σ54 -Dependent regulator DVU2956 switches Desulfovibrio vulgaris from biofilm formation to planktonic growth and regulates hydrogen sulfide production.

Microbiologically influenced corrosion causes $100 billion in damage per year, and biofilms formed by sulfate-reducing bacteria (SRB) are the major culprit. However, little is known about the regulation of SRB biofilm formation. Using Desulfovibrio vulgaris as a model SRB organism, we compared the transcriptomes of biofilm and planktonic cells and identified that the gene for σ54 -dependent regulator DVU2956 is repressed in biofilms. Utilizing a novel promoter that is primarily transcribed in biofilms (Pdvu0304 ), we found production of DVU2956 inhibits biofilm formation by 70%. Corroborating this result, deleting dvu2956 increased biofilm formation, and this biofilm phenotype could be complemented. By producing proteins in biofilms from genes controlled by DVU2956 (dvu2960 and dvu2962), biofilm formation was inhibited almost completely. A second round of RNA-seq for the production of DVU2956 revealed DVU2956 influences electron transport via an Hmc complex (high-molecular-weight cytochrome c encoded by dvu0531-dvu0536) and the Fe-only hydrogenase (encoded by dvu1769, hydA and dvu1770, hydB) to control H2 S production. Corroborating these results, producing DVU2956 in biofilms decreased H2 S production by half, deleting dvu2956 increased H2 S production by 131 ± 5%, and producing DVU2956 in the dvu2956 strain reduced H2 S production. Therefore, DVU2956 maintains SRB in the planktonic state and reduces H2 S formation.

Rhamnolipids from Pseudomonas aeruginosa disperse the biofilms of sulfate-reducing bacteria.

Biofilm formation is an important problem for many industries. Desulfovibrio vulgaris is the representative sulfate-reducing bacterium (SRB) which causes metal corrosion in oil wells and drilling equipment, and the corrosion is related to its biofilm formation. Biofilms are extremely difficult to remove since the cells are cemented in a polymer matrix. In an effort to eliminate SRB biofilms, we examined the ability of supernatants from Pseudomonas aeruginosa PA14 to disperse SRB biofilms. We found that the P. aeruginosa supernatants dispersed more than 98% of the biofilm. To determine the biochemical basis of this SRB biofilm dispersal, we examined a series of P. aeruginosa mutants and found that mutants rhlA, rhlB, rhlI, and rhlR, defective in rhamnolipids production, had significantly reduced levels of SRB biofilm dispersal. Corroborating these results, purified rhamnolipids dispersed SRB biofilms, and rhamnolipids were detected in the P. aeruginosa supernatants. Hence, P. aeruginosa supernatants disperse SRB biofilms via rhamnolipids. To determine the genetic basis of how the P. aeruginosa supernatants disperse SRB biofilms, a whole transcriptomic analysis was conducted (RNA-seq); based on this analysis, we identified four proteins (DVUA0018, DVUA0034, DVUA0066, and DVUA0084) of the D. vulgaris megaplasmid that influence biofilm formation, with production of DVUA0066 (a putative phospholipase) reducing biofilm formation 5.6-fold. In addition, the supernatants of P. aeruginosa dispersed the SRB biofilms more readily than protease in M9 glucose minimum medium and were also effective against biofilms of Escherichia coli and Staphylococcus aureus.

Glycoside hydrolase DisH from Desulfovibrio vulgaris degrades the N-acetylgalactosamine component of diverse biofilms.

Biofilms of sulfate-reducing bacteria (SRB) produce H2 S, which contributes to corrosion. Although bacterial cells in biofilms are cemented together, they often dissolve their own biofilm to allow the cells to disperse. Using Desulfovibrio vulgaris as a model SRB, we sought polysaccharide-degrading enzymes that disperse its biofilm. Using a whole-genome approach, we identified eight enzymes as putative extracellular glycoside hydrolases including DisH (DVU2239, dispersal hexosaminidase), an enzyme that we demonstrated here, by utilizing various p-nitrooligosaccharide substrates, to be an N-acetyl-ß-D-hexosaminidase. For N-acetyl-ß-D-galactosamine (GalNAc), Vmax was 3.6 µmol of p-nitrophenyl/min (mg protein)-1 and Km was 0.8 mM; the specific activity for N-acetyl ß-D-glucosamine (GlcNAc) was 7.8 µmol of p-nitrophenyl/min (mg protein)-1 . Since GalNAc is one of the three exopolysaccharide matrix components of D. vulgaris, purified DisH was found to disperse 63 ± 2% biofilm as well as inhibit biofilm formation up to 47 ± 4%. The temperature and pH optima are 60°C and pH 6, respectively; DisH is also inhibited by copper and is secreted. In addition, since polymers of GalNAc and GlcNAc are found in the matrix of diverse bacteria, DisH dispersed biofilms of Pseudomonas aeruginosa, Escherichia coli and Bacillus subtilis. Therefore, DisH has the potential to inhibit and disperse a wide-range of biofilms.

Dispersal and inhibitory roles of mannose, 2-deoxy-d-glucose and N-acetylgalactosaminidase on the biofilm of Desulfovibrio vulgaris.

Biofilms of sulfate-reducing bacteria (SRB) are often the major cause of microbiologically influenced corrosion. The representative SRB Desulfovibrio vulgaris has previously been shown to have a biofilm that consists primarily of protein. In this study, by utilizing lectin staining, we identified that the biofilm of D. vulgaris also consists of the matrix components mannose, fucose and N-acetylgalactosamine (GalNAc), with mannose predominating. Based on these results, we found that the addition of mannose and the nonmetabolizable mannose analog 2-deoxy-d-glucose inhibits the biofilm formation of D. vulgaris as well as that of D. desulfuricans; both compounds also dispersed the SRB biofilms. In addition, the enzyme N-acetylgalactosaminidase, which degrades GalNAc, was effective in dispersing D. vulgaris biofilms. Therefore, by determining composition of the SRB biofilm, effective biofilm control methods may be devised.

An oxygen-sensitive toxin-antitoxin system.

The Hha and TomB proteins from Escherichia coli form an oxygen-dependent toxin-antitoxin (TA) system. Here we show that YmoB, the Yersinia orthologue of TomB, and its single cysteine variant [C117S]YmoB can replace TomB as antitoxins in E. coli. In contrast to other TA systems, [C117S]YmoB transiently interacts with Hha (rather than forming a stable complex) and enhances the spontaneous oxidation of the Hha conserved cysteine residue to a -SOxH-containing species (sulfenic, sulfinic or sulfonic acid), which destabilizes the toxin. The nuclear magnetic resonance structure of [C117S]YmoB and the homology model of TomB show that the two proteins form a four-helix bundle with a conserved buried cysteine connected to the exterior by a channel with a diameter comparable to that of an oxygen molecule. The Hha interaction site is located on the opposite side of the helix bundle.

Living biofouling-resistant membranes as a model for the beneficial use of engineered biofilms.

Membrane systems are used increasingly for water treatment, recycling water from wastewater, during food processing, and energy production. They thus are a key technology to ensure water, energy, and food sustainability. However, biofouling, the build-up of microbes and their polymeric matrix, clogs these systems and reduces their efficiency. Realizing that a microbial film is inevitable, we engineered a beneficial biofilm that prevents membrane biofouling, limiting its own thickness by sensing the number of its cells that are present via a quorum-sensing circuit. The beneficial biofilm also prevents biofilm formation by deleterious bacteria by secreting nitric oxide, a general biofilm dispersal agent, as demonstrated by both short-term dead-end filtration and long-term cross-flow filtration tests. In addition, the beneficial biofilm was engineered to produce an epoxide hydrolase so that it efficiently removes the environmental pollutant epichlorohydrin. Thus, we have created a living biofouling-resistant membrane system that simultaneously reduces biofouling and provides a platform for biodegradation of persistent organic pollutants.

The HigB/HigA toxin/antitoxin system of Pseudomonas aeruginosa influences the virulence factors pyochelin, pyocyanin, and biofilm formation.

Toxin/antitoxin (TA) systems are prevalent in most bacterial and archaeal genomes, and one of the emerging physiological roles of TA systems is to help regulate pathogenicity. Although TA systems have been studied in several model organisms, few studies have investigated the role of TA systems in pseudomonads. Here, we demonstrate that the previously uncharacterized proteins HigB (unannotated) and HigA (PA4674) of Pseudomonas aeruginosa PA14 form a type II TA system in which antitoxin HigA masks the RNase activity of toxin HigB through direct binding. Furthermore, toxin HigB reduces production of the virulence factors pyochelin, pyocyanin, swarming, and biofilm formation; hence, this system affects the pathogencity of this strain in a manner that has not been demonstrated previously for TA systems.

DNA-crosslinker cisplatin eradicates bacterial persister cells.

For all bacteria, nearly every antimicrobial fails since a subpopulation of the bacteria enter a dormant state known as persistence, in which the antimicrobials are rendered ineffective due to the lack of metabolism. This tolerance to antibiotics makes microbial infections the leading cause of death worldwide and makes treating chronic infections, including those of wounds problematic. Here, we show that the FDA-approved anti-cancer drug cisplatin [cis-diamminodichloroplatinum(II)], which mainly forms intra-strand DNA crosslinks, eradicates Escherichia coli K-12 persister cells through a growth-independent mechanism. Additionally, cisplatin is more effective at killing Pseudomonas aeruginosa persister cells than mitomycin C, which forms inter-strand DNA crosslinks, and cisplatin eradicates the persister cells of several pathogens including enterohemorrhagic E. coli, Staphylococcus aureus, and P. aeruginosa. Cisplatin was also highly effective against clinical isolates of S. aureus and P. aeruginosa. Therefore, cisplatin has broad spectrum activity against persister cells. Biotechnol. Bioeng. 2016;113: 1984-1992. © 2016 Wiley Periodicals, Inc.

Complete Genome of Salmonella enterica Serovar Typhimurium T5-Like Siphophage Stitch.

Salmonellosis, caused by Salmonella, is a leading cause of food poisoning worldwide. With the continuing rise of bacterial antibiotic resistance, efforts are focused on seeking new approaches for treatment of bacterial infections, namely, bacteriophage therapy. Here, we report the complete genome of S. Typhimurium siphophage Stitch.

Complete Genome of Salmonella enterica Serovar Typhimurium Myophage Maynard.

Salmonella enterica serovar Typhimurium is a pathogenic bacterium that has been a major concern for food and public safety. Phages infecting S. Typhimurium may prove to be useful therapeutics against this harmful bacterium. Here, we announce the complete genome of S. Typhimurium T4-like myophage Maynard and describe its features.

Complete Genome of Acinetobacter baumannii Podophage Petty.

Acinetobacter baumannii is an emerging pathogen that was isolated from wounded soldiers in military treatment facilities in Iraq but has since become a problem in civilian hospitals. Here, we announce and describe the complete genome of the KMV-like A. baumannii podophage Petty.

Complete Genome of Acinetobacter baumannii N4-Like Podophage Presley.

Acinetobacter baumannii is an emerging multidrug-resistant nosocomial pathogen. Bacteriophages may be useful as an alternative method of treatment against this and other multidrug-resistant bacteria. Here, we present the complete genome sequence of A. baumannii phage Presley, an N4-like podophage.

Complete Genome of Salmonella enterica Serovar Enteritidis Myophage Marshall.

Salmonella enterica serovar Enteritidis is a food-borne pathogen that causes salmonellosis in the United States. Bacteriophages are emerging as viable biocontrol agents against this pathogen. Here, we present the complete annotated genome sequence of Salmonella Enteritidis T4-like myophage Marshall, which has potential as a phage therapy agent.

The KaiA protein of the cyanobacterial circadian oscillator is modulated by a redox-active cofactor.

The circadian rhythms exhibited in the cyanobacterium Synechococcus elongatus are generated by an oscillator comprised of the proteins KaiA, KaiB, and KaiC. An external signal that commonly affects the circadian clock is light. Previously, we reported that the bacteriophytochrome-like protein CikA passes environmental signals to the oscillator by directly binding a quinone and using cellular redox state as a measure of light in this photosynthetic organism. Here, we report that KaiA also binds the quinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), and the oxidized form of DBMIB, but not its reduced form, decreases the stability of KaiA in vivo, causes multimerization in vitro, and blocks KaiA stimulation of KaiC phosphorylation, which is central to circadian oscillation. Our data suggest that KaiA directly senses environmental signals as changes in redox state and modulates the circadian clock.

Elevated ATPase activity of KaiC applies a circadian checkpoint on cell division in Synechococcus elongatus.

A circadian clock coordinates physiology and behavior in diverse groups of living organisms. Another major cyclic cellular event, the cell cycle, is regulated by the circadian clock in the few cases where linkage of these cycles has been studied. In the cyanobacterium Synechococcus elongatus, the circadian clock gates cell division by an unknown mechanism. Using timelapse microscopy, we confirm the gating of cell division in the wild-type and demonstrate the regulation of cytokinesis by key clock components. Specifically, a state of the oscillator protein KaiC that is associated with elevated ATPase activity closes the gate by acting through a known clock output pathway to inhibit FtsZ ring formation at the division site. An activity that stimulates KaiC phosphorylation independently of the KaiA protein was also uncovered. We propose a model that separates the functions of KaiC ATPase and phosphorylation in cell division gating and other circadian behaviors.