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Genetic analysis of alanine synthesis in the model genetic organism Escherichia coli has implicated avtA, the still uncharacterized alaA and alaB genes, and probably other genes. We identified a/aA as yfbQ. We then transferred mutations in several transaminase genes into a yJbQ mutant and isolated a mutant that required alanine for optimal growth. For cells grown with carbon sources other than pyruvate, the major alanine-synthesizing transaminases are AvtA, YfbQ (AlaA), and YfdZ (which we designate MAC). Growth with pyruvate as the carbon source and multicopy suppression suggest that several other transaminases can contribute to alanine synthesis. Expression studies showed that alanine modestly repressed avtA and yJbQ but had no effect on yfdZ. The leucine-responsive regulatory protein (Lrp) mediated control by alanine. We purified YfbQ and YfdZ and showed that both are dimers with [K.sub.m]S for pyruvate within the intracellular range of pyruvate concentration. doi: 10.1128/JB.00738-10

Antibiotic biosynthesis in the streptomycetes is a complex and highly regulated process. Here, we provide evidence for the contribution of a novel genetic locus to antibiotic production in Streptomyces coelicolor. The overexpression of a gene cluster comprising four protein-encoding genes (abeABCD) and an antisense RNA-encoding gene ([alpha]-abeA) stimulated the production of the blue-pigmented metabolite actinorhodin on solid medium. Actinorhodin production also was enhanced by the overexpression of an adjacent gene (abeR) encoding a predicted Streptomyces antibiotic regulatory protein (SARP), while the deletion of this gene impaired actinorhodin production. We found the abe genes to be differentially regulated and controlled at multiple levels. Upstream of abeA was a promoter that directed the transcription of abeABCD at a low but constitutive level. The expression of abeBCD was, however, significantly upregulated at a time that coincided with the initiation of aerial development and the onset of secondary metabolism; this expression was activated by the binding of AbeR to four heptameric repeats upstream of a promoter within abeA. Expressed divergently to the abeBCD promoter was [alpha]-abeA, whose expression mirrored that of abeBCD but did not require activation by AbeR. Instead, [alpha]-abeA transcript levels were subject to negative control by the double-strand-specific RNase, RNase III. doi: 10.1128/JB.00681-10

In this study, we report the identification of genes required for the biosynthesis of the core lipopolysaccharides (LPSs) of two strains of Proteus mirabilis. Since P. mirabilis and Klebsiella pneumoniae share a core LPS carbohydrate backbone extending up to the second outer-core residue, the functions of the common P. mirabilis genes was elucidated by genetic complementation studies using well-defined mutants of K. pneumoniae. The functions of strain-specific outer-core genes were identified by using as surrogate acceptors LPSs from two well-defined K. pneumoniae core LPS mutants. This approach allowed the identification of two new heptosyl-transferases (WamA and WamC), a galactosyltransferase (WamB), and an N-acetylglucosaminyltransferase (WamD). In both strains, most of these genes were found in the so-called waa gene cluster, although one common core biosynthetic gene (wabO) was found outside this cluster. doi: 10.1128/JB.00494-10

Acclimation to high salt concentrations involves concerted changes in gene expression. For the majority of salt-regulated genes, the mechanism underlying the induction process is not known. The gene ggpS (sll1566), which encodes the glucosylglycerol-phosphate synthase responsible for the synthesis of the compatible solute glucosylglycerol (GG), is specifically induced by salt in the cyanobacterial model strain Synechocystis sp. strain PCC 6803. To identify mechanisms mediating this salt-specific gene regulation, the ggpS promoter was analyzed in more detail. 5' rapid amplification of cDNA ends (5'-RACE) experiments revealed that the adjacent open reading frame (ORF), which is annotated as unknown protein Ssl3076, overlaps with the transcriptional start site of the ggpS gene. Reporter gene expression analyses indicated an essential role for the intact ssl3076 gene in the salt-regulated transcription of a gfp reporter gene. Promoter fragments containing a mutated ssl3076 lost the salt regulation; similarly, a frameshift mutation in ssl3076 resulted in a high level of ggpS expression under low-salt conditions, thereby establishing this small ORF, named ggpR, as a negative regulator of ggpS. Interestingly, small ORFs were also found adjacent to ggpS genes in the genomes of other GG-accumulating cyanobacteria. These results suggest that the GgpR protein represses ggpS expression under low-salt conditions, whereas in salt-shocked and salt-acclimated cells a stress-proportional ggpS expression occurs, leading to GG accumulation. doi: 10.1128/JB.00481-10

We report the design, synthesis, and assembly of the 1.08--mega--base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including 'watermark' sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication. 10.1126/science.1190719

A combination of small molecule chemistry, biosynthetic analysis, and genome mining has revealed the unexpected conservation of polycyclic tetramate macrolactam biosynthetic loci in diverse bacteria. Initially our chemical analysis of a Streptomyces strain associated with the southern pine beetle led to the discovery of frontalamides A and B, two previously undescribed members of this antibiotic family. Genome analyses and genetic manipulation of the producing organism led to the identification of the frontalamide biosynthetic gene cluster and several biosynthetic intermediates. The biosynthetic locus for the frontalamides' mixed polyketide/amino acid structure encodes a hybrid polyketide synthase nonribosomal peptide synthetase (PKS-NRPS), which resembles iterative enzymes known in fungi. No such mixed iterative PKSNRPS enzymes have been characterized in bacteria. Genomemining efforts revealed strikingly conserved frontalamide-like biosynthetic clusters in the genomes of phylogenetically diverse bacteria ranging from proteobacteria to actinomycetes. Screens for environmental actinomycete isolates carrying frontalamide-like biosynthetic loci led to the isolation of a number of positive strains, the majority of which produced candidate frontalamide-like compounds under suitable growth conditions. These results establish the prevalence of frontalamide-like gene clusters in diverse bacterial types, with medicinally important Streptomyces species being particularly enriched. biosynthesis | frontalamide | genome-mining Streptomyces | tetramic acid doi/ 10.1073/pnas.1001513107

Mycobacterium tuberculosis synthesizes isoprenoids via the nonmevalonate or DOXP pathway. Previous work demonstrated that three enzymes in the pathway (Dxr, IspD, and IspF) are all required for growth in vitro. We demonstrate the essentiality of the key genes dxs1 and gcpE, confirming that the pathway is of central importance and that the second homolog of the synthase (dxs2) cannot compensate for the loss of dxs1. We looked at the effect of overexpression of Dxr, Dxs1, Dxs2, and GcpE on viability and on growth in M. tuberculosis. Overexpression of dxs1 or dxs2 was inhibitory to growth, whereas overexpression of dxr or gcpE was not. Toxicity is likely to be, at least partially, due to depletion of pyruvate from the cells. Overexpression of dxs1 or gcpE resulted in increased flux through the pathway, as measured by accumulation of the metabolite 4-hydroxy-3-methyl-but-2-enyl pyrophosphate. We identified the functional translational start site and promoter region for dxr and demonstrated that it is expressed as part of a polycistronic mRNA with gcpE and two other genes. Increased expression of this operon was seen in cells overexpressing Dxs1, indicating that transcriptional control is effected by the first enzyme of the pathway via an unknown regulator. doi: 10.1128/JB.01402-09

Myxococcus xanthus, a Gram-negative soil bacterium, undergoes multicellular development when nutrients become limiting. Aggregation, which is part of the developmental process, requires the surface motility of this organism. One component of M. xanthus motility, the social (S) gliding motility, enables the movement of cells in close physical proximity. Previous studies demonstrated that the cell surface-associated exopolysaccharide (EPS) is essential for S motility and that the Dif proteins form a chemotaxis-like pathway that regulates EPS production in M. xanthus. DifA, a homologue of methyl-accepting chemotaxis proteins (MCPs) in the Dif system, is required for EPS production, S motility and development. In this study, a spontaneous extragenic suppressor of a difA deletion was isolated in order to identify additional regulators of EPS production. The suppressor mutation was found to be a single base pair insertion in cheW7 at the che7 chemotaxis gene cluster. Further examination indicated that mutations in cheW7 may lead to the interaction of Mcp7 with DifC (CheW-like) and DifE (CheA-like) to reconstruct a functional pathway to regulate EPS production in the absence of DifA. In addition, the cheW7 mutation was found to partially suppress a pilA mutation in EPS production in a [difA.sup.+] background. Further deletion of difA from the pilA cheW7 double mutant resulted in a triple mutant that produced wildtype levels of EPS, implying that DifA (MCP-like) and Mcp7 compete for interactions with DifC and DifE in the modulation of EPS production. DOI 10.1099/mic.0.031070-0

Sialic acids (Sias) are nonulosonic acid (NulO) sugars prominently displayed on vertebrate cells and occasionally mimicked by bacterial pathogens using homologous biosynthetic pathways. It has been suggested that Sias were an animal innovation and later emerged in pathogens by convergent evolution or horizontal gene transfer. To better illuminate the evolutionary processes underlying the phenomenon of Sia molecular mimicry, we performed phyIogenomic analyses of biosynthetic pathways for Sias and related higher sugars derived from 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids. Examination of [approximately equal to]1,000 sequenced microbial genomes indicated that such biosynthetic pathways are far more widely distributed than previously realized. Phylogenetic analysis, validated by targeted biochemistry, was used to predict NulO types (i.e., neuraminic, legionaminic, or pseudaminic acids) expressed by various organisms. This approach uncovered previously unreported occurrences of Sia pathways in pathogenic and symbiotic bacteria and identified at least one instance in which a human archaeal symbiont tentatively reported to express Sias in fact expressed the related pseudaminic acid structure. Evaluation of targeted phylogenies and protein domain organization revealed that the 'unique' Sia biosynthetic pathway of animals was instead a much more ancient innovation. Pathway phylogenies suggest that bacterial pathogens may have acquired Sia expression via adaptation of pathways for legionaminic acid biosynthesis, one of at least 3 evolutionary paths for de novo Sia synthesis. Together, these data indicate that some of the long-standing paradigms in Sia biology should be reconsidered in a wider evolutionary context of the extended family of NulO sugars. legionaminic acid I phylogeny | pseudaminic acid | neuraminic acid | biosynthetic pathway

Aeromonas caviae Sch3N possesses a small genomic island that is involved in both flagellin glycosylation and lipopolysaccharide (LPS) O-antigen biosynthesis. This island appears to have been laterally acquired as it is flanked by insertion element-like sequences and has a much lower G+C content than the average aeromonad G+C content. Most of the gene products encoded by the island are orthologues of proteins that have been shown to be involved in pseudaminic acid biosynthesis and flagellin glycosylation in both Campylobacter jejuni and Helicobacter pylori. Two of the genes, 1st and lsg, are LPS specific as mutation of them results in the loss of only a band for the LPS O-antigen. Lsg encodes a putative Wzx flippase, and mutation of Lsg affects only LPS; this finding supports the notion that flagellin glycosylation occurs within the cell before the flagellins are exported and assembled and not at the surface once the sugar has been exported. The proteins encoded by flmA, flmB, neuA, flmD, and neuB are thought to make up a pseudaminic acid biosynthetic pathway, and mutation of any of these genes resulted in the loss of motility, flagellar expression, and a band for the LPS O-antigen. Furthermore, pseudaminic acid was shown to be present on both flagellin subunits that make up the polar flagellum filament, to be present in the LPS O-antigen of the A. caviae wild-type strain, and to be absent from the A. caviae flmD mutant strain.

Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only 10% of all bacteria are predicted to synthesize PC. In Rhizobiaceae, including the phytopathogenic bacterium Agrobacterium tumefaciens, PC is essential for the establishment of a successful host-microbe interaction. A. tumefaciens produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The responsible genes, pmtA (coding for a phospholipid N-methyltransferase) and pcs (coding for a PC synthase), are located on the circular chromosome of A. tumefaciens C58. Recombinant expression of pmtA and pcs in Escherichia coli revealed that the individual proteins carry out the annotated enzyme functions. Both genes and a putative ABC transporter operon downstream of PC are constitutively expressed in A. tumefaciens. The amount of PC in A. tumefaciens membranes reaches around 23% of total membrane lipids. We show that PC is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased biofilm formation, two processes known to be involved in virulence. Our work documents the critical importance of membrane lipid homeostasis for diverse cellular processes in A. tumefaciens.

Curcuminoids, which are produced specifically by plants of the order Zingiberales, have long been used as food additives because of their aromatic, stimulant and colouring properties and as traditional Asian medicines because of their anti-tumour, antioxidant and hepatoprotective activities. Curcuminoids are therefore attractive targets for metabolic engineering. An artificial curcuminoid biosynthetic pathway, including reactions of phenylalanine ammonia-lyase (PAL) from the yeast Rhodotorula rubra, 4-coumarate : CoA ligase (4CL) from Lithospermum erythrorhizon and curcuminoid synthase (CUS) from rice (Oryza sativa), a type III polyketide synthase, was constructed in Escherichia coli for the production of curcuminoids. Cultivation of the recombinant E. coli cells in the presence of tyrosine or phenylalanine, or both, led to production of bisdemethoxycurcumin, dicinnamoylmethane and cinnamoyl-p-coumaroylmethane. Another E. coli system carrying 4CL and CUS genes was also used for high-yield production of curcuminoids from exogenously supplemented phenylpropanoid acids: p-coumaric acid, cinnamic acid and ferulic acid. The yields of curucminoids were up to ~100 mg [l.sup.-1]. Furthermore, this system gave approximately 60 mg curcumin [l.sup.-1] from 10 g rice bran pitch, an industrial waste discharged during rice edible oil production, as a source of ferulic acid.

Wall teichoic acids are anionic phosphate-rich polymers that are part of the complex meshwork of carbohydrates that make up the gram-positive cell wall. These polymers are essential to the proper rod-shaped morphology of Bacillus subtilis and have been shown to be an important virulence determinant in the nosocomial opportunistic pathogen Staphylococcus aureus. Together, sequence-based studies, in vitro experiments with biosynthetic proteins, and analyses of the chemical structure of wall teichoic acid have begun to shed considerable light on our understanding of the biogenesis of this polymer. Nevertheless, some paradoxes remain unresolved. One of these involves a putative duplication of genes linked to CDP-ribitol synthesis (tarI'J' and tarIJ) as well as pnly(ribitol phosphate) polymerization (tarK and tarL) in S. aureus. In the work reported here, we performed careful studies of the dispensability of each gene and discovered a functional redundancy in the duplicated gene clusters. We were able to create mutants in either of the putative ribitol phosphate polymerases (encoded by tarK and tarL) without affecting teichoic acid levels in the S. aureus cell wall. Although genes linked to CDP-ribitol synthesis are also duplicated, a null mutant in only one of these (tarI'J') could be obtained, while tarIJ remained essential. Suppression analysis of the tarIJ null mutant indicated that the mechanism of dysfunction in tarI'J' is due to poor translation of the TaIJ' enzyme, which catalyzes the rate-limiting step in CDP-ribitol formation. This work provides new insights into understanding the complex synthetic steps of the ribitol phosphate polymer in S. aureus and has implications on specifically targeting enzymes involved in polymer biosynthesis for antimicrobial design.

The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti possesses the Sin quorum-sensing system based on N-acyl homoserine lactones (AHLs) as signal molecules. The Sin system consists of SinI, the AHL synthase, and SinR, the LuxR-type regulator. This system regulates the expression of a multitude of S. meliloti genes through ExpR, another LuxR-type regulator. Analysis of the activity of the sinI promoter showed that the expression of sinI is dependent on sinR and enhanced by a combination of expR and Sin AHLs. The characterization of the ExpR binding site upstream of sinI and the identification of binding sites upstream of the galactoglucan biosynthesis genes wgaA (expA1) and wgeA (expE1) allowed the definition of a consensus sequence for these binding sites. Based on this consensus, two additional ExpR binding sites in the promoter regions of exol and exsH, two genes related to the production of succinoglycan, were found. The specific binding of ExpR to the wgaA and wgeA promoters was enhanced in the presence of oxo-[C.sub.l4]-HL. Positive regulation of the galactoglucan biosynthesis genes by ExpR was shown to be dependent on WggR (ExpG) and influenced by MucR, both of which are previously characterized regulators of these genes. Based on these results, a reworked model of the Sin-ExpR quorum-sensing regulation scheme of galactoglucan production in S. meliloti is suggested.

The RelA, RelP, and RelQ enzymes are responsible for the production of the alarmone (p)ppGpp in Streptococcus mutans. A strain lacking all three synthetases (ArelAPQ) does not grow in minimal medium lacking the branched-chain amino acids (BCAA) leucine or valine but grows well if isoleucine is also omitted. Here, we investigated whether there was a correlation between growth in the absence of leucine and valine with (p)ppGpp pools and the activation of CodY. By using a combination of single, double, and triple mutants lacking the (p)ppGpp synthetase enzymes, we demonstrated that the ability to grow in the absence of leucine or valine required basal levels of (p)ppGpp production by RelP and RelQ. The introduction of a codY mutation into the [DELTA]relAPQ strain fully restored growth in medium lacking leucine or valine, revealing that the growthdefective phenotype of [DELTA]relAPQ was directly linked to CodY. Lowering GTP levels through the addition of decoyinine did not alleviate CodY repression or affect the expression of genes involved in BCAA biosynthesis, suggesting that S. mutans CodY is not activated by GTP. The results of phenotypic studies revealed that the codY mutant had a reduced capacity to form biofilms and that its growth was more sensitive to low pH, showing a role for CodY in two key virulence properties of S. mutans. Microarray results revealed the extent of the CodY regulon. Notably, the identification of putative CodY-binding boxes upstream of genes that were downregulated in the codY mutant indicates that CodY may also function as a transcriptional activator in S. mutans.

Depending on the phosphate concentration encountered in the environment Sinorhizobium meliloti 2011 synthesizes two different exopolysaccharides (EPS). Galactoglucan (EPS II) is produced under phosphate starvation but also in the presence of extra copies of the transcriptional regulator WggR (ExpG) or as a consequence of a mutation in mucR. The galactoglucan biosynthesis gene cluster contains the operons wga (expA), wge (expE), wgd (expD), and wggR (expG). Two promoters, differentially controlled by WggR, PhoB, and MucR, were identified upstream of each of these operons. The proximal promoters of the wga, wge, and wgd transcription units were constitutively active when separated from the upstream regulatory sequences. Promoter activity studies and the positions of predicted PhoB and WggR binding sites suggested that the proximal promoters are cooperatively induced by PhoB and WggR. MucR was shown to strongly inhibit the distal promoters and bound to the DNA in the vicinity of the distal transcription start sites. An additional inhibitory effect on the distal promoter of the structural galactoglucan biosynthesis genes was identified as a new feature of WggR in a mucR mutant. A regulatory model of the fine-tuning of galactoglucan production is proposed.

The central metabolic model for Geobacter sulfurreducens included a single pathway for the biosynthesis of isoleucine that was analogous to that of Escherichia coil, in which the isoleucine precursor 2-oxobutanoate is generated from threonine. [sup.13]C labeling studies performed in G. sulfurreducens indicated that this pathway accounted for a minor fraction of isoleucine biosynthesis and that the majority of isoleucine was instead derived from acetyl-coenzyme A and pyruvate, possibly via the citramalate pathway. Genes encoding citramalate synthase (GSU1798), which catalyzes the first dedicated step in the citramalate pathway, and threonine ammonia-lyase (GSU0486), which catalyzes the conversion of threonine to 2-oxobutanoate, were identified and knocked out. Mutants lacking both of these enzymes were auxotrophs for isoleucine, whereas single mutants were capable of growth in the absence of isoleucine. Biochemical characterization of the single mutants revealed deficiencies in citramalate synthase and threonine ammonia-lyase activity. Thus, in G. sulfurreducens, 2-oxobutanoate can be synthesized either from citramalate or threonine, with the former being the main pathway for isoleucine biosynthesis. The citramalate synthase of G. suifurreducens constitutes the first characterized member of a phylogenetically distinct clade of citramalate synthases, which contains representatives from a wide variety of microorganisms.

The actinomycete Nonomuraea sp. strain ATCC 39727 produces the glycopeptide A40926, the precursor of the novel antibiotic dalbavancin. Previous studies have shown that phosphate limitation results in enhanced A40926 production. The A40926 biosynthetic gene (dbv) cluster, which consists of 37 genes, encodes two putative regulators, Dbv3 and Dbv4, as well as the response regulator (Dbv6) and the sensor-kinase (Dbv221 of a putative two-component system. Reverse transcription-PCR (RT-PCR) and real-time RT-PCR analysis revealed that the dbv14-dbv8 and the dbv30-dbv35 operons, as well as dbv4, were negatively influenced by phosphate. Dbv4 shows a putative helix-turn-helix DNA-binding motif and shares sequence similarity with StrR, the transcriptional activator of streptomycin biosynthesis in Streptomyces griseus. Dbv4 was expressed in Eseherichia cog as an N-terminal [His.sub.6]-tagged protein. The purified protein bound the dbvI4 and dbv30 upstream regions but not the region preceding dbv4. Bbr, a Dbv4 ortholog from the gene cluster for the synthesis of the glycopeptide balhimycin, also bound to the dbv14 and dbv30 upstream regions, while Dbv4 bound appropriate regions from the balhimycin cluster. Our results provide new insights into the regulation of glycopeptide antibiotics, indicating that the phosphate- controlled regulator Dbv4 governs two key steps in A40926 biosynthesis: the biosynthesis of the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine and critical tailoring reactions on the heptapeptide backbone.

In Salmonella enterica serovar Typhimurium, gmk encodes guanylate kinase, an essential enzyme involved in the synthesis and salvage of guanine nucleotides. Here we report the isolation of a mutation in gmk that results in a nutritional requirement for adenine at low temperature. Comparisons of kinetic parameters from the wild-type and mutant Gmk enzymes revealed that the mutant enzyme had a more than 20-fold-higher [K.sub.m] for ATP than the wild-type enzyme. The growth dependence of the mutant on temperature and/or adenine could not be explained as a direct result of this kinetic difference. We propose a model in which previously described regulatory effects of GMP are responsible for these phenotypes.

On solid media, the reproductive growth of Streptomyces involves antibiotic biosynthesis coincident with the erection of filamentous aerial hyphae. Following cessation of growth of an aerial hypha, multiple septation occurs at the tip to form a chain of unigenomic spores. A gene, crgA, that coordinates several aspects of this reproductive growth is described. The gene product is representative of a well-conserved family of small actinomycete proteins with two C-terminal hydrophobic-potential membrane-spanning segments. In Streptomyces avermitilis, crgA is required for sporulation, and inactivation of the gene abolished most sporulation septation in aerial hyphae. Disruption of the orthologous gene in Streptomyces coelicolor indicates that whereas CrgA is not essential for sporulation in this species, during growth on glucose-containing media, it influences the timing of the onset of reproductive growth, with precocious erection of aerial hyphae and antibiotic production by the mutant. Moreover, CrgA subsequently acts to inhibit sporulation septation prior to growth arrest of aerial hyphae. Overexpression of CrgA in S. coelicolor, uncoupling any nutritional and growth phase-dependent regulation, results in growth of nonseptated aerial hyphae on all media tested, consistent with a role for the protein in inhibiting sporulation septation.

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable [V.sub.max]/[K.sub.m] values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.

rRNA synthesis is the rate-limiting step in ribosome synthesis in Escherichia coli. Its regulation has been described in terms of a negative-feedback control loop in which rRNA promoter activity responds to the amount of translation. The feedback nature of this control system was demonstrated previously by artificially changing ribosome synthesis rates and observing responses of rRNA promoters. However, it has not been demonstrated previously that the initiating nucleoside triphosphate (iNTP) and guanosine 5'-diphosphate 3'-diphosphate (ppGpp), the molecular effectors responsible for controlling rRNA promoters in response to changes in the nutritional environment, are responsible for altering rRNA promoter activities under these feedback conditions. Here, we show that most feedback situations result in changes in the concentrations of both the iNTP and ppGpp and that the directions of these changes are consistent with a role for these two small-molecule regulators in feedback control of rRNA synthesis. In contrast, we observed no change in the level of DNA supercoiling under the feedback conditions examined.

We report the isolation and partial characterization of three new mutants of Streptomyces coelicolor that are defective in morphogenesis and antibiotic production. The genes identified by the mutations were located and cloned by using a combination of Tn5 in vitro mutagenesis, cotransformation, and genetic complementation. Mutant SE69 produces lower amounts of antibiotics than the wild type produces, produces spores only after prolonged incubation on rich media, and identifies a gene whose predicted protein product is similar to the GntR family of transcriptional regulators; also, production of aerial mycelia on both rich and poor media is significantly delayed in this mutant. Mutant SE293 is defective in morphogenesis, overproduces antibiotics on rich media, fails to grow on minimal media, and identifies a gene whose predicted protein product is similar to the TetR family of transcriptional regulators. Preliminary evidence suggests that the SE293 gene product may control a molybdopterin binding protein located immediately adjacent to it. Mutant SJ175 sporulates sooner and more abundantly than the wild type and overproduces antibiotics on rich media, and it identifies a gene whose predicted protein product contains regions of predominantly hydrophobic residues similar to those of integral membrane proteins.

Research describes development of a new biocatalyst for the production of D-lactic acid in the form of engineered Escherichia coli. Results show that D-lactic acid produced by these E. coli approach the maximum yield of two molecules per glucose molecule with a chemical and optical purity of approximately 98% and 100%, respectively.

Research describes the construction of an Escherichia coli mutant that cannot metabolize mannose but can takeup labeled mannose, mediated by plasmid-encoded enzyme, and preferentially convert to GDP-mannoseand 2-O-alpha-D-mannosyl-D-glycerate preserving specific isotopic enrichment.

A previously unknown open reading frame coding for the protein YfcX is required for medium-chain-length polyhydroxyalkanoate formation using fatty acids in an E. coli fadB mutant. YfcX is a multifunctional enzyme encoding hydratase and dehydrogenase activities.

The genes involved in the 2,4-dinitrotoluene degradative pathway are localized to a 27-kilobase region of DNA cloned from Burkholderia cepacia R34. Transposon remnants and vestigial genes which are found in the bacteria suggest that the 2,4-DNT degradation pathway evolved relatively recently.

A mutant strain of Pseudomonas putida (LU2201) expressing a novel ethylphenylacetate (EPA) esterase was isolated after mutagenesis of P. putida LU6456. The DNA fragment conferring the EPA+ phenotype was cloned and characterized. It included the structural genes of the 2,3-butanediol catabolic pathway, including acoC, which catalyzes the acetyl transfer step (i.e., dihydrolipoamide acetyltransferase). The acoC gene product from LU2201 was expressed in E. coli and shown to be responsible for the novel EPA-esterase activity. Biochemical characterization revealed that the enzyme still retained the natural dihydrolipoamide acetyltransferase activity. Sequence comparison with the corresponding wild-type gene indicated a single-base pair change leading to a I238V replacement in the respective protein. Biochemical characterization of wild-type AcoC enzyme showed that it too was able to catalyze EPA hydrolysis despite the apparent absence of this activity in cells grown in the presence of EPA. Unlike the wild type, EPA serves as an inducer in the mutant strain. Inverse PCR was used to clone the putative regulator acoR from the aco operon in both wild type and mutant strains. Sequence analyses indicated an S14R exchange in the AcoR from the P. putida mutant. This mutation was located in the N-terminal region responsible for regulatory functions, presumably by interaction with signal molecules. This may account for the ability of EPA to induce expression of acoC only in the mutant strain. Key words: Pseudomonas putida, ethylphenylacetate esterase, acetoin cleaving system, acoC gene, dihydrolipoamide acetyl-transferase, E2 component, acoR gene.

Research describes the isolation of a true bifunctional protein in enterococci bacterria. The mvaE gene encodes a 86.5 kilodalton protein that catalyzes both the acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A reductase reactions.

Genomic sequence data indicate that absA two-component system resides within the calcium-dependent antibiotic gene cluster in Streptomyces coelicolor. Results discuss the role of absA in the regulation of antibiotic biosynthesis clusters with reference to its location in the calcium-dependent antibiotic gene cluster.

Mutational studies indicate that the homologous polysaccharide biosynthetic protein WbjA adds the terminal glucose to O-antigen unit of the Pseudomonas aeruginosa serogroup O11 lipopolysaccharide.

Results reveal that ferE gene codes a 91-kilodaltton heme-containing outer membrane protein, which supports the anaerobic growth of Shewanella putrefaciens on iron(III) and manganese(IV).