With the advent of cheaper, high-throughput sequencing technologies, the ability to survey biodiversity in previously unexplored niches and geographies has expanded massively. Within Anaplasma, a genus containing several intra-hematopoietic pathogens of medical and economic importance, at least 25 new species have been proposed since the last formal taxonomic organization. Given the obligate intracellular nature of these bacteria, none of these proposed species have been able to attain formal standing in the nomenclature per the International Code of Nomenclature of Prokaryotes rules. Many novel species’ proposals use sequence data obtained from targeted or metagenomic PCR studies of only a few genes, most commonly the 16S rRNA gene. We examined the utility of the 16S rRNA gene sequence for discriminating Anaplasma samples to the species level. We find that while the genetic diversity of the genus Anaplasma appears greater than appreciated in the last organization of the genus, caution must be used when attempting to resolve to a species descriptor from the 16S rRNA gene alone. Specifically, genomically distinct species have similar 16S rRNA gene sequences, especially when only partial amplicons of the 16S rRNA are used. Furthermore, we provide key bases that allow classification of the formally named species of Anaplasma.

Proline utilization (Put) systems have been described in a number of bacteria; however, the importance and functionality of the Put system in the intracellular pathogen Brucellaabortus has not been explored. Generally, bacterial Put systems are composed of the bifunctional enzyme proline dehydrogenase PutA and its transcriptional activator PutR. Here, we demonstrate that the genes putA (bab2_0518) and putR (bab2_0517) are critical for the chronic infection of mice by B. abortus, but putA and putR are not required for the survival and replication of the bacteria in naive macrophages. Additionally, in vitro experiments revealed that putR is necessary for the ability of the bacteria to withstand oxidative stress, as a DputR deletion strain is hypersensitive to hydrogen peroxide exposure. Quantitative reverse transcription-PCR and putA-lacZ transcriptional reporter studies revealed that PutR acts as a transcriptional activator of putA in Brucella, and electrophoretic mobility shift assays confirmed that PutR binds directly to the putA promoter region. Biochemical analyses demonstrated that a purified recombinant B. abortus PutA protein possesses quintessential proline dehydrogenase activity, as PutA is capable of catalysing the conversion of proline to glutamate. Altogether, these data are the first to reveal that the Put system plays a significant role in the ability of B. abortus to replicate and survive within its host, as well as to describe the genetic regulation and biochemical activity of the Put system in Brucella.

Dietary mercury exposure is associated with suppressed immune responsiveness in birds. This study examined the immune-responsiveness of domestic zebra finches (Taeniopygia guttata) experimentally exposed to mercury through their diet. We used the phytohemagglutinin (PHA) skin-swelling test to assay the effect of two modes of mercury exposure. Some finches received exposure to mercury only after reaching sexual maturity, while others were maintained on a mercury-dosed diet throughout life, including development. Each bird received one of five dietary concentrations of methylmercury cysteine (0.0, 0.3, 0.6, 1.2 or 2.4 ppm). In contrast to a study on wild songbirds at a mercury-contaminated site, we detected no relationship between mercury level and immunological response to PHA, regardless of mode of exposure. This result represents the first major difference found by our laboratory between wild birds exposed to environmental mercury and captive birds experimentally exposed to mercury.