Low Salt Medium Research Articles

Peptidoglycan modulation by Francisella tularensis

HAYDEN HESS, STUART CANTLAY, JOSEPH HORZEMPA, Department of Biomedical Sciences, West Liberty University, West Liberty, WV 26074 and MELANIE SAL, Department of Biology, West Virginia Wesleyan College, Buckhannon, WV 26201. Peptidoglycan modulation by Francisella tularensis. Peptidoglycan, a crucial component of bacterial cell walls, plays a fundamental role in maintaining cell shape, integrity, and resistance to osmotic pressure. Mycoplasmas are the only bacteria that do not produce peptidoglycan cell walls. Other peptidoglycan-producing bacteria are capable of transiently existing without a peptidoglycan as an “L-form”, but only on solid media. Preliminary observations made by our laboratory suggested that Francisella tularensis bacteria do not produce detectible levels of peptidoglycan while growing in liquid media. However, increasing salt concentration induces the production of peptidoglycan in all bacteria. In this study we confirmed and extended these observations by incubating bacteria with fluorescent D-alanine (which integrates into the peptidoglycan during synthesis). Fluorescence microscopy revealed that very few F. tularensis bacteria produce detectable peptidoglycan in low salt media, while seemingly all individual bacteria produce this layer in high salt media. Interestingly, the genome of F. tularensis encodes two distinct beta-lactamases. Here we show that both the wild-type F. tularensis LVS and beta-lactamase null mutant were both resistant to high levels of beta-lactam antibiotics in low salt liquid media. However, only the wild type and complemented mutant strains were resistant to beta-lactam antibiotics in high salt media while the beta lactamase null mutant was susceptible. These data further support the conclusion that F. tularensis does not produce, nor need, peptidoglycan in low salt liquid media. Future studies will investigate whether peptidoglycan modulation is required to avoid detection by the innate immune system.

Mycobacterial FtsEX-RipC interaction is required for normal growth and cell morphology in rifampicin and low ionic strength conditions.

Mycobacterial cell growth and division are coordinated with regulated peptidoglycan hydrolysis. Understanding cell wall gene complexes that govern normal cell division and elongation will aid in the development of tools to disarm the ability of mycobacteria to survive immune-like and antibiotic stresses. We combined genetic analyses and scanning electron microscopy to analyze morphological changes of mycobacterial FtsEX and RipC mutants in stressful conditions. We demonstrate that FtsE, FtsX, FtsEX, and RipC are conditionally required for the survival of Mycobacterium smegmatis during rifampicin treatment and in low-salt conditions. Growth defects in these conditions were characterized by short and bulgy cells as well as elongated filamentous cells. We also show that the FtsEX-RipC interaction is essential for the survival of M. smegmatis in rifampicin. Our results suggest that FtsE, FtsX, and RipC are required for normal cell wall regulation and ultimately for survival in stressful conditions.

The effect of high‐pressure homogenization on rabbit myofibrillar proteins in dilute sodium chloride solutions: weak ionic shielding promotes the depolymerisation

SummaryThe study attempts to determine the effect of ionic strength on the structure and solubility of rabbit myofibrillar proteins (MPs) and improves MPs dissolution in low salt medium. There was a significant effect of ionic strength on the structure and dissolution characteristics of MPs in low salt medium range (0–200 mM NaCl), increased ionic strength led to MPs aggregation and reduced solubility (P < 0.05), while decreased ionic strength weakened charge shielding effect, resulted in stronger electrostatic repulsion and weaker hydrophobic interaction between molecules, thus promoting MPs structural swell and separation between filaments. The modification effect of HPH on the MPs also depended on the ionic strength. Upon HPH, due to the difference in charge shielding effect and hydrophobic interactions, there was a progressive improvement in MPs solubility (from 23.51% to 85.25%) and dispersion stability at 0 mM salt medium (I = 0.0217) while limited increase in solubility at medium higher than 13 mM (from 4.39% to 7.20% at 20 mM). Decreasing ionic strength promoted the depolymerisation of HPH on MPs, inhibited the myosin assembly into filaments after HPH. The modification of ionic strength on the surface charge and hydrophobicity of MPs were further amplified, also contributed to the improved solubility and dispersion stability.

Disparate Regions of the Human Chemokine CXCL10 Exhibit Broad-Spectrum Antimicrobial Activity against Biodefense and Antibiotic-Resistant Bacterial Pathogens.

CXCL10 is a pro-inflammatory chemokine produced by the host in response to microbial infection. In addition to canonical, receptor-dependent actions affecting immune-cell migration and activation, CXCL10 has also been found to directly kill a broad range of pathogenic bacteria. Prior investigations suggest that the bactericidal effects of CXCL10 occur through two distinct pathways that compromise the cell envelope. These observations raise the intriguing notion that CXCL10 features a separable pair of antimicrobial domains. Herein, we affirm this possibility through peptide-based mapping and structure/function analyses, which demonstrate that discrete peptides derived from the N- and C-terminal regions of CXCL10 mediate bacterial killing. The N-terminal derivative, peptide P1, exhibited marked antimicrobial activity against Bacillus anthracis vegetative bacilli and spores, as well as antibiotic-resistant clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecium, and Staphylococcus aureus, among others. At bactericidal concentrations, peptide P1 had a minimal degree of chemotactic activity, but did not cause red blood cell hemolysis or cytotoxic effects against primary human cells. The C-terminal derivative, peptide P9, exhibited antimicrobial effects, but only against Gram-negative bacteria in low-salt medium─conditions under which the peptide can adopt an α-helical conformation. The introduction of a hydrocarbon staple induced and stabilized α-helicity; accordingly, stapled peptide P9 displayed significantly improved bactericidal effects against both Gram-positive and Gram-negative bacteria in media containing physiologic levels of salt. Together, our findings identify and characterize the antimicrobial regions of CXCL10 and functionalize these novel determinants as discrete peptides with potential therapeutic utility against difficult-to-treat pathogens.

Effect of high pressure homogenization on aggregation, conformation, and interfacial properties of bighead carp myofibrillar protein.

Fish myofibrillar protein is underutilized due to the formation of insoluble aggregates in low salt media. High pressure homogenization (HPH) at 20, 40, and 60MPa for four passes was applied on bighead carp myofibrillar protein in order to modify its structure and interfacial properties. Changes in aggregation, conformation, solubility, emulsifying and foaming properties of myofibrillar protein were investigated. The aggregates of myofibrillar protein were obviously disrupted by HPH treatment. The size of myofibrillar protein aggregates became smaller and more uniform as the treating pressure increased, accompanied by notable decreases of cross-sectional height and Rq value in AFM image. Furthermore, the conformation of HPH-treated myofibrillar protein was unfolded into a flexible and open structure. α-helix and β-sheet were converted into β-turn and random coil. Surface hydrophobicity and zeta potential were strengthened, along with the exposure of sulfhydryl groups onto molecule surface. On the other hand, solubility, emulsifying activity index (EAI) and foaming capacity (FC) of HPH-treated myofibrillar protein were markedly enhanced with the increasing pressure. Especially after HPH treatment at 60MPa, myofibrillar protein was almost dissolved in low salt media (solubility 91.86%) with 4.92 fold for EAI and 3.52 fold for FC. But there was little variation in emulsifying and foaming stabilities. These results suggested that HPH treatment has interesting potential to induce the dissociation and unfolding of myofibrillar protein in low salt media, therefore improving its interfacial properties. PRACTICAL APPLICATION: Carp myofibrillar protein was treated by high pressure homogenization (HPH). Aggregates of myofibrillar protein were disrupted into smaller size form. Conformation of myofibrillar protein was unfolded into open and loose structure. Emulsifying and foaming capacities of myofibrillar protein were improved. HPH treatment modified the structure and interfacial properties of myofibrillar protein.

Inoculum Preparation Conditions Influence Adherence of Salmonella enterica Serovars to Red Leaf Lettuce (Lactuca sativa)

Salmonella enterica has been increasingly linked to outbreaks involving consumption of fresh produce. Although researchers have identified genes whose products are involved in mediating S. enterica-plant interactions, the use of various experimental approaches, serovars, and plant types has generated variable and conflicting data. The purpose of this study was to determine whether conditions under which inocula are prepared for in vitro plant interaction studies influence the outcome of these studies. Seven S. enterica serovars were grown in media that differed in salinity and physical state with incubation at 25 or 37°C. These cultures were then used to inoculate red leaf lettuce, and adherent microbes were subsequently recovered. Although all Salmonella serovars were influenced by inoculum preparation conditions, the amount of variation differed. Analysis of pooled serovar data revealed that inocula prepared from either agar plates or biphasic cultures had higher levels of interaction with red leaf lettuce than those prepared from broth cultures. Incubation at 37°C enhanced adherence after 30 s or 5 days of contact time, and adherence after 1 h of contact time was increased in low-salt medium. Broth inoculum cultures were highly influenced by medium salinity and incubation temperature, whereas plate and biphasic inoculum cultures were only minimally affected. Therefore, inocula prepared from bacteria grown on plates or in biphasic culture would be most suitable for evaluation of strategies used to interfere with plant-Salmonella interactions. However, pooled data mask serovar-specific responses, and care should be taken when extrapolating these findings to individual serovars. The previous association of a serovar with outbreaks involving leafy greens was not correlated with levels of interaction with red leaf lettuce, suggesting that the occurrence of these serovars in or on these commodities does not reflect their fitness in the plant environment.

Fast and easy quantitative characterization of methanotroph-photoautotroph cocultures.

Recent research has demonstrated that synthetic methanotroph-photoautotroph cocultures offer a highly promising route to convert biogas into value-added products. However, there is a lack of techniques for fast and accurate characterization of cocultures, such as determining the individual biomass concentration of each organism in real-time. To address this unsolved challenge, we propose an experimental-computational protocol for fast, easy, and accurate quantitative characterization of the methanotroph-photoautotroph cocultures. Besides determining the individual biomass concentration of each organism in the coculture, the protocol can also obtain the individual consumption and production rates of O2 and CO2 for the methanotroph and photoautotroph, respectively. The accuracy and effectiveness of the proposed protocol was demonstrated using two model coculture pairs, Methylomicrobium alcaliphilum 20ZR-Synechococcussp. PCC7002 that prefers high pH high salt condition, and Methylococcus capsulatus-Chlorella sorokiniana that prefers low salt and neutral pH medium. The performance of the proposed protocol was compared with a flow cytometry-based cell counting approach. The experimental results show that the proposed protocol is much easier to carry out and delivers faster and more accurate results in measuring individual biomass concentration than the cell counting approach without requiring any special equipment.

Combining metabolic engineering and evolutionary adaptation in Klebsiella oxytoca KMS004 to significantly improve optically pure D-(-)-lactic acid yield and specific productivity in low nutrient medium.

In this study, K. oxytoca KMS004 (ΔadhE Δpta-ackA) was further reengineered by the deletion of frdABCD and pflB genes to divert carbon flux through D-(-)-lactate production. During fermentation of high glucose concentration, the resulted strain named K. oxytoca KIS004 showed poor in growth and glucose consumption due to its insufficient capacity to generate acetyl-CoA for biosynthesis. Evolutionary adaptation was thus employed with the strain to overcome impaired growth and acetate auxotroph. The evolved K. oxytoca KIS004-91T strain exhibited significantly higher glucose-utilizing rate and D-(-)-lactate production as a primary route to regenerate NAD+. D-(-)-lactate at concentration of 133g/L (1.48M), with yield and productivity of 0.98g/g and 2.22g/L/h, respectively, was obtained by the strain. To the best of our knowledge, this strain provided a relatively high specific productivity of 1.91g/gCDW/h among those of other previous works. Cassava starch was also used to demonstrate a potential low-cost renewable substrate for D-(-)-lactate production. Production cost of D-(-)-lactate was estimated at $3.72/kg. Therefore, it is possible for the KIS004-91T strain to be an alternative biocatalyst offering a more economically competitive D-(-)-lactate production on an industrial scale. KEY POINTS: • KIS004-91T produced optically pure D-(-)-lactate up to 1.48M in a low salts medium. • It possessed the highest specific D-(-)-lactate productivity than other reported strains. • Cassava starch as a cheap and renewable substrate was used for D-(-)-lactate production. • Costs related to media, fermentation, purification, and waste disposal were reduced.

CTP synthase forms cytoophidia in archaea

CTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of nucleotide CTP de novo synthesis. Since 2010, a series of studies have demonstrated that CTPS can form filamentous structures in bacteria and eukaryotes, which are termed cytoophidia. However, it is unknown whether cytoophidia exist in the third domain of life, archaea. Using Haloarcula hispanica as a model system, here we demonstrate that CTPS forms distinct intracellular compartments in archaea. Under stimulated emission depletion microscopy, we find that the structures of H. hispanica CTPS are elongated, similar to cytoophidia in bacteria and eukaryotes. When Haloarcula cells are cultured in low-salt medium, the occurrence of cytoophidia increases dramatically. In addition, treatment of H. hispanica with a glutamine analog or overexpression of CTPS can promote cytoophidium assembly. Our study reveals that CTPS can form cytoophidia in all three domains of life, suggesting that forming cytoophidia is an ancient property of CTPS.

Surviving salt fluctuations: stress and recovery in Halobacterium salinarum, an extreme halophilic Archaeon

Halophilic proteins subjected to below about 15% salt in vitro denature through misfolding, aggregation and/or precipitation. Halobacteria, however, have been detected in environments of fluctuating salinity such as coastal salterns and even around fresh water springs in the depths of the Dead Sea. In order to identify the underlying mechanisms of low salt survival, we explored the reactivation capacity of Halobacterium (Hbt) salinarum sub-populations after incubation in low salt media and recovery in physiological salt. Respiratory oxygen consumption was assessed in stressed cells and cell viability was estimated by Live/Dead staining and flow cytometry. In vivo neutron scattering experiments showed that the recovery of Hbt salinarum sub-populations exposed to severe low salt conditions is related to a rapid retrieval of functional molecular dynamics in the proteome. In the hypothesis that the observations on Hbt salinarum have wider relevance, they could be of key ecological significance for the dispersion of extremophiles when environmental fluctuations become severe.

Identification and functional characterization of the astacidin family of proline-rich host defence peptides (PcAst) from the red swamp crayfish (Procambarus clarkii, Girard 1852)

This study reports the identification of four novel proline-rich antimicrobial peptides (PR-AMP) from the transcriptome of the red swamp crayfish Procambarus clarkii. The newly identified putative peptides (PcAst-1b, -1c, −2 and −3), which are related with the previously identified hemocyte-specific PR-AMP astacidin-1, are encoded by the multi-genic astacidin gene family. The screening of available and proprietary transcriptomes allowed to define the taxonomical range of distribution of this gene family to Astacoidea and Parastacoidea. The antimicrobial properties of three synthetic PcAst peptides (PcAst-1a, -1b/c and −2), were characterized against reference bacteria or multidrug resistant clinical isolates, and their cytotoxicity was evaluated towards human transformed cell lines. The antimicrobial activity ranged from potent and broad-spectrum, in low-salt medium, to poor, whereas it was generally low in full nutrient broth. No significant toxic effects were observed on cultured human cells. RNA-seq data from 12 different tissues indicated a strong specificity for haemocytes under naïve physiological condition, with moderate expression (5-fold lower) in gills. Quantitative real time PCR revealed a rapid (within 2 h) and significant up-regulation of PcAst-1a (Astacidin 1) and PcAst-2 expression in response to LPS injection. Due to the variation in antimicrobial potency and inducibility, the roles of the other astacidins (PcAst-1b, -1c and −3) need to be further investigated to determine their significance to the immune responses of the red swamp crayfish.

Evaluation of quantity and quality of chitosan produce from Rhizopus oryzae by utilizing food product processing waste whey and molasses

Low cost whey salt medium (WSM) and molasses salt medium (MSM) have been constructed utilizing food processing byproduct whey and molasses for the production of bio-polysaccharide chitosan from Rhizopus oryzae and subsequently comprehensive physico-chemical characterization of the fungal chitosan has been carried out using various analytical tools to apprehend its biochemical utility. Same has been repeated with chitosan from conventional potato dextrose broth (PDB) for comparison purpose. The yields of chitosan in three different media were 0.62 (WSM), 0.39 (MSM) and 0.63 (PDB) g/L respectively. Molecular weights of the chitosans were in the range of 100–300 kDa. WSM-chitosan and MSM-chitosan were less polydispersed, possessed more hydrated polymorph and loose crystal packing than PDB-chitosan. This indicate that WSM-chitosan and MSM-chitosan are highly exposed to the external reagent hence more reactive to the external reagents with compare to PDB-chitosan. Literature suggest isolated chitosans are useful for specific drug delivery applications.

Lytic transglycosylases RlpA and MltC assist in Vibrio cholerae daughter cell separation.

The cell wall is a crucial structural feature in the vast majority of bacteria and comprises a covalently closed network of peptidoglycan (PG) strands. While PG synthesis is important for survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by 'autolysins', enzymes that break down PG. Cell division, for example, requires extensive PG remodeling, especially during separation of daughter cells, which depends heavily upon the activity of amidases. However, in Vibrio cholerae, we demonstrate that amidase activity alone is insufficient for daughter cell separation and that lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low-salt media. The division-specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum cleave PG strands that are crosslinked beyond the reach of the highly regulated activity of the amidase and clear PG debris that may block the completion of outer membrane invagination.

Exploring the piezotolerant/piezophilic microbial community and genomic basis of piezotolerance within the deep subsurface Deccan traps.

The deep biosphere is often characterized by multiple extreme physical-chemical conditions, of which pressure is an important parameter that influences life but remains less studied. This geomicrobiology study was designed to understand the response of a subterranean microbial community of the Deccan traps to high-pressure conditions and to elucidate their genomic properties. Groundwater from a deep basaltic aquifer of the Deccan traps was used to ascertain the community response to 25MPa and 50MPa pressure following enrichment in high-salt and low-salt organic media. Quantitative PCR data indicated a decrease in bacterial and archaeal cell numbers with increasing pressure. 16S rRNA gene sequencing displayed substantial changes in the microbial community in which Acidovorax appeared to be the most dominant genus in the low-salt medium and Microbacteriaceae emerged as the major family in the high-salt medium under both pressure conditions. Genes present in metagenome-associated genomes which have previously been associated with piezotolerance include those related to nutrient uptake and extracytoplasmic stress (omp, rseC), protein folding and unfolding (dnaK, groEL and others), and DNA repair mechanisms (mutT, uvr and others). We hypothesize that these genes facilitate tolerance to high pressure by certain groups of microbes residing in subsurface Deccan traps.

Antimicrobial Activity of NCR Plant Peptides Strongly Depends on the Test Assays.

The symbiosis specific NCR247 and NCR335 cationic plant peptides of Medicago truncatula have been shown to exert antimicrobial activity against a wide range of microbes. However, their antimicrobial efficiency is clearly limited by divalent cations. Here, the antibacterial and antifungal activities of NCR247 and NCR335 peptides were compared to those of the well-characterized peptide antibiotics polymyxin B and the aminoglycoside streptomycin on three model microbes, Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae as representatives of Gram-negative and Gram-positive bacteria as well as eukaryotic fungi. The aim of the study was to assess how the killing efficiency of these peptides depends on various, widely used antimicrobial susceptibility assays. Validated resazurin microdilution assay was used to determine minimal growth inhibitory concentrations in three general test media (MHB, MHBII and low-salt medium LSM). Bactericidal/fungicidal activities were determined by the commonly used drop plate assay. The natural plant peptides showed distinct characteristics, NCR247 had a generally high sensitivity for Ca2+ and Mg2+ in the medium, while NCR335 proved to be a robust and strong antimicrobial agent with comparable efficiency values to polymyxin B. Activity data were confirmed visually, both NCR247 and NCR335 treatments at minimal bactericidal concentrations induced complete disruption of the membranes and provoked cell lysis on all tested microorganisms as observed by scanning electron microscopy.

Rapid and complete degradation of diclofenac by native soil microorganisms

Abstract Diclofenac one of the widespread xenobiotics, among other organic micropollutants, is persistent accumulating in different habitats like earth, water, plants and even mammalians. Natural microbial communities in soil and water play a key role in fundamental ecological processes such as regulating the fate of pollution released in the environment. As presented in this study varying concentrations of diclofenac between 0.1 and 1.0 g/L solubilized in a low salt medium could be aerobically degraded by forest soil within less than 10 days. In the course of full degradation, a carboxylated diclofenac intermediate could be isolated and identified by LC-MS/MS-TOF. The carboxylated diclofenac might be a key intermediate to enable a complete biodegradation of diclofenac via 2,6-dichloroaniline and carboxylated 2-hydroxyphenylacetic acid by a microbial consortium.

The inner membrane protein YhiM is necessary for Escherichia coli growth at high temperatures and low osmolarity.

To survive, Escherichia coli must be able to survive in rapidly changing environmental conditions including changes in temperature and osmolarity. We have studied the role of the inner membrane protein YhiM in changing environmental conditions. Our data indicate that YhiM is required for normal growth at 37 and 41°C but not 21°C. YhiM-deficient cells grown at high temperatures spend more time in lag phase and stop growing at lower cell densities in comparison with their wild-type counterparts. They also have growth defects in low NaCl medium at 37°C and do not grow at all at 41°C. The effects of low NaCl can be rescued by addition of KCl or sucrose to the low salt medium. Finally, YhiM-deficient cells fail to grow in dilute medium at 41°C. These data suggest that YhiM may be important in protecting the cells from changes in temperature and osmolarity.

Identification of Haloferax volcanii Pilin N-Glycans with Diverse Roles in Pilus Biosynthesis, Adhesion, and Microcolony Formation

N-Glycosylation is a post-translational modification common to all three domains of life. In many archaea, the oligosacharyltransferase (AglB)-dependent N-glycosylation of flagellins is required for flagella assembly. However, whether N-glycosylation is required for the assembly and/or function of the structurally related archaeal type IV pili is unknown. Here, we show that of six Haloferax volcanii adhesion pilins, PilA1 and PilA2, the most abundant pilins in pili of wild-type and ΔaglB strains, are modified under planktonic conditions in an AglB-dependent manner by the same pentasaccharide detected on H. volcanii flagellins. However, unlike wild-type cells, which have surfaces decorated with discrete pili and form a dispersed layer of cells on a plastic surface, ΔaglB cells have thick pili bundles and form microcolonies. Moreover, expressing PilA1, PilA2, or PilA6 in ΔpilA[1-6]ΔaglB stimulates microcolony formation compared with their expression in ΔpilA[1-6]. Conversely, expressing PilA3 or PilA4 in ΔpilA[1-6] cells results in strong surface adhesion, but not microcolony formation, and neither pilin stimulates surface adhesion in ΔpilA[1-6]ΔaglB cells. Although PilA4 assembles into pili in the ΔpilA[1-6]ΔaglB cells, these pili are, unlike wild-type pili, curled, perhaps rendering them non-functional. To our knowledge, this is the first demonstration of a differential effect of glycosylation on pilus assembly and function of paralogous pilins. The growth of wild-type cells in low salt media, a condition that decreases AglB glycosylation, also stimulates microcolony formation and inhibits motility, supporting our hypothesis that N-glycosylation plays an important role in regulating the transition between planktonic to sessile cell states as a response to stress.

A D, D-carboxypeptidase is required for Vibrio cholerae halotolerance.

The biological roles of low molecular weight penicillin-binding proteins (LMW PBP) have been difficult to discern in Gram-negative organisms. In Escherichia coli, mutants lacking these proteins often have no phenotype, and cells lacking all seven LMW PBPs remain viable. In contrast, we report here that Vibrio cholerae lacking DacA-1, a PBP5 homologue, displays slow growth, aberrant morphology and altered peptidoglycan (PG) homeostasis in Luria-Bertani (LB) medium, as well as a profound plating defect. DacA-1 alone among V. cholerae's LMW PBPs is critical for bacterial growth; mutants lacking the related protein DacA-2 and/or homologues of PBP4 or PBP7 displayed normal growth and morphology. Remarkably, the growth and morphology of the dacA-1 mutant were unimpaired in LB media containing reduced concentrations of NaCl (100 mM or less), and also within suckling mice, a model host for the study of cholera pathogenesis. Peptidoglycan from the dacA-1 mutant contained elevated pentapeptide levels in standard and low salt media, and comparative analyses suggest that DacA-1 is V. cholerae's principal DD-carboxypeptidase. The basis for the dacA-1 mutant's halosensitivity is unknown; nonetheless, the mutant's survival in biochemically uncharacterized environments (such as the suckling mouse intestine) can be used as a reporter of low Na(+) content.

An ideal media for the in-vitro propagation of Ipomoea palmata Forssk. [Synonym – Ipomoea cairica. L. Sweet] Convolulaceae

In order to study its development in-vitro, an investigation was carried out to determine and establish a suitable media for the growth of Ipomoea palmata Forssk, (Ipomoea. cairica). Aerial parts of a healthy plant were chosen as explants. Callogenesis of these leaf, stem and flower explants was initiated on three different media namely Murashige and Skoog (1962) media, Gamborg or B5 (1968) media and White's media, (1963). These basal media were supplemented with a quantity of coconut water and with various combinations of growth hormones- 2,4-Dichlorophenoxyacetic acid (2,4- D), Indole Acetic acid (IAA), Indole Butyric Acid ( IBA) and 6-Benzyl amino purine (BA). The observation showed that the explants regenerated into profuse calli which eventually proliferated into multiple shoots. Root formation from the callus was also observed. The highest response in callogenesis and morphogenesis was observed in M.S basal media supplemented with 4mg/l BA and 1mg/l 2,4-D. However development was first noticed in B5 basal media supplemented with 4mg/l and 1mg/l 2,4-D. I. palmata responded positively recording rapid growth, thereby, demonstrating both Re and De differentiation. Among the three different media, the low-salt media i.e. White's media showed a relatively poor response in callus and plantlet formation. Interestingly I.palmata seemed to have a great affinity for both MS and B5 media specifically, the hormonal concentration mentioned above. This report of a positive in-vitro response of I.palmata has not been published before.