Octominin: An antibacterial and anti-biofilm peptide for controlling the multidrug resistance and pathogenic Streptococcus parauberis.

Streptococcus parauberis is a pathogenic gram-positive bacterium that causes streptococcosis infection in fish. Since S. parauberis is becoming resistant to multiple antibiotics, the development of alternatives, such as antimicrobial peptides, has gained great attention. Octominin, derived from the defense protein of Octopus minor, showed a significant antimicrobial activity against multidrug resistance S. parauberis, with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 50 and 100 µg/mL, respectively. Furthermore, time-kill kinetics, agar diffusion, and bacterial viability assays confirmed the concentration-dependent antibacterial activity of Octominin against S. parauberis. Field emission scanning electron microscopy analysis showed morphological and ultra-structural changes in S. parauberis upon Octominin treatment. Moreover, Octominin treatment demonstrated changes in membrane permeability, induced reactive oxygen species (ROS), and its binding ability to genomic DNA, suggesting its strong bactericidal activity with multiple modes of action. We confirmed the inhibition of biofilm formation and the eradication of existing biofilms in a concentration-dependent manner. Additionally, Octominin on S. parauberis at transcriptional level exhibited downregulation of membrane formation (pgsA and cds1), DNA repairing (recF), biofilm formation (pgaB and epsF) genes, while upregulation of ROS detoxification (sodA) and DNA protecting (ahpF) related genes. An in vivo study confirmed a significantly (P < 0.05) higher relative percentage survival in Octominin-treated larval zebrafish exposed to S. parauberis (93.3%) compared to the control group (20.0%). Collectively, our results confirm that Octominin could be a potential antibacterial and anti-biofilm agent against S. parauberis.

Vaginal isolation of beta-haemolytic Streptococcus from bitches with and without neonatal deaths in the litters.

The aim of the study was to identify beta-haemolytic streptococci in the vagina of bitches who had delivered healthy litters and bitches who had delivered litters in which neonatal deaths occurred. Fifty-one bitches divided into two groups were used. Group 1 (G1) included 28 bitches that had delivered healthy litters and group 2 (G2) included 23 bitches that had delivered puppies who died in the neonatal period. Two vaginal samples were taken, one in proestrus and the other at the end of gestation (EG). Beta-haemolytic Streptococcus (BS) was isolated from 16 bitches (57%) in G1 and from 21 bitches (91%) in G2. The bacteriological cultures, serological tests (Streptex® ) and PCR assay allowed identification of Streptococcus canis and Streptococcus dysgalactiae in G1 and G2. Ultramicroscopic studies allowed the observation of M Protein and capsules in strains of S. dysgalactiae and S. canis in G1 and G2. The S. canis strains isolated from G2 showed thicker capsules than S. canis strains isolated from G1 (234 ± 24.2 vs 151.23 ± 28.93 nm; p < .001.). No differences were observed in capsule thickness between strains of S. dysgalactiae isolated from G1 and G2 (210 ± 13.54 vs 211.66 ± 19.67 nm; p > .70). All strains of beta-haemolytic Streptococcus isolated were penicillin sensitive. Penicillin was administered from EG to 5 days post-partum in 10 G2 females with isolation of BS (G2A). Saline solution was administered in eleven G2 females with isolation of BS (G2B). Ninety per cent of the puppies survived in G2A and 25% survived in G2B. Our results suggest BS is involved in canine neonatal deaths.

Single cell stochastic regulation of pilus phase variation by an attenuation-like mechanism.

The molecular triggers leading to virulence of a number of human-adapted commensal bacteria such as Streptococcus gallolyticus are largely unknown. This opportunistic pathogen is responsible for endocarditis in the elderly and associated with colorectal cancer. Colonization of damaged host tissues with exposed collagen, such as cardiac valves and pre-cancerous polyps, is mediated by appendages referred to as Pil1 pili. Populations of S. gallolyticus are heterogeneous with the majority of cells weakly piliated while a smaller fraction is hyper piliated. We provide genetic evidences that heterogeneous pil1 expression depends on a phase variation mechanism involving addition/deletion of GCAGA repeats that modifies the length of an upstream leader peptide. Synthesis of longer leader peptides potentiates the transcription of the pil1 genes through ribosome-induced destabilization of a premature stem-loop transcription terminator. This study describes, at the molecular level, a new regulatory mechanism combining phase variation in a leader peptide-encoding gene and transcription attenuation. This simple and robust mechanism controls a stochastic heterogeneous pilus expression, which is important for evading the host immune system while ensuring optimal tissue colonization.

Characterization of Streptococcus tigurinus small-colony variants causing prosthetic joint infection by comparative whole-genome analyses.

Small-colony variants (SCVs) of bacteria are associated with recurrent and persistent infections. We describe for the first time SCVs of Streptococcus tigurinus in a patient with a prosthetic joint infection. S. tigurinus is a novel pathogen of the Streptococcus mitis group and causes invasive infections. We sought to characterize S. tigurinus SCVs using experimental methods and find possible genetic explanations for their phenotypes. The S. tigurinus SCVs were compared with the wild-type (WT) isolate using phenotypic methods, including growth under different conditions, autolysis, and visualization of the cell ultrastructure by use of transmission electron microscopy (TEM). Furthermore, comparative genome analyses were performed. The S. tigurinus SCVs displayed reduced growth compared to the WT and showed either a very stable or a fluctuating SCV phenotype. TEM analyses revealed major alterations in cell separation and morphological abnormalities, which were partially explained by impaired autolytic behavior. Intriguingly, the SCVs were more resistant to induced autolysis. Whole-genome sequencing revealed mutations in the genes involved in general cell metabolism, cell division, stringent response, and virulence. Clinically, the patient recovered after a 2-stage exchange of the prosthesis. Comparative whole-genome sequencing in clinical strains is a useful tool for identifying novel genetic signatures leading to the most persistent bacterial forms. The detection of viridans streptococcal SCVs is challenging in a clinical laboratory due to the small colony size. Thus, it is of major clinical importance for microbiologists and clinicians to be aware of viridans streptococcal SCVs, such as those of S. tigurinus, which lead to difficult-to-treat infections.

D-alanylation of lipoteichoic acids confers resistance to cationic peptides in group B streptococcus by increasing the cell wall density.

Cationic antimicrobial peptides (CAMPs) serve as the first line of defense of the innate immune system against invading microbial pathogens. Gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids (TAs) with D-alanine, but the exact mechanism of resistance is not fully understood. Here, we utilized various functional and biophysical approaches to investigate the interactions of the human pathogen Group B Streptococcus (GBS) with a series of CAMPs having different properties. The data reveal that: (i) D-alanylation of lipoteichoic acids (LTAs) enhance GBS resistance only to a subset of CAMPs and there is a direct correlation between resistance and CAMPs length and charge density; (ii) resistance due to reduced anionic charge of LTAs is not attributed to decreased amounts of bound peptides to the bacteria; and (iii) D-alanylation most probably alters the conformation of LTAs which results in increasing the cell wall density, as seen by Transmission Electron Microscopy, and reduces the penetration of CAMPs through the cell wall. Furthermore, Atomic Force Microscopy reveals increased surface rigidity of the cell wall of the wild-type GBS strain to more than 20-fold that of the dltA mutant. We propose that D-alanylation of LTAs confers protection against linear CAMPs mainly by decreasing the flexibility and permeability of the cell wall, rather than by reducing the electrostatic interactions of the peptide with the cell surface. Overall, our findings uncover an important protective role of the cell wall against CAMPs and extend our understanding of mechanisms of bacterial resistance.

Gap2 promotes the formation of a stable protein complex required for mature Fap1 biogenesis.

Serine-rich repeat glycoproteins (SRRPs) are important bacterial adhesins conserved in streptococci and staphylococci. Fap1, a SRRP identified in Streptococcus parasanguinis, is the major constituent of bacterial fimbriae and is required for adhesion and biofilm formation. An 11-gene cluster is required for Fap1 glycosylation and secretion; however, the exact mechanism of Fap1 biogenesis remains a mystery. Two glycosylation-associated proteins within this cluster--Gap1 and Gap3--function together in Fap1 biogenesis. Here we report the role of the third glycosylation-associated protein, Gap2. A gap2 mutant exhibited the same phenotype as the gap1 and gap3 mutants in terms of Fap1 biogenesis, fimbrial assembly, and bacterial adhesion, suggesting that the three proteins interact. Indeed, all three proteins interacted with each other independently and together to form a stable protein complex. Mechanistically, Gap2 protected Gap3 from degradation by ClpP protease, and Gap2 required the presence of Gap1 for expression at the wild-type level. Gap2 augmented the function of Gap1 in stabilizing Gap3; this function was conserved in Gap homologs from Streptococcus agalactiae. Our studies demonstrate that the three Gap proteins work in concert in Fap1 biogenesis and reveal a new function of Gap2. This insight will help us elucidate the molecular mechanism of SRRP biogenesis in this bacterium and in pathogenic species.

Genomic characterization, expression analysis, and antimicrobial function of a glyrichin homologue from rock bream, Oplegnathus fasciatus.

Antimicrobial peptides are important innate effector molecules, playing a vital role in antimicrobial immunity in all species. Glyrichin is a transmembrane protein and an antibacterial peptide, exerting its functions against a wide range of pathogenic bacteria. In this study, cDNA and a BAC clone harboring the glyrichin gene were identified from rock bream and characterized. Genomic characterization showed that the OfGlyrichin gene exhibited a 3 exon-2 intron structure. OfGlyrichin is a 79-amino-acid protein with a transmembrane domain at (22)GFMMGFAVGMAAGAMFGTFSCLR(44). Pairwise and multiple sequence alignments showed high identity and conservation with mammalian orthologues. Phylogenetic analysis showed a close relationship with fish species. Higher levels of OfGlyrichin transcripts were detected in the liver from healthy rock bream which were induced by immunogens like lipopolysaccharide, poly I:C, rock bream irido virus, Edwardsiella tarda and Streptococcus iniae. The synthetic peptide (pOf19) showed antibacterial activity against Escherichia coli, E. tarda, and S. iniae. Analysis of the bacterial morphological features after pOf19 peptide treatment showed breakage of the cell membrane, affirming that antibacterial function is accomplished through membrane lysis. The pOf19 peptide also showed antiviral activity against RBIV infection. The high conservation of the genomic structure and protein, together with the antimicrobial roles of OfGlyrichin, provide evidence for the evolutionary existence of this protein playing a vital role in innate immune defense in rock bream.

Antimicrobial activity of ethanol extracts of Laminaria japonica against oral microorganisms.

Laminaria japonica is a brown alga, which is consumed widely in Korea, Japan, and China. This study investigated the antimicrobial activity of ethanol extracts of L. japonica against oral microbial species to assess the possible application of L. japonica extracts in dental care products. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined in culture medium using a microdilution method. The MICs of ethanol extracts of L. japonica with oral streptococci were 62.5-500 µg/ml and the MBCs were 125-1000 µg/ml. The MICs of Actinomyces naeslundii and Actinomyces odontolyticus were 250 and 62.5 µg/ml, respectively. The MBCs of A. naeslundii and A. odontolyticus were 500 and 250 µg/ml, respectively. The MICs were 250 and 62.5 µg/ml for Fusobacterium nucleatum and Porphyromonas gingivalis, respectively. The killing of Streptococcus mutans and P. gingivalis was dependent on the incubation time. The killing of S. mutans, A. odontolyticus, and P. gingivalis was significantly dependent on the extract concentration. Bacterial treatment with L. japonica extracts changed the cell surface texture of S. mutans, A. odontolyticus, and P. gingivalis. The results of this study suggest that L. japonica extracts may be useful for the development of antimicrobial agents to combat oral pathogens.

Structural insights into serine-rich fimbriae from Gram-positive bacteria.

The serine-rich repeat family of fimbriae play important roles in the pathogenesis of streptococci and staphylococci. Despite recent attention, their finer structural details and precise adhesion mechanisms have yet to be determined. Fap1 (Fimbriae-associated protein 1) is the major structural subunit of serine-rich repeat fimbriae from Streptococcus parasanguinis and plays an essential role in fimbrial biogenesis, adhesion, and the early stages of dental plaque formation. Combining multidisciplinary, high resolution structural studies with biological assays, we provide new structural insight into adhesion by Fap1. We propose a model in which the serine-rich repeats of Fap1 subunits form an extended structure that projects the N-terminal globular domains away from the bacterial surface for adhesion to the salivary pellicle. We also uncover a novel pH-dependent conformational change that modulates adhesion and likely plays a role in survival in acidic environments.

Prevalence and different characteristics of two serotypes of Streptococcus parauberis isolated from the farmed olive flounder, Paralichthys olivaceus (Temminck and Schlegel), in Korea.

The prevalence of two serotypes of Streptococcus parauberis isolated from the olive flounder, Paralichthys olivaceus, was evaluated in a total of 29 isolates between 2003 and 2010 in Korea. Streptococcus parauberis isolates were divided into two serologically distinct types (serotype 1 and serotype 2), except for one strain (S1091), using an agglutination assay with rabbit antiserum, and serotype 1 was identified as the dominant type (24 of 29 isolates) in this study. To identify the characteristics of the two serotypes of S. parauberis, we conducted a biochemical test using the API 20 Strep kit, a transmission electron microscopy (TEM) assay, sequence analysis of 16S-23S rRNA intergenic spacer region (ISR) and a pathogenicity test. In TEM, both serotypes possessed polysaccharide capsule layers around the cell surface when bacterial cells were treated with a homologous serotype of rabbit antiserum. However, we were unable to discriminate serotype-specific biochemical characteristics and genetic characteristics of 16S-23S rRNA ISR between the two serotypes. In the pathogenicity test, the serotype 1 strains induced significantly higher mortality than the serotype 2 strains in olive flounder when experimentally inoculated via the intraperitoneal route.

Nonspecific complement activation by streptococcal structures. II. Properdin-independent initiation of the alternate pathway.

Complement consumption by isolated membranes and walls from Group A streptococci and various other gram-positive microbes has been tested. These microbial structures were found to activate the alternate complement pathway. However, unlike endotoxin, inulin, or other plant polysaccharides, activation of complement by our material was found to bypass properdin. The activating factor(s) also differs from cobra venom in its/their requirement for factor D. Preliminary experiments suggest this factor isolated from membranes to be a protein and to have a mol wt greater than 40-60,000 daltons. Our studies have led us to speculate that the phylogenetic role of the alternate complement pathway may be the primordial nonspecific defense system which has retained certain fundamental aspects up to the present time.

A group B streptococcal pilus protein promotes phagocyte resistance and systemic virulence.

Group B Streptococcus (GBS) is a major cause of invasive bacterial infections in newborns and certain adult populations. Surface filamentous appendages known as pili have been recently identified in GBS. However, little is known about the role of these structures in disease pathogenesis. In this study we sought to probe potential functional role(s) of PilB, the major GBS pilus protein subunit, by coupling analysis of an isogenic GBS pilB knockout strain with heterologous expression of the pilB gene in the nonpathogenic bacterium Lactococcus lactis. We found the knockout GBS strain that lacked PilB was more susceptible than wild-type (WT) GBS to killing by isolated macrophages and neutrophils. Survival was linked to the ability of PilB to mediate GBS resistance to cathelicidin antimicrobial peptides. Furthermore, the PilB-deficient GBS mutant was more readily cleared from the mouse bloodstream and less-virulent in vivo compared to the WT parent strain. Strikingly, overexpression of the pilB gene alone in L. lactis enhanced resistance to phagocyte killing, increased bloodstream survival, and conferred virulence in a mouse challenge model. Together these data demonstrate that the pilus backbone subunit, PilB, plays an integral role in GBS virulence and suggests a novel role for gram-positive pili in thwarting the innate defenses of phagocyte killing.

Antimicrobial efficacy of the supernatant of Streptococcus dentisani against microorganisms implicated in root canal infections.

The present study aimed to test the antimicrobial activity of Streptococcus dentisani (S. dentisani) supernatant against a collection of microorganisms implicated in dental root infections, and to analyze morphological changes induced in a selection of the tested microorganisms. A total of 22 microbial species were selected, and their growth was monitored by spectrophotometry in the presence and absence of the supernatant of S. dentisani at different assay concentrations (0.2×, 1×, 2×). The generation time and maximum growth rates were evaluated under every tested condition. Scanning electron microscope (SEM) images were obtained to assess the effect on the cell surface following incubation of the pathogens with the concentrated (2×) supernatant of S. dentisani. The supernatant of S. dentisani was found to exert effective inhibitory activity against most of the studied microorganisms implicated in dental root infections (20 out of 22). Total growth inhibition was observed in the case of Streptococcus oralis, Streptococcus sobrinus, Streptococcus salivarius, Prevotella intermedia, and Streptococcus mutans, while the rest of the microorganisms showed an increase in the generation time (between 30 min and 4 h). SEM images revealed structural changes in the membrane consistent with bacteriocin activity, although the effects were heterogeneous among the different species tested.

Identification of critical residues in Gap3 of Streptococcus parasanguinis involved in Fap1 glycosylation, fimbrial formation and in vitro adhesion.

BACKGROUND: Streptococcus parasanguinis is a primary colonizer of human tooth surfaces and plays an important role in dental plaque formation. Bacterial adhesion and biofilm formation are mediated by long peritrichous fimbriae that are composed of a 200 kDa serine rich glycoprotein named Fap1 (fimbriae-associated protein). Glycosylation and biogenesis of Fap1 are modulated by a gene cluster downstream of the fap1 locus. A gene encoding a glycosylation-associated protein, Gap3, was found to be important for Fap1 glycosylation, long fimbrial formation and Fap1-mediated biofilm formation. RESULTS: Deletion and site-directed mutagenesis were employed to dissect the regions within Gap3 that were important for its function in Fap1 glycosylation and biogenesis. A deletion of 6 consecutive amino acids, PDLPIL, eliminated the production of the mature 200 kDa Fap1 protein and gave rise instead to a 470 kDa Fap1 intermediate that was only partially glycosylated. Site-directed mutagenesis of the 6 amino acids revealed that only three of these amino acids were required. Mutants in these amino acids (L64R, P65R and L67T) produced the premature 470 kDa Fap1 intermediate. Mutants in the remaining amino acids produced the mature form of Fap1. Cell surface expression of the Fap1 precursor among L64R, P65R and L67T mutants was reduced to levels consistent with that of a gap3 insertional mutant. Electron micrographs showed that these 3 mutants lost their long peritrichous fimbriae. Furthermore, their in vitro adhesion ability to saliva-coated hydroxylapatite (SHA) was inhibited. CONCLUSION: Our data suggest that 3 highly conserved, hydrophobic residues L64, P65 and L67 in Gap3 are essential for Gap3 function and are important for complete glycosylation of Fap1, fimbrial formation and bacterial adhesion.

Evaluation of the potential of animal streptococcal isolates belonging to serogroups C and G to elicit acute rheumatic fever.

OBJECTIVE: To determine whether groups C and G streptococci (GCS-GGS) isolated from animals have rheumatogenic traits associated with human GCS-GGS isolates, particularly the potential of the bacteria to interact with human collagen type IV (collagen-IV), known to be targeted during acute rheumatic fever (ARF). SAMPLE POPULATION: 64 GCS and GGS bacterial strains isolated from infected animals. PROCEDURES: Bacteria were analyzed for their ability to bind and aggregate collagen-IV and for the presence of collagen binding factors, such as the hyaluronic acid capsule, cne gene, and emm gene. RESULTS: Collagen-IV binding ability was detected in 19% (n = 12) of the isolates studied. Of the collagen-IV binding strains, 5 expressed hyaluronic acid capsule. Furthermore, emm was detected in the genome of 1 isolate, whereas all remaining collagen-IV binding isolates possessed the cne gene. Of the collagen binding factors investigated, the hyaluronic capsule was the only factor for which collagen-IV interaction could be detected. Investigation of the potential of these strains to aggregate collagen-IV revealed that animal isolates had a nonaggregating phenotype. CONCLUSIONS AND CLINICAL RELEVANCE: Despite efficiently binding collagen-IV via hyaluronic acid, animal isolates lacked the ability to initiate aggregation of this protein. Because collagen-IV aggregation is associated with all collagen-IV-binding rheumatogenic strains, this suggested a lack of rheumatogenic potential among animal-derived GCS and GGS and, therefore, a low chance of acquiring ARF through animal contact.

Capsule expression regulated by a two-component signal transduction system in Streptococcus iniae.

Streptococcus iniae causes disease in fish and humans and the presence of capsule is associated with virulence. Tn917 transposon mutagenesis was performed to identify capsule-associated genes and a mutant was isolated, with an insertion in a genetic locus encoding a two-component signal transduction system (TCS), which we termed sivS/R. sivS and sivR encode a 506-amino-acid (aa) putative histidine kinase and a 223-aa putative response regulator, respectively. In order to investigate the role of sivS/R, a deletion-insertion mutant was constructed using a PCR ligation technique. Real-time PCR showed that transcription of cpsA, the first gene in the S. iniae capsule operon, was reduced in the mutant, indicating that sivS/R regulates expression of this gene at the transcriptional level. Whole human blood killing assays demonstrated that unlike the parent, the mutant was susceptible to phagocytosis. Transmission electron microscopy showed exopolysaccharide on the surface of the parent strain but not the mutant which showed aberrant asymmetric septae that resulted in clumps of abnormal-shaped cells. Exponential growth rates of the mutant and parent strain were similar, although the mutant exhibited a longer lag phase. We conclude that sivS/R regulates capsule expression, thus affecting the ability to evade phagocytosis.

In Vitro and In Vivo Biofilm Formation by Pathogenic Streptococci.

This manuscript presents novel approaches to grow and evaluate Streptococcal biofilm formation using the human respiratory pathogen Streptococcus pneumoniae (the pneumococcus) as the main model organism on biological surfaces in vitro and in vivo. Most biofilm models are based on growth on abiotic surfaces, which is relevant for many pathogens whose growth on surfaces or medical devices is a major cause of disease transmission and infections, especially in hospital environments. However, most infections with commensal organisms require biofilm formation on biological surfaces in the host at the site of colonization or infection. In vitro model systems incorporating biological components from the host and taking into account the host environment of the infectious site are not well described.In a series of publications, we have shown that S. pneumoniae form complex biofilms in the nasopharynx of mice and have devised methodology to evaluate the biofilm structure and function in this environment. We have also been able to recapitulate this biofilm phenotype in vitro by incorporating crucial factors associated with the host environment. Although the protocols presented in this manuscript are focused on S. pneumoniae, the same methodology can and has been used for other Streptococcal species that form biofilms on mucosal surfaces.

Pilus biogenesis of Gram-positive bacteria: Roles of sortases and implications for assembly.

Successful adherence, colonization, and survival of Gram-positive bacteria require surface proteins, and multiprotein assemblies called pili. These surface appendages are attractive pharmacotherapeutic targets and understanding their assembly mechanisms is essential for identifying a new class of 'anti-infectives' that do not elicit microbial resistance. Molecular details of the Gram-negative pilus assembly are available indepth, but the Gram-positive pilus biogenesis is still an emerging field and investigations continue to reveal novel insights into this process. Pilus biogenesis in Gram-positive bacteria is a biphasic process that requires enzymes called pilus-sortases for assembly and a housekeeping sortase for covalent attachment of the assembled pilus to the peptidoglycan cell wall. Emerging structural and functional data indicate that there are at least two groups of Gram-positive pili, which require either the Class C sortase or Class B sortase in conjunction with LepA/SipA protein for major pilin polymerization. This observation suggests two distinct modes of sortase-mediated pilus biogenesis in Gram-positive bacteria. Here we review the structural and functional biology of the pilus-sortases from select streptococcal pilus systems and their role in Gram-positive pilus assembly.

Effects of mot gene expression on the structure of the flagellar motor.

Direct freezing procedures have enabled us to visualize distinctive intramembrane particle ring structures in the cytoplasmic membranes of peritrichously flagellated bacteria by means of freeze-fracture electron microscopy. These structures were identified as flagellar motor components because their distribution matched that of flagella, and because they were absent in non-flagellated mutants of Escherichia coli. Particle rings were present in both the Gram-positive Streptococcus and the Gram-negative E. coli. In E. coli, a non-functional mocha operon produced flagellated but immotile cells lacking the particle rings. Simultaneous introduction of the motA and motB genes, led to recovery of both motility and the ring structures but neither gene alone was sufficient. The concomitant loss of the rings and motility is consistent with the ring particles having a central role in the flagellar motor.

The surface stress theory of microbial morphogenesis.

From the physics of the situation, one might conclude that the osmotic pressure within most prokaryotes creates a sufficiently high tension in the wall that organisms are at risk of ripping themselves apart. The Surface Stress Theory holds that they avoid this, and are able to carry out certain morphogenetic processes by linking the cleavages of appropriate bonds to enzymes that are sensitive to the stress in the bonds under attack. This tends to maintain the internal pressure and couples wall growth to cytoplasmic growth. Mechanisms with widely different geometry function for different organisms, but they have in common the requirement that new murein be covalently linked, and usually in an unextended conformation. Organisms differ in the site of wall addition and site of cleavage. In the Gram-positive Streptococcus, septum formation, and septal splitting occurs with little stretching of the unsplit septum. In Gram-positive bacilli, the cylinder grows by the inside-to-outside mechanism, and the poles appear to be formed by a split-and-stretch mechanism. Gram-negative rods, with their much thinner wall, resist a spherical shape and are capable of cell division by altering the biochemical mechanism so that initially one-third to one-fifth of the pressure-volume work required to increase the area of the side wall is needed to increase that in a developing pole. The growth of hyphae is a separate case; it requires that much less work is needed to force growth of the apex relative to the side wall. Some other bacterial shapes also can be explained by the theory. But at present, it is only a theory, although it is gradually becoming capable of accounting for current observations in detail. Its importance is that it prescribes many experiments that now need to be done.