Hiroshi Kadokura

Hop-Resistant Lactobacillus brevis Contains a Novel Plasmid Harboring a Multidrug Resistance-Like Gene

The bitter-tasting compounds derived from the flowers of the hop plant (Humulus lupulus L.) protect beer from bacterial spoilage. However, a few lactic acid bacteria, especially lactobacilli, are resistant to these compounds and sometimes cause serious spoilage in the beer industry. It is important to elucidate the mechanisms of hop-resistance in lactic acid bacteria. We selected mutants of Lactobacillus brevis resistant to high concentrations of the hop compounds. The parental strain, L. brevis ABBC45, carries several plasmids. The copy number of one plasmid, termed pRH45, was remarkably increased in one of the hop-resistant mutants compared with that in the wild-type strain. pRH45 (15.0 kb) contains an open reading frame of 1749 b, termed horA, the deduced protein of which includes six putative transmembrane domains and an ATP-binding domain. The amino acid sequence of this putative protein is significantly homologous to half molecules of a mammalian multidrug resistance gene product, P-glycoprotein, and to several bacterial ABC transporters. Furthermore, the hop-resistant mutant was found to be weakly resistant to novobiocin and ethidium bromide, which are structurally and functionally unrelated to the hop compounds. A possible role of the potential drug efflux pump gene in the hop-resistance of L. brevis is discussed.

Biochemical characterization of horA-independent hop resistance mechanism in Lactobacillus brevis

We isolated a strain from hop-resistant Lactobacillus hrevis ABBC45, which had lost a plasmid (pRH45) harboring a putative hop resistance gene, horA. The hop resistance level of this horA-deficient strain, named ABBC45(C), was initially low but gradually induced by repeated growth in media containing progressively increasing levels of hop compounds. Although the hop resistance level was substantially lower than that of the hop-adapted wild type strain, hop-adapted ABBC45(C) (ABBC45(CR)) was still capable of growing in beer, suggesting ABBC45 possesses at least two hop resistance mechanisms. Hop resistance acquired by ABBC45(CR) gradually diminished to the pre-adapted level, when the strain was grown repeatedly in the absence of hop compounds. ABBC45(CR) was found to be cross-resistant to several structurally unrelated drugs, including ethidium bromide, daunomycin and nisin. In addition, ABBC45(CR) was shown to extrude ethidium in an energy-dependent manner, while ABBC45(C) did not show such activity. This indicates that the efflux pump was induced by adaptation to hop compounds. The efflux activity of ethidium was reduced by the addition of hop compounds, suggesting hop compounds are also the substrate of the efflux pump. It was also shown that the efflux activity was completely dissipated with the abolition of proton motive force (PMF). These results, taken together, suggest the hop resistance mechanism of ABBC45(C) is mediated by PMF-dependent multidrug efflux pump.

Immunological and molecular comparison of polyphenol oxidase in Rosaceae fruit trees

An antibody raised against apple polyphenol oxidase (PPO) cross-reacted with PPOs from Japanese pear (Pyrus pyrifolia), pear (Pyrus communis), peach (Prunus persica), Chinese quince (Pseudocydonia sinensis) and Japanese loquat (Eriobotrya japonica). Core fragments (681 bp) of the corresponding PPO genes were amplified and characterized. The deduced protein sequences showed identities of 85.3 to 97.5%. Chlorogenic acid oxidase activity of these PPOs showed higher activities when assayed at pH 4 than at pH 6. These results indicate that PPOs in Rosaceae plants are structurally and enzymatically similar.

Production and product quality assessment of human hepatitis B virus pre-S2 antigen in submerged and solid-state cultures of Aspergillus oryzae

Pre-S2 is a diagnostically important antigen of human hepatitis B virus (HBV). In order to produce pre-S2 antigen in Aspergillus oryzae, the gene [pre-S2]3, which encodes a tandemly triplicated repeat of pre-S2 polypeptides was fused with the partial glaA gene encoding glucoamylase lacking the starch-binding domain. In submerged culture, A. oryzae transformants carrying glaA-[pre-S2]3 secreted a heterogeneously glycosylated form of the fusion protein that was partially degraded. Contrarily, utilization of a wheat brain solid-state culture system resulted in the secretion of a homogeneous glycosylated form of the whole fusion protein. This is the first report of a dissimilarity in glycosylated modification between submerged and solid-state culture conditions in heterologous protein production in A. oryzae.

Enhancement of protein secretion by optimizing protein synthesis: isolation and characterization of Escherichia coli mutants with increased secretion ability of alkaline phosphatase

Overexpression of the Escherichia coli phoA gene, coding for alkaline phosphatase, on multicopy plasmids caused the accumulation of the precursor form of alkaline phosphatase. The cells lost their viability by a half-life of 60 min and exhibited high sensitivity to 1% sodium dodecyl sulfate (SDS), suggesting that the assembly of the surface proteins were affected by overexpression of the phoA gene. From the cells exhibiting resistance to 1% SDS, we obtained 20 mutants that secrete more alkaline phosphatase into the periplasmic space. Three representatives of the mutants accumulated no precursor molecules and secreted alkaline phosphatase by five- to six-fold that of the wild-type cells carrying multicopy phoA. In all of the three mutants, the amount of phoA transcripts were two to four times less than those in the wild-type cells, indicating that the ability to secrete a large amount of alkaline phosphatase was conferred by decreasing the synthetic rates of the phoA gene product. When the promoter of phoA was replaced with the tacI promoter and the expression level of the phoA gene was regulated with isopropyl-1-thio-β-d-galactoside, the secretion of alkaline phosphatase into the periplasm decreased as the synthetic rate of the phoA gene product increased over a threshold. All these results indicate that overproduction of the phoA gene product causes defects in the secretion of alkaline phosphatase and that the regulation of the expression level is essential for efficient translocation.

Cloning and sequencing of a gene encoding a novel salt stress-induced membrane protein from Rhodobacter sphaeroides f. sp. denitrificans

Abstract. The gene encoding a membrane protein, SspA, induced under salt stress conditions was cloned and sequenced from a photosynthetic bacterium, Rhodobacter sphaeroides f. sp. denitrificans IL106. A single open reading frame consisting of 972 base pairs that encoded a polypeptide composed of a signal peptide of 24 amino acids and a mature protein of 300 amino acids (Mr 33,386) was found. A database search failed to detect any highly homologous sequences, indicating that SspA is a novel protein. The protein was present in the outer membrane as a transmembrane protein and was specifically induced by salt stress, but not by heat shock.

Secretion of Bacillus subtilis α-amylase in the periplasmic space of Escherichia coli

SUMMARY: The Bacillus subtilis α-amylase structural gene (amyE) lacking its own signal peptide coding sequence was joined to the end of the Escherichia coli alkaline phosphatase (phoA) signal peptide coding sequence by using the technique of oligonucleotide-directed site-specific deletion. On induction of the phoA promoter, the B. subtilis α-amylase was expressed and almost all the activity was found in the periplasmic space of E. coli. The sequence of the five amino-terminal amino acids of the secreted polypeptide was Glu-Thr-Ala-Asn-Lys-, and thus the fused protein was correctly processed by the E. coli signal peptidase at the end of the phoA signal peptide.

Expression of the pre-S(2) gene of hepatitis B virus inEscherichia coli

SummaryThe pre-S(2) antigen of hepatitis B virus was produced as a fused protein with theE.coli alkaline phosphatase. The product remained in precipitable materials but could be solubilized by 8M urea treatment. The amount of the bacterial antigen was equivalent to 160 μg of serum HBsAg per ml of culture.