Changes in membrane functions during short-term starvation of Vibrio fluvialis strain NCTC 11328

The marine bacterium Vibrio fluvialis strain NCTC 11328 responded to starvation conditions by forming ultramicrocells of dwarf bacteria. The viability of starved cells began to decrease after 2–3 days. During this time the respiratory potential of the bacteria decreased by four- or fivefold, most probably as a result of a decrease in the specific activity of NADH and succinate dehydrogenases. Although respiratory potential in starving cells was lower than in growing cells, bacteria starved for 1 or 2 days maintained a proton motive force that was slightly larger than that of growing bacteria. Starved bacteria contained substantial concentrations of ATP although the UTP and GTP concentrations were much lower in starved than in growing cells. Two or three proteins that were not present in membranes of growing cells, were evident in the membranes of starved bacteria.Abbreviations MMS modified Morita's salts - MMSGC modified Morita's salts plus 20 mM glucose and 0.1% (w/v) casamino acids - MMST modified Morita's salts buffered with 50 mM tricine, (pH 8.5) - NM broth nutrient modified Morita's salts - CFU colony-forming unit - TPP tetraphenylphosphonium - STM 0.1 M tricine, (pH 8.0) plus 0.25 M sucrose and 0.02 M magnesium acetate - DCPIP dichlorophenolindophenol - CCCP carbonyl cyanidem-chlorophenylhydrazone - PMF proton motive force     

Effects of respiratory activity on starvation survival of marine vibrios

The marine bacterium Vibrio fluvialis NCTC11328 responded to nutrient depletion by a reduction in cell volume, and this was prevented by conditions that eliminated respiration as a source of energy. Addition of the protonophore, CCCP, removal of oxygen and introduction of mutations leading to defects of the respiratory chain prevented size reduction during periods of nutrient limitation. Further, survival of the wild-type strain during starvation was reduced under anaerobic conditions and survival of respiratory mutants under aerobic conditions was reduced compared with that of the parent strain. Removal by mutation of the respiratory Na⁺ pump from Vibrio alginolyticus did not inhibit size reduction or lead to reduced viability in starved cultures.Abbreviations ANa medium A containing 0.4 M sodium chloride - ANaS ANa containing 50 mM sodium succinate - CCCP carbonyl cyanide-m-chlorophenylhydrazone - CFU colony forming units - FMNH flavine mononucleotide - MMS modified Morita's salts solution - MMSGC MMS containing 20 mM glucose and 0.1% (w/v) casamino acids - MMST MMS buffered to pH 8.5 with 50 mM tricine - NM nutrient Morita's broth - Ox oxidase - DH dehydrogenase - NTG N-methyl-N'-nitro-N-nitrosoguanidine     

Effect of Interfaces on Small, Starved Marine Bacteria

The copiotrophic marine Vibrio sp. strain DW1, shown previously in batch culture to increase in numbers at the onset of starvation and then to form viable small cells with low endogenous respiration, appears to have a survival advantage at interfaces. Vibrio sp. strain DW1 behaved differently at interfaces compared with the aqueous phase under starvation conditions: (i) small cells were observed at an air-water interface without nutrients, (ii) nutrients added to the air-water interface quickly produced larger cells at the surface, (iii) motility persisted many hours longer at the solid-water interface of a dialysis membrane in a microchamber at the onset of starvation, and (iv) regrowth and division at the solid-liquid interface occurred quickly and at nutrient concentrations too low to permit growth in the aqueous phase. It was concluded that, if small starved cells from copiotrophic bacteria can reach an interface, additional survival mechanisms become available to them: (i) interfaces constitute areas of favorable nutrient conditions, and (ii) interfaces lacking a sufficient amount of nutrient, nevertheless, trigger cells to become smaller, thus increasing their surface/volume ratio and the packing density.     

Ammonium assimilation in Proteus vulgaris,Bacillus pasteurii,and Sporosarcina ureae

No active uptake of ammonium was detected in Proteus vulgaris, Bacillus pasteurii, and Sporosarcina ureae, which indicates that these bacteria depend on the passive diffusion of ammonia across the cell membrane. In P. vulgaris the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway and glutamate dehydrogenase (GDH) were present, and these enzymes exhibited high affinities for ammonium. In B. pasteurii and S. ureae, however, no GS activity was detected, and GOGAT activity was only present in S. ureae. GDH enzymes were present in these two organisms, but showed only low affinity for ammonium, with apparent K _m-values of 55.2 mM in B. pasteurii and 36.7 mM in S. ureae, repectively. These observations explain why P. vulgaris is able to grow at neutral pH and low ammonium concentration (2 mM), while B. pasteurii and S. ureae require high ammonium concentration (40 mM) and alkaline pH for growth.Non-standard abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - GT glutamyl transferase - MA methylammonium - NB nutrient broth - YE yeast extract - NA nocotinic acid     

Utilization of surface localized substrate by non-adhesive marine bacteria

Thirty-four marine bacteria were isolated from the eluate of seawater passed through a column of glass beads coated with stearic acid. Irreversible attachment of these isolates to stearic acid-coated glass surfaces ranged from 7.6–100% of the total attached population, with 7 isolates exhibiting less than 10% irreversible adhesion. All 14 isolates tested were able to utilize surface bound¹⁴C-stearic acid, even though some showed mostly reversible adhesion to the surface. More detailed studies were made comparing the reversibly adheringVibrio MH3 with the irreversibly adheringPseudomonas NCMB2021. MH3 cells were readily removed from the surface by a gentle shear force, and a significant degree of¹⁴C-labeling of MH3 cells, but not of NCMB2021 cells, in the bulk phase was observed. The ecological significance of nutrient scavenging at solid surfaces by reversibly attached bacteria is considered.     

Growth characteristics of low-nutrient bacteria from the north-east and central Pacific Ocean

Abstract Low-nutrient, or oligotrophic, marine bacteria, defined operationally as those which grow well in the dilute nutrient concentrations typical of natural seawaters, were found in waters of the eastern and central Pacific Ocean. All isolates tested grew in sterile unsuplemented seawater, ultraviolet-irradiated seawater, artificial seawater and charcoal-treated artificial seawater. Under optimal conditions, doubling times for low-nutrient bacteria were generally 2–5 h. Low-nutrient bacteria are heterotrophs responding to added organic substrates including glucose, proline, acetate, peptone, algal extract and yeast extract. Organic substrate concentrations ( K _s values) necessary to yield half the maximum growth rates of low-nutrient bacteria ranged from 0.8–12 μg · 1⁻¹ (0.2–4.9 μg C · 1⁻¹). Bacterial inoculum size had little effect on doubling times, whereas low oxygen tensions and low temperatures reduced growth rates. Surfaces (glass beads) increased growth rates and maximum cell densities. The results of these laboratory studies indicate that low-nutrient bacteria play a major role in marine heterotrophic transformations of dissolved organic matter.     

Statistic evaluation of the influence of species variation,culture conditions,surface wettability and fluid shear on attachment and biofilm development of marine bacteria

A budding coccoid bacterium, (CH1), a Vibrio sp. and a Pseudomonas sp. were investigated for factors governing their attachment to glass surfaces in static batch culture and laminar flow continuous culture systems. An analysis of variance showed that the three species exhibited very different responses. For CH1 attachment was dependent on cell density, incubation time and nutrient concentration. The Vibrio sp. was affected by nutrient concentration while the attachment of the Pseudomonas sp. was independent of cell density, incubation time and nutrient concentration. A comparison of attachment to hydrophilic and hydrophobic surfaces showed that attachment of the Vibrio sp. and CH1 to hydrophilic surfaces was 3 and 10 times greater respectively than to hydrophobic surfaces while Pseudomonas attached in equal numbers to both surfaces. The continuous culture system with defined flow hydrodynamics and growth conditions at steady state revealed a random sampling effect 3 times smaller than the batch culture system did. When the biofilm development of Pseudomonas sp. was followed during 46 h at different fluid shear under laminar and turbulent flow conditions, the former biofilm reached 3.3·10⁸ cells·cm^-2 and the latter 8.2·10⁷ cells·cm^-2.Non-common abbreviation NSS Nine salt solution     

Chloride dependence of endospore germination in Halobacillus halophilus

The ion requirement for germination and outgrowth of endospores from the moderately halophilic salt marsh bacterium Halobacillus halophilus was studied. Germination and outgrowth of endospores plated onto nutrient broth was dependent on the salt concentration in the artificial seawater used as the source of ions. Maximal germination and outgrowth were observed when double-concentrated artificial seawater was used. Replacement of chloride salts in the artificial seawater by other salts resulted in a complete loss of germination and outgrowth that was restored upon addition of chloride. To analyze the role of chloride more directly and quantitatively, a defined growth medium was used in which the artificial seawater was substituted by a solution of magnesium sulfate and sodium chloride. Spore germination and outgrowth were strictly dependent on the chloride concentration; maximal germination and outgrowth were observed at ≈ 1.3 M Cl^–. Chloride could be substituted by bromide, but not by sulfate or nitrate. Microscopic examinations of single spores clearly showed that germination is the chloride-dependent step. This first report on chloride dependence of spore germination in any endospore-forming bacterium adds another function to chloride in H. halophilus apart from its being essential for the physiology of the vegetative cell. Received: 21 May 1999 / Accepted: 26 July 1999     

Optimization of medium for the production of a novel aquaculture probiotic,<Emphasis Type="Italic">Micrococcus</Emphasis> MCCB 104 using central composite design

A marine isolate ofMicrococcus MCCB 104 has been identified as an aquaculture probiotic antagonistic toVibrio. In the present study different carbon and nitrogen sources and growth factors in a mineral base medium were optimized for enhanced biomass production and antagonistic activity against the target pathogen,Vibrio harveyi, following response surface methodology (RSM). Accordingly the minimum and maximum limits of the selected variables were determined and a set of fifty experiments programmed employing central composite design (CCD) of RSM for the final optimization. The response surface plots of biomass showed similar pattern with that of antagonistic activity, which indicated a strong correlation between the biomass and antagonism. The optimum concentration of the carbon sources, nitrogen sources, and growth factors for both biomass and antagonistic activity were glucose (17.4 g/L), lactose (17 g/L), sodium chloride (16.9 g/L). ammonium chloride (3.3 g/L), and mineral salts solution (18.3 mL/L).     

Microbiological studies of Tokyo Bay

The generic composition of the heterotrophic bacterial population of Tokyo Bay, which is now highly polluted and eutrophic, was compared with that of the adjacent, less polluted regions of Sagami Bay and Suruga Bay. Members of Vibrionaceae predominated in the bacterial flora of seawater and zooplankton samples from Sagami Bay, Suruga Bay, and the mouth of Tokyo Bay. However,Vibrio spp. formed only a small proportion of the bacterial population of the water and sediment samples from the inner Tokyo Bay; there the Gram-negative, nonmotile, nonpigmented bacteria, which were tentatively identified asAcinetobacter, were predominant. The result of experiments, in which seawater samples from Tokyo Bay were incubated under various experimental conditions, indicated that two significant factors apparently control the growth ofVibrio spp. in seawater; (1) a direct antagonism betweenVibrios and phytoplankton undergoing rapid growth, and (2) a limiting organic nutrient forvibrios.     

Bacterial corrosion of iron in seawater in situ, and in aerobic and anaerobic model systems

Abstract A screening of twenty-two marine isolates was made to examine their effects on corrosion of carbon steel ASTM A619. In batch cultures, sixteen of the isolates gave a lower corrosion than the control. Aerobic and anaerobic biofilm populations were formed by immersing iron coupons in natural seawater under aerobic and anaerobic conditions. The effects of the biofilms depended on a balance between the presence of oxygen and the type of population. An anaerobic population attached to the surface increased the corrosion rate if immersed in a suspension of Vibrio sp. DW1. The vibrio population probably 'protected' the anaerobic population from oxygen and may have provided nutrients, thereby creating conditions that allowed production of corrosive metabolites close to the metal. In contrast, coupons without a biofilm showed a decrease in the corrosion when immersed in the same vibrio suspension. The protective effect of a dense suspension of bacteria found earlier [5,6] was tested in situ in seawater. Iron coupons were immersed in dialysis bags with a suspension of Vibrio sp. DW1. Coupons immersed in dialysis bags with DW1 showed a lower degree of corrosion than coupons immersed in bags with seawater.     

Maintaining the ionic permeability of a cellulose ester membrane

This paper reports the results of a series of experiments designed to test conditions that would permit NaCl to diffuse through 100 Da molecular weight cut-off (MWCO) and 1,000 Da MWCO membranes. For the 100 Da MWCO membrane, the membrane becomes completely impermeable to NaCl when dialyzed against distilled water (DW), but inclusion of one of a variety of different salts in the dialyzing solution maintains membrane permeability to NaCl. A titration experiment revealed that a minimum concentration of 0.1 mM of a salt such as KH2PO4 is required to sustain membrane permeability. In contrast, diffusion through the 1,000 Da MWCO membrane was slightly higher when DW was used as the dialysate. We conclude that the 100 Da MWCO membrane works well for a variety of dialysis applications provided that a maintenance salt is included in all dialyzing solutions.     

Influence of Sulfate-Reducing Bacteria on Outdoor Hydrogen Production by Photosynthetic Bacterium with Seawater

The application of seawater for bacterial fermentative production is a cost-effective technology. Hydrogen production by marine photosynthetic bacterium with seawater failed to continue after more than 10 days, and was accompanied by the formation of hydrogen sulfide and a change in culture color from red to black. However, substrate consumption in the blackish culture was comparable to that in a hydrogen-producing culture. A decrease in hydrogen production occurred upon the addition of sodium sulfide at concentrations of 1.5 mM or higher. PCR analysis targeted at the 16S rDNA sequence selective for sulfate-reducing bacteria revealed the existence of sulfate-reducing bacteria in inoculation cultures of the phototrophic bacterium and medium for hydrogen production. Hence, the high sulfate concentration of seawater, the low oxidation-reduction potential under hydrogen-producing conditions, and the presence of electron donors such as acetate might promote the metabolic activities of sulfate-reducing bacteria, resulting in the deterioration of hydrogen production with seawater. Received: 15 September 1999 / Accepted: 14 October 1999     

Continuous observations of bacterial gliding motility in a dialysis microchamber: The effects of inhibitors

Use of a dialysis microchamber has allowed continuous observations on the same set of gliding bacteria during changes in the composition of the perfused medium. This procedure has revealed the presence of an adaptive, cyanide-insensitive metabolic pathway, which allows cyanide-treated Flexibacter BH3 to begin gliding again at a reduced rate when glucose is the substrate. In addition, it has revealed that individual flexibacter cells can maintain their gliding motility for up to 20 h in the absence of exogenous substrate.Gliding in Flexibacter BH3 was prevented by those inhibitors blocking the electron transport process. Inhibitors of glucose metabolism did not prevent motility, since the flexibacters obviously metabolize endogenous substrate under such circumstances. Proton ionophores, which induce membrane depolarization, rapidly inhibited gliding in Flexibacter BH3. This inhibition was irreversible in the case of gramicidin S. Gliding was not inhibited by cytochalasin B or antiactin antibody. High concentrations of Ca²⁺ were particularly inhibitory to the gliding process. The significance of these results is discussed in relation to a possible mechanism of gliding involving the generation of rhythmical contractions in the outer cell membrane of Flexibacter BH3.Abbreviations used CCCP carbonyl cyanide m-chlorophenyl hydrazone - DNP p-dinitrophenol - GMCS gramicidin S - HQNO 2-heptyl-4-hydroxyquinoline N-oxide - PCMB p-chloromercuribenzoate - CM complete Lewin's medium - BS Lewin's basal salts     

Adhesion: A tactic in the survival strategy of a marine vibrio during starvation

The mesophilic marineVibrio DW1 increases in number and decreases rapidly in size within 5 h of exposure to starvation conditions. The decrease in cell size continues with further starvation, but is accompanied by a rapid decline in viability. Starvation-induced dwarfs show an enhanced rate of adhesion to siliconized glass surfaces, an effect that is related to the appearance of bridging polymer at the cell surface. It is suggested that the adhesive properties of dwarf forms may enhance their chance of survival in oligotrophic marine conditions.     

Variation of environmental features and microbial populations with salt concentrations in a multi-pond saltern

A multi-pond saltern that creates a gradient of salt concentrations has been studied with respect to some characteristics of the resulting environments and their microbial populations. The increase in salt concentration was correlated with increase in diurnal temperature and biomass present and with decrease in oxygen concentrations. Many types of organisms below 15% (w/v) total salts, were found, many of them normal inhabitants of seawater and even freshwater. Most organisms over 15% salts were halophilic. The salt concentrations comprised two ranges, each characterized by different microbial populations. First, between 15 and 30% salts, the populations ofDunaliella increased, reaching large numbers; moderately halophilic eubacteria and some fast-growing halobacteria predominated as heterotrophic microorganisms and, among the first, thePseudomonas-Alteromonas-Alcaligenes group andVibrio were the more abundant taxonomic groups; and gram-positive cocci appeared mainly over 25% salts. Phototrophic bacteria, both oxygenic and anoxygenic, were also found in this range, and among the anoxygenic type,Chromatium species andRodospirillum salexigens were probably predominant. Second, over 30% salts the diversity decreased greatly, all organisms found at the lower salt concentrations disappeared, and instead large populations of halobacteria developed. Over 50% salts, only three species of halobacteria were found.     

Iron Acquisition and Transport in Staphylococcus aureus

Pathogenic Gram-positive bacteria encounter many obstacles in route to successful invasion and subversion of a mammalian host. As such, bacterial species have evolved clever ways to prevent the host from clearing an infection, including the production of specialized virulence systems aimed at counteracting host defenses or providing protection from host immune mechanisms. Positioned at the interface of bacteria/host interactions is the bacterial cell wall, a dynamic surface organelle that serves a multitude of functions, ranging from physiologic processes such as structural scaffold and barrier to osmotic lysis to pathogenic properties, for example the deposition of surface molecules and the secretion of cytotoxins. In order to succeed in a battle with host defenses, invading bacteria need to acquire the nutrient iron, which is sequestered within host tissues. A cell-wall based iron acquisition and import pathway was uncovered in Staphylococcus aureus. This pathway, termed the isd or iron-responsive surface determinant locus, consists of a membrane transporter, cell wall anchored heme-binding proteins, heme/haptoglobin receptors, two heme oxygenases, and sortase B, a transpeptidase that anchors substrate proteins to the cell wall. Identification of the isd pathway provides an additional function to the already bountiful roles the cell wall plays in bacterial pathogenesis and provides new avenues for therapeutics to combat the rise of antimicrobial resistance in S. aureus. This review focuses on the molecular attributes of this locus, with emphasis placed on the mechanism of iron transport and the role of such a system during infection.     

Vibrio cyclitrophicus population-specific biofilm formation and epibiotic growth on marine copepods

Vibrio spp. form a part of the microbiome of copepods—an abundant component of marine mesozooplankton. The biological mechanisms of the Vibrio-copepod association are largely unknown. In this study we compared biofilm formation of V. cyclitrophicus isolated from copepods (L-strains related to other particle-associated strains) and closely related strains originating from seawater (S-strains), and visualized and quantified their attachment and growth on copepods. The S- and L-strains formed similar biofilms in the presence of complete sea salts, suggesting previously unknown biofilm mechanisms in the S-strains. No biofilms formed if sodium chloride was present as the only salt but added calcium significantly enhanced biofilms in the L-strains. GFP-L-strain cells attached to live copepods at higher numbers than the S-strains, suggesting distinct mechanisms, potentially including calcium, support their colonization of copepods. The cells grew on live copepods after attachment, demonstrating that copepods sustain epibiotic V. cyclitrophicus growth in situ. The results demonstrate that in spite of their 99.1% average nucleotide identity, these V. cyclitrophicus strains have a differential capacity to colonize marine copepods. The introduced V. cyclitrophicus—A. tonsa model could be informative in future studies on Vibrio-copepod association.     

The relative abundance and seawater requirements of gram-positive bacteria in near-shore tropical marine samples

The relative abundance of gram-positive bacteria in a variety of near-shore marine samples was determined using the KOH method. Gram-positive bacteria accounted for 14%, 25%, 31 %, and 12%, respectively, of the colony-forming bacteria obtained from seawater, sediments, and the surfaces of algae and invertebrates. A total of 481 gram-positive strains were isolated representing a wide range of morphological groups including regular and irregular rods, cocci, and actinomycetes. Seventy-seven percent of the strains characterized did not form spores and were aerobic, catalase-positive rods with regular to irregular cell morphologies. Eighty-two percent of the strains tested showed an obligate requirement of seawater for growth. None of the cocci tested required seawater or sodium for growth. This is the first report documenting that gram-positive bacteria can compose a large percentage of the culturable, heterotrophic bacteria associated with the surfaces of tropical marine algae. Correspondence to: P.R. Jensen     

Kinetic mechanism for the ability of sulfate reducers to out-compete methanogens for acetate

Methanosarcina barkeri and Desulfobacter postgatei are ubiquitous anaerobic bacteria which grow on acetate or acetate plus sulfate, respectively, as sole energy sources. Their apparent K _s values for acetate were determined and found to be approximately 0.2 mM for the sulfate-reducing bacterium and 3 mM for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V _max) the rate of acetate consumption by D. postgatei approached 15-fold the rate of M. barkeri at low acetate concentrations. The apparent inhibition of methanogenesis was of the same order as expected from the different K _s value for acetate. Difference in substrate affinities can thus account for the inhibition of methanogenesis from acetate in sulfate-rich environments, where the acetate concentration is well below 1 mM.