巴西固氮螺菌Yu62由水稻和烟草根部向茎、叶的迁移运动(英文)

巴西固氮螺菌(Azospirillum brasilence)是重要的植物促生内生菌之一。用gfp基因标记固氮螺菌后接种无菌的水稻和烟草幼苗的根部,限菌培养一定时间后,用共聚焦激光显微镜观察,结果表明:除了根内部有发荧光的螺菌定殖外,螺菌还分布在茎、叶的表皮细胞,皮层细胞和维管系统组织的细胞和细胞间隙。从根、茎、叶器官分离固氮螺菌,都存在有较高的螺菌群体密度。这一结果证明螺菌在植物内存在着从根部向茎、叶顶端的迁移现象。这一发现为研究巴西固氮螺菌在宿主植物体内的迁移运动的机制、与植物细胞间的分子相互作用及其对植物的促生作用奠定了生态学和细胞形态学的基础,也为实际应用提供了进一步的科学依据,具有重要的科学和实践意义。     

A loss of resistance to avirulent bacterial pathogens in tobacco is associated with the attenuation of a salicylic acid-potentiated oxidative burst

The role of salicylic acid (SA) in events occurring before cell death during the hypersensitive reaction (HR) was investigated in leaves of wild-type tobacco Samsun NN and in transgenic lines expressing salicylate hydroxylase (35S-SH-L). Challenge of 35S-SH-L tobacco with avirulent strains of Pseudomonas syringae gave rise to symptoms resembling those normally associated with a compatible response to virulent strains in terms of visible phenotype, kinetics of bacterial multiplication, and escape from the infection site. Compared with responses in wild-type tobacco, both the onset of plant cell death and the induction of an active oxygen species-responsive promoter (AoPR1-GUS) were delayed following challenge of 35S-SH-L plants with avirulent bacteria. The oxidative burst occurring after challenge with avirulent bacteria was visualized histochemically and quantified in situ. H2O2 accumulation at reaction sites was evident within 1 h after inoculation in wild-type tobacco, whereas in 35S-SH-L plants the onset of H2O2 accumulation was delayed by 2-3 h. The delay in H2O2 generation was correlated with a reduction in the transient rise in SA that usually occurred within 1-2 h following inoculation in wild-type plants. Our data indicate that an early transient rise in SA potentiates the oxidative burst, with resultant effects on accumulation of H2O2, plant cell death and also defence-gene induction, factors that together may determine the outcome of plant-pathogen interactions.     

巴西固氮螺菌Yu62由水稻和烟草根部向茎、叶的迁移运动

巴西固氮螺菌(Azospirillrm brasilence)是重要的植物促生内生菌之一.用gfp基因标记固氮螺菌后接种无菌的水稻和烟草幼苗的根部,限制培养一定时间后,用共聚焦激光显微镜观察,结果表明:除了根部有发荧光的螺菌定殖外,螺菌还分布在茎、叶的表皮细胞,皮层细胞和维管系统组织的细胞间隙.从根、茎、叶器官分离固氮螺菌,都存在有较高的螺菌群体密度.这一结果证明螺菌在植物内存在着从根部向茎、叶顶端的迁移现象.这一发现为研究巴西固氮螺菌在窠主植物体内的迁移运动的机制、与植物细胞间的分子相互作用及其对植物的促生作用奠定了生态学和细胞形态学的基础,也为实际应用提示了进一步的科学依据,具有重要的科学和实践意义.     

Expression of a human lactoferrin cDNA in tobacco cells produces antibacterial protein(s).

A suspension tobacco (Nicotiana tabacum L.) cell line was transformed to express human lactoferrin, an iron-binding glycoprotein. The transgenic calli produced a protein that was significantly smaller than the full-length lactoferrin protein. Total protein extracts made from transgenic tobacco callus exhibited much higher antibacterial activity than commercially available purified lactoferrin as determined by the decrease of colony-forming units when tested with four phytopathogenic species of bacteria. Introduction of the lactoferrin gene in crop plants may provide resistance against phytopathogenic bacteria.     

Interactions of Pseudomonas syringae pv. glycinea with host and nonhost plants in relation to temperature and phytotoxin synthesis

Pseudomonas syringae pv. glycinea PG4180 causes bacterial blight of soybean and produces the phytotoxin coronatine (COR) in a temperature-dependent manner. COR consists of a polyketide, coronafacic acid (CFA), and an amino acid derivative, coronamic acid, and is produced optimally at 18 degrees C whereas no detectable synthesis occurs at 28 degrees C. We investigated the impact of temperature on PG4180 during compatible and incompatible interactions with soybean and tobacco plants, respectively. After spray inoculation, PG4180 caused typical bacterial blight symptoms on soybean plants when the bacteria were grown at 18 degrees C prior to inoculation but not when derived from cultures grown at 28 degrees C. The disease outcome was quantified by determination of bacterial populations in planta. The temperature effect was not observed when PG4180 was artificially infiltrated into soybean leaves, indicating that the pre-inoculation temperature and phytotoxin synthesis were important for bacterial invasion via natural plant openings. In the incompatible interaction, PG4180 elicited the hypersensitive response (HR) on tobacco plants regardless of the bacterial pre-inoculation temperature. However, the HR was significantly delayed when tobacco plants were treated with cells of the CFA-overproducing derivative, PG4180.N9, which were derived from cultures grown at 18 degrees C, compared with parallels incubated at 28 degrees C. CFA biosynthesis by PG4180.N9 was optimal at 18 degrees C and negligible at 28 degrees C. The impact of CFA synthesis on the HR was studied with different growth media, mutants, and transconjugants of PG4180, indicating that the amount of synthesized CFA but not that of COR influenced the outcome of the HR. Feeding experiments with purified coronafacoyl compounds suggested that the observed delay of the HR was mediated by CFA, shedding further light on CFA's putative role as a molecular mimic of the plant signaling molecule, jasmonic acid.     

Early Detection of Bacterium-induced Basal Resistance in Tobacco Leaves with Diaminobenzidine and Dichlorofluorescein Diacetate

The present study demonstrate that in tobacco leaves the diaminobenzidine (DAB) and 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) staining is a useful indicator of the basal (also known as general or innate) defence‐associated reactions, especially of the early developing form of basal resistance (EBR). DAB and DCFH‐DA, in the presence of H₂O₂ and peroxidase converts to a brown polymer and fluorescent DCF respectively. In the present study, the hypersensitive response (HR)‐inducing avirulent Pseudomonas syringae pv. syringae 61, its HR‐negative hrp/hrc mutants and even non‐pathogenic bacteria such as P. fluorescens and Escherichia coli caused DAB and DCFH‐DA staining, if the dyes were injected 3–4 h after bacterial inoculation into tobacco leaves. The conditions that enable the staining of plant leaves infiltrated with HR‐negative bacteria were persisted for 1 to several days depending on the physiological state of the plant, and plant activity was required to the development of the staining. The live virulent P. syringae pv. tabaci was able to suppress the development of the staining reaction. Bacteria that induced more intensive staining reaction triggered stronger local resistance response, which was verified by its ability to inhibit the HR by challenging avirulent bacteria and by expression analysis of genes that are activated during the basal defence response. The peroxidase enzyme activity increased in bacterially treated tobacco tissue, and inhibition of peroxidase activity blocked the development of the staining. The results showed that in tobacco leaves the staining reactions were associated with the general recognition and basal defence reaction of tobacco plant and can be used as markers in tobacco leaves for testing the occurrence of this type of defence.     

Physiological Diversity of Rhizoplane Diazotrophs of the Saltmeadow Cordgrass,Spartina patens: Implications for Host Specific Ecotypes

Diazotrophic bacteria are important contributors to salt marsh productivity, but the biotic and abiotic factors that influence their distributions and function and the extent of their diversity cannot be understood in the absence of physiological information. Here we examine the physiological diversity and distribution patterns of diazotrophic bacteria associated with the rhizoplane of the saltmeadow cordgrass, Spartina patens, in comparison with diazotrophs from other intertidal grasses (tall and short form Spartina alterniflora and Juncus roemerianus) from the same salt marsh. S. patens plants were collected from two distinct habitats, and a total of 115 strains (111 Gram negative and 4 Gram positive strains) were isolated into pure culture by stab inoculating roots and rhizomes into combined nitrogen-free semisolid media. Most strains were microaerophilic and approximately one-half were motile. API test strips were used to eliminate redundancy within the culture collection, resulting in 21 physiologically different API groups (17 Gram negative and 4 Gram positive groups). A representative strain from each API group was selected for dot blot hybridization with a nifH specific probe and 16 strains (13 Gram negative and 3 Gram positive) were scored as positive. The nifH positive API group representative strains were characterized further using BIOLOG test plates. Substrate utilization potentials defined two S. patens strain clusters, and only one S. patens strain was physiologically similar to any other strain from a different host plant origin. No distinctions could be made based on the different S. patens habitats, suggesting that the host plant may have a greater impact than abiotic environmental conditions on the distributions of the rhizoplane diazotrophs recovered.     

Flagellin glycosylation island in Pseudomonas syringae pv. glycinea and its role in host specificity

The deduced amino acid sequences of the flagellins of Pseudomonas syringae pv. tabaci and P. syringae pv. glycinea are identical; however, their abilities to induce a hypersensitive reaction are clearly different. The reason for the difference seems to depend on the posttranslational modification of the flagellins. To investigate the role of this posttranslational modification in the interactions between plants and bacterial pathogens, we isolated genes that are potentially involved in the posttranslational modification of flagellin in P. syringae pv. glycinea (glycosylation island); then defective mutants with mutations in these genes were generated. There are three open reading frames in the glycosylation island, designated orf1, orf2, and orf3. orf1 and orf2 encode putative glycosyltransferases, and mutants with defects in these open reading frames, deltaorf1 and deltaorf2, secreted nonglycosylated and slightly glycosylated flagellins, respectively. Inoculation tests performed with these mutants and original nonhost tobacco leaves revealed that deltaorf1 and deltaorf2 could grow on tobacco leaves and caused symptom-like changes. In contrast, these mutants failed to cause symptoms on original host soybean leaves. These data indicate that putative glycosyltransferases encoded in the flagellin glycosylation island are strongly involved in recognition by plants and could be the specific determinants of compatibility between phytopathogenic bacteria and plant species.     

Processes of plant colonization by Methylobacterium strains and some bacterial properties

The pink-pigmented facultative methylotrophic bacteria (PPFMB) of the genus Methylobacterium are indespensible inhabitants of the plant phyllosphere. Using maize Zea mays as a model, the ways of plant colonization by PPFMB and some properties of the latter that might be beneficial to plants were studied. A marked strain, Methylobacterium mesophilicum APR-8 (pULB113), was generated to facilitate the detection of the methylotrophic bacteria inoculated into the soil or applied to the maize leaves. Colonization of maize leaves by M. mesophilicum APR-8 (pULB113) occurred only after the bacteria were applied onto the leaf surface. In this case, the number of PPFMB cells on inoculated leaves increased with plant growth. During seed germination, no colonization of maize leaves with M. mesophilicum cells occurred immediately from the soil inoculated with the marked strain. Thus, under natural conditions, colonization of plant leaves with PPFMB seems to occur via soil particle transfer to the leaves by air. PPFMB monocultures were not antagonistic to phytopathogenic bacteria. However, mixed cultures of epiphytic bacteria containing Methylobacterium mesophilicum or M. extorquens did exhibit an antagonistic effect against the phytopathogenic bacteria studied (Xanthomonas camprestris, Pseudomonas syringae, Erwinia carotovora, Clavibacter michiganense, and Agrobacterium tumifaciens). Neither epiphytic and soil strains of Methylobacterium extorquens, M. organophillum, M. mesophilicum, and M. fujisawaense catalyzed ice nucleation. Hence, they cause no frost injury to plants. Thus, the results indicate that the strains of the genus Methylobacterium can protect plants against adverse environmental factors.     

Influence of Immigration on Epiphytic Bacterial Populations on Navel Orange Leaves

Factors that influenced the increase in epiphytic bacterial population size on navel orange leaves during winter months were investigated to test the assumption that such populations were the result of multiplication on orange leaves. The population sizes of bacteria of different kinds, including ice nucleation-active (Ice(sup+)) bacteria, were from 6- to 30-fold larger on leaves of navel orange trees adjacent to other plant species than on trees growing near other citrus species. Total and Ice(sup+) bacterial population sizes on other plant species growing near navel orange trees were from 18- to 60-fold and 2- to 18,000-fold larger, respectively, than on navel orange trees. About twice the number of bacterial cells of a given type were deposited onto petri dishes opened simultaneously in navel orange orchards with other plant species nearby as in orchards surrounded by citrus trees. Epiphytic bacteria and airborne bacteria were more numerous near the upwind edge of orchards bordering on other plant species, but not in orchards adjacent to other citrus trees, and decreased with distance from other plant species. Navel orange leaves also exhibited progressive increases in the ability to supercool as a function of increasing distance from the upwind edge of orchards adjacent to other plant species but not in orchards adjacent to other citrus trees. While the population size of three different bacterial strains remained nearly constant for 60 days after inoculation, total bacterial populations increased more than 50-fold during this period. These results suggest that immigration of bacteria from plants having high epiphytic bacterial populations could account for most, if not all, of the seasonal increase in bacterial populations on navel orange leaves and have important implications for procedures to modify bacterial communities on leaves.     

A peroxiredoxin Q homolog from gentians is involved in both resistance against fungal disease and oxidative stress

An antifungal protein (GtAFP1) showing antimicrobial activity against phytopathogenic fungi was purified from leaves of Gentiana triflora. The deduced amino acid sequence of the cDNA of the corresponding gene, GtAFP1, showed 94, 75, 72 and 63% amino acid identities with peroxiredoxin Q from Populus balsamifera x P. deltoides subsp. trichocarpa, Sedum lineare, Suaeda maritima and Arabidopsis thaliana, respectively. The GtAFP1 gene is suggested to be present in the genome in one to two copies and was expressed in the leaves, roots and stems. Expression of GtAFP1 was induced by treatment with salicylic acid, but not methyl jasmonate. Recombinant GtAFP1 protein showed not only antifungal activity but also thioredoxin-dependent peroxidase activity. Overexpression of GtAFP1 in tobacco plants improved tolerance not only against fungal diseases but also against oxidative stress. These results indicate that GtAFP1 might act as a disease and oxidative stress defensive gene in plants and could be useful for engineering stress-resistant plants.     

Copper Oxide Nanomaterials Derived from Zanthoxylum armatum DC. and Berberis lycium Royle Plant Species: Characterization,Assessment of Free Radical Scavenging and Antibacterial Activity

Copper oxide nanomaterials were synthesized by a facile sustainable biological method using two plant species (Zanthoxylum armatum DC. and Berberis lycium Royle ). The formation of materials was confirmed by FT‐IR, ATR, UV‐visible, XRD, TEM, SEM, EDX, TGA and PL. The antibacterial activity was evaluated by agar well diffusion method to ascertain the efficacy of plant species extract and extract derived copper oxide nanomaterials against six Gram‐positive bacteria namely Staphylococcus aureus, Streptococcus mutans, Streptococcus pyogenes, Corynebacterium diphtheriae, Corynebacterium xerosis, Bacillus cereus and four Gram‐negative bacteria such as Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris against the standard drug, Ciprofloxacin for Gram‐positive and Gentamicin for Gram‐negative bacteria, respectively. In both cases, copper oxide nanomaterials were found to be sensitive in all the bacterial species. Sensitivity of copper oxide nanomaterials shows an be higher as compared to plant species extract against different bacteria. Scavenging activity of plant extracts along with nanomaterials have been accessed using previously reported protocols employing ascorbic acid as standard. Scavenging activity of copper oxide nanomaterials shows an increase with increase in concentration. The biological activity (bactericidal and scavenging efficiency) of plant derived copper oxide nanomaterials revealed that these materials can be used as potent antimicrobial agent and DPPH scavengers in industrial as well as pharmacological fields.     

Antifungal and phytostimulating characteristics of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Ch-13 rhizospheric strain,producer of bioprepations

Bacillus subtilis Ch-13 industrial strain was shown to have a wide spectrum of antagonistic activities against different species of phytopathogenic fungi and bacteria. The B. subtilis Ch-13 strain produces lytic enzymes; cyanide and other antifungal metabolites; stimulates plant growth, producing phytohormones—auxin derivatives. This strain by 2.5 times reduced the quantity of tomato plants infected with phytopathogenic fungus Fusarium oxysporum during inoculation. Fungi abundance on roots with bacterial inoculation was 6.9 times less than in the absence of inoculation. The application of detected antifungal metabolites as biochemical markers for the strain enables to control the stability of physiologic and biochemical characteristics of the producer, and ensures a rapid quality assay of biopreparations with high performance liquid chromatography (HPLC).     

Role of polyisoprenoids in tobacco resistance against biotic stresses

Infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. tabaci induced accumulation of polyisoprenoid alcohols, solanesol and a family of polyprenols [from polyprenol composed of 14 isoprene units (Pren-14) to -18, with Pren-16 dominating] in the leaves of resistant tobacco plants Nicotiana tabacum cv. Samsun NN. Upon TMV infection, solanesol content was increased seven- and eight-fold in the inoculated and upper leaves, respectively, while polyprenol content was increased 2.5- and 2-fold in the inoculated and upper leaves, respectively, on the seventh day post-infection. Accumulation of polyisoprenoid alcohols was also stimulated by exogenously applied hydrogen peroxide but not by exogenous salicylic acid (SA). On the contrary, neither inoculation of the leaves of susceptible tobacco plants nor wounding of tobacco leaves caused an increase in polyisoprenoid content. Taken together, these results indicate that polyisoprenoid alcohols might be involved in plant resistance against pathogens. A putative role of accumulated polyisoprenoids in plant response to pathogen attack is discussed. Similarly, the content of plastoquinone (PQ) was increased two-fold in TMV-inoculated and upper leaves of resistant plants. Accumulation of PQ was also stimulated by hydrogen peroxide, bacteria ( P.  syringae ) and SA. The role of PQ in antioxidant defense in cellular membranous compartments is discussed in the context of the enzymatic antioxidant machinery activated in tobacco leaves subjected to viral infection. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase was found in inoculated leaves of resistant tobacco plants but not in susceptible plants.     

Characteristics of infection of plants and their cultured tissue with cyanobacterial-bacterial symbiotic associations]

The infection of tobacco, nightshade, rice plants, and their tissue cultures with the cyanobacteria-bacteria symbiotic associations (CBSA) isolated from natural syncyanoses (the ferns Azolla pinnata and Azolla sp. and the cycad Encephalartos ferox) was studied. The inoculation of the intact plants or their cuttings with CBSA led to the colonization of the plant roots, stems, and leaves by cyanobacteria and their bacterial symbionts (referred to as satellite bacteria, SB). The sites of the long-term contact of plant organs with cyanobacteria were characterized by the formation of copious slime. On the roots of infected plants, one could observe the callus growth of cortical parenchyma cells and the formation of pseudonodules, in which SB cells gradually accumulated. In mixed cultures of plant callus tissues and the CBSA isolated from the ferns A. pinnata and Azolla sp., the callus tissue specifically influenced the growth of the CBSA components, causing (depending on the plant species and strain) either their balanced growth, or their cyclic growth, or the predominant growth of one of the CBSA components (either cyanobacteria or satellite bacteria). This phenomenon is proposed to be used for the dissociation of stable multicomponent natural symbiotic complexes and the selection of their particular components.     

Factors associated with detrimental effects of rhizobacteria on plant growth

Some of the factors interfering with the specific response of young common bean plants to two rhizosphere fluorescent pseudomonads were studied. These two bacterial strains produced symptoms in foliar plant parts and reduced yield in beans and several other plant species when inoculated on roots. Sensitivity in the plants subjected to bacterial application was highest at early growth stages (up to ten days old plants) giving typical symptoms for each strain in first expanding leaves and typically stunted plant growth. Symptoms to some extent also appeared on plants treated at the age of 2–3 weeks and the fresh shoot weight was affected also in such plants. Deleterious effects of the tested bacteria were found to be related to the level of inoculum (cfu/ml) used and presence or absence of certain nutrients in the bacterial suspensions used for inoculation. The two tested strains differed in nutritional requirements for affecting plant growth. One strain needed sucrose only. The other tested strain required peptone or yeast extract in addition to sucrose to induce typical symptoms and significant yield reductions. Supply of peptone as the only nutrient source to the bacterial suspensions eliminated the deleterious effects of both bacterial strains as tested on beans under nonsterile conditions. The two strains are assumed to affect test plants by different modes of action.     

Evaluation and Risk Management by Using Ocimum bacillicum Against Waterborne Pathogens

Food and medicinal plants have been used for centuries against human diseases. The methanolic and aqueous extracts of different parts of Ocimum bacillicum Lam. (Niazboo or Sweet basil) of family Lamiaceae were investigated in vitro for their antibacterial properties against waterborne pathogens. The antibacterial properties of different parts of Ocimum bacillicum samples were assessed against Gram positive and Gram negative bacteria, which were isolated from water samples and identified by the API 20E method. The extracts from leaves and seeds of Ocimum bacillicum showed more antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa, and E. coli at 37°C than those obtained from shoots, roots, and buds. The lowest minimum inhibitory concentrations and minimum bactericidal concentrations were observed for leaves and seeds as compared to other parts of the plant. No activity of alum was observed against the tested bacterial strains. The seeds of Ocimum bacillicum were associated with higher coagulation activity in comparison to other parts of the plant and were comparable with alum. These results suggest that parts of a plant offer a potential for antimicrobial treatment of drinking water, but needs further exploration.     

Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses.

The invasive properties of Azoarcus sp. strain BH72, an endorhizospheric isolate of Kallar grass, on gnotobiotically grown seedlings of Oryza sativa IR36 and Leptochloa fusca (L.) Kunth were studied. Additionally, Azoarcus spp. were localized in roots of field-grown Kallar grass. To facilitate localization and to assure identity of bacteria, genetically engineered microorganisms expressing beta-glucuronidase were also used as inocula. beta-Glucuronidase staining indicated that the apical region of the root behind the meristem was the most intensively colonized. Light and electron microscopy showed that strain BH72 penetrated the rhizoplane preferentially in the zones of elongation and differentiation and colonized the root interior inter- and intracellularly. In addition to the root cortex, stelar tissue was also colonized; bacteria were found in the xylem. No evidence was obtained that Azoarcus spp. could reside in living plant cells; rather, plant cells were apparently destroyed after bacteria had penetrated the cell wall. A common pathogenicity test on tobacco leaves provided no evidence that representative strains of Azoarcus spp. are phytopathogenic. Compared with the control, inoculation with strain BH72 significantly promoted growth of rice seedlings. This effect was reversed when the plant medium was supplemented with malate (0.2 g/liter). N2 fixation was apparently not involved, because the same response was obtained with a nifK mutant of strain BH72, which has a Nif- phenotype. Also, Western blot (immunoblot) analysis of protein extracts from rice seedlings gave no indication that nitrogenase was present. PCR and Western immunoblotting, using primers specific for eubacteria and antibodies recognizing type-specific antigens, respectively, indicated that strain BH72 could colonize rice plants systemically, probably mediated by longitudinal spreading through vessels.     

Simultaneous fluorescent gram staining and activity assessment of activated sludge bacteria

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.