Calcium (Ca2+) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca2+ and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca2+ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca2+ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca2+ at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca2+ is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca2+ alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca2+ to eDNA thereby mediating bacterial aggregation and biofilm formation.

Dehalobacter sp. strain UNSWDHB can dechlorinate up to 4 mM trichloromethane at a rate of 0.1 mM per day to dichloromethane and 1,1,2-trichloroethane (1 mM, 0.1 mM per day) with the unprecedented product profile of 1,2-dichloroethane and vinyl chloride. 1,1,1-trichloroethane and 1,1-dichloroethane were slowly utilized by strain UNSWDHB and were not completely removed, with minimum threshold concentrations of 0.12 mM and 0.07 mM respectively under growth conditions. Enzyme kinetic experiments confirmed strong substrate affinity for trichloromethane and 1,1,2-trichloroethane (Km  = 30 and 62 µM respectively) and poor substrate affinity for 1,1,1-trichloroethane and 1,1-dichloroethane (Km  = 238 and 837 µM respectively). Comparison of enzyme kinetic and growth data with other trichloromethane respiring organisms (Dehalobacter sp. strain CF and Desulfitobacterium sp. strain PR) suggests an adaptation of strain UNSWDHB to trichloromethane. The trichloromethane RDase (TmrA) expressed by strain UNSWDHB was identified by BN-PAGE and functionally characterized. Amino acid comparison of homologous RDases from all three organisms revealed only six significant amino acid substitutions/deletions, which are likely to be crucial for substrate specificity. Furthermore, strain UNSWDHB was shown to grow without exogenous supply of cobalamin confirming genomic-based predictions of a fully functional cobalamin synthetic pathway.

Bacteria capable of dechlorinating the toxic environmental contaminant dichloromethane (DCM, CH2Cl2) are of great interest for potential bioremediation applications. A novel, strictly anaerobic, DCM-fermenting bacterium, “DCMF”, was enriched from organochlorine-contaminated groundwater near Botany Bay, Australia. The enrichment culture was maintained in minimal, mineral salt medium amended with dichloromethane as the sole energy source. PacBio whole genome SMRTTM sequencing of DCMF allowed de novo, gap-free assembly despite the presence of cohabiting organisms in the culture. Illumina sequencing reads were utilised to correct minor indels. The single, circularised 6.44 Mb chromosome was annotated with the IMG pipeline and contains 5,773 predicted protein-coding genes. Based on 16S rRNA gene and predicted proteome phylogeny, the organism appears to be a novel member of the Peptococcaceae family. The DCMF genome is large in comparison to known DCM-fermenting bacteria. It includes an abundance of methyltransferases, which may provide clues to the basis of its DCM metabolism, as well as potential to metabolise additional methylated substrates such as quaternary amines. Full annotation has been provided in a custom genome browser and search tool, in addition to multiple sequence alignments and phylogenetic trees for every predicted protein, http://www.slimsuite.unsw.edu.au/research/dcmf/.

The role of bacteria and zerovalent iron (Fe(0)) in the degradation of chlorinated solvents in subsurface environments is of interest to researchers and remediation practitioners alike. Fe(0) used in reactive iron barriers for groundwater remediation positively interacted with enrichment cultures containing Dehalobacter strains in the transformation of halogenated methanes. Chloroform transformation and dichloromethane formation was up to 8-fold faster and 14 times higher, respectively, when a Dehalobacter-containing enrichment culture was combined with Fe(0) compared with Fe(0) alone. The dichloromethane-fermenting culture transformed dichloromethane up to three times faster with Fe(0) compared to without. Compound-specific isotope analysis was employed to compare abiotic and biotic chloroform and dichloromethane degradation. The isotope enrichment factor for the abiotic chloroform/Fe(0) reaction was large at -29.4 ± 2.1‰, while that for chloroform respiration by Dehalobacter was minimal at -4.3 ± 0.45‰. The combined abiotic/biotic dechlorination was -8.3 ± 0.7‰, confirming the predominance of biotic dechlorination. The enrichment factor for dichloromethane fermentation was -15.5 ± 1.5‰; however, in the presence of Fe(0) the factor increased to -23.5 ± 2.1‰, suggesting multiple mechanisms were contributing to dichloromethane degradation. Together the results show that chlorinated methane-metabolizing organisms introduced into reactive iron barriers can have a significant impact on trichloromethane and dichloromethane degradation and that compound-specific isotope analysis can be employed to distinguish between the biotic and abiotic reactions involved.

The chloroform-respiring bacterium Dehalobacter UNSWDHB was isolated from subsurface soil contaminated with a mixture of organohalides, including chloroform. Here, we present its 3.2-Mb genome.