[Microbial Community Structure of Activated Sludge for Total Nitrogen Upgrading Project].

The activated sludge of a biochemical unit (WLK_OD) and an advanced denitrification unit (WLK_AD) were collected from a municipal wastewater treatment plant (WWTP), in which the TN concentration of effluent was less than 1.5 mg·L-1, and their microbial community structure and function profiles were analyzed using 16S rRNA gene high-throughput sequencing. The microorganisms in WLK_AD had lower evenness compared with that in WLK_OD, which was attributed to environmental selection. Furthermore, PCoA revealed that different incoming wastewaters had an impact on microbial community structure. At the phylum level, Proteobacteria (70.11%) was enriched in WLK_AD. At the genus level, Thauera, Flavobacterium, Hydrogenophaga, and Zoogloea served as distinct-dominant denitrifying bacteria in WLK_AD; however, Trichococcus (3.50%) and Terrimonas (1.10%) were enriched in WLK_OD. Through the comparison between groups (P<0.05), the biomarkers detected in each WWTP were different. Furthermore, the results of the co-occurrence network showed that the bacteria from module I had a higher proportion in WLK_AD; the bacteria from module II had a higher proportion in WLK_OD, and they were common microorganisms in WWTPs, implying that wastewater environments drpve the differences in the microbial community structure. Among the types of environmental parameters, the removal efficiency of COD and TN had the greatest impact on the microbial community by the RDA. The removal efficiency of COD was positively correlated with the dominant bacteria from WLK_OD, such as Saccharibacteria, Thermomarinilinea, Terrimonas, and Comamonas; the removal efficiency of TN was positively correlated with the denitrifying bacteria from WLK_AD, such as Dokdonella, Thauera, Flavobacterium, and Zoogloea. WLK_AD was enriched with Novosphingobium, Dokdonella, Thauera, and Sphingomonas, which synergistically removed TN, leading to the TN of the effluent being less than 1.5 mg·L-1. Moreover, based on the results of function prediction, WLK_AD had a higher proportion of genes that could code the denitrification enzymes.

Engineering potassium activation into biosynthetic thiolase.

Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation-π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation-π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.

Genome analysis provides insights into microaerobic toluene-degradation pathway of Zoogloea oleivorans BucT.

Zoogloea oleivorans, capable of using toluene as a sole source of carbon and energy, was earlier found to be an active degrader under microaerobic conditions in aquifer samples. To uncover the genetic background of the ability of microaerobic toluene degradation in Z. oleivorans, the whole-genome sequence of the type strain BucT was revealed. Metatranscriptomic sequence reads, originated from a previous SIP study on microaerobic toluene degradation, were mapped on the genome. The genome (5.68 Mb) had a mean G + C content of 62.5%, 5005 protein coding gene sequences and 80 RNA genes. Annotation predicted that 66 genes were involved in the metabolism of aromatic compounds. Genome analysis revealed the presence of a cluster with genes coding for a multicomponent phenol-hydroxylase system and a complete catechol meta-cleavage pathway. Another cluster flanked by mobile-element protein coding genes coded a partial catechol meta-cleavage pathway including a subfamily I.2.C-type extradiol dioxygenase. Analysis of metatranscriptomic data of a microaerobic toluene-degrading enrichment, containing Z . oleivorans as an active-toluene degrader revealed that a toluene dioxygenase-like enzyme was responsible for the ring-hydroxylation, while enzymes of the partial catechol meta-cleavage pathway coding cluster were responsible for further degradation of the aromatic ring under microaerobic conditions. This further advances our understanding of aromatic hydrocarbon degradation between fully oxic and strictly anoxic conditions.

Nitrogen Removal Characteristics of a Newly Isolated Indigenous Aerobic Denitrifier from Oligotrophic Drinking Water Reservoir, Zoogloea sp. N299.

Nitrogen is considered to be one of the most widespread pollutants leading to eutrophication of freshwater ecosystems, especially in drinking water reservoirs. In this study, an oligotrophic aerobic denitrifier was isolated from drinking water reservoir sediment. Nitrogen removal performance was explored. The strain was identified by 16S rRNA gene sequence analysis as Zoogloea sp. N299. This species exhibits a periplasmic nitrate reductase gene (napA). Its specific growth rate was 0.22 h-1. Obvious denitrification and perfect nitrogen removal performances occurred when cultured in nitrate and nitrite mediums, at rates of 75.53%±1.69% and 58.65%±0.61%, respectively. The ammonia removal rate reached 44.12%±1.61% in ammonia medium. Zoogloea sp. N299 was inoculated into sterilized and unsterilized reservoir source waters with a dissolved oxygen level of 5-9 mg/L, pH 8-9, and C/N 1.14:1. The total nitrogen removal rate reached 46.41%±3.17% (sterilized) and 44.88%±4.31% (unsterilized). The cell optical density suggested the strain could survive in oligotrophic drinking water reservoir water conditions and perform nitrogen removal. Sodium acetate was the most favorable carbon source for nitrogen removal by strain N299 (p<0.05). High C/N was beneficial for nitrate reduction (p<0.05). The nitrate removal efficiencies showed no significant differences among the tested inoculums dosage (p>0.05). Furthermore, strain N299 could efficiently remove nitrate at neutral and slightly alkaline and low temperature conditions. These results, therefore, demonstrate that Zoogloea sp. N299 has high removal characteristics, and can be used as a nitrogen removal microbial inoculum with simultaneous aerobic nitrification and denitrification in a micro-polluted reservoir water ecosystem.

Zoogloea oleivorans sp. nov., a floc-forming, petroleum hydrocarbon-degrading bacterium isolated from biofilm.

A floc-forming, Gram-stain-negative, petroleum hydrocarbon-degrading bacterial strain, designated Buc(T), was isolated from a petroleum hydrocarbon-contaminated site in Hungary. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Buc(T) formed a distinct phyletic lineage within the genus Zoogloea. Its closest relative was found to be Zoogloea caeni EMB43(T) (97.2% 16S rRNA gene sequence similarity) followed by Zoogloea oryzae A-7(T) (95.9%), Zoogloea ramigera ATCC 19544(T) (95.5%) and Zoogloea resiniphila DhA-35(T) (95.4%). The level of DNA-DNA relatedness between strain Buc(T) and Z. caeni EMB43(T) was 31.6%. Cells of strain Buc(T) are facultatively aerobic, rod-shaped, and motile by means of a polar flagellum. The strain grew at temperatures of 5-35 °C (optimum 25-28 °C), and at pH 6.0-9.0 (optimum 6.5-7.5). The predominant fatty acids were C16:0, C10 : 0 3-OH, C12:0 and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The major respiratory quinone was ubiquinone-8 (Q-8) and the predominant polar lipid was phosphatidylethanolamine. The genomic DNA G+C content was 63.2 mol%. On the basis of the chemotaxonomic, molecular and phenotypic data, isolate Buc(T) is considered to represent a novel species of the genus Zoogloea, for which the name Zoogloea oleivorans sp. nov. is proposed. The type strain is Buc(T) ( =DSM 28387(T) =NCAIM B 02570(T)).

Impacts of single-walled carbon nanotubes on microbial community structure in activated sludge.

AIMS: Single-walled carbon nanotubes (SWNTs) are likely to become increasingly widespread and yet their environmental impact is not well understood. The purpose of the current study was to evaluate the impact of SWNTs on microbial communities in a 'sentinel' environmental system, activated sludge batch-scale reactors. METHODS AND RESULTS: Triplicate batch reactors were exposed to SWNTs and compared to control reactors exposed to impurities associated with SWNTs. Automated ribosomal intergenic spacer analysis (ARISA) was used to assess bacterial community structure in each reactor. SWNT exposure was found to impact microbial community structure, while SWNT-associated impurities had no effect, compared to controls. 16S rRNA gene sequence analysis indicated that dominant phylotypes detected by ARISA included members of the families Sphingomonadaceae and Cytophagacaceae and the genus Zoogloea. ARISA results indicated an adverse impact of SWNTs on the sphingomonad relative to other community members. Changes in community structure also occurred in both SWNT-exposed and control reactors over the experimental time period and with the date on which activated sludge was obtained from a wastewater treatment facility. CONCLUSIONS: These results indicate that SWNTs differentially impact members of the activated sludge reactor bacterial community. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding that community structure was affected by SWNTs indicates that this emerging contaminant differentially impacted members of the activated sludge bacterial community and raises the concern that SWNTs may also affect the services it provides.

Zoogloea caeni sp. nov., a floc-forming bacterium isolated from activated sludge.

Two floc-forming, nitrogen-fixing bacteria, strains EMB43(T) and EMB61, obtained from activated sludge of a domestic wastewater treatment plant in Korea, were characterized. The two strains were very closely related, sharing 99.7 % 16S rRNA gene sequence similarity and showing a level of DNA-DNA relatedness of 93 %, which suggests that they represent members of a single species. Phylogenetic analysis based on 16S rRNA gene sequences showed that the two novel isolates formed a distinct phyletic lineage within the genus Zoogloea and were related most closely to Zoogloea resiniphila DhA-35(T) and Zoogloea oryzae A-7(T), with sequence similarities of 97.2 %. Levels of DNA-DNA relatedness between strain EMB43(T) and Z. resiniphila DhA-35(T) and Z. oryzae A-7(T) were 12.8 and 7.4 %, respectively. Cells of strains EMB43(T) and EMB61 were facultatively aerobic, rod-shaped, Gram-negative and motile by means of a polar flagellum. The strains grew at temperatures of 15-40 degrees C (optimum: 25-30 degrees C) and at pH 6.0-9.0 (optimum: pH 6.5-7.5). The predominant fatty acids were C(16 : 0), C(10 : 0) 3-OH and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH), and the predominant polar lipid was phosphatidylethanolamine. The genomic DNA G+C content was 64.9-65.0 mol% and the major isoprenoid quinone was ubiquinone-8 (Q-8). On the basis of phenotypic, chemotaxonomic and molecular data, the isolates are considered to represent a novel species of the genus Zoogloea, for which the name Zoogloea caeni sp. nov. is proposed. The type strain is EMB43(T) (=KCTC 22084(T)=DSM 19389(T)).

Zoogloea oryzae sp. nov., a nitrogen-fixing bacterium isolated from rice paddy soil, and reclassification of the strain ATCC 19623 as Crabtreella saccharophila gen. nov., sp. nov.

Two strains of free-living diazotrophs isolated from soil from a rice paddy field were characterized by using a polyphasic approach. The novel strains, A-7T and A-4, were found to be very closely related, with 99.9% 16S rRNA gene sequence similarity and a DNA-DNA hybridization value of 89.5%, suggesting that they represent a single species. 16S rRNA gene sequence analyses indicated that the two strains fell within the Zoogloea lineage, with less than 96.7 % sequence similarity to other Zoogloea species. Chemotaxonomic characteristics of the novel strains, including DNA G + C content (65.1 mol%), the major quinone system (Q-8), predominant fatty acids (16:1omega7c and 16:0) and major hydroxy fatty acids (3-OH 10:0 and 3-OH 12:0), are similar to those of the genus Zoogloea. The novel strains showed positive results for floc formation which is accepted as confirmatory for species of the genus Zoogloea. However, the novel strains can be distinguished from the other species of Zoogloea by physiological characteristics. The name Zoogloea oryzae sp. nov. is therefore proposed for the novel strains with strain A-7T (= IAM 15218T = CCTCC AB 2052005T) as the type strain. Phylogenetic and chemotaxonomic analyses indicate that strain ATCC 19623, designated as a reference strain of Zoogloea ramigera, does not belong to the genus Zoogloea but to a new genus of Alphaproteobacteria. The name Crabtreella saccharophila gen. nov., sp. nov. is proposed for strain ATCC 19623T (= IAM 12669T).

Identification and characterization of poly-3-hydroxybutyrate granule-associated protein, PGA12 and PGA16 in Zoogloea ramigera I-16-M.

Poly-3-hydroxybutyrate (PHB) granules of Zoogloea ramigera I-16-M contained two major PHB granule-associated proteins (PGA12 and PGA16) as revealed by sodium dodecyl sulfate-polyacrylamide gel elecrophoresis. N-terminal amino acid sequences of these proteins were determined. The genes encoding these proteins were cloned and sequenced. The structural genes of PGA12 and PGA16 were 351 and 447 bp long, which encode polypeptides with deduced molecular masses of 12.3 and 16.0 kDa, respectively. PGA12 and PGA16 were expressed in Escherichia coli. PHB granules were isolated from cells of recombinant strains of E. coli JM109, which harbored and expressed the PHB-synthetic genes of Ralstonia eutropha H16 and PGA12 or PGA16. These PHB granules contained PGA12 or PGA16 as a major protein. The presence of pga12 or pga16 did not affect the amount of PHB synthesized in E. coli. PGA12 and PGA16 bound to crystalline and amorphous PHB granules.

Immunological methods for the study of Zoogloea strains in natural environments.

Since Zoogloea ramigera has been considered to be important in aerobic wastewater treatment, we have evaluated several methods for detecting and enumerating Z. ramigera in water and wastewater samples. Indirect immunoassay methods for the detection of Zoogloea strains were developed using polyclonal antibodies against the cells or the isolated exocellular polymer (EP) of the neotype Zoogloea ramigera strain 106 (ATCC 19544). The primary antibodies reacted with the cells and the exopolymer associated with finger-like zoogloeal projections, but not with other bacteria from natural samples. These antibodies allowed detection of Z. ramigera in environmental samples. Scanning electron microscopy (SEM) was used to show that the cells and the exocellular polymer of naturally occurring zoogloeal projections are antigenically and structurally related to those of Z. ramigera 106. Both immunological procedures and probes complementary to regions on the 16S rRNA could detect Z. ramigera in natural samples but the immunological procedures were easier to use. RT-PCR was also used to detect Z. ramigera in natural samples. These methods were also used to identify Z. ramigera in biofilms that developed over wastewater samples as part of an MPN procedure that was used to quantitate Z. ramigera at different stages of the wastewater treatment process and in different lakes. Z. ramigera could be found in all stages of wastewa ter treatment processes, from raw wastewater to chlorinated effluent, The highest concentration of Z. ramigera was found in the mixed liquor stage of the a wastewater treatment plant. Additionally, Z. ramigera was found in all eutrophic and mesotrophic lakes and in some oligotrophic lakes.

A biosynthetic thiolase in complex with a reaction intermediate: the crystal structure provides new insights into the catalytic mechanism.

BACKGROUND: Thiolases are ubiquitous and form a large family of dimeric or tetrameric enzymes with a conserved, five-layered alphabetaalphabetaalpha catalytic domain. Thiolases can function either degradatively, in the beta-oxidation pathway of fatty acids, or biosynthetically. Biosynthetic thiolases catalyze the biological Claisen condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in a wide range of biosynthetic pathways, including those that generate cholesterol, steroid hormones, and various energy-storage molecules. RESULTS: The crystal structure of the tetrameric biosynthetic thiolase from Zoogloea ramigera has been determined at 2.0 A resolution. The structure contains a striking and novel 'cage-like' tetramerization motif, which allows for some hinge motion of the two tight dimers with respect to each other. The protein crystals were flash-frozen after a short soak with the enzyme's substrate, acetoacetyl-CoA. A reaction intermediate was thus trapped: the enzyme tetramer is acetylated at Cys89 and has a CoA molecule bound in each of its active-site pockets. CONCLUSIONS: The shape of the substrate-binding pocket reveals the basis for the short-chain substrate specificity of the enzyme. The active-site architecture, and in particular the position of the covalently attached acetyl group, allow a more detailed reaction mechanism to be proposed in which Cys378 is involved in both steps of the reaction. The structure also suggests an important role for the thioester oxygen atom of the acetylated enzyme in catalysis.

Proposal to reclassify Zoogloea ramigera IAM 12670 (P. R. Dugan 115) as Duganella zoogloeoides gen. nov., sp. nov.

The taxonomic position of a misclassified strain, Zoogloea remigera IAM 12670T (= ATCC 25925T = P. R. Dugan 115T), was reevaluated. A phylogenetic analysis based on 16S ribosomal rDNA sequences revealed that this organism was located in the beta subclass of the class Proteobacteria with members of the genus Telluria as its closest relatives. On the basis of phenotypic and phylogenetic information, we propose that this organism should be reclassified in a new taxon with the name Duganella zoogloeoides gen. nov., sp. nov.

Diversity and distribution of subterranean bacteria in groundwater at Oklo in Gabon, Africa, as determined by 16S rRNA gene sequencing.

This paper describes how ground water was sampled, DNA extracted, amplified and cloned and how information available in the ribosomal 16S rRNA gene was used for mapping diversity and distribution of subterranean bacteria in groundwater at the Bangombé site in the Oklo region. The results showed that this site was inhabited by a diversified population of bacteria. Each borehole was dominated by species that did not dominate in any of the other boreholes; a result that probably reflects documented differences in the geochemical environment. Two of the sequences obtained were identified at genus level to represent Acinetobacter and Zoogloea, but most of the 44 sequences found were only distantly related to species in the DNA database. The deepest borehole, BAX01 (105 m), had the highest number of bacteria and also total organic carbon (TOC). This borehole harboured only Proteobacteria beta group sequences while sequences related to Proteobacteria beta, gamma and delta groups and Gram-positive bacteria were found in the other four boreholes. Two of the boreholes, BAX02 (34 m) and BAX04 (10 m) had many 16S rRNA gene sequences in common and also had similar counts of bacteria, content of TOC, pH and equal conductivity, suggesting a hydraulic connection between them.

Bacteria obtained from a sequencing batch reactor that are capable of growth on dehydroabietic acid.

Eleven isolates capable of growth on the resin acid dehydroabietic acid (DhA) were obtained from a sequencing batch reactor designed to treat a high-strength process stream from a paper mill. The isolates belonged to two groups, represented by strains DhA-33 and DhA-35, which were characterized. In the bioreactor, bacteria like DhA-35 were more abundant than those like DhA-33. The population in the bioreactor of organisms capable of growth on DhA was estimated to be 1.1 x 10(6) propagules per ml, based on a most-probable-number determination. Analysis of small-subunit rRNA partial sequences indicated that DhA-33 was most closely related to Sphingomonas yanoikuyae (Sab = 0.875) and that DhA-35 was most closely related to Zoogloea ramigera (Sab = 0.849). Both isolates additionally grew on other abietanes, i.e., abietic and palustric acids, but not on the pimaranes, pimaric and isopimaric acids. For DhA-33 and DhA-35 with DhA as the sole organic substrate, doubling times were 2.7 and 2.2 h, respectively, and growth yields were 0.30 and 0.25 g of protein per g of DhA, respectively. Glucose as a cosubstrate stimulated growth of DhA-33 on DhA and stimulated DhA degradation by the culture. Pyruvate as a cosubstrate did not stimulate growth of DhA-35 on DhA and reduced the specific rate of DhA degradation of the culture. DhA induced DhA and abietic acid degradation activities in both strains, and these activities were heat labile. Cell suspensions of both strains consumed DhA at a rate of 6 mumol mg of protein-1 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)

The abundance of Zoogloea ramigera in sewage treatment plants.

Zoogloea ramigera has long been considered the typical activated sludge bacterium responsible for the formation of activated sludge flocs. On the basis of the results of a comparative sequence analysis, we designed three oligonucleotide probes complementary to characteristic regions of the 16S rRNAs of Z. ramigera ATCC 19544T (T = type strain) and two misclassified strains, Z. ramigera ATCC 25935 and ATCC 19623. Dissociation temperatures were determined, and probe specificities, as well as the potential of probes for whole-cell hybridization, were evaluated by using numerous reference organisms. Several activated sludge samples were examined with these probes by using both the in situ and dot blot hybridization methods. Only the type strain probe hybridized to cells that accumulated in the typical branched gelatinous matrices, the so-called Zoogloea fingers. This probe revealed cells in most of the activated sludge samples studied. We found that relatively high levels of Z. ramigera cells (up to approximately 10% of the total number of cells) and typical morphology tended to be linked to overloading of sewage plants. The probe directed to rejected type strain Z. ramigera ATCC 19623 bound to only a few cells. Cells that reacted with the probe complementary to Z. ramigera ATCC 25935, which was originally isolated from a trickling filter, were not observed in activated sludge.

Molecular systematics of the genus Zoogloea and emendation of the genus.

Phylogenetic relationships among strains of Zoogloea and related taxa were determined by 16S rDNA sequencing and genomic DNA hybridization techniques. The 16S rRNA gene was amplified by the polymerase chain reaction with a pair of eubacterial consensus primers and sequenced directly by using an automated fluorescent DNA sequencer. Sequence comparisons and distance matrix tree analysis revealed that Zoogloea ramigera IAM 12136 (= N. C. Dondero 106, type strain) and Zoogloea sp. ATCC 19324 formed a lineage with Rhodocyclus purpureus in the beta subclass of Proteobacteria. Z. ramigera IAM 12670 (= P. R. Dugan 115) was shown to belong to another cluster with Alcaligenes eutrophus and Pseudomonas cepacia in the beta subclass. In contrast, Z. ramigera IAM 12669 (= K. Crabtree I-16-M) proved to be a member of the alpha subclass of the Proteobacteria, closely related to Agrobacterium tumefaciens. Genomic DNA hybridization studies also showed that there is genetic diversity among the strains currently designated Z. ramigera, but typical Zoogloea strains, characterized by their production of rhodoquinones, are highly related to each other and can be regarded as a single species. On the basis of the molecular data, together with the early phenotypic and chemotaxonomic information, we have emended the generic description of Zoogloea.

The NH2-terminal 14-16 amino acids of mitochondrial and bacterial thiolases can direct mature ornithine carbamoyltransferase into mitochondria.

Unlike most mitochondrial matrix proteins, the mitochondrial 3-oxoacyl-CoA thiolase [EC 2.3.1.16] is synthesized with no cleavable presequence and possesses information for mitochondrial targeting and import in the mature protein. This mitochondrial thiolase is homologous with the mature portion of peroxisomal 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase [EC 2.3.1.9] of Zoogloea ramigera along the entire sequence. A hybrid gene encoding the NH2-terminal 16 residues (MALLRGVFIVAAKRTP) of the mitochondrial thiolase fused to the mature portion of rat ornithine carbamoyltransferase [EC 2.1.3.3] (lacking its own presequence) was transfected into COS cells, and subcellular localization of the fusion protein was analyzed. Cell fractionation and immunocytochemical analyses showed that the fusion protein was localized in the mitochondria. These results indicate that the NH2-terminal 16 residues of the mitochondrial thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. The fusion protein containing the NH2-terminal 14 residues (MSTPSIVIASARTA) of the bacterial thiolase was also localized in the mitochondria. On the other hand, the fusion protein containing the corresponding portion (MQASASDVVVVHGQRTP) of the peroxisomal thiolase appeared not to be localized to the mitochondria. These results show that the import signal of mitochondrial 3-oxoacyl-CoA thiolase originated from the NH2-terminal portion of the ancestral thiolase. The ancestral enzyme might have already possessed a mitochondrial import activity when mitochondria appeared first, or that it might have acquired the import activity during evolution by accumulation of point mutations in the NH2-terminal portion of the enzyme.

Fine structural analysis of the Zoogloea ramigera phbA-phbB locus encoding beta-ketothiolase and acetoacetyl-CoA reductase: nucleotide sequence of phbB.

A series of expression plasmids containing either the complete insert from plasmid pUCDBK1 (Peoples et al., 1987) or sub-fragments thereof were constructed in a tac promoter vector. Analysis of protein lysates of induced cultures of these clones identified the gene encoding NADPH-specific acetoacetyl-CoA reductase in the 2.3kb of sequence located downstream from the beta-ketothiolase gene in plasmid pUCDBK1. The complete nucleotide sequence (2.1kb) of this region was determined. An open reading frame was located 88bp downstream from the stop codon of the thiolase gene encoding a potential polypeptide of Mr 25,000, which is in good agreement with that observed for the overexpressed protein on SDS-PAGE. N-terminal protein sequence data obtained by Edman degradation of the purified Mr = 25,000 polypeptide were used to identify the correct start of the NADPH-specific acetoacetyl-CoA reductase gene. Hence in Z. ramigera, the genes encoding beta-ketothiolase (phbA) and NADPH-specific acetoacetyl-CoA reductase (phbB) are organized as phbA-phbB. S1-nuclease analysis of Z. ramigera RNA identified a transcription start site 85 bp upstream from the phbA structural gene locating the promoter region.

Isolation of Zoogloea ramigera I-16-M exopolysaccharide biosynthetic genes and evidence for instability within this region.

The genetics of the biosynthesis of an exocellular polysaccharide (EPS) from Zoogloea ramigera I-16-M is being investigated. Tn5 insertion mutants deficient in EPS production were isolated by screening for the absence of fluorescence on plates containing the dye Cellufluor (Polysciences Chemicals). Complementation of these mutations was achieved with a Z. ramigera I-16-M gene library constructed in a broad-host-range cosmid vector and introduced into the I-16-M mutants by conjugation. Four recombinant plasmids able to restore EPS production to all of these mutants were found to contain at least 14 kilobases of common insert DNA. Subcloning of the common region and restriction mapping the locations of Tn5 insertions have identified two complementation groups contained within a chromosomal segment of DNA that is between 4.6 and 6.5 kilobases in size. We have clearly demonstrated genetic instability in this region which leads to spontaneous deletions and possibly rearrangements resulting in the loss of EPS production.