Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria.

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

As the decade progresses this prediction is rapidly being realized, and microbial enzymes are becoming increasingly important in such diverse fields as medicine, brewing, and timber preservation. The genus Bacillus has played a major role in this development as evidenced by the distribution of the papers read at the Fifth International Fermentation Symposium, 1976 (64). Of 23 papers in the session devoted to "Microbial Enzymes of Industrial Interest" no less than ten were concerned with enzymes from bacilli. Reasons for the predominance of these bacteria in this area of study are several. First, they comprise a group of chemoorganotrophs that can be easily maintained and cultivated and yet are markedly heterogeneous in character. Psychrophiles, mesophiles, and thermophiles, in addition to alkalophilic, neutrophilic, and acidophilic species are well represented. Furthermore, virtually all 48 species of the genus listed in Bergey's Manual ofDeterminative Bacteriology (92) secrete a variety of soluble extracellular enzymes, which reflects the diversity of the parental habitats. Amylases that can liquefy starch under pressure at 11000 (194) and proteases that are stable and active at pH 12.0 (6) are extreme examples of enzyme adaption. This article will attempt to review the recent literature concerned with the characterization and properties of the exoenzymes synthesized by the bacilli and the control and mechanisms of their synthesis. It is restricted to this genus because the commercial importance of extracellular enzymes and academic interest in the process of sporulation have prompted a considerable amount of research into this general area. Nevertheless, in the final section I have attempted to equate our present knowledge of exoenzyme synthesis in procaryotes other than

Extracellular enzyme synthesis in the genus Bacillus.

Secretory proteins perform a variety of important "remote-control" functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which approximately 90 extracellular proteins were identified. Analysis of these proteins disclosed various "secrets of the secretome," such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only approximately 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.

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Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome

Bacillus thuringiensis and related insect pathogens.

Mutations affecting sucrose metabolism have been mapped by PBS1 transduction on the Bacillus subtilis chromosome in seven loci sacA, sacB, sacQ, sacR, sacS, sacT and sacU. sacA and sacB are presumed to be the structural genes of a sucrase and a levansucrase respectively. sacR, sacS and sacT correspond to groups of mutations leading to constitutive synthesis of sucrase or both sucrase and levansucrase. In sacQ, sacS and sacU are located either mutations increasing the level of synthesis of levansucrase specifically (sacQ

 h
 , sacS

 h
 , sacU

 h
 ) or mutations abolishing specifically the synthesis of levansucrase (sacU

 −
 ). sacA, sacS and sacT map to the left of purA16. sacQ is located to the left of thr5, sacB and sacR between cysB3 and hisA1 and sacU between uvr1 and gtaB.

Chromosomal location of mutations affecting sucrose metabolism in Bacillus subtilis Marburg.

The mechanism of action of levansucrase of Bacillus subtilis was investigated by initial velocity measurements of reactions catalyzed by this enzyme and initial velocity of isotopic exchange at equilibrium. Comparison of experimental results with theoretical results derived from various postulated mechanisms, supports a “ping-pong” mechanism, involving the intermediate participation of a fructosyl-enzyme. Rate constants for each step of this sequential mechanism were estimated.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1974.tb03269.x

Kinetic Studies of Levansucrase of Bacillus subtilis

The sacUh, amyB and pap mutations are identical with respect to their pleiotropic phenotype and their genetic location. Strains bearing these mutations overproduce several exocellular enzymes: α-amylase, lavansucrase and proteases, they are poorly or not at all transformable and most of them are devoid of flagella. These mutations are tightly linked to the sacU- mutations by transformation and therefore lie between the hisA1 and gtaB290 markers. It is possible that the sacUh, amyB and pap mutations on one hand and the sacU- mutations on the other are two different classes of alterations of the same regulatory gene controlling the synthesis of some exocellular enzymes and several other cellular functions. Furthermore an amy- mutation, leading to the lack of α-amylase activity, was mapped between the lin2 and aroI906 markers which are not linked to the sacU locus.

Mapping of mutations affecting synthesis of exocellular enzymes in Bacillus subtilis. Identity of the sacUh, amyB and pap mutations.

The biogenesis of the crystalline inclusion of Bacillus thuringiensis has been studied in the more general context of sporulation and in regard to the formation of some spore constituents. It has been shown that crystal protein is essentially formed during stages III and IV, before the acquisition of octanol resistance. One fraction of protein spore coats which is solubilized by a reducing agent associated with urea or guanidine hydrochloride, is synthesised at the same time; a more insoluble cystine-rich fraction is formed later, during stage V, just before the heat resistance is reached.



The analysis of bacterial extracts at intervals during sporogenesis indicates preferential synthesis of insoluble proteins (or proteins bound to membraneous structures), crystal protein represents a large proportion of these proteins. Immunological characterisation of crystal protein, with autoradiographic experiments reveals at least three antigens produced simultaneously after t4 and continuously throughout the biogenesis.



The sporulation of a mutant non crystal bearing strain was studied; it has been shown that this mutant accumulates soluble proteins having some of the properties of crystal constituents; in this case the synthesis and maturation of spore coats arise earlier in the course of sporulation than in the wild strain.



All results suggest a relation between crystal protein and a protein fraction of the spore coats.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1971.tb01620.x

Biogenesis of the Crystalline Inclusion of Bacillus thuringiensis during Sporulation

A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA).poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid. Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such at thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec- recipient strain of B. subtilis was used.

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Raymond Dedonder

Papers

Chromosomal location of mutations affecting sucrose metabolism in Bacillus subtilis Marburg.

Kinetic Studies of Levansucrase of Bacillus subtilis

Mapping of mutations affecting synthesis of exocellular enzymes in Bacillus subtilis. Identity of the sacUh, amyB and pap mutations.

Biogenesis of the Crystalline Inclusion of Bacillus thuringiensis during Sporulation

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.