Myxobacteria are single-celled, but social, eubacterial predators. Upon starvation they build multicellular fruiting bodies using a developmental program that progressively changes the pattern of cell movement and the repertoire of genes expressed. Development terminates with spore differentiation and is coordinated by both diffusible and cell-bound signals. The growth and development of Myxococcus xanthus is regulated by the integration of multiple signals from outside the cells with physiological signals from within. A collection of M. xanthus cells behaves, in many respects, like a multicellular organism. For these reasons M. xanthus offers unparalleled access to a regulatory network that controls development and that organizes cell movement on surfaces. The genome of M. xanthus is large (9.14 Mb), considerably larger than the other sequenced δ-proteobacteria. We suggest that gene duplication and divergence were major contributors to genomic expansion from its progenitor. More than 1,500 duplications specific to the myxobacterial lineage were identified, representing >15% of the total genes. Genes were not duplicated at random; rather, genes for cell–cell signaling, small molecule sensing, and integrative transcription control were amplified selectively. Families of genes encoding the production of secondary metabolites are overrepresented in the genome but may have been received by horizontal gene transfer and are likely to be important for predation.

Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.

The seqA gene negatively modulates replication initiation at the E. coli origin, oriC. seqA is also essential for sequestration, which acts at oriC and the dnaA promoter to ensure that replication initiation occurs exactly once per chromosome per cell cycle. Initiation is promoted by full methylation of GATC sites clustered in oriC; sequestration is specific to the hemimethylated forms generated by replication. SeqA protein purification and DNA binding are described. SeqA interacts with fully methylated oriC strongly and specifically. This reaction requires multiple molecules of SeqA and determinants throughout oriC, including segments involved in open complex formation. SeqA interacts more strongly with hemimethylated DNA; in this case, oriC and non-oriC sequences are bound similarly. Also, binding of hemimethylated oriC by membrane fractions is due to SeqA. Direct interaction of SeqA protein with the replication origin is likely to be involved in both replication initiation and sequestration.

The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of the Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three biovars, based on physiological and biochemical properties. The genome of a biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study, the genomes of the biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the biovar III strain A. vitis S4, a narrow-host-range strain that infects grapes and invokes a hypersensitive response on nonhost plants, were fully sequenced and annotated. Comparison with other sequenced members of the Alphaproteobacteria provides new data on the evolution of multipartite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select Beta-and Gammaproteobacteria species. Together, these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second-chromosome formation among bacteria.The family Rhizobiaceae (order Rhizobiales) of the Alphaproteobacteria includes the plant pathogens of the genus Agrobacterium and the nitrogen-fixing plant mutualists of the genera Rhizobium and Sinorhizobium. Members house single and multiple chromosome arrangements, linear replicons, and plasmids of various sizes. Genes of pathogenicity, mutualism, and other symbiotic properties are primarily encoded on large transmissible plasmids. Given the promiscuous nature of these elements, different genomic lineages within the Rhizobiaceae exhibit a variety of symbiotic phenotypes that range from pathogenesis to nitrogen-fixing mutualism.Agrobacterium taxonomy and phylogeny display a marked disparity. Empirically, organisms of the genus Agrobacterium are grouped into five species based on the disease phenotype associated with the resident disease-inducing plasmid: A. tumefaciens causes crown gall on dicotyledonous plants, including stone fruit and nut trees; A. rubi causes crown gall on raspberries; A. vitis causes gall formation that is limited to grapes; A. rhizogenes causes hairy root disease; and A. radiobacter is avirulent. An alternative classification scheme

Leaf-cutter ants are one of the most important herbivorous insects in the Neotropics, harvesting vast quantities of fresh leaf material. The ants use leaves to cultivate a fungus that serves as the colony's primary food source. This obligate ant-fungus mutualism is one of the few occurrences of farming by non-humans and likely facilitated the formation of their massive colonies. Mature leaf-cutter ant colonies contain millions of workers ranging in size from small garden tenders to large soldiers, resulting in one of the most complex polymorphic caste systems within ants. To begin uncovering the genomic underpinnings of this system, we sequenced the genome of Atta cephalotes using 454 pyrosequencing. One prediction from this ant's lifestyle is that it has undergone genetic modifications that reflect its obligate dependence on the fungus for nutrients. Analysis of this genome sequence is consistent with this hypothesis, as we find evidence for reductions in genes related to nutrient acquisition. These include extensive reductions in serine proteases (which are likely unnecessary because proteolysis is not a primary mechanism used to process nutrients obtained from the fungus), a loss of genes involved in arginine biosynthesis (suggesting that this amino acid is obtained from the fungus), and the absence of a hexamerin (which sequesters amino acids during larval development in other insects). Following recent reports of genome sequences from other insects that engage in symbioses with beneficial microbes, the A. cephalotes genome provides new insights into the symbiotic lifestyle of this ant and advances our understanding of host–microbe symbioses.

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome's predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy.

The poly-I-hydroxybutyrate (PHB) biosynthetic pathway from Alcaligenes eutrophus H16 has been cloned and expressed in Escherichia coli. Initially, an A. eutrophus H16 genomic library was constructed by using cosmid pVK102, and cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for the first enzyme of the pathway, I-ketothiolase. Six enzyme-positive clones were identified. Three of these clones manifested acetoacetyl coenzyme A reductase activity, the second enzyme of the biosynthetic pathway, and accumulated PHB. PHB was produced in the cosmid clones at approximately 50% of the level found in A. eutrophus. One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.2-kilobase KpnI-EcoRI fragment. This fragment, when cloned into small multicopy vectors, can direct the synthesis of PHB in E. coli to levels approaching 80% of the bacterial cell dry weight.Poly-p-hydroxybutyrate (PHB), a homopolymer of D-(-)-3-Hydroxybutyrate, is a storage material produced by a variety of bacteria in response to environmental stress. The presence of PHB in bacteria was first recognized by Lemoigne in 1926 (11), and it has since been identified in more than 20 bacterial genera, including Azotobacter, Bacillus, Beijerinckia, Alcaligenes, Pseudomonas, Rhizobium, and Rhodospirillum (4).The PHB pathway and its regulation have been studied extensively in Alcaligenes eutrophus H16 and Azotobacter beijerinckii (3,4,9,10,15,(18)(19)(20)(21)24). The pathway consists of a biosynthetic portion and a degradative portion and is made up of five enzymes. One of these enzymes, 0-ketothiolase, is both the entry point and the exit point of the cycle (4). In the biosynthetic part of the pathway, ,-ketothiolase catalyzes the reversible condensation of two acetyl coenzyme A (CoA) molecules to acetoacetyl-CoA. Acetoacetyl-CoA is subsequently reduced to D-(-)-3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase, and PHB is then produced by the polymerization of 0-hydroxybutyrylCoA via the action of PHB synthetase.Studies in A. eutrophus H16 and in Azotobacter beijerinckii have shown the PHB pathway to be regulated in response to several types of environmental limitation. These limitations include oxygen deprivation, nitrogen deprivation, sulfate limitation, and magnesium limitation (3,4,10,15,(18)(19)(20)(21)24). Under limiting environmental conditions, PHB may constitute as much as 80% of the dry cell weight. When limiting conditions are relaxed, PHB quantities decrease to preinduction levels (4). Induction studies in which 1-ketothiolase and acetoacetyl-CoA reductase were studied have revealed that both enzymatic activities increase markedly in response to PHB-stimulating limitations (4,10,15,19).These experiments indicate that the PHB pathway may exhibit a mode of transcriptional control that is similar to that of other metabolic pathways that are induced by environmental stress. Examples of such pathways include the heat shock regulon, the pho regulon, and the carbon starvati...

Members of the genus Xenorhabdus are entomopathogenic bacteria that associate with nematodes. The nematode-bacteria pair infects and kills insects, with both partners contributing to insect pathogenesis and the bacteria providing nutrition to the nematode from available insect-derived nutrients. The nematode provides the bacteria with protection from predators, access to nutrients, and a mechanism of dispersal. Members of the bacterial genus Photorhabdus also associate with nematodes to kill insects, and both genera of bacteria provide similar services to their different nematode hosts through unique physiological and metabolic mechanisms. We posited that these differences would be reflected in their respective genomes. To test this, we sequenced to completion the genomes of Xenorhabdus nematophila ATCC 19061 and Xenorhabdus bovienii SS-2004. As expected, both Xenorhabdus genomes encode many anti-insecticidal compounds, commensurate with their entomopathogenic lifestyle. Despite the similarities in lifestyle between Xenorhabdus and Photorhabdus bacteria, a comparative analysis of the Xenorhabdus, Photorhabdus luminescens, and P. asymbiotica genomes suggests genomic divergence. These findings indicate that evolutionary changes shaped by symbiotic interactions can follow different routes to achieve similar end points.