Mycelial nutrient transfer promotes bacterial co-metabolic organochlorine pesticide degradation in nutrient-deprived environments.

Biotransformation of soil organochlorine pesticides (OCP) is often impeded by a lack of nutrients relevant for bacterial growth and/or co-metabolic OCP biotransformation. By providing space-filling mycelia, fungi promote contaminant biodegradation by facilitating bacterial dispersal and the mobilization and release of nutrients in the mycosphere. We here tested whether mycelial nutrient transfer from nutrient-rich to nutrient-deprived areas facilitates bacterial OCP degradation in a nutrient-deficient habitat. The legacy pesticide hexachlorocyclohexane (HCH), a non-HCH-degrading fungus (Fusarium equiseti K3), and a co-metabolically HCH-degrading bacterium (Sphingobium sp. S8) isolated from the same HCH-contaminated soil were used in spatially structured model ecosystems. Using 13C-labeled fungal biomass and protein-based stable isotope probing (protein-SIP), we traced the incorporation of 13C fungal metabolites into bacterial proteins while simultaneously determining the biotransformation of the HCH isomers. The relative isotope abundance (RIA, 7.1-14.2%), labeling ratio (LR, 0.13-0.35), and the shape of isotopic mass distribution profiles of bacterial peptides indicated the transfer of 13C-labeled fungal metabolites into bacterial proteins. Distinct 13C incorporation into the haloalkane dehalogenase (linB) and 2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase (LinC), as key enzymes in metabolic HCH degradation, underpin the role of mycelial nutrient transport and fungal-bacterial interactions for co-metabolic bacterial HCH degradation in heterogeneous habitats. Nutrient uptake from mycelia increased HCH removal by twofold as compared to bacterial monocultures. Fungal-bacterial interactions hence may play an important role in the co-metabolic biotransformation of OCP or recalcitrant micropollutants (MPs).

Draft Genome Sequences of Two Sphingobium Species Associated with Hexachlorocyclohexane (HCH) Degradation Isolated from an HCH-Contaminated Soil.

The draft genome sequences of two Sphingobium strains that are hexachlorocyclohexane (HCH) degraders are presented. The strains were isolated from HCH-contaminated soil in Kitengela, Kenya. Both genomes possess the lin genes responsible for HCH degradation and gene clusters for degradation of other xenobiotic compounds.

Draft Genome Sequence of Fusarium equiseti K3, a Fungal Species Isolated from Hexachlorocyclohexane-Contaminated Soil.

We present the draft genome sequence of Fusarium equiseti strain K3, a fungus isolated from a hexachlorocyclohexane (HCH)-contaminated soil (Kitengela, Kenya). The 37.88-Mb draft genome sequence consists of 206 contigs, 12,311 predicted protein-coding sequences, and 261 tRNA sequences. This genome sequence contributes to our understanding of fungal-bacterial interactions during hexachlorocyclohexane degradation.

Characterization and engineering of two new GH9 and GH48 cellulases from a Bacillus pumilus isolated from Lake Bogoria.

OBJECTIVES: To search for new alkaliphilic cellulases and to improve their efficiency on crystalline cellulose through molecular engineering RESULTS: Two novel cellulases, BpGH9 and BpGH48, from a Bacillus pumilus strain were identified, cloned and biochemically characterized. BpGH9 is a modular endocellulase belonging to the glycoside hydrolase 9 family (GH9), which contains a catalytic module (GH) and a carbohydrate-binding module belonging to class 3 and subclass c (CBM3c). This enzyme is extremely tolerant to high alkali pH and remains significantly active at pH 10. BpGH48 is an exocellulase, belonging to the glycoside hydrolase 48 family (GH48) and acts on the reducing end of oligo-ß1,4 glucanes. A truncated form of BpGH9 and a chimeric fusion with an additional CBM3a module was constructed. The deletion of the CBM3c module results in a significant decline in the catalytic activity. However, fusion of CBM3a, although in a non native position, enhanced the activity of BpGH9 on crystalline cellulose. CONCLUSIONS: A new alkaliphilic endocellulase BpGH9, was cloned and engineered as a fusion protein (CBM3a-BpGH9), which led to an improved activity on crystalline cellulose.

Alkaliphilic Enzymes and Their Application in Novel Leather Processing Technology for Next-Generation Tanneries.

Leather manufacturing involves conversion of raw skin and hides into leather (stable material) through series of mechanical and chemical operations. The leather industry has attracted public outcry due to severe environmental degradation, pollution and health and safety risks. Currently the industry faces serious sustainability challenge due to extensive use of toxic chemicals and generation of hazardous waste. This chapter describes the polluting chemicals consumed in different stages of conventional leather processing and the nature of waste generated. In order to overcome the hazards caused by toxic chemicals in tanneries and protect the environment, enzymes have been identified as a realistic alternate for chemicals used in beam house operation and waste management. Alkaline active proteases of alkaliphiles offer advantages over the use of conventional chemical catalysts for numerous reasons, for example, they exhibit high catalytic activity and high degree of substrate specificity, can be produced in large amounts and are economically viable. This is because the enzymes of these alkaliphiles are capable of catalysing reactions at the extremes of pH, temperature and salinity of leather-manufacturing processes.The chapter describes how alkaliphilic enzyme can effectively be used in soaking, dehairing, bating and degreasing operations to prevent waste generation, help in recovery of valuable by-products, reduce cost and increase leather quality. It is worth noting that protease has the capability to replace sodium sulphide in the dehairing process. In addition, alkaline proteases have shown remarkable ability in bioremediation of waste generated during the industrial processes. Intensive efforts are being directed towards chemical-based industries to use viable clean technology in their operation to reduce their negative impact on the environment. Similarly, leather industry should adopt the use of eco-friendly reagents such as enzymes to achieve long-term sustainability and clean environment and avert health hazards. Application of enzyme technology in clean leather processing strongly depends on legislation, political will and allocation of financial resources in research, development and implementation of this potentially powerful technology. Graphical Abstract.

Viral load, CD4+ T-lymphocyte counts and antibody titres in HIV-1 infected untreated children in Kenya; implication for immunodeficiency and AIDS progression.

BACKGROUND: There are limited reports on HIV-1 RNA load, CD4+ T-lymphocytes and antibody responses in relation to disease progression in HIV-1 infected untreated children in Africa. METHODS: To describe the relationships between these parameters, we conducted a longitudinal cohort study involving 51 perinatally HIV-1 infected children aged between 1 and 13 years. HIV status was determined by ELISA and confirmed by western blot and PCR. Antibodies were quantified by limiting dilution ELISA, plasma HIV-1 RNA load by RT-PCR and CD4+ T-lymphocytes by FACSCount. RESULTS: Asymptomatic and symptomatic disease had, respectively, a rise in median HIV-1 RNA load from 1,195 to 132,543 and from 42,962 to 1,109,281 copies/ml in children below 6 years. The increase in viral load was 10-fold higher for asymptomatic compared to other categories and 2-fold faster for children less than 6 years than those above. Similarly, symptomatic children below 6 years had initial median CD4+ T-lymphocyte counts of 647 (22%) cells/muL, declining to 378 (20%) while those above 6 years had initial values of below 335 (15%) but which increased to 428 (17%). Median viral load correlated significantly with median CD4+ T-lymphocyte percentage in children above 6 years (p=0.026) but not below. CONCLUSIONS: Viral load is lower in older than younger children and correlates significantly with percentage CD4+ T-lymphocytes. Survival by HIV-1 infected children requires a competent immune response early in infection to counter the rapidly replicating virus. Interventions aimed at boosting the naïve immune system may prolong survival in these children.

Molecular characterization of a tsetse fly midgut proteolytic lectin that mediates differentiation of African trypanosomes.

Differentiation of bloodstream-form trypanosomes into procyclic (midgut) forms is an important first step in the establishment of an infection within the tsetse fly. This complex process is mediated by a wide variety of factors, including those associated with the vector itself, the trypanosomes and the bloodmeal. As part of an on-going project in our laboratory, we recently isolated and characterized a bloodmeal-induced molecule with both lectin and trypsin activities from midguts of the tsetse fly, Glossina longipennis [Osir, E.O., Abubakar, L., Imbuga, M.O., 1995. Purification and characterization of a midgut lectin-trypsin complex from the tsetse fly, Glossina longipennis. Parasitol. Res. 81, 276-281]. The protein (lectin-trypsin complex) was found to be capable of stimulating differentiation of bloodstream trypanosomes in vitro. Using polyclonal antibodies to the complex, we screened a G. fuscipes fuscipes cDNA midgut expression library and identified a putative proteolytic lectin gene. The cDNA encodes a putative mature polypeptide with 274 amino acids (designated Glossina proteolytic lectin, Gpl). The deduced amino acid sequence includes a hydrophobic signal peptide and a highly conserved N-terminal sequence motif. The typical features of serine protease trypsin family of proteins found in the sequence include the His/Asp/Ser active site triad with the conserved residues surrounding it, three pairs of cysteine residues for disulfide bridges and an aspartate residue at the specificity pocket. Expression of the gene in a bacterial expression system yielded a protein (M(r) approximately 32,500). The recombinant protein (Gpl) bound d(+) glucosamine and agglutinated bloodstream-form trypanosomes and rabbit red blood cells. In addition, the protein was found to be capable of inducing transformation of bloodstream-form trypanosomes into procyclic forms in vitro. Antibodies raised against the recombinant protein showed cross-reactivity with the alpha subunit of the lectin-trypsin complex. These results support our earlier hypothesis that this molecule is involved in the establishment of trypanosome infections in tsetse flies.

Differential scanning calorimetric studies of a Bacillus halodurans alpha-amylase.

The thermal unfolding of Amy 34, a recombinant alpha-amylase from Bacillus halodurans, has been investigated using differential scanning calorimetry (DSC). The denaturation of Amy 34 involves irreversible processes with an apparent denaturation temperature (T(m)) of 70.8 degrees C at pH 9.0, with four transitions, as determined using multiple Gaussian curves. The T(m) increased by 5 degrees C in the presence of 100-fold molar excess of CaCl2 while the aggregation of Amy 34 was observed in the presence of 1000-fold molar excess of CaCl2. Increase in the calcium ion concentration from 1- to 5-fold molar excess resulted in an increase in calorimetric enthalpy (DeltaH(cal)), however, at higher concentrations of CaCl2 (up to 100-fold), DeltaH(cal) was found to decrease, accompanied by a decrease in entropy change (DeltaS), while the T(m) steadily increased. The presence of 100-fold excess of metal chelator, EDTA, resulted in a decrease in T(m) by 10.4 degrees C. T(m) was also decreased to 61.1 degrees C and 65.9 degrees C at pH 6.0 and pH 11.0, respectively.

Starch hydrolysing Bacillus halodurans isolates from a Kenyan soda lake.

Fourteen obligate alkaliphilic and halotolerant bacterial isolates, exhibiting extracellular amylase activity at 55 degrees C and pH 10, were isolated from hot springs around Lake Bogoria, Kenya. From 16S rDNA sequence analysis, nine isolates shared 100% identity with Bacillus halodurans strain DSM 497T, while the rest shared 99% identity with alkaliphilic Bacillus species A-59. PCR of the intergenic spacer region between 16S and 23S rRNA genes (ISR-PCR) divided the isolates into two groups, while tDNA-PCR divided them into three groups. Bacillus halodurans DSM 497T had a different ISR pattern from the isolates, while it had a tDNA-PCR profile similar to the group that shared 99% identity with alkaliphilic Bacillus species A-59. All isolates hydrolysed soluble starch as well as amylose, amylopectin and pullulan. The amylase activity (1.2-1.8 U ml(-1)) in the culture broths had an optimum temperature of 55-65 degrees C, was stimulated by 1 mm Ca2+, and was either partially (16-30%) or completely inhibited by 1 mM EDTA. Activity staining of the cell-free culture supernatant from the isolates revealed five alkaline active amylase bands.