( 1993 )
Putrescine oxidase of Micrococcus rubens: primary structure and Escherichia coli.
PMID : 8473854 : DOI : 10.1099/00221287-139-3-425
The flavin adenine dinucleotide (FAD)-containing putrescine oxidase of Micrococcus rubens catalyses the oxidative deamination of putrescine. The amino acid sequences of the NH2-termini of the mature enzyme and lysyl-endopeptidase-generated fragments were determined for preparation of synthetic oligonucleotides as hybridization probes for cloning. A 4.4 kb BamHI fragment which contained DNA sequences hybridizing to the probes was cloned in pUC19 in Escherichia coli. The nucleotide sequence together with the determined amino acid sequences revealed that this enzyme consists of 480 amino acids (M(r) 52,000) and contains an FAD-binding consensus sequence at its NH2-terminal portion. In front of the transcriptional start point, which is 28 bases upstream of the initiation codon as determined by primer extension, -35 and -10 sequences similar to typical prokaryotic promoter consensus sequences are present. E. coli JM109 containing the putrescine oxidase gene just downstream of the lac promoter in pUC18 produced a large amount of this protein when grown at 37 degrees C but in the enzymically inactive form of inclusion bodies. However, cultivation of the recombinant E. coli cells at temperatures below 30 degrees C led to production of active enzyme (20 times as much as produced by the original M. rubens strain).
Ben Maamar S,
( 2019 )
Impacts of indoor surface finishes on bacterial viability.
PMID : 30980566 : DOI : 10.1111/ina.12558
Microbes in indoor environments are constantly being exposed to antimicrobial surface finishes. Many are rendered non-viable after spending extended periods of time under low-moisture, low-nutrient surface conditions, regardless of whether those surfaces have been amended with antimicrobial chemicals. However, some microorganisms remain viable even after prolonged exposure to these hostile conditions. Work with specific model pathogens makes it difficult to draw general conclusions about how chemical and physical properties of surfaces affect microbes. Here, we explore the survival of a synthetic community of non-model microorganisms isolated from built environments following exposure to three chemically and physically distinct surface finishes. Our findings demonstrated the differences in bacterial survival associated with three chemically and physically distinct materials. Alkaline clay surfaces select for an alkaliphilic bacterium, Kocuria rosea, whereas acidic mold-resistant paint favors Bacillus timonensis, a Gram-negative spore-forming bacterium that also survives on antimicrobial surfaces after 24 hours of exposure. Additionally, antibiotic-resistant Pantoea allii did not exhibit prolonged retention on antimicrobial surfaces. Our controlled microcosm experiment integrates measurement of indoor chemistry and microbiology to elucidate the complex biochemical interactions that influence the indoor microbiome.
( 2017 )
Identification and heterologous expression of the kocurin biosynthetic gene cluster.
PMID : 28942758 : DOI : 10.1099/mic.0.000538
The antibiotically bioactive thiopeptide compound kocurin was identified in extracts from a newly isolated Kocuria rosea strain. The axenic strain was retrieved from a soil sample of the intertidal area at the Paracas National Park, Peru. The genetic basis of this promising natural product with activity against methicillin-resistant Staphylococcus aureus (MRSA) strains was revealed by comparative genome analysis of this new isolate and other reported thiopeptide producer strains. The functionality of the predicted gene locus was experimentally proven by heterologous expression in Streptomyces coelicolor M1146. Expression of the gene cluster under the control of a constitutive promoter enabled the transgenic strain to produce kocurin in selected media. The kocurin biosynthetic gene cluster comprises nine open reading frames and spans around 12 kbp of the genome.