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Archives of Microbiology (v.189, #1)

Populations under microevolutionary scrutiny: what will we gain? by Johannes Sikorski (pp. 1-5).

Understanding the evolution of biodiversity and the function of biological systems are burning and linked questions in biology. Evolution of biodiversity begins at the level of microevolution, with the differentiation of individuals in populations. The study of this process splits into two conceptually different approaches (1) the concept of functional biology of testing hypothesis by precisely controlled and forward-directed experiments (digital and experimental evolution), and (2) the concept of a theory-based historical narrative (testing hypothesis on events in the past for their suitability to best explain the present). Here, I discuss and emphasize the benefits of the study of natural bacterial populations for a deeper understanding of prokaryotic biology. Also, I adress current problems in taxonomy at the ‘species’ level which obviously need discussion and clarification. I exemplify this with a natural model population for such studies, Bacillus simplex from “Evolution Canyon”, Israel.

Keywords: Microevolution; Species concept; Evolutionary lineage; Intraspecies diversity; Bacillus simplex ; Evolution Canyon

Entry and intracellular location of Mycoplasma hominis in Trichomonas vaginalis by Ricardo Gomes Vancini; Marlene Benchimol (pp. 7-18).

The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. The coexistence of different sexually transmitted diseases in the same individual is very common, such as vaginal infections by T. vaginalis in association with Mycoplasma fermentans or Mycoplasma hominis. However, the consequences and behavior of mycoplasma during trichomonad infections are virtually unknown. This study was undertaken to elucidate whether mycoplasmas enter and leave trichomonad cells and if so how. M. hominis was analyzed in different trichomonad isolates and the process of internalization and the pathway within the parasite was studied. Parasites naturally and experimentally infected with mycoplasmas were used and transmission electron microscopy, cytochemistry and PCR analyses were performed. The results show that: (1) M. hominis enters T. vaginalis cells by endocytosis; (2) some mycoplasmas use a terminal polar tip as anchor to the trichomonad plasma membrane; (3) some trichomonad isolates are able to digest mycoplasmas, mainly when the trichomonads are experimentally infected; (4) some fresh virulent isolates are able to maintain mycoplasmas as cohabitants in the cell’s interior; (5) some mycoplasmas are able to escape from the vacuole to the trichomonad cytosol, and trichomonad plasma membrane budding suggested that mycoplasmas could leave the parasite cell.

Keywords: Trichomonas vaginalis ; Mycoplasmas; Mycoplasma hominis

Preliminary phenotypic characterization of newly isolated halophilic microorganisms by footprinting: a rapid metabolome analysis by Berna Sarıyar-Akbulut; Aydan Salman-Dilgimen; Selim Ceylan; Sinem Perk; Akın A. Denizci; Dilek Kazan (pp. 19-26).

The emerging need for rapid screening and identification methods for microbiological purposes necessitates the combined uses of high-tech instruments. In this work, electrospray ionization mass spectrometry was used to visualize the relation of ten newly isolated moderately halophilic microorganisms, to Halomonas salina DSMZ 5928 and Halomonas halophila DSMZ 4770. The method was based on the global analysis of the metabolites in culture media and is termed as metabolic footprinting. Since it was not possible to gain insight into the similarities solely based on the visual inspection of the chromatograms, principal component (PC) analysis was applied on the data. Three PCs alone were able to explain 99% of the information in the data set. The score plots revealed the relation of the new isolates to the two type strains whereas the loading plots gave important clues on the significant ions responsible for the observed clustering. Loading plots also indicated inversely correlated ions that give clues on differing metabolic pathways. The work described here offers a potentially useful way for preliminary rapid phenotypic characterization of new and closely related isolates and a method for screening of similar microorganisms for different and valuable secondary metabolites.

Keywords: Footprinting; ESI-MS; Principal component analysis; Halomonas sp.; Phenotype

Growth phase-associated changes in the transcriptome and proteome of Streptococcus pyogenes by Michelle A. Chaussee; Alexander V. Dmitriev; Eduardo A. Callegari; Michael S. Chaussee (pp. 27-41).

Streptococcus pyogenes is responsible for approximately 500,000 deaths each year worldwide. Many of the associated virulence factors are expressed in a growth phase-dependent manner. To identify growth phase-associated changes in expression on a genomescale, the exponential and stationary phase transcriptomes and proteomes of S. pyogenes strain NZ131 (serotype M49) were compared by using Affymetrix NimbleExpress gene chips and two-dimensional gel electrophoresis. At the transcript level, the expression of 689 genes, representing approximately 40% of the chromosome, differed by twofold or more between the two growth phases. The majority of transcripts that were more abundant in the early-stationary phase encoded proteins involved in energy conversion, transport, and metabolism. At the protein level, an average of 527 and 403 protein spots were detected in the exponential and stationary phases of growth, respectively. Tandem mass spectrometry was used to identify 172 protein spots, 128 of which were growth phase regulated. Enzymes involved in glycolysis and pyruvate metabolism and several stress-responsive proteins were more abundant in the stationary phase of growth. Overall, the results identified growth phase-regulated genes in strain NZ131 and revealed significant post-transcriptional complexity associated with pathogen adaptation to the stationary phase of growth.

Keywords: 2-DE; Proteomics; Transcriptome; Growth phase; Group A streptococcus; Bacterial pathogenesis

Alkaline phosphatase as a reporter of σ^S levels and rpoS polymorphisms in different E. coli strains by Beny Spira; Thomas Ferenci (pp. 43-47).

σ^S is responsible for the transcriptional regulation of genes related to protection against stresses and bacterial survival and it accumulates in the cell under conditions of stress, such as nutrient limitation. An increase in the levels of σ^S causes a reduction in the expression of genes that are transcribed by RNA polymerase associated with the principal sigma factor, σ⁷⁰. phoA, that encodes alkaline phosphatase (AP) is expressed under phosphate shortage conditions, and is also repressed by σ^S. Here we show that in a Pi-limited chemostat, accumulation of rpoS mutations is proportional to the intrinsic level of σ^S in the cells. Acquisition of mutations in rpoS relieves repression of the PHO genes. We also devised a non-destructive method based on the rpoS effect on AP that differentiates between rpoS ⁺ and rpoS mutants, as well as between high and low-σ^S producers. Using this method, we provide evidence that σ^S contributes to the repression of AP under conditions of Pi excess and that AP variation among different strains is at least partly due to intrinsic variation in σ^S levels. Consequently, a simple and non-destructive AP assay can be employed to differentiate between strains expressing different levels of σ^S on agar plates.

Keywords: rpoS ; Sigma factor; Alkaline phosphatase; PHO regulon

Growth, osmotic downshock resistance and differentiation of Bacillus subtilis strains lacking mechanosensitive channels by Paul G. Wahome; Peter Setlow (pp. 49-58).

Previous work has shown that the mechanosensitive (MS) channel of large conductance (MscL) is essential for preventing lysis of Bacillus subtilis log phase cells upon a rapid, severe osmotic downshock. Growing cells of B. subtilis strains lacking MscL and one or more putative MS channel proteins of small conductance (YhdY, YkuT and YfkC) showed even higher sensitivity to an osmotic downshock. The effect was greatest for a strain lacking MscL and YkuT, and a strain lacking all four MS channel proteins had a similar phenotype. These defects were complemented by expression of either MscL or YkuT in trans. All MS channel mutant strains ultimately became resistant to osmotic downshock in stationary phase but at varying times, with mscL ykuT strains taking the longest time to become resistant. Expression of β-galactosidase from gene fusions to lacZ showed modest expression of ykuT and lower levels of expression of yhdY and yfkC when strains were grown in medium containing high salt. Sporulation of all MS channel mutant strains was normal, and the mutant spores germinated normally with l-alanine or dodecylamine.

Keywords: Bacillus ; Mechanosensitive channels; Osmoregulation; Osmotic downshock; Sporulation; Spore germination

In vitro studies indicate a quinone is involved in bacterial Mn(II) oxidation by Hope A. Johnson; Bradley M. Tebo (pp. 59-69).

Manganese(II)-oxidizing bacteria play an integral role in the cycling of Mn as well as other metals and organics. Prior work with Mn(II)-oxidizing bacteria suggested that Mn(II) oxidation involves a multicopper oxidase, but whether this enzyme directly catalyzes Mn(II) oxidation is unknown. For a clearer understanding of Mn(II) oxidation, we have undertaken biochemical studies in the model marine α-proteobacterium, Erythrobacter sp. strain SD21. The optimum pH for Mn(II)-oxidizing activity was 8.0 with a specific activity of 2.5 nmol × min⁻¹ × mg⁻¹ and a K _m = 204 μM. The activity was soluble suggesting a cytoplasmic or periplasmic protein. Mn(III) was an intermediate in the oxidation of Mn(II) and likely the primary product of enzymatic oxidation. The activity was stimulated by pyrroloquinoline quinone (PQQ), NAD⁺, and calcium but not by copper. In addition, PQQ rescued Pseudomonas putida MnB1 non Mn(II)-oxidizing mutants with insertions in the anthranilate synthase gene. The substrate and product of anthranilate synthase are intermediates in various quinone biosyntheses. Partially purified Mn(II) oxidase was enriched in quinones and had a UV/VIS absorption spectrum similar to a known quinone requiring enzyme but not to multicopper oxidases. These studies suggest that quinones may play an integral role in bacterial Mn(II) oxidation.

Keywords: Manganese oxidation; PQQ; Erythrobacter; Multicopper oxidase

Properties of Bacillus anthracis spores prepared under various environmental conditions by Renu B. Baweja; Mohd S. Zaman; Abid R. Mattoo; Kirti Sharma; Vishwas Tripathi; Anita Aggarwal; Gyanendra P. Dubey; Raj K. Kurupati; Munia Ganguli; N. K. Chaudhury; Somdutta Sen; Taposh K. Das; Wasudev N. Gade; Yogendra Singh (pp. 71-79).

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.

Keywords: Bacillus anthracis ; Spore sensitivity; Sporulation; Germination; Dipicolinic acid

Regulation of cuticle-degrading subtilisin proteases from the entomopathogenic fungi, Lecanicillium spp: implications for host specificity by Natasha J. Bye; A. Keith Charnley (pp. 81-92).

The ability to produce cuticle-degrading proteases to facilitate host penetration does not distinguish per se entomopathogenic fungi from saprophytes. However, adapted pathogens may produce host-protein specific enzymes in response to cues. This possibility prompted an investigation of the regulation of isoforms of the subtilisin Pr1-like proteases from five aphid-pathogenic isolates of Lecanicillium spp. Significant differences were found in substrate specificity and regulation of Pr1-like proteases between isoforms of the same isolate and between different isolates. For example, the pI 8.6 isoform from KV71 was considerably more active against aphid than locust cuticle and was induced specifically by N-acetylglucosamine (NAG). Isoform pI 9.1 from the same isolate was only produced on insect cuticle while most other isoforms were more prominent on chitin containing substrates but not induced by NAG. The ability to regulate isoforms independently may allow production at critical points in host penetration. Appearance of proteases (not subtilisins) with pI 4.2 and 4.4 only on aphid cuticle was a possible link with host specificity of KV71. The absence of C or N metabolite repression in subtilisins from KV42 is unusual for pathogen proteases and may help to account for differences in virulence strategy between aphid-pathogenic isolates of Lecanicillium longisporum (unpublished data).

Keywords: Protease; Regulation; Fungus; Pathogen; Specificity; Lecanicillium ; Verticillium ; Insect; Aphid

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Archives of Microbiology (v.189, #1)