Genetic organization of genes encoding phenol hydroxylase,benzoate 1,2-dioxygenase alpha subunit and its regulatory proteins in Acinetobacter calcoaceticus PHEA-2

Acinetobacter calcoaceticus PHEA-2 is a phenol-degrading bacterium isolated from the wastewater from an oil refinery. A 10-kb XhoI fragment consisting of nine complete Open Reading Frames (ORFs) and one partial ORF was screened from a lambda library of PHEA-2 by Southern hybridization. The sequence analyses revealed that ORF2-ORF7, designated mphKLMNOP, are homologous to dmpKLMNOP of Pseudomonas sp. CF600 and mopKLMNOP of Acinetobacter calcoaceticus NCIB8250, sharing 38%-72% and 58.5%-93.5% respectively. The products encoded by dmp and mop genes convert phenol to catechol. The mph-operon and downstream ORFs, ORF9 and ORF10, sharing high identities to benM and benA, which encode ben-operon regulatory protein and benzoate 1,2-dioxygenase alpha subunit respectively, are separated by ORF8, whose function is unknown. The organization of the mph and ben operons is different from that described previously.     

Targeted gene inactivation for the elucidation of deoxysugar biosynthesis in the erythromycin producer Saccharopolyspora erythraea

The production of erythromycin A by Saccharopolysporaerythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-α-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV ), ORF17 (eryCIV ) and ORF7 (eryBII ) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates. Received: 29 July 1997 / Accepted: 16 October 1997     

Single-culture aerobic granules with <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

Aerobic granules are cultivated by a single bacterial strain, Acinetobacter calcoaceticus, in a sequencing batch reactor (SBR). This strain presents as a good phenol reducer and an efficient auto coagulator in the presence of phenol, mediated by heat-sensitive adhesins proteins. Stable 2.3-mm granules were formed in the SBR following a 7-week cultivation. These granules exhibit excellent settling attributes and degrade phenol efficiently at concentrations of 250–2,000 mg l⁻¹. The corresponding phenol degradation rate reached 993.6 mg phenol g⁻¹ volatile suspended solids (VSS) day⁻¹ at 250 mg l⁻¹ phenol and 519.3 mg phenol g⁻¹ VSS day⁻¹ at 2,000 mg l⁻¹ phenol concentration. Meanwhile, free A. calcoaceticus cells were fully inhibited at phenol >1,500 mg l⁻¹. Denaturing gradient gel electrophoresis fingerprint profile demonstrated no genetic modification in the strain during aerobic granulation. The present single-strain granules showed long-term structural stability and performed high phenol degrading capacity and high phenol tolerance. The confocal laser scanning microscopic test revealed that live A. calcoaceticus cells principally distributed at 200–250 μm beneath the outer surface, with an extracellular polymeric substance layer covering them to defend phenol toxicity. Autoaggregation assay tests demonstrated the possibly significant role of secreted proteins on the formation of single-culture A. calcoaceticus granules.     

Channa nox, a new channid fish lacking a pelvic fin from Guangxi, China

 A new species of channid fish, genus Channa, is described from 7 specimens collected from the vicinity of Hepu, Guangxi Province, southern China. The new species, Channa nox, is distinguished from all other channid species by the following combination of characters: absence of pelvic fins, small rounded head (22.1%–26.8% SL), narrow interorbital width (19.6%–26.7% HL), short snout length (3.6%–5.1% SL), predorsal and prepectoral lengths (26.9%–28.4% SL and 24.8%–28.3% SL, respectively), 47–51 dorsal fin rays, 31–33 anal fin rays, 55–63 lateral line scales, 5.5–6.5 scales above lateral line, 9–13 cheek scales, 53–55 total vertebrae, 1 or 2 scale(s) on each side of lower jaw undersurface, the black upper half of body with 8–11 irregular (often anteriorly pointed V-shaped) bands or blotches, a large white-rimmed black ocellus on caudal peduncle and sparse white spots on the dark brown body and dorsal and caudal fins, as well as the shape of the hyomandibular process of the suprabranchial organs. Channa nox is sympatrically distributed with its morphologically most similar congener, C. asiatica. Received: January 18, 2001 / Revised: November 2, 2001 / Accepted: December 12, 2001     

<Emphasis Type="Italic">Acinetobacter brisouii</Emphasis> sp. nov., isolated from a wetland in Korea

A bacterial strain 5YN5-8^T was isolated from peat layer on Yongneup in Korea. Cells of strain 5YN5-8^T were strictly aerobic, Gram-negative, coccobacilli, non-spore forming, and non-motile. The isolate exhibited optimal growth at 28°C, pH 7.0, and 0–1% NaCl. Results of 16S rRNA gene sequence analyses indicated a close relationship of this isolate to Acinetobacter calcoaceticus (97.8% similarity for strain DSM 30006^T). It also exhibited 94.4–97.8% 16S rRNA gene sequence similarities to the validly published Acinetobacter species. The value for DNA-DNA hybridization between strain 5YN5-8^T and other members of the genus Acinetobacter ranged from 16 to 28%. Predominant cellular fatty acids were C_18:1 ω9c, summed feature 4 containing C_15:0 iso 2-OH and/or C_16:1 ω7c, and C_16:0. The DNA G+C content was 43.9 mol%. Phylogenetic, phenotypic, and chemotaxonomic data accumulated in this study revealed that the isolate could be classified in a novel species of the genus Acinetobacter. The name Acinetobacter brisouii sp. nov. is proposed for the novel species, with 5YN5-8^T (=KACC 11602^T = DSM 18516^T) as the type strain.     

Characterization of alginate lyase gene using a metagenomic library constructed from the gut microflora of abalone

A metagenomic fosmid library was constructed using a genomic DNA mixture extracted from the gut microflora of abalone. The library gave an alginate lyase positive clone (AlyDW) harboring a 31.7-kbp insert. The AlyDW insert consisted of 22 open reading frames (ORFs). The deduced amino acid sequences of ORFs 11–13 were similar to those of known alginate lyase genes, which are found adjacent in the genome of Klebsiella pneumoniae subsp. aerogenes, Vibrio splendidus, and Vibrio sp. belonging to the phylum Gammaproteobacteria. Among the three recombinant proteins expressed from the three ORFs, alginate lyase activity was only observed in the recombinant protein (AlyDW11) coded by ORF 11. The expressed protein (AlyDW11) had the highest alginate lyase activity at pH 7.0 and 45°C in the presence of 1 mM AgNO₃. The alginate lyase activity of ORF 11 was confirmed to be endolytic by thin-layer chromatography. AlyDW11 preferred poly(β-d-mannuronate) as a substrate over poly(α-l-guluronate). AlyDW11 contained three highly conserved regions, RSEL, QIH, and YFKAGVYNQ, which may act to stabilize the three-dimensional conformation and function of the alginate lyase.     

Sequence analysis of segment 8 of five Chinese isolates of Rice gall dwarf virus and expression of a main outer capsid protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8 (S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%–98.8% and 97.3%–99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%–95.6% and 95.0%–96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta (DE3) II cells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression. Foundation item: National natural science foundation of China (30370929) and Guangdong province natural science foundation (C036845)     

Isolation and Characterization of Arsenic-Resistant Bacteria from Contaminated Water-Bodies in West Bengal,India

Bengal Basin is known for severe arsenic contamination. In the present study, we have isolated six bacteria from the arsenic contaminated surface water of Bengal Basin. 16S rDNA sequence analysis identified them as Microbacterium oleivorans, Acinetobacter soli, Acinetobacter venetianus, Acinetobacter junii, Acinetobacter baumannii, Acinetobacter calcoaceticus. All the isolates possess arsenic accumulation potential and high molecular weight plasmid (>10 kb). PCR amplification indicated the presence of arsenic-resistance genes (arsB and aoxB) either in the genome or plasmid or in both in the isolated bacteria (except in Acinetobacter venetianus). Exposure to arsenic affected bacterial growth and induced alteration in cytoplasmic membrane integrity.     

Influence of growth phase and carbon source on the content of rubredoxin in Acinetobacter calcoaceticus

The rubredoxin content of Acinetobacter calcoaceticus in dependence on the carbon source (acetate, n-alkanes, succinate, L-malate) and on the growth phase was studied by means of a radioimmunoassay. The method used was specific for rubredoxin from Acinetobacter calcoaceticus. The formation of rubredoxin increased with time up to the end of the logarithmic phase when n-alkanes were the sole carbon source. After growth of Acinetobacter calcoaceticus on non-hydrocarbon substrates, rubredoxin was not detected.     

A Novel Gene Cluster soxSRT Is Essential for the Chemolithotrophic Oxidation of Thiosulfate and Tetrathionate by Pseudaminobacter salicylatoxidans KCT001

Chemolithotrophic sulfur oxidation (Sox) in the α-proteobacterium Pseudaminobacter salicylatoxidans KCT001 was found to be governed by the gene cluster soxSRT–soxVWXYZABCD. Independent transposon-insertion mutations in the genes soxB, soxC, soxD, and also in a novel open reading frame (ORF), designated as soxT, afforded revelation of the entire sox locus of this bacterium. The deduced amino acid sequence of the novel ORF soxT comprised 362 residues and exhibited significant homology with hypothetical proteins of diverse origin, including a permease-like transport protein of Escherichia coli. Two contiguous ORFs, soxR and soxS, immediately preceded the soxT gene. The gene cluster soxSRT was located upstream of soxVWXYZABCD and was transcribed divergently with respect to the latter. Chemolithotrophic utilization of both thiosulfate and tetrathionate was observed to have been impaired in all of these Sox⁻ mutants, implicating the involvement of the gene cluster soxSRT–soxVWXYZABCD in the oxidation of both thiosulfate and tetrathionate. Pradosh Roy Deceased     

Characterization of interleukin-8 receptors in non-human primates

Interleukin-8 is a chemokine with a potent neutrophil chemoatractant activity. In humans, two different cDNAs encoding human IL8 receptors designated IL8RA and IL8RB have been cloned. IL8RA binds IL8, while IL8RB binds IL8 as well as other α-chemokines. Both human IL8Rs are encoded by two genes physically linked on chromosome 2. TheIL8RA andIL8RB genes have open reading frames (ORF) lacking introns. By direct sequencing of the polymerase chain reaction products, we sequenced theIL8R genes of cell lines from four non-human primates: chimpanzee, gorilla, orangutan, and macaca. TheIL8RB encodes an ORF in the four non-human primates, showing 95%–99% similarity to the human IL8RB sequence. TheIL8RA homologue in gorilla and chimpanzee consisted of two ORF 98%–99% identical to the human sequence. The macaca and orangutanIL8RA homologues are pseudogenes: a 2 base pair insertion generated a sequence with several stop codons. In addition, we describe the physical linkage of these genes in the four non-human primates and discuss the evolutionary implications of these findings. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence databases and have assigned the accession numbers X91109 (PTIL8RA), X91110 (GGIL8RA), X91111 (PPIL8Ra), X91112 (MMIL8RA), X91113 (PTIL8RB), X9114 (GGIL8RB), X91115 (PPIL8RB), and X91116 (MMIL8RB)     

Biodegradation of mixture containing monohydroxybenzoate isomers by <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

A bacterial strain capable of utilizing a mixture containing 2-hydroxybenzoic acid (2-HBA), 3-hydroxybenzoic acid (3-HBA) and 4-hydroxybenzoic (4-HBA) acid was isolated through enrichment from a soil sample. Based on 16SrDNA sequencing, the microorganism was identified as Acinetobacter calcoaceticus. The sequence of biodegradation of the three isomers when provided as a mixture (0.025%, w/v each) was elucidated. The dihydroxylated metabolites formed from the degradation of 2-HBA, 3-HBA and 4-HBA were identified as catechol, gentisate and protocatechuate, respectively, using the cell-free supernatant and cell-free crude extracts. Monooxygenases and dioxygenases that were induced in the cells of Acinetobacter calcoaceticus in response to growth on mixture containing 2-HBA, 3-HBA and 4-HBA could be detected in cell-free extracts. These data revealed the pathways operating in Acinetobacter calcoaceticus for the sequential metabolism of monohydroxybenzoate isomers when presented as a mixture.     

Organization and Sequence of Histidine Biosynthesis Genes hisH, -A, -F, and -IE in Thermoanaerobacter ethanolicus

Nucleotide sequence analysis of a 3.5-kb chromosomal fragment from the low G + C Gram-positive bacterium Thermoanaerobacter ethanolicus revealed a cluster of five contiguous open reading frames (ORFs) designated hisH, hisA, hisF, hisIE, and ORF5. The first four ORFs showed homology to genes of the histidine biosynthesis pathway, and ORF5 encoded a product with no significant similarities to polypeptides presently known. The hisH ORF was partial (truncated by cloning) and ORF5 was adjacent to xylF, which codes for a xylose-binding periplasmic protein. The five genes encoded putative proteins of >104, 237, 254, 216, and 169 amino acids, respectively. Amino acid sequence comparison of the four his gene products indicated closely related homologs in prokaryotes, varying from low G + C Gram-positive bacteria to archaea. This is the first report of his anabolic genes in a thermophilic anaerobic bacterium. Received: 8 July 1999 / Accepted: 23 August 1999