Cloning and Production of a Novel Bacteriocin, Lactococcin K, from Lactococcus lactis Subsp. lactis MY23

A gene encoding the antimicrobial peptide, lactococcin K, was isolated from Lactococcus lactis subsp. lactis MY23 then cloned and expressed in Escherichia coli. Because the expressed lactococcin K was formed as an inclusion body in recombinant E. coli, a fusion protein containing lactococcin K and maltose-binding protein (MBP) was produced in a soluble form. For high-level production of lactococcin K, we performed a pH-stat fed-batch culture to produce 43,000 AU lactococcin K ml⁻¹ in 12 h. Revisions requested 3 November 2005; Revisions received 7 December 2005     

The Occurrence of Antibiotic Resistance Genes in Taq Polymerases and a Decontamination Method Applied to the Detection of Genetically Modified Crops

Different antibiotic resistance (AR) genes, such as Bla, Tet and NPTII, contaminate commercially available Taq polymerases. The specificity of the AR gene PCR can be increased when using a restriction enzyme-based decontamination of polymerase. The elimination of Taq polymerase contamination allows the use of PCR tests to screen seeds (corn) and processed food for the presence of genetically modified organisms (GMO) based on the detection of AR genes. Without a decontamination procedure for AR genes, PCR screening tests should be interpreted with caution. Revisions requested 1 November 2005; Revisions received 28 November 2005     

Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (<Emphasis Type="Italic">R</Emphasis>)-4-chloro-3-hydroxybutanoate with two co-existing,recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis> strains

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP⁺/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM substrate. Revisions requested 27 July 2004/23 September 2004; Revisions received 21 September 2004/29 November 2004     

Taxonomic and functional diversity of atrazine‐degrading bacterial communities enriched from agrochemical factory soil

Aims: To characterize atrazine‐degrading potential of bacterial communities enriched from agrochemical factory soil by analysing diversity and organization of catabolic genes. Methods and Results: The bacterial communities enriched from three different sites of varying atrazine contamination mineralized 65–80% of ¹⁴C ring‐labelled atrazine. The presence of trzN‐atzBC‐trzD, trzN‐atzABC‐trzD and trzN‐atzABCDEF‐trzD gene combinations was determined by PCR. In all enriched communities, trzN‐atzBC genes were located on a 165‐kb plasmid, while atzBC or atzC genes were located on separated plasmids. Quantitative PCR revealed that catabolic genes were present in up to 4% of the community. Restriction analysis of 16S rDNA clone libraries of the three enrichments revealed marked differences in microbial community structure and diversity. Sequencing of selected clones identified members belonging to Proteobacteria (α‐, β‐ and γ‐subclasses), the Actinobacteria, Bacteroidetes and TM7 division. Several 16S rRNA gene sequences were closely related to atrazine‐degrading community members previously isolated from the same contaminated site. Conclusions: The enriched communities represent a complex and diverse bacterial associations displaying heterogeneity of catabolic genes and their functional redundancies at the first steps of the upper and lower atrazine‐catabolic pathway. The presence of catabolic genes in small proportion suggests that only a subset of the community has the capacity to catabolize atrazine. Significance and Impact of the Study: This study provides insights into the genetic specificity and the repertoire of catabolic genes within bacterial communities originating from soils exposed to long‐term contamination by s‐triazine compounds.     

Degradation of phenanthrene by different bacteria: evidence for novel transformation sequences involving the formation of 1-naphthol

Four polycyclic aromatic hydrocarbon (PAH)- degrading bacteria, namely Arthrobacter sulphureus RKJ4, Acidovorax delafieldii P4-1, Brevibacterium sp. HL4 and Pseudomonas sp. DLC-P11, capable of utilizing phenanthrene as the sole source of carbon and energy, were tested for its degradation using radiolabelled phenanthrene. [9-¹⁴C]Phenanthrene was incubated with microorganisms containing 100 mg/l unlabelled phenanthrene and the evolution of ¹⁴CO₂ was monitored: within 18 h of incubation, 30.1, 35.6, 26.5 and 2.1% of the recovered radiolabelled carbon was degraded to ¹⁴CO₂ by RKJ4, P4-1, HL4 and DLC-P11, respectively. When mixtures of other PAHs such as fluorene, fluoranthene and pyrene, in addition to phenanthrene, were added as additional carbon sources, there was a 36.1 and 20.6% increase in ¹⁴CO₂ production from [9-¹⁴C]phenanthrene in the cases of RKJ4 and HL4, respectively, whereas P4-1 and DLC-P11 did not show any enhancement in ¹⁴CO₂ production. Although, a combination of many bacteria enhances the degradation of organic compounds, no enhancement in the degradation of [9-¹⁴C]phenanthrene was observed in mixed culture involving all four microorganisms together. However, when different PAHs, as indicated above, were used in mixed culture, there was a 68.2% increase in ¹⁴CO₂ production. In another experiment, the overall growth rate of P4-1 on phenanthrene could be enhanced by adding the non-ionic surfactant Triton X-100, whereas RKJ4, HL4 and DLC-P11 did not show any enhancement in growth. Pathways for phenanthrene degradation were also analysed by thin-layer chromatography, gas chromatography and gas chromatography-mass spectrometry. Common intermediates such as o-phthalic acid and protocatechuic acid were detected in the case of RKJ4 and o-phthalic acid was detected in the case of P4-1. A new intermediate, 1-naphthol, was detected in the cases of HL4 and DLC-P11. HL4 degrades phenanthrene via 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, whereas DLC-P11 degrades phenanthrene via the formation of 1-hydroxy-2-naphthoic acid, 1-naphthol and o-phthalic acid. Both transformation sequences are novel and have not been previously reported in the literature. Mega plasmids were found to be present in RKJ4, HL4 and DLC-P11, but their involvement in phenanthrene degradation could not be established. Received: 25 May 1999 / Received revision: 16 July 1999 / Accepted: 1 August 1999     

Lactose-induced Production of Human Soluble B Lymphocyte Stimulator (hsBLyS) in E. coli with Different Culture Strategies

Over-production of human soluble B lymphocyte stimulator (hsBLyS) was carried out with four different fed-batch culture strategies using lactose as inducer, instead of IPTG, in a fed-batch culture of Escherichia coli. As lactose acted as both inducer and carbon source, the best and simplest culture strategy was direct feeding of lactose after batch culture, thereby giving hsBLyS at 3.7 g l⁻¹ and a productivity of 0.11 g l⁻¹ h⁻¹. Revisions requested 1 September 2005 and 11 November 2005; Revisions received 7 November 2005 and 4 January 2006     

Genetics of naphthalene and phenanthrene degradation by Comamonas testosteroni

Naphthalene and phenanthrene have long been used as model compounds to investigate the ability of bacteria to degrade polycyclic aromatic hydrocarbons. The catabolic pathways have been determined, several of the enzymes have been purified to homogeneity, and genes have been cloned and sequenced. However, the majority of this work has been performed with fast growing Pseudomonas strains related to the archetypal naphthalene-degrading P. putida strains G7 and NCIB 9816-4. Recently Comamonas testosteroni strains able to degrade naphthalene and phenanthrene have been isolated and shown to possess genes for polycyclic aromatic hydrocarbon degradation that are different from the canonical genes found in Pseudomonas species. For instance, C. testosteroni GZ39 has genes for naphthalene and phenanthrene degradation which are not only different from those found in Pseudomonas species but are also arranged in a different configuration. C. testosteroni GZ42, on the other hand, has genes for naphthalene and phenanthrene degradation which are arranged almost the same as those found in Pseudomonas species but show significant divergence in their sequences. Received 10 August 1997/ Accepted in revised form 15 August 1997     

Microbial characterization and hydrocarbon biodegradation potential of natural bilge waste microflora

Shipping operations produce oily wastes that must be managed properly to avoid environmental pollution. The aim of this study was to characterize microorganisms occurring in ship bilge wastes placed in open lagoons and, particularly, to assess their potential to degrade polycyclic aromatic hydrocarbons (PAHs). A first-order kinetic was suitable for describing hydrocarbon biodegradation after 17 days of treatment. The calculated rate constants were 0.0668 and 0.0513 day^–1 with a corresponding half-life of 10.3 and 13.5 days for the aliphatic and aromatic hydrocarbon fractions, respectively. At day 17, PAH removal percentages were: acenaphtylene 100, fluorene 95.2, phenanthrene 93.6, anthracene 70.3, and pyrene 71.5. Methyl phenanthrene removals were lower than that of their parent compound (3-methyl phenanthrene 83.6, 2-methyl phenanthrene 80.8, 1-methyl phenanthrene 77.3, 9-methyl phenanthrene 75.1, and 2,7-dimethyl phenanthrene 76.6). Neither pure cultures nor the microbial community from these wastes showed extracellular biosurfactant production suggesting that the addition of an exogenously produced biosurfactant may be important in enhancing hydrocarbon bioavailability and biodegradation. DNA analysis of bilge waste samples revealed a ubiquitous distribution of the nahAc genotype in the dump pools. Although almost all of the isolates grew on naphthalene as sole carbon source, only some of them yielded nahAc amplification under the experimental conditions used. The variety of PAHs in bilge wastes could support bacteria with multiple degradation pathways and a diversity of catabolic genes divergent from the classical nah-like type.     

Polycyclic Aromatic Hydrocarbon (PAH) Degradation Coupled to Methanogenesis

Baltimore Harbor (Baltimore, MD) sediments were utilized to initiate anaerobic enrichment cultures with polycyclic aromatic hydrocarbons (PAHs) in the absence of supplementary electron acceptors. Cultures amended with naphthalene and phenanthrene exhibited sustained, transferable degradation of the PAHs. Bromoethanesulfonic acid, a selective inhibitor of methanogenesis, inhibited the degradation of 200 μm naphthalene and phenanthrene; molecular characterization based on 16S rRNA sequences confirmed that methanogenic Archaea were eliminated, thus providing evidence that methanogenesis is involved in the degradation pathway. Revisions requested 16 November 2005; Revisions received 14 December 2005     

Characterization of pbpA and pbp2 Encoding Penicillin-binding Proteins Located on the Downstream of Clavulanic Acid Gene Cluster in Streptomyces clavuligerus

Two genes, pbpA (orf18) and pbp2 (orf19) located on the downstream of clavulanic acid (CA) gene cluster of Streptomyces clavuligerus were cloned into pET-28a(+), and confirmed to encode a family of high molecular-weight penicillin-binding proteins (PBPs). Both genes were amplified from genomic DNA by PCR and expressed in E. coli BL21 (DE3). Hydropathy plots of the proteins revealed a single stretch of hydrophobic amino acids indicating them to be transmembrane proteins. Pbp2 had lower affinity to penicillin G compared to PbpA, and was essential to the cell growth in contrast to PbpA. Revisions requested 3 November 2005; Revisions received 13 December 2005     

Quantification of S-adenosyl Methionine in Microbial Cell Extracts

A sensitive method for quantification of S-adenosyl methionine (SAM) in microbial cell extracts was developed and applied to Corynebacterium glutamicum. The method is based on SAM being completely hydrolyzed into ¹⁸O-homoserine when extracted in boiling H₂¹⁸O and thus can be clearly distinguished by GC-MS analysis from naturally labeled homoserine present in the cell extract. Additional quantification of the total homoserine pool, representing both SAM and homoserine, via HPLC allows separate determination of both metabolites. Received 23 August 2005; Revisions requested 30 August 2005; Revisions received 26 September 2005; Accepted 31 October 2005     

Differential Gene Expression of Human CD34+ Hematopoietic Stem and Progenitor Cells Before and After Culture

Ex vivo expanded CD34⁺ hematopoietic stem and progenitor cells (HSPCs) have compromised homing and engraftment capacities. To investigate underlying mechanisms for functional changes of expanded HSPCs, we compared gene expression profiling of cultured and fresh CD34⁺ cells derived from cord blood using SMART-PCR and cDNA array: 20 genes were up-regulated while 25 genes were down-regulated in cultured CD34⁺ HSPCs. These differentially expressed genes are involved primarily in proliferation, differentiation, apoptosis, and homing. Revisions requested 27 September 2005; Revisions received 14 December 2005     

The catabolic pathways of in situ rhizosphere PAH degraders and the main factors driving PAH rhizoremediation in oil-contaminated soil

Rhizoremediation is a potential technique for polycyclic aromatic hydrocarbon (PAH) remediation; however, the catabolic pathways of in situ rhizosphere PAH degraders and the main factors driving PAH rhizoremediation remain unclear. To address these issues, stable-isotope-probing coupled with metagenomics and molecular ecological network analyses were first used to investigate the phenanthrene rhizoremediation by three different prairie grasses in this study. All rhizospheres exhibited a significant increase in phenanthrene removal and markedly modified the diversity of phenanthrene degraders by increasing their populations and interactions with other microbes. Of all the active phenanthrene degraders, Marinobacter and Enterobacteriaceae dominated in the bare and switchgrass rhizosphere respectively; Achromobacter was markedly enriched in ryegrass and tall fescue rhizospheres. Metagenomes of ¹³C-DNA illustrated several complete pathways of phenanthrene degradation for each rhizosphere, which clearly explained their unique rhizoremediation mechanisms. Additionally, propanoate and inositol phosphate of carbohydrates were identified as the dominant factors that drove PAH rhizoremediation by strengthening the ecological networks of soil microbial communities. This was verified by the results of rhizospheric and non-rhizospheric treatments supplemented with these two substances, further confirming their key roles in PAH removal and in situ PAH rhizoremediation. Our study offers novel insights into the mechanisms of in situ rhizoremediation at PAH-contaminated sites.     

Identification and Genetic Characterization of Phenol-Degrading Bacteria from Leaf Microbial Communities

Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O−, but not the C23O−, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.     

Structural and Functional Variation in Genetic Systems for Catabolism of Polycyclic Aromatic Hydrocarbons in Pseudomonas putida Strains

The genetic control of naphthalene, phenanthrene, and anthracene biodegradation was studied in three Pseudomonas putida strains isolated from coal tar- and oil-contaminated soils. These strains isolated from different geographical locations contained similar catabolic plasmids controlling the first steps of naphthalene conversion to salicylate (the nah1operon), functionally inoperative salicylate hydroxylase genes, and genes of the metha-pathway of catechol degradation (the nah2 operon). Salicylate oxidation in these strains is determined by genes located in trans-position relative to the nah1 operon: in strains BS202 and BS3701, they are located on the chromosome, and in the strain BS3790, on the second plasmid.     

Suspension culture of gametophytes of transgenic kelp in a photobioreactor

Transgenic Laminaria japonica gametophytes producing a recombinant tissue-type plasminogen activator (rtPA) protein, which is an effective third-generation thrombolytic agent for acute myocardial infarction (AMI), were cultured in an illuminated bubble column bioreactor. A maximum final dry cell weight of 1120 mg l⁻¹ was obtained in batch culture with an initial dry cell weight of 126 mg l⁻¹ and with aeration rate of 1.2 l air min⁻¹ l⁻¹ culture, nitrate at 1.5 mM and phosphate at 0.17 mM. The yield of rtPA was 56 μg g⁻¹ dry cell wt. This is the first report regarding cultivation of a transgenic macroalga in a bioreactor.Revisions requested 27 January 2005 and 14 April 2005; Revisions received 6 April 2005 and 17 May 2005     

Conidia production by <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (for the biocontrol of a diamondback moth) during solid-state fermentation in a packed-bed bioreactor

Conidia of Beauveria bassiana CS-1, which have the potential for the control of the diamondback moth (Plutella xylostella), were produced by solid-state fermentation (SSF) using a packed-bed bioreactor with rice straw and wheat bran. As the packing density and the bed height were increased, the production of conidia decreased. In a packed-bed bioreactor under no aeration and no addition of polypropylene (PP) foam (control), the total average of conidia was 4.9 × 10⁸ g^-1. The production of conidia was affected more by the addition of PP foam as an inert support than forced aeration and was approx. 23 times higher than that of the control. The total average of conidia produced by B. bassiana was 1.1–1.2 × 10¹⁰ g^-1 . Revisions requested 6 September 2004/2 November 2004; Revisions received 1 November 2004/8 December 2004     

Distribution and Biogeochemical Importance of Bacterial Populations in a Thick Clay-Rich Aquitard System

To investigate the distribution of microbial biomass, populations and activities within a clay-rich, low hydraulic conductivity (10⁻¹¹ to 10⁻¹² m s⁻¹) aquitard complex, cores were aseptically obtained from a series of overlying clayey deposits; a fractured till, unfractured till (20–30 ka BP), a disturbed interfacial zone (20–30 ka BP), and a Cretaceous clay aquitard (71–72 Ma BP). The results of confocal microscopy studies, culture methods, molecular approaches, and extractive fatty acid analyses all indicated low bacterial numbers that were non-homogeneously distributed within the sediments. Various primers for catabolic genes were used to amplify extracted DNA. Results indicated the presence of eubacterial 23S rDNA, and thenarH gene for nitrate reductase and ribulose-1,5-biphosphate carboxylase (RuBP carboxylase). Although there was no evidence of limitation by electron acceptors or donors, sulfate-reducing bacteria were not detected below the fractured till zone, using PCR, enrichment, or culture techniques. Denaturing gradient gel electrophoresis (DGGE) analyses indicated differences in community composition and abundance between the various geologic units. Results of FAME analyses of sediments yielded detectable extractable fatty acids throughout the aquitard complex. Bacterial activities were demonstrated by measuring mineralization of (¹⁴C) glucose. Porewater chemistry and stable isotope data were in keeping with an environment in which extremely slow growing, low populations of bacteria exert little impact. The observations also support the contention that in low permeability sediments bacteria may survive for geologic time periods.