Characterization of a Stress-Induced Alternate Sigma Factor, RpoS, of Coxiella burnetii and Its Expression during the Development Cycle

Coxiella burnetii is an obligate intracellular bacterium that resides in an acidified phagolysosome and has a remarkable ability to persist in the extracellular environment. C. burnetii has evolved a developmental cycle that includes at least two morphologic forms, designated large cell variants (LCV) and small cell variants (SCV). Based on differential protein expression, distinct ultrastructures, and different metabolic activities, we speculated that LCV and SCV are similar to typical logarithmic- and stationary-phase growth stages. We hypothesized that the alternate sigma factor, RpoS, a global regulator of genes expressed under stationary-phase, starvation, and stress conditions in many bacteria, regulates differential expression in life cycle variants of C. burnetii. To test this hypothesis, we cloned and characterized the major sigma factor, encoded by an rpoD homologue, and the stress response sigma factor, encoded by an rpoS homologue. The rpoS gene was cloned by complementation of an Escherichia coli rpoS null mutant containing an RpoS-dependent lacZ fusion (osmY::lacZ). Expression of C. burnetii rpoS was regulated by growth phase in E. coli (induced upon entry into stationary phase). A glutathione S-transferase–RpoS fusion protein was used to develop polyclonal antiserum against C. burnetii RpoS. Western blot analysis detected abundant RpoS in LCV but not in SCV. These results suggest that LCV and SCV are not comparable to logarithmic and stationary phases of growth and may represent a novel adaptation for survival in both the phagolysosome and the extracellular environment

Differential Expression of Translational Elements by Life Cycle Variants of Coxiella burnetii

Coxiella burnetii replicates as distinct morphological forms, which may allow potential life cycle variants to survive the harsh environment of the phagolysosome. Monoclonal antibodies (MAbs) were compared by Western blotting for reactivity with large cell variant (LCV) and small cell variant (SCV) antigens to characterize proteins differentially expressed by C. burnetii. MAb NM7.3 reacted with a ∼32-kDa LCV-upregulated antigen, and MAb NM183 reacted with a ∼45-kDa LCV-specific antigen. MAb NM7.3 was used to screen a λZapII C. burnetii DNA expression library, and an immunoreactive clone was identified with sequence similarity to the Escherichia coli tsf gene, which encodes elongation factor Ts (EF-Ts). Since a similar screen with MAb NM183 did not identify immunoreactive clones, an alternate strategy was devised to clone the reactive antigen based on observations of cross-reactivity with the 45-kDa elongation factor Tu (EF-Tu) protein from Chlamydia trachomatis. The highly conserved nature of EF-Tu among eubacteria allowed PCR amplification of a tuf gene fragment (encoding ∼95% of the predicted EF-Tu open reading frame) from C. burnetii using degenerate primers. The product of the cloned tuf gene fragment reacted with MAb NM183 in Western blot analysis, confirming the identity of the 45-kDa LCV-specific antigen. Identification of two proteins differentially expressed by C. burnetii, EF-Tu and EF-Ts, both essential components of the translational machinery of the cell, supports the hypothesis that LCVs are metabolically more active than SCVs

CAMERA: A Community Resource for Metagenomics

The CAMERA (Cyberinfrastructure for Advanced Marine Microbial Ecology Research and Analysis) community database for metagenomic data deposition is an important first step in developing methods for monitoring microbial communities

High-quality permanent draft genome sequence of Bradyrhizobium sp Th.b2, a microsymbiont of Amphicarpaea bracteata collected in Johnson City, New York

Bradyrhizobium sp. Th.b2 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing root nodule of Amphicarpaea bracteata collected in Johnson City, New York. Here we describe the features of Bradyrhizobium sp. Th.b2, together with high-quality permanent draft genome sequence information and annotation. The 10,118,060 high-quality draft genome is arranged in 266 scaffolds of 274 contigs, contains 9,809 protein-coding genes and 108 RNA-only encoding genes. This rhizobial genome was sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project

High-quality permanent draft genome sequence of Bradyrhizobium sp Ai1a-2; a microsymbiont of Andira inermis discovered in Costa Rica

Bradyrhizobium sp. Ai1a-2 is is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen fixing root nodule of Andira inermis collected from Tres Piedras in Costa Rica. In this report we describe, for the first time, the genome sequence information and annotation of this legume microsymbiont. The 9,029,266 bp genome has a GC content of 62.56% with 247 contigs arranged into 246 scaffolds. The assembled genome contains 8,482 protein-coding genes and 102 RNA-only encoding genes. This rhizobial genome was sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project proposal

High-quality permanent draft genome sequence of Bradyrhizobium sp Tv2a.2, a microsymbiont of Tachigali versicolor discovered in Barro Colorado Island of Panama

Bradyrhizobiumsp. Tv2a.2 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen-fixing root nodule of Tachigali versicolor collected in Barro Colorado Island of Panama. Here we describe the features of Bradyrhizobiumsp. Tv2a.2, together with high-quality permanent draft genome sequence information and annotation. The 8,496,279 bp high-quality draft genome is arranged in 87 scaffolds of 87 contigs, contains 8,109 protein-coding genes and 72 RNA-only encoding genes. This rhizobial genome was sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project

High-quality permanent draft genome sequence of the Mimosa asperata - nodulating Cupriavidus sp strain AMP6

Cupriavidus sp. strain AMP6 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Mimosa asperata collected in Santa Ana National Wildlife Refuge, Texas, in 2005. Mimosa asperata is the only legume described so far to exclusively associates with Cupriavidus symbionts. Moreover, strain AMP6 represents an early-diverging lineage within the symbiotic Cupriavidus group and has the capacity to develop an effective nitrogen-fixing symbiosis with three other species of Mimosa. Therefore, the genome of Cupriavidus sp. strain AMP6 enables comparative analyses of symbiotic trait evolution in this genus and here we describe the general features, together with sequence and annotation. The 7,579,563 bp high-quality permanent draft genome is arranged in 260 scaffolds of 262 contigs, contains 7,033 protein-coding genes and 97 RNA-only encoding genes, and is part of the GEBA-RNB project proposal

High-quality permanent draft genome sequence of the Mimosa asperata - nodulating Cupriavidus sp strain AMP6

Cupriavidus sp. strain AMP6 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Mimosa asperata collected in Santa Ana National Wildlife Refuge, Texas, in 2005. Mimosa asperata is the only legume described so far to exclusively associates with Cupriavidus symbionts. Moreover, strain AMP6 represents an early-diverging lineage within the symbiotic Cupriavidus group and has the capacity to develop an effective nitrogen-fixing symbiosis with three other species of Mimosa. Therefore, the genome of Cupriavidus sp. strain AMP6 enables comparative analyses of symbiotic trait evolution in this genus and here we describe the general features, together with sequence and annotation. The 7,579,563 bp high-quality permanent draft genome is arranged in 260 scaffolds of 262 contigs, contains 7,033 protein-coding genes and 97 RNA-only encoding genes, and is part of the GEBA-RNB project proposal