Lolita Daneo-Moore

PROBLEMS OF CELL WALL AND MEMBRANE GROWTH, ENLARGEMENT, AND DIVISION*

Not many years ago, growth and division of bacteria was thought of as a rather casual process. Cells merely increased in size and, after a more or less appropriate increase, divided into two smaller units. In fact, the observations of Henrici (published in 1928) were not fully appreciated until relatively recently. In cultures of Escherichia coli, Henrici observed that cells from the lag phase were both larger on the average and less uniform in size than cells from the exponential or stationary phases of growth. More recent observations, of balanced exponentially growing cultures, showed that both cellular composition and the average size of bacterial cells are dependent on growth rate.?, Such observations served as a basis for defining some of the factors involved in the regulation of the synthesis of informational macromo1ecules.3 Later, it became apparent that the processes that lead to cell division also must be coupled to, and interrelated with, the synthesis of informational macromolec u l e ~ . ~ ~ In addition to the cell division process itself, the enlargement of the bacterial cell surface (cell wall and membrane) also must be somehow related to the synthesis of other cellular constituents. I t is necessary for the cell surface not only to expand in area to enclose an increasing volume of protoplasm but also to undergo a series of changes in shape (morphogenesis), while maintaining the osmotic integrity of the cell. As cells increase in mass and volume, all of the cellular dimensions do not increase proportionately. During balanced growth of rod-shaped species, such as E. coli, at any specific growth rate the diameter of the cylinder remains fixed,9 so that increases in cell volume can be attributed to an increase in cell length. In the process of cylindrical elongation, there is a continuous decrease in the surface area to volume ratio.lo Such potential changes in surface area to volume ratio are illustrated in FIGURE 1. The formation of new poles and cell division result in the surface area to volume ratio reverting to that at the start of the cycle. Immediate separation of newly formed cross walls, to become one of the poles of each of the daughter cells, is not, however, essential to the process. When newly divided cells remain in chains, the potential surface area of their poles remains hidden, so that the ratio of cellular surface area to volume is considerably less than that of fully separated cells. Therefore, consideration must be given not only to exposed surface area but also to total

Persistence of Streptococcus mutans in Stationary-Phase Batch Cultures and Biofilms

ABSTRACT Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (±8) days, and an average of 2.7 × 105 CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.

Differential Protein Synthesis in Candida albicans during Blastospore Formation at 24.5 C and during Germ Tube Formation at 37 C

SUMMARY: To identify proteins synthesized during blastospore to germ tube transformation in Candida albicans, membrane and cytoplasmic protein fractions labelled with 14C were analysed by SDS-PAGE and autoradiography. Four cytoplasmic proteins were detected in pulse-labelled lysates prepared from cells forming blastospores and germ tubes at 37 °C, but not in pulse-labelled lysates prepared from cells forming blastospores at 24.5 °C. Three proteins detectable in 24.5 °C lysates were diminished in 37 °C lysates.

Biochemical Differentiation of Certain Oral Streptococci

A reliable scheme for the differentiation of certain species of oral streptococci has been developed. It employs a commercial biochemical test system (API 50L) modified to accommodate some of the oral streptococci. The scheme relies primarily on eight biochemical reactions, which demonstrate consistent results. Compared to Shklair and Keenes very useful biochemical scheme, our scheme employs additional definitive tests, is a much simpler system technically, and can differentiate a proposed new species, S. ferus.

Effect of growth rate on lipid and lipoteichoic acid composition in Streptococcus faecium.

The lipid composition of Streptococcus faecium (S. faecalis ATCC 9790) was analyzed at various growth rates. Diphosphatidylglycerol and the non-ionic lipid fraction containing diacylglycerols and neutral glycolipids appeared to accumulate relative to cellular mass as the culture mass doubling time increased from 30 to 80 min. Within the same range of doubling times the non-ionic lipid fraction appeared to become substantially enriched with diacylglycerols. All lipid species and cellular lipoteichoic acid accumulated relative to the cellular mass at doubling times exceeding 80 min, although diacylglycerol accumulation exceeded that of all other compounds studied.

Transposon-916-like elements in clinical isolates of Enterococcus faecium.

Tetracycline (Tc) resistance was found in nine out of ten clinical isolates of Enterococcus faecium. Conjugative transposons, designated Tn5031, Tn5032 and Tn5033, were present in the chromosome of three isolates. The transposons were similar both structurally and functionally to Tn916 containing the tetM determinant. A large non-conjugative plasmid found in a fourth isolate contained an element homologous to Tn916. The four isolates containing the element showing homology to Tn916 exhibited a substantially higher level of Tc resistance than the remaining five Tc-resistant isolates. Tc-resistance genes which have not been identified are apparently responsible for the low-level Tc resistance in five clinical isolates.

Effects of culture density on the kinetics of germ tube formation in Candida albicans

The relationship between culture density or phase of growth at 24.5 degrees C and the ability of Candida albicans to form germ tubes when shifted to 37 degrees C was investigated. Evidence is presented demonstrating germ tube production from liquid synthetic medium cultures at all phases of growth. Previous studies reported that only cells from stationary phase cultures were competent to form germ tubes. Comparisons between exponential and stationary phase cultures indicate more rapid and more synchronous germ tube production from cells growing in the exponential phase.

A rapid, quantitative, and selective estimation of radioactively labeled peptidoglycan in gram-positive bacteria

Abstract The Park and Hancock procedure for the isolation of bacterial cell wall peptidoglycans has been modified. The method developed permits rapid and accurate determination of small quantities of radioactively labeled material. The principal modifications consist of carrying out the procedure on glass-fiber filters after a vigorous hydrolysis of the cells with TCA (96°C, 30 min) and using Pronase in place of trypsin.

Peptidoglycan (Murein) Hydrolases: Unusual Enzymes for Unusual Substrates

A broad variety of both Gram-positive and Gram-negative bacteria have been shown to have the ability to dissolve themselves (autolyze), particularly under “adverse” conditions (see Ghuysen and Shockman, 1973; Daneo-Moore and Shockman, 1977; Rogers et al., 1980; Shockman and Barrett, 1983; Doyle and Koch, 1987; Holtje and Tuomanen, 1991, for reviews). This process is now known to be initiated by the action of endogenous enzymes that hydrolyze specific bonds in the insoluble, osmotically protective, shape-maintaining and essential, peptidoglycan (murein) polymer of the bacterial cell wall. Hydrolysis of a sufficient number of bonds in a restricted area of the two- or three-dimensional peptidoglycan network, or a larger number of bonds in a broader area of the wall, creates a weak spot (or a generalized weakness in the structure), so that the wall can no longer protect the protoplast from its own internal osmotic pressure, so that the protoplast then explodes out through the weakened structure. Endogenous enzymes are now known that can hydrolyze virtually every bond in the peptidoglycan including: (i) glycosidases and transglycosidases, such as N-acetylmuramoylhydrolases (muramidases) and N-acetylglucosaminidases, (ii) N-acetylmuramoyl-L-alanine amidases, and (iii) various peptidases and transpeptidases, including DD- and LD-carboxypeptidases (Rogers et al., 1980).

A truncated Tn916-like element in a clinical isolate of Enterococcus faecium.

A 58.7-kb nonconjugative plasmid (pKQ1) previously reported in a clinical isolate of Enterococcus faecium was found to contain both a tetM and an erythromycin resistance (erm) determinant. The plasmid contained a region homologous to the A, F, H, and G HincII fragments of Tn916. However, the 4.8-kb B fragment of Tn916 which contained the tetM determinant was replaced by a 7.3-kb fragment, and the 3.6-kb HincII C fragment of Tn916 was missing. An element homologous to Tn917 was juxtaposed to the truncated Tn916-like element. The Tn917-like element was similar in size to the erm transposon Tn917 as determined by a ClaI restriction digest which spanned approximately 99% of the transposon. When Bacillus subtilis or Streptococcus sanguis were transformed with pKQ1, no zygotically induced transposition of the tetM element was detected. Similarly no transposition of the Tn917-like element was detected.