Inducible xylitol dehydrogenases in enteric bacteria.

Morganella morganii ATCC 25829, Providencia stuartii ATCC 25827, Serratia marcescens ATCC 13880, and Erwinia sp. strain 4D2P were found to induce a xylitol dehydrogenase when grown on a xylitol-containing medium. The xylitol dehydrogenases were partially purified from the four strains, and those from M. morganii ATCC 25829, P. stuartii ATCC 25827, and S. marcescens ATCC 13880 were all found to oxidize xylitol to D-xylulose. These three enzymes had KmS for xylitol of 7.1 to 16.4 mM and molecular weights ranging from 130,000 to 155,000. In contrast, the xylitol dehydrogenase from Erwinia sp. strain 4D2P oxidized xylitol at the C-4 position to produce L-xylulose, had a Km for xylitol of 72 mM, and had a molecular weight of 102,000.     

Genetics of lipopolysaccharide biosynthesis in enteric bacteria.

From a historical perspective, the study of both the biochemistry and the genetics of lipopolysaccharide (LPS) synthesis began with the enteric bacteria. These organisms have again come to the forefront as the blocks of genes involved in LPS synthesis have been sequenced and analyzed. A number of new and unanticipated genes were found in these clusters, indicating a complexity of the biochemical pathways which was not predicted from the older studies. One of the most dramatic areas of LPS research has been the elucidation of the lipid A biosynthetic pathway. Four of the genes in this pathway have now been identified and sequenced, and three of them are located in a complex operon which also contains genes involved in DNA and phospholipid synthesis. The rfa gene cluster, which contains many of the genes for LPS core synthesis, includes at least 17 genes. One of the remarkable findings in this cluster is a group of several genes which appear to be involved in the synthesis of alternate rough core species which are modified so that they cannot be acceptors for O-specific polysaccharides. The rfb gene clusters which encode O-antigen synthesis have been sequenced from a number of serotypes and exhibit the genetic polymorphism anticipated on the basis of the chemical complexity of the O antigens. These clusters appear to have originated by the exchange of blocks of genes among ancestral organisms. Among the large number of LPS genes which have now been sequenced from these rfa and rfb clusters, there are none which encode proteins that appear to be secreted across the cytoplasmic membrane and surprisingly few which encode integral membrane proteins or proteins with extensive hydrophobic domains. These data, together with sequence comparison and complementation experiments across strain and species lines, suggest that the LPS biosynthetic enzymes may be organized into clusters on the inner surface of the cytoplasmic membrane which are organized around a few key membrane proteins.     

Mechanisms of siderophore iron transport in enteric bacteria.

Uptake of 55Fe- and 3H-labeled siderophores and their chronic analogues have been studied in Salmonella typhimurium LT-2 and Escherichia coli K-12. In S. typhimurium LT-2, at least two different mechanisms for siderophore iron transport may be operative. Uptake of 55Fe- and 3H-labeled ferrichrome and kinetically inert lambda-cis-chromic [3H]deferriferrichrome by the S. typhimurium LT-2 enb7 mutant, which is defective in the production of its native siderophore, enterobactin, appears to occur by two concurrent mechanisms. The first mechanism is postulated to involve either rapid uptake of iron released from the ferric complex by cellular reduction without penetration of the complex or ligand or dissociation of the complex and simultaneous uptake of both ligand and iron coupled with simultaneous expulsion of the ligand. The second mechanism appears to consist of slower uptake of the intact ferric complex.     

Detergent-shock response in enteric bacteria

Our work on bacterial detergent resistance started with the realization that bacteria growing in a sink full of soap must be resistant to the detergents in that soap. We chose sodium dodecyl sulphate (SDS) as a model detergent and decided to see how much SDS the bacterium growing in the sink could tolerate. The research program thus initiated has shown that bacteria such as Enterobacter cloacae can grow in up to 25% SDS and that SDS-shock proteins constitute c. 8% of the proteins synthesized by SDS-grown Escherichia coli. It has also provided explanations why enteric bacteria are oxidase negative, and how pyrroloquinoline quinone (PQQ) enters the periplasmic space. Finally, for E. coli, it has provided evidence for an alternate, phosphate-limited, aquatic life style which places greater emphasis on the Entner-Doudoroff pathway. Detergent resistance is important both medically and ecologically, e.g. entry of pathogens via bile-salt-containing intestinal tracts and biodegradation of detergent-like pollutants such as those resulting from oil spills. Our current research is focused on SDS-induced modifications of the cytoplasmic membrane and the presence of SDS in the periplasm.     

Evolution of aromatic amino acid biosynthesis and application to the fine-tuned phylogenetic positioning of enteric bacteria.

A comprehensive phylogenetic tree for virtually the entire assemblage of enteric bacteria is presented. Character states of aromatic amino acid biosynthesis are used as criteria, and the results are compared with partial trees based upon sequencing of 16S rRNA, 5S rRNA, and tryptophan leader peptide. Three major clusters are apparent. Enterocluster 1 possesses a gene fusion (trpG-trpD) encoding anthranilate synthase: anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase of tryptophan biosynthesis. This cluster includes the genera Escherichia, Shigella, Citrobacter, Salmonella, Klebsiella, and Enterobacter. The remaining two clusters lack the trpG-trpD gene fusion, but differ in the presence (enterocluster 2) or absence (enterocluster 3) of the three-step overflow pathway to L-phenylalanine. Enterocluster 2 consists of the genera Serratia and Erwinia. Enterocluster 3 includes the genera Cedecea, Kluyvera, Edwardsiella, Hafnia, Yersinia, Proteus, Providencia, and Morganella. Within these three major clusters, a tentative hierarchy of subcluster ordering is formulated on the basis of all data available. This hierarchical framework is proposed as a general working basis for continued refinement of the phylogenetic relationships of enteric bacteria.     

Survival of enteric bacteria in seawater

Enteric bacteria exposed to the marine environment simultaneously encounter a variety of abiotic and biotic challenges. Among the former, light appears to be critical in affecting seawater survival; previous growth history plays a major part in preadaptation of the cells, and stationary phase cells are generally more resistant than exponentially growing ones. Predation, mostly by protozoa, is probably the most significant biotic factor. Using Escherichia coli as a model, a surprisingly small number of genes was found that, when mutated, significantly affect seawater sensitivity of this bacterium. Most prominent among those is rpoS, which was also dominant among genes induced upon transfer to seawater.     

Modelling enteric bacteria survival in aquatic systems

We measured the uptake of carbon and inorganic nitrogen in nutrient-enriched water samples during 15-days incubation in summer in Lake Nakanuma, Japan. We calculated daily variations in neutral sugars and amino acids of the phytoplankton and estimated the efficiency of the increases in sugars and amino acids relative to photosynthetic activities. Only a small portion of carbon incorporated via photosynthesis was used for synthesis of sugars and amino acids during the incubation periods. The percentage increase in neutral sugars plus amino acids compared to photosynthetic rates ranged from 3.7 to 26.9% with an average of 12.8%. These findings suggest that large amounts of photosynthates were not used for the synthesis of cell components of phytoplankton and were lost through processes such as excretion and respiration.     

Modified assay procedures for the phosphotransferase system in enteric bacteria.

Conditions for the assay of individual components of the bacterial phosphotransferase system (PTS) are presented wich offer two important improvements over earlier methods. First, a lactate dehydrogenase-coupled assay for phosphocarrier proteins (HPr, FPr, and Factor III^Gle) which permits their measurement in either pure or partially pure form was developed. Quantitation by this assay does not rely on the level of activity of the enzymes used. Second, conditions under which Enzyme I activity was proportional to enzyme concentration are given. With these methods levels of PTS components have been measured that are 2-to 20-fold higher than those previously reported. These levels can now account for various PTS functions measured in vivo. Further, we have shown that the phosphocarrier proteins HPr and Factor III^Gle are substrates for their respective enzymes which show typical Michaelis-Menten kineties. In addition, a method for the partial purification of Enzyme II-B^Gle essentially free of Enzyme II^Man activity is presented.     

The rate of adaptive evolution in enteric bacteria

Here we estimate the rate of adaptive substitution in a set of 410 genes that are present in 6 Escherichia coli and 6 Salmonella enterica genomes. We estimate that more than 50% of amino acid substitutions in this set of genes have been fixed by positive selection between the E. coli and S. enterica lineages. We also show that the proportion of adaptive substitutions is uncorrelated with the rate of amino acid substitution or gene function but that it may be correlated with levels of synonymous codon usage bias.     

Occurrence of airborne enteric bacteria in Mexico city

Summary An investigation of microbial air quality in the area of the National Autonomous University of Mexico, located in the southern part of Mexico City, was conducted for one year. Ambient outdoor concentrations and size distribution of airborne bacteria were measured, 130 samples were taken at noon, using an Andersen 6 stage sampler, located 2 m above ground level. Concentration ranges and colony-forming units per cubic meter of air (CFU m^–3) found, were as follows:14 to 12999 for total bacteria, No growth (NG) to 55 for coliform bacteria, NG to 11 for fecal coliform and NG to 10 for fecal Streptococci.Bacteria associated with the potentially respirable fraction (0.65 to 4.7 µm) averaged 37% and 9% for total bacteria and coliform bacteria respectively. In 23% of the samples, coliform bacteria were recovered, with higher incidences during dry season. The most common of these were:Escherichia coli (15%), followed bySerratia (13%) andEnterobacter (10%),The total bacteria correlated significatively (p<0.05) with the following parameters: particulate matter smaller than 10 µm (PM₁₀) (r=0.40), total suspended particulates (TSP) (r=0.26), daily variation of temperature (r=0.18), and vapor pressure (r=–0.16). These relationships indicate that fecal soil pollution could affect air quality with potential health risks.     

Influence of culture medium of the fatty-acid profile in enteric bacteria.

Enteric bacteria having a high content of cyclopropane fatty acids steeply increase their synthesis when grown on insufficiently propitious culture media (meat-peptone agar or modified Drobot'ko synthetic medium) as compared with bacteria grown under more favourable conditions (meat-peptone broth). Simultaneously, a decrease in monounsaturated fatty acids and increase in palmitic acid are observed. One of the main factors underlying the change in the proportion of fatty acids in bacteria grown on synthetic medium is an increase in medium pH in the process of their growth. Enteric bacteria containing minute amounts/or not containing cyclopropane fatty acids at all (under the experimental conditions used) change their fatty-acid profile little if the culture medium is changed. When grown under insufficiently favourable conditions, these bacteria mainly display an enhanced content of palmitic acid and a lowered content of octadacenoic acid as compared with bacteria grown under more favourable conditions. Of the culture media used, meat-peptone broth, which affords the most favourable conditions for eneteric bacteria growth, is the most suitable medium for obtaining data of taxonomic value.     

Occurrence of multiple-antibiotic-resistant enteric bacteria in domestic sewage and oxidation lagoons.

The coliform bacterial population in the Grand Forks, N.Dak. sewage system was examined for multiple-antibiotic-resistant organisms over a 1-year period. Multiple-antibiotic-resistant coliforms were found to be common in the sewage, and their numbers remained fairly constant relative to the total coliform population throughout the year. Resistance to kanamycin, tetracycline, and ampicillin was found to be transferable at variable rates. Transfer rates were found to be temperature sensitive and were optimal at 35 degrees C. Although 75% of the multiple-antibiotic-resistant coliforms were capable of transferring resistance at some level, only 25% were capable of transferring resistance at rates greater than 10(-3) transconjugants per initial donor.