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Copper (Cu) is a key transition metal that is involved in many important biological processes in a cell. Cu is also utilized by the immune system to hamper pathogen growth during infection. However, genome-level knowledge on the mechanisms involved in adaptation to Cu stress is limited. Here, we report the results of a genome-wide reverse genetic screen for Cu-responsive phenotypes in Escherichia coli. Our screen has identified novel genes involved in adaptation to Cu stress in E. coli. We detected multiple genes involved in the biosynthesis and uptake of enterobactin, a siderophore utilized for high-affinity TonB-dependent acquisition of iron (Fe), as critical players in survival under Cu intoxication. We demonstrated the specificity of Cu-dependent killing by chelation of Cu and by genetic complementation of tonB. Notably, TonB is involved in protection from Cu in both laboratory and uropathogenic strains of E. coli. Cu stress leads to increased expression of the genes involved in Fe uptake, indicating that Fur regulon is derepressed during exposure to excess Cu. Trace element analyses revealed that Fe homeostasis is dysregulated during Cu stress. Taken together, our data supports a model in which lack of enterobactin-dependent Fe uptake leads to exacerbation of Cu toxicity, and elucidates the intricate connection between the homeostasis of Cu and Fe in a bacterial cell., Graphical Abstract Graphical AbstractCopper Stress and Iron Homeostasis in E. coli.

Surface-enhanced Raman spectroscopy (SERS) enables multiplex detection of analytes using simple, portable equipment consisting of a single excitation source and detector. Thus, in theory, SERS is ideally suited to replace fluorescence in assays that screen for numerous deoxyribonucleic acid (DNA) targets, but in practice, SERS-based assays have suffered from complexity and elaborate processing steps. Here, we report an assay in which a simple inkjetfabricated plasmonic paper device enables SERS-based detection of multiple DNA targets within a single polymerase chain reaction (PCR). In prior work, we demonstrated the principles of chromatographic separation and SERS-based detection on inkjet-fabricated plasmonic paper. The present work extends that capability for post-PCR gene sequence detection. In this design, hydrolysis DNA probes with 5′ Raman labels are utilized; if the target is present, the probe is hydrolyzed during PCR, freeing the reporter. After applying the PCR sample to a paper SERS device, an on-device chromatographic separation and concentration is conducted to discriminate between hydrolyzed and intact probes. SERS is then used to detect the reporter released by the hydrolyzed probes. This simple separation and detection on paper eliminates the need for complex sample processing steps. In this work, we simultaneously detect the methicillin-resistant Staphylococcus aureus genes mecA and femB to illustrate the concept. We envision that this approach could contribute to the development of multiplex DNA diagnostic tests enabling screening for several target sequences within a single reaction, which is necessary for cases in which sample volume and resources are limited.

An alkaline active xylanase, XynBYG, was purified from an alkaliphilic Bacillus pumilus BYG, which was newly isolated from paper mill effluent. It had an optimum pH of 8.0-9.0, and showed good stability after incubated at pH 9.0 for 120 min. The optimum temperature for the activity was 50°C, and the enzyme retained below 55% of its original activity for 30 min at 55°C. The gene coding for XynBYG consists of 687 bp and encodes 229 amino acids. Similarity analysis indicated that XynBYG belong to family 11 glycosyl hydrolases. Site-directed mutagenesis was performed to replace five sites (Tyr/Ser) to Arg/Glu and the results demonstrated that the optimum temperature of the mutant Y7 (S39R-T146E) increased 5°C and the half-life of inactivation (T1/2) at 60 and 65°C was 1 h and 25 min, respectively. Thus, it provides a potential xylanase that can meet the harsh conditions in the industrial applications.

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N 2 fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase ( nifH ) gene probing, and bacterial isolations. In situ N 2 fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH , of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH . In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N 2 -fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N 2 fixation (0.87 to 4.90 mg of N liter −1 day −1 ) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.

Multiple independent approaches were applied for monitoring the abundance and identity of sulfate-reducing bacteria (SRB) in pulp of a paper-recycling plant suffering from excessive sulfide emission. The methods applied included most-probable-number (MPN) enumeration of cultivable SRB, rate measurements, FISH and PCR-based retrieval of the functional marker genes dsrA and B (encoding the two major subunits of dissimilatory bisulfite reductase) and 16S rRNA genes. The SRB community was composed of phylogenetically highly different lineages all of low abundance relative to the total microbial community in the pulp, which hampered the applicability of FISH. It was also demonstrated that dsrA- or B-targeted PCR primers commonly used for denaturing gradient gel electrophoresis and real-time PCR analyses were biased. However, using a novel approach combining MPN-PCR and terminal restriction fragment length polymorphism analysis of dsrAB amplicons generated from serially diluted DNA extracts allowed the enumeration and identification of the quantitatively most important members of the SRB community. For fast quantification of SRB in the pulp, the dsrAB-MPN-PCR assay and sulfate reduction rate measurements were found to be most suitable.

The microflora isolated from food-packaging board is dominated by paenibacilli; a number of these micro-organisms have been characterized using a polyphasic approach. The highest 16S rRNA gene similarity was found between these isolates and Paenibacillus azotofixans ATCC 35681(T) (97.7 %). The main fatty acid of the paperboard isolates was C(16 : 0) (34-45 %); straight-chain fatty acids made up 41-60 % of the total cellular fatty acids, thus distinguishing these strains from other Paenibacillus species. The paperboard isolates produced cyclodextrins from starch. The spore surface had a characteristic ribbed ornamentation. Spores and vegetative cells frequently had pilus-like appendages. Based on phylogenetic data and phenotypic and chemotaxonomic characteristics, it is proposed that the isolates represent a novel species, Paenibacillus stellifer sp. nov., with IS 1(T) (=DSM 14472(T)=CCUG 45566(T)) as the type strain.

Escherichia coli KO11 was previously constructed to produce ethanol from acid hydrolysates of hemicellulose (pentoses and hexoses) by the chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB). Klebsiella oxytoca P2 was constructed in an analogous fashion for the simultaneous saccharification and fermentation of cellulose and contains PTS enzymes for cellobiose. In this study, KO11 was further engineered for the fermentation of cellulose by adding the K. oxytoca casAB genes encoding Enzyme IIcellobiose and phospho-beta-glucosidase. Although the two K. oxytoca genes were well expressed in cloning hosts such as DH5 alpha, both were expressed poorly in E. coli KO11, a derivative of E. coli B. Spontaneous mutants which exhibited more than 15-fold-higher specific activities for cellobiose metabolism were isolated. The mutations of these mutants resided in the plasmid rather than the host. Three mutants were characterized by sequence analysis. All contained similar internal deletions which eliminated the casAB promoter and operator regions and placed the lacZ Shine-Dalgarno region immediately upstream from the casA Shine-Dalgarno region. KO11 harboring mutant plasmids (pLOI1908, pLOI1909, or pLOI1910) rapidly fermented cellobiose to ethanol, and the yield was more than 90% of the theoretical yield. Two of these strains were used with commercial cellulase to ferment mixed-waste office paper to ethanol.