Low first-trimester hemoglobin and low birth weight, preterm birth and small for gestational age newborns.

OBJECTIVE: To examine the relationship between first-trimester hemoglobin (Hb) concentration and risk of low birth weight (LBW), preterm birth and small for gestational age (SGA). METHODS: Data were obtained from a population-based prenatal care program in China. A total of 88,149 women who delivered during 1995-2000 and had their Hb measured in the first trimester were selected as study subjects. RESULTS: The prevalence of anemia (Hb<110 g/L) was 22.1% in the first trimester. The risk of LBW, preterm birth and SGA was increased steadily with the decrease of first-trimester Hb concentration. After controlling for confounding factors, women with Hb 80-99 g/L had significantly higher risk for LBW (OR=1.44, 95% CI 1.17-1.78), preterm birth (OR=1.34, 95% CI 1.16-1.55) and SGA (OR=1.13, 95% CI 0.98-1.31) than women with Hb 100-119 g/L. No elevated risk was noted for women with Hb> or =120 g/L. CONCLUSION: Low first-trimester Hb concentration increases the risk of LBW, preterm birth and SGA.

Microbial nitrogen cycles: physiology, genomics and applications.

Genes and pathways involved in inorganic nitrogen cycles have been found in traditional as well as unusual microorganisms. These pathways or enzymes play a very important role in the adaptation or survival of these microorganisms under a variety of environmental conditions. Microbial nitrogen metabolism has industrial applications ranging from wastewater treatment to bioremediation and potential future use in biocatalysis for chemical production.

Biodegradation of 2,4,5-trichlorophenoxyacetic acid by Burkholderia cepacia strain AC1100: evolutionary insight.

Many microorganisms in nature have evolved new genes which encode catabolic enzymes specific for chlorinated aromatic substrates, allowing them to utilize these compounds as sole sources of carbon and energy. An understanding of the evolutionary mechanisms involved in the acquisition of such genes may facilitate the development of microorganisms with enhanced capabilities of degrading highly chlorinated recalcitrant compounds. A number of studies have been based on microorganisms isolated from the environment which utilize simple chlorinated substrates. In our laboratory, a selective technique was used to isolate microorganisms capable of degrading highly chlorinated compounds, such as 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), as sole sources of carbon and energy. This article summarizes the genetic and biochemical information obtained regarding the pathway of degradation, the mechanism of recruitment of new genes, and the organization of the degradative genes. In addition, we discuss the potential practical application of such microorganisms in the environment.

Diversity of oxygen and N-oxide regulation of nitrite reductases in denitrifying bacteria.

We examined alpha, beta and gamma Proteobacteria with Cu and heme-type dissimilatory nitrite reductases for patterns of nir regulation. Six of seven strains expressed nitrite reductase under aerobic growth conditions. In only one strain, G-179, was it stringently regulated by O2. Growth with NO-3 or NO-2 enhanced nitrite reductase production in four of seven strains under anaerobic growth conditions, but in only one strain, Pseudomonas aeruginosa PA01, under aerobic conditions. In this strain the nitrite reductase production was primarily regulated by an anr gene when grown under anaerobic conditions, but when grown under aerobic conditions it was regulated by both an anr gene and nitrogen oxide. Constitutive production of nitrite reductase was a common phenomenon rather than the exception among denitrifiers from the environment, which helps explain the prevalence of denitrifying enzymes in aerobic soils.

Applications of DNA microarrays in microbial systems.

DNA microarray technology allows a parallel analysis of RNA abundance and DNA homology for thousands of genes in a single experiment. Over the past few years, this powerful technology has been used to explore transcriptional profiles and genome differences for a variety of microorganisms, greatly facilitating our understanding of microbial metabolism. With the increasing availability of complete microbial genomes, DNA microarrays are becoming a common tool in many areas of microbial research, including microbial physiology, pathogenesis, epidemiology, ecology, phylogeny, pathway engineering and fermentation optimization.

Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide.

The algC gene from Pseudomonas aeruginosa has been shown to encode phosphomannomutase (PMM), an essential enzyme for biosynthesis of alginate and lipopolysaccharide (LPS). This gene was overexpressed under control of the tac promoter, and the enzyme was purified and its substrate specificity and metal ion effects were characterized. The enzyme was determined to be a monomer with a molecular mass of 50 kDa. The enzyme catalyzed the interconversion of mannose 1-phosphate (M1P) and mannose 6-phosphate, as well as that of glucose 1-phosphate (G1P) and glucose 6-phosphate. The apparent Km values for M1P and G1P were 17 and 22 microM, respectively. On the basis of Kcat/Km ratio, the catalytic efficiency for G1P was about twofold higher than that for M1P. PMM also catalyzed the conversion of ribose 1-phosphate and 2-deoxyglucose 6-phosphate to their corresponding isomers, although activities were much lower. Purified PMM/phosphoglucomutase (PGM) required Mg2+ for maximum activity; Mn2+ was the only other divalent metal that showed some activation. The presence of other divalent metals in addition to Mg2+ in the reaction inhibited the enzymatic activity. PMM and PGM activities could not be detected in nonmucoid algC mutant strain 8858 and in LPS-rough algC mutant strain AK1012, while they were present in the wild-type strains as well as in algC-complemented mutant strains. This evidence suggests that AlgC functions as PMM and PGM in vivo, converting phosphomannose and phosphoglucose in the biosynthesis of both alginate and LPS.

Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. strain G-179, which contains a copper nitrite reductase.

Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-). The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot). Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase. The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O). The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction. NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide. These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.

The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification.

Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria. Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined. To analyze the function of the periplasmic nitrate reductase in Pseudomonas sp. strain G-179, the nap gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis. Mutation in the nap region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor. No nitrate reduction activity was detected in the Nap- mutant, but that activity could be restored by complementation with the nap region. Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide. Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate. These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.

Nucleotide sequence and functional analysis of the genes encoding 2,4,5-trichlorophenoxyacetic acid oxygenase in Pseudomonas cepacia AC1100.

Pseudomonas cepacia AC1100 is able to use the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as the sole source of carbon and energy. One of the early steps in this pathway is the conversion of 2,4,5-T to 2,4,5-trichlorophenol (2,4,5-TCP). 2,4,5-TCP accumulates in the culture medium when AC1100 is grown in the presence of 2,4,5-T. A DNA region from the AC1100 genome has been subcloned as a 2.7-kb SstI-XbaI DNA fragment, which on transfer to Pseudomonas aeruginosa PAO1 allows the conversion of 2,4,5-T to 2,4,5-TCP. We have determined the directions of transcription of these genes as well as the complete nucleotide sequences of the genes and the number and sizes of the polypeptides synthesized by pulse-labeling experiments. This 2.7-kb DNA fragment encodes two polypeptides with calculated molecular masses of 51 and 18 kDa. Proteins of similar sizes were seen in the T7 pulse-labeling experiment in Escherichia coli. We have designated the genes for these proteins tftA1 (which encodes the 51-kDa protein) and tftA2 (which encodes the 18-kDa protein). TftA1 and TftA2 have strong amino acid sequence homology to BenA and BenB from the benzoate 1,2-dioxygenase system of Acinetobacter calcoaceticus, as well as to XylX and XylY from the toluate 1,2-dioxygenase system of Pseudomonas putida. The Pseudomonas aeruginosa PAO1 strain containing the 2.7-kb SstI-XbaI fragment was able to convert not only 2,4,5-T to 2,4,5-TCP but also 2,4-dichlorophenoxyacetic acid to 2,4-dichlorophenol and phenoxyacetate to phenol.

Mutants of Pseudomonas fluorescens deficient in dissimilatory nitrite reduction are also altered in nitric oxide reduction.

Five Tn5 mutants of Pseudomonas fluorescens AK-15 deficient in dissimilatory reduction of nitrite were isolated and characterized. Two insertions occurred inside the nitrite reductase structural gene (nirS) and resulted in no detectable nitrite reductase protein on a Western immunoblot. One mutant had Tn5 inserted inside nirC, the third gene in the same operon, and produced a defective nitrite reductase protein. Two other mutants had insertions outside of this nir operon and also produced defective proteins. All of the Nir- mutants characterized showed not only loss of nitrite reductase activity but also a significant decrease in nitric oxide reductase activity. When cells were incubated with 15NO in H2(18)O, about 25% of the oxygen found in nitrous oxide exchanged with H2O. The extent of exchange remained constant throughout the reaction, indicating the incorporation of 18O from H2(18)O reached equilibrium rapidly. In all nitrite reduction-deficient mutants, less than 4% of the 18O exchange was found, suggesting that the hydration and dehydration step was altered. These results indicate that the factors involved in dissimilatory reduction of nitrite influenced the subsequent NO reduction in this organism.

H218O isotope exchange studies on the mechanism of reduction of nitric oxide and nitrite to nitrous oxide by denitrifying bacteria. Evidence for an electrophilic nitrosyl during reduction of nitric oxide.

Reduction of NO and NO2-by whole cells of eight strains of denitrifying bacteria known to contain either heme cd1 or copper-containing nitrite reductases (NiRs) has been examined in the presence of H218O. All organisms containing heme cd1 NiRs exhibited relatively large extents of exchange between NO2- and H218O (39-100%), as monitored by the 18O content of product N2O. Organisms containing copper NiRs gave highly variable results, with Achromobacter cycloclastes and Pseudomonas aureofaciens exhibiting no 18O incorporation and Rhodopseudomonas sphaeroides and Alcaligenes entrophus exhibiting complete exchange between NO2- and H218O. Organisms containing heme cd1 NiRs exhibited significant but lower levels of exchange between NO and H218O than between NO2- and H218O, while organisms containing copper NiRs gave significantly higher amounts of 18O incorporation than observed for the heme cd1 organisms. These results demonstrate the existence of an NO-derived species capable of undergoing O-atom exchange with H218O during the reduction of NO. Trapping experiments with 15NO, 14N3-, and crude extracts of R. sphaeroides support the electrophilic nature of this intermediate and suggest its formulation as an enzyme nitrosyl, E-NO+, analogous to that observed during reduction of NO2-. The observation of lower levels of 18O incorporation with NO2- than with NO as substrate for A. cycloclastes and P. aureofaciens indicates that, for these organisms at least, a sequential pathway involving free NO as an intermediate is significantly less important than a direct pathway in which N2O is formed via reaction of two NO2- ions on a single enzyme.

Global gene expression profiles of Bacillus subtilis grown under anaerobic conditions.

Bacillus subtilis can grow under anaerobic conditions, either with nitrate or nitrite as the electron acceptor or by fermentation. A DNA microarray containing 4,020 genes from this organism was constructed to explore anaerobic gene expression patterns on a genomic scale. When mRNA levels of aerobic and anaerobic cultures during exponential growth were compared, several hundred genes were observed to be induced or repressed under anaerobic conditions. These genes are involved in a variety of cell functions, including carbon metabolism, electron transport, iron uptake, antibiotic production, and stress response. Among the highly induced genes are not only those responsible for nitrate respiration and fermentation but also those of unknown function. Certain groups of genes were specifically regulated during anaerobic growth on nitrite, while others were primarily affected during fermentative growth, indicating a complex regulatory circuitry of anaerobic metabolism.

Critical nucleotides in the interaction of a LysR-type regulator with its target promoter region. catBC promoter activation by CatR.

We have used site-directed mutagenesis to analyze the importance of nucleotides in the catBC promoter region for the binding of CatR, a member of the LysR family of DNA-binding proteins and for the in vivo activation of the catBC operon. The binding affinity of CatR for its binding site in the catBC promoter region was shown to increase about 2.2-fold in the presence of the inducer, cis,cis-muconate. Nucleotides were targeted for mutagenesis on the basis of previous footprinting data and sequence analysis of CatR binding sites. Critical nucleotides for CatR binding were found within an imperfect inverted repeat. Previous studies have indicated that the LysR family of DNA-binding proteins shares a consensus T-N11-A binding motif (Goethals, K., Van Montagu, M., and Holsters, M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1646-1650), but the CatR binding site sequence has located within the imperfect inverted repeat a G-N11-A instead of the predicted T. Mutagenesis of the G to a T resulted in an increase in both the binding of CatR to the catBC promoter and in the in vivo activation. Nucleotides within the -35 and -10 regions of the catBC promoter were found to be important for promoter activity but not for CatR binding.

Gene expression in Escherichia coli biofilms.

DNA microarrays were used to study the gene expression profile of Escherichia coli JM109 and K12 biofilms. Both glass wool in shake flasks and mild steel 1010 plates in continuous reactors were used to create the biofilms. For the biofilms grown on glass wool, 22 genes were induced significantly (p< or =0.05) compared to suspension cells, including several genes for the stress response ( hslS, hslT, hha, and soxS), type I fimbriae ( fimG), metabolism ( metK), and 11 genes of unknown function ( ybaJ, ychM, yefM, ygfA, b1060, b1112, b2377, b3022, b1373, b1601, and b0836). The DNA microarray results were corroborated with RNA dot blotting. For the biofilm grown on mild steel plates, the DNA microarray data showed that, at a specific growth rate of 0.05/h, the mature biofilm after 5 days in the continuous reactors did not exhibit differential gene expression compared to suspension cells although genes were induced at 0.03/h. The present study suggests that biofilm gene expression is strongly associated with environmental conditions and that stress genes are involved in E. coli JM109 biofilm formation.

Substrate diversity and expression of the 2,4,5-trichlorophenoxyacetic acid oxygenase from Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid as a sole source of carbon and energy. The genes encoding the proteins involved in the first step (tftA and tftB [previously designated tftA1 and tftA2, respectively]) have been cloned and sequenced. The oxygenase, TftAB, is capable of converting not only 2,4,5-trichlorophenoxyacetic acid to 2,4,5-trichlorophenol but also a wide range of chlorinated aromatic phenoxyacetates to their corresponding phenolic derivatives, as shown by whole-cell and cell-free assays. The rate of substrate utilization by TftAB depends upon the extent of chlorination of the substrate, the positions of the chlorines, and the phenoxy group. These results indicate a mechanistic similarity between TftAB and the 2,4-dichlorophenoxyacetic acid/alpha-ketoglutarate-dependent dioxygenase, TfdA, from Alcaligenes eutrophus JMP134. The promoter of the oxygenase genes was localized by promoter-probe analysis, and the transcriptional start site was identified by primer extension. The beta-galactosidase activity of the construct containing the promoter region cloned upstream of the beta-galactosidase gene in the promoter-probe vector pKRZ-1 showed that this construct is constitutively expressed in Escherichia coli and in AC1100. The -35 and -10 regions of the oxygenase genes show significant sequence identity to typical Escherichia coli sigma 70 promoters.

Sigma factor-anti-sigma factor interaction in alginate synthesis: inhibition of AlgT by MucA.

Conversion from the nonmucoid to the mucoid phenotype is a typical feature of Pseudomonas aeruginosa strains causing chronic pulmonary infections in cystic fibrosis patients. One of the key genetic controls in this conversion to mucoidy is from the algT(U)-mucA-mucB(algN) locus, located at 67.5 min on the standard P. aeruginosa chromosomal map. The algT gene promotes conversion to mucoidy and encodes an alternative sigma factor (sigma E) which belongs to the ECF (for extracytoplasmic function) family. On the other hand, the mucA and mucB (algN) genes suppress conversion to mucoidy. Loss-of-function mutations in mucA have been postulated to be the cause of mucoidy in some P. aeruginosa strains isolated from cystic fibrosis patients. We expressed and purified the protein products from the mucA and mucB open reading frames. The purified MucA protein abolishes the in vitro transcription specified by AlgT and the ability of AlgT to compete with an Escherichia coli sigma factor, FliA, suggesting that inhibiting AlgT-dependent transcription could be the mechanism by which mucA suppresses mucoidy in vivo. Enzyme-linked immunosorbent assay and glycerol density gradient sedimentation experiments suggest that MucA physically interacts with AlgT.

Characterization of the structural gene encoding a copper-containing nitrite reductase and homology of this gene to DNA of other denitrifiers.

A copper-containing nitrite reductase gene (nirU) from Pseudomonas sp. strain G-179 was found in a 1.9-kb EcoRI-BamHI DNA fragment. The coding region contained information for a polypeptide of 379 amino acids. The encoded protein had 78% identity in amino acid sequence to the nitrite reductase purified from Achromobacter cycloclastes. The ligands for type 1 copper- and type 2 copper-binding sites found in A. cycloclastes were also found in Pseudomonas sp. strain G-179, suggesting that these binding sites are conserved. Upstream from the promoter, two putative fnr boxes were found, suggesting that an FNR-like protein may be involved in regulation of the nitrite reductase gene under anaerobic conditions. When the 1.9-kb clone was used to probe Southern blots for similar sequences in DNAs from different denitrifiers, hybridization bands were seen for 15 of 16 denitrifiers known to have nitrite reductase containing copper. Except for Pseudomonas stutzeri JM300, all denitrifiers tested that have nitrite reductases containing heme c,d1 showed no or weak hybridization to this probe. Thus, this structural gene may be useful as a probe to detect denitrifiers with copper-containing nitrite reductases.

Purification of cytochrome cd1 nitrite reductase from Pseudomonas stutzeri JM300 and reconstitution with native and synthetic heme d1.

Cytochrome cd1 nitrite reductase has been purified from Pseudomonas stutzeri strain JM 300. This enzyme appears to be a dimer with a subunit molecular mass of 54 kDa and its isoelectric point is determined to be 5.4. The N terminus of amino acid sequence has strong homology with that of nitrite reductase from P. aeruginosa. The apoprotein of this enzyme has been reconstituted with native and synthetic heme d1. The nitrite reductase activity measured by NO and N2O gas evolution can be restored to 82% of the activity of the original enzyme when the protein was reconstituted with the native heme d1 and to 77% of the activity when reconstituted with the synthetic heme d1. The absorption spectra of both reconstituted enzymes are essentially identical to that of the original nitrite reductase. These results further substantiate the novel dione structure of heme d1 as proposed. The loss of NO2- reducing activity in the absence of heme d1 and its restoration by addition of heme d1 provides further evidence that heme d1 plays a key role in the conversion of NO2- to NO and N2O.

The algT (algU) gene of Pseudomonas aeruginosa, a key regulator involved in alginate biosynthesis, encodes an alternative sigma factor (sigma E).

Chronic infection by alginate-producing (mucoid) Pseudomonas aeruginosa is the leading cause of mortality among cystic fibrosis (CF) patients. During the course of sustained infection, the production of an alginate capsule protects the bacteria and allows them to persist in the CF lung. One of the key regulators of alginate synthesis is the algT (algU) gene encoding a putative alternative sigma factor (sigma E). AlgT was hyperproduced and purified from Escherichia coli. The N-terminal sequence of the purified protein matched perfectly with that predicted from the DNA sequence. The purified protein, in the presence of E. coli RNA polymerase core enzyme, was able to initiate transcription of an algT promoter. Deletion of the -35 region of this promoter abolished this activity in vitro as well as in vivo. These data indicate that the algT gene encodes a sigma factor that is autoregulatory.