Post-transcriptional control of the essential enzyme MurF by a small regulatory RNA in Brucella abortus.

Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.

Proline utilization system is required for infection by the pathogenic α-proteobacterium Brucella abortus.

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 ΔputR 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.

PHA-stimulated immune-responsiveness in mercury-dosed zebra finches does not match results from environmentally exposed songbirds.

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.

The Use and Limitations of the 16S rRNA Sequence for Species Classification of Anaplasma Samples.

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.

A comprehensive review of small regulatory RNAs in Brucella spp.

Brucella spp. are Gram-negative bacteria that naturally infect a variety of domesticated and wild animals, often resulting in abortions and sterility. Humans exposed to these animals or animal products can also develop debilitating, flu-like disease. The brucellae are intracellular pathogens that reside predominantly within immune cells, typically macrophages, where they replicate in a specialized compartment. This capacity of Brucella to survive and replicate within macrophages is essential to their ability to cause disease. In recent years, several groups have identified and characterized small regulatory RNAs (sRNAs) as critical factors in the control of Brucella physiology within macrophages and overall disease virulence. sRNAs are generally < 300 nucleotides in length, and these independent sRNA transcripts are encoded either next to (i.e., cis-encoded) or at a distant location to (i.e., trans-encoded) the genes that they regulate. Trans-encoded sRNAs interact with the mRNA transcripts through short stretches of imperfect base pairing that often require the RNA chaperone Hfq to facilitate sRNA-mRNA interaction. In many instances, these sRNA-mRNA interactions inhibit gene expression, usually by occluding the ribosome-binding site (RBS) and/or by decreasing the stability of the mRNA, leading to degradation of the transcript. A number of sRNAs have been predicted and authenticated in Brucella strains, and a variety of approaches, techniques, and means of validation have been employed in these efforts. Nonetheless, some important issues and considerations regarding the study of sRNA regulation in Brucella need to be addressed. For example, the lack of uniform sRNA nomenclature in Brucella has led to difficulty in comparisons of sRNAs across the different Brucella species, and there exist multiple names in the literature for what are functionally the same sRNA. Moreover, even though bona fide sRNAs have been discovered in Brucella, scant functional information is known about the regulatory activities of these sRNAs, or the extent to which these sRNAs are required for the intracellular life and/or host colonization by the brucellae. Therefore, this review summarizes the historical context of Hfq and sRNAs in Brucella; our current understanding of Brucella sRNAs; and some future perspectives and considerations for the field of sRNA biology in the brucellae.