Species Delimitation, Phylogenetic Relationships, and Temporal Divergence Model in the Genus Aeromonas.

The definition of species boundaries constitutes an important challenge in biodiversity studies. In this work we applied the Generalized Mixed Yule Coalescent (GMYC) method, which determines a divergence threshold to delimit species in a phylogenetic tree. Based on the tree branching pattern, the analysis fixes the transition threshold between speciation and the coalescent process associated with the intra-species diversification. This approach has been widely used to delineate eukaryote species and establish their diversification process from sequence data. Nevertheless, there are few examples in which this analysis has been applied to a bacterial population. Although the GMYC method was originally designed to assume a constant (Yule) model of diversification at between-species level, it was later evaluated simulating other conditions. Our aim was therefore to determine the species delineation in Aeromonas using the GMYC method and asses which model best explains the speciation process in this bacterial genus. The application of the GMYC method allowed us to clearly delineate the Aeromonas species boundaries, even in the controversial groups, such as the A. veronii or A. media species complexes.

Evolutionary Roots and Diversification of the Genus Aeromonas.

Despite the importance of diversification rates in the study of prokaryote evolution, they have not been quantitatively assessed for the majority of microorganism taxa. The investigation of evolutionary patterns in prokaryotes constitutes a challenge due to a very scarce fossil record, limited morphological differentiation and frequently complex taxonomic relationships, which make even species recognition difficult. Although the speciation models and speciation rates in eukaryotes have traditionally been established by analyzing the fossil record data, this is frequently incomplete, and not always available. More recently, several methods based on molecular sequence data have been developed to estimate speciation and extinction rates from phylogenies reconstructed from contemporary taxa. In this work, we determined the divergence time and temporal diversification of the genus Aeromonas by applying these methods widely used with eukaryotic taxa. Our analysis involved 150 Aeromonas strains using the concatenated sequences of two housekeeping genes (approximately 2,000 bp). Dating and diversification model analyses were performed using two different approaches: obtaining the consensus sequence from the concatenated sequences corresponding to all the strains belonging to the same species, or generating the species tree from multiple alignments of each gene. We used BEAST to perform a Bayesian analysis to estimate both the phylogeny and the divergence times. A global molecular clock cannot be assumed for any gene. From the chronograms obtained, we carried out a diversification analysis using several approaches. The results suggest that the genus Aeromonas began to diverge approximately 250 millions of years (Ma) ago. All methods used to determine Aeromonas diversification gave similar results, suggesting that the speciation process in this bacterial genus followed a rate-constant (Yule) diversification model, although there is a small probability that a slight deceleration occurred in recent times. We also determined the constant of diversification (λ) values, which in all cases were very similar, about 0.01 species/Ma, a value clearly lower than those described for different eukaryotes.

Buffering capacity and H+ membrane conductance of gram-negative bacteria.

Buffering capacity and membrane H+ conductance were examined in seven Gram-negative species: Aquaspirillum serpens, Pseudomonas aeruginosa, Alcaligenes faecalis, Escherichia coli, Salmonella typhimurium, Proteus mirabilis and Aeromonas hydrophila. All strains of Enterobacteriaceae studied here showed a decrease in both parameters as the external pH increased, over the pH range studied. The other four species presented an increase in buffering capacity and membrane conductance to protons as the external pH increased from 5.5 to 7.0.

Modification by analgesics of the susceptibility to antibiotics in Serratia marcescens.

The influence of analgesics at low concentrations on permeability and beta-lactamase expression have been studied "in vitro". The effect of these drugs on major outer membrane proteins and the lipopolysaccharide was evaluated. Acetylsalicylate and paracetamol induced modifications in susceptibility to a large variety of antibiotics when tested at therapeutic concentrations. The results suggested that when analgesics and antibiotics are administered simultaneously, the interaction between both kinds of drugs can alter the response of microorganisms to antibiotics.

Buffering capacity and membrane H+ conductance of Halobacterium halobium.

Buffering capacity and membrane H+ conductance were measured in Halobacterium halobium suspensions in the light and in the dark over a wide range of external pH. The values of both variables for this archaeobacterium were significantly higher than those found for eubacteria in other reports. It appears from our results that the special chemical composition of the cell envelope and the movement of ions, mainly protons, may influence the magnitude of the buffering power and the H+ membrane conductance of these cells.

[Effect of glucose concentration on the biosynthesis of prodigiosin by serratia marcescens (author's transl)]. / Efecto de la concentración de glucosa en la biosíntesis de prodigiosina por Serratia marcescens.

Serratia marcescens is an enterobacteria which produces a characteristic red pigment denominated prodigiosin. To study the effect of glucose on the kinetics of this secondary metabolite, cultures of Serratia marcescens S10 were incubated at 30 degrees C in the mineral medium GL, with glucose (2 g/l) as the carbon source. Prodigiosin production in relation to glucose consumption is studied, and parallel-wise, the effect of various concentrations of glucose on prodigiosin production. The kinetics data show the close correlation between glucose consumption and the synthesis of prodigiosin. This substrate inhibits the synthesis of pigment in cultures grown on solid medium GL with concentrations of glucose up to 15 g/l.

Buffering capacity and membrane H+ conductance of neutrophilic and alkalophilic gram-positive bacteria.

Buffering capacity and membrane H+ conductance were examined in three gram-positive bacteria, Staphylococcus aureus, Bacillus subtilis, and Bacillus alcalophilus. An acid pulse technique was used to measure both parameters. The buffering capacity and membrane H+ conductance of B. alcalophilus are influenced by the pH of the medium and the culture conditions. Suspensions of B. alcalophilus cells from both H. A. medium and L-malate medium cultures grown at pH 10.5 exhibited higher values for these parameters than cells grown at pH 8.5. B. alcalophilus grown aerobically had a lower buffering capacity and a lower membrane conductance for protons than the neutrophilic bacteria S. aureus and B. subtilis. Fermenting cells exhibited significantly higher values for both variables than respiring cells.

The effect of pH on prodigiosin production by non-proliferating cells of Serratia marcescens.

The synthesis of prodigiosin by non-proliferating cells of Serratia marcescens was examined at various pH values between 5.5 and 9.5. During incubation in unbuffered medium, pH changed and prodigiosin production was similar regardless of the initial pH. Variations in pigment production were noted when buffers were employed in cultures of non-proliferating cells. The optimum pH for prodigiosin production was 8.0-8.5. Proline oxidase was also measured. The results suggest that the effect of pH may be related to the amount of proline which can be incorporated into prodigiosin.

Induction of Yellow Pigmentation in Serratia marcescens.

The appearance of yellow pigmentation in nonpigmented strains of Serratia sp. has been demonstrated to be due to the production of a muconic acid, 2-hydroxy-5-carboxymethylmuconic acid semialdehyde. The 3,4-dihydroxyphenylacetate 2,3-dioxygenase responsible for the synthesis of this muconic acid was induced in all strains tested. Another muconic acid, the beta-cis-cis-carboxymuconic acid, could also be synthesized from 3,4-dihydroxybenzoate, but this product was not colored. Mutants that were unable to grow on tyrosine and produced yellow pigment were isolated from nonpigmented strains. These mutants had properties similar to those of the yellow-pigmented strains. The ability to produce pigment may be more widespread among Serratia marcescens strains than is currently known.

Buffering Capacity of Pigmented and Nonpigmented Strains of Serratia marcescens.

The pigmented strain Serratia marcescens ATCC 274 had a higher buffering capacity and a higher membrane H conductance than S. marcescens GP, a spontaneous nonpigmented mutant of ATCC 274. The data suggest that mutations which apparently affect only the synthesis of a secondary metabolite can modify buffering capacity and passive H conductance.

Analysis of microcalorimetric curves for bacterial identification.

A numeric method is suggested for the treatment of microcalorimetric curves of bacterial growth to provide a new tool for their automatic identification. In this method the microcalorimetric curves are searched against certain reference profiles (stored in a library) by means of a cross-correlation analysis and a parametric comparison. The matching between the new curve and each reference profile is evaluated by means of a specific identification coefficient which provides an objective criterion for the identification of each species. The reliability of the method is discussed.

Glucose-6-phosphate dehydrogenase alloenzymes and their relationship to pigmentation in Serratia marcescens.

A comparative study of environmental and clinical isolates of Serratia marcescens was undertaken with regard to glucose-6-phosphate dehydrogenase (G6PD) electrophoretic mobility and the production of prodigiosin. Two electromorphs of G6PD with electrophoretic mobilities of 0.22 and 0.30 were detected. G6PD electrophoretic type showed a good correlation with the ability to produce prodigiosin.

Isolation from urine of two Serratia marcescens strains excreting a diffusible yellow pigment.

Two bacterial strains excreting a yellow pigment were isolated from human urine and identified as Serratia marcescens. The pigment was produced in the late exponential and early stationary phases of growth. Minimal media supplemented with tyrosine, phenylalanine, 3,4-dihydroxyphenylacetate or tryptophan, as well as complex media, induced pigment production. UV-visible spectra of the extracted pigment had peaks characteristic of 2-hydroxy-5-carboxymethylmuconate semialdehyde, produced from meta-cleavage of 3,4-dihydroxyphenylacetate by the enzyme 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15). This enzyme was active when the bacteria were grown under conditions promoting pigment production. The kinetics and factors affecting pigment production are also reported.

Rapid extracellular acidification induced by glucose metabolism in non-proliferating cells of Serratia marcescens.

The addition of glucose or other sugars to resting cells of Serratia maurcescens induced rapid acidification of the extracellular medium. This acidification was due to the catabolism of sugars. The rate of acidification depended on the carbon source and its concentration. HPLC analysis of the supernatants demonstrated that the progressive fall in pH resulted from the rapid production of lactic, acetic, pyruvic and citric acids. Other microorganisms were tested for their ability to produce this rapid acidification of the medium. This study may provide a rapid and simple method for metabolism studies.

Brown pigmentation in Serratia marcescens cultures associated with tyrosine metabolism.

Serratia marcescens produced a brown pigment when grown in minimal medium in the presence of tyrosine and high concentrations of copper(II) ion. The pigment was not related to the melanin pigments, but was similar to the pigment produced by autooxidation and polymerization of 3,4-dihydroxyphenylacetate, which is synthesized in S. marcescens from tyrosine through the 3,4-dihydroxyphenylacetate catabolic pathway. The enzymes of this pathway were induced under pigment production conditions; however, 3,4-dihydroxyphenylacetate 2,3-dioxygenase remained at low activity levels, permitting the accumulation and excretion of the substrate. Mutants unable to use tyrosine as a sole carbon and energy source were able to produce brown pigments only if the step blocked by the mutation was after the synthesis of 3,4-dihydroxyphenylacetate. The ability to produce brown pigments was common to all the S. marcescens strains tested.

Lipopolysaccharide is the receptor for kappa phage in Serratia marcescens.

Kappa phage active on Serratia marcescens can form plaques on white and red strains with identical efficiencies. To identify the kappa phage receptor, the inactivation of the phage was studied after incubation with several bacterial subcellular fractions. The experiments demonstrated that kappa phage adsorbs to outer membrane fractions of susceptible cells. Proteinase K did not affect the rate of inactivation. Lipopolysaccharide proved to be the primary receptor for kappa phage. Prodigiosin content of the lipopolysaccharide fraction was low.

Discriminant analysis of microcalorimetric data of bacterial growth.

In this work a bacterial classification method based on the discriminant analysis of the microcalorimetric data provided by the growth power-time (p-t) curves is developed. This method is applied to classify several species of Enterobacteria of different origins, and the results are compared with those obtained by conventional techniques. The proposed analysis allows us to classify bacteria into species and discriminate among strains of the same species. The classification is carried out using one run of each isolate after standardization of inocula and growth conditions. The discrimination power of available microcalorimetric data is also discussed, and the most discriminant set of data is proposed as the input variables of the analysis. Finally, the advantages of microcalorimetry as a taxonomical technique are discussed.

Lipopolysaccharide recovery restores susceptibility levels towards beta-lactams in Serratia marcescens.

O-side chain-defective spontaneous mutants of Serratia marcescens, selected by phage resistance, showed lower MICs against various beta-lactams than did their parental strains. The recovery of their ability to produce O-antigen restored the original MIC values, as well as phage susceptibility. The permeability coefficients of wild-type, O- mutants, and revertants, demonstrated that O-antigen modifies the permeability of antibiotics in S. marcescens.

Clonality of multidrug-resistant nontypeable strains of Haemophilus influenzae.

The genetic structure of a population of multidrug-resistant nontypeable (unencapsulated) Haemophilus influenzae strains isolated at a hospital in Barcelona, Spain, was investigated by using multilocus enzyme electrophoresis to determine the allelic variation in 15 structural loci. In our study we have also included some antimicrobial agent-susceptible strains isolated at the same hospital. All enzymes were polymorphic for two to eight electromorphs, and the analysis revealed 43 distinct electrophoretic types among the 44 isolates. The mean genetic diversity of the entire population was 0.55. Multilocus linkage disequilibrium analysis of the isolates revealed a strong association between alleles, suggesting little possibility of recombination. Furthermore, the dendrogram and the allele mismatch distribution are typical of a population with no extensive genetic mixing.