Nontypeable Haemophilus influenzae (NTHI), an opportunistic pathogen that is commonly found in the human upper respiratory tract, has only four identified two-component signal transduction systems. One of these, an ortholog to the QseBC (quorum-sensing Escherichia coli) system, was characterized. This system, designated firRS, was found to be transcribed in an operon with a gene encoding a small, predicted periplasmic protein with an unknown function, ygiW. The ygiW-firRS operon exhibited a unique feature with an attenuator present between ygiW and firR that caused the ygiW transcript level to be 6-fold higher than the ygiW-firRS transcript level. FirRS induced expression of ygiW and firR, demonstrating that FirR is an autoactivator. Unlike the QseBC system of E. coli, FirRS does not respond to epinephrine or norepinephrine. FirRS signal transduction was stimulated when NTHI cultures were exposed to ferrous iron or zinc but was unresponsive to ferric iron. Notably, the ferrous iron-responsive activation only occurred when a putative iron-binding site in FirS and the key phosphorylation aspartate in FirR were intact. FirRS was also activated when cultures were exposed to cold shock. Mutants in ygiW, firR, and firS were attenuated during pulmonary infection, but not otitis media. These data demonstrate that the H. influenzae strain 2019 FirRS is a two-component regulatory system that senses ferrous iron and autoregulates its own operon.

Brucella strains are exposed to potentially toxic levels of H 2 O 2 both as a consequence of their aerobic metabolism and through the respiratory burst of host phagocytes. To evaluate the relative contributions of the sole catalase KatE and the peroxiredoxin AhpC produced by these strains in defense against H 2 O 2 -mediated toxicity, isogenic katE, ahpC, and katE ahpC mutants were constructed and the phenotypic properties of these mutants compared with those of the virulent parental strain B. abortus 2308. The results of these studies indicate that AhpC is the primary detoxifier of endogenous H 2 O 2 generated by aerobic metabolism. KatE, on the other hand, plays a major role in scavenging exogenous and supraphysiologic levels of H 2 O 2 , although this enzyme can play a supporting role in the detoxification of H 2 O 2 of endogenous origin if AhpC is absent. B. abortus ahpC and katE mutants exhibit wild-type virulence in C57BL/6 and BALB/c mice, but the B. abortus ahpC katE double mutant is extremely attenuated, and this attenuation is not relieved in derivatives of C57BL/6 mice that lack NADPH oxidase (cybb) or inducible nitric oxide synthase (Nos2) activity. These experimental findings indicate that the generation of endogenous H 2 O 2 represents a relevant environmental stress that B. abortus 2308 must deal with during its residence in the host and that AhpC and KatE perform compensatory roles in detoxifying this metabolic H 2 O 2 .

Irr and RirA, rather than Fur, serve as the major iron-responsive regulators in the alphaproteobacteria. With only a few exceptions, however, the relative contributions of these transcriptional regulators to the differential expression of specific iron metabolism genes in Brucella strains are unclear. The gene encoding the outer membrane heme transporter BhuA exhibits maximum expression in Brucella abortus 2308 during growth under iron-deprived conditions, and mutational studies indicate that this pattern of bhuA expression is mediated by the iron-responsive regulator Irr. Specifically, a bhuA-lacZ transcriptional fusion does not produce elevated levels of ␤-galactosidase in response to iron deprivation in the isogenic irr mutant BEA5, and, unlike the parental strain, B. abortus BEA5 cannot utilize heme as an iron source in vitro and is attenuated in mice. A derivative of the bhuA-lacZ transcriptional fusion lacking the predicted Irr binding site upstream of the bhuA promoter does not produce elevated levels of ␤-galactosidase in response to iron deprivation in the parental B. abortus 2308 strain, and a direct and specific interaction between a recombinant version of the Brucella Irr and the bhuA promoter region was observed in an electrophoretic mobility shift assay. Despite the fact that it lacks the heme regulatory element linked to the iron-responsive degradation of its counterpart in Bradyrhizobium japonicum, readily detectable levels of Irr were found only in B. abortus 2308 cells by Western blot analysis following growth under iron-deprived conditions.

The baculovirus expression vector system (BEVS) is a widely used platform for the production of recombinant eukaryotic proteins. However, the BEVS has limitations in comparison to other higher eukaryotic expression systems. First, the insect cell lines used in the BEVS cannot produce glycoproteins with complex-type N-glycosylation patterns. Second, protein production is limited as cells die and lyse in response to baculovirus infection. To delay cell death and lysis, we transformed several insect cell lines with an expression plasmid harboring a vankyrin gene (P-vank-1), which encodes an anti-apoptotic protein. Specifically, we transformed Sf9 cells, Trichoplusia ni High Five™ cells, and SfSWT-4 cells, which can produce glycoproteins with complex-type N-glycosylation patterns. The latter was included with the aim to increase production of glycoproteins with complex N-glycans, thereby overcoming the two aforementioned limitations of the BEVS. To further increase vankyrin expression levels and further delay cell death, we also modified baculovirus vectors with the P-vank-1 gene. We found that cell lysis was delayed and recombinant glycoprotein yield increased when SfSWT-4 cells were infected with a vankyrin-encoding baculovirus. A synergistic effect in elevated levels of recombinant protein production was observed when vankyrin-expressing cells were combined with a vankyrin-encoding baculovirus. These effects were observed with various model proteins including medically relevant therapeutic proteins. In summary, we found that cell lysis could be delayed and recombinant protein yields could be increased by using cell lines constitutively expressing vankyrin or vankyrin-encoding baculovirus vectors.

Artificial insemination (AI) is an assisted reproductive technique that is implemented successfully in humans as a fertility treatment, performed extensively for commercial breeding of livestock, and is also successful in laboratory rodents. AI in the mouse may be especially useful for breeding of transgenic or mutant mice with fertility problems, expansion of mouse colonies, and as an alternative to in vitro fertilization. Nonsurgical AI techniques for the mouse have been described previously but are not often implemented due to technical difficulties. Here we compare various protocols for preparation of CD1 recipients prior to AI for naïve (in estrus), ovulation-induced, and superovulated females. Timing of hormone administration relative to sperm delivery is also compared. An improved protocol for nonsurgical AI in mice is described, which incorporates a convenient hormone administration schedule for female recipients and rapid, non-stressful sperm transfer without the need for anesthesia or analgesia.