Chromosome and cell wall segregation in Streptococcus faecium ATCC 9790.

Segregation was studied by measuring the positions of autoradiographic grain clusters in chains formed from single cells containing on average less than one radiolabeled chromosome strand. The degree to which chromosomal and cell wall material cosegregated was quantified by using the methods of S. Cooper and M. Weinberger, dividing the number of chains labeled at the middle. This analysis indicated that in contrast to chromosomal segregation in Escherichia coli and, in some studies, to that in gram-positive rods, chromosomal segregation in Streptococcus faecium was slightly nonrandom and did not vary with growth rate. Results were not significantly affected by strand exchange. In contrast, labeled cell wall segregated predominantly nonrandomly.     

Variation in buoyant density of whole cells and isolated cell walls of Streptococcus faecium (ATCC 9790).

The buoyant density of whole cells of Streptococcus faecium varies with growth rate and during the cell cycle. Two possible explanations for this were explored: (i) the density of cell walls may vary, and (ii) the proportions of wall and cytoplasm may vary. We tested the first possibility by isolating walls from chilled, unfixed populations of S. faecium cells and fractionating them on Percoll density gradients. Mean cell wall density averaged 4% less than whole-cell density and did not vary significantly with growth rate. In addition, walls isolated from heavy and light fractions of a population of cells did not differ significantly in density. Thus, variation in the density of isolated cell walls could not account for the observed variation in whole-cell density within or between populations. Using previously published measurements of the physical dimensions of S. faecium cells, we also found that the relative proportions of wall and cytoplasm (see the second possibility above) could not account for the observed changes in whole-cell buoyant density.     

Division blocks in temperature-sensitive mutants of Streptococcus faecium (S. faecalis ATCC 9790).

Two hundred nine temperature-sensitive growth or division (or both) mutants of Streptococcus faecium ATCC 9790 were isolated. These strains were examined for timing of the division block in the cell division cycle. About 42% of the isolates were blocked at terminal stages of cell division. A second large group appeared to be blocked at various stages of septation. Only five of the temperature-sensitive isolates were blocked at a stage before the completion of chromosome replication. Thirty temperature-sensitive isolates lysed after one or more doublings at the nonpermissive temperature.     

Dodecylglycerol. A new type of antibacterial agent which stimulates autolysin activity in Streptococcus faecium ATCC 9790

Dodecylglycerol has a minimum inhibitory concentration of 4 micrograms/ml compared to 9 micrograms/ml for monolaurin (dodecanoylglycerol) with Streptococcus faecium ATCC 9790 as the test organism. The greater potency of dodecylglycerol can be correlated to its greater retention by the cell. Gram-positive bacteria were more susceptible than Gram-negative bacteria to dodecylglycerol. The antibacterial action of dodecylglycerol is not through the physical dissolution of cell walls, but rather as an enzymatic effector. The autolysin activity of whole cells of S. faecium was greatly stimulated by dodecylglycerol. The stimulation of autolytic activity and inhibition of growth respond in parallel to different concentrations of dodecylglycerol, to dodecylglycerol versus some poorer effector such as monolaurin or a glycerol alkyl ether with a longer or shorter fatty alkyl side chain than dodecanol, and to the antagonistic effects of diphosphatidlyglycerol. This close relationship implies that the stimulation of autolysin activity could be a primary, but not necessarily the only, mechanism by which dodecylglycerol and related compounds exert their antibacterial activity. However, the autolysin activity is not stimulated by a direct interaction between the enzyme and dodecylglycerol. A non-wall entity, such as a proteinase, has been implicated as an intermediary (Ved, H. S., Gustow, E., and Pieringer, R. A. (1984) J. Biol. Chem. 259, 8122-8124).     

Inhibition of beta-lactam antibiotics at two different times in the cell cycle of Streptococcus faecium ATCC 9790.

Treatment of Streptococcus faecium ATCC 9790 with sublytic concentrations of beta-lactam antibiotics revealed two different division blocks in the cell division cycle. One block, induced by N-formimidoyl thienamycin and methicillin, occurred before the completion of chromosome replication, whereas the other, induced by cefoxitin and cephalothin, took place later in the cycle. In addition, these antibiotics gave rise to distinct morphological forms; the antibiotics acting at the earlier block point produced mainly "dumbbells," whereas those affecting the later time formed "lemons." When used in combination N-formimidoyl thienamycin and cefoxitin exerted synergistic killing on this strain. These data suggest that beta-lactam antibiotics have at least two sites of action in S. faecium.     

Interactions between beta-lactam antibiotics and isolated membranes of Streptococcus faecalis ATCC 9790.

The DD-carboxypeptidase-exchange membrane-bound enzyme in Streptococcus faecalis ATCC 9790 reacts with beta-lactam antibiotics to form complexes with rather long half-lives. Depending upon the antibiotic, the second-order rate constants for complex formation range from 0.75-560 M-1 S-1 (at 37 degrees C and in water) and the first-order rate constants for complex breakdown range from 1.3 to 26 x 10(-5) s-1 (at 37 degrees C and in 5 mM phosphate buffer pH 7.5). There are about 30 pmol of DD-carboxypeptidase-exchange enzyme per mg of membrane protein. The degradation products arising from benzylpenicillin are phenylacetylglycine and probably N-formyl-D-penicillamine. Isolated membranes also contain other penicillin binding sites (about 70 pmol/mg membrane protein). That part of benzylpenicillin which reacts with at least some of these latter sites is slowly degraded into penicilloic acid. Normal functioning of the DD-carboxypeptidase-exchange membrane-bound enzyme is important, if not essential, for cell growth. With the beta-lactam antibiotics tested inhibition of cell growth is mainly related to the rates of formation of the inactive enzyme-antibiotic complexes. The relationship, however, is not a direct one probably due to the competitive effect exerted by the other penicillin binding sites.     

The involvement of the proteinase of Streptococcus faecium ATCC 9790 in the stimulation of its autolysin activity by dodecylglycerol

Treatment of Streptococcus faecium ATCC 9790 with 3.5 micrograms/ml of dodecylglycerol produces a nonwall entity found in the 25,000 X g supernatant cell fraction which activates the autolysin activity of S. faecium. The stimulation of the autolysin activity by dodecylglycerol mimics the activation of the autolysin from a latent to an active form by trypsin and other proteolytic enzymes. This stimulation of autolytic activity by dodecylglycerol can be reversed by specific proteinase inhibitors. Dodecylglycerol also markedly stimulates the proteinase activity endogenous to S. faecium, and this stimulation can be reversed by several proteinase inhibitors. It is concluded that one primary antibacterial mode of action of dodecylglycerol is to stimulate the proteinase of S. faecium which activates the cell's autolysin and thereby prevents bacterial growth.     

Streptococcus faecium ATCC 9790 penicillin-binding proteins and penicillin sensitivity are heavily influenced by growth conditions: proposal for an indirect mechanism of growth inhibition by beta-lactams

The effects of variations in growth conditions on the penicillin response of Streptococcus faecium ATCC 9790 were studied. Changes in the growth temperature and medium composition were found to cause striking changes in the bacterial generation time, cellular penicillin sensitivity (minimum inhibitory concentration), sensitivity of peptidoglycan synthesis to inhibition by penicillin, rate of autolysis, and labeling pattern of penicillin-binding proteins. However, no constant relationship between these parameters and the minimum inhibitory concentration could be observed. Similar electrophoretic patterns for penicillin-binding proteins were observed in cells grown in different media at the optimal growth temperature. Inhibition of cell division by penicillin in cells grown at this temperature (but not at higher or lower temperatures) caused filamentation of the bacteria. In cells grown in a chemically defined medium at the optimal temperature (but not at temperatures above or below), complete inhibition of cell division was associated with only partial inhibition (34% after 150 min) of peptidoglycan synthesis. It is suggested that the status and physiological importance of individual penicillin-binding proteins in S. faecium are heavily influenced by growth conditions. Depending on the growth conditions, different penicillin-binding proteins may perform the cellular function, indispensible for bacterial growth.     

Cytokine-inducing glycolipids in the lipoteichoic acid fraction from Enterococcus hirae ATCC 9790

Abstract Five high molecular weight glycolipids capable of stimulating human peripheral whole-blood cell cultures to cause interleukin 6 (IL-6) and tumor necrosis factor (TNF)-α induction were isolated from one of the lipoteichoic acid fractions (LTA-2) extracted from Enterococcus hirae ATCC 9790 (Tsutsui et al., (1991) FEMS Microbiol. Immunol. 76, 211–218) by a combination of hydrophobic interaction and anion-exchange chromatographies. This purification procedure resulted in a remarkable increase in the cytokine-inducing activities on the weight basis of isolated glycolipids (a maximum of 36- and 17-fold increases of IL-6 and TNF-α induction, respectively). The total yield of these bioactive glycolipids amounted to 6 wt% of the parent LTA-2 fraction, while the recovery rate in terms of the cytokine-inducing activities was estimated to be sufficient. The chemical composition and the profile, using SDS-PAGE, revealed that all of the isolated bioactive components were high molecular weight glycolipids, which were distinct from each other and from the parent LTA-2 fraction. These findings suggest that the IL-6 and TNF-α-inducing activities previously noted in the parent LTA-2 fraction are not attributable to a chemical entity, the structure of which had been proposed elsewhere (Fischer, W. (1990) in Glycolipids, Phosphoglycolipids and Sulfoglycolipids (Kates, M. ed.) pp. 123–234, Plenum Press, New York), but to the other high molecular weight glycolipids described here.     

Detection of penicillin-binding proteins immunologically related to penicillin-binding protein 5 of Enterococcus hirae ATCC 9790 in Enterococcus faecium and Enterococcus faecalis

Penicillin-binding protein (PBP) 5 of Enterococcus hirae ATCC 9790 belongs to the class of the high-molecular mass, low-affinity PBPs which have been correlated with penicillin resistance in most Enterococcus species. Polyclonal antibodies were raised against PBP 5 and used to detect immunologically related membrane proteins in E. faecium and E. faecalis strains. Several strains of both species were found to have a membrane protein of similar molecular mass to E. hirae PBP 5 which reacted with the antibodies. Some E. faecium strains did not react with antibodies but their derivatives with increased penicillin minimal inhibitory concentrations did. In some E. faecalis strains the lack of a PBP 5-related protein was associated with failure to select stable penicillin-resistant derivatives.     

磷胁迫下大豆酸性磷酸酶活性变化及磷效率基因型差异分析

在3种磷水平处理后,对23个大豆品种的叶片和根尖酸性磷酸酶活性、根冠比、干物质量、全磷含量及磷效率进行测定、比较和分析。结果表明,叶片或根尖酸性磷酸酶活性、叶片或根尖酸性磷酸酶活性相对值、根冠比,磷效率、磷效率相对值之间具有极显著差异(P相似文献   

Transfer of a miniplasmid determining bacteriocin production and bacteriocin immunity in Streptococcus faecium

Abstract Streptococcus faecium strain 3 produced a bacteriocin (enterococcin Sf3) and contained a homogeneous species of plasmid DNA (pJK3) with an M _r of 3.5 · 10⁶. Plasmid pJK3 was transferable in a filter mating procedure to S. faecium M16. The non-bacteriocinogenic strain M16 was susceptible to enterococcin Sf3 and harboured a non-selftransferable 19.1 MDa plasmid, which was responsible for erythromycin resistance. Transcipient cells of S. faecium M16 contained the 19.1 MDa and the pJK3 plasmid, produced the enterococcin Sf3 and were resistant against the inhibitory action of this bacteriocin.     

Co-transfer of two plasmids determining bacteriocin production and sucrose utilization in Streptococcus faecium

Abstract Streptococcus faecium strain 25 produced a bacteriocin (enterococcin Sf25), metabolized sucrose and contained three plasmids of 2.4, 4.7 and 13.0 MDa. Plasmids Sfp2.4 and Sfp4.7 were cotransferred in a filter mating procedure to sucrose negative and bacteriocin negative S. faecium strain M16. Strain M16 harboured a nonselftransferable plasmid Sfp 19.1 MDa, which was responsible for erythromycin resistance. Transcipient cells of S. faecium M16 contained the 19.1-MDa plasmid and plasmids Sfp2.4 and Sfp4.7, produced the enterococcin Sf25 and gained the ability to degrade sucrose.     

高效广谱猪链球菌7型前噬菌体裂解酶的挖掘及活性研究

【目的】噬菌体具有防控耐药性病原菌的抗菌应用潜力,但是有些病原菌噬菌体的获得非常困难,研究发现大多数病原菌存在前噬菌体(prophage),且由前噬菌体编码的裂解酶(endolysin)具有良好的抗菌应用前景,本研究将挖掘猪链球菌前噬菌体及其编码的裂解酶。【方法】通过对GenBank中登录的数株猪链球菌前噬菌体裂解酶的基因信息分析,挖掘出一株猪链球菌7型菌株前噬菌体编码的裂解酶,研究其生物学活性。以猪链球菌7型菌株7917的基因组为模板,采用PCR技术扩增获得裂解酶基因ly7917,将其克隆至质粒pET28a(+)并转化大肠杆菌DH5α细胞,挑选基因序列正确的阳性克隆、抽提质粒、转化表达菌株大肠杆菌BL21,经IPTG诱导可高效表达裂解酶Ly7917。【结果】平板裂解试验结果显示Ly7917具有高效裂菌活性,能够裂解猪链球菌2型高致病力菌株HA9801;浊度递减试验结果显示该裂解酶能够高效裂解猪链球菌2型、7型、9型和马链球菌兽疫亚种参考株、金黄色葡萄球菌(包括耐甲氧西林金黄色葡萄球菌)等多种革兰氏阳性菌。【结论】基于前噬菌体挖掘的裂解酶Ly7917,具有高效广谱裂菌活性,为临床上利用裂解酶治疗耐药菌的混合感染提供了可能。     

Relationship between changes in buoyant density and formation of new sites of cell wall growth in cultures of streptococci (Enterococcus hirae ATCC 9790) undergoing a nutritional shift-up.

When the glutamate concentration of cultures of Enterococcus hirae was raised from 20 to 300 micrograms/ml, the mass doubling time decreased from ca. 85 to 45 min in 9 min, but balanced growth was not reestablished for 30 to 40 min. During the unbalanced period of growth, RNA and protein synthesis proceeded more rapidly than did peptidoglycan synthesis, buoyant density increased from ca. 1.1024 to 1.1075 g/ml, and the rate of formation of new cell wall growth sites transitorily accelerated above the new growth rate. When studied as a function of cell size, all cultures showed buoyant density to decrease around cell separation, increase as cells increased in size, and then plateau when cells reached large volumes. Greater increases in buoyant density as a function of cell size were seen after shift-up, with the greatest increases observed at 15 to 20 min after shift-up, when the rate of formation of new sites was also maximal. In a population of cells examined by electron microscopy 15 min after shift-up, buoyant density and the frequency of cells with new sites increased as old sites approached the size of two poles. These data were consistent with a model whereby buoyant density increases in the terminal stages of the cell cycle when the surface grows slower than the cytoplasm. The greater the difference in the rates of inside to outside growth, the greater the increase in buoyant density and the more frequently new sites will be initiated.     

Efficient synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose by using new 2-deoxy-d-ribose-5-phosphate aldolase from Streptococcus suis with moderate activity and aldehyde tolerance

The enzyme 2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a useful tool for synthesizing statin side-chain intermediates. In this work, we identified the DERA from Streptococcus suis (SsDERA) by structural and sequence alignment and highly expressed it in Escherichia coli BL21. The recombinant SsDERA had a specific activity of 18.2 U mg⁻¹, K_M of 0.8 mM, and V_max of 32.9 μmol min⁻¹ mg⁻¹ toward 2-deoxy-d-ribose-5-phosphate under the optimal conditions: 40 °C and pH 7.0. The enzyme retained 23.3 % activity after incubation in 200 mM acetaldehyde for 2 h and 58.2 % activity in 100 mM chloroacetaldehyde for 2 h. The enzyme showed moderate activity and aldehyde tolerance compared with reported DERAs. The SsDERA-catalyzed reaction between 200 mM acetaldehyde and 100 mM chloroacetaldehyde generated (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose in 76 % yield in 8 h. This work provides a new DERA for the synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose, which is a potential candidate for the industrial synthesis of statin intermediates.     

Relationship between cellular autolytic activity, peptidoglycan synthesis, septation, and the cell cycle in synchronized populations of Streptococcus faecium.

Synchronized, slowly growing (TD = 70 to 80 min) cultures were used to study several wall-associated parameters during the cell cycle: rate of peptidoglycan synthesis, septation, and cellular autolytic activity. The rate of peptidoglycan synthesis per cell declined during most of the period of chromosome replication (C), but increased during the latter part of C and into the period between chromosome termination and cell division (D). An increase in cellular septation was correlated with the increased rate of peptidoglycan synthesis. Cellular autolytic capacity increased during the early portion of C, reached a maximum late in C or early in D, and declined during D. Inhibition of DNA synthesis during C prevented the decline in autolytic capacity at the end of the cell cycle, caused a slight reduction in the rate of peptidoglycan synthesis, delayed but did not prevent septation, and prevented the impending cell division by inhibiting cell separation. Inhibition of DNA synthesis during D did not prevent the increase in autolytic capacity during the next C phase, but, once again, prevented the decline at the end of the subsequent cycle. Thus, increased autolytic capacity at the beginning of the cell cycle did not seem to be related to chromosome initiation, whereas decreased autolytic capacity at the end of the cell cycle seemed to be related to chromosome termination. The data presented are consistent with the role of autolytic enzyme activity in the previously proposed model for cell division of S. faecium (G.D. Shockman et al., Ann. N.Y Acad. Sci. 235:161-197, 1974).     

Critical thermal limits in Mediterranean ant species: trade-off between mortality risk and foraging performance

1. In Mediterranean ant communities, a close relationship has been found between activity rhythm in the period of maximum activity and position in the dominance hierarchy: subordinate species are active during the day, when conditions are more severe, while dominants are active during the afternoon and the night.
2. Results obtained in this study confirmed that the species foraging at higher temperatures were closer to their critical thermal limits than the species foraging at lower temperatures.
3. This enabled two extreme strategies of foraging in relation to temperature to be distinguished: (1) heat-intolerant ant species behaved as risk-averse species, foraging at temperatures very far from their critical thermal limits; and (2) heat-tolerant ant species behaved as risk-prone species, foraging very near their critical thermal limits and running a high heat mortality risk.
4. Heat-tolerant species benefited from this strategy by having better foraging performance at high temperatures.
5. This wide range of thermal niches may be one reason why Mediterranean ant faunas are so diverse in the face of limited diversity in vegetation and habitat structure: the daily range of temperature may be sufficiently great to meet the requirement both of heat-adapted and cold-adapted species as well as a spectrum of intermediate forms.