Comparison of invasion of fibroblasts and macrophages by high- and low-virulence Leptospira strains: colonization of the host-cell nucleus and induction of necrosis by the virulent strain

The infection cycle of low- and high-virulence strains of Leptospira interrogans was compared in fibroblasts and macrophages. L. interrogans serovar Lai strain Lai was used as a representative high-virulence strain, while L. interrogans serovars Pomona strain Luo was used as a low-virulence strain. L. biflexa serovar Patoc strain Patoc I, a nonparasitic strain of Leptospira, was used as a control. Both the high- and low-virulence strains could adhere to fibroblasts and macrophages using one or both ends of the spirochete, which was followed by phagocytosis of both strains. Both strains adhered more strongly to macrophages than fibroblasts. However, the high-virulence strain could invade the host-cell nucleus, while the low-virulence strain remained in phagosomes. The L. biflexa strain neither adhered to nor invaded either cell type. Both of the L. interrogans strains also induced cell death (mostly necrosis) of macrophages, whether or not the spirochetes were viable, suggesting that leptospiral virulence is unrelated to macrophage death. However, the high-virulence strain induced mainly necrosis in fibroblasts, while the low-virulence strain induced more apoptosis. Thus, the main feature distinguishing the two L. interrogans strains is the ability of the high-virulence strain to invade the host-cell nucleus and induce pro-inflammatory necrosis in fibroblasts.     

Comparisons of Characteristics of Mercury-Tolerant Bacterium Pseudomonas oleovorans G-1 and Its Mercury-Sensitive Mutant Strain

An Hg²⁺-sensitive mutant strain was isolated from an Hg²⁺-tolerant bacterium Pseudomonas oleovorans G-1 strain by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine. The Hg²⁺-sensitive mutant strain was about 10-times as sensitive to Hg²⁺ as the parent strain. Moreover, the mutant strain was considerably more sensitive to Cr⁶⁺ than the parent strain, but it did not show an appreciable change in sensitivity to Cd²⁺ and Cu²⁺. The mutant strain was considerably more sensitive to antibiotics achromycin, chloramphenicol and streptomycin than the parent strain. A more rigid structure was observed in the cell envelope of the mutant strain than the parent strain under transmission electron microscope. Higher amounts of DNA but less protein and RNA were found in the mutant strain compared to the parent strain. Disc electrophoretic patterns showed some differences in protein bands between the parent and mutant strain.     

Suitability of Selected Bacteria and Yeasts for Growing the Estuarine Heterotrich Ciliate Fabrea salina (Henneguy)1

ABSTRACT. Twenty-six species of bacteria and seven species of yeasts were aseptically presented separately as potential food sources to the estuarine heterotrich ciliate, Fabrea salina, under standardized conditions of cell number, medium, and temperature. The bacteria and yeasts were classified according to their effect on the intrinsic growth rate of the ciliate: Nutritious bacteria: Photobacterium fisherii, Vibrio neresis, V. natriegens, Flavobacterium sp. strain ASN16, V. harveyi, Xanthomonas sp. strain ASN22; Maintainer bacteria: V. vulnificus, Vibrio sp. strain V344, V. alginolyticus, V. anguillarum, Bacillus sp. strain ST₃32B₂, Vibrio sp. strain V415, Acinetobacter sp. strain BHT8, Flectobacillus marinus, and Enterobacter aerogenes; Maintainer yeasts: Candida albicans and Cryptococcus marcerans strain 2; Nonnutriuous bacteria: V. parahaemolyticus, Planococcus sp. strain ASN13, P. mandapapanensis, Hyphomicrobium vulgari, Pseudomonas sp. strain CNS1, an unidentified gram-negative, yellow-pigmented, motile bacillus strain IG9A2, Thiobacillus thioparus, Escherichia coli, Corynebacterium sp. strain BR 17, Achromobacter sp. strain 23030, and Oceanospirillum beijerinckii; Nonnutriuous yeasts: C. marcerans strain 1, Saccharomyces cerevisiae, Debaryomyces hansenii, an unidentified black yeast, and C. laurentii. Under the conditions specified, bacteria appear to have a certain minimal value for the growth of Fabrea while yeast have little to none.     

Efficiency of insecticidal crystal protein production in a Bacillus thuringiensis mutant with derepressed expression of the terminal oxidase aa 3 during sporulation

A Bacillus thuringiensis respiratory mutant (AB1 strain) that shows premature sporulation and insecticidal crystal protein (ICP) production was isolated. The mutant strain harbours the cryIC and cryID insecticidal genes and could be important for the production of ICP highly toxic to Spodoptera sp. The mutant was selected by its increased capacity to oxidize. N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). In this strain, cytochrome aa ₃ expression is not repressed during the sporulation phase, in contrast with the wild-type strain. The growth, spore production, dissolved O₂, O₂ consumption, CO₂ evolution rate and ICP production were recorded as a function of time. The AB1 mutant strain has a similar growth yield to the wild-type strain, but begins sporulation at least 4 h earlier. The AB1 strain consumes 74.5% more O₂ than the wild-type strain, during the fermentation process. The mutation on strain AB1 has an important positive effect on ICP production. This procedure shows that ICP production could be increased during fermentation by increasing the respiration capacity of Bacillus thuringiensis. Correspondence to: A. Bravo     

Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue

Structural genes encoding an uptake hydrogenase of Nostoc sp. strain PCC 73102 were isolated. From partial libraries of genomic DNA, two clones (pNfo01 and pNfo02) were selected and sequenced, revealing the complete sequence of both a hupS (960 bases) and a hupL (1,593 bases) homologue in Nostoc sp. strain PCC 73102. A comparison between the deduced amino acid sequences of HupS and HupL of Nostoc sp. strain PCC 73102 and Anabaena sp. strain PCC 7120 showed that the HupS proteins are 89% identical and the HupL proteins are 91% identical. However, the noncoding region between the genes in Nostoc sp. strain PCC 73102 (192 bases) is longer than that of Anabaena sp. strain PCC 7120 and of many other microorganisms. Southern hybridizations using DNA from both N₂-fixing and non-N₂-fixing cells of Nostoc sp. strain PCC 73102 and different probes from within hupL clearly demonstrated that, in contrast to Anabaena sp. strain PCC 7120, there is no rearrangement within hupL of Nostoc sp. strain PCC 73102. Indeed, 6 nucleotides out of 16 within the potential recombination site are different from those of Anabaena sp. strain PCC 7120. Furthermore, we have recently published evidence demonstrating the absence of the bidirectional/reversible hydrogenase in Nostoc sp. strain PCC 73102. The present knowledge, in combination with the unique characteristics, makes Nostoc sp. strain PCC 73102 an interesting candidate for the study of deletion mutants lacking the uptake-type enzyme. Received: 20 August 1997 / Accepted: 24 November 1997     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S. sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey models, vaccinated animals were protected against the challenges of wild S. flexneri 2a strain 2457T and S. sonnei strain S9.     

Description of a Distinctive Aflatoxin-Producing Strain of <Emphasis Type="Italic">Aspergillus nomius</Emphasis> that Produces Submerged Sclerotia

A new distinctive strain of Aspergillus nomius that produces the potent mycotoxins, aflatoxins, is described from pistachio, pecan, and fig orchards in California. Similar to the typical strain of A. nomius (as represented by the ex-type), the O strain produced both B and G aflatoxins but not cyclopiazonic acid, had similar conidial ornamentation, and grew poorly at 42°C. Furthermore, previous published DNA sequence supports that the new strain is very closely related to the ex-type of A. nomius. However, the O strain differs from the ex-type in several morphological characters. The ex-type was initially described as producing “indeterminate sclerotia” that appear as large (up to 3 mm long) elongated sclerotia on surfaces of media. The O strain produces only small spherical sclerotia (mean diameter <0.3 mm) submerged in the medium. In addition, the O strain has predominantly uniseriate conidial heads, whereas the typical strain of A. nomius has predominantly biseriate heads. The O strain colony color on both Czapek solution agar and Czapek yeast extract agar was more yellowish than the ex-type of A. nomius and other common aflatoxin-producing fungi. Isolates of the O strain reported here from several orchards represent the first report of A. nomius in California.     

Comparative analysis of the genomes of <Emphasis Type="Italic">Bombyx mandarina</Emphasis> and <Emphasis Type="Italic">Bombyx mori</Emphasis> nucleopolyhedroviruses

The Bombyx mandarina nucleopolyhedrovirus (BomaNPV) S1 strain can infect the silkworm, Bombyx mori, but is significantly less virulent than B. mori nucleopolyhedrovirus (BmNPV) T3 strain. The complete nucleotide sequence of the S1 strain of BomaNPV was determined and compared with the BmNPV T3 strain. The circular, double stranded DNA genome of the S1 strain was 126,770 nucleotides long (GenBank accession no. FJ882854), with a G+C content of 40.23%. The genome contained 133 potential ORFs. Most of the putative proteins were more than 96% identical to homologs in the BmNPV T3 strain, except for bro-a, lef-12, bro-c, and bro-d. Compared with the BmNPV T3 strain, however, this genome did not encode the bro-b and bro-e genes. In addition, hr1 lacked two repeat units, while hr2L, hr2R, hr3, hr4L, hr4R, and hr5 were similar to the corresponding hrs in the T3 strain. The sequence strongly suggested that BomaNPV and BmNPV are variants with each other, and supported the idea that baculovirus strain heterogeneity may often be caused by variation in the hrs and bro genes.     

Characterization of Neurotoxigenic Clostridium butyricum Strain by DNA Hybridization Test and by in vivo and in vitro Germination Tests of Spores

The germination of spores of a neurotoxigenic Clostridium butyricum strain (BL 6340), which was isolated from infant botulism in Italy, and that of a non-toxigenic C. butyricum type strain (NCIB 7423) were studied. The spores of BL 6340 strain were killed at 80 C for 10 min, and required the mixture of L-alanine, L-lactate, glucose and bicarbonate for their optimal germination. These characteristics are the same as those of Clostridium botulinum type E strain, but different from those of NCIB 7423 strain. In a hybridization test, however, the labeled DNAs extracted from NCIB 7423 strain highly (98%) hybridized to the DNAs of the BL 6340 strain, but little (45%) to the DNAs of C. botulinum type E strain. The biochemical properties of the BL 6340 and NCIB 7423 strains were identical, but different from those of C. botulinum type E. These data confirmed that the BL 6340 strain belongs to C. butyricum species, but that only its characteristics of toxin production, its minimum requirements for germination, and the behavior of its spores to heat treatment are the same as those of C. botulinum type E. When conventionally raised suckling mice were injected with 5 × 10⁷ spores of BL 6340 strain intra- or orogastrically, botulism was not observed. However, 8- to 13-day-old mice had type E botulinum toxin in the large intestine 3 days after introduction of its spores.     

The involvement of DNA polymerase I in the postreplication repair of ultraviolet radiation-induced damage in Escherichia coli K-12.

Summary A deficiency in DNA polymerase I increased the ultraviolet (UV) radiation sensitivity of a uvrA strain of Escherichia coli K-12 when plated on minimal growth medium. The slope of the survival curve for the uvrA polA strain was 2.0-times greater than that for the uvrA strain. The fluence-dependent yield of unrepaired deoxyribonucleic acid (DNA) parental-strand breaks following UV irradiation and incubation in minimal growth medium was similar in both strains. However, the fluence-dependent yield of unrepaired DNA daughter-strand gaps observed following UV irradiation was 1.8-fold greater in the uvrA polA strain than in the uvrA strain. These results suggest that DNA polymerase I is involved in the filling of at least some daughter-strand gaps during postreplication repair. Also, the uvrA polA strain was sensitized by a post-UV treatment with chloramphenicol (CAP) to a similar extent as was the uvrA strain, indicating that DNA polymerase I is not involved in the CAP-inhibitable pathway of postreplication repair.     

枯草芽孢杆菌L-脯氨酸合成途径中glnA、proB、proA基因功能探究

【目的】从基因水平探究枯草芽孢杆菌渗透压调节因子L-脯氨酸合成途径中glnA、proB、proA基因的功能,通过分子改造实现对代谢途径的人工扰动。【方法】从枯草芽孢杆菌WB600出发,通过向胞内引入一系列基因敲除或过表达,分别构建了proB和proA基因过表达的重组菌WB601和WB602、glnA基因缺失的重组菌WB603以及在此基础之上过表达proB基因的重组菌WB604。借助菌株胞外和胞内游离脯氨酸积累的表型分析影响途径的关键节点。【结果】在非胁迫条件下,重组菌WB601和WB602胞外脯氨酸含量分别是原始菌的2.21倍和2.82倍,单位细胞胞外脯氨酸得率分别是原始菌的4.09倍和9.80倍,胞内游离脯氨酸含量分别是原始菌的1.91倍和3.34倍;重组菌WB603胞外脯氨酸含量上升至1221.43 mg/L,是原始菌的6.28倍,单位细胞胞外和胞内游离脯氨酸得率分别为原始菌的9.13倍和3.66倍;而重组菌WB604胞外脯氨酸含量最高达1391.65 mg/L,相比菌株WB603,其胞外脯氨酸含量及单位细胞得率分别提高了13.94%和14.10%,且胞内游离脯氨酸含量提高了32.60%。在5%Na Cl胁迫条件下,重组菌WB601和WB602的胞外脯氨酸含量分别是原始菌的1.94倍和1.54倍,单位细胞胞外脯氨酸得率分别是原始菌的2.15倍和2.19倍;重组菌WB603胞外脯氨酸含量及其单位细胞得率分别是原始菌的4.16倍和7.29倍;相同条件下,相比于重组菌WB603,重组菌WB604的胞外脯氨酸含量及其单位细胞得率分别提高了32.61%和5.54%。此外,实验组菌株的胞内游离脯氨酸含量均高于非胁迫时,并达到相对平衡状态。【结论】proB和proA基因的过表达均能显著提升细胞合成脯氨酸的能力,并且能增强细胞的耐盐性;glnA基因的缺失能增强脯氨酸合成途径,提高脯氨酸的积累;两种效应的正向叠加可进一步提升细胞脯氨酸合成能力。     

一株支气管炎鲍特杆菌国家标准菌种的再鉴定

通过生化试验、质谱鉴定、16S rRNA基因序列分析、全基因组序列测定及生物信息学分析等方法对国家标准菌种CMCC(B)40001进行再鉴定。对菌株的毒力、耐药和MLST进行分析，并对鲍特杆菌（Bordetella spp.）的群体进化进行分析。结合不同方法，CMCC(B)40001再鉴定为支气管炎鲍特杆菌（Bordetella bronchiseptica），且其作为标准菌种能够发挥国家标准描述的质控作用。同时获取该菌株的新ST型，在进化关系上，其与百日咳鲍特杆菌(Bordetella pertussis)和副百日咳鲍特杆菌(Bordetella parapertussis)遗传距离近。本研究再鉴定了国家标准菌种CMCC(B)40001为支气管炎鲍特杆菌，并分析了其遗传特征，为国家标准菌种的应用提供资源描述支持。     

New haplotype and inter‐strain reproductive compatibility of Wolbachia‐uninfected alfalfa weevil,Hypera postica (Coleoptera: Curculionidae), in Japan

Hypera postica is a univoltine invasive pest of alfalfa, Medicago sativa, in North America. In Japan, H. postica was first found in 1982 from Fukuoka and Okinawa Prefectures and became a serious pest of Chinese milk vetch, Astragalus sinicus, cultivated as a honey source for humans and green manure for rice. In North America, three strains, Western, Eastern and Egyptian, have been identified and the Western strain is infected with Wolbachia, which causes complete inter‐strain reproductive incompatibility. In contrast, only Western and Egyptian strains had been reported throughout Japan and none of the Western strain examined for the Fukuoka populations in northern Kyushu was infected with Wolbachia. First, we screened populations from northern Kyushu collected since 1982 for geographical and chronological distribution of the Eastern strain. The Eastern strain has been found at low frequencies since 1985 and is still present in 2014. Second, we experimentally tested our hypothesis that inter‐strain crosses between uninfected Western‐strain males and Egyptian‐strain females should produce viable offspring. We crossbred virgin adults reared individually from field‐collected larvae and confirmed that the F₁ eggs of crosses between the Western‐strain males and the Egyptian‐strain females develop successfully into larvae.     

Cell wall thickness and the molecular mechanism of heterogeneous vancomycin-intermediate Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) with reduced sensitivity to vancomycin (VAN) has caused many clinical cases of VAN treatment failure, but the molecular mechanism underlying the reduced sensitivity to VAN is still unclear. We isolated a heterogeneous VAN-intermediate Staphylococcus aureus (hVISA), which was also a MRSA strain with reduced sensitivity to VAN. To investigate the molecular mechanism underlying the reduced sensitivity to VAN exhibited by the hVISA strain, we compared the hVISA strain with a VAN-sensitive MRSA strain, known as the N315 strain. The images captured by transmission electron microscopy showed that the cell wall of the hVISA strain was significantly thicker than that of the N315 strain (36·72 ± 1·04 nm vs 28·15 ± 1·25 nm, P < 0·05), and the results of real-time quantitative PCR analysis suggested that the expression levels of the cell wall thickness related genes (glmS, vraR/S, sgtB, murZ and PBP4) of the hVISA strain were significantly higher than those of the N315 strain (P < 0·05). In conclusion, this study indicated that the upregulation of the expression of the genes related to cell wall synthesis might be the molecular mechanism underlying the cell wall thickening of the hVISA strain and might be related to its resistance to VAN.     

Occurrence of potato virus X strain group 1 in seed stocks of potato cultivars lacking resistance genes

Potato virus X (PVX) isolates were obtained from a simple seed potato production scheme or from ware potatoes produced by seed potatoes obtained from it. In this scheme, PVX infection is widespread in seed stocks and most of the potatoes grown lack PVX resistance genes. Thirteen PVX isolates were typed to strain group by inoculation to potato cultivars containing different combinations of hypersensitivity genes Nx and Nb. Six failed to overcome either gene and therefore belonged to strain group 1, four overcame Nb only and were placed in strain group 3 and three were mixtures of the two. All 13 isolates failed to overcome extreme resistance/immunity gene Rx. Naturally infected cultivars of genotype nx.nb contained strain group 1 alone or strain groups 1 and 3, while those of genotype nx:Nb contained only strain group 3. The widespread occurrence of strain group 1 contrasts with the predominant occurrence of strain group 3 in potatoes in the UK. However, it resembles the UK situation before sophisticated seed potato production schemes were introduced and before PVX hypersensitivity genes Nx and Nb were deliberately exploited in potato breeding. Prior infection with potato leafroll virus (PLRV) did not affect expression of hypersensitivity to PVX in inoculated leaves of an nx:Nb genotype.     

Identification and utilization of a 1,3-propanediol oxidoreductase isoenzyme for production of 1,3-propanediol from glycerol in Klebsiella pneumoniae

In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.     

Antimutagenic activity of soybeans fermented with basidiomycetes in Ames/Salmonella test

Soybeans fermented with either Phellinus igniarius or Agrocybe cylindracea inhibited the mutagenicity of the directly-acting mutagens: 4-nitro-o-phenylenediamine on Salmonella typhimurium strain TA 98 and NaN₃ on S. typhimurium strain TA 100; and indirectly-acting mutagens, 2-aminofluorene using strain TA 98 and benzo[a]pyrene using strain TA 100, in the presence of a supernatant solution from mammalian hepatic microsomes.     

Discrimination ofSUC gene fromMAL-constitutive gene by the fermentability of fructooligosaccharide in the yeastSaccharomyces cerevisiae

Since the yeastSaccharomyces cerevisiae carrying eitherSUC gene orMAL-constitutive gene ferments sucrose, these two genes could not be distinguished unless extracellular invertase activity was determined. The present work shows the strain carryingSUC fermented fructooligosaccharide, and the strain carryingMAL-constitutive did not. We applied these findings to genetic analysis of YOY10–13D, a haploid strain derived from a baker's yeast. The segregation of sucrose, maltose, and fructooligosaccharide fermentability in the tetrads of the cross between YOY10–13D and the tester strain showed that this strain carried oneSUC and oneMAL-constitutive.     

Construction of an engineered strain free of antibiotic resistance gene markers for simultaneous mineralization of methyl parathion and ortho-nitrophenol

Pseudomonas sp. strain NyZ402, a native soil organism that grows on para-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of methyl parathion (MP) and ortho-nitrophenol (ONP) by integrating mph (methyl parathion hydrolase gene) from Pseudomonas sp. strain WBC-3 and onpAB (ONP 2-monooxygenase and ONP o-benzoquinone reductase genes) from Alcaligenes sp. strain NyZ215 into the genome of strain NyZ402. Methyl parathion hydrolase (MPH), ONP 2-monooxygenase (OnpA) and o-benzoquinone reductase (OnpB) were constitutively expressed in the engineered strain NyZ-MO. Strain NyZ-MO was free of exogenous antibiotic resistance gene markers and the introduced genes were genetically stable. Degradation experiments showed that strain NyZ-MO could utilize MP or ONP as the sole carbon and energy source, and mineralize 0.1 mM MP–0.1 mM ONP simultaneously. This method may serve as a useful strategy for the construction of engineered strains in the degradation of multiple environmental pollutants.