<Emphasis Type="Italic">Bacillus subtilis</Emphasis> CinA is a stationary phase–induced protein that localizes to the nucleoid and plays a minor role in competent cells

CinA is a conserved bacterial protein that has been reported to play an important role during competence in Streptococcus pneumoniae by recruiting the RecA protein to the cell membrane. Here, we provide information on the homologous CinA in Bacillus subtilis. We found that the synthesis of CinA is upregulated during stationary phase in all cells. The loss of CinA has a mild effect during competence, but it has no influence on the localization of RecA. CinA was observed to be associated with the nucleoid in the cell, and not with the cell membrane, as shown for S. pneumoniae. Purified CinA is a soluble protein, probably forming trimers, like other homologues, which share a domain with CinA that has been reported to be involved in molybdopterine biosynthesis. Our results suggest that CinA plays a nucleoid-associated general role in cells entering stationary phase that is not specific to competence in B. subtilis and possibly in many other bacteria.     

Interspecific plasmid transfer between Streptococcus pneumoniae and Bacillus subtilis

Summary The streptococcal plasmids pMV158 and pLS1, grown in Streptococcus pneumoniae, were transferred to Bacillus subtilis by DNA-mediated transformation. The plasmids were unchanged in the new host; no deletions were observed in 80 instances of transfer. Their copy number was similar to that in S. pneumoniae. Two B. subtilis plasmids, pUB110 and pBD6, could not be transferred to S. pneumoniae. Hybrid plasmids were produced by recombining the EcoRI fragment of pBD6 that confers Km^r with EcoRI-cut pLS1, which confers Tc^r. The simple hybrid, pMP2, was transferable to both species and expressed Tc^r and Km^r in both. A derivative, pMP5, which contained an insertion in the pBD6 component, expressed a higher level of kanomycin resistance and was more easily selected in S. pneumoniae. Another derivative, pMP3, which contained an additional EcoRI fragment, presumably of pneumococcal chromosomal DNA, could not be transferred to B. subtilis. Previous findings that monomeric plasmid forms could transform S. pneumoniae but not B. subtilis were confirmed using single plasmid preparations. Although plasmids extracted from either species were readily transferred to S. pneumoniae, successive passage in B. subtilis increased the ability of plasmid extracts to transfer the plasmid to a B. subtilis recipient. This adaptation was tentatively ascribed to an enrichment of multimeric forms in extracts of B. subtilis as compared to S. pneumoniae. A review of host ranges exhibited by plasmids of Gram-positive bacteria suggested differences in their ability to use particular host replication functions. The pMP5 plasmid, with readily selectable Km^r and Tc^r markers in both hosts, and with the potential for inactivation of Km^r by insertion in the Bg/II site, could be a useful shuttle vector for cloning in S. pneumoniae and B. subtilis.     

Peculiarities of luminescent response of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> recombinant strain bearing cloned <Emphasis Type="Italic">Vibro harveyi lux AB</Emphasis> genes to the action of thermostable blood serum compounds

A strain of Bacillus subtilis previously used as the test-organism in bacteriological and nephelometry methods for detection of thrombocytes cation protein (TCP) has been transformed by a plasmid pLFlux containing cloned luxAB genes of a sea luminescent bacterium Vibro harveyi. The designed luminescent biosensor B. subtilis of The All-Russia Collection of Industrial Microorganisms (Moscow) B-10191 demonstrated specific response to the thermostable fraction of guinea pig blood serum. Sensitivity to chromatographically purified TCP as part of the system has been analyzed and the correlation between luminescence inhibition and direct bacterial effect on the target cells has been demonstrated. The obtained results are considered as the first stage of the design of the bioluminescent technology for TCP detection in biological liquids with complicate composition of the components.     

Antibacterial activity of stevioside towards food‐borne pathogenic bacteria

This study determines the inhibitory effect of Stevia rebaudiana leaf extracts and its purified bioactive compound ‘stevioside’ against food‐related pathogens. The S. rebaudiana solvent extracts (1000 μg/mL) displayed antibacterial activity to Serratia marcescens, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, B. subtilis, Alcaligenes denitrificans and Salmonella typhimurium. Of the six solvents, ethanol and acetone extracts displayed the highest zone of inhibition. The bioactive compound from S. rebaudiana was purified by solvent extraction, thin‐layer chromatography followed by structural characterization by spectroscopy evidence. Purified stevioside prevented the growth of tested bacterial species, i.e. B. subtilis, K. pneumoniae and S. typhimurium. Significant zone of inhibition (12 mm) was observed against B. cereus which proposes potential application of stevioside in foods to increase their shelf life.     

Glycogen from Klebsiella pneumoniae M 5 al and Escherichia coli K 12

Summary 1. A continuous two stage cultivation method for two strains of Klebsiella pneumoniae and Escherichia coli yielding high cell mass and relatively high glycogen contents is described. The stage 1 cells (carbon-limited) were fed with the nitrogen source ammonia (which also neutralized simultaneously) soly via the pH-stat. In stage 2, the cells grew nitrogen-limited, a small excess of the carbon source was maintained by continuous addition of a glucose solution.2. Through the action of lysozyme, the glycogen could be quantitatively solubilized from the alkali-insoluble cell material obtained through alkaline hydrolysis of the cells in dimethylsulfoxide-3M aqueous potassium hydroxide. The possibility that the glycogen is in part covalently linked to the peptidoglycan and localized in the periplasm is discussed. 3. Analytical data for the glycogens isolated from the two bacterial strains is given.     

Pherotypes are driving genetic differentiation within Streptococcus pneumoniae

Background  

The boundaries of bacterial species and the mechanisms underlying bacterial speciation are matters of intense debate. Theoretical studies have shown that recombination acts as a strong cohesive force preventing divergence in bacterial populations. Streptococcus pneumoniae populations have the telltale signs of high recombination with competence implicated as the major driving force behind gene exchange. Competence in S. pneumoniae is triggered by a quorum-sensing mechanism controlled by the competence-stimulating peptide pheromone.     

Survival of Plasmid-Containing Bacillus subtilis Released into Mushroom Compost

Abstract The survival of a plasmid-containing Bacillus subtilis released into mushroom compost was investigated. The indigenous Bacillus population of mushroom compost exhibited an antibiotic-resistance profile that was distinguished by almost complete absence of chloramphenicol resistance. Bacillus subtilis containing the chloramphenicol-resistance plasmid pC194 was released into mushroom compost microcosms and populations were monitored at different incubation temperatures. The organism colonized both sterile and untreated compost at 37°C, and to a lesser extent at 50°C, but was eliminated after 30 d at 65°C. Although sporulation of the B. subtilis population occurred within compost, the population was maintained for up to 13 weeks at 50°C, largely as vegetative cells. Experiments in which the B. subtilis host strain, without plasmid, was released demonstrated that plasmid carriage had no effect on the ability of the bacterium to colonize and survive in compost. Furthermore, the size and composition of the indigenous bacterial population was unaffected by the presence of the introduced B. subtilis strain. Virtually no loss of plasmid pC194 from the B. subtilis population in compost was observed, and experiments at low growth rates in chemostats confirmed the stability of this host/vector system in the absence of positive selection pressure. Received: 9 July 1997; Accepted: 20 October 1997     

Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis,Streptococcus pneumoniae,and Lactococcus lactis for Live Cell Imaging

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis, S. pneumoniae, and Lactococcus lactis, using promoter-gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.     

FINE-SCALE GENETIC AND PHENOTYPIC STRUCTURE IN NATURAL POPULATIONS OF BACILLUS SUBTILIS AND BACILLUS LICHENIFORMIS: IMPLICATIONS FOR BACTERIAL EVOLUTION AND SPECIATION

The genetic and phenotypic structure of sympatric populations of wild bacteria traditionally identified as Bacillus subtilis and B. licheniformis was analyzed. Small soil samples were taken from a single, tiny site in the Sonoran Desert of Arizona, USA, to provide a true population analysis, in contrast to many analyses of genetic structure using bacterial strain collections of widely heterogeneous origin. Genetic analyses of isolates used multilocus enzyme electrophoresis, mismatches in restriction fragment length polymorphism, and variants from Southern hybridization with B. subtilis DNA probes. Phenotypic analyses of isolates used the API test system for detection of growth and acid production on specific carbon sources. The two species were distinct both phenotypically and genetically, despite their known potential for genetic exchange in laboratory experiments. Genic and genotypic diversity were high in both species, and only 16% of observed allozyme variants might possibly be common to both species. Hence, there is probably modest genetic exchange, if any, between the species in nature. Clear hierarchies of population-genetic structure were found for both species. Different types of genetic data yield concordant population structures for B. subtilis. For both species, two-locus and multilocus statistical analyses of linkage demonstrated modest to strong disequilibrium at the species level but truly panmictic subunits within each species. The evidence for extensive genetic recombination within these fine-scale subdivisions is unequivocal, indicating that the sexuality of these bacteria can be well expressed in nature. The relation of these results to processes of bacterial evolution and speciation is discussed.     

Role of nutrient limitation in the competition between uropathogenic strains of Klebsiella pneumoniae and Escherichia coli in mixed biofilms

Klebsiella pneumoniae and Escherichia coli form mixed species biofilms in catheter-associated urinary tract infections. Recently, a detrimental effect of K. pneumoniae over E. coli was observed in mixed species biofilms grown in an artificial urine medium. The mechanism behind this competitive interaction was studied. K. pneumoniae partially outcompeted E. coli in early-stage batch-fed biofilms, whereas both microorganisms co-exist at longer times (K. pneumoniae:E. coli ratio, 55:1), as shown by cell counts and confocal microscopy. E. coli cells were scattered along the K. pneumoniae biofilm. Biofilm supernatants did not appear to contain either antimicrobial or anti-biofilm activities against E. coli. Biofilms grown under continuous flow prevented interspecies competition. K. pneumoniae showed both increased siderophore production and better growth in iron-limited media compared to E. coli. In summary, these results indicate the importance of nutrient (particularly iron) competition in the modulation of the bacterial composition of mixed species biofilms formed by uropathogenic K. pneumoniae and E. coli.     

Bacillus subtilis as a host for mosquitocidal toxins production

Aedes albopictus transmits several arboviral infections. In the absence of vaccines, control of mosquito populations is the only strategy to prevent vector-borne diseases. As part of the search for novel, biological and environmentally friendly strategies for vector control, the isolation of new bacterial species with mosquitocidal activity represents a promising approach. However, new bacterial isolates may be difficult to grow and genetically manipulate. To overcome these limits, here we set up a system allowing the expression of mosquitocidal bacterial toxins in the well-known genetic background of Bacillus subtilis. As a proof of this concept, the ability of B. subtilis to express individual or combinations of toxins of Bacillus thuringiensis israelensis (Bti) was studied. Different expression systems in which toxin gene expression was driven by IPTG-inducible, auto-inducible or toxin gene-specific promoters were developed. The larvicidal activity of the resulting B. subtilis strains against second-instar Ae. albopictus larvae allowed studying the activity of individual toxins or the synergistic interaction among Cry and Cyt toxins. The expression systems here presented lay the foundation for a better improved system to be used in the future to characterize the larvicidal activity of toxin genes from new environmental isolates.     

Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Phospholipid biosynthesis commences with the acylation of glycerol-3-phosphate (G3P) to form 1-acyl-G3P. This step is catalyzed by the PlsB protein in Escherichia coli. The gene encoding this protein has not been identified, however, in the majority of bacterial genome sequences, including that of Bacillus subtilis. Recently, a new two-step pathway catalyzed by PlsX and PlsY proteins for the initiation of phospholipid formation in Streptococcus pneumoniae has been reported.     

Whole-genome analysis of Klebsiella pneumoniae from bovine mastitis milk in the U.S.

Dairy cattle mastitis has long been one of the most common and costly diseases in the dairy industry worldwide, due to its significant impact on milk production and animal welfare. Among all mastitis causing bacterial pathogens, Klebsiella pneumoniae causes the largest milk loss. To better understand the genomic features of this population, 180 K. pneumoniae strains isolated from dairy cattle mastitis milk in 11 U.S. states were sequenced. The phylogenetic analysis classified all mastitis-causing K. pneumoniae into two major phylogroups, with exclusive predominance in phylogroup KpI. Analysis of more than 61 sequence types, 51 capsular types and 12 lipopolysaccharide O-antigen types revealed great genomic diversity of this K. pneumoniae population. Approximately 100 gene units in accessory genomes were detected with significantly higher prevalence in bovine mastitis strains, compared to human-sourced or dairy environmental strains. The most notable genes were identified associated with ferric citrate uptake, lactose fermentation and resistance to heavy metals. The acquired antimicrobial resistance genes were identified in sporadic mastitis strains. This comprehensive genomic epidemiological study provides insights for a better understanding of the virulence of mastitis-causing K. pneumoniae strains and may lead to the development of novel diagnostic tools and preventive strategies.     

肺炎克雷伯菌噬菌体解聚酶的研究进展

肺炎克雷伯菌(Klebsiella pneumoniae)是在临床引起多种感染的常见条件致病菌之一。多重耐药肺炎克雷伯菌株的出现,给防控细菌感染带来了巨大阻力。肺炎克雷伯菌噬菌体编码的解聚酶是一种稳定性高、特异性强的生物酶,具有分解细菌胞外多糖、限制细菌生长等多种功能。解聚酶可为防控肺炎克雷伯菌感染提供新思路,在抗菌应用中具有广阔前景。本文就肺炎克雷伯菌噬菌体解聚酶的研究进展进行综述。     

The role of bacteria in retting of desiccated flax during damp weather

Summary The bacteria present on samples of desiccated flax stems were Bacillus mycoides, B. subtilis, Erwinia carotovora, Pseudomonas fluorescens, P. putida and Micrococcus sp. and the fungi present were Cladosporium herbarum, Fusarium culmorum, Botrytis cineria, Epicoccum nigrum and yeast. When inoculated on autoclaved stems or in liquid culture, B. subtilis produced mainly pectin-lyase and xylanase. However, only pectin-lyase was detected in significant levels in autoclaved stem sections or in liquid cultures inoculated with E. carotovora. Enhanced pectin-lyase and xylanase levels were detected in field-retted stems sprayed with B. subtilis compared with enzyme levels in stems sprayed with E. carotovora or the control stem tissues. Increases in the fungal population coincided with a reduction in the bacterial population on treated stems at the later part of retting. Enhanced retting was observed in stems sprayed with B. subtilis and consequently the stems produced finer fibres than fibres from E. carotovora-sprayed or control stems.     

Two distinct sensing pathways allow recognition of Klebsiella pneumoniae by Dictyostelium amoebae

Recognition of bacteria by metazoans is mediated by receptors that recognize different types of microorganisms and elicit specific cellular responses. The soil amoebae Dictyostelium discoideum feeds upon a variable mixture of environmental bacteria, and it is expected to recognize and adapt to various food sources. To date, however, no bacteria‐sensing mechanisms have been described. In this study, we isolated a Dictyostelium mutant (fspA KO) unable to grow in the presence of non‐capsulated Klebsiella pneumoniae bacteria, but growing as efficiently as wild‐type cells in the presence of other bacteria, such as Bacillus subtilis. fspA KO cells were also unable to respond to K. pneumoniae and more specifically to bacterially secreted folate in a chemokinetic assay, while they responded readily to B. subtilis. Remarkably, both WT and fspA KO cells were able to grow in the presence of capsulated LM21 K. pneumoniae, and responded to purified capsule, indicating that capsule recognition may represent an alternative, FspA‐independent mechanism for K. pneumoniae sensing. When LM21 capsule synthesis genes were deleted, growth and chemokinetic response were lost for fspA KO cells, but not for WT cells. Altogether, these results indicate that Dictyostelium amoebae use specific recognition mechanisms to respond to different K. pneumoniae elements.     

Galactocerebroside biosynthesis pathways of Mycoplasma species: an antigen triggering Guillain–Barré–Stohl syndrome

Infection by Mycoplasma pneumoniae has been identified as a preceding factor of Guillain–Barré–Stohl syndrome. The Guillain–Barré–Stohl syndrome is triggered by an immune reaction against the major glycolipids and it has been postulated that M. pneumoniae infection triggers this syndrome due to bacterial production of galactocerebroside. Here, we present an extensive comparison of 224 genome sequences from 104 Mycoplasma species to characterize the genetic determinants of galactocerebroside biosynthesis. Hidden Markov models were used to analyse glycosil transferases, leading to identification of a functional protein domain, termed M2000535 that appears in about a third of the studied genomes. This domain appears to be associated with a potential UDP-glucose epimerase, which converts UDP-glucose into UDP-galactose, a main substrate for the biosynthesis of galactocerebroside. These findings clarify the pathogenic mechanisms underlining the triggering of Guillain–Barré–Stohl syndrome by M. pneumoniae infections.     

Cell Surface Display of the Chlamydial Glycolipid Exoantigen (GLXA) Demonstrated by Antibody-Dependent Complement-Mediated Cytotoxicity

The chlamydial species are Gram-negative bacterial pathogens critical to human health. Their developmental cycle is associated with the formation and release of the broadly conserved glycolipid exoantigen (GLXA), which has been implicated in the chlamydial elementary body–host cell interaction. This study examines the potential surface display of this glycolipid by chlamydiae-infected cells and the ability of the GLXA they secrete to associate with the plasma membranes of uninfected cells, a prerequisite for exerting influence on them. The sequential incubation of anti-GLXA antibody and complement with Chlamydia trachomatis serovar K or C. pneumoniae AR-39-infected HeLa 229 or macrophage cells resulted in significant cellular cytotoxicity, which preceded the formation of mature elementary bodies. For uninfected cells, co-incubation of GLXA, purified from supernatants of either C. trachomatis or C. pneumoniae-infected HeLa 229 cells, followed by the successive addition of mouse anti-GLXA antibody and complement, yielded similar levels of cellular cytotoxicity. Thus, GLXA indeed is displayed on the surface of infected cells and, therefore, if antibody of appropriate specificity were present, this GLXA could serve to target these infected cells for elimination. Furthermore, released GLXA can associate with uninfected cells and therefore would be positioned to influence their behavior, especially in the context of infection.     

Essentiality and function of WalK/WalR two-component system: the past,present, and future of research*

The WalK/WalR two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved in gram-positive bacteria, including several important pathogens. The WalK/WalR TCS appears to be involved in the growth of most bacterial species encoding it. Previous studies have indicated conserved functions of this system, defining this signal transduction pathway as a crucial regulatory system for cell wall metabolism. Because of such effects on essential functions, this system is considered a potential target for anti-infective therapeutics. In this review, we discuss the role of WalK/WalR TCS in different bacterial cells, focusing on the function of the genes in its regulon as well as the variations in walRK operon structure, its auxiliary proteins, and the composition of its regulon. We also discuss recent experimental data addressing its essential function and the potential type of signal being sensed by B. subtilis. This review also focuses on the potential future research.     

Role of interspecies interactions in dual-species biofilms developed in vitro by uropathogens isolated from polymicrobial urinary catheter-associated bacteriuria

Most catheter-associated urinary tract infections are polymicrobial. Here, uropathogen interactions in dual-species biofilms were studied. The dual-species associations selected based on their prevalence in clinical settings were Klebsiella pneumoniae–Escherichia coli, E. coli–Enterococcus faecalis, K. pneumoniae–E. faecalis, and K. pneumoniae–Proteus mirabilis. All species developed single-species biofilms in artificial urine. The ability of K. pneumoniae to form biofilms was not affected by E. coli or E. faecalis co-inoculation, but was impaired by P. mirabilis. Conversely, P. mirabilis established a biofilm when co-inoculated with K. pneumoniae. Additionally, E. coli persistence in biofilms was hampered by K. pneumoniae but not by E. faecalis. Interestingly, E. coli, but not K. pneumoniae, partially inhibited E. faecalis attachment to the surface and retarded biofilm development. The findings reveal bacterial interactions between uropathogens in dual-species biofilms ranged from affecting initial adhesion to outcompeting one bacterial species, depending on the identity of the partners involved.