The HtrB protein was first identified in Escherichia coli as a protein required for cell viability at high temperature, but its expression was not regulated by temperature. We isolated an htrB homologue from nontypable Haemophilus influenzae strain (NTHi) 2019, which was able to functionally complement the E. coli htrB mutation. The promoter for the NTHi 2019 htrB gene overlaps the promoter for the rfaE gene, and the two genes are divergently transcribed. The deduced amino acid sequence of NTHi 2019 HtrB had 56% homology to E. coli HtrB. In vitro transcription-translation analysis confirmed production of a protein with an apparent molecular mass of 32-33 kDa. Primer extension analysis revealed that htrB was transcribed from a 70 -dependent consensus promoter and its expression was not affected by temperature. The expression of htrB and rfaE was 2.5-4 times higher in the NTHi htrB mutant B29 than in the parental strain. In order to study the function of the HtrB protein in Haemophilus, we generated two isogenic htrB mutants by shuttle mutagenesis using a mini-Tn3. The htrB mutants initially showed temperature sensitivity, but they lost the sensitivity after a few passages at 30°C and were able to grow at 37°C. They also showed hypersensitivity to deoxycholate and kanamycin, which persisted on passage. SDS-polyacrylamide gel electrophoresis analysis revealed that the lipo-oligosaccharide (LOS) isolated from these mutants migrated faster than the wild type LOS and its color changed from black to brown as has been described for E. coli htrB mutants. Immunoblotting analysis also showed that the LOS from the htrB mutants lost reactivity to a monoclonal antibody, 6E4, which binds to the wild type NTHi 2019 LOS. Electrospray ionization-mass spectrometry analysis of the Odeacylated LOS oligosaccharide indicated a modification of the core structure characterized in part by a net loss in phosphoethanolamine. Mass spectrometric analysis of the lipid A of the htrB mutant indicated a loss of one or both myristic acid substitutions. These data suggest that HtrB is a multifunctional protein and may play a controlling role in regulating cell responses to various environmental changes. Lipopolysaccharide (LPS)1 is a component of the outer membrane of Gram-negative bacteria. It consists of lipid A linked by 2-keto-3-deoxyoctulosonic acid (KDO) to a heterogeneous sugar polymer and repeating O-antigen units. LPS plays an important role in pathogenicity and virulence. It also serves as a building block for the outer membrane and permeation barrier to hydrophobic compounds (1). Salmonella typhimurium LPS deep core mutants show increased sensitivity to various hydrophobic reagents and to elevated temperatures.The htrB gene was first identified in Escherichia coli as encoding a protein essential for cell viability at a temperature above 33°C (2). Unlike other heat shock proteins, however, its expression is not regulated by temperature (3). Bacteria with a mutation in the htrB gene, when exposed to nonpermissive temperatures in rich medi...

The htrB gene product of Haemophilus influenzae contributes to the toxicity of the lipooligosaccharide. The htrB gene encodes a 2-keto-3-deoxyoctulosonic acid-dependent acyltransferase which is responsible for myristic acid substitutions at the hydroxy moiety of lipid A ␤-hydroxymyristic acid. Mass spectroscopic analysis has demonstrated that lipid A from an H. influenzae htrB mutant is predominantly tetraacyl and similar in structure to lipid IV A , which has been shown to be nontoxic in animal models. We sought to construct a Salmonella typhimurium htrB mutant in order to investigate the contribution of htrB to virulence in a welldefined murine typhoid model of animal pathogenesis. To this end, an r ؊ m ؉ galE mutS recD strain of S. typhimurium was constructed (MGS-7) and used in inter-and intrastrain transduction experiments with both coliphage P1 and Salmonella phage P22. The Escherichia coli htrB gene containing a mini-Tn10 insertion was transduced from E. coli MLK217 into S. typhimurium MGS-7 via phage P1 and subsequently via phage P22 into the virulent Salmonella strain SL1344. All S. typhimurium transductants showed phenotypes similar to those described for the E. coli htrB mutant. Mass spectrometric analysis of the crude lipid A fraction from the lipopolysaccharide of the S. typhimurium htrB mutant strain showed that for the dominant hexaacyl form, a lauric acid moiety was lost at one position on the lipid A and a palmitic acid moiety was added at another position; for the less abundant heptaacyl species, the lauric acid was replaced with palmitoleic acid.Lipooligosaccharide (LOS), a major component of the outer membrane of nontypeable Haemophilus influenzae, is a complex molecule that requires the functions of many genes for proper assembly. One gene, htrB, has recently been shown to play a role in the acylation of the lipid A portion of the LOS of H. influenzae (19). The lipid A of H. influenzae is typically hexaacyl, containing ester-and amide-linked 4-hydroxymyristic acids. The two ␤-hydroxymyristic acids on the second glucosamine are replaced at their hydroxy group with myristic acid. In contrast, the lipid A of the H. influenzae htrB mutant is approximately 90% tetraacyl, with only four hydroxymyristic acid ester-and amide-linked fatty acids. This mutant lipid A is similar in structure to lipid IV A . The remaining 10% of the htrB lipid A is pentaacyl, with a single myristic acid substitution.Recent studies of nontypeable H. influenzae have indicated that changes in the LOS structure affect bacterial virulence. The similarity of the LOS from the nontypeable H. influenzae htrB mutant to lipid IV A suggests that it binds to the CD14 receptor but does not initiate signalling which results in a macrophage cytokine response (18). Experimental evidence has shown that LOS from the nontypeable H. influenzae htrB mutant has reduced toxicity in an infant-rat model and elicits less tumor necrosis factor alpha from human macrophages than does wild-type LOS (unpublished data). In addition, Somerville et al. (3...

Neisseria meningitidis is the etiologic agent of epidemic bacterial meningitis. Lipooligosaccharide (LOS) is a principal virulence factor associated with the organism, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of LOS has demonstrated that there is considerable microheterogeneity in the molecule. To begin our understanding of the nature of this heterogeneity, we identified a Tn916-generated LOS mutant of N. meningitidis NMB (serotype L3, monoclonal antibodies 3F11 ؉ , 6B4 ؉ , and 4C4 ؊) that was designated NMB-SS3 (monoclonal antibodies 3F11 ؊ , 6B4 ؊ , and 4C4 ؉). The transposon insertion was localized to the amino terminus of the functional copy of the UDP-Glc 4-epimerase gene (galE). UDP-Glc 4-epimerase (EC 5.1.3.2) activity was present in N. meningitidis NMB but not in NMB-SS3, indicating that the Tn916 insertion had abolished this activity. Mass spectrometric analysis of the LOS from strain NMB revealed multiple species of LOS, which is consistent with extensive microheterogeneity. While the most predominant structure was consistent with a terminal lacto-N-neotetrose structure found in other strains of N. meningitidis, Gal␤13 4GlcNAc␤133Gal␤134Glc3(GlcNAc)3Hep 2 PEA3KDO 2 (where Hep is heptose, PEA is phosphoethanolamine, and KDO is 2-keto-3-deoxymannooctulosonic acid), structures containing repetitive hexoses which are not precursors of this structure were also identified. Compositional analysis of LOS from strain NMB-SS3 revealed that there were no galactoses present in the structure. Mass spectrometric analysis of O-deacylated LOS revealed the presence of multiple species, with the predominant LOS species in this mutant strain formed by the Hex3(HexNAc)3Hep 2 PEA3KDO 2 (where Hex is hexose and HexNAc is N-acetylhexosamine) structure. However, LOS structures with repetitive hexoses, e.g., Hex n 3(HexNAc)3Hep 2 PEA3KDO 2 (n ‫؍‬ 2, 3, or 4), emanating from one or both heptoses were also identified. Since this mutant cannot synthesize UDP-Gal, these structures must be repetitive glucoses. These data suggest that NMB has a glycosyltransferase capable of polymerizing glucose moieties as an alternative biosynthetic pathway to the wild-type lacto-N-neotetrose structure.

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