Inhibition of Swarming of Proteus by Sodium Tetradecyl Sulfate, beta-Phenethyl Alcohol, and p-Nitrophenylglycerine

The effects of sodium tetradecyl sulfate (STS), β-phenethyl alcohol (PEA), and p-nitrophenylglycerol (PNPG) on motility, swarming, flagellation, and growth of Proteus were examined. Growth-inhibitory concentrations (GIC) and swarming-inhibitory concentrations (SIC) were determined. A characterization of the swarming-inhibitory efficacy of these compounds was based on their GIC/SIC ratio and their concentration inhibition curves. Using the homologous series of sodium alkyl sulfates as a standard reference, we showed that PNPG was more effective than STS, which was the most effective of the homologous series. PEA was less effective than sodium decyl sulfate but more effective than sodium octyl sulfate. Motility tests in liquid medium and electron microscope investigations indicated that the modes of action of the three compounds, all of which effectively inhibit the swarming of Proteus, are different. Whereas STS and PEA inhibit swarming by inhibition of motility, PNPG seems to act on the swarming mechanism sensu strictori, without impairment of motility. STS immobilizes by inhibition of flagellum formation or by some lytic action on the flagella already synthesized. PEA acts by impairing flagellar function, but leaves the flagella morphologically intact.     

Abolition of Swarming of Proteus by p-Nitrophenyl Glycerin: General Properties

Para-nitrophenyl glycerin (PNPG) was shown to be an effective agent to abolish the swarming of Proteus mirabilis and Proteus vulgaris on predried solid culture media. The level required to abolish swarming varied with the strain of Proteus, the components of the medium, and also with the conditions of incubation. Generally 0.1 to 0.2 mM PNPG effectively abolished swarming for at least 24 h with aerobic incubation. Levels of PNPG that abolished swarming showed no effect upon the growth of the cells, little or no effect upon the motility characteristics of the organisms, and no effect upon the cellular morphology. PNPG was found to be freely water soluble, stable to autoclaving, and to retain biological activity for at least one month in prepared culture media stored under refrigeration.     

Regulation of the Swarming Inhibitor disA in Proteus mirabilis

The disA gene encodes a putative amino acid decarboxylase that inhibits swarming in Proteus mirabilis. 5′ rapid amplification of cDNA ends (RACE) and deletion analysis were used to identify the disA promoter. The use of a disA-lacZ fusion indicated that FlhD₄C₂, the class I flagellar master regulator, did not have a role in disA regulation. The putative product of DisA, phenethylamine, was able to inhibit disA expression, indicating that a negative regulatory feedback loop was present. Transposon mutagenesis was used to identify regulators of disA and revealed that umoB (igaA) was a negative regulator of disA. Our data demonstrate that the regulation of disA by UmoB is mediated through the Rcs phosphorelay.     

Inhibition of Babesia spp. by plastics.

Five commercially available plastic containers were compared with glass for toxicity toward Babesia rodhaini and Babesia bigemina. Comparisons were made by using infectivity tests in mice (B. rodhaini) and cattle (B. bigemina). Low-density polypropylene and polystyrene containers were not toxic, but two of the three polyvinyl chloride containers tested significantly reduced the viability of both species of Babesia. Plasticizer present in various amounts on the surface of the toxic containers was most likely the inhibitory material.     

Swarming characteristics of Proteus mirabilis under anaerobic and aerobic conditions

Nutrients have a pronounced effect on the growth and swarming behaviour of Proteus mirabilis 7002. Iron, zinc, amino acids, and dioxygen are important for rapid growth and normal swarming. Anaerobically grown cultures of P. mirabilis 7002 were unable to swarm on anaerobically maintained rich nutrient agar. Upon exposure to aerobic conditions, P. mirabilis 7002 resumed swarming behaviour. Scanning electron microscopy was used to demonstrate the presence of community organization and mature rafts during normal swarming. These results support the importance of dioxygen and redox status in cell differentiation.     

The Dienes Phenomenon: Competition and Territoriality in Swarming Proteus mirabilis

When two different strains of swarming Proteus mirabilis encounter one another on an agar plate, swarming ceases and a visible line of demarcation forms. This boundary region is known as the Dienes line and is associated with the formation of rounded cells. While the Dienes line appears to be the product of distinction between self and nonself, many aspects of its formation and function are unclear. In this work, we studied Dienes line formation using clinical isolates labeled with fluorescent proteins. We show that round cells in the Dienes line originate exclusively from one of the swarms involved and that these round cells have decreased viability. In this sense one of the swarms involved is dominant over the other. Close cell proximity is required for Dienes line formation, and when strains initiate swarming in close proximity, the dominant Dienes type has a significant competitive advantage. When one strain is killed by UV irradiation, a Dienes line does not form. Killing of the dominant strain limits the induction of round cells. We suggest that both strains are actively involved in boundary formation and that round cell formation is the result of a short-range killing mechanism that mediates a competitive advantage, an advantage highly specific to the swarming state. Dienes line formation has implications for the physiology of swarming and social recognition in bacteria.The gram-negative bacterium Proteus mirabilis is well known for its ability to differentiate into hyperflagellated, motile, and elongated swarmer cells that rapidly spread over a surface. When cultured on a nutrient agar plate, a strain of P. mirabilis typically is able to colonize the whole plate within 24 h. This phenomenon is both of interest in terms of the differentiation and survival strategy of the organism and of practical importance, as contamination of agar plates by swarming P. mirabilis is a common problem in diagnostic microbiology. When two different strains of P. mirabilis swarm on the same agar plate, a visible demarcation line with lower cell density forms at the intersection, and this line is known as a Dienes line (5, 6). A Dienes line is seen when both strains are swarming; it is not a property of the smaller vegetative cells (5). When two identical isolates meet, the swarming edges merge without formation of a Dienes line. This phenomenon has been used in epidemiological typing of clinical isolates (4, 20, 23, 28) and raises interesting questions concerning its mechanism and biological importance. Dienes typing in the clinical environment suggests that the number of Dienes types is large; 81 types were found in one study alone (25). Incompatibility between swarming strains may not be unique to P. mirabilis; a comparable process also appears to occur in Pseudomonas aeruginosa (19). Like many other bacteria, some P. mirabilis strains produce bacteriocins, termed proticines (3). It has been shown that Dienes line formation is not directly caused by a proticine, nor has any other secreted substance or cellular lysate been found to contribute to this phenomenon (27). There does, however, seem to be a circumstantial link between proticine production and Dienes line formation. In the 1970s Senior typed strains of P. mirabilis based on their proticine production and sensitivity (23, 24). He found that proticine production is not related to proticine sensitivity (apart from strains being resistant to their own proticine) but that there is a good correlation between a combined proticine production-sensitivity (P/S) type and Dienes line formation. Strains with the same P/S type do not form a Dienes line, whereas strains with different P/S types do. The more closely related the P/S types of two strains, the less clearly defined the Dienes line is, suggesting that relatedness of strains plays a central role. In contrast, a strain of Proteus vulgaris has been shown to be Dienes compatible with a strain of P. mirabilis with the same P/S type (24). Furthermore, Dienes incompatibility between otherwise identical strains can be triggered by phage lysogeny (2). In contrast to P/S type, the polysaccharide (O) or flagellar (H) serotype has been shown to have no relationship to Dienes line formation (25, 27).Research into the mechanism governing Dienes line formation in Proteus has revealed some important features. The Dienes line region contains many large and often rounded cells (5). The nature of these cells remains controversial. Dienes suggested that round cells originated from both strains but that viable round cells always originated from one of the two intersecting strains. He concluded from this that nonviable round cells should therefore be cells of the other strain (6). In contrast, Wolstenhome suggested that the round cells originated mostly from one of the two swarms involved and that only 50% of these cells were viable (32). It has also been noted that round cells occasionally seem to develop into stable L forms lacking a full cell wall and that they can grow to form tiny L-type colonies (5). Additionally, extracellular DNase has been found at the site of the Dienes line (1). The presence of this enzyme has been interpreted to be a result of cell lysis, as P. mirabilis is known to contain large amounts of DNase (22, 26). Recently, a cluster of six genes, termed ids (identification of self), has been linked to the incompatibility in interpenetration between two strains (and therefore formation of the visible demarcation line), although the function and expression of these genes are not understood yet (8).Despite the fact that Dienes line formation has been known for over 50 years, many questions concerning this phenomenon remain unanswered. Recent advances in imaging, molecular biology, and genomics offer new ways of investigating it. In this work, clinical isolates expressing fluorescent proteins were used to observe the cells involved in Dienes line formation in real time, to evaluate the fate of the round cells, and to test the role of extracellular material and direct cell-cell contact in this phenomenon. Furthermore, the biological role of the Dienes phenomenon in the competition between strains in different situations was investigated.     

Abolition of Swarming of Proteus by p-Nitrophenyl Glycerin: Application to Blood Agar Media

Comparative plate counts were made of Staphylococcus aureus and Streptococcus pyogenes growing on blood agar supplemented with individual chemicals to abolish the swarming of Proteus. B-phenylethanol, sodium azide, and p-nitrophenyl glycerin (PNPG) were used as anti-swarm agents. Each anti-swarm agent effectively abolished swarming for 24 h, but azide failed to control swarming for longer periods of incubation. In addition, azide displayed growth inhibition towards the staphylococci and streptococci resulting in no hemolysis and reduced viable cell numbers with the streptococci. Phenylethanol showed reduced viable cell numbers with the streptococci and unreliable hemolytic reactions. At 0.1 to 0.3 mM, PNPG proved to be a superior anti-swarm agent in that it showed no growth inhibition and allowed normal hemolysis, but abolished swarming for extended periods of time. When laboratory strains of Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa. Listeria monocytogenes, and Vibrio cholerae were screened on a blood agar medium containing 0.1 mm PNPG, they displayed similar growth and hemolytic characteristics to the identical medium without PNPG.     

Loss of FliL Alters Proteus mirabilis Surface Sensing and Temperature-Dependent Swarming

Proteus mirabilis is a dimorphic motile bacterium well known for its flagellum-dependent swarming motility over surfaces. In liquid, P. mirabilis cells are 1.5- to 2.0-μm swimmer cells with 4 to 6 flagella. When P. mirabilis encounters a solid surface, where flagellar rotation is limited, swimmer cells differentiate into elongated (10- to 80-μm), highly flagellated swarmer cells. In order for P. mirabilis to swarm, it first needs to detect a surface. The ubiquitous but functionally enigmatic flagellar basal body protein FliL is involved in P. mirabilis surface sensing. Previous studies have suggested that FliL is essential for swarming through its involvement in viscosity-dependent monitoring of flagellar rotation. In this study, we constructed and characterized ΔfliL mutants of P. mirabilis and Escherichia coli. Unexpectedly and unlike other fliL mutants, both P. mirabilis and E. coli ΔfliL cells swarm (Swr⁺). Further analysis revealed that P. mirabilis ΔfliL cells also exhibit an alteration in their ability to sense a surface: e.g., ΔfliLP. mirabilis cells swarm precociously over surfaces with low viscosity that normally impede wild-type swarming. Precocious swarming is due to an increase in the number of elongated swarmer cells in the population. Loss of fliL also results in an inhibition of swarming at <30°C. E. coli ΔfliL cells also exhibit temperature-sensitive swarming. These results suggest an involvement of FliL in the energetics and function of the flagellar motor.     

Characterization of Proteus mirabilis Precocious Swarming Mutants: Identification of rsbA, Encoding a Regulator of Swarming Behavior

Proteus mirabilis swarming behavior is characterized by the development of concentric rings of growth that are formed as cyclic events of swarmer cell differentiation, swarming migration, and cellular differentiation are repeated during colony translocation across a surface. This cycle produces the bull’s-eye colony often associated with cultures of P. mirabilis. How the cells communicate with one another to coordinate these perfectly synchronized rings is presently unknown. We report here the identification of a genetic locus that, when mutated, results in a precocious swarming phenotype. These mutants are defective in the temporal control of swarming migration and start swarming ca. 60 min sooner than wild-type cells. Unlike the wild type, precocious swarming mutants are also constitutive swarmer cells and swarm on minimal agar medium. The defects were found to be localized to a 5.4-kb locus on the P. mirabilis genome encoding RsbA (regulator of swarming behavior) and the P. mirabilis homologs to RcsB and RcsC. RsbA is homologous to membrane sensor histidine kinases of the two-component family of regulatory proteins, suggesting that RsbA may function as a sensor of environmental conditions required to initiate swarming migration. Introduction of a rsbA mutation back into the wild type via allelic-exchange mutagenesis reconstructed the precocious swarming phenotype, which could be complemented in trans by a plasmid-borne copy of rsbA. Overexpression of RsbA in wild-type cells resulted in precocious swarming, suggesting that RsbA may have both positive and negative functions in regulating swarming migration. A possible model to describe the role of RsbA in swarming migration is discussed.     

Colistin-induced surface changes in the cells of Proteus spp.

The effects of colistin on some strains of Proteus mirabilis, P. morganii and P. vulgaris are described. Two strains of P. mirabilis , NCTC 60 and NCTC 4199, took up greater amounts of colistin from solution than did the other strains. Pretreatment of strains 60 and 4199 resulted in osmotic instability and in increased susceptibility to Tris and deoxycholate. Colistin-induced lysis in Tris could be overcome by means of 0–16 mol/l sodium chloride or 0–33 mol/l sucrose. Pre-treated, but not control, cells of these two strains exposed to Tris (0–05 mol/l, pH 9) developed surface protuberances (blebs).     

Transformation of Glycyrrhizic Acid by Aspergillus spp.

Glycryrrhizic acid was metabolized to 3-oxo-18β-glycyrrhetinic acid via 18β-glycyrrhetinic acid by Aspergillus niger, A. oryzae, A. sojae, and A. tamarii. Two methyl esters were derived from these two metabolites and identified by their ¹³C-NMR spectra and MS data.     

一株奇异变形杆菌对铜绿假单胞菌多细胞行为的抑制作用

目的：抗生素耐药性成为了全球性的健康问题。研究发现病原菌的多细胞行为在抗生素的耐药性中起着至关重要的作用（尤其是生物膜）,因而通过抑制多细胞行为而控制耐药性成为当务之急。本文以奇异变形杆菌（Proteus mirabilis）为研究对象,考察它的发酵滤液对一种机会致病菌——铜绿假单胞菌（Pseudomonas aeruginosa）多细胞行为的作用,以期得到一株多细胞行为抑制菌：在不影响Paerugiliosa生长的前提下,抑制生物膜形成、EPS产生以及定向丛集运动,解除保护,减缓扩散,为降低Paemgi—nosa耐药性,增强抗生素作用效果提供可能。方法：采用结晶紫生物膜测定法、蒽酮一硫酸法、平板检测法,探究Pmirabilis发酵滤液对Paemginosa生物膜、胞外多聚物、定向丛集运动和生长的影响。结果：Pmirabilis发酵滤液能显著抑制Paeruginosa生物膜量,在体积百分比浓度为1％时,抑制率可达60．9％。该菌的发酵滤液还能阻碍Paeruginosa的定向丛集运动,减弱它的吸附和扩散运动;同时,也减少了Pacrugillosa胞外多聚物的产量,在滤液体积百分比浓度为1％时,抑制率达到45．9％。更重要的是,固体平板实验证明该发酵滤液对P．aemginosa的生长没有影响。结论：Pmirabilis在不影响病原菌生长的前提下,对病原菌的多细胞行为有一定的控制作用。其发酵滤液中存在着抑制微生物膜、定向丛集运动等的成分,在治疗细菌感染性疾病和降低抗生素耐药性方面有潜在应用价值。     

IAA-oxidase in Impatiens balsamina Affected by GA3 and Tannic Acid

The activity of IAA-oxidase increased in the leaves of Impatiensbalsamina plants receiving inductive photoperiodic cycles andin plants receiving treatments with gibberellic acid (GA₃) and/ortannic acid (TA), even under non-inductive photoperiods; theactivity also increased in the stem receiving inductive photoperiodiccycles (8 h). Treatment with GA₃ and TA mimics the effect ofSD cycles in the development of some isoenzymes of IAA-oxidase.Thus a new isoenzyme at R_f 0.48 developed in the leaves andone at R_f 0.82 developed in both the stem and the leaves ofall plants receiving inductive treatments – photoperiodicor chemical – but not in water-treated controls undernon-inductive photoperiods. Another isoenzyme at R_f 0.68 developedonly in the stems. Flowering, gibberellic acid, IAA oxidase, Impatiens, phenols, photoperiod     

Inhibition of Spores of Clostridium spp. by Sodium Nitrite

S ummary . Sodium nitrite heated in a laboratory medium was more inhibitory to spores of Clostridium spp. than nitrite added as a filter-sterilized solution to the same medium. Most spores remained refractile after inhibition for >3 months and some proved viable when inoculated into fresh nitrite-free medium. The inhibitory activity of heated nitrite medium was not stable indefinitely, growth sometimes occurred on re-inoculation with vegetative cells.