Summary Important pathogens in the genus∞ C, however, the three yersiniae synthesized LPS containing predominantly hexa-acylated lipid A. This more complex lipid A stimulated human monocytes to secrete tumour necrosis factor-a a a a , whereas the lipid A synthesized by the three species at 37 ∞ ∞ ∞ ∞ C did not. The Y. pestis phoP gene was required for aminoarabinose modification of lipid A, but not for the temperaturedependent acylation changes. The results suggest that the production of a less immunostimulatory form of LPS upon entry into the mammalian host is a conserved pathogenesis mechanism in the genus Yersinia , and that species-specific lipid A forms may be important for life cycle and pathogenicity differences.

Yersinia pestis causes bubonic plague, characterized by an enlarged, painful lymph node, termed a bubo, that develops after bacterial dissemination from a fleabite site. In susceptible animals, the bacteria rapidly escape containment in the lymph node, spread systemically through the blood, and produce fatal sepsis. The fulminant progression of disease has been largely ascribed to the ability of Y. pestis to avoid phagocytosis and exposure to antimicrobial effectors of innate immunity. In vivo microarray analysis of Y. pestis gene expression, however, revealed an adaptive response to nitric oxide (NO)-derived reactive nitrogen species and to iron limitation in the extracellular environment of the bubo. Polymorphonuclear neutrophils recruited to the infected lymph node expressed abundant inducible NO synthase, and several Y. pestis homologs of genes involved in the protective response to reactive nitrogen species were up-regulated in the bubo. Mutation of one of these genes, which encodes the Hmp flavohemoglobin that detoxifies NO, attenuated virulence. Thus, the ability of Y. pestis to destroy immune cells and remain extracellular in the bubo appears to limit exposure to some but not all innate immune effectors. High NO levels induced during plague may also influence the developing adaptive immune response and contribute to septic shock. inducible nitric oxide synthase ͉ reactive nitrogen species

A n important property of bacterial endospores is their ability to survive high doses of UV radiation, which is due to the packaging of DNA and the unique photochemistry that this packaging imparts on dormant spores (1-3). The major photoproduct in DNA of UV-irradiated dormant spores is the unique thymine dimer 5-thyminyl-5,6-dihydrothymine, informally called spore photoproduct or SP (4, 5). SP can be removed from DNA during spore germination by the general nucleotide excision repair (NER) pathway (6, 7). In addition to NER, spores possess an SP-specific enzyme called SP lyase that can directly reverse SP to two thymines in situ without excision from DNA (6,8,9). Analysis of the deduced amino acid sequence encoded by the SP lyase gene (splB) cloned from Bacillus subtilis revealed a limited region with similarity to DNA photolyases (10), although early indirect experiments indicated that SP lyase caused direct reversal of SP back to two thymines in situ in a light-independent process (8, 9). These observations led to the question as to the mechanism SP lyase utilizes to reverse SP to two thymines. Inspection of the 342-aa deduced SplB sequence (10) revealed that the protein contains four cysteines, three of which are clustered at residues 91, 95, and 98, and the fourth at residue 141. The region from C91 to C98 was similar to the signature cysteine clusters present in the recently dubbed ''radical SAM'' protein superfamily (11), which includes enzymes such as anaerobic ribonucleotide reductase (RNR), pyruvate formate-lyase (PFL), lysine-2,3-aminomutase (KAM), and biotin synthase (BioB) (12). In radical SAM enzymes, the clustered cysteines serve as ligands in the formation of iron-sulfur ([Fe-S]) centers, and these enzymes use S-adenosylmethionine (S-AdoMet) as a cofactor to generate an adenosyl radical (13-16). We constructed a version of SplB protein containing an N-terminal tag of 10 histidines [(10His)SplB] and subsequently determined that (10His)SplB (i) contained both iron and acid-labile sulfur in a stoichiometric ratio of 1-2 atoms of iron or sulfur per (10His)SplB subunit (17); (ii) exhibited a UV-visible absorption spectrum consistent with other [Fe-S] proteins (17); (iii) required both anaerobic reducing conditions and S-AdoMet for activity in vitro (17, 18); and (iv) was inactive for SP cleavage unless its (10His) tag was first removed proteolytically (17, 18). The above evidence led us to the hypothesis that SP lyase carries out catalysis using an [Fe-S] center to cleave S-AdoMet, thus generating a 5Ј-adenosyl radical that could participate either directly or indirectly in SP cleavage. Support for this hypothesis has recently been obtained by Mehl and Begley (19). Using synthetic analogues of SP, they proposed a mechanistic scenario for SP reversal to two thymines in which the radical generated from S-AdoMet cleavage by electron donation from the [4Fe-4S] center can abstract a proton from C6Ј of SP, effectively generating an SP radical that fragments readily back to two thymines (Fig. 6) (19). The...

Yersinia pestis is an important human pathogen that is maintained in flea-rodent enzootic cycles in many parts of the world. During its life cycle, Y. pestis senses host-specific environmental cues such as temperature and regulates gene expression appropriately to adapt to the insect or mammalian host. For example, Y. pestis synthesizes different forms of lipid A when grown at temperatures corresponding to the in vivo environments of the mammalian host and the flea vector. At 37°C, tetra-acylated lipid A is the major form; but at 26°C or below, hexa-acylated lipid A predominates. In this study, we show that the Y. pestis msbB (lpxM) and lpxP homologs encode the acyltransferases that add C 12 and C 16:1 groups, respectively, to lipid IV A to generate the hexa-acylated form, and that their expression is upregulated at 21°C in vitro and in the flea midgut. A Y. pestis ⌬msbB ⌬lpxP double mutant that did not produce hexa-acylated lipid A was more sensitive to cecropin A, but not to polymyxin B. This mutant was able to infect and block fleas as well as the parental wild-type strain, indicating that the low-temperature-dependent change to hexa-acylated lipid A synthesis is not required for survival in the flea gut.The cell envelope of gram-negative bacteria includes two lipid bilayers, an inner membrane composed primarily of phospholipids, and an outer membrane containing primarily phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. Tight stacking of the long-chain fatty acids of lipid A, the hydrophobic anchor of LPS, creates a permeability barrier against toxic compounds encountered in the environment and the host (25). In Escherichia coli and Salmonella enterica serovar Typhimurium, the final steps of lipid A synthesis occur in the inner membrane, where two acyl groups are added to the tetra-acylated Kdo-lipid IV A before the mature hexa-acylated lipid A is exported to the outer membrane. At normal growth temperatures, the late acyltransferases HtrB (LpxL) and MsbB (LpxM) consecutively add lauroyl (C 12 ) and myristoyl (C 14 ) groups to the tetra-acylated intermediate (7,8,31). At 12°C, however, the cold-temperature-specific late acyltransferase LpxP acts instead of LpxL to add palmitoleate (C 16:1 ) (4). Mutation of htrB and msbB leads to growth defects and hypersensitivity to rifampin and vancomycin in E. coli, and to decreased virulence and resistance to macrophage killing in E. coli and S. enterica (37).Yersinia pestis, the zoonotic agent of bubonic and pneumonic plague in humans, is primarily a pathogen of rodents that is transmitted by fleas (27). Upon transmission to a mammalian host and an increase in temperature to 37°C, Y. pestis upregulates the expression of virulence factors that aid colonization and invasion. Conversely, after Y. pestis is taken up in a blood meal by a flea vector, the decrease in temperature results in downregulation of virulence factors and activation of genes and gene products important for survival in the flea midgut and transmission to a new mammal...