Borrelia burgdorferi, the Lyme disease spirochete, undergoes dramatic changes in antigenic composition as it cycles between its arthropod and mammalian hosts. A growing body of evidence suggests that these changes reflect, at least in part, the need for spirochetes to adapt to the physiological stresses imposed by abrupt changes in environmental conditions and nutrient availability. -PCR), we demonstrated that, in contrast to its ortholog (rpoS Ec ) in Escherichia coli, rpoS Bb was expressed at significant levels in B. burgdorferi throughout all phases of growth following temperature shift. By comparing a B. burgdorferi strain 297 rpoS Bb mutant to its wild-type counterpart, we determined that RpoS Bb was not required for survival following exposure to a wide range of environmental stresses (i.e., temperature shift, serum starvation, increased osmolality, reactive oxygen intermediates, and increased or reduced oxygen tension), although the mutant was more sensitive to extremes of pH. While B. burgdorferi strains lacking RpoS were able to survive within intraperitoneal dialysis membrane chambers at a level equivalent to that of the wild type, they were avirulent in mice. Lastly, RT-PCR analysis of the ospE-ospF-elp paralogous lipoprotein families complements earlier findings that many temperature-inducible borrelial loci are controlled in an RpoS Bbindependent manner. Together, these data point to fundamental differences between the role(s) of RpoS in B. burgdorferi and that in E. coli. Rather than functioning as a master regulator, RpoS Bb appears to serve as a stress-responsive activator of a subset of virulence determinants that, together with the RpoS-independent, differentially expressed regulon, encompass the spirochete's genetic programs required for mammalian host adaptation.

PCR primers that target the bacterial 16S rRNA genes (or the tuf gene for the genus Enterococcus) were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were investigated. Microbial samples from the root canals of 24 teeth with necrotic pulp were included in the study. PCR with universal bacterial primers identified bacterial DNA in 22 specimens; the remaining 2 specimens were from intact teeth that had been traumatized 6 months prior to treatment. PCR with specific primers showed that preoperative symptoms were significantly associated with the presence of Streptococcus spp. (P < 0.001 by chi-square analysis). There was also a nonsignificant trend for symptoms to be associated with Fusobacterium nucleatum and Porphyromonas gingivalis (odds ratio, >2) and for diabetes mellitus to be associated with P. gingivalis and Porphyromonas endodontalis (odds ratio, >2). Cloning and sequencing of the universal PCR product in one specimen revealed the presence of an organism related to the genus Olsenella, which has not previously been described in endodontic infections.

f Acinetobacter baumannii is a nosocomial opportunistic pathogen that can cause severe infections, including hospital-acquired pneumonia, wound infections, and sepsis. Multidrug-resistant (MDR) strains are prevalent, further complicating patient treatment. Due to the increase in MDR strains, the cationic antimicrobial peptide colistin has been used to treat A. baumannii infections. Colistin-resistant strains of A. baumannii with alterations to the lipid A component of lipopolysaccharide (LPS) have been reported; specifically, the lipid A structure was shown to be hepta-acylated with a phosphoethanolamine (pEtN) modification present on one of the terminal phosphate residues. Using a tandem mass spectrometry platform, we provide definitive evidence that the lipid A isolated from colistin-resistant A. baumannii MAC204 LPS contains a novel structure corresponding to a diphosphoryl hepta-acylated lipid A structure with both pEtN and galactosamine (GalN) modifications. To correlate our structural studies with clinically relevant samples, we characterized colistin-susceptible and -resistant isolates obtained from patients. These results demonstrated that the clinical colistin-resistant isolate had the same pEtN and GalN modifications as those seen in the laboratory-adapted A. baumannii strain MAC204. In summary, this work has shown complete structure characterization including the accurate assignment of acylation, phosphorylation, and glycosylation of lipid A from A. baumannii, which are important for resistance to colistin.

The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod-or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHIand macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI-and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHBgrown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa ␣ 2 homodimer containing, on average, 0.7 iron atoms/subunit. Mö ssbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K 3 (Fe(CN) 6 ), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A 280 /A 656 ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.