Isolation of heavy metal-resistant Staphylococcus epidermidis strain TWSL_22 and evaluation of heavy metal bioremediation potential of recombinant E. coli cloned with isolated cadD.

A heavy metal-resistant bacterial strain, TWSL_22 was isolated from an industrial effluent sample and tested for heavy metal tolerance and resistance. The strain was molecularly characterized as Staphylococcus epidermidis based on 16S rDNA gene analysis and the sequence was deposited in the NCBI repository (accession number KT184893.1). Metal removal activity (P < .001) of TWSL_22 was 99.99 ± 0.001%, 74.43 ± 2.51%, and 51.16 ± 4.17% for Cd, Pb, and Cu, respectively. Highest MIC was observed for Cd. Antibiotic susceptibility assays revealed the strain TWSL_22 to be resistant to several antibiotics. The strain was screened for possible heavy metal-resistant genes and presence of cadA, copA, and cadD was confirmed by PCR. A DNA fragment containing complete sequence of cadD (618 bp) was isolated and cloned into pET 21a(+), transformed into E. coli BL21 and designated as E. coli/cadDET. E. coli/cadDET showed high metal tolerance capacity and could remove over 82% of heavy metals (Zn2+, Cd2+, Cu2+, and Cr3+) in the industrial effluent.

Reduction of lag in crude oil degradation by Aspergillus when it is in synergy with Bacillus in biofilm mode.

A major hindrance to the effective use of fungi in bioremediation is their inherent slow growth. Despite this, Aspergillus spp. may be used effectively. Our experiments demonstrate that bacteria, although inefficient in hydrocarbon degradation, may be effectively used in a consortium to overcome the lag in fungal utilization of petroleum hydrocarbons. Crude petroleum oil (160 mg; at 8 g/L) in minimal medium was inoculated with a previously isolated biofilm-forming consortium (Aspergillus sp. MM1 and Bacillus sp. MM1) as well as monocultures of each organism and incubated at 30 ℃ under static conditions. Residual oil was analyzed by GC-MS. Crude oil utilization of Aspergillus-Bacillus biofilm was 24 ± 1.4% in 3 days, increased to 66 ± 7% by day 5 and reached 99 ± 0.2% in 7 days. Aspergillus sp. MM1 monoculture degraded only 14 ± 6% in 5 days. However, at the end of 7 days, it was able to utilize 98 ± 2%. Bacillus sp. MM1 monoculture utilized 20 ± 4% in 7 days. This study indicates that there is a reduction of the fungal lag in bioremediation when it is in association with the bacterium. Although in monoculture, Bacillus sp. MM1 is inefficient in crude oil degradation, it synergistically enhances the initial rate of crude petroleum oil degradation of the fungus in the consortium. The rapid initial removal of as much crude oil as possible from contaminated sites is vital to minimize detrimental impacts on biodiversity.

The study of antimicrobial, anti-cancer, anti-inflammatory and α-glucosidase inhibitory activities of Nigronapthaphenyl, isolated from an extract of Nigrospora sphaerica.

A new compound, nigronapthaphenyl, was extracted from the endophytic fungus Nigrospora sphaerica isolated from a mangrove plant Bruguiera gymnorrhyza. The structure of the compound was elucidated by analysis of 1D and 2D NMR spectra and mass spectrometric data. It was tested in vitro for its antimicrobial activity, cytotoxicity, anti-inflammatory activity and for its ability to inhibit α-glucosidase. Nigronapthaphenyl showed antibacterial activities against Bacillus subtilis TISTR 088 and Bacillus cereus TISTR 688 with MIC values of 4 and 2 µg/mL respectively. Cytotoxicity against colon cancer cell line HCT 116 was found to be an IC50 value of 9.62 ± 0.5 µM . This further showed potential anti-inflammatory activity amounting to an IC50 of 6.2 ± 0.5 µM and also α-glucosidase inhibitory activity, with an IC50 value of 6.9 ± 0.5 µM.

Isolation of ftsI and murE genes involved in peptidoglycan synthesis from Corynebacterium glutamicum.

Corynebacterium glutamicum is known to excrete large amounts of L-glutamic acid upon treatment by penicillin. However, the mechanism of L-glutamate overproduction by penicillin treatment is still unknown. A 5.3-kb HindIII fragment was isolated by directly introducing the C. glutamicum (Brevibacterium lactofermentum) ATCC 13869 gene library into the temperature-sensitive Escherichia coli murE mutant and selecting temperature resistant clones. Two open reading frames (ORFs) were found in this fragment: (1) murE, encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase, and (2)ftsI, encoding septum-peptidoglycan synthetase, one of the targets of penicillin (penicillin-binding protein 3). Both ORFs were involved in peptidoglycan synthesis. Proteins were synthesized from the C. glutamicum murE and ftsI genes, 55 kDa and 73 kDa respectively, in an in vitro protein synthesis system, using E. coli S30 extracts.

A murC gene from coryneform bacteria.

The upstream flanking region of the ftsQ and ftsZ genes of Brevibacterium flavum MJ233, which belongs to the coryneform bacteria, was amplified by the inverse polymerase chain reaction method and cloned in Escherichia coli. Complementation analysis of E. coli mutant with a defective cell-wall synthesis mechanism with the cloned fragment and its DNA sequencing indicated the presence of the murC gene, encoding UDP-N-acetylmuramate:L-alanine ligase involved in peptidoglycan synthesis, just upstream from the ftsQ gene. The B. flavum murC gene could encode a protein of 486 amino acid residues with a calculated molecular mass of 51 198 Da. A 50-kDa protein was synthesized by the B. flavum murC gene in an in vitro transcription/translation system using E. coli S30 lysate. These results indicate that the genes responsible for cell-wall synthesis and cell division are located as a cluster in B. flavum similar to the E. coli mra region.