Testing the biological activity of Vibrio cholerae on cell subcultures

Testing the supernatants of ctx(+) strains of V. cholerae eltor and V. cholerae O139 on cell subcultures confirmed the possibility of the synthesis of hemolysin by V. cholerae under the condition of growing them in tripton medium lacking FeCl3. At the same time ctx(+) strains of V. cholerae of both serogroups retained, simultaneously with hemolysin production, their capacity for the synthesis of cholera toxin.     

Synthesis of protective antigens during submerged cultivation of Vibrio cholerae

The effectiveness of dot immunoanalysis for evaluating the dynamics of the synthesis of O-antigen, cholera toxin, neuraminidase, adhesin CFA1 in the process of the reactor cultivation of V. cholerae used for the production of oral chemical cholera vaccine is shown. The established regularities of the synthesis of the protective antigens of V. cholerae in the process of scaled-up cultivation are discussed.     

The cultivation of Vibrio cholerae with green algae in an experiment

Relationships between Vibrio cholerae of different origin and some serogroups with green algae Scenedesmus quadricauda in mineral medium at two temperatures have been experimentally studied. Differences in the relationships of various strains with green algae under the above-mentioned experimental conditions have been established. The study has shown that a decrease in the concentration and the death of vct+ and vct- vibrios of all strains under study occur in the linear phase of the development of algae. 3 V. cholerae strains, serogroups O139 (vct+) and O50 (vct-), have been shown to be capable of survival under the conditions mix cultivation with algae for 50-100 days. The perish of green algae is supposed to increase the survival time and multiplication of V. cholerae under experimental conditions.     

不同温度条件下霍乱弧菌生存能力的实验研究

目的通过在不同温度条件下对霍乱弧菌生存能力的观察,探索大连市水域中霍乱弧菌在流行＂间歇期＂如何存活和越冬。方法在自然海水和湖水中分别接种本市分离出的埃尔托型霍乱弧菌流行株小川1b型（大E940014）和埃尔托型霍乱弧菌非流行株小川32l（大E940011）,在1～2℃、5～6℃及10～12℃3个温度条件下检测霍乱弧菌生存能力。结果流行株（小川1b型）在1～2℃海水存活26 d,5～6℃存活28 d,10～12℃存活46 d。可见,10～12℃生存时间明显高于1～2℃或5～6℃（P〈0.05）。结论两类菌株的生存对环境温度均很敏感,温度越低死亡越快。在自然水体中,非流行株生存时间略长于流行株;而在除菌水体中两类菌存活能力基本一致。     

Experimental non-culturable Vibrio cholerae eltor and its biological properties

In experiments with the cultivation of V. cholerae eltor under the conditions of high salt concentration, as well as low temperature and deficiency in nutrient substances, uncultivable forms (UF) of toxigenic and nontoxigenic vibrios were obtained. The absence of growth of seeded vibrios after the filtration of samples (with a filter of 0.22 micron), the preservation of specific antigenic determinants and the initial set of genes, changes in the morphology of cells (small size, coccoid form with the flagella retained) confirm the transition of V. cholerae eltor under study into the uncultivable state which, under unfavorable conditions, more rapidly develops in toxigenic vibrios than in nontoxigenic ones. The analysis of the INT-reductase activity of UF disintegrates revealed that they had endogenic respiration whose activity increased (4.5- to 6.5-fold) in the presence of the exogenic intermediates of the Krebs cycle. The uncultivable forms of the vibrios retain genes responsible for pathogenicity, as well as their antigenic determinants.     

Dependence of magnetic suspectibility of various biological objects on their physiological conditions and viability

The magnetic susceptibility of seeds of cereals and worker honey bees was studied. A correlation between the magnetic susceptibility of bioobjects and their physiological state was established. The theory of magnetic orientation of migrating animals is discussed.     

Replication of Vibrio cholerae Chromosome I in Escherichia coli: Dependence on Dam Methylation

We successfully substituted Escherichia coli''s origin of replication oriC with the origin region of Vibrio cholerae chromosome I (oriCI_Vc). Replication from oriCI_Vc initiated at a similar or slightly reduced cell mass compared to that of normal E. coli oriC. With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation from oriC and oriCI_Vc were similar. Since Hda is involved in the conversion of DnaA^ATP (DnaA bound to ATP) to DnaA^ADP (DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting for V. cholerae chromosome I replication, which similar to what is observed for E. coli. No hda homologue has been identified in V. cholerae yet. In V. cholerae, dam is essential for viability, whereas in E. coli, dam mutants are viable. Replacement of E. coli oriC with oriCI_Vc allowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation from oriCI_Vc. We show that when E. coli''s origin of replication is substituted by oriCI_Vc, dam, but not seqA, becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly at oriCI_Vc in E. coli. In this model, methylation at oriCI_Vc would ease DNA melting. This is supported by the fact that the requirement for dam can be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.The genomes of Vibrio cholerae and several related Vibrio spp. are distributed between two circular chromosomes. Characterization of the origins of replication of V. cholerae chromosomes I and II (oriCI_Vc and oriCII_Vc, respectively) has shown that oriCI_Vc is similar to the origin of replication of the Escherichia coli chromosome, oriC, whereas oriCII_Vc is completely different (20). Like oriC, oriCI_Vc has five R-type DnaA boxes (53) as well as boxes conforming to the I and τ types (52, 61), and the DnaA protein is the rate-limiting factor in the initiation of replication in both cases (18). In E. coli, DnaA associates with both ATP and ADP, and the ATP-bound form is absolutely required for initiation to take place (reviewed in reference 60). When reaching a critical level, DnaA^ATP (DnaA bound to ATP) protein is proposed to form a helical filament, anchored at one or more R-boxes (54, 69), in which origin DNA wraps around the outside of the DnaA core (21) or where the DnaA wraps around oriC (61). In both cases, the topology of the DnaA-oriC nucleoprotein complex leads to formation of compensatory negative supercoiling that facilitates unwinding of the adjacent AT-rich region resulting in initiation. In both models, DnaA^ATP is absolutely required for initiation, and in agreement with this, DnaA^ATP was found to be the rate-limiting factor for initiation in vivo (69).The V. cholerae oriCI_Vc also resembles oriC in having many potential sites for methylation by DNA adenine methyltransferase (Dam), although the number and position of the GATC sites differ slightly (see Fig. ​Fig.1).1). The role of Dam in initiation of chromosome replication has been studied mainly in E. coli. After initiation of DNA replication has occurred on a fully methylated oriC, the newly replicated hemimethylated origins are sequestered from the Dam methyltransferase and from reinitiation for approximately one-third of a doubling time. During this time interval, the activity and amount of DnaA available for initiation are reduced to prevent immediate reinitiation (reviewed in references 57 and 83). The sequestration is carried out by the SeqA protein that binds hemimethylated oriC GATC sequences with high affinity (48). In the absence of Dam methylation or SeqA, the same origin can be reinitiated in the same cell cycle, and initiations become asynchronous (9, 48).Open in a separate windowFIG. 1.Alignment of the E. coli minimal oriC with the corresponding region from V. cholerae chromosome I. The AT-rich sequence and the three 13-mer repeats L, M, and R found in E. coli (5) are indicated above the alignment. The 6-mer (A/T)GATCT boxes (80) are underlined. Other DnaA binding sites, i.e., R-boxes (53), I-boxes (52), and τ-boxes (61), are shown as boxed regions. Dam methylation sites (GATC) are shaded gray. The experimentally defined binding sites for integration host factor (IHF) (22) and factor for inversion stimulation (FIS) (65) in E. coli are indicated, and bases that match the consensus sequence are in boldface type. The single base difference between oriCI_Vc and oriCI_Vc* (see Materials and Methods) in the minimal origin region is shown below the two sequences. A gap introduced to maximize alignment of the two sequences is indicated by a dash in the sequence. Nucleotides that are identical in the two sequences are indicated by an asterisk below the two sequences.Genes encoding a Dam homologue and a SeqA homologue are present on Vibrio genomes, but there appear to be some differences between the functions of the proteins in E. coli and V. cholerae. dam has been found to be an essential gene in V. cholerae (33, 15), which is not the case in E. coli (48, 51). Conflicting data exist concerning the essentiality of seqA in V. cholerae (15, 72). The roles of Dam and SeqA in oriCI_Vc replication have been studied using minichromosomes, i.e., plasmids replicating exclusively from a cloned copy of oriCI_Vc (20). oriCI_Vc-based minichromosomes can replicate in wild-type E. coli cells but were unable to replicate in dam, seqA, and seqA dam mutants (20). The extrachromosomal existence of minichromosomes is dependent on their ability to initiate replication in synchrony with the chromosomal origin (46, 75). In E. coli cells mutated in dam or seqA, incompatibility exists between the oriC carried on minichromosomes and that of the chromosome due to origin competition (13), and when minichromosomes are maintained under selective pressure, they integrate into the origin region of the host chromosome (46, 75). Minichromosomes based on oriCI_Vc may also compete with the E. coli oriC for initiations in dam or seqA mutant cells. However, due to limited sequence identity, they may not be able to integrate into the E. coli chromosome. This could provide an explanation for the failure to introduce oriCI_Vc minichromosomes into dam and seqA mutant cells (20). Both dam and seqA genes could therefore be required for viability of V. cholerae for reasons not related to chromosome replication. In addition to its role in DNA replication, roles for Dam methylation in gene regulation and DNA repair have also been demonstrated in a number of bacteria (for reviews, see references 11, 45, 47, and 50). For V. cholerae as well as for Salmonella spp. and Yersinia pseudotuberculosis, Dam plays a role in virulence possibly through regulation of virulence gene expression (33). Less is known about the functions of seqA apart from its role in E. coli replication, but it has been suggested that SeqA functions as a nucleoid-organizing protein (for a review, see reference 83), and the E. coli chromosome has been demonstrated to have increased supercoiling in a seqA strain (85).Here we describe the first in vivo evidence that Dam plays an important role in the initiation of replication by facilitating the replication initiation at oriCI_Vc in E. coli. In addition, we show that SeqA does not carry an essential role in the initiation of replication.     

TolC affects virulence gene expression in Vibrio cholerae

A Vibrio cholerae tolC mutant showed increased toxT expression in M9 medium, but not in the presence of four amino acids that induce cholera toxin production, and in LB with high osmolarity but not high pH or temperature. TolC did not affect expression of other regulatory genes in the ToxR regulon.     

Proteome comparison of Vibrio cholerae cultured in aerobic and anaerobic conditions

The pathogen Vibrio cholerae causes severe diarrheal disease in humans. This environmental inhabitant has two distinct life cycles, in the environment and in the human small intestine, in which it differs in its multiplication behavior and virulence expression. Anaerobiosis, limitation of some nutrient elements, and excess burden from host metabolism reactants are the major stresses for V. cholerae living in intestine, in comparison to conditions in the environment and laboratory medium. For an insight into the response of V. cholerae to different microenvironments, we cultured the bacteria in aerobic and anaerobic conditions, and compared the whole cell proteome by two-dimensional electrophoresis. Among the protein spots identified, some protein species involved in aerobic respiration and the nutrient carbohydrate transporters were found to be more abundant in aerobic conditions, and some enzymes for anaerobic respiration and some stress response proteins were found more abundant in anaerobic culture. One spot corresponding to flagellin B subunit was decreased in anaerobic conditions, which suggests correlation with the meticulous regulation of bacterial motility during infection in the host intestine. This proteome analysis is the starting point for in-depth understanding of V. cholerae behavior in different environments.     

Toxins of Vibrio cholerae

Surveyed in the paper are published data on properties, biological activity, genetic determinants and action mechanisms of recently known toxins produced by different strains of Vibrio cholerae irrespectively of their capacity for the synthesis of choleric toxin--the main virulence factor. Their possible importance both for the general clinical pattern of cholera provoked by cholerogenic agents and as independent virulence factors causing diarrhea without cholera is elucidated. The sets and levels of expression of additional toxins can differ for different pathogenic clones and they can correspondingly condition degrees of their epidemic and etiological safety.     

The Zymovars of Vibrio cholerae: multilocus enzyme electrophoresis of Vibrio cholerae

Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest. Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia. The mean genetic diversity was 0.339. It is shown that the same O antigen (both O1 and non-O1) may be present in several genetically diverse (different zymovars) strains. Conversely the same zymovar may contain more than one serogroup. It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase). Here it is shown that this rare allele is present in 1 V. mimicus and 4 non-O1 V. cholerae. Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars. The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V. cholerae.