Rugosity in Grimontia hollisae

Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30 degrees C form rugose colonies, while the same cells grown at 37 degrees C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30 degrees C produced more biofilm material than rugose colonies grown at 37 degrees C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.     

Salmonella enterica Serovar Typhimurium DT104 Displays a Rugose Phenotype

Rugose phenotypes, such as those observed in Vibrio cholerae, have increased resistance to chlorine, oxidative stress, and complement-mediated killing. In this study we identified and defined a rugose phenotype in Salmonella enterica serovar Typhimurium DT104 and showed induction only on certain media at 25°C after 3 days of incubation. Incubation at 37°C resulted in the appearance of the smooth phenotype. Observation of the ultrastructure of the rugose form and a stable smooth variant (Stv), which was isolated following a series of passages of the rugose cells, revealed extracellular substances only in cells from the rugose colony. Observation of the extracellular substance by scanning electron microscopy (SEM) was correlated with the appearance of corrugation during development of rugose colony morphology over a 4-day incubation period at 25°C. In addition, the cells also formed a pellicle in liquid broth, which was associated with the appearance of interlacing slime and fibrillar structures, as observed by SEM. The pellicle-forming cells were completely surrounded by capsular material, which bound cationic ferritin, thus indicating the presence of an extracellular anionic component. The rugose cells, in contrast to Stv, showed resistance to low pH and hydrogen peroxide and an ability to form biofilms. Based on these results and analogy to the rugose phenotype in V. cholerae, we propose a possible role for the rugose phenotype in the survival of S. enterica serovar Typhimurium DT104.     

Growth Characteristics, Bile Sensitivity, and Freeze Damage in Colonial Variants of Lactobacillus acidophilus

Rough (R) and smooth (S) colonial variants were isolated from a heterogeneous culture of Lactobacillus acidophilus RL8K. R and S types were stable upon repeated transfer on agar, but revertant colonies did appear after broth transfers. When propagated in commercial MRS broth, R and S cultures showed similar growth characteristics, and both cell types were insensitive to freezing and frozen storage at −20°C. Alternatively, during growth in scratch MRS broth, R cultures shifted to a reduced rate of growth during the late logarithmic phase. R cells grown under these conditions were susceptible to death by freezing and injury at −20°C. Microscopically, R cells were observed as long gram-positive rods with small nonstainable blebs protruding from the cell wall. In bile sensitivity studies of R and S cells plated on MRS agar plus oxgall, the S culture was resistant to 1% bile, whereas the R culture was sensitive to 0.6% bile. Differences in the bile resistance and freeze damage of R and S cells suggest that colonial and cellular morphologies are important considerations for the selection of Lactobacillus strains as dietary adjuncts and for the development of growth conditions for preparing frozen concentrated cultures from either cell type.     

Capsular Polysaccharide Surrounds Smooth and Rugose Types of Salmonella enterica serovar Typhimurium DT104

The biofilms and rugose colony morphology of Salmonella enterica serovar Typhimurium strains are usually associated with at least two different exopolymeric substances (EPS), curli and cellulose. In this study, another EPS, a capsular polysaccharide (CP) synthesized constitutively in S. enterica serovar Typhimurium strain DT104 at 25 and 37°C, has been recognized as a biofilm matrix component as well. Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis indicated that the CP is comprised principally of glucose and mannose, with galactose as a minor constituent. The composition differs from that of known colanic acid-containing CP that is isolated from cells of Escherichia coli and other enteric bacteria grown at 37°C. The reactivity of carbohydrate-specific lectins conjugated to fluorescein isothiocyanate or gold particles with cellular carbohydrates demonstrated the cell surface localization of CP. Further, lectin binding also correlated with the FACE analysis of CP. Immunoelectron microscopy, using specific antibodies against CP, confirmed that CP surrounds the cells. Confocal microscopy of antibody-labeled cells showed greater biofilm formation at 25°C than at 37°C. Since the CP was shown to be produced at both 37°C and 25°C, it does not appear to be significantly involved in attachment during the early formation of the biofilm matrix. Although the attachment of S. enterica serovar Typhimurium DT104 does not appear to be mediated by its CP, the capsule does contribute to the biofilm matrix and may have a role in other features of this organism, such as virulence, as has been shown previously for the capsules of other gram-negative and gram-positive bacteria.     

Radiation Resistance and Injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25°C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and −30°C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at −20°C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at −20°C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at −20°C, nor did storage at −20°C alter the cell's resistance to irradiation at 25°C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36°C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36°C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5°C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36°C for 1 day than at 5°C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Photosynthetic and respiratory rates of two psychrophilic diatoms

The photosynthetic rates in two psychrophilic diatoms, Chaetoceros sp. strain K3-10 and Nitzschia sp. K3-3 for cells grown at 0°C were 8 to 10 microliters O₂ evolved per milligram dry weight per hour, and 10-fold higher, about 80 for cells grown at 10°C. The respiration rates followed the same pattern, with a value of around 1 microliter dark uptake per milligram dry weight per hour for both organisms grown at 0°C, and 6 to 10 for cells grown at 10°C. When cells grown at 0°C were immediately shifted to 10°C or cells grown at 10°C were shifted to 0°C, the respiratory rates quickly adapted to values characteristic of cells grown at the shift temperature. On the other hand, the light-saturated rate of O₂ evolution showed much less immediate adaptation, especially on the up shift, 0° to 10°C. The chlorophyll a content of 0°C grown cells was about 0.5% of dry weight, in 10°C grown cells 1.3% (strain K3-10) and 2.2% (strain K3-3). In addition to a diminished chlorophyll a content in 0°C grown cells, there seemed proportionally (by absorbance and calculation) less c to a than in 10°C grown cells. The relative fluorescence excitation spectra of 680-nm emission also showed a lower contribution by both chlorophyll c and fucoxanthin in 0°C grown cells of Chaetoceros sp. strain K3-10 as compared to 10°C grown cells. The data at hand suggest that in psychrophilic diatoms continuously growing at 0°C there may be problems associated with synthesis of an effective accessory pigment system, and as a working hypothesis it is suggested this is related to restriction of synthesis of one or several accessory pigment proteins.     

VpsR, a Member of the Response Regulators of the Two-Component Regulatory Systems, Is Required for Expression of vps Biosynthesis Genes and EPS(ETr)-Associated Phenotypes in Vibrio cholerae O1 El Tor

The rugose colonial variant of Vibrio cholerae O1 El Tor produces an exopolysaccharide (EPS(ETr)) that enables the organism to form a biofilm and to resist oxidative stress and the bactericidal action of chlorine. Transposon mutagenesis of the rugose variant led to the identification of vpsR, which codes for a homologue of the NtrC subclass of response regulators. Targeted disruption of vpsR in the rugose colony genetic background yielded a nonreverting smooth-colony morphotype that produced no detectable EPS(ETr) and did not form an architecturally mature biofilm. Analysis of two genes, vpsA and vpsL, within the vps cluster of EPS(ETr) biosynthesis genes revealed that their expression is induced above basal levels in the rugose variant, compared to the smooth colonial variant, and requires vpsR. These results show that VpsR functions as a positive regulator of vpsA and vpsL and thus acts to positively regulate EPS(ETr) production and biofilm formation.     

Thermal Tolerance of Zymomonas mobilis: Temperature-Induced Changes in Membrane Composition

The membrane composition of Zymomonas mobilis changed dramatically in response to growth temperature. With increasing temperature, the proportion of vaccenic acid declined with an increase in myristic acid, the proportion of phosphatidylcholine and cardiolipin increased with decreases in phosphatidylethanolamine and phosphatidylglycerol, and the phospholipid/protein ratio of the membrane declined. These changes in membrane composition were correlated with changes in thermal tolerance and with changes in membrane fluidity. Cells grown at 20°C were more sensitive to inactivation at 45°C than were cells grown at 30°C, as expected. However, cells grown at 41°C (near the maximal growth temperature for Z. mobilis) were hypersensitive to thermal inactivation, suggesting that cells may be damaged during growth at this temperature. When cells were held at 45°C, soluble proteins from cells grown at 41°C were rapidly lost into the surrounding buffer in contrast to cells grown at lower temperatures. The synthesis of phospholipid-deficient membranes during growth at 41°C was proposed as being responsible for this increased thermal sensitivity.     

Genetic Analysis and Prevalence Studies of the brp Exopolysaccharide Locus of Vibrio vulnificus

Phase variation in the Gram-negative human pathogen Vibrio vulnificus involves three colonial morphotypes- smooth opaque colonies due to production of capsular polysaccharide (CPS), smooth translucent colonies as the result of little or no CPS expression, and rugose colonies due to production of a separate extracellular polysaccharide (EPS), which greatly enhances biofilm formation. Previously, it was shown that the brp locus, which consists of nine genes arranged as an operon, is up-regulated in rugose strains in a c-di-GMP-dependent manner, and that plasmid insertions into the locus resulted in loss of rugosity and efficient biofilm production. Here, we have used non-polar mutagenesis to assess the involvement of individual brp genes in production of EPS and related phenotypes. Inactivation of genes predicted to be involved in various stages of EPS biosynthesis eliminated both the rugose colonial appearance and production of EPS, while knockout of a predicted flippase function involved in EPS transport resulted in a dry, lightly striated phenotype, which was associated with a reduction of brp-encoded EPS on the cell surface. All brp mutants retained the reduced motility characteristic of rugose strains. Lastly, we provide evidence that the brp locus is highly prevalent among strains of V. vulnificus.     

Population Regulation in Magellanic Penguins: What Determines Changes in Colony Size?

Seabirds are often studied at individual colonies, but the confounding effects of emigration and mortality processes in open populations may lead to inappropriate conclusions on the mechanisms underlying population changes. Magellanic penguin (Spheniscus magellanicus) colonies of variable population sizes are distributed along the Argentine coastline. In recent decades, several population and distributional changes have occurred, with some colonies declining and others newly established or increasing. We integrated data of eight colonies scattered along ∼ 600 km in Northern Patagonia (from 41°26´S, 65°01´W to 45°11´S, 66°30´W, Rio Negro and Chubut provinces) and conducted analysis in terms of their growth rates, production of young and of the dependence of those vital rates on colony age, size, and location. We contrasted population trends estimated from abundance data with those derived from population modeling to understand if observed growth rates were attainable under closed population scenarios. Population trends were inversely related to colony size, suggesting a density dependent growth pattern. All colonies located in the north—which were established during the last decades—increased at high rates, with the smallest, recently established colonies growing at the fastest rate. In central-southern Chubut, where colonies are the oldest, the largest breeding aggregations declined, but smaller colonies remained relatively stable. Results provided strong evidence that dispersal played a major role in driving local trends. Breeding success was higher in northern colonies, likely mediated by favorable oceanographic conditions. However, mean foraging distance and body condition of chicks at fledging were influenced by colony size. Recruitment of penguins in the northern area may have been triggered by a combination of density dependence, likely exacerbated by less favorable oceanographic conditions in the southern sector. Our results reaffirm the idea that individual colony trends do not provide confident indicators of population health, highlighting the need to redefine the scale for the study of population changes.     

Asexual reproduction and vegetative growth of Bionectria ochroleuca in response to temperature and photoperiod

Growth and reproduction are two essential life‐history traits for fungi. Understanding life‐history strategies provides insight into the environmental adaption of species. Here, we investigated the colonial morphology, vegetative growth, and asexual reproduction of the ascomycete fungus Bionectria ochroleuca in response to a variety of environmental conditions. We demonstrated that the increased temperature from 15 to 25°C induced mycelial growth and conidiation in B. ochroleuca. We also found that the optimal temperatures for mycelial growth and conidial formation in this fungus species were 25 and 30°C, respectively. However, as the temperature increased from 25 to 30°C, mycelial growth was suppressed, but the total number of conidia was significantly increased. The shift in light–dark cycles dramatically changed the morphological features of the colonies and affected both vegetative growth and asexual reproduction. Under incubation environments of alternating light and dark (16:8 and 8:16 light:dark cycles), conidiophores and conidia in the colonies formed dense‐sparse rings and displayed synchronous wave structures. When the light duration was prolonged in the sequence of 0, 8, 16, and 24 hr per day, mycelial growth was suppressed, but conidiation was promoted. Together, our results indicate that temperature and light period may trigger a trade‐off between vegetative growth and asexual reproduction in B. ochroleuca.     

Factors Influencing the Chlorine Susceptibility of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30°C was increased when cells were exposed to chlorine at 40°C compared to susceptibility after exposure at 30°C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.     

A Flow-Cytometric Gram-Staining Technique for Milk-Associated Bacteria

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50°C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at −18°C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5°C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.     

Analysis of gyrB and toxR Gene Sequences of Vibrio hollisae and Development of gyrB- and toxR-Targeted PCR Methods for Isolation of V. hollisae from the Environment and Its Identification

Isolation of Vibrio hollisae strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identification of V. hollisae. The two genes are presumed to be conserved among the bacterial species (gyrB) or among the species of the genus Vibrio (toxR). A portion of the gyrB sequence of V. hollisae was cloned by PCR using a set of degenerate primers. The sequence showed 80% identity with the corresponding Vibrio parahaemolyticus gyrB sequence. The toxR gene of V. hollisae was cloned utilizing a htpG gene probe derived from the V. parahaemolyticus htpG gene, which is known to be linked to the toxR gene in V. hollisae. The coding sequence of the cloned V. hollisae toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of V. hollisae indicated that these gene sequences could be utilized for differentiation of V. hollisae from other Vibrio species and from microorganisms found in marine fish. PCR methods targeting the two gene sequences were established. Both PCR methods were shown to specifically detect the respective target sequences of V. hollisae but not other organisms. A strain of V. hollisae added at a concentration of 1 to 10² CFU/ml to alkaline peptone water containing a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37°C followed by quick DNA extraction with an extraction kit and 35-cycle PCR specific for the V. hollisae toxR gene. We conclude that screening of seafood samples by this 35-cycle, V. hollisae toxR-specific PCR, followed by isolation on a differential medium and identification by the above htpG- and toxR-targeted PCR methods, can be useful for isolation from the environment and identification of V. hollisae.     

Small Changes in Environmental Parameters Lead to Alterations in Antibiotic Resistance,Cell Morphology and Membrane Fatty Acid Composition in Staphylococcus lugdunensis

Staphylococcus lugdunensis has emerged as a major cause of community-acquired and nosocomial infections. This bacterium can rapidly adapt to changing environmental conditions to survive and capitalize on opportunities to colonize and infect through wound surfaces. It was proposed that S. lugdunensis would have underlying alterations in metabolic homeostasis to provide the necessary levels of adaptive protection. The aims of this project were to examine the impacts of subtle variations in environmental conditions on growth characteristics, cell size and membrane fatty acid composition in S. lugdunensis. Liquid broth cultures of S. lugdunensis were grown under varying combinations of pH (6–8), temperature (35–39°C) and osmotic pressure (0–5% sodium chloride w/w) to reflect potential ranges of conditions encountered during transition from skin surfaces to invasion of wound sites. The cells were harvested at the mid-exponential phase of growth and assessed for antibiotic minimal inhibitory concentration (MIC), generation time, formation of small colony variants, cell size (by scanning electron microscopy) and membrane fatty acid composition. Stress regimes with elevated NaCl concentrations resulted in significantly higher antibiotic resistance (MIC) and three of the combinations with 5% NaCl had increased generation times (P<0.05). It was found that all ten experimental growth regimes, including the control and centroid cultures, yielded significantly different profiles of plasma membrane fatty acid composition (P<0.0001). Alterations in cell size (P<0.01) were also observed under the range of conditions with the most substantial reduction occurring when cells were grown at 39°C, pH 8 (514±52 nm, mean ± Standard Deviation) compared with cells grown under control conditions at 37°C with pH 7 (702±76 nm, P<0.01). It was concluded that S. lugdunensis responded to slight changes in environmental conditions by altering plasma membrane fatty acid composition, growth rates and morphology to achieve optimal adaptations for survival in changing environments.     

Role of Glucose in Enhancing the Temperature-Dependent Growth Inhibition of Escherichia coli O157:H7 ATCC 43895 by a Pseudomonas sp.

Growth of Escherichia coli O157:H7 strain ATCC 43895 was monitored at 5, 10, 15, and 25°C in both pure and mixed (1:1) cultures with a gluconate-producing Pseudomonas sp. found in meat to evaluate the effect of the absence and presence of 1% glucose in broth on temperature-dependent competition. The number of colonies of the Pseudomonas strain exceeded 9 log CFU/ml under all conditions tested. The pathogen grew better as the temperature increased from 10 to 15 and 25°C and grew better in pure culture than in mixed cultures. Pseudomonas sp. inhibited E. coli O157:H7 in cocultures with glucose at 10°C, while at 15°C the pathogen exhibited a biphasic pattern of growth with an intermediate inactivation period. Pathogen inhibition was much weaker in cocultures grown without glucose at 10 to 15°C and, irrespective of glucose, at 25°C. These results indicate that glucose enhances the growth inhibition of E. coli O157:H7 by some Pseudomonas spp., potentially due to its rapid uptake and conversion to gluconate, at low (≤15°C) temperatures.     

Growth Temperature-Induced Alterations in the Thermotropic Properties of Nerium oleander Membrane Lipids

The temperature boundary for phase separation of membrane lipids extracted from Nerium oleander leaves was determined by analysis of spin label motion using electron spin resonance spectroscopy and by analysis of polarization of fluorescence from the probe, trans-parinaric acid. A discontinuity of the temperature coefficient for spin label motion, and for trans-parinaric acid fluorescence was detected at 7°C and −3°C with membrane lipids from plants grown at 45°C/32°C (day/night) and 20°C/15°C, respectively. This change was associated with a sharp increase in the polarization of fluorescence from trans-parinaric acid indicating that significant domains of solid lipid form below 7°C or −3°C in these preparations but not above these temperatures. In addition, spin label motion indicated that the lipids of plants grown at low temperatures are more fluid than those of plants grown at higher temperatures.

A change in the molecular ordering of lipids was also detected by analysis of the separation of the hyperfine extrema of electron spin resonance spectra. This occurred at 2°C and 33°C with lipids from the high and low temperature grown plants, respectively. According to previous interpretation of spin label data the change at 29°C (or 33°C) would have indicated the temperature for the initiation of the phase separation process, and the change at 7°C (or −3°C) its completion. Because of the present results, however, this interpretation needs to be modified.

Differences in the physical properties of membrane lipids of plants grown at the hot or cool temperatures correlate with differences in the physiological characteristics of plants and with changes in the fatty acid composition of the corresponding membrane lipids. Environmentally induced modification of membrane lipids could thus account, in part, for the apparently beneficial adjustments of physiological properties of this plant when grown in these regimes.