Shawn R. Starkenburg

Complete Genome Sequence of Nitrosospira multiformis, an Ammonia-Oxidizing Bacterium from the Soil Environment

ABSTRACT The complete genome of the ammonia-oxidizing bacterium Nitrosospira multiformis (ATCC 25196T) consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2,827 putative proteins. Of the 2,827 putative proteins, 2,026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and Nitrosomonas eutropha were the best match for 42% of the predicted genes in N. multiformis. The N. multiformis genome contains three nearly identical copies of amo and hao gene clusters as large repeats. The features of N. multiformis that distinguish it from N. europaea include the presence of gene clusters encoding urease and hydrogenase, a ribulose-bisphosphate carboxylase/oxygenase-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced genomes of ammonia-oxidizing bacteria. Gene clusters encoding proteins associated with outer membrane and cell envelope functions, including transporters, porins, exopolysaccharide synthesis, capsule formation, and protein sorting/export, were abundant. Numerous sensory transduction and response regulator gene systems directed toward sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate, and cyanophycin storage and utilization were identified, providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.

Genome sequence of the chemolithoautotrophic nitrite-oxidizing bacterium Nitrobacter winogradskyi Nb-255

ABSTRACT The alphaproteobacterium Nitrobacter winogradskyi (ATCC 25391) is a gram-negative facultative chemolithoautotroph capable of extracting energy from the oxidation of nitrite to nitrate. Sequencing and analysis of its genome revealed a single circular chromosome of 3,402,093 bp encoding 3,143 predicted proteins. There were extensive similarities to genes in two alphaproteobacteria, Bradyrhizobium japonicum USDA110 (1,300 genes) and Rhodopseudomonas palustris CGA009 CG (815 genes). Genes encoding pathways for known modes of chemolithotrophic and chemoorganotrophic growth were identified. Genes encoding multiple enzymes involved in anapleurotic reactions centered on C2 to C4 metabolism, including a glyoxylate bypass, were annotated. The inability of N. winogradskyi to grow on C6 molecules is consistent with the genome sequence, which lacks genes for complete Embden-Meyerhof and Entner-Doudoroff pathways, and active uptake of sugars. Two gene copies of the nitrite oxidoreductase, type I ribulose-1,5-bisphosphate carboxylase/oxygenase, cytochrome c oxidase, and gene homologs encoding an aerobic-type carbon monoxide dehydrogenase were present. Similarity of nitrite oxidoreductases to respiratory nitrate reductases was confirmed. Approximately 10% of the N. winogradskyi genome codes for genes involved in transport and secretion, including the presence of transporters for various organic-nitrogen molecules. The N. winogradskyi genome provides new insight into the phylogenetic identity and physiological capabilities of nitrite-oxidizing bacteria. The genome will serve as a model to study the cellular and molecular processes that control nitrite oxidation and its interaction with other nitrogen-cycling processes.

Complete genome sequence of Nitrobacter hamburgensis X14 and comparative genomic analysis of species within the genus Nitrobacter.

ABSTRACT The alphaproteobacterium Nitrobacter hamburgensis X14 is a gram-negative facultative chemolithoautotroph that conserves energy from the oxidation of nitrite to nitrate. Sequencing and analysis of the Nitrobacter hamburgensis X14 genome revealed four replicons comprised of one chromosome (4.4 Mbp) and three plasmids (294, 188, and 121 kbp). Over 20% of the genome is composed of pseudogenes and paralogs. Whole-genome comparisons were conducted between N. hamburgensis and the finished and draft genome sequences of Nitrobacter winogradskyi and Nitrobacter sp. strain Nb-311A, respectively. Most of the plasmid-borne genes were unique to N. hamburgensis and encode a variety of functions (central metabolism, energy conservation, conjugation, and heavy metal resistance), yet ∼21 kb of a ∼28-kb “autotrophic” island on the largest plasmid was conserved in the chromosomes of Nitrobacter winogradskyi Nb-255 and Nitrobacter sp. strain Nb-311A. The N. hamburgensis chromosome also harbors many unique genes, including those for heme-copper oxidases, cytochrome b561, and putative pathways for the catabolism of aromatic, organic, and one-carbon compounds, which help verify and extend its mixotrophic potential. A Nitrobacter “subcore” genome was also constructed by removing homologs found in strains of the closest evolutionary relatives, Bradyrhizobium japonicum and Rhodopseudomonas palustris. Among the Nitrobacter subcore inventory (116 genes), copies of genes or gene clusters for nitrite oxidoreductase (NXR), cytochromes associated with a dissimilatory nitrite reductase (NirK), PII-like regulators, and polysaccharide formation were identified. Many of the subcore genes have diverged significantly from, or have origins outside, the alphaproteobacterial lineage and may indicate some of the unique genetic requirements for nitrite oxidation in Nitrobacter.

Importance of Type II Secretion for Survival of Legionella pneumophila in Tap Water and in Amoebae at Low Temperatures

ABSTRACT Legionella pneumophila type II secretion mutants showed reduced survival in both tap water at 4 to 17°C and aquatic amoebae at 22 to 25°C. Wild-type supernatants stimulated the growth of these mutants, indicating that secreted factors promote low-temperature survival. There was a correlation between low-temperature survival and secretion function when 12 additional Legionella species were examined.

Pathogen comparative genomics in the next-generation sequencing era: genome alignments, pangenomics and metagenomics

As soon as whole-genome sequencing entered the scene in the mid-1990s and demonstrated its use in revealing the entire genetic potential of any given microbial organism, this technique immediately revolutionized the way pathogen (and many other fields of) research was carried out. The ability to perform whole-genome comparisons further transformed the field and allowed scientists to obtain information linking phenotypic dissimilarities among closely related organisms and their underlying genetic mechanisms. Such comparisons have become commonplace in examining strain-to-strain variability, as well as comparing pathogens to less, or nonpathogenic near neighbors. In recent years, a bloom in novel sequencing technologies along with continuous increases in throughput has occurred, inundating the field with various types of massively parallel sequencing data and further transforming comparative genomics research. Here, we review the evolution of comparative genomics, its impact in understanding pathogen evolution and physiology and the opportunities and challenges presented by next-generation sequencing as applied to pathogen genome comparisons.

Siderophore activity among members of the Legionella genus.

Members of the Legionella genus are ubiquitous aquatic bacteria and the etiologic agents of Legionnaires’ disease, a potentially fatal form of pneumonia. Using the chrome azurol S (CAS) assay, we previously determined that Legionella pneumophila secretes a siderophore (legiobactin) when it is grown in a low-iron, chemically defined medium (CDM). In the present study, we examined 29 other species of Legionella for their ability to produce CAS-reactive material when grown in deferrated CDM. Although some of the species did not grow in CDM, the majority replicated and secreted CAS reactivity, suggesting that siderophores are conserved among the legionellae.

D-Lactate metabolism and the obligate requirement for CO2 during growth on nitrite by the facultative lithoautotroph Nitrobacter hamburgensis

Nitrobacter hamburgensis X14 is a facultative lithoautotroph that conserves energy from the oxidation of nitrite (NO(-)2) and fixes carbon dioxide (CO(2)) as its sole source of carbon. The availability of the N. hamburgensis X14 genome sequence initiated a re-examination of its mixotrophic and organotrophic potential, as genes encoding three flavin-dependent oxidases were identified that may function to oxidize lactate, providing energy and carbon for growth. The response of N. hamburgensis to D- and L-lactate in the presence (mixotrophy) and absence (organotrophy) of NO(-)2 was examined. L-lactate did not support organotrophic growth or stimulate mixotrophic growth. In contrast, D-lactate enhanced the growth rate and yield of N. hamburgensis in the presence of NO(-)2, and served as the sole carbon and energy source for growth in the absence of NO(-)2 with ammonium as the sole nitrogen source. Lithoautotrophically grown cells immediately consumed D-lactate, suggesting that a lactate metabolic pathway is constitutively expressed. Nevertheless, a physiological adaptation to lactate occurred, as D-lactate-grown cells consumed and assimilated lactate at a faster rate than NO(-)2-grown cells, and the D-lactate-dependent O(2) uptake rate was significantly greater in cells grown either organotrophically or mixotrophically compared with cells grown lithoautotrophically. Although D-lactate was assimilated and metabolized to CO(2) in the presence or absence of NO(-)2, exposure to atmospheric CO(2) or the addition of 0.75 mM sodium carbonate was required for mixotrophic growth and for optimum organotrophic growth on D-lactate.

Responses of plant and bird communities to prescribed burning in tallgrass prairies

Historic losses and fragmentation of tallgrass prairie habitat to agriculture and urban development have led to declines in diversity and abundance of plants and birds associated with such habitat. Prescribed burning is a management strategy that has potential for restoring and rejuvenating prairies in fragmented landscapes, and through such restoration, might create habitat for birds dependent upon prairies. To provide improved data for management decision-making regarding the use of prescribed fire in tallgrass prairies, we compared responses of plant and bird communities on five burned and five unburned tallgrass prairie fragments at the DeSoto National Wildlife Refuge, Iowa, USA, from 1995 to 1997. Overall species richness and diversity were unaffected by burning, but individual species of plants and birds were affected by year-treatment interactions, including northern bobwhite (Colinus virginianus) and ring-necked pheasant (Phasianus colchicus), which showed time-delayed increases in density on burned sites. Analyses of species/area relationships indicated that, collectively, many small sites did make significant contributions to plant biodiversity at landscape levels, supporting the overall conservation value of prairie fragments. In contrast, most birds species were present on larger sites. Thus, higher biodiversity in bird communities which contain area-sensitive species might require larger sites able to support larger, more stable populations, greater habitat heterogeneity, and greater opportunity for niche separation.