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...

We show that a collection of 93 E. coli mutations which map between thr and leu and which block phage lambda DNA replication define two closely linked cistrons. Work published in the accompanying paper shows that these mutations also affect host DNA replication, so we designate them dnaJ and dnaK; the gene order is thr--dnaK--dnaJ--leu. Demonstration of two cistrons was possible with the isolation of lambda transducing phages carrying one or the other or both of the dna genes. These phages were employed in phage vs bacterial complementation studies which unambiguously show that dnaK and dnaJ are different cistrons.

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The htrB mutant of Haemophilus influenzae (strain B29) has been shown to lack secondary (nonhydroxylated) acyl groups in its lipid A. We have determined through in vitro biochemical assays that the HtrB protein acts as a specific acyltransferase in the late stages of lipid A biosynthesis and that the preferred acyl group donor is myristoyl-acyl carrier protein. Under the conditions employed, the Escherichia coli precursor, Kdo 2 -lipid IV A , functions as a myristate acceptor. Introduction of the Haemophilus htrB gene into an E. coli mutant lacking htrB complements the biochemical and physiological defects associated with the E. coli htrB mutation.Tumor necrosis factor α (TNFα) assays using murine and human macrophage cells indicated that nontypeable H. influenzae (NtHi) strain 2019 and H. influenzae type b strain A2 elicit levels of expression of TNFα that are 30-40 times greater than levels induced by the isogenic htrB mutants (B29 and A2B29). Studies using cell-free LOS indicated that the LOS from wild type strain 2019 elicits levels of TNFα expression that are 6-8-fold higher than those of B29. In situ hybridization studies of a primary human bronchial epithelial cell line demonstrated a greater increase of TNFα message produced in the presence of 2019 LOS than in the presence of B29 LOS. TNFα levels of the cell supernatant of cells stimulated with 2019 LOS were found to be 7-8-fold higher than levels in B29 stimulated supernatants. Using the Limulus amoebocyte lysate for assessment of endotoxic activity, we found that wild type LOS was 8-fold higher in endotoxic activity compared with the mutant LOS. In virulence assays using intraperitoneal inoculation of infant rats, the htrB isogenic strain caused bacteremia at 50% the frequency of the wild type strain. In intranasal inoculation studies, the htrB mutant strain was unable to cause bacteremia whereas the wild type b parent produced bacteremia in 40-60% of the animals. These findings suggest that the htrB gene of H. influenzae is important for virulence and that host TNFα expression is attenuated in response to htrB mutant strains.

Some strains of Escherichia coli contain retroelements (retrons) that encode genes for reverse transcriptase and branched, multicopy, single-stranded DNA (msDNA) linked to RNA. However, the origin of retrons is unknown. A P4-like cryptic prophage was found that contains a retroelement (retron Ec73) for msDNA-Ec73 in an E. coli clinical strain. The entire genome of this prophage, named phi R73, is 12.7 kilobase pairs and is flanked by 29-base pair direct repeats derived from the 3' end of the selenocystyl transfer RNA gene (selC). P2 bacteriophage caused excision of the phi R73 prophage and acted as a helper to package phi R73 DNA into an infectious virion. The newly formed phi R73 closely resembled P4 as a virion and in its lytic growth. Retronphage phi R73 lysogenized a new host strain, reintegrating its genome into the selC gene of the host chromosome and enabling the newly formed lysogens to produce msDNA-Ec73. Hence, retron Ec73 can be transferred intercellularly as part of the genome of a helper-dependent retronphage.