Alterations in the cell envelope composition of Proteus mirabilis during the development of swarmer cells.

Long, swarming cells of Proteus mirabilis had different proportions of some lipopolysaccharide components when compared to short cells, either agar grown or broth grown. Fluorescence spectrophotometry of antibody binding, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that the change was in the proportion of lipopolysaccharide with long O-antigenic sidechains, swarmer lipopolysaccharide relative to short sidechain lipopolysaccharide than the non-swarming cells. The proteins and phospholipids of the envelop remained the same during swarmer development. The results are discussed in relation to the increase in flagella synthesis and permeability to some antibacterial agents during swarmer development.     

Capsulation and virulence in Erwinia amylovora

Evidence is presented that capsulation may be one virulence determinant for Erwinia amylovora, the fireblight pathogen. When 15 virulent and seven avirulent strains were grown on a medium containing asparagine as the only source of carbon and nitrogen, or yeast peptone agar, or on a sugar medium containing an inorganic source of nitrogen, capsule production and virulence were not correlated. However, if a sugar or sugar alcohol was added to the asparagine medium or to yeast peptone agar all the virulent strains produced some or many capsulated cells whereas six of the avirulent ones did not. Capsules were also produced by all the virulent strains during infection. The existence of a seventh avirulent strain which was capsulated on all media except unsupplemented asparagine agar, suggested that capsule production was not the only virulence determinant.     

Nitrogen-fixing bacteria of the genusBeijerinckia Derx in the rhizosphere of sugar cane

Summary and Conclusions The rhizosphere effect of sugar cane on nitrogen fixing bacteria of the genusBeijerinckia Derx was studied under field conditions, during two growing periods of the cane. Counts ofBeijerinckia in soil samples from the rhizosphere and from the rhizoplan (soil from root surface) showed an increase in this nitrogen fixer of up to 20 times in the rhizosphere and up to 50 times in the rhizoplan. Bacteria, actinomyces, and fungi developing on egg-albumin agar decreased in the rhizosphere of sugar cane.This work was in part, supported by the Conselho Nacional de pesquisas.     

Hapten-specific murine colony-forming B cells. II. Delineation of a tolerogen-sensitive subpopulation of colony-forming B cells

B cell subpopulations were studied by using B cell cloning procedures and an in vitro tolerance induction model. Fluorescein- (FL) specific B cells from normal spleens were isolated by using FL gelatin plates and were then cultured in semisolid agar in the presence or absence of tolerogen. Hapten-specific cells grew in soft agar to form discrete colonies. Colony growth is dependent on "mitogens" present in agar, sheep red blood cells (SRBC), and lipopolysaccharide (LPS). For example, SRBC plus LPS potentiate the growth of an increased number of colony-forming B cells (CFU-B) compared to either additive alone. These CFU-B could be triggered by a specific antigen to yield plaque-forming cells (PFC). With tolerogen (FL-sheep gamma-globulin) present in the agar, the number of FL-specific CFU-B was reduced by 25 to 50%. The ability of the remaining colonies to form PFC upon antigenic stimulation was also reduced. This reduction in CFU-B numbers, however, was observed only when the agar contained both SRBC and LPS as mitogenic potentiators of growth; no effect of tolerogen on CFU-B numbers was seen when cells were grown with either additive alone. Interestingly, the effect of tolerogen on CFU-B numbers was abrogated when peritoneal macrophages, in addition to SRBC plus LPS, were present during cloning. It is postulated that unique subpopulations of B cells form colonies under varied cloning conditions and that those CFU-B grown with SRBC plus LPS display an increased sensitivity to growth inhibition by tolerogen.     

Role of sugars in regulating transfer cell development in cotyledons of developing Vicia faba seeds

Summary. Transfer cell formation in cotyledons of developing faba bean (Vicia faba L.) seeds coincides with an abrupt change in seed apoplasm composition from one dominated by hexoses to one in which sucrose is the principal sugar. On the basis of these observations, we tested the hypothesis that sugars induce and/or sustain transfer cell development. To avoid confounding effects of in planta developmental programs, we exploited the finding that adaxial epidermal cells of cotyledons, which do not become transfer cells in planta, can be induced to form functional transfer cells when cotyledons are cultured on an agar medium. Growth rates of cotyledons cultured on hexose or sucrose media were used to inform choice of sugar concentrations. The same proportion of adaxial epidermal cells of excised cotyledons were induced to form wall ingrowths independent of sugar species and concentration supplied. In all cases, induction of wall ingrowths coincided with a marked increase in the intracellular sucrose-to-hexose ratio. In contrast, further progression of wall ingrowth deposition was correlated positively with intracellular sucrose concentrations that varied depending upon external sugar species and supply. Sucrose symporter induction and subsequent maintenance behaved identically to wall ingrowth formation in response to an external supply of hexoses or sucrose. However, in contrast to wall ingrowth formation, induction of sucrose symporter activity was delayed. We discuss the possibility of intracellular sugars functioning both as signals and substrates that induce and control subsequent development of transfer cells. Correspondence and reprints: School of Environmental and Life Sciences, Biology Building, University of Newcastle, Callaghan, NSW 2308, Australia.     

Gene expression patterns during swarming in Salmonella typhimurium: genes specific to surface growth and putative new motility and pathogenicity genes

Swarming is a specialized form of surface motility displayed by several flagellated bacterial genera, which shares features with other surface phenomenon such as biofilm formation and host invasion. Swarmer cells are generally more flagellated and longer than vegetative cells of the same species propagated in liquid media, and move within an encasement of polysaccharide 'slime'. Signals and signalling pathways controlling swarm cell differentiation are largely unknown. In order to test whether there is a genetic programme specific to swarming, we have determined global gene expression profiles of Salmonella typhimurium over an 8 h time course during swarming, and compared the microarray data with a similar time course of growth in liquid media as well as on harder agar where the bacteria do not swarm. Our data show that bacteria growing on the surface of agar have a markedly different physiology from those in broth, as judged by differential regulation of nearly one-third of the functional genome. The large number of genes showing surface-specific upregulation included those for lipopolysaccharide synthesis, iron metabolism and type III secretion. Although swarming-specific induction of flagellar gene expression was not generally apparent, genes for iron metabolism were strongly induced specifically on swarm agar. Surface-dependent regulation of many virulence genes suggests that growth on an agar surface could serve as a model for gene expression during the initial stages of host infection. Based on cluster analysis of distinctive expression patterns, we report here the identification of putative new genes involved in motility and virulence.     

Factors affecting the colonization of isolated rabbit intestinal epithelial cells by Vibrio cholerae 01 in vitro

The adhesive capability of Vibrio cholerae 01 strains to isolated rabbit intestinal epithelial cells was maximally expressed when the bacteria were grown in synthetic broth and was enhanced by the presence of Ca2+ in the growth media. N-Acetyl-D-glucosamine could inhibit the adhesion of the bacteria to rabbit intestinal epithelial cells as could lipopolysaccharide O-antigen from Vibrio cholerae 01 and lectin from Triticum vulgaris. Since the lipopolysaccharide is known to contain N-acetyl-D-glucosamine and because the lectin from Triticum vulgaris shows specificity for this sugar, it is probable that N-acetyl-D-glucosamine is actively involved in the adhesion of Vibrio cholerae 01 to isolated rabbit intestinal epithelial cells.     

Hapten-specific murine colony-forming B cells. III. Delineation of functional B cell subpopulations grown under different conditions

The influence of anti-immunoglobulin M (IgM) and anti-IgD on the ability of fluorescein (FL)-specific B cells to proliferate in a colony-forming assay, and of their progeny to further differentiate in response to different FL-antigens was studied. Splenic FL-specific B cells were purified on FL-gelatin plates and were then cultured in semisolid agar in the presence or absence of anti-mu, and anti-delta, or both. Experiments were performed under conditions of either sheep red blood cell (SRBC)-potentiated or SRBC + lipopolysaccharide (LPS)-potentiated colony growth. The resulting colonies were then tested in secondary filler cell-dependent microcultures for the ability to be triggered by different classes of FL-antigens to yield plaque-forming cells (PFC). Anti-delta inhibited 47% of colony growth under both agar culture conditions. Anti-mu inhibited 55% of colony growth in SRBC + LPS-potentiated agar cultures, and inhibited 72% if only SRBC was present. If anti-delta and anti-mu were added together, inhibition was nearly additive. When anti-Ig-treated colonies were tested for PFC responses against FL-polymerized flagellin (POL), both normal and anti-delta resistant colonies, grown under both agar culture conditions, responded well. Anti-mu resistant colonies were refractory to FL-POL challenge. Only normal or anti-delta resistant colonies grown in SRBC + LPS agar cultures were able to respond well to FL-Ficoll, whereas even normal SRBC-potentiated colonies responded poorly. All except SRBC-potentiated, anti-mu treated colonies were able to respond to nonspecific signals present in cultures containing FL-KLH and activated T cell help. These data suggest that addition of specific anti-Ig antibodies, and variation of agar culture conditions, can select for B cell subpopulations responsive only to certain types of antigens.     

A determinant of resistance of Neisseria gonorrhoeae to killing by human phagocytes: an outer membrane lipoprotein of about 20 kDa with a high content of glutamic acid

A protein of about 20 kDa was extracted by sodium cholate (1%, w/v) from outer membranes of a strain of Neisseria gonorrhoeae, BS4 (agar), which is resistant to killing by human phagocytes. When the protein was purified by repeated fractionation on Sephadex G75, contamination with other outer-membrane proteins and lipopolysaccharide was negligible. The protein contained a full complement of amino acids, with high levels of glutamic acid. Carbohydrate, detected by the anthrone method and by sugar and hexosamine analysis, was present, but at very low levels. There was a significant content of fatty acids (about 5.7% of the protein), indicating a lipoprotein. The 20 kDa lipoprotein: (1) neutralized the ability of antiserum against whole organisms of BS4 (agar) to reduce the resistance of this strain to phagocyte killing; (2) evoked in mice an antiserum which reduced this resistance and immunoblotted only with 20 kDa lipoprotein in the cholate extract of outer membranes; and (3) promoted resistance to intracellular killing of an otherwise phagocyte susceptible gonococcal strain (BSSH). This is strong evidence that it is a determinant of gonococcal resistance to phagocyte killing.     

Oxidase testing from Kligler's iron agar and triple sugar iron agar slants

Many members of the familyVibrionaceae have been implicated as causative agents of diarrhea. Most of these organisms are non-lactose fermenters, and all are oxidase-positive. If the oxidase test could be reliably performed on growth from the surface of Kligler's iron agar and/or triple sugar iron agar slants, it would aid in the screening of potential stool pathogens. Forty-six isolates from the generaAeromonas, Plesiomonas, andVibrio were inoculated onto Kligler's iron agar and triple sugar iron agar slants, incubated overnight, and tested for oxidase activity. All 46 isolates produced alkaline over acid, with or without gas, Kligler's iron agar slants and were oxidase-positive. On triple sugar iron agar slants, 13 isolates produced these same patterns, and all were oxidase-positive. Acid over acid, with gas, triple sugar iron agar slants were produced by 18 isolates, and all were oxidase-positive. Acid over acid, without gas, triple sugar iron agar slants were produced by 15 isolates, and all were oxidase-negative. These negative oxidase tests were due to low pH. Oxidase tests performed from the surface of Kligler's iron agar and triple sugar iron agar slants used to screen stool isolates were reliable, provided the slants were acid over acid with gas, or alkaline over acid with or without gas. Kligler's iron agar is recommended with this procedure, since most potential stool pathogens of both theEnterobacteriaceae and theVibrionaceae will produce an alkaline over acid, with or without gas, slant, and false negative oxidase tests will be minimized.     

冷鲜牛肉中蜂房哈夫尼亚菌(Hafnia alvei)的分离与鉴定

在出口冷鲜牛肉的沙门氏菌检验过程中,从HE琼脂平板上分离到1株蜂房哈夫尼亚菌,该菌三糖铁斜面产碱,底层产酸,不产生H2S,不产气,革兰阴性。经BBL Crystal微生物半自动检测仪检测,同时结合伯杰细菌鉴定手册的生化试验结果鉴定为蜂房哈夫尼亚菌(Hafnia alvei)。     

COMPARISONS OF DIFFERENT MEDIA FOR COUNTING SUGAR TOLERANT YEASTS IN CONCENTRATED ORANGE JUICE

SUMMARY: To select and count the sugar tolerant yeasts which ferment sixfold concentrated orange juice, a high sugar agar medium was developed which contains 50% of glucose, 1% of citric acid and 1% of Tryptone; it is incubated for 4–5 days at 25°.
The medium has disadvantages: it is troublesome to prepare, and colonies grow slowly and are translucent. These properties result directly' from the high sugar concentration, on which the selective action of the medium depends.
Counts on this medium have been compared with those on potato dextrose or nutrient dextrose agars (with 2% and 1% of glucose respectively), with yeasts isolated from fermenting concentrate, in pure culture, and under various practical conditions. As a rule, the counts were virtually the same on the different media; nutrient dextrose agar occasionally failed to record small numbers of these yeasts. If the two low sugar media were acidified to pH 3·5 the counts were reduced.
Potato dextrose agar recorded, besides the above yeasts, sugar intolerant yeasts entering from dirty machines or through bad canning practice: nutrient dextrose agar recorded bacteria in addition. The difference between parallel plating on these media and on the high sugar medium thus yielded useful information about sources of casual contamination.
It is suggested that the above would also be largely applicable to other sugar-rich concentrates of not less than 50° Brix.     

荷兰菊组织培养快繁技术研究

本文利用组织培养技术研究荷兰菊的快速繁殖,为大量培育荷兰菊种苗提供可靠方法。通过多种培养基的试验筛选,找出较满意的培养方法、三种培养基依次使用,可以达到快速繁殖目的。三种培养基分别是：ＭＳ＋ＫＴＯ·ｌｍｇ／ｌ＋糖２０９／１＋琼脂７ｇ／ｌ、ＭＳ＋６—ＢＡ５ｍｇ／ｌ＋ＮＡＡＯ．０１ｍｇ／ｌ＋糖２０９／ｌ＋琼脂７ｇ／ｌ、１／ＺＭＳ＋ＮＡＡＯ．ｌｍｇ／ｌ＋糖２０ｇ／ｌ＋琼脂７ｇ／ｌ·三种培养基ｐＨ均为６。     

Correlation between bizarre colony morphology and metastatic potential of tumor cells

The morphology of colonies developing from cells of UV-2237 fibrosarcoma clones grown in an unattached state correlated with their experimental metastatic potential in vivo. The frequency of bizarre-shaped (non-symmetrical) colonies was increased in colonies that developed from cells of highly metastatic clones growing in an overlay of media on agar, in 1.3% Methocel, or in 0.3% Noble agar. These bizarre-shaped colonies rarely developed from cells of clones with low metastatic frequency. In addition, when tested for in vivo experimental metastasis, the cells from the bizarre colonies were highly metastatic.     

Two-Dimensionality of Yeast Colony Expansion Accompanied by Pattern Formation

Yeasts can form multicellular patterns as they expand on agar plates, a phenotype that requires a functional copy of the FLO11 gene. Although the biochemical and molecular requirements for such patterns have been examined, the mechanisms underlying their formation are not entirely clear. Here we develop quantitative methods to accurately characterize the size, shape, and surface patterns of yeast colonies for various combinations of agar and sugar concentrations. We combine these measurements with mathematical and physical models and find that FLO11 gene constrains cells to grow near the agar surface, causing the formation of larger and more irregular colonies that undergo hierarchical wrinkling. Head-to-head competition assays on agar plates indicate that two-dimensional constraint on the expansion of FLO11 wild type (FLO11) cells confers a fitness advantage over FLO11 knockout (flo11Δ) cells on the agar surface.     

An improved tetrazolium agar medium for testing sugar fermentation in lactobacilli]

An improved tetrazolium agar medium for testing sugar fermentation in lactobacilli is described. Basal medium 86 was essentially a modified MRS broth with the omission of glucose. The standard formula was 30 micrograms/ml of 2,3,5-triphenyltetrazolium chloride, 2% sugar to be tested, and 2% agar in medium 86. Plates were incubated anaerobically for 2 days at 37C or 5 days at 30C, depending on the strain. With three strains each of group II and III lactobacilli, colorless, fermentation-positive colonies were clearly differentiated from red, fermentation-negative colonies. For three strains of group I lactobacilli, this medium was not satisfactory because they grew poorly on it unless supplemented with a sugar.     

Biomass estimation in solid state fermentation I. Manual biochemical methods

Summary We examined the changes in three biomass constituents (glucosamine, ergosterol, total sugar), sucrose consumption and conidia formation, during cultivation of Beauveria bassiana on agar plates or clay granules. We showed that glucosamine can be considered a good biomass indicator on condition that the media had the same constituents but not necessarily the same C/N ratio. Total sugar was not constant during the different growth phases and cannot accurately represent biomass changes. The ergosterol amounts changed during the different growth phases but, for a fixed process, it can be a good indicator of conidiation. Good correlations were obtained between glucosamine and sucrose consumption allowing biomass yield calculations.Offprint requests to: C. Desgranges     

Presence of 3-O-methyl-l-xylose in the lipopolysaccharide of Pseudomonas maltophilia N.C.T.C. 10257.

3-O-Methyl-L-xylose was isolated from whole cells of Pseudomonas maltophilia N.C.T.C. 10257. The sugar is a component of lipopolysaccharide from which a polysaccharide also containing L-rhamnose and L-xylose was released by mild acid hydrolysis. 3-O-Methyl-L-xylose was absent from five other strains of Ps. maltophilia and one strain of Pseudomonas geniculata.     

Comparative studies of the neutral sugar composition of cell wall polysaccharides between cultured cells and mother plant tissues of soybean and carrot

The cell walls obtained from cultured cells grown under variousconditions and their mother plant tissues or organs were comparedwith regard to the neutral sugar composition of polysaccharides.In the pectic fraction of cell walls from soybean hypocotyl,greater amounts of galactose than other neutral sugars weredetected, and the relative sugar ratio did not change in theelongating and non-elongating portions of the tissue. On theother hand, more arabinose than other sugars was detected inthe pectic fraction of cell walls from cultured cells from soybeanhypocotyl tissue, and no significant change in the sugar ratioof polysaccharides was found in this fraction during the cultureperiod. A higher arabinose content in cell walls from culturedcells from root tissues as compared to the mother tissues ororgans was also detected in carrot. The neutral sugar ratioof cell walls from cultured cells was similar in cells grownon agar or liquid medium, with indoleacetic acid, or with orwithout 2,4-dichlorophenoxyacetic acid (2,4-D). These factssuggest that the high arabinose content of cell walls is notcommon to the cell walls of young, growing, undifferentiatedcells, and that 2,4-D is not required for the maintenance ofthe higher content of arabinose in cell walls of cultured cells. ¹A preliminary report of this work was presented for the annualmeeting of the American Society of Plant Physiologist at OhioState University, August, 1979. [Plant Physiol., 63(5) 118 (1979)]. ⁴Department of Agricultural Chemistry, Washington State University,Pullman, Washington 99164, U.S.A. (Received October 6, 1979; )     

Expression of a new porin 'OmpE' by strains of Salmonella enteritidis

Abstract Strains of Salmonella enteritidis expressed a novel porin with a subunit size of 35.5 kDa as shown by SDS-PAGE. This protein was expressed as a major outer membrane protein (MOMP) when grown on nutrient agar, McConkey agar or blood agar, or in Tris-succinate medium; but was only produced in trace amounts when strains were grown in nutrient broth. This OMP was produced by all strains of S. enteritidis examined, regardless of phage type, and expression was not related to the possession of a 38-MDa mouse-virulence plasmid or the ability of strains to make long-chain lipopolysaccharide. This new porin has been tentatively called OmpE.