The pattern of biodegradation and the chemical changes occurring in the macromolecular fraction of domestic sludge during autothermal thermophilic aerobic digestion (ATAD) was monitored and ...characterised via solid-state (13)C-NMR CP-MAS. Major indexes such as aromaticity, hydrophobicity and alkyl/O-alkyl ratios calculated for the ATAD processed biosolids were compared by means of these values to corresponding indexes reported for sludges of different origin such as manures, soil organic matter and certain types of compost. Given that this is the first time that these techniques have been applied to ATAD sludge, the data indicates that long-chain aliphatics are easily utilized by the microbial populations as substrates for metabolic activities at all stages of aerobic digestion and serve as a key substrate for the temperature increase, which in turn results in sludge sterilization. The ATAD biosolids following treatment had a prevalence of O-alkyl domains, a low aromaticity index (10.4%) and an alkyl/O-alkyl ratio of 0.48 while the hydrophobicity index of the sludge decreased from 1.12 to 0.62 during the treatment. These results have important implications for the evolution of new ATAD modalities particularly in relation to dewatering and the future use of ATAD processed biosolids as a fertilizer, particularly with respect to hydrological impacts on the soil behaviour.
Adsorption studies were carried out on a red mud modified sawdust biochar material to assess its capacity in the removal of vanadium (V) from aqueous solution. In this study, a number of parameters ...which can potentially influence V(V) adsorption were investigated including equilibrium V(V) solution concentration, contact time for effective V(V) removal, temperature of the adsorption process, solution pH and ionic strength. The uptake of V(V) was shown to be heavily influenced by solution pH with maximum uptake (16.45 mg g−1) achieved in the pH range of 3.5 - 5.5. The adsorption process was best described by the Langmuir model. The kinetics of the adsorption process indicated that V(V) uptake occurred within 60 min of contact and that pseudo-second order kinetics best described the kinetics of the overall adsorption process. There was a clear increase in V(V) uptake with increasing temperature (range 293–343 K) indicating an endothermic adsorption process and the level of uptake remained largely unchanged at solution salt concentrations of up to 0.1 M NaCl and competing cation concentrations of up to 2000 mg L-1 of sodium and 200 mg L-1 aluminium. The relatively weak interaction between V(V) and the modified biochar surface may indicate a possibility of recovery of the bound V(V) and subsequent regeneration of the adsorbent.
The enterobacterial mobile genetic element R391, the prototype ICE (integrating-conjugative element) of the SXT/R391 family, shows increased conjugative transfer following UV irradiation. This is ...dependent on a functioning R391 orf4 gene, which is adjacent to the element encoded integrase gene, int. orf4 mutants fail to form a detectable circular transfer intermediate, do not show UV induced transfer and show a much reduced general transfer ability. The orf4 gene product, termed Jef (IncJ excision factor), shows little homology to anything currently in the nucleotide or protein databases. It is predicted to encode a 66 amino acid, 8.03 kDa, basic, DNA-binding protein with an iso-electric point of pH 8.1: these characteristics being similar to those of recombinational directionality factors involved in excision. Jef expression is up-regulated upon UV irradiation as demonstrated by real-time reverse transcriptase PCR and is controlled by two element encoded genes orf90 and orf91, which show similarity to the transcriptional activator complex FlhC and FlhD. orf4, orf90 and orf91 are conserved in all the SXT/R391-like elements sequenced to date including SXT, ICESpuPO1 and ICEVchMex1. orf4 is also conserved in other SXT/R391 family members such as R997, R392, R705 and pMERPH as shown by sequencing amplicons from these ICEs generated using orf4 specific primers.
The IncJ group of enterobacterial mobile genetic elements, which include R391, R392, R705, R997 and pMERPH, have been shown to be site-specific integrating elements encoding variable antibiotic and ...heavy metal resistance genes. They insert into a specific 17-bp site located in the
prfC gene, encoding peptide release factor 3, in
Escherichia coli and other hosts. A key feature of known IncJ elements is the presence of a site-specific recombination module consisting of an attachment site on the element and an integrase-encoding gene of the tyrosine recombinase class, which promotes integration between the attachment site on the element and a similar site on the host chromosome. We have cloned and sequenced the integrases from a number of known IncJ elements and designed PCR primers for specific amplification of this gene. Using conserved regions of enterobacterial
prfC genes upstream and downstream of the insertion site, and conserved sequences at the ends of the integrated IncJ elements, we have designed specific primers to amplify across the integrated IncJ
attL and
attR junction fragments. Alignment of over 30 enterobacterial
prfC-like genes indicates that the primers designed to amplify
attR junction would amplify IncJ element: host junctions from a wide variety of hosts. The IncJ elements have been shown to sensitise
recA
+
E. coli K12 strains to UV irradiation. A simple and rapid procedure for demonstrating this effect is described. These tools should enable the rapid detection of such elements in clinical and environmental settings.
Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the ...primarily free-living, phylogenetically related
spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic
spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or
; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and
genera separate to avoid further bewilderment and harm.
Human and animal pathogenic bacteria are constantly being released into the environment through human activity. Many of these organisms can harbour genes such as virulence genes, antibiotic ...resistance and heavy metal resistance genes that are inserted into plasmids, transposons and Integrating Conjugating elements (ICEs), leading to potential spread. Such spread can be detected among water and soil communities and in particular in wastewater treatment plants. This makes wastewater and treatment plants a potential reservoir for mobile genetic elements including SXT/R391 ICEs, commonly detected amongst enterobacterial genera. Many plasmid and ICE genomes have been detected serendipitously from clinical sources but few have been identified without selection. Here we examined a domestic wastewater treatment plant to identify, isolate and characterize SXT/R391 ICE’s without selection. Standard microbial replica plating in conjunction with ICE specific (conserved integrase gene) PCR techniques were employed to identify an SXT/R391 ICE MGE using a range of enterobactial selective media. A Novel SXT/R391 ICE MGE was identified from a wastewater Proteus mirabilisstrain. Whole genome sequencing using Ilumina sequencing technology revealed a novel 81 kb element which on annotation contained 75 open reading frames. The “hotspot regions”, which contain adaptive genes, encoded a novel bacteriophage defence mechanisms but lacked other selectable determinants. With the continuous arms race between bacteria and phage, bacteria have developed novel resistance mechanism systems that protect the bacteria from phage. Such systems may be key adaptive mechanisms harboured by ICEs particularly in wastewater systems which will contain large phage populations.
The use of photosynthetic autotrophs and in particular the model organism Synechocystis PCC6803 is receiving much attention for the production of sustainable biofuels and other economically useful ...products through metabolic engineering. Optimisation of metabolic-engineered organisms for high-level sustained production of product is a key element in the manipulation of this organism. A limitation to the utilisation of metabolically-engineered Synechocystis PCC6803 is the availability of strong controllable promoters and stable gene dosage methods for maximising gene expression and subsequent product formation following genetic manipulation.
A native Synechocystis PCC6803 small plasmid, pCA2.4, is consistently maintained at a copy level of up to 7 times that of the polyploid chromosome. As this plasmid is stable during cell division, it is potentially an ideal candidate for maximising gene dosage levels within the organism. Here, we describe the construction of a novel expression vector generated from the native plasmid, pCA2.4. To investigate the feasibility of this new expression system, a yellow fluorescent protein (YFP) encoding gene was cloned downstream of the strong Ptrc promoter and integrated into a predicted neutral site within the pCA2.4 plasmid. The stability of the integrated construct was monitored over time compared to a control strain containing an identical YFP-expressing construct integrated at a known neutral site in a chromosomal location.
A significantly higher fluorescence level of the yellow fluorescent protein was observed when its encoded gene was integrated into the pCA2.4 native plasmid when compared to the isogenic chromosomally integrated control strain. On average, a minimum of 20-fold higher fluorescence level could be achieved from integration into the native plasmid. Fluorescence was also monitored as a function of culture time and demonstrated to be stable over multiple sub-cultures even after the removal of selective pressure. Therefore, the native small plasmid, pCA2.4 may be utilised to stably increase gene expression levels in Synechocystis PCC6803. With the complementary utilisation of an inducible promoter system, rapid generation of commodity-producing Synechocystis PCC6803 strains having high level, controlled expression may be more achievable.
The enteric conjugative transposon-like IncJ elements R391, R392, R705, R706 and pMERPH, all demonstrated increased conjugative transfer upon UV irradiation. The transfer frequency increased on ...average from its basal rate of 10
−5 to 10
−3 per recipient, upon pre-exposure to UV irradiation. However, the transfer frequency of R997, which was higher than the other IncJ elements at 10
−3 per donor, showed a smaller increase. This effect was shown to be
recA
+ dependent in all cases. Using PCR primers directed outwards from the ends of the integrated R391 element it was observed that a circular intermediate of the element forms within the host, which has been proposed to be a transfer intermediate. Using real-time PCR, it was determined that the amount of the circular intermediate produced increased substantially upon UV irradiation.
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