In Bacillus subtilis a null mutation of the relA gene, whose gene product is involved in the synthesis and/or hydrolysis of (p)ppGpp, causes a growth defect that can be suppressed by mutation(s) of yjbM and/or ywaC coding for small (p)ppGpp synthetases. All 35 suppressor mutations newly isolated were classified into two groups, either yjbM or ywaC, by mapping and sequencing their mutations, suggesting that there are no (p)ppGpp synthetases other than RelA, YjbM, and YwaC in B. subtilis. In order to understand better the relation between RelA and rRNA synthesis, we studied in the relA mutant the transcriptional regulation of seven rRNA operons (rrnO, -A, -J, -I, -E, -D, or -B) individually after integration of a promoter-and terminatorless cat gene. We identified the transcriptional start sites of each rrn operon (a G) and found that transcription of all rrn operons from their P1 promoters was drastically reduced in the relA mutant while this was almost completely restored in the relA yjbM ywaC triple mutant. Taken together with previous results showing that the intracellular GTP concentration was reduced in the relA mutant while it was restored in the triple mutant, it seems likely that continuous (p)ppGpp synthesis by YjbM and/or YwaC at a basal level causes a decrease in the amounts of intracellular GTP.

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The hemagglutinin (HA) of avian influenza viruses plays a very important role in the infection of host cells. In this study, the HA gene of the highly pathogenic avian influenza H5N1 virus was cloned and expressed in silkworm larvae. The expressed recombinant HA (rHA) was purified using fetuin-agarose chromatography and Superdex 200 10/300 GL gel filtration chromatography, and the identity of purified rHA was confirmed by SDS-PAGE and Western blot. Approximately 500 μg of purified rHA was obtained from a total of 30 silkworm larvae, suggesting the high efficiency of the silkworm expression system. The purified rHA bound to a rabbit polyclonal antibody against influenza A virus H5N1 (avian flu) HA, suggesting its antigenicity and potential application in vaccine development. Gel filtration chromatography showed that purified HA was present in the void volume fractions, indicating that rHA may form an oligomer. The rHA bound to poly{Neu5Acα2,3LacNAcβ-O[(CH₂)₅NHCO]₂(CH₂)₅NH-/γ-PGA}, which mimics an avian type receptor, but did not bind to γ-polyglutamic acid or human type receptor mimic, poly{Neu5Acα2,6LacNAcβ-O[(CH₂)₅NHCO]₂(CH₂)₅NH-/γ-PGA}, suggesting that it could be utilized as a blocking agent against infection by highly pathogenic influenza viruses.