Streptomycetes, soil-dwelling mycelial bacteria that form sporulating aerial branches, have an exceptionally large number of predicted secreted proteins, including many exported via the twin-arginine transport system. Their use of noncatalytic substrate-binding proteins and hydrolytic enzymes to obtain soluble nutrients from carbohydrates such as chitin and cellulose enables them to interact with other organisms. Some of their numerous secreted proteases participate in developmentally significant extracellular cascades, regulated by inhibitors, which lead to cannibalization of the substrate mycelium biomass to support aerial growth and sporulation. They excrete many secondary metabolites, including important antibiotics. Some of these play roles in interactions with eukaryotes. Surprisingly, some antibiotic biosynthetic enzymes are extracellular. Antibiotic production is often regulated by extracellular signalling molecules, some of which also control morphological differentiation. Amphipathic proteins, assembled with the help of cellulose-like material, are required for both hyphal attachment to surfaces and aerial reproductive growth. Comparative genomic analysis suggests that the acquisition of genes for extracellular processes has played a huge part in speciation. The rare codon TTA, which is present in the key pleiotropic regulatory gene adpA and many pathway-specific regulatory genes for antibiotic production, has a particular influence on extracellular biology.

We report the complete genome sequence of Zymomonas mobilis ZM4 (ATCC31821), an ethanologenic microorganism of interest for the production of fuel ethanol. The genome consists of 2,056,416 base pairs forming a circular chromosome with 1,998 open reading frames (ORFs) and three ribosomal RNA transcription units. The genome lacks recognizable genes for 6-phosphofructokinase, an essential enzyme in the Embden-Meyerhof-Parnas pathway, and for two enzymes in the tricarboxylic acid cycle, the 2-oxoglutarate dehydrogenase complex and malate dehydrogenase, so glucose can be metabolized only by the Entner-Doudoroff pathway. Whole genome microarrays were used for genomic comparisons with the Z. mobilis type strain ZM1 (ATCC10988) revealing that 54 ORFs predicted to encode for transport and secretory proteins, transcriptional regulators and oxidoreductase in the ZM4 strain were absent from ZM1. Most of these ORFs were also found to be actively transcribed in association with ethanol production by ZM4.Growing environmental concerns over the use and depletion of nonrenewable energy resources, together with the recent price increases and instabilities in the international oil markets have stimulated an increasing interest in the use of fermentation processes for the large-scale production of alternative fuels such as ethanol. As such, ethanol-producing microorganisms, such as the Gram-negative bacterium Z. mobilis, have potential for the production of fuel ethanol.Z. mobilis, which is used in the tropics to produce pulque and alcoholic palm wines, uses the Entner-Doudoroff (ED) pathway to metabolize glucose, which results in only 1 mole of ATP being produced per mole of glucose 1 . The potential advantages of using Z. mobilis for ethanol production include: (i) its high and specific rates of sugar uptake and ethanol production, (ii) its production of ethanol at yields close to the theoretical maximum with relatively low biomass formation, (iii) its high ethanol tolerance of up to 16% (vol/vol) and (iv) its facility for genetic manipulation 2-6 . However, wild strains of Z. mobilis can use only glucose, fructose and sucrose as carbon substrates, so recent research has focused on the development of recombinant strains capable of using pentose sugars 7,8 for the conversion of cheaper lignocellulosic hydrolysates to ethanol. Improved mutants 9-11 as well as the application of metabolic flux analysis, sitedirected mutagenesis, specific gene deletion/insertion and metabolic engineering for strain developlment 12,13 have also been reported. A physical map of Z. mobilis ZM4 genome and the ribosomal transcriptional unit have been previously reported 14,15 . In the current paper, the features of the complete sequence of the Z. mobilis ZM4 genome are presented and genomic characters are compared with those of another Z. mobilis strain, ZM1.

SummaryThe emergence and dissemination of extended-spectrum (ES) b -lactamases induce therapeutic failure and a lack of eradication of clinical isolates even by thirdgeneration b -lactam antibiotics like ceftazidime. CMY-10 is a plasmid-encoded class C b -lactamase with a wide spectrum of substrates. Unlike the well-studied class C ES b -lactamase from Enterobacter cloacae GC1, the W -loop does not affect the active site conformation and the catalytic activity of CMY-10. Instead, a three-amino-acid deletion in the R2-loop appears to be responsible for the ES activity of CMY-10. According to the crystal structure solved at 1.55 Å resolution, the deletion significantly widens the R2 active site, which accommodates the R2 side-chains of blactam antibiotics. This observation led us to demonstrate the hydrolysing activity of CMY-10 towards imipenem with a long R2 substituent. The forced mutational analyses of P99 b -lactamase reveal that the introduction of deletion mutations into the R2-loop is able to extend the substrate spectrum of class C non-ES b -lactamases, which is compatible with the isolation of natural class C ES enzymes harbouring deletion mutations in the R2-loop. Consequently, the opening of the R2 active site by the deletion of some residues in the R2-loop can be considered as an operative molecular strategy of class C b -lactamases to extend their substrate spectrum.

The objectives of the current studies were to determine the roles of key enzymes in central carbon metabolism in the context of increased production of antibiotics in Streptomyces coelicolor. Genes for glucose-6-phosphate dehydrogenase and phosphoglucomutase (Pgm) were deleted and those for the acetyl coenzyme A carboxylase (ACCase) were overexpressed. Under the conditions tested, glucose-6-phosphate dehydrogenase encoded by zwf2 plays a more important role than that encoded by zwf1 in determining the carbon flux to actinorhodin (Act), while the function of Pgm encoded by SCO7443 is not clearly understood. The pgm-deleted mutant unexpectedly produced abundant glycogen but was impaired in Act production, the exact reverse of what had been anticipated. Overexpression of the ACCase resulted in more rapid utilization of glucose and sharply increased the efficiency of its conversion to Act. From the current experiments, it is concluded that carbon storage metabolism plays a significant role in precursor supply for Act production and that manipulation of central carbohydrate metabolism can lead to an increased production of Act in S. coelicolor.The wide occurrence of multiply antibiotic-resistant bacterial pathogens of humans has made it urgent to develop new antibiotics. Although over 6,000 different antibiotics have been identified from actinomycetes, these microorganisms are still considered likely to be an important source of further new antibiotics (7). To develop a new antibiotic for clinical application, the compound must be synthesized in sufficient quantities. Random mutation and selection is the tried and tested way to obtain strains producing increased amounts of the targeted compound (11). However, with the advances in genetics, rational or target-directed gene manipulation methods have been developed, and those provide more targeted ways of increasing productivity and discovering novel compounds (12). Such predictive manipulation of central metabolism is likely to be particularly useful at early stages in the testing of new compounds made by genetic manipulation of biosynthetic gene sets in well-characterized surrogate hosts, such as Streptomyces coelicolor or Streptomyces lividans, but the preparation of sufficient quantities of the new compound may be a significant obstacle to further progress.Antibiotics identified from metabolites of microorganisms are classified into several families, such as polyketides, polyethers, macrolides, and ␤-lactams, based on chemical structure similarity and common biosynthetic pathways. The provision of intermediates or precursors from primary/intermediary metabolism is a prerequisite for the biosynthesis of secondary metabolites, and the availability of those molecules is a key factor determining the productivity of antibiotics. These precursors are generally formed through the catabolism of various carbon substrates. S. coelicolor produces blue (actinorhodin [Act])-and red (undecylprodiginines [Red])-pigmented antibiotics, which are synthesized at least in part from the...

Streptomyces clavuligerus is an important industrial strain that produces a number of antibiotics, including clavulanic acid and cephamycin C. A high-quality draft genome sequence of the S. clavuligerus NRRL 3585 strain was produced by employing a hybrid approach that involved Sanger sequencing, Roche/454 pyrosequencing, optical mapping, and partial finishing. Its genome, comprising four linear replicons, one chromosome, and four plasmids, carries numerous sets of genes involved in the biosynthesis of secondary metabolites, including a variety of antibiotics.Streptomyces clavuligerus is a bacterium of industrial and clinical importance producing the ␤-lactamase inhibitor clavulanic acid (4), as well as cephamycin C (a ␤-lactam), clavams, tunicamycin, and holomycin (5,9,14,17). S. clavuligerus is intriguing in regard to its genetic structure and mechanism of cephamycin C, clavulanic acid, and clavam biosynthesis (19,20,21). Here, we present a draft genome sequence of the type strain NRRL 3585 (ATCC 27064). The specific clone used, SC2, is a single-spore isolate from the stock of NRRL 3585 that showed good sporulation and metabolite production, a typical example of the wild-type strain.The genome sequence was determined by Sanger paired-end sequencing (7) and Roche/454 pyrosequencing (12). Sanger reads at 4.9-fold coverage were produced from 4-or 40-kb genomic libraries, followed by 454 reads at 61.6-fold coverage. Sanger reads (14-fold coverage) provided by the Broad Institute were also utilized. The Sanger paired-end reads and the Newbler-assembled 454 contigs were assembled with a PCAP assembler (8). Optical mapping (Opgen, Inc.) was performed to confirm the assembly output and to assign contigs into each replicon. Gap closure was attempted using gap-spanning clones and PCR products. Coding sequences were predicted by the combined use of Glimmer (6), GeneMark (3), and CRITICA (2). Automatic functional annotation results obtained by AutoFACT (10) and the Rapid Annotation using Subsystem Technology (RAST) server (1) were compiled and validated with Artemis.The genome consists of one linear chromosome (58 contigs in 4 scaffolds, 6,736,475 bp, 72.69% GϩC) and four linear plasmids, pSCL1 (3 contigs in 2 scaffolds, 10,266 bp, 71.96% GϩC), pSCL2 (2 contigs in 2 scaffolds, 149,326 bp, 70.07% GϩC), pSCL3 (15 contigs in 2 scaffolds, 442,792 bp, 70.77% GϩC), and pSCL4 (11 contigs in 3 scaffolds, 1,796,117 bp, 71.85% GϩC). The 6.7-Mb chromosome is the smallest of the completely sequenced Streptomyces species. At least six rRNA operons and 66 tRNA genes as well as 7,898 protein-coding genes were annotated. Recently, a draft sequence of S. clavuligerus ATCC 27064 describing the 1.8-Mb megaplasmid was reported (13). The sequences of the chromosome and pSCL4 were nearly identical to our sequences. However, three other plasmids (15,22) are present only in our data, suggesting that our clone has preserved the genome in its intact form.A plethora of genes related to biosynthesis of secondary metabolites were discovered. The super...

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SCO4677 is one of a large number of similar genes in Streptomyces coelicolor that encode proteins with an HATPase_c domain resembling that of anti-sigma factors such as SpoIIAB of Bacillus subtilis. However, SCO4677 is not located close to genes likely to encode a cognate sigma or anti-anti-sigma factor. SCO4677 was found to regulate antibiotic production and morphological differentiation, both of which were significantly enhanced by the deletion of SCO4677. Through protein-protein interaction screening of candidate sigma factor partners using the yeast two-hybrid system, SCO4677 protein was found to interact with the developmentally specific Bacteria of the genus Streptomyces are highly adapted to living in soil environments and produce various valuable secondary metabolites. Their complex life cycle of morphological and physiological differentiation requires elaborate regulatory systems (6). The genome of the model species Streptomyces coelicolor A3(2) encodes 7,825 theoretical proteins, with more than 12% (Ն900 gene products) encoding transcriptional regulators. The 65 sigma factors of S. coelicolor strikingly point to the complexity of its gene regulation (ScoDB at http: //strepdb.streptomyces.org.uk) (2).Sigma factors have been found to be important for differentiation. BldN and N are required for formation of aerial hyphae (3,8), while WhiG initiates the formation of spores from aerial hyphae (5): bldN, sigN, and whiG appear to be transcribed throughout aerial mycelium formation and development. On the other hand, F is involved in the later stages of spore formation, and sigF transcription was detected only when sporulation started, and depended upon whiG and other early sporulation genes (whiA, whiB, whiH, whiI, and whiJ) (28). A sigF-null mutant formed smaller spores compared to the wild-type strain, with the spore wall being much thinner, and the chromosomal DNA of the sigF mutant was much less condensed (43). Among the seven other

Three proteases, namely chymotrypsin-like protease (CTP), trypsin-like protease (TLP) and metalloprotease (MTP), were identified in cultures of Streptomyces albidoflaws SMF301. The dynamics of protease production were determined and the roles of the proteases in morphological differentiation were deduced to be as follows: CTP is essential for hydrolysing the proteinaceous nitrogen source for mycelium growth; TLP plays a role in the formation of thickened mycelium in submerged culture and of aerial mycelium in solid culture; MTP may participate in the maturation of spores. The unique thickened mycelia in submerged culture are thought to be an intermediate form between mycelium and spores. TLP (molecular mass 32 kDa) and MTP (molecular mass 18 kDa) were purified and their enzymic properties were determined.