.

The cellulosome is a supramolecular multienzyme complex formed via species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Here, we report a comparative analysis of cellulosomes prepared from the thermophilic anaerobic bacteria Clostridium (Ruminiclostridium) clariflavum DSM 19732 and Clostridium (Ruminiclostridium) thermocellum ATCC 27405 grown on delignified rice straw. The results indicate that the isolated C. clariflavum cellulosome exhibits lower activity for insoluble cellulosic substrates and higher activity for hemicellulosic substrates, especially for xylan, compared to the isolated C. thermocellum cellulosome. The C. clariflavum cellulosome was separated into large and small complexes by size exclusion chromatography, and the high xylanase activity of the intact complex is mainly attributed to the small complex. Furthermore, both C. clariflavum and C. thermocellum cellulosomes efficiently converted delignified rice straw into soluble sugars with different compositions, whereas a mixture of these cellulosomes exhibited essentially no synergy for the saccharification of delignified rice straw. This is the first study to report that isolated C. clariflavum cellulosomes exhibit greater xylanase activity than isolated C. thermocellum cellulosomes. We also report the effect of a combination of intact cellulosome complexes isolated from different species on the saccharification of plant biomass.

Role and Function of Class III LitR, a Photosensor Homolog from Burkholderia multivorans.

The LitR/CarH protein family is an adenosyl B12 (AdoB12)-dependent photoreceptor family with DNA-binding activity, and its homologs are widely distributed in the genomes of diverse bacterial genera. In this investigation, we studied the role and functions of a LitR homolog from a Gram-negative soil bacterium, Burkholderia multivorans, which does not possess an AdoB12-binding domain. Transcriptome analysis indicated the existence of 19 light-induced genes, including folE2, cfaB, litS, photolyase gene phrB2, and cryB, located in the region flanking litR Disruption of litR caused constitutive expression of all the light-inducible genes, while mutation in the light-induced sigma factor gene, litS, abolished the transcription of the phrB2 operon and the cfa operon, indicating that LitR and LitS play a central role in light-inducible transcription. A gel shift assay showed that recombinant protein LitR specifically binds to the promoter regions of litR and the folE2 operon, and its binding was weakened by UV-A illumination. LitR absorbs light at maximally near 340 nm and exhibited a photocyclic response and light-dependent dissociation of multimer into tetramer. The litR mutant produced a 20-fold-higher intracellular level of folate than that of the wild-type strain. Thus, the evidence suggests that LitR light-dependently regulates the transcription of litR itself and the folE2 operon, resulting in the production of folate, and then the expressed RNA polymerase complex containing σLitS directs the transcription of the phrB2 operon and the cfa operon. These light-dependent characteristics suggest that class III LitR, in complex with a UV-A-absorbing molecule, follows a novel light-sensing mechanism.IMPORTANCE Members of the LitR/CarH family are adenosyl B12-based photosensory transcriptional regulator involved in light-inducible carotenoid production in nonphototrophic bacteria. Our study provides the first evidence of the involvement of a class III LitR, which lacks an adenosyl B12-binding domain in the light response of Burkholderia multivorans belonging to betaproteobacteria. Our biochemical analysis suggests that class III LitR protein exhibits features as a photosensor including absorption of light at the UV-A region (λmax = ca. 340 nm), photocyclic response, and light-dependent dissociation. This suggests that class III LitR associates with a UV-A-absorbing molecule, and it has a photosensing mechanism distinguishable from that of the B12-based type.

High production of a class III lantipeptide AmfS in Streptomyces griseus.

AmfS, a class III lantipeptide serves as a morphogen in Streptomyces griseus. Here, we constructed a high production system of AmfS in S. griseus. We isolated S. griseus Grd1 strain defective in glucose repression of aerial mycelium formation and found it suitable for the overproduction of AmfS. Two expression vectors carrying the strong and constitutive ermE2 promoter were constructed using a multicopy number plasmid, pIJ702. The use of the Grd1 strain combined with the expression vectors enabled high production of AmfS by S. griseus into its culture broth. The expression system was also effective for the generation of abundant AmfS derived from Streptomyces avermitilis. In addition, site-directed mutagenesis revealed the amino acid residues essential for the morphogen activity of AmfS. These results indicate that the constructed system enables efficient production of class III lantipeptides by Streptomyces.

Whole-Genome Sequence of Filimonas lacunae, a Bacterium of the Family Chitinophagaceae Characterized by Marked Colony Growth under a High-CO2 Atmosphere.

We report here the genome sequence of Filimonas lacunae, a bacterium of the family Chitinophagaceae characterized by high-CO2-dependent growth. The 7.81-Mb circular genome harbors many genes involved in carbohydrate degradation and related genetic regulation, suggesting the role of the bacterium as a carbohydrate degrader in diverse environments.

Role and Function of LitR, an Adenosyl B12-Bound Light-Sensitive Regulator of Bacillus megaterium QM B1551, in Regulation of Carotenoid Production.

UNLABELLED: The LitR/CarH family of proteins is a light-sensitive MerR family of transcriptional regulators that contain an adenosyl B12 (coenzyme B12 or AdoB12)-binding domain at the C terminus. The genes encoding these proteins are found in phylogenetically diverse bacterial genera; however, the biochemical properties of these proteins from Gram-positive bacteria remain poorly understood. We performed genetic and biochemical analyses of a homolog of the LitR protein from Bacillus megaterium QM B1551, a Gram-positive endospore-forming soil bacterium. Carotenoid production was induced by illumination in this bacterium. In vivo analysis demonstrated that LitR plays a central role in light-inducible carotenoid production and serves as a negative regulator of the light-inducible transcription of crt and litR itself. Biochemical evidence showed that LitR in complex with AdoB12 binds to the promoter regions of litR and the crt operon in a light-sensitive manner. In vitro transcription experiments demonstrated that AdoB12-LitR inhibited the specific transcription of the crt promoter generated by a σ(A)-containing RNA polymerase holoenzyme under dark conditions. Collectively, these data indicate that the AdoB12-LitR complex serves as a photoreceptor with DNA-binding activity in B. megaterium QM B1551 and that its function as a transcriptional repressor is fundamental to the light-induced carotenoid production. IMPORTANCE: Members of the LitR/CarH family are AdoB12-based photosensors involved in light-inducible carotenoid production in nonphototrophic Gram-negative bacteria. Our study revealed that Bacillus LitR in complex with AdoB12 also serves as a transcriptional regulator with a photosensory function, which indicates that the LitR/CarH family is generally involved in the light-inducible carotenoid production of nonphototrophic bacteria.

Analysis of the tryptophanase expression in Symbiobacterium thermophilum in a coculture with Geobacillus stearothermophilus.

The tryptophanase-positive Symbiobacterium thermophilum is a free-living syntrophic bacterium that grows effectively in a coculture with Geobacillus stearothermophilus. Our studies have shown that S. thermophilum growth depends on the high CO2 and low O2 condition established by the precedent growth of G. stearothermophilus. The use of an anoxic atmosphere containing high CO2 allows S. thermophilum to grow independently of G. stearothermophilus, but the cellular yield is ten times lower than that achieved in the coculture. In this study, we characterized the coculture-dependent expression and activity of tryptophanase in S. thermophilum. S. thermophilum cells accumulated a marked amount of indole in a coculture with G. stearothermophilus, but not in the bacterium's pure culture irrespective of the addition of tryptophan. S. thermophilum cells accumulated indole in its pure culture consisting of conditioned medium (medium supplied with culture supernatant of G. stearothermophilus). Proteomic analysis identified the protein specifically produced in the S. thermophilum cells grown in conditioned medium, which was a tryptophanase encoded by tna2 (STH439). An attempt to isolate the tryptophanase-inducing component from the culture supernatant of G. stearothermophilus was unsuccessful, but we did discover that the indole accumulation occurs when 10 mM bicarbonate is added to the medium. RT-PCR analysis showed that the addition of bicarbonate stimulated transcription of tna2. The transcriptional start site, identified within the tna2 promoter, was preceded by the -24 and -12 consensus sequences specified by an alternative sigma factor, σ(54). The evidence suggests that the transcription of some genes involved in amino acid metabolism is σ(54)-dependent, and that a bacterial enhancer-binding protein containing a PAS domain controls the transcription under the presence of high levels of bicarbonate.

Description of Symbiobacterium ostreiconchae sp. nov., Symbiobacterium turbinis sp. nov. and Symbiobacterium terraclitae sp. nov., isolated from shellfish, emended description of the genus Symbiobacterium and proposal of Symbiobacteriaceae fam. nov.

Three novel moderately anaerobic, thermophilic, rod-shaped bacterial strains, KY38(T), KY46(T) and KA13(T), were isolated from shellfish collected on the Pacific coastline of Enoshima, Japan. Phylogenetic analysis of the 16S rRNA gene sequences indicated that these bacteria belong to the genus Symbiobacterium, sharing sequence similarities of 97.8% (KY38(T)), 96.4% (KY46(T)) and 93.3% (KA13(T)) with the type strain of Symbiobacterium thermophilum, the only species of the genus with a validly published name. These isolates reduced nitrate and grew optimally at 55-60 °C. Strains KY38(T) and KA13(T) formed endospore-like structures in the terminal or subterminal part of their cells at low frequencies. Genomic DNA G+C contents were 68.8 (KY38(T)), 67.2 (KY46(T)) and 67.1 (KA13(T)) mol%. The isolates all presented the predominant menaquinone MK-6, major fatty acids iso-C15:0, C16:0 and iso-C17:0 and the major polar lipids phosphatidylglycerol, phosphatidylethanolamine and unknown glycol-containing phospholipids. On the basis of their morphological, physiological and phylogenetic properties, strains KY38(T), KY46(T) and KA13(T) represent three novel species, for which the names Symbiobacterium ostreiconchae sp. nov. (type strain KY38(T) = DSM 27624(T) = KCTC 4567(T) = JCM 15048(T)), Symbiobacterium turbinis sp. nov. (type strain KY46(T) = DSM 27625(T) = KCTC 4568(T) = JCM 15996(T)) and Symbiobacterium terraclitae sp. nov. (type strain KA13(T) = DSM 27138(T) = KCTC 4569(T) = JCM 15997(T)) are proposed. An emended description of the genus Symbiobacterium is also presented. The phylogenetic distinctiveness of the genus Symbiobacterium indicates its affiliation with a novel family, for which the name Symbiobacteriaceae fam. nov. is proposed.

Preparation of optically-active 3-pyrrolidinol and its derivatives from 1-benzoylpyrrolidinol by hydroxylation with Aspergillus sp. and stereo-selective esterification by a commercial lipase.

An effective preparation scheme for optically-active 3-pyrrolidinol and its derivatives based on biological transformation is proposed. Aspergillus sp. NBRC 109513 hydroxylated 1-benzoylpyrrolidine, yielding (S)-1-benzoyl-3-pyrrolidinol with 66 % ee. Kinetic resolution of 1-benzoyl-3-pyrrolidinol by Amano PS-IM lipase formed optically-active 1-benzoyl-3-pyrrolidinol with >99 % ee. (S)-1-Benzoyl-3-pyrrolidinol was successfully converted to 3-pyrrolidinol and its derivatives with by chemical reactions (>99 % ee).

Amide-transforming activity of Streptomyces: possible application to the formation of hydroxy amides and aminoalcohols.

To develop an efficient bioconversion process for amides, we screened our collection of Streptomyces strains, mostly obtained from soil, for effective transformers. Five strains, including the SY007 (NBRC 109343) and SY435 (NBRC 109344) of Streptomyces sp., exhibited marked conversion activities from the approximately 700 strains analyzed. These strains transformed diverse amide compounds such as N-acetyltetrahydroquinoline, N-benzoylpyrrolidine, and N-benzoylpiperidine into alcohols or N,O-acetals with high activity and regioselectivity. N,O-acetal was transformed into alcohol by serial tautomerization and reduction reactions. As such, Streptomyces spp. can potentially be used for the efficient preparation of hydroxy amides and aminoalcohols.

Divergent effects of desferrioxamine on bacterial growth and characteristics.

Desferrioxamines (DF's) are siderophores produced by some groups of bacteria. Previously, we discovered that DFE, produced by Streptomyces griseus, induced divergent developmental phenotypes in various Streptomyces isolates. In this study, we isolated bacteria whose phenotype was affected by the presence of 0.1 mM DFB from soil samples, and studied their phylogenetic position via 16 S rRNA gene-based analysis. Isolates belonging to Microbacterium grew only in the presence of DFB in the medium. DFB promoted growth of some isolates, while significantly inhibiting that of other divergent bacteria. Different groups of isolates were affected, not because of growth-related changes, but because of changes in the colony morphology based on possible stimulation of motility. An isolate affiliated with Janthinobacterium was stimulated for violacein production as well as for pilus formation. The wide and divergent effects of DFB suggest that availability of siderophores significantly affect the structure of microbial community.

Longispora fulva sp. nov., isolated from a forest soil, and emended description of the genus Longispora.

A novel actinomycete, strain KZ0017(T), was isolated from a forest soil collected in Ohnuma, Fukushima, Japan. Strain KZ0017(T) formed spore chains borne on top of short sporophores arising from vegetative hyphae. Spores were non-motile and cylindrical with smooth surfaces. Strain KZ0017(T) contained meso-diaminopimelic (A(2)pm) acid, 3-OH A(2)pm, d-glutamic acid, glycine and l-alanine in the cell-wall peptidoglycan, and xylose, mannose, galactose, rhamnose and ribose in cell-wall hydrolysates. The acyl type of the cell-wall polysaccharides was glycolyl. The predominant menaquinones were MK-10(H(4)) and MK-10(H(6)); MK-10(H(8)) was a minor component. The polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, phosphatidylinositol and several unknown lipids and glycolipids. The major fatty acids were iso-C(16 : 0), 10-methyl-C(17 : 0) and iso-C(17 : 1)ω9c. The DNA G+C content was 70.7 mol%. The 16S rRNA gene sequence of the isolate formed a monophyletic cluster with the single member of the genus Longispora in the family Micromonosporaceae. On the basis of morphological, chemotaxonomic and phylogenetic properties, strain KZ0017(T) represents a novel species of the genus Longispora, for which the name Longispora fulva sp. nov. is proposed; the type strain is KZ0017(T) ( = NBRC 105670(T) = DSM 45356(T)).