Gene Ontology annotations and resources.

The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bioinformatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources.

Microbial competition: Escherichia coli mutants that take over stationary phase cultures.

Many microorganisms, including Escherichia coli, can survive extended periods of starvation. The properties of cells that survived prolonged incubation in stationary phase were studied by mixture of 10-day-old (aged) cultures with 1-day-old (young) cultures of the same strain of Escherichia coli. Mutants from the aged cultures that could grow eventually took over the population, which resulted in the death of the cells from the young cultures. This phenotype was conferred by mutations in rpoS, which encodes a putative stationary phase-specific sigma factor. These rapid population shifts have implications for the studies of microbial evolution and ecology.

A functional domain of bacteriophage lambda terminase for prohead binding.

Terminase is a multifunctional protein complex involved in DNA packaging during bacteriophage lambda assembly. Terminase is made of gpNul and gpA, the products of the phage lambda Nu1 and A genes. Early during DNA packaging terminase binds to lambda DNA to form a complex called complex I. Terminase is required for the binding of proheads by complex I to form a DNA: terminase: prohead complex known as complex II. Terminase remains associated with the DNA during encapsidation. The other known role for terminase in packaging is the production of staggered nicks in the DNA thereby generating the cohesive ends. Lambdoid phage 21 has cohesive ends identical to those of lambda. The head genes of lambda and 21 show partial sequence homology and are analogous in structure, function and position. The terminases of lambda and 21 are not interchangeable. At least two actions of terminase are involved in this specificity: (1) DNA binding; (2) prohead binding. The 1 and 2 genes at the left end of the 21 chromosome were identified as coding for the 21 terminase. gp1 and gp2 are analogous to gpNu1 and gpA, respectively. We have isolated a phage, lambda-21 hybrid 33, which is the product of a crossover between lambda and 21 within the terminase genes. Lambda-21 hybrid 33 DNA and terminase have phage 21 packaging specificity, as determined by complementation and helper packaging studies. The terminase of lambda-21 hybrid 33 requires lambda proheads for packaging. We have determined the position at which the crossover between lambda DNA and 21 DNA occurred to produce the hybrid phage. Lambda-21 hybrid 33 carries the phage 21 1 gene and a hybrid phage 2/A gene. Sequencing of lambda-21 hybrid 33 DNA shows that it encodes a protein that is homologous at the carboxy terminus with the 38 amino acids of the carboxy terminus of lambda gpA; the remainder of the protein is homologous to gp2. The results of these studies define a specificity domain for prohead binding at the carboxy terminus of gpA.

The terminase of bacteriophage lambda. Functional domains for cosB binding and multimer assembly.

Terminase is a protein complex involved in lambda DNA packaging. The subunits of terminase, gpNul and gpA, are the products of genes Nul and A. The actions of terminase include DNA binding, prohead binding and DNA nicking. Phage 21 is a lambdoid phage that also makes a terminase, encoded by genes 1 and 2. The terminases of 21 and lambda are not interchangeable. This specificity involves two actions of terminase; DNA binding and prohead binding. In addition, the subunits of lambda terminase will not form functional multimers with the subunits of 21 terminase. lambda-21 hybrid phages can be produced as a result of recombination. We describe here lambda-21 hybrid phages that have hybrid terminase genes. The packaging specificities of the hybrids and the structure of their genes were compared in order to identify functional domains of terminase. The packaging specificities were determined in vivo by complementation tests and helper packaging experiments. Restriction enzyme site mapping and sequencing located the sites at which recombination occurred to produce the hybrid phages. lambda-21 hybrid 51 carries the lambda A gene, and a hybrid 1/Nul gene. The crossover that produced this phage occurred near the middle of the 1 and Nul genes. The amino-terminal portion of the hybrid protein is homologous to gp1 and the carboxy-terminal portion is homologous to gpNul. It binds to 21 DNA and forms functional multimers with gpA, providing evidence that the amino-terminal portion of gpNul is involved in DNA binding and the carboxy-terminal portion of gpNul is involved in the interaction with gpA. lambda-21 hybrid 54 has a hybrid 2/A gene. The amino terminus of the hybrid protein of lambda-21 hybrid 54 is homologous with gp2. This protein forms functional multimers only with gp1, providing evidence that the amino terminus of gpA is involved in the interaction with gpNul. These studies identify three functional domains of terminase.

Mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that increase expression of the lac operon in the absence of CAP-cAMP.

We have isolated a new class of mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that alter the transcription initiation properties of RNA polymerase holoenzyme. The rpoD(Lac) mutations increase expression of the lac operon in the absence of CAP-cAMP, allowing a strain lacking adenyl cyclase to grow on lactose. Four of the six alleles isolated have three- to fivefold increases in the amount of lac mRNA and beta-galactosidase per cell. We show that these four mutations increase transcription initiation from the same promoter used by wild-type RNA polymerase. The mutations were mapped and sequenced. One mutation occurs in the codon for amino acid 389 of the sigma 70 polypeptide. The remaining five mutations are clustered, affecting residues 570, 571 and 575. These five mutations are within or near a proposed helix-turn-helix motif in the C terminus of sigma 70.

Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase.

We have systematically assayed the in vivo promoter recognition properties of 13 mutations in rpoD, the gene that encodes the sigma 70 subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of sigma 70 and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of sigma 70, which is similar to the helix-turn-helix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the -35 region, and that a sequence internal to sigma 70, in a region which is highly conserved among sigma factors, recognizes the -10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the -35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effect on promoter recognition.

Cosmid DNA packaging in vivo.

The packaging of cosmid DNA into phage particles during phage lambda growth is described. Evidence is presented supporting the work of others that cosmid transducing phages contain linear multimers of cosmid DNA in which the number of cosmid copies is that required to make a packagable DNA length (greater than 0.77 of the lambda DNA length). The yield of cosmid transducing phages declines sharply as the number of cosmid copies required to make a packagable DNA length increases. The cosmid DNA replication that produces the packaging substrate shares with lambda rolling-circle replication a dependence on the lambda gam gene product.

Isolation and characterization of an Escherichia coli mutant defective in resuming growth after starvation.

To understand the mechanisms that allow the enteric bacterium Escherichia coli to make the transitions between growth and stationary phase and to maintain cell viability during starvation, we have looked for mutants defective in stationary-phase survival (a Sur- phenotype). In this paper we describe a conditional E. coli mutant, surB1, that grows normally and remains viable during stationary phase but is unable to exit stationary phase and resume aerobic growth at high temperature. Thus, the surB gene product is not required for cell survival per se but, rather, it is required for starved cells to reinitiate growth under restrictive conditions. Once growth has started, SurB function is no longer required. Mutant cells sense and respond to fresh medium but appear to arrest growth before the first cell division. The surB gene was mapped to 19.5 min on the E. coli chromosome, cloned, and sequenced. The surB gene product is predicted to be an integral membrane protein with multiple membrane-spanning regions and is homologous to the ATP-binding cassette (ABC) family of transporters, a large family of transport proteins found in both prokaryotic and eukaryotic cells. An open reading frame, designated ybjA, was found immediately upstream of surB and may be in an operon with surB. The predicted ybjA gene product is also homologous to the ABC transporter family and SurB and YbjA may function together in a common transport pathway. Either surB or ybjA may be the same gene as cydC, a gene described previously whose function is needed for the production of functional cytochrome d oxidase complexes. Consistent with this prediction, surB1 mutant cells were found to lack functional cytochrome d oxidase. However, the SurB- phenotype is not simply attributable to the absence of cytochrome d oxidase. Thus, the surB gene product may have an additional role in the cell.

Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations.

Gene expression from plasmids containing the araBAD promoter can be regulated by the concentration of arabinose in the growth medium. Guzman et al. [Guzman, L.-M., Belin, D., Carson, M. J. & Beckwith, J. (1995) J. Bacteriol. 177, 4121-4130] showed that expression of a cloned gene could be modulated over several orders of magnitude in cultures grown in the presence of subsaturating concentrations of arabinose. We constructed plasmids expressing a fast-folding mutant Aequorea victoria green fluorescent protein from the araBAD promoter to examine the distribution of expressed gene products in individual cells at intermediate induction levels. Microscopic examination of cells grown at low arabinose concentrations shows mixtures of brightly fluorescent and dark cells, suggesting that intermediate expression levels in cultures reflect a population average of induced and uninduced cells. The kinetics of green fluorescent protein induction suggest that this reflects an "autocatalytic" induction mechanism due to accumulation of the inducer by active transport. This mechanism, which is analogous to the induction of the lac operon at subsaturating inducer concentrations in lacY+ cells, was described 40 years ago by Novick and Weiner [Novick, A. & Weiner, M. (1957) Proc. Natl. Acad. Sci. USA 43, 553-566].

Escherichia coli proteins synthesized during recovery from starvation.

Proteins synthesized in Escherichia coli during recovery from starvation were resolved by two-dimensional polyacrylamide gel electrophoresis. Nine outgrowth-specific proteins, which appeared in two kinetic groups, that were not detected in either starved or exponential-phase cells were synthesized. Five other proteins whose rate of synthesis during outgrowth was > or = 5-fold higher than during exponential growth were observed.

Genetic analysis of the stationary phase-induced mcb operon promoter in Escherichia coli.

A combination of deletion analysis and random mutagenesis was used to identify regulatory elements in Pmcb, the stationary phase-induced promoter of the mcb operon. Our results indicate that Pmcb is controlled by at least three different factors, two previously identified and at least one unknown factor, which act at four different sites in the promoter. Sequences between -344 and -164 upstream of the transcriptional start site were required for wild-type levels of mcb transcription in stationary phase. More dramatic reductions in both exponential and stationary phase expression were observed when sequences from -164 to -54 were deleted. Point mutations located between -105 and -138 decreased both exponential and stationary phase expression. All but one of these mutations decreased OmpR-dependent activation of Pmcb transcription. EmrR, also known as MprA, acts directly or indirectly at sequences downstream of -54 to repress Pmcb. A minimal promoter containing sequences from -34 to +79 was still induced > or = 10-fold in stationary phase. Point mutations within this region identified sequences at -8, -11, -30, -31 and -32 as important for Pmcb activity. These bases are in the gearbox sequence, present in Pmcb and several other stationary phase-induced Escherichia coli promoters.

The stationary phase of the bacterial life cycle.

In the natural environment bacteria seldom encounter conditions that permit periods of exponential growth. Rather, bacterial growth is characterized by long periods of nutritional deprivation punctuated by short periods that allow fast growth, a feature that is commonly referred to as the feast-or-famine lifestyle. In this chapter we review the recent advances made in our understanding of the molecular events that allow some gram-negative bacteria to survive prolonged periods of starvation. After an introductory description of the properties of starved gram-negative bacteria, the review presents three aspects of stationary phase: entry into stationary phase, responses during prolonged starvation, and reentry into the growth cycle.

The stationary-phase-exit defect of cydC (surB) mutants is due to the lack of a functional terminal cytochrome oxidase.

The surB gene was identified as a gene product required for Escherichia coli cells to exit stationary phase at 37 degrees C under aerobic conditions. surB was shown to be the same as cydC, whose product is required for the proper assembly and activity of cytochrome d oxidase. Cytochrome d oxidase, encoded by the cydAB operon, is one of two alternate terminal cytochrome oxidases that function during aerobic electron transport in E. coli. Mutations inactivating the cydAB operon also cause a temperature-sensitive defect in exiting stationary phase, but the phenotype is not as severe as it is for surB mutants. In this study, we examined the phenotypes of surB1 delta(cydAB) double mutants and the ability of overexpression of cytochrome o oxidase to suppress the temperature-sensitive stationary-phase-exit defect of surB1 and delta(cydAB) mutants and analyzed spontaneous suppressors of surB1. Our results indicate that the severe temperature-sensitive defect in exiting stationary phase of surB1 mutants is due both to the absence of terminal cytochrome oxidase activity and to the presence of a defective cytochrome d oxidase. Membrane vesicles prepared from wild-type, surB1, and delta(cydAB) strains produced superoxide radicals at the same rate in vitro. Therefore, the aerobic growth defects of the surB1 and delta(cydAB) strains are not due to enhanced superoxide production resulting from the block in aerobic electron transport.

Cloning and sequencing of Escherichia coli murZ and purification of its product, a UDP-N-acetylglucosamine enolpyruvyl transferase.

The Escherichia coli gene murZ, encoding the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase, has been cloned and sequenced. Identified by screening an E. coli genomic library for clones that conferred phosphomycin resistance, murZ encoded a 419-amino-acid polypeptide and was mapped to 69.3 min on the E. coli chromosome. MurZ protein was purified to near homogeneity and found to have the expected UDP-N-acetylglucosamine enolpyruvyl transferase activity. Sequence analysis of the predicted product revealed 44% identity to OrfR from Bacillus subtilis (K. Trach, J.W. Chapman, P. Piggot, D. LeCoq, and J.A. Hoch, J. Bacteriol. 170:4194-4208, 1988), suggesting that orfR may also encode a UDP-N-acetylglucosamine enolpyruvyl transferase enzyme. MurZ is also homologous to the aromatic amino acid biosynthetic enzyme enolpyruvyl shikimate phosphate synthase, the other enzyme known to catalyze an enolpyruvyl transfer.

Global analysis of Escherichia coli gene expression during the acetate-induced acid tolerance response.

The ability of Escherichia coli to survive at low pH is strongly affected by environmental factors, such as composition of the growth medium and growth phase. Exposure to short-chain fatty acids, such as acetate, proprionate, and butyrate, at neutral or nearly neutral pH has also been shown to increase acid survival of E. coli and Salmonella enterica serovar Typhimurium. To investigate the basis for acetate-induced acid tolerance in E. coli O157:H7, genes whose expression was altered by exposure to acetate were identified using gene arrays. The expression of 60 genes was reduced by at least twofold; of these, 48 encode components of the transcription-translation machinery. Expression of 26 genes increased twofold or greater following treatment with acetate. This included six genes whose products are known to be important for survival at low pH. Five of these genes, as well as six other acetate-induced genes, are members of the E. coli RpoS regulon. RpoS, the stress sigma factor, is known to be required for acid tolerance induced by growth at nonlethal low pH or by entry into stationary phase. Disruption of the rpoS gene by a transposon insertion mutation also prevented acetate-induced acid tolerance. However, induction of RpoS expression did not appear to be sufficient to activate the acid tolerance response. Treatment with either NaCl or sodium acetate (pH 7.0) increased expression of an rpoS::lacZ fusion protein, but only treatment with acetate increased acid survival.

Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA.

The sigma 70 subunit of E. coli RNA polymerase is required for sequence-specific recognition of promoter DNA. Genetic studies and sequence analysis have indicated that sigma 70 contains two specific DNA-binding domains that recognize the two conserved portions of the prokaryotic promoter. However, intact sigma 70 does not bind to DNA. Using C-terminal and internal polypeptides of sigma 70, carrying one or both putative DNA-binding domains, we demonstrate that sigma 70 does contain two DNA-binding domains, but that N-terminal sequences inhibit the ability of intact sigma 70 to bind to DNA. Thus, we propose that sigma 70 is a sequence-specific DNA-binding protein that normally functions through an allosteric interaction with the core subunits of RNA polymerase.

The head genes of bacteriophage 21.

Physical and genetic maps of the head genes of lambdoid phage 21 have been made and compared with the head gene map of lambda. Because 21 and lambda have partial sequence homology throughout the head genes it was expected that the head genes of 21 would be analogous to those of lambda. Eight head genes of 21 have been identified and it was found that each of the genes is analogous in position, structure, and/or function to a lambda head gene. Phage 21 genes analogous to the lambda D and FI genes were not identified by mutation. Complementation studies between phage 21 and lambda mutants indicate that only gpFII (the protein product of a gene is referred to as gp (gene product] is fully interchangeable, gpW and gpD are partially interchangeable, and the rest of the head morphogenetic proteins are phage specific. In analogy with phage lambda, it is found that the gpNu3 analog (gp6) of phage 21 is synthesized from the same reading frame as the gpC analog (gp5), resulting in a protein identical to the carboxy terminus of gp5.