Richard A. Morton

Recent Developments in the Molecular Biology of Extremely Halophilic Bacteria

(1978). Recent Developments in the Molecular Biology of Extremely Halophilic Bacteria. CRC Critical Reviews in Microbiology: Vol. 6, No. 2, pp. 151-206.

Expression of the Escherichia coli NRZ nitrate reductase is highly growth phase dependent and is controlled by RpoS, the alternative vegetative sigma factor

In the absence of oxygen, many bacteria preferentially use nitrate as a terminal electron acceptor for anaerobic respiration. In Escherichia coli, there are two membrane‐bound, differentially regulated nitrate reductases. While the physiological basis for this metabolic redundancy is not completely understood, during exponential growth, synthesis of NRA is greatly induced by anaerobiosis plus nitrate, whereas NRZ is expressed at a low level that is not influenced by anaerobiosis or nitrate. In the course of identifying genes controlled by the stationary phase regulatory factor RpoS (σs), we found that the expression of NRZ is induced during entry into stationary phase and highly dependent on this alternative sigma factor. Expression studies, using operon fusions and nitrate reductase assays, revealed that the NRZ operon is controlled mainly at the level of transcription and is induced 10‐fold at the onset of stationary phase in rich media. Consistent with previous reports of RpoS expression, the RpoS dependency of NRZ in minimal media was very high (several hundredfold). We also observed a fivefold stationary phase induction of NRZ in an rpoS background, indicating that other regulatory factors, besides RpoS, are probably involved in transcriptional control of NRZ. The RpoS dependence of NRZ expression was confirmed by Northern analyses using RNA extracted from wild‐type and rpoS− strains sampled in exponential and stationary phase. In toto, these data indicate that RpoS‐mediated regulation of NRZ may be an important physiological adaptation that allows the cell to use nitrate under stress‐associated conditions.

Genetic and biochemical properties of an alkaline phosphatase PhoX family protein found in many bacteria

We report on the biochemical, phylogenetic and genetic regulation of PhoX, the major alkaline phosphatase protein from the soil bacterium Sinorhizobium meliloti. The protein is shown to be a member of a recently identified family of PhoX alkaline phosphatase proteins that is distinct from the well-characterized PhoA family. The mature S. meliloti PhoX protein is located in the periplasm and lacks a 76-amino-acid N-terminal Tat signal peptide. Its phosphatase activity was stimulated by Ca(+2) and was optimal at pH 9-11. Except for phytic acid and phosphatidic acid, the enzyme was active against a wide range of phosphorylated substrates (77 nucleotides, phosphorylated carbohydrates and amino acids) and thus exhibited low substrate specificity for C-O-P bonds. No C-P bond substrate was dephosphorylated while the protein was active with two of six phosphoramidate substrates (N-P bond) tested. Sinorhizobium meliloti phoX was induced when cells were starved for phosphorous and the induction was dependent on the PhoB-regulatory protein. We demonstrate by in vitro analysis that PhoB protein binds to two tandem 22 nt PhoB binding sites located 64-21 nt upstream from the phoX transcription start site. Analysis of 95 PhoX orthologues from diverse bacteria revealed two distinct phylogenetic groups of PhoX proteins. The two groups differed in having a conserved glycine (PhoX-I) or asparagine (PhoX-II) next to their putative catalytic Ca(+2) binding site. Analysis of the phoX promoter regions from many of these bacteria also revealed the presence of PhoB binding sites. Alkaline phosphatase proteins of either the PhoX or PhoA family (but rarely both) are found in many bacteria, thus it appears that these are functionally equivalent.

Control of hydroxyproline catabolism in Sinorhizobium meliloti

Hydroxyproline (Hyp) in decaying organic matter is a rich source of carbon and nitrogen for microorganisms. A bacterial pathway for Hyp catabolism is known; however, genes and function relationships are not established. In the pathway, trans‐4‐hydroxy‐l‐proline (4‐l‐Hyp) is epimerized to cis‐4‐hydroxy‐d‐proline (4‐d‐Hyp), and then, in three enzymatic reactions, the d‐isomer is converted via Δ‐pyrroline‐4‐hydroxy‐2‐carboxylate (HPC) and α‐ketoglutarate semialdehyde (KGSA) to α‐ketoglutarate (KG). Here a transcriptional analysis of cells growing on 4‐l‐Hyp, and the regulation and functions of genes from a Hyp catabolism locus of the legume endosymbiont Sinorhizobium meliloti are reported. Fourteen hydroxyproline catabolism genes (hyp), in five transcripts hypR, hypD, hypH, hypST and hypMNPQO(RE)XYZ, were negatively regulated by hypR. hypRE was shown to encode 4‐hydroxyproline 2‐epimerase and a hypRE mutant grew with 4‐d‐Hyp but not 4‐l‐Hyp. hypO, hypD and hypH are predicted to encode 4‐d‐Hyp oxidase, HPC deaminase and α‐KGSA dehydrogenase respectively. The functions for hypS, hypT, hypX, hypY and hypZ remain to be determined. The data suggest 4‐Hyp is converted to the tricarboxylic acid cycle intermediate α‐ketoglutarate via the pathway established biochemically for Pseudomonas. This report describes the first molecular characterization of a Hyp catabolism locus.

Separating the effects of mutation and selection in producing DNA skew in bacterial chromosomes

BackgroundMany bacterial chromosomes display nucleotide asymmetry, or skew, between the leading and lagging strands of replication. Mutational differences between these strands result in an overall pattern of skew that is centered about the origin of replication. Such a pattern could also arise from selection coupled with a bias for genes coded on the leading strand. The relative contributions of selection and mutation in producing compositional skew are largely unknown.ResultsWe describe a model to quantify the contribution of mutational differences between the leading and lagging strands in producing replication-induced skew. When the origin and terminus of replication are known, the model can be used to estimate the relative accumulation of G over C and of A over T on the leading strand due to replication effects in a chromosome with bidirectional replication arms. The model may also be implemented in a maximum likelihood framework to estimate the locations of origin and terminus. We find that our estimations for the origin and terminus agree very well with the location of genes that are thought to be associated with the replication origin. This indicates that our model provides an accurate, objective method of determining the replication arms and also provides support for the hypothesis that these genes represent an ancestral cluster of origin-associated genes.ConclusionThe model has several advantages over other methods of analyzing genome skew. First, it quantifies the role of mutation in generating skew so that its effect on composition, for example codon bias, can be assessed. Second, it provides an objective method for locating origin and terminus, one that is based on chromosome-wide accumulation of leading vs lagging strand nucleotide differences. Finally, the model has the potential to be utilized in a maximum likelihood framework in order to analyze the effect of chromosome rearrangements on nucleotide composition.

A reanalysis of protein polymorphism in Drosophila melanogaster, D. simulans, D. sechellia and D. mauritiana: effects of population size and selection

Comparison of synonymous and nonsynonymous variation/substitution within and between species at individual genes has become a widely used general approach to detect the effect of selection versus drift. The sibling species group comprised of two cosmopolitan (Drosophila melanogaster and Drosophila simulans) and two island (Drosophila mauritiana and Drosophila sechellia) species has become a model system for such studies. In the present study we reanalyzed the pattern of protein variation in these species, and the results were compared against the patterns of nucleotide variation obtained from the literature, mostly available for melanogaster and simulans. We have mainly focused on the contrasting patterns of variation between the cosmopolitan pair. The results can be summarized as follows: (1) As expected the island species D. mauritiana and D. sechellia showed much less variation than the cosmopolitan species D. melanogaster and D. simulans. (2) The chromosome 2 showed significantly less variation than chromosome 3 and X in all four species which may indicate effects of past selective sweeps. (3) In contrast to its overall low variation, D. mauritiana showed highest variation for X-linked loci which may indicate introgression from its sibling, D. simulans. (4) An average population of D. simulans was as heterozygous as that of D. melanogaster (14.4% v.s. 13.9%) but the difference was large and significant when considering only polymorphic loci (37.2% v.s. 26.1%). (5) The species-wise pooled populations of these two species showed similar results (all loci = 18.3% v.s. 20.0%, polymorphic loci = 47.2% v.s. 37.6%). (6) An average population of D. simulans had more low-frequency alleles than D. melanogaster, and the D. simulans alleles were found widely distributed in all populations whereas the D. melanogaster alleles were limited to local populations. As a results of this, pooled populations of D. melanogaster showed more polymorphic loci than those of D. simulans (48.0% v.s. 32.0%) but the difference was reduced when the comparison was made on the basis of an average population (29.1% v.s. 21.4%). (7) While the allele frequency distributions within populations were nonsignificant in both D. melanogaster and D. simulans, melanogaster had fewer than simulans, but more than expected from the neutral theory, low frequency alleles. (8) Diallelic loci with the second allele with a frequency less than 20% had similar frequencies in all four species but those with the second allele with a frequency higher than 20% were limited to only melanogaster: the latter group of loci have clinal (latitudinal) patterns of variation indicative of balancing selection. (9) The comparison of D. simulans/D. melanogaster protein variation gave a ratio of 1.04 for all loci and 1.42 for polymorphic loci, against a ratio of approximately 2-fold difference for silent nucleotide sites. This suggests that the species ratios of protein and silent nucleotide polymorphism are too close to call for selective difference between silent and allozyme variation in D. simulans. In conclusion, the contrasting levels of allozyme polymorphism, distribution of rare alleles, number of diallelic loci and the patterns of geographic differentiation between the two species suggest the role of natural selection in D. melanogaster, and of possibly ancient population structure and recent worldwide migration in D. simulans. Population size differences alone are insufficient as an explanation for the patterns of variation between these two species.

Mate Choice and the Origin of Menopause

Human menopause is an unsolved evolutionary puzzle, and relationships among the factors that produced it remain understood poorly. Classic theory, involving a one-sex (female) model of human demography, suggests that genes imparting deleterious effects on post-reproductive survival will accumulate. Thus, a ‘death barrier’ should emerge beyond the maximum age for female reproduction. Under this scenario, few women would experience menopause (decreased fertility with continued survival) because few would survive much longer than they reproduced. However, no death barrier is observed in human populations. Subsequent theoretical research has shown that two-sex models, including male fertility at older ages, avoid the death barrier. Here we use a stochastic, two-sex computational model implemented by computer simulation to show how male mating preference for younger females could lead to the accumulation of mutations deleterious to female fertility and thus produce a menopausal period. Our model requires neither the initial assumption of a decline in older female fertility nor the effects of inclusive fitness through which older, non-reproducing women assist in the reproductive efforts of younger women. Our model helps to explain why such effects, observed in many societies, may be insufficient factors in elucidating the origin of menopause.

Loss of malic enzymes leads to metabolic imbalance and altered levels of trehalose and putrescine in the bacterium Sinorhizobium meliloti.

BackgroundMalic enzymes decarboxylate the tricarboxylic acid (TCA) cycle intermediate malate to the glycolytic end-product pyruvate and are well positioned to regulate metabolic flux in central carbon metabolism. Despite the wide distribution of these enzymes, their biological roles are unclear in part because the reaction catalyzed by these enzymes can be by-passed by other pathways. The N2-fixing alfalfa symbiont Sinorhizobium meliloti contains both a NAD(P)-malic enzyme (DME) and a separate NADP-malic enzyme (TME) and to help understand the role of these enzymes, we investigated growth, metabolomic, and transcriptional consequences resulting from loss of these enzymes in free-living cells.ResultsLoss of DME, TME, or both enzymes had no effect on growth with the glycolytic substrate, glucose. In contrast, the dme mutants, but not tme, grew slowly on the gluconeogenic substrate succinate and this slow growth was further reduced upon the addition of glucose. The dme mutant strains incubated with succinate accumulated trehalose and hexose sugar phosphates, secreted malate, and relative to wild-type, these cells had moderately increased transcription of genes involved in gluconeogenesis and pathways that divert metabolites away from the TCA cycle. While tme mutant cells grew at the same rate as wild-type on succinate, they accumulated the compatible solute putrescine.ConclusionsNAD(P)-malic enzyme (DME) of S. meliloti is required for efficient metabolism of succinate via the TCA cycle. In dme mutants utilizing succinate, malate accumulates and is excreted and these cells appear to increase metabolite flow via gluconeogenesis with a resulting increase in the levels of hexose-6-phosphates and trehalose. For cells utilizing succinate, TME activity alone appeared to be insufficient to produce the levels of pyruvate required for efficient TCA cycle metabolism. Putrescine was found to accumulate in tme cells growing with succinate, and whether this is related to altered levels of NADPH requires further investigation.

Integrating Sinorhizobium meliloti Genomics: An Expression Library of the Genome

through Sustainable Agriculture.

Phosphate-Dependent Gene Expression in Free-Living and Symbiotic Sinorhizobium meliloti

Growth of Sinorhizobium meliloti under Pi-deficient conditions induces many genes involved in both P acquisition and in a general response to P-limitation. PhoB regulates genes induced by P limitation by binding to Pho-regulon promoters with a Pho-Box. PhoR is the histidine-protein kinase that activates PhoB by phosphorylation. Pi-PhoB interacts with the 70 subunit of RNA polymerase. Using a whole genome in silico analysis of known Pho-box sequences, we identified 34 genes as putative members of the Pho regulon. Thirty-one genes were induced and three repressed by P-starvation in a PhoB-dependent manner (Yuan et al., 2006a; Krol and Becker, 2004), including genes with no obvious relation to P-metabolism, e.g., for iron transporters and katA, which encodes catalase and has two independent promoters regulated by OxyR and PhoB (Yuan et al., 2005). Pho regulon members in S.meliloti include the pstSCAB and phoCDET operons, encoding ABC-type high-affinity Pi-transporters (Bardin et al., 1998; Voegele et al., 1997; Yuan et al., 2006b), and the orfA-pit operon, which encodes a low-affinity Pi-transport system negatively regulated by PhoB (Bardin et al., 1998). Expression of these S. meliloti Pi transporters in root nodules, using promoter-gusA fusion plasmids in wild type and phoB S. meliloti, showed that the orfA-pit system was highly expressed in nodules, whereas very little phoCDET or pstSCAB expression was detected in either strain. There was no zone-specific expression of orfA-pit::gusA, and neither phoC nor pstS expression was detected in any nodule zone. These and other (Yuan et al., 2005, 2006b) data suggest that bacteroid metabolism in alfalfa nodules is not Pi limited.