Next‐Generation Microbial Workhorses: Comparative Genomic Analysis of Fast‐Growing Vibrio Strains Reveals Their Biotechnological Potential

Vibrio is a recognized fast‐growing bacterial genus, which is considered to be attractive for the development of next‐generation biotechnological workhorses. Here, three Vibrio strains FA1, FA2, and FA3, capable of growing rapidly in cost‐effective media, are isolated and systematically evaluated. Genome sequencing and comparative genomic analyses are performed to reveal the underlying genetic differences between the strains and estimate their biotechnological potential. Studies of their phylogenetic tree, colinear visualization, and orthology uncover some difference in the gene content related to cell growth of the four Vibrio strains FA1, FA2, FA3, and ATCC 14048, which may explain growth superiority of the isolated strains. It is noted that there are more copies of several genes related to the DNA replication in the FA2 genome than in the other compared Vibrio strains. Furthermore, the genes responsible for amino synthesis are found, such as asD, within strains FA1 and FA2. Gene cluster cadABC, which relates to cell adaptation at acidic pH, only exists in strains FA1, FA2, and FA3. Finally, the wide spectra of substrates and genetic operability of these three isolated Vibrio strains are initially verified. This study provides excellent candidates for the development of next‐generation fast‐growing microbial workhorses, which may be very useful in synthetic biology.     

Serological studies with Agrobacterium radiobacter,A. tumefaciens,and Rhizobium strains

Summary Antisera were prepared against cell material from 3 strains of A. radiobacter and 6 of A. tumefaciens. Agar diffusion and immune absorption techniques revealed 3 antigens common to each strain of these organisms. However, 5 different lipopolysaccharide antigens occurred in the 9 test strains. There was no obvious species differences in the distribution of these antigens. Mannose, and possibly glucuronic acid were immunologically active in one of the lipopolysaccharides.The agrobacterial antisera were further crosstested with antigenic material for 34 strains of Rhizobium. Fast-growing rhizobia showed extensive cross-reaction, but only one of 7 R. lupini strains tested reacted with any antiserum.     

Nutritional Study of Three Strains of Naegleria gruberi*

Naegleria gruberi strains cloned from amebas isolated from a Vero cell culture (“TS”), a sewer drainage ditch (“PD”), and an established laboratory line (“S”) were morphologically identical except for differences in size and flagellate transforming ability. Cultivation on a Trypticase-yeast extract-glucose medium (“TYG”) fortified with autoclaved E. coli resulted in increased cell size of 2 strains. Differences also were noted in growth rates and optimal growth temperatures. The autoclaved E. coli in TYG medium was replaceable with serum only for strains TS and PD. A basal salts medium + autoclaved E. coli supported growth of all 3 strains, but the basal salts medium + serum would not support growth of any of the strains.     

Influence of <Emphasis Type="Italic">Daphnia</Emphasis> infochemicals on functional traits of <Emphasis Type="Italic">Microcystis</Emphasis> strains (Cyanobacteria)

We conducted a laboratory experiment to investigate the influence of Daphnia infochemicals on growth rate, microcystin production, colony formation and cell size of eight Microcystis strains isolated from two lakes. The strains were characterized genetically by their 16S-23S rDNA ITS sequence. The experiment was composed of four treatments: (1) a control using filtered WC medium, (2) addition of Scenedesmus obliquus culture medium filtrate, (3) addition of Daphnia magna culture medium filtrate and (4) addition of sodium octyl sulphate, a commercially available Daphnia infochemical. Our results showed that sympatric strains differed strongly for the measured functional traits, while no correlations between traits were found. Between-strain differences in growth rate, microcystin production, colony formation and cell size were generally larger than the differences in phenotypes observed between treatments. Despite this, several strains reacted to the infochemicals by changing functional trait values. Daphnia culture medium filtrate and, to a lesser extent, sodium octyl sulphate had a negative influence on the growth rate of half of the strains and stimulated microcystin production in one strain, but the latter effect was not Daphnia-specific as Scenedesmus culture medium filtrate had the same effect. Daphnia culture medium filtrate also induced colony formation in one strain. Our data suggest that Daphnia infochemicals generally have a weak influence on growth rate, microcystin production and colony formation of Microcystis strains as compared to the inter-strain variability, while existing inducible effects are highly strain-specific.     

An ultrastructural comparison of the cell envelopes of selected strains of Nocardia asteroides and Nocardia brasiliensis

Growth curves were determined for three strains each ofNocardia asteroides andNocardia brasiliensis. Two strains ofN. brasiliensis and one strain ofN. asteroides had longer lag periods of growth than the remaining three strains. All strains had generation times of approximately 5.5 hours.The ultrastructure of the cell envelope of eachNocardia strain in early stationary phase growth was also examined. All the strains had typical trilaminar cell walls and cell membranes. The thickness of the cell wall layers, especially the inner peptidoglycan layer, varied from strain to strain. The inner layer of two strains ofN. brasiliensis and one strain ofN. asteroides was 12 nm or more in thickness, while that of the remaining three strains was 7 nm thick. These observed differences in growth patterns and/or thickness of the cell wall layers could be correlated to the varying degress of virulence as well as the divergent pathologies exhibited by these organisms.     

Acid and heat tolerance of persistent and nonpersistent Listeria monocytogenes food plant strains

Aims: Acid and heat tolerance of 17 persistent and 23 nonpersistent Listeria monocytogenes strains, recovered from three meat‐processing plants, were investigated. Methods and Results: The isolates were genotyped by pulsed‐field gel electrophoresis and categorized into persistent strains according to the frequency of the strain and duration of the contamination. The persistent and nonpersistent strains were challenged to acidic conditions (pH 2·4 for 2 h, 1 mol l^?1 HCl were used to acidify the suspension) and to heat (55°C for 40 min) to receive a reduction in cell count. Listeria monocytogenes strains showed large variation in acid tolerance (over 6 log units) and in heat tolerance (3 log units). The persistent strains showed higher tolerance to acidic conditions than the nonpersistent strains (Student’s t‐test, P = 0·02), but significant differences in heat tolerance between persistent and nonpersistent strains were not observed. Conclusions: The results indicate that acid tolerance may have an effect on the persistence of L. monocytogenes contamination. Significance and Impact of the Study: This study highlights the fact that there are great differences in acid and heat tolerances between L. monocytogenes strains, and the preventive measures should be designed to be effective against the most tolerant strains.     

Gel shift analysis of the <Emphasis Type="Italic">empA</Emphasis> promoter region in <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

Background  

The induction of metalloprotease encoded by empA in Vibrio anguillarum occurs at high cell density in salmon intestinal mucus. Previously we have shown that there are significant differences in empA expression in two strains of V. anguillarum, M93Sm and NB10. It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements.     

Proteomic Adaptation of Australian Epidemic Bordetella pertussis

Bordetella pertussis causes whooping cough. The predominant strains in Australia changed to single nucleotide polymorphism (SNP) cluster I (pertussis toxin promoter allele ptxP3/pertactin gene allele prn2) from cluster II (non‐ptxP3/non‐prn2). Cluster I was mostly responsible for the 2008–2012 Australian epidemic and was found to have higher fitness compared to cluster II using an in vivo mouse competition assay, regardless of host's immunization status. This study aimed to identify proteomic differences that explain higher fitness in cluster I using isobaric tags for relative and absolute quantification (iTRAQ), and high‐resolution multiple reaction monitoring (MRM‐hr). A few key differences in the whole cell and secretome were identified between the cluster I and II strains tested. In the whole cell, nine proteins were upregulated (>1.2 fold change, q < 0.05) and three were downregulated (<0.8 fold change, q < 0.05) in cluster I. One downregulated protein was BP1569, a TLR2 agonist for Th1 immunity. In the secretome, 12 proteins were upregulated and 1 was downregulated which was Bsp22, a type III secretion system (T3SS) protein. Furthermore, there was a trend of downregulation in three T3SS effectors and other virulence factors. Three proteins were upregulated in both whole cell and supernatant: BP0200, molybdate ABC transporter (ModB), and tracheal colonization factor A (TcfA). Important expression differences in lipoprotein, T3SS, and transport proteins between the cluster I and II strains were identified. These differences may affect immune evasion, virulence and metabolism, and play a role in increased fitness of cluster I.     

Cell Surface Properties of Pseudomonas syringae pv. phaseolicola Wild-type and hrp Mutants

The cell surface hydrophobicity and charge as well as surface polysaccharides of eight independent prototrophic hrp::-Tn5 mutants (Lindgren et al., J. Bacteriol. 168 , 512–522, 1986) were compared to the wild-type parent strain NPS3121 of Pseudomonas syringae pv. phaseolicola. No significant differences were found in cell surface charge, but mutant strain NPS4005 exhibited significantly lower cell surface hydrophobicity than the wild-type and the other mutant strains. The mutant strains all retained the ability to produce the exopolysaccharides (EPS) levan, a neutral fructan, and alginate, an acidic polymer. Relative amounts of EPS produced in vitro was dependent on culture conditions. Lipopolysaccharide (LPS) chemotypes were similar for all nine strains. Chemical as well as ¹³C-NMR analyses of the O-antigens from four wild-type strains of P. s. pv. phaseolicola representing two physiological races as well as the O-antigens of two strains of P. s. pv. syringae which belong to the same serogroup as P. s. pv. phaseolicola indicated that all of the O-antigens were very similar if not identical. LPS of three strains of P. s. pv. phaseolicola produced in vitro or in planta were also compared and no significant differences were detected. The altered phenotype of the Tn5 mutants of P. s. pv. phaseolicola does not appear to be due to changes in the ability to produce exopolysaccharides or to an altered composition of cell surface polysaccharides (LPS and EPS). However, a change in an unidentified cell surface component(s) leading to lowered cell surface hydrophobicity of mutant strain NPS4005 may be important.     

Chromosomal changes in eight cell strains of the tasmanian rat-kangaroo,Potorous tridactylis (Marsupialia). Male and female heart fibroblasts cultured in vitro

Eight cell strains, derived from the hearts of a male and a female Motorous, were followed during their in vitro cultivation.All three male cell strains started as normal diploid cell strains. One of them, 2Hpo stayed diploid until passage 59, when the cells were frozen and stored at –96°C. After a period of growth retardation, that lasted two months, 1Hpo showed aneuploidy, the cells having 22–24 chromosomes. The cells with 23 chromosomes formed about 30% of the population. These cells predominantly missed the chromosomes 2, 3 and Y₁, from the tetraploid set. In the other cells no consistent pattern was observed. The cell strain 4Hpo did not show aneuploidy after three months of growth retardation. At the last passage (nr. 24) before death, it showed 25% diploid cells and 40% tetraploid cells.Three female strains were initiated on fibrin clot, two on plasm clot. No differences in growth and chromosomal changes, due to the different embedding media, were observed. All the strains started as diploid (2n=12) cell strains. The chromosomal changes that occurred showed many differences. Three cell strains (5Hf, 7Hp, 52Hf) died without showing any pattern in the aneuploid cells. One cell strain (53Hf) showed an aneuploid cell population with a stemline of 14 chromosomes. The cell strain (8Hp) showed different changes in ploidy. After 50 passages, it changed from diploid to aneuploid (19 chromosomes per cell in the stemline). Twenty passages later diploid cells started to dominate the population again (80% at passage 85). Then a new aneuploid population with a stemline of 18 chromosomes (30% triploid cells) arose, and the strain survived as a permanent line.The work was carried out, in part, under the association between Euratom and the University of Leiden, contract Nr. 052-64-I BIAN, and it also received support form the Foundation for Basic Medical Research (FUNGO).     

Intraspecific variation in the response of the cyanobacterium Nodularia spumigena to moderate UV-B radiation

Floating and nodularin-producing strains of Nodularia spumigena from the Baltic Sea are regarded as belonging to one species. However, intraspecific variation in the response of N. spumigena to environmental factors has been commonly overlooked. As blooms of N. spumigena occur in late summer, a period with strong light and stable water-column stratification, the cells can be expected to also be exposed to ultraviolet-B radiation (UV-B, 280–320 nm). The UV-B tolerance of four different strains of N. spumigena, isolated from the Baltic Sea, was investigated in the laboratory for 8 days, by measuring photosynthesis, growth and pigment composition. Variables included maximum quantum yield of photosynthesis (F_v/F_m, PAM fluorometry), growth rate (cell counts) and photosynthetic pigments, as well as mycosporine-like amino acids (HPLC). Intraspecific differences regardless of treatment were found for cell dimension, growth rate, F_v/F_m and pigment concentrations. UV-B related effects differed between strains. By Day 8 one of the four strains showed a lower F_v/F_m when treated with UV-B; in another strain the growth rate and cell numbers were lower. In three strains, UV-B exposure resulted in higher cell concentrations of carotenoids and chlorophyll a. In all strains, the concentrations of total mycosporine-like amino acids were 60–130% higher in the UV-B treated samples compared with samples shielded from UV-B. Although strain-specific differences in UV-B tolerance were observed, it is concluded that N. spumigena is a species that is not generally negatively affected by moderate levels of UV-B radiation.     

BCG strain S4-Jena: An early BCG strain is capable to reduce the proliferation of bladder cancer cells by induction of apoptosis

Background  

Intravesical immunotherapy with Mycobacterium bovis bacillus Calmette-Guérin has been established as the most effective adjuvant treatment for high risk non-muscle-invasive bladder cancer (NMIBC). We investigated the differences between the S4-Jena BCG strain and commercially available BCG strains. We tested the genotypic varieties between S4-Jena and other BCG strains and analysed the effect of the BCG strains TICE and S4-Jena on two bladder cancer cell lines.     

Patterns of survival and volatile metabolites of selected <Emphasis Type="Italic">Lactobacillus</Emphasis> strains during long-term incubation in milk

The focus of this study was to monitor the survival of populations and the volatile compound profiles of selected Lactobacillus strains during long-term incubation in milk. The enumeration of cells was determined by both the Direct Epifluorescent Filter Technique using carboxyfluorescein diacetate (CFDA) staining and the plate method. Volatile compounds were analysed by the gas-chromatography technique. All strains exhibited good survival in cultured milks, but Lactobacillus crispatus L800 was the only strain with comparable growth and viability in milk, assessed by plate and epifluorescence methods. The significant differences in cell numbers between plate and microscopic counts were obtained for L. acidophilus strains. The investigated strains exhibited different metabolic profiles. Depending on the strain used, 3 to 8 compounds were produced. The strains produced significantly higher concentrations of acetic acid, compared to other volatiles. Lactobacillus strains differed from one another in number and contents of the volatile compounds.     

SEROLOGICAL STUDIES OF MALE AND FEMALE STRAINS OF EUDORINA ELEGANS (CHLOROPHYCEAE)1,2

Male and female strains of Eudorina elegans Ehrenberg were examined by serological methods for flagellar and cellular antigenic differences. No significant differences could be detected between the strains by using agglutination, immobilization and diffusion tests. However, differentiated sperm were neither agglutinated nor immobilized by anti-vegetative cell sera.     

Phylogenetic relationship withinFusarium sambucinum Fuckel sensu lato,determined from ribosomal RNA sequences

Partial ribosomal RNA nucleotide sequences were determined for 11 strains ofFusarium sambucinum Fuckelsensu lato to assess by molecular genetic means, Nirenberg's recent morphotaxonomic interpretation which split the species into three distinct taxa:F. sambucinum sensu stricto, F. torulosum, and one other species, as yet unnamed (Fusarium species nova). Four sequence patterns were identified among the 11 strains. Two sequences that varied at one site were found among strains ofF. sambucinum, strains ofF. torulosum andFusarium sp. nov. showed no intraspecific variation. Interspecific comparisons revealed nucleotide sequence differences of 3–9 substitutions in the ca. 240 nucleotide rRNA segment examined. Although interspecific differences are not large in terms of percent nucleotide substitution, they are much larger than the observed intraspecific variation and support the morphological interpretation distinguishing three taxa. When the data were analysed using parsimony and bootstrapping, the three taxon tree was well supported. The phylogenetic arrangement of these strains is congruent with secondary metabolite profile similarities.     

NITROGEN LIMITATION INCREASES BREVETOXINS IN KARENIA BREVIS (DINOPHYCEAE): IMPLICATIONS FOR BLOOM TOXICITY1

Laboratory and field measurements of the toxin content in Karenia brevis cells vary by >4‐fold. These differences have been largely attributed to genotypic variations in toxin production among strains. We hypothesized that nutrient limitation of growth rate is equally or more important in controlling the toxicity of K. brevis, as has been documented for other toxic algae. To test this hypothesis, we measured cellular growth rate, chlorophyll a, cellular carbon and nitrogen, cell volume, and brevetoxins in four strains of K. brevis grown in nutrient‐replete and nitrogen (N)‐limited semi‐continuous cultures. N‐limitation resulted in reductions of chlorophyll a, growth rate, volume per cell and nirtogen:carbon (N:C) ratios as well as a two‐fold increase (1%–4% to 5%–9%) in the percentage of cellular carbon present as brevetoxins. The increase in cellular brevetoxin concentrations was consistent among genetically distinct strains. Normalizing brevetoxins to cellular volume instead of per cell eliminated much of the commonly reported toxin variability among strains. These results suggest that genetically linked differences in cellular volume may affect the toxin content of K. brevis cells as much or more than innate genotypic differences in cellular toxin content per unit of biomass. Our data suggest at least some of the >4‐fold difference in toxicity per cell reported from field studies can be explained by limitation by nitrogen or other nutrients and by differences in cell size. The observed increase in brevetoxins in nitrogen limited cells is consistent with the carbon:nutrient balance hypothesis for increases in toxins and other plant defenses under nutrient limitation.     

Nutrient conditions determine the localization of Bacillus thuringiensis Vip3Aa protein in the mother cell compartment

Vip3Aa was first identified as a protein secreted during the vegetative growth phase of Bacillus thuringiensis (Bt) bacteria and which shows high insecticidal toxicity against lepidopteran insect pests (Estruch et al., 1996). Bt strains formulated as bio-insecticides only had low amounts of Vip3Aa secreted to the medium. Here, we report that Vip3Aa proteins produced by three different Bt strains, including an industrial strain, were indeed not secreted to the culture solution when grown in sporulation medium, but were retained in the mother cell compartment. In order to further investigate the Vip3Aa secretion and location, we grew the strains in rich medium. We found that in rich medium, a fraction of Vip3Aa was secreted, suggesting that Vip3Aa secretion is nutrient-dependent. Regardless of the growth conditions, we found that Vip3Aa retained in cell pellets exhibited high toxicity against Spodoptera frugiperda larvae. Hence, we speculate that the accumulation of Vip3Aa protein in the mother cell compartment under sporulation conditions could still be used as an efficient strategy for industrial production in commercial Bt strains.     

Microarray genomotyping of key experimental strains of Neisseria gonorrhoeae reveals gene complement diversity and five new neisserial genes associated with Minimal Mobile Elements.

Background  

There are four widely used experimental strains of N. gonorrhoeae, one of which has been sequenced and used as the basis for the construction of a multi-strain, mutli-species pan-neisserial microarray. Although the N. gonorrhoeae population structure is thought to be less diverse than N. meningitidis, there are some recognized gene-complement differences between strains, including the 59 genes of the Gonococcal Genetic Island. In this study we have investigated the three experimental strains that have not been sequenced to determine the extent and nature of their similarities and differences.