A nascent cellulosic ethanol industry is struggling to become costcompetitive against corn ethanol and gasoline. Millions of dollars are spent on nitrogen supplements to make up for the low nitrogen content of the cellulosic feedstock. Here we show for the first time to our knowledge that the ethanol-producing bacterium, Zymomonas mobilis, can use N 2 gas in lieu of traditional nitrogen supplements. Despite being an electron-intensive process, N 2 fixation by Z. mobilis did not divert electrons away from ethanol production, as the ethanol yield was greater than 97% of the theoretical maximum. In a defined medium, Z. mobilis produced ethanol 50% faster per cell and generated half the unwanted biomass when supplied N 2 instead of ammonium. In a cellulosic feedstock-derived medium, Z. mobilis achieved a similar cell density and a slightly higher ethanol yield when supplied N 2 instead of the industrial nitrogen supplement, corn steep liquor. We estimate that N 2 -utilizing Z. mobilis could save a cellulosic ethanol production facility more than $1 million/y.Zymomonas mobilis | nitrogenase | ethanol | cellulosic | biofuel

Bacteriophages isolated on Mycobacterium smegmatis mc2155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous free-standing HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution.

Zymomonas mobilis produces ethanol from glucose near the theoretical maximum yield, making it a potential alternative to the yeast Saccharomyces cerevisiae for industrial ethanol production. A potentially useful industrial feature is the ability to form multicellular aggregates called flocs, which can settle quickly and exhibit higher resistance to harmful chemicals than single cells. While spontaneous floc-forming Z. mobilis mutants have been described, little is known about the natural conditions that induce Z. mobilis floc formation or about the genetic factors involved. Here we found that wild-type Z. mobilis forms flocs in response to aerobic growth conditions but only in a minimal medium. We identified a cellulose synthase gene cluster and a single diguanylate cyclase that are essential for both floc formation and survival in a minimal aerobic medium. We also found that NADH dehydrogenase 2, a key component of the aerobic respiratory chain, is important for survival in a minimal aerobic medium, providing a physiological role for this enzyme, which has previously been found to be disadvantageous in a rich aerobic medium. Supplementation of the minimal medium with vitamins also promoted survival but did not inhibit floc formation. IMPORTANCE The bacterium Zymomonas mobilis is best known for its anaerobic fermentative lifestyle, in which it converts glucose into ethanol at a yield surpassing that of yeast. However, Z. mobilis also has an aerobic lifestyle, which has confounded researchers with its attributes of poor growth, accumulation of toxic acetic acid and acetaldehyde, and respiratory enzymes that are detrimental for aerobic growth. Here we show that a major Z. mobilis respiratory enzyme and the ability to form multicellular aggregates, called flocs, are important for survival, but only during aerobic growth in a medium containing a minimum set of nutrients required for growth. Supplements, such as vitamins or yeast extract, promote aerobic growth and, in some cases, inhibit floc formation. We propose that Z. mobilis likely requires aerobic respiration and floc formation in order to survive in natural environments that lack protective factors found in supplements such as yeast extract.

The bacterium Zymomonas mobilis naturally produces ethanol at near theoretical maximum yields, making it of interest for industrial ethanol production. Zymomonas mobilis requires the vitamin pantothenate for growth. Here we characterized the genetic basis for the Z. mobilis pantothenate auxotrophy. We found that this auxotrophy is due to the absence of a single gene, panD, encoding aspartate-decarboxylase. Heterologous expression of Escherichia coli PanD in Z. mobilis or supplementation of the growth medium with the product of PanD activity, β-alanine, eliminated the need for exogenous pantothenate. We also determined that Z. mobilis IlvC, an enzyme better known for branched-chain amino acid synthesis, is required for pantothenate synthesis in Z. mobilis, as it compensates for the absence of PanE, another pantothenate synthesis pathway enzyme. In addition to contributing to an understanding of the nutritional requirements of Z. mobilis, our results have led to the design of a more cost-effective growth medium.

Zymomonas mobilis produces ethanol from glucose near the theoretical maximum yield, making it a potential alternative to yeast for industrial ethanol production. A potentially useful industrial feature is the ability to form multicellular aggregates called flocs, which can settle 15 quickly and exhibit higher resistance to harmful chemicals. While spontaneous floc-forming Z. mobilis mutants have been described, little is known about the natural conditions that induce Z. mobilis floc formation and the genetic factors involved. Here we found that wild-type Z. mobilis forms flocs in response to aerobic growth conditions but only in a minimal medium. We identified a cellulose synthase gene cluster and a single diguanylate cyclase that are essential 20 for both floc formation and survival in an aerobic minimal medium. We also found that NADH dehydrogenase 2, a key component of the aerobic respiratory chain, is important for survival in an aerobic minimal medium, providing a physiological role for this enzyme which has previously been found to be disadvantageous in aerobic rich media. Supplementation of the minimal medium with vitamins also promoted survival but did not inhibit floc formation. 25