Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a freeliving bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes. N utrient requirements are directly linked to biomass production, and any potential increased improvement in the scale of biomass yield will necessitate a proportional increase in the demand for essential nutrients. For all photosynthetic organisms (photoautotrophs such as land plants, algae, and cyanobacteria) with requisite light energy and water, nitrogen is the most limiting and expensive nutrient input for aquaculture and agricultural production alike (1). A majority of our current nitrogen fertilizer production is tied to the burning of fossil fuels to generate ammonia from molecular nitrogen (N 2 gas) through the Haber-Bosch process, which accounts for 3 to 5% of world natural gas consumption, or about 1 to 2% of total worldwide energy expenditures (1-3). In developed countries, industrial nitrogen production is accompanied by a huge economic and energetic cost overall (2), while this key nutrient limits agricultural productivity in developing countries, where energy and infrastructure costs prohibit the utilization of the Haber-Bosch process to produce ammonia from atmospheric nitrogen on a large scale.The development of biological approaches to improve biof...

ABSTRACTA novel phenotype was recently identified in which specific strains ofEscherichia coliinhibit competingE. colistrains via a mechanism that was designated “proximity-dependent inhibition” (PDI). PDI-expressing (PDI+)E. coliis known to inhibit susceptible (PDI−)E. colistrains, including several enterohemorrhagic (EHEC) and enterotoxigenic (ETEC)E. colistrains. In this study, every strain from a genetically diverse panel ofE. coliO157:H7 (n= 25) and additional strains ofE. coliserovar O26 were susceptible to the PDI phenotype. LIVE/DEAD staining was consistent with inhibition by killing of susceptible cells. Comparative genome analysis identified the genetic component of PDI, which is composed of a plasmid-borne (Incl1) operon encoding a putative microcin and associated genes for transport, immunity, and microcin activation. Transfer of the plasmid to a PDI−strain resulted in transfer of the phenotype, and deletion of the genes within the operon resulted in loss of the inhibition phenotype. Deletion of chromosomally encodedtolCalso resulted in loss of the inhibitory phenotype, and this confirmed that the putative microcin is most likely secreted via a type I secretion pathway. Deletion of an unrelated plasmid gene did not affect the PDI phenotype. Quantitative reverse transcription (RT)-PCR demonstrated that microcin expression is correlated with logarithmic-phase growth. The ability to inhibit a diversity ofE. colistrains indicates that this microcin may influence gut community composition and could be useful for control of important enteric pathogens.

The microcin PDI inhibits a diverse group of pathogenic Escherichia coli strains. Coculture of a single-gene knockout library (BW25113; n ‫؍‬ 3,985 mutants) against a microcin PDI-producing strain (E. coli 25) identified six mutants that were not susceptible (⌬atpA, ⌬atpF, ⌬dsbA, ⌬dsbB, ⌬ompF, and ⌬ompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts in E. coli O157:H7 Sakai. Heterologous expression of E. coli ompF conferred susceptibility to Salmonella enterica and Yersinia enterocolitica strains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K 47 G 48 N 49 region within the first extracellular loop of E. coli OmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator for ompF, and consequently loss of susceptibility by the ⌬ompR strain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. In trans expression of ompF in the ⌬dsbA and ⌬dsbB strains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI. E scherichia coli strain 25 (E. coli 25; cattle origin) has an in vitro and in vivo competitive advantage against other E. coli strains that is linked to the production of the microcin PDI (MccPDI) (1-3). The inhibitory phenotype was first observed in vitro (4) and later called "proximity-dependent inhibition" (PDI), because inhibition occurred only when competing cells were in close proximity to sensitive cells (1). MccPDI appears to be most closely related to class IIa microcins, and the cluster of genes that encode MccPDI and associated immunity, activation, and export are located on a conjugative plasmid (2).E. coli produces various antimicrobial bacteriocins that are classified as colicins or microcins. Microcins are distinguished by their lower molecular mass (Ͻ10 kDa) and require active transport across the membrane of producing cells. Microcins typically have a narrow spectrum of activity that is mediated through specific receptors expressed on the surface of susceptible bacteria. To date, 16 microcins have been described, including MccPDI. The receptors for seven of these microcins have been identified and include the outer membrane proteins Cir, FepA, Fiu, FhuA, and OmpF, all of which normally function in iron and other nutrient uptake (5-7).While the gene encoding MccPDI has been identified and gene knockout and complementation studies have confirmed its inhibitory activity (2), little is known about how this microcin functi...